The conclusion is drawn that Glomus maculosum should also be ... An epitype is defined to aid in the circumscription of G. claroideum. ..... Moonstone. Beach.
Annals of Botany 82 : 601–624, 1998 Article No. bo980714
Synonymy Amongst the Arbuscular Mycorrhizal Fungi : Glomus claroideum, G. maculosum, G. multisubstenum and G. fistulosum C H R I S T O P H E R W A L K E R* and M A U R I T Z V E S T B E R G† * Biological Research and Imaging Laboratory, 2 Penny Hedge, New Milton, Hampshire BH25 7TB UK and † Agricultural Research Centre of Finland, MTT, Laukaa Research and Elite Plant Laboratory, Antinniementie 1, FIN-41330 Vihtauori, Finland Received : 19 March 1998
Returned for revision : 11 June 1998
Accepted : 29 June 1998
Ex-type material of Glomus fistulosum was re-established in pot culture and differences from the description in the protologue noted. Type specimens and other cultures from Europe, and North and South America showed the original species description was incorrect. The species is re-described from pot cultured specimens. The holotype of Glomus claroideum was also examined. The two species were morphologically identical. This was confirmed by comparisons with other specimens of G. claroideum. One culture, used in the protologue, but not designated as a paratype, had smaller spores than those from other G. fistulosum or G. claroideum cultures, but could not otherwise be separated. They are therefore synonymized. Type material of Glomus maculosum and specimens of Glomus manihotis from Colombia were studied. The latter came from a heavily parasitized pot culture, and its spores possessed the maculiferous ‘ ornamentation ’ that supposedly characterized the former. Similar structures were noted in the isotype of G. claroideum. The feature is probably environmentally induced and should not be used as a taxonomic characteristic. The conclusion is drawn that Glomus maculosum should also be synonymized with G. claroideum. Glomus multisubstensum is considered from its protologue, and this too should be synonymized with G. claroideum. An epitype is defined to aid in the circumscription of G. claroideum. Less comprehensive comparisons were also made with other similar species, G. albidum, G. przelewicense, G. lamellosum, G. pustulatum and G. diaphanum. It is concluded that G. lamellosum is inadequately defined, and its validity as a species needs clarification. It is also suggested that Glomus albidum and G. przelewicense may be conspecific. # 1998 Annals of Botany Company Key words : Epitype, Glomus fistulosum, Glomus claroideum, Glomus maculosum, Glomus multisubstensum, G. albidum, G. przelewicense, G. lamellosum, G. pustulatum, G. diaphanum, Glomales, arbuscular mycorrhiza, synonymy, taxonomy.
INTRODUCTION During a survey of arbuscular mycorrhizal fungi in Finland, a species was found and established in pot culture from 63 locations (Vestberg, 1995). Nineteen of these cultures were studied in detail, and similar fungi from Scotland, the Czech Republic and Ireland were also examined to determine whether they were morphologically distinguishable. Whilst attempting to identify the Finnish fungi, comparisons were made with existing descriptions of members of the Glomales, and it was at first thought that they represented a taxon new to science. However, species descriptions of fungi in the Glomales are sometimes inaccurate or incomplete, and the type material of species that might be similar to our fungi were examined where possible. Included in this examination was the culture from which the type material of the fungus Glomus fistulosum Skou & Jakobsen was derived. The specimens from this culture had characteristics at variance with the description in the * E-mail walker!globalnet.co.uk † For correspondence. E-mail Mauritz.Vestberg!MTT.Fi
0305-7364}98}11060124 $30.00}0
protologue (Skou and Jakobsen, 1989), so we examined the holotype material to satisfy ourselves that the type culture remained uncontaminated by other species of Glomus, and that our interpretation of the morphology was correct. Spores from cultures of fungi assigned to the species Glomus claroideum Schenck & Smith were also examined, since the diagnostic characteristics of that species (Schenk and Smith, 1982) fitted the morphological characteristics of G. fistulosum, though not its original description. Similarly, the species Glomus maculosum Miller & Walker was reexamined because it shared some characteristics (Miller and Walker, 1986) with the aforementioned species. A culture of Glomus manihotis Howeler, Sieverding & Schenck from Colombia, showing some of the characteristics of Glomus maculosum, was examined to compare it with the other fungi we had studied. Spores of Glomus przelewicense Błaszkowski were examined from the type material, since the description and illustrations in its protologue (Błaszkowski, 1988) also bore some similarity to that of G. claroideum. Specimens of Glomus multisubstensum Mukerji, Bhattacharjee & Tewari were not available for examination, but its species description (Mukerji, Bhattacharjee and Tewari, 1983) was studied for comparison with the above species. Other similar species, G. albidum Walker & Rhodes, # 1998 Annals of Botany Company
602
Walker and Vestberg—Glomus claroideum Emended
G. lamellosum Dalpe! , Koske & Tews, G. pustulatum Koske et al., and G. diaphanum Morton & Walker were also compared. The taxonomic system of wall structure originally defined by Walker (1983) and later extended by Berch and Koske (1986), Morton (1986), Spain, Sieverding and Schenck (1989) and Walker (1986), was intended only as an artificial system to be used in species description (Walker, 1992), regardless of misrepresentative statements to the contrary (Bentivenga and Morton, 1995 ; Stu$ rmer and Morton, 1997). As such, it is useful for the purposes of comparing and cataloguing the fungi in the Glomales, but is not so likely to be adequate for providing information about homologous developmental characteristics. Nevertheless, it has provided a solid base for critical examination of spores of arbuscular mycorrhizal fungi, and pending a fully scientific evaluation of the wall components in the group, will continue, perhaps with further amendments, to be useful in species descriptions. We therefore have maintained its use, with slight modification, in the following species descriptions. We have substituted the functional term ‘ flexible ’ (Morton, 1995) for ‘ membranous ’ (Walker, 1983). Although the taxonomy of the Glomales is based mainly on light microscopy of intact or lightly crushed spores, there are fundamental difficulties in their study. The compound microscope is intended for observation of thin sections. The depth of focus is limited, and it is often possible to view such sections without serious artefacts. Interpretation of the image is consequently relatively straightforward. In contrast, examining relatively large objects such as spores of arbuscular mycorrhizal fungi, is subject to many problems caused by refraction and refringence, resulting from their shape and thickness. Interpretation of the image must therefore be carried out with these limitations in mind.
MATERIALS AND METHODS Fungal isolation Open pot cultures (Gilmore, 1968) were produced over several years in various ways. Most of the cultures used were produced from more than one spore, and thus cannot be unquestionably assigned to a single genet. Only cultures produced from a single spore are described as isolates because the use of more than one spore as inoculum can produce a culture originating from more than one ortet (Walker, 1992). Nevertheless, with three exceptions, an extype pot culture of Glomus lamellosum, an un-purified pot culture from field soil and a contaminated pot culture of Glomus mosseae, only one morphotype of spore was produced in each culture. In addition to the pot-cultured specimens, some spores obtained directly from field soil were examined.
Specimen preparation Spores were extracted from the soil or pot culture substrate by centrifugation and sugar flotation (Walker, Mize and McNabb, 1982) or by wet sieving and decanting (Gerdemann
and Nicolson, 1963). Specimens were then selected and placed in a dish of water for examination under the dissecting microscope at magnifications up to 50¬ with illumination by incident light from a fibre-optic quartzhalogen light source with a colour temperature of 3200 K (Walker, Gianinazzi-Pearson and Marion-Espinasse, 1993).
Record maintenance Details of original collection and isolation, resultant cultures and subcultures, and herbarium specimens (Tables 1 and 2) have been recorded in a database developed specifically for the purpose. Original soil or plant samples are given a unique registration number. Each culture pot is given an ‘ Attempt ’ number (unique to each culture attempt made from any one original sample) and culture number (sequential for sub-cultures from a successful attempt). Information about cultures and field collections from any collection can be entered into the database, hence attempt and culture numbers do not necessarily indicate that we have maintained the culture ourselves. Semi-permanent microscope slides and, where available, spores in either 5 % formaldehyde or 0±025 % aqueous sodium azide solution, were kept. Voucher specimens of all fresh material have been accessed into the personal herbarium of the first author, each individual collection being given an accession number (‘ W ’ number). Voucher numbers may have been given to specimens borrowed from other collections (e.g. from a herbarium) and subsequently returned. For example, the culture from which the type material Glomus claroideum was obtained was designated Attempt 622–3, (Attempt 622, Culture 3) and the two samples examined from it were accessed as W947 and W2955. The former was a sample received directly from Dr N. C. Schenck, whereas the latter was the holotype loaned by the curator of Oregon State University (OSC). The three subcultures of Glomus fistulosum examined in most detail for this study were Attempts 5–6 (herbarium specimens W1779 and W1840), 5–8 (W1841), and 5–19 (W2844). Further details of the database, which is available by arrangement, can be obtained from the first author.
Morphological features For some of the fungi, taxonomic features were studied from both young (14 weeks) and old (44 weeks or more) growing cultures, allowing observation of developmental sequences in spore wall components and changes in colour with advancing age, although no formal experimental approach was taken to sequencing the spore development. For others, only mature cultures, or cultures of unknown age, were available. Spores extracted from dried or refrigerated pot culture substrate were also examined for some cultures. Spores were examined in water (Spain, 1990), in polyvinyl alcohol lacto-glycerol (PVLG) (Omar, Bolland and Heather, 1979) and PVLG with Melzer’s reagent, 5 : 1 v}v (PVLG}Melzer’s) (Walker et al., 1993) through a compound microscope with bright field and Nomarski differential interference contrast illumination. Spore
Voucher
W1498 W1579 W1779 W1840 W1759 W1841 W2371 W2427 W2780 W2835 W2837 W2844 W2374 W1853 W2115 W1843 W2027 W2269 W1520 W2110 W2111 W2871 W1628
W1688 W1842 W1912 W2029 W1839 W1947 W1972 W2020 W2902 W1872 W2849 W2112 W2113 W2114 W2850
W2851 W2370 W2840 W2852 W2853
Attempt– culture
5–4 5–4 5–6 5–6 5–7 5–8 5–9 5–10 5–10 5–12 5–17 5–19 43–1 74–3 79–2 79–3 79–4 79–6 223–0 223–1 223–3 269–3 271–2
271–4 271–5 271–5 271–5 271–7 271–11 313–2 313–2 313–4 335–3 383–3 420–1 421–0 422–1 435–3
480–3 564–3 632–3 634–3 635–3
Vestberg, Finland O’Neill, Ireland Vestberg, Finland Vestberg, Finland Vestberg, Finland
Gryndler, Czech Republic Walker, Scotland Walker, Scotland Walker, Scotland Gryndler, Czech Republic Gianinazzi-Pearson, France Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland
Jakobsen, Denmark Jakobsen, Denmark Walker, Scotland Walker, Scotland Vestberg, Finland Walker, Scotland Walker, Scotland Walker, Scotland Walker, Scotland Merryweather, England Merryweather, England Vestberg, Finland Walker, Scotland Vestberg, Finland Vestberg, Finland Vestberg, Finland Walker, Scotland Gianinazzi-Pearson, France Vestberg, Finland Vestberg, Finland Vestberg, Finland Vestberg, Finland Gryndler, Czech Republic
Cultures and location
Vestberg Vestberg
Soil trap U multi spore Soil trap U multi spore Multi spore from soil
Soil Soil Soil Soil Soil
Soil Soil Soil Soil Soil Soil
spore spore spore spore spore spore spore spore spore spore spore
trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi trap U multi
Vestberg O’Neill Vestberg Vestberg Vestberg
Vestberg Vestberg Vestberg Vestberg Vestberg Vestberg
Vestberg
Walker Vestberg Vestberg
Soil trap Soil trap U multi spore
1 root fragment U multi spore
Jakobsen
Original collector
Multi spore from soil
Original culture method
V184 Irish. UCDI V12 V187 V198
V13 V170 V138 V107 V127 V174
V112a
V151 23A
V92
Foul1 V14b V128
No. 21 Ex-type
Other identifier and notes
Kuhmo Peta$ ja$ vesi Ja$ msa$ Uurainen Laukaa Jyva$ skyla$ n maalaiskunta Ja$ msa$ Ardattin Nurmes Ja$ msa$ Korpilahti
Orivesi
Korpilahti Novy Bydzov
Na$ rpes
East Newton Ka$ rsa$ ma$ ki Laukaa
Askov lermark
Town
Finland Ireland Finland Finland Finland
Finland Finland Finland Finland Finland Finland
Finland
Finland Czech Republic
Finland
UK Finland Finland
Denmark
Country
Europe Europe Europe Europe Europe
Europe Europe Europe Europe Europe Europe
Europe
Europe Europe
Europe
Europe Europe Europe
Europe
Continent
T 1. Geographic and culturing information for specimens of Glomus fistulosum examined in a comparatie study of similar species in the arbuscular mycorrhizal genus Glomus
Walker and Vestberg—Glomus claroideum Emended 603
Voucher
Culturer and location
Originally assigned species name Specimens of Glomus claroideum 57–6 W1404 Camprubi, Spain 57–7 W1467 Walker, Scotland 57–9 W2466 Walker, Scotland 105–2 W2709 Walker, Scotland 193–2 W1871 Rosendahl, Denmark 193–4 W2417 Rosendahl, Denmark 193–8 W2499 Dodd, England 193–9 W2721 Walker, Scotland 251–1 W2839 Merryweather, England 251–1 W2847 Merryweather, England 474–0 W388 Daft, Scotland 597–5 W2503 Morton, USA 597–6 W2504 Merryweather, England 597–6 W2846 Merryweather, England 598–5 W2506 Bentivenga, USA 598–6 W2507 Merryweather, England 598–6 W2845 Merryweather, England 622–3 W947 Schenck, USA 622–3 W2595 Schenck, USA None W538 Field collected None W1149 Field collected None W2191 Field collected None W2984 Field collected Specimens of Glomus sp. (W2494) 12–8 W2494 Walker, England Specimens of Glomus maculosum 491–0 W505 Miller, USA 491–0 W568 Miller, USA 491–1 W531 Miller, USA 491–2 W765 Miller, USA
Attempt– culture
Błaszkowski Rosendahl
Walker
Skipper
Ming Lin
Schenck Koske Walker Walker Walker
Soil trap Unknown
Soil trap U multi spore Pot culture contaminant Pot culture contaminant
Unknown
Unknown Field Field Field Field
Miller
Soil trap Soil trap U multi spore Soil trap U multi spore
Newsham
Plant trap U single spore
collection collection collection collection
Unknown
Original collector
Unknown
Original culture method
Isotype Ex-type Ex-type Ex-type
‘ lemchrome ’
Isotype Holotype Koske 432
Authenticated
Other identifier and notes
Sturgeon Bay
Mildenhall
Assateague Island Roslin Tabernas Burascund
Sanford
Campinas
Dundee Clemson
East Newton
Leba Unknown
Unknown
Town
USA
England
USA Scotland Spain India
USA
Brazil
Scotland USA
Scotland
Poland Denmark
Scotland
Country
North America
Europe
North America Europe Europe India
North America
South America
UK North America
Europe
Europe Europe
Europe
Continent
T 2. Geographic and culturing information for specimens of Glomus spp. examined in comparison to Glomus fistulosum in a study of similar species in the arbuscular mycorrhizal genus Glomus
604 Walker and Vestberg—Glomus claroideum Emended
Unknown
Dalpe!
Specimens 288–1 288–1 288–1 288–4 456–0 None Specimens None Specimens 627–0 Specimens 641–0 641–0
of Glomus W79 W169 W170 W258 W282 W179 of Glomus W2957 of Glomus W2828 of Glomus W949 W952
albidum Rhodes, USA Rhodes, USA Rhodes, USA Walker, USA Sanders, England Field collected przelewicense Field collected, Poland manihotis Dodd, UK diaphanum Rhodes
Sanders Walker Błaszkowski Dodd Morton
Multi spore from soil
Soil trap Field collection Field collection Multi spore Multi spore
244–4 W2117 Walker, Scotland 244–5 W2392 Dodd, England 244–4 W2432 Walker, Scotland 244–6 W2712 Walker, Scotland 244–6 W2868 Walker, Scotland 472–0 W385 Daft, Scotland Unknown Daft Specimens of Glomus pustulatum 368–0 W2066 Walker, Scotland Soil trap Walker None W655 Field collected, USA Field collection Koske None W1064 Field collected, USA Field collection Friese Specimens of Glomus multisubstensum considered from protologue description only None None Field collected, India Field collection Bhattacharjee
Specimens of Glomus lamellosum 244–3 W1707 Dalpe! , Canada
Delhi
Holotype. DU}KMB 500
Morton 73
Colombia
No. 578
Paratype
India
Scotland USA USA
UK
Canada
Elkins
Unknown
Przelewice
Bramham Rhodes
USA
Colombia
Poland
England USA
Washington Township, Ohio USA
Cambuslang Moonstone Beach Moonstone Beach
Hallside Isotype. Koske 534 Isotype. Friese 33
Ex-type Isotype Ex-type Ex-type
Dundee
Wasaga Beach Provincial Park
Culture contaminant
Ex-type
North America
South America
Europe
Europe North America
North America
India
Europe North America North America
Europe
North America
Walker and Vestberg—Glomus claroideum Emended 605
606
Walker and Vestberg—Glomus claroideum Emended
measurements were made from newly made microscope slides of spores in PVLG. Spore colour was determined under the dissecting microscope from spores suspended in water. Colours were matched by name and number [e.g. sienna (11)] with a colour identification chart (Anon, 1969) illuminated simultaneously with a split fibre optic from the source used to illuminate the spores. The paler colours on the chart lack names, so they have been assigned the following : 1, white ; 2, ivory ; 3, pale yellowish cream ; 4, pale pinkish cream ; 5, yellowish cream ; 6, pale ochraceous ; (7, white, repeated) ; 8, ochraceous ; 9, ochre. Description of spore wall structures follow the conventions of Walker (1983) with some modification to accommodate recent arguments about the nature of spore wall components. There has been much discussion about the use of the terms ‘ wall ’ and ‘ wall layer ’ in the taxonomy of the arbuscular mycorrhizal fungi (Berch, 1986 ; Walker, 1992 ; Stu$ rmer and Morton, 1997), stimulated by the inability to define with certainty the true nature of the spore in the Glomales. It is unlikely that all the spore types formed by members of this order are homologous. Some may indeed be simple asexual spores, whereas others are very complex in structure, and may be sporangia or sexual structures. Until this controversy is resolved, it can be no more correct to use one term than the other. We have therefore chosen to use the term ‘ component ’, as a neutral choice, when describing the elements of wall structure in glomalean spores, and to retain the use of the term ‘ group ’ when one or more of these components separate into an assemblage in crushed spores on microscope slides. Measurements of spore dimensions were made with eyepiece graticules calibrated with a stage-micrometer. For statistical analysis, it is necessary to prevent bias in selecting specimens for measurement. To achieve this, the spores were extracted and placed in a dish of water under a dissecting microscope. The dish was marked into squares corresponding with the field of view at 50¬magnification, and scanned systematically from top left to bottom right. All spores in each field were picked out and placed in a watch glass of water. They were then transferred, with a pipette or fine forceps, to microscope slides. The slides were scanned in a similar manner to the dishes until 100 had been measured, so that no biased selection of specimens could take place. For a few of the samples, more than 100 spores had been measured during previous work. In these instances, the entire set of measurements was taken and random observations were deleted until 100 remained. Some samples were of fewer than 100 spores. These, and the data excluded from sets of more than 100 measurements, have been included in general consideration of the species and in determination of maximum and minimum spore dimensions, but have not been used in statistical analysis. At least one set of measurements was made for each attempt examined, and in some instances, two or three samples were measured, either from the same, or from different cultures. The convention that length includes all spore wall components, and is measured perpendicular to the spore base (subtending hypha) is followed (resulting in the possibility of spores being ‘ broader than long ’). The width is taken as a line across the broadest point at right angles to the length.
Reaction to Melzer’s reagent The reaction of the spore wall components to PVLG} Melzer’s was routinely assessed when fresh specimens were prepared on microscope slides. By the nature of pot cultures, most of these were from mature spores, although some immature spores were inevitably also included. An experimental approach was taken for Glomus fistulosum : 100 fresh and 100 dry spores from each of five cultures were mounted in PVLG}Melzer’s and individually examined. Germination characteristics Germination characteristics were noted, where information was available, from the protologues and other published descriptions. In addition, where germinating spores could be seen in other collections, their characteristics were noted. In an experimental assessment, spores (from Attempt 627 in Vestberg’s database) were retrieved from air-dried pot culture substrate which had either been stored at 8 °C (three trials), or had been frozen to ®20 °C (six trials) for 2 or 6 weeks. Those spores that had been frozen were gradually thawed at 4 °C for 24 h, after which apparently healthy spores were selected under a dissecting microscope, surface disinfested for 2 min in 0±5 % NaOCl, and washed in three changes of sterile water. Excess liquid was then absorbed on sterile filter paper, and the spores were transferred to 1±5 % Difco water agar (pH 5±5), and incubated in darkness for up to 8 weeks. The number of spores used in different trials varied between 16 and 40. Spore germination recording began 7 d after starting the incubation. For observation of germination characteristics and for photography, germinated spores were stained in lactophenol-methyl blue. Specimens examined All the species currently described in the genus Glomus with species descriptions similar to that of G. fistulosum were considered. Where possible, specimens, both preserved material and fresh cultures, were examined, and compared with their protologues and with G. fistulosum. Some species were unavailable as specimens, and so they are considered solely from their descriptions. As well as the fresh material specifically produced for this study, relevant preserved material in Walker’s personal herbarium was included (Tables 1 and 2). Glomus fistulosum Skou & Jakobsen. Mycotaxon 36, 274 (1989) The holotype of Glomus fistulosum (Attempt 5–4) was studied at the herbarium of the University of Copenhagen. It consists of microscope slide preparations, from pot number 21, a multi-spore culture from the Agricultural Research Department at the Risø National Laboratory, Denmark (Skou and Jakobsen, 1989). No voucher number was given to this collection. In addition to the type material, 12 samples from nine different ex-type subcultures (including
Walker and Vestberg—Glomus claroideum Emended two from the ‘ type ’ pot culture itself) were examined (Table 1). Two cultures assigned to this species were donated by colleagues. One, from the Czech Republic, was established by inoculating a host plant in sterile substrate with 20 morphologically similar spores from soil (Gryndler, 1995). From this culture, seven samples from a total of five pot cultures were examined (Table 1). A sample from an Irish isolate (W2370) started with a single-spore from a tree nursery soil trap, was studied from a closed pot culture (Walker and Vestberg, 1994). One other sample of G. fistulosum (W2374) was examined from a mixed-species soil trap (Attempt 43–1) from a site in Scotland. All except one of the 21 Finnish arbuscular mycorrhizal fungi (Table 1) were baited out in open pot culture (Vestberg, 1995) by mixing soil from the field with a sterile substrate in a pot, and sowing with a suitable host (soil trap culture). This was followed by establishment as a single-taxon culture by extracting spores from the pots which were then placed with a suitable host in sterile substrate (multi spore cultures). The exception, Attempt 313–2 was produced by inoculating a mycorrhiza-free host plant with a single root fragment. A multi spore culture was subsequently made from the resultant pot culture, producing samples W1972 and 2020. The fungus has been recorded as mycorrhizal with a large number of plant hosts. Glomus claroideum Schenk & Smith. Mycologia 74, 84 (1982) Among the samples of Glomus claroideum examined (Table 2), were an isotype sample (W947, from Dr N. C. Schenck) and the holotype W2595 (¯ OSC accession number 40252), loaned by the herbarium of Oregon State University. The spores of W2959 were either preserved in lactophenol, or had been mounted on lactophenol slides. The slides had dried out and were re-made with PVLG. In addition, new PVLG-mounted slides were made from the lactophenol-preserved material. All living material of this culture seems to be lost, so another culture was obtained from Dr J. Merryweather, University of York, UK (dried inoculum of INVAM SC186 ; W2503) later examined from an active pot culture (UY1053, W2504 and W2846). This fungus was specifically mentioned in the protologue, but not formally designated as a paratype. Although lacking any formal taxonomic status, by merit of its special mention in the protologue, this isolate provides living evidence of the species as conceived by Schenck and Smith (1982). This fungus originated as a pot culture contaminant from South Carolina, and was first cultured by Dr H. Skipper, of Clemson University, North Carolina. Another organism found contaminating a pot culture of Glomus mosseae (Nicolson & Gerdemann) Gerdemann & Trappe contributed by Dr M. J. Daft, University of Dundee, UK (W388), was found to fit the description of G. claroideum. A second culture was provided by York University, both as dried inoculum from INVAM (BR-147A, W2506) and from the substrate of an active pot culture (UY1052, W2507 and W2845). This fungus, which originated from Brazil, and
607
Skipper’s North Carolina culture, were used in a recent informal re-description of Glomus claroideum (Stu$ rmer and Morton, 1997). Two cultures had origins in Scotland. Attempt 57 came, via Rothamsted Experiment Station, to Cabrils, Spain (contributed by Dr A. Camprubi). Other than that it originated from peat (pH 6±0) from ‘ a hillside in Scotland ’, nothing else is known about this culture. Two cultures of this fungus were examined, represented by the voucher specimens W1404 and W2466. The second Scottish culture, made and purified by Dr J. Merryweather as UY73, was produced from a sample taken near the type locality of Scutellospora calospora (Nicolson & Gerdemann) Walker and Sanders, at Denhead of Foulis, Perthshire. Specimens were studied from an original soil trap (W2839) and a subsequent multi-spore, single-species culture (W2847). Another culture of G. claroideum was provided from Denmark by Dr S. Rosendahl, but without details of its origins or culturing history. Four different subcultures of this were examined. Specimens were retained as W1871, W2417, W2499 and W2721. Another mainland European culture from a Polish soil trap culture established by Dr J. Błaszkowski was studied (W2709). In addition to the cultured samples, four field collections yielded specimens of Glomus claroideum. One, which came from Assateague Island, Maryland, USA, was donated by Dr R. E. Koske (W538). Two were from Europe (Scotland, W1149 and Spain, W2191), and the fourth was from India (W2894). Mycorrhizas are formed with a large range of host plants. Glomus maculosum Miller & Walker. Mycotaxon 25, 218 (1986) As with G. claroideum, no living material of the soil trap type culture or the multi-spore subculture of G. maculosum could be found, but preserved material (W505) of the isotype and ex-type sub-cultures (W531, W568 and W765) (Table 2) was examined. Locating living material of these cultures proved impossible ; they were apparently lost from Iowa State University and the INVAM collection in Florida. This fungus, originally cultured from an apple orchard in Wisconsin, USA has been grown in pot culture with Malus domestica, Sorghum sudanense and Coleus x hybridus. Glomus lamellosum DalpeU , Koske & Tews. Mycotaxon 43, 289 (1992) Six samples from among four ex-type cultures (Table 2) (with Allium porrum) of G. lamellosum (W1707, W2117, W2432, W2392, W2712 and W2868) were examined. It is not known if the original culture was from a soil trap or a purification through selected spores. Glomus pustulatum Koske, Friese, Walker & DalpeU . Mycotaxon 26, 143 (1986) Two voucher collections of this were examined. One was from Koske (W655) and the other by Friese (W1064) on the same day. Later enquiries revealed that these were from the same sample, and they were both subsamples of the isotype.
608
Walker and Vestberg—Glomus claroideum Emended
The species was also found and pot-cultured in Canada, but we have not seen specimens. It was also recovered from a soil trap pot culture (Plantago lanceolata L.) started from an old coal mine spoil heap in Cambuslang, Scotland (W2066). In addition to being symbiotic with P. lanceolata, this species is known, from the paratype, to form arbuscular mycorrhizas with Lathyrus maritumus (Koske et al., 1986).
it was thought that this was an example of G. maculosum, but later examination showed it to be a heavily parasitized G. manihotis (W2828). It is included in this study (Table 2) because some of its characteristics cast light on to the comparison of G. maculosum with G. claroideum.
RESULTS Glomus multisubstensum Mukerji, Bhattacharjee & Tewari. Transactions of the British Mycological Society 81, 3 (1983) No specimens were available for examination. Enquiries of the herbarium at Delhi (DU), and of Drs Mukerji and Tewari have revealed that the type material has been lost and no ex-type culture can be found. The protologue was used to study the characteristics of this fungus, which was reportedly mycorrhizal with Zea mays (Mukerji et al., 1983). Glomus albidum Walker & Rhodes. Mycotaxon 12, 509 (1981) As with so many of the valuable cultures from the past, all known ex-type pot cultures have been lost or discarded. Cultures existed at Iowa State University, Ohio State University and INVAM, Florida, but none could be located despite repeated enquiries. Consequently, only preserved specimens have been examined. Four samples of the original type culture [W79, W169 (isotype), W170 and W258] were studied. Two other collections, one from a field collection in Iowa, USA (W179), and one from a soil trap culture from Yorkshire, England (W282) were examined (Table 2). The fungus has been grown in symbiosis with Zea mays, Sorghum ulgare and Populus x euramericana. Glomus przelewicense Błaszkowski. Bulletin of the Polish Academy of Sciences. Biological Sciences 36, 272 (1988) The holotype collection, consisting of 20 microscope slides, was kindly made available for examination by Dr J. Błaszkowski. All except one (which had two spores) of the slides, contained only a single spore mounted in PVLG. It is known only from a field collection of soil beneath Thuja occidentalis (Błaszkowski, 1988). Glomus diaphanum Morton & Walker. Mycotaxon 21, 433 (1984) Isotype (W949) and ex-type (W952) material from the original soil trap culture with Sorghum sudanense was studied from existing microscope slides in polyvinyl alcohol lactophenol (PVL). No fresh material was examined. Glomus manihotis Howeler, Sieerding & Schenck in Schenck et al. Mycologia 76, 695 (1984) A sample of a multi spore culture (host unknown) from Colombia was provided by Dr J. C. Dodd, International Institute of Biotechnology, Canterbury, Kent, UK. At first,
Colour In general appearance, the spores of G. fistulosum, G. claroideum and G. maculosum (except for the spotted appearance of older spores) differ only slightly (Figs 1–5), and there was as much variation within cultures as among them. Their appearance also changed over time with storage in different preservatives, mainly because of changes in colour and degradation of the outer wall components. In the protologue, the colour of the spores of G. fistulosum was described as pale yellow, and no mention is made of variation. Colours of stored and fresh specimens from the ex-type cultures in Attempt 5 of G. fistulosum varied, but could loosely be described as shades of yellow. The palest spores were ivory (2), and the darkest normally ochre (9), though in one sample they were pale apricot (pale 47). They also varied with time of storage, gradually becoming much darker once detached from their living host. For example, samples from Attempt 5–19 were uniformly straw (50) in colour, even after 9 d in water at room temperature, but after a further 9 d, they varied between straw and ochraceous (8). Colour of other isolates assigned to G. fistulosum also varied (Table 3), but still within a broad definition of various shades of yellow, and with the palest being ivory (2) and the darkest usually ochre (9), but occasionally apricot (47). Because the spores in the type material of G. claroideum have been preserved in lactophenol they have discoloured. No culture of this isolate could be located to determine the colour of living spores. The protologue describes them as ‘… hyaline to yellow, becoming yellow-brown with age …’, but no reference is made to any colour standard. This range is consistent with our observations, in which we found the spores to be (rarely) hyaline or white (1), and normally various shades of yellow, from ivory (2) to pale yellowish cream (3) to ochre (9) through yellowish cream (5) to pale ochraceous (6) to ochraceous (8). The colour of G. maculosum spores from different samples of the same culture was not consistent. However, those from Attempt 491–0 [yellowish cream to pale ochraceous to ochraceous (5–6–8)], were assessed after storage in 5 % formaldehyde solution. Attempt 491–1, assessed from freshly extracted spores, was pale straw (50) to ochraceous (8), but both results can be broadly interpreted as various shades of yellow. The protologue of G. lamellosum gives the colour of these spores as hyaline (when young), lemon yellow to light yellow at maturity (No. 86 on the ISCC NBS Color Name Charts), equivalent to between yellowish cream and pale ochraceous (5 and 6) on the RBG chart. One of the samples, W385 (the pot culture contaminant from Attempt 472–0), was recorded only as an unmatched colour, pale yellow. The
Walker and Vestberg—Glomus claroideum Emended first ex-type sample (W1707, Attempt 244–3), had spores that were ivory (2) when young, and pale ochraceous to ochraceous (6–8) at maturity. A sample W2117, from Attempt 244–4 had yellowish cream to straw (5–50) spores. Spores (W2432) from the same pot culture almost a year later were white to yellowish cream to pale ochraceous (1–5–6). Two more samples of this fungus, both from Attempt 244–6, were assessed. In the first, W2712, in which spores were described as hyaline to pale yellow, no chart was used. Specimens from a later sample, W2868, were ivory, pale ochraceous, ochraceous, or, rarely, ochre (2–6–8–9). We have not seen fresh spores of G. pustulatum, but they are described in the protologue as ‘ … pale yellow to yellowbrown or orange-brown … ’, though no chart was used to provide colour matching. Similarly, there was no fresh material of G. multisubstensum available for examination. Its spores are described as ‘ light brown ’, and the wall layers as ‘ brown ’ or ‘ pale yellow brown ’ in the species description. No colour chart matching was carried out on living material of G. albidum, but from original records spores are hyaline when young, white to off-white at maturity, appearing yellowish to brownish yellow by transmitted light. This colour is maintained in samples preserved in 5 % formaldehyde solution. According to the original species description, spores of G. przelewicense are yellow (2.5Y 8}8), equivalent to ochraceous on the RBG chart. In the original description, Blaszkowski describes a darkening of the colour to reddish yellow (5YR 6}6) when viewed through a compound microscope. A similar characteristic was recorded for G. albidum (Walker and Rhodes, 1981). Spores of G. diaphanum are consistently hyaline, in contrast to all fungi studied here, which, whilst sometimes being hyaline when immature, are pigmented at maturity.
Spore dimensions For G. fistulosum mean spore dimensions ranged widely (Table 4, Figure 29), but generally could not be separated statistically when considered as a group (Table 5). Both length and width measurements for two different samples of G. fistulosum from the Czech Republic (Attempts 271–5 and 271–7) were significantly different (P % 0±05). Sizes of spores in G. fistulosum are given as 67–220¬78–200 µm in the protologue, and from our studies this can be modified slightly, including the protologue measurements to 60– 220¬60–243 µm. The size range of G. claroideum is quoted as 70–180 µm diameter (mean 130) when globose, and 59–126¬72–145 µm when subglobose to irregular. Our measurements of type material (n ¯ 34) 95–175¬92–205 (mean 130¬133), and (n ¯ 100) (63)–81–181¬(51)–86–188 (mean 129¬130) µm, extend the maximum spore dimensions somewhat. Stu$ rmer and Morton (1997) give a size range of (100)–120–140 (–180) µm diameter, falling within our new range, but erroneously implying that the spores are all globose. Many spores had an apparent break in development of laminated wall component, giving the impression of two separate laminated components. Our other measurements (Table 3)
609
result in a composite measurement of 63–199¬70–226 µm. Spores of the type are globose to subglobose, with a remarkably narrow size range and little variation. Of 100 measured spores in the holotype, W2595, 74 were globose, 13 broader than long, and 13 longer than broad. A number of spores (11 out of 100) had two subtending hyphae. Spore dimensions of G. maculosum have the range (95)–102–178 (–220)¬(95)–102–178 (–220) µm. This is slightly changed from the protologue as a result of new measurements. From 100 spores of W505, the mean is 138¬139 µm. The subtending hypha behaves in the same way as that of the G. claroideum and G. fistulosum cultures examined. In the protologue, dimensions of G. lamellosum spores are given as (98)–106–142¬122–162 µm. Measurements of 100 spores from an ex-type culture (W2868 from Attempt 244–6) resulted in the dimensions of 84–160¬81–174 (mean 219¬174) µm extending the upper range somewhat. The ‘ volunteer ’ specimen from a pot culture in Scotland, W385, was 100–107¬82–169 (mean 130¬131) µm. The spore sizes for G. pustulatum are quoted as (43)– 86–140¬(60)–86–140 µm in the protologue, and no other measurements are available. Glomus multisubstensum, also from the protologue only, has spore dimensions of 100– 150 µm, which are within the dimensions of G. claroideum. Dimensions in the protologue of G. albidum are given as (85)–95–168 (®198)¬(85)–95–168 (®177) µm. New measurements on preserved material extend the lower range somewhat, resulting in (68)–95–168 (®198)¬(50)–95–168 (®177) µm. All spores in type of G. przelewicense are crushed, and consequently cannot be re-measured. They are described in the protologue as mostly globose to subglobose, (120)–137 (®160) µm diameter, or ovoid and 160¬100 µm. The spores of G. diaphanum are mainly globose, but can also be ellipsoid, particularly in roots. Their dimensions, combined from the species description are (36)–74–86¬(39)–64 (–132) µm.
Wall structure The wall structure of the ex-type material of G. claroideum and G. fistulosum did not correspond with the original species description, though the fistules, used to characterize the latter, were evident in moribund spores of both species. The spores of G. fistulosum have a muronym (Walker, 1986) of A(EL)B(F), which is much simpler than that originally described. The innermost wall component is unitary, though its flexibility and consequent collapse and folding in the mounting medium can give an erroneous impression of multiple layering, and in many specimens is completely separated from the other wall components (Figs 19–21). In contrast, the wall structure description of G. claroideum is slightly more complex than described, being the same as G. fistulosum. Because of its age and state of preservation, the evanescent component can be detected only from remnants, and the innermost, flexible component is quite difficult to see (Fig. 11). The laminations have, on some specimens, separated to give an erroneous impression of multiple laminated components (Fig. 9). On those few
610
1
Walker and Vestberg—Glomus claroideum Emended
500 µm
500 µm
2
100 µm
15
100 µm
16
50 µm
3
500 µm
500 µm
500 µm
250 µm
6
5
50 µm
18
17
4
100 µm
19
Gt
i
25 µm
100 µm
20
s 100 µm 7
150 µm
100 µm
8
22
21
Gt
50 µm
Gt
Gt
50 µm 50 µm
50 µm 10
9
24
23 25 µm
V
150 µm
3 2 50 µm 12
11
150 µm
1
50 µm
V 26
25
150 µm
100 µm
Lv
13
100 µm
Scar 14
27
28
F 1–5. Spores in a dish of water, all at the same magnification. Unless otherwise stated, the samples are freshly extracted from a pot culture. The epithets in the legends are those used when first identified. Fig. 1. An ex-type sample of Glomus fistulosum. Fig. 2. Part of the holotype of G. claroideum after preservation in lactophenol. The preservative has caused considerable darkening of the specimens. Fig. 3. The culture of G. claroideum from South Carolina relied on in a partial redefinition of the species by Stu$ rmer and Morton (1997). Fig. 4. A Brazilian culture of G. claroideum, also used by Stu$ rmer and Morton (1997). Fig. 5. Part of the isotype of G. maculosum after preservation in 5 % formaldehyde solution.
Walker and Vestberg—Glomus claroideum Emended spores where the subtending hypha can be seen, this flexible structure is completely separate from the subtending hypha, although it may bulge into it. For the living specimens of G. claroideum, the wall structure of the spores had a muronym of A(EL)B(F) in all specimens examined. Studying the living cultures, we found the mature wall structure to be of three or four components (depending on interpretation) in two groups. The first group (group 1), which forms the main structure of the spore, consists of one thin, hyaline component that disappears as the spores age (designated E in the muronym), and a thicker, pigmented, laminated component (L). The evanescent component may also be partly covered by a mucigel-like substance, interpreted by Stu$ rmer and Morton (1997) as a layer of the outermost wall of the spore, but on our specimens this is a rare occurrence. This (and the evanescent component) may or may not be present on both immature and mature spores, and we do not consider that the evidence is sufficiently strong to ascertain whether it is really part of the wall structure, or an exudation similar to that produced in G. iscosum Nicolson (Walker et al., 1995). It is unlikely that light microscopy will be capable of resolving this, and its clarification will probably require ultrastructural examination of spores, subtending hypha and sporogenous mycelium. The second group is a single, flexible component which does not appear layered under the light microscope. In the freshly extracted, living material, it is easier to observe the manner of its formation than in preserved, fixed specimens. It appears to be produced only after completion of group 1, and by light
611
microscopy, appears to form directly from the cytoplasm bounded by group 1. From our light-microscope observations, we consider that it is not continuous with either component of group 1, but forms a distinct entity which might be termed an ‘ endospore ’. In G. maculosum, the spores begin growth and appear to reach their ultimate size, with a simple, two layered structural wall, consisting of an evanescent component and a very finely laminated component. In an identical manner to G. fistulosum and G. claroideum, as the spore develops, a flexible innermost component becomes evident. At maturity, this third component appears to be completely separated from the subtending hypha and to form independently of it. Sometimes, there is a thin septum in the subtending hypha, near the spore base, but it is not consistently formed, and it is continuous with the innermost laminae of the laminated wall component. The same type of structure (Fig. 21) can be seen in both G. fistulosum and G. claroideum. The voucher collection of G. lamellosum (W1707) was obtained directly from Canada as authenticated ex-type material. Spores have a wall structure of A(EL)B(F), although because of fractures or separations in the laminated component and loss of others, it could be interpreted as A(ELL)B(F) or A(LF) or even just A(L). The outermost wall on some specimens consists of two layers. This could be considered to be two different laminated components, but is probably just a split lamina, since most spores have only one. The inner wall component seems to form an endosporelike structure, but is very difficult to detect on most
F. 6. Spores produced sparsely in the cortex of a root from an ex-type pot culture of G. fistulosum. F 7–8. Spore formation from two subtending hyphae. Fig. 7. Two adjacent subtending hyphae from an ex-type pot culture of G. fistulosum. Fig. 8. Two opposite subtending hyphae from the isotype of G. claroideum. F 9–12. Wall structure of Glomus claroideum (9–11 from the isotype). Fig. 9. Separation (arrow) of the layers in the laminated wall component giving the erroneous impression of two separate components. Fig. 10. Early development of the inner, flexible wall group (wall component 3) (arrow). Fig. 11. A later stage in development. Wall component 3 (arrow) is fully developed and thickened, and, despite the specimen being crushed, remains close to the laminated component as would normally be the case in an intact spore. Fig. 12. A young spore of G. fistulosum from Finland showing all three wall components (1, 2 and 3). Group B, consisting of component 3, has separated from the laminated structural component of wall group A. F 13–14. Formation of internal maculae. Fig. 13. A spore from the G. maculosum isotype, showing the maculae from which it was named. Fig. 14. A spore of the G. claroideum isotype, showing structures identical to those used to define G. maculosum. F 15–17. Subtending hyphae of G. claroideum and spores originally identified as G. fistulosum. Fig. 15. An immature spore, from Finland, showing the persistent subtending hypha (arrow). Fig. 16. A mature spore of G. claroideum (from the isotype) showing the normal considerable reduction of subtending hypha (arrow). Fig. 17. Detail of the spore base of a maturing specimen from Finland. The subtending hypha, still persistent in this specimen, is somewhat recurved. The outer evanescent wall component is arrowed near the spore base. F 18–21. Inner wall group (wall component 3), all from Finnish collections identified originally as G. fistulosum. Fig. 18. This specimen shows the intrusion of the innermost wall component to give the erroneous impression of a septum (arrow). Fig. 19. Here, the innermost wall group appears as an ‘ endospore ’ after gentle crushing on a microscope slide. Fig. 20. In this specimen, the innermost wall group has separated as an ‘ endospore ’ and collapsed after heavy crushing. The point of intrusion into the subtending hypha is arrowed. Fig. 21. At higher magnification all wall components can be seen. Remnants of the evanescent component can be seen externally (bottom), and the septum sometimes formed by the laminated component is evident (s, arrowed). The flexible component shows the intrusion into the subtending hypha (i, arrowed) at a higher magnification than Fig. 20. F 22–24. Germination of spores all from Finnish cultures originally identified as G. fistulosum. Fig. 22. Multiple germination tubes emerging directly through the spore wall (Gt, arrowed), stained in methyl blue. Fig. 23. A germ tube (Gt) emerging by regrowth from the subtending hypha, generally regarded as the normal method for species of Glomus. Fig. 24. Greater detail of germ tubes (Gt) emerging directly through the spore wall. F 25–26. Roots, cleared and stained to reveal typical Glomus vesicular arbuscular mycorrhizas, with vesicles labelled (V). Fig. 25. Mycorrhiza with Paspalum notatum from the G. claroideum isotype collection. Fig. 27. Mycorrhiza with Plantago lanceolata from a Finnish pot culture originally identified as G. fistulosum. F 27–28. Evidence of contamination in type material of Glomus claroideum. Fig. 27. Mycorrhiza on P. notatum with a lobed vesicle, probably of Acaulospora laeis. Fig. 28. A spore of A. laeis showing the scar (arrowed) at the point of spore formation typical of members of this genus.
612
Walker and Vestberg—Glomus claroideum Emended T 3. Colours of Glomus claroideum spores and of other species compared for similarity
Epithet
Voucher
Attempt–culture
claroideum claroideum claroideum claroideum claroideum claroideum claroideum claroideum claroideum
W2191 W538 W1149 W1404 W1467 W2709 W2499 W2721 W2839
Field sample Field sample Field sample 57–6 57–7 105–2 193–8 193–9 251–1
claroideum
W2847
251–1
claroideum claroideum claroideum
W388 W2503 W2846
474–0 597–5 597–6
claroideum claroideum claroideum claroideum
W2056 W2845 W2507 W947
598–5 598–6 598–6 622–3
claroideum
W2595
622–3
fistulosum fistulosum
W1498 W1779
5–4 5–6
fistulosum fistulosum fistulosum
W1840 W1759 W1841
5–6 5–7 5–8
fistulosum
W2371
5–9
fistulosum fistulosum fistulosum fistulosum
W2427 W2835 W2837 W2844
5–10 5–12 5–17 5–19
fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum
W2374 W1853 W1843 W2027 W2269 W1520 W2871
43–1 74–3 79–3 79–4 79–6 223–0 269–3
fistulosum
W1912
271–5
fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum
W2029 W1839 W1947 W1972 W2020 W2902 W1872 W2849
271–5 271–7 271–11 313–2 313–2 313–4 335–3 383–3
fistulosum fistulosum fistulosum fistulosum fistulosum
W2112 W2114 W2850 W2851 W2370
420–1 422–1 435–3 480–3 564–3
Colour White (1) (Hyaline—one spore) to ochre to fulvous (Hyaline–9–12). Hyaline to pale yellowish cream (Hyaline–3) Hyaline or white to pale ochraceous when older (Hyaline or 1–6). Hyaline to white (Hyaline–1) Hyaline to pale yellowish cream. Pale yellowish cream to pinkish cream to yellowish cream (3–4–5) White to ivory to pale yellowish cream (1–2–3) Hyaline to ivory when fresh. Ivory to pale ochraceous after 16 d in 0±025 % sodium azide solution. (Hyaline–2 or 2–6) Ivory to pale yellowish cream to yellowish cream to pale ochraceous (2–3–5–6) Pale ochraceous to ochraceous (6–8) White to pale yellow (no chart used) (Ivory to) pale yellowish cream to yellowish cream to pale ochraceousochraceous (to ochre) [(2–)3–5–6 (–8)] Ivory to pale yellowish cream to yellowish cream (2–3–4) Pale yellowish cream (4) Ivory to pale yellowish cream (2–3) Hyaline to light yellow, becoming brown (after protologue, no chart used) Sienna to brick in these discoloured lactophenol-stored specimens. (11–15) Yellowish cream to pale ochraceous, a few pale apricot [5–6 (–47)] Cream to yellowish cream to pale ochraceous (to ochraceous) [3–5–6 (–8)] Ivory to yellowish cream to ochraceous (2–5–6) Cream to yellowish cream to pale ochraceous (3–5–6) Ivory to yellowish cream to pale ochraceous rarely ochraceous [2–5–6 (–8)] Ivory to pale yellowish cream to yellowish cream to ochraceous to ochre (2–3–5–6–8–9) Yellowish cream to ochraceous (5–8) Ivory to yellowish cream (to pale ochraceous) [2–5 (–6)] Ivory to pale yellowish cream to yellowish cream (2–3–5) Straw (50) after 9 d. Straw to ochraceous (50–8) after 16 d Pale yellowish cream to pale ochraceous (3–6) Ivory to pale pinkish cream to yellowish cream (2–4–5) Ivory to pale yellowish cream (2–3) Ivory to pale ochraceous (2–6) Ivory to yellowish cream (2–5) Pale ochraceous to buff a few spores apricot [6–52 (–47)] Dried sample ; ochraceous (8) Fresh sample ; ivory to pale yellowish cream (2–3) Ivory to pale yellowish cream to yellowish cream to pale ochraceous (2–3–5–6) Ivory to ochre but not pinkish cream (2–9 excluding 4) Ivory to cream to pale ochraceous (2–3–6) Yellowish cream to pale ochraceous to ochraceous to ochre (5–6–8–9) Very pale yellowish cream to yellowish cream (3–5) Pale yellowish cream to yellowish cream (3–5) Pale ochraceous to ochraceous (6–8) Yellowish cream (5). No variation Pale yellowish cream to yellowish cream to pale ochraceous to ochraceous (3–5–6–8) Ivory (2) Pale yellowish cream to pale pinkish cream (3–4) Ivory to pale yellowish cream to yellowish cream (2–3–5) Ivory to pale yellowish cream (2–3) Ivory to pale yellowish cream to yellowish cream to pale ochraceous to ochraceous (2–3–5–6–8)
Observer Walker C Walker C Walker C Walker C Walker C Walker C Walker C Broome A Walker C Walker C Walker C Walker C Walker C Walker C Walker C Walker C Schenck N Walker C Walker C Walker C Walker C Vestberg M Walker C Broome A Broome A Walker C Walker C Vestberg M Walker C Vestberg M Vestberg M Walker C Walker C Vestberg M Walker C Walker C Walker C Walker C Walker C Vestberg M Vestberg M Walker C Walker C Vestberg M Vestberg M Vestberg M Vestberg M Vestberg M Walker C
Walker and Vestberg—Glomus claroideum Emended
613
T 3. (cont.) Epithet
Voucher
Attempt–culture
fistulosum fistulosum fistulosum
W2840 W2852 W2853
632–3 634–3 635–3
maculosum
W505
491–0
maculosum pustulatum pustulatum lamellosum lamellosum lamellosum lamellosum lamellosum lamellosum albidum albidum albdium albdium albidum albidum
W531 W655 W1064 W1707 W2117 W2432 W2712 W2868 W385 W179 W79 W169 W170 W258 W282
491–1 Field sample Field sample 244–3 244–4 244–4 244–6 244–6 472–0 Field sample 288–1 288–1 288–1 288–4 456–0
Colour
Observer
Ivory to pale yellowish cream (2–3) Pale yellowish cream to pale pinkish cream to yellowish cream (3–4–5) Ivory to pale yellowish cream to pale pinkish cream to yellowish cream (2–3–4–5) Yellowish cream to pale ochraceous to ochraceous (5–6–8) after formaldehyde Pale straw to ochraceous (50–8) Sienna to cinnamon (11–10) Pale yellow to yellow brown or orange brown Pale ochraceous to ochraceous (6–8). Ivory when young (2) Yellowish cream to straw (5–50) White, yellowish cream to pale ochraceous (5–6) Hyaline to pale yellow Ivory to pale ochraceous (to ochre) [2–6–8 (–9)] Pale yellow Dull white Hyaline to white (to off-white when mature) Hyaline to white (to off-white when mature) Hyaline to white (to off-white when mature) Dull white White
Vestberg M Vestberg M Vestberg M Miller D Walker C Walker C Koske R Walker C Walker C Broome A Walker C Walker C Walker C Walker C Walker C Walker C Walker C Walker C Walker C
T 4. Spore dimensions (mean and ranges of length and widths in µm) measured in a comparatie study of Glomus claroideum and similar fungi. Unless otherwise stated, measurements are from 100 spores in each sample Spore length (µm) Original identification
Culture
G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.
BR147 BR147 SC-09 SC186 SC186 Foul2 Foul2 Holotype Isotype G23a G23a Ex-type Ex-type Ex-type Ex-type V12 V13 V112a V127 V128 V138 V14b V151 V170 V174 V184 V187 V198 V92 Irish. UCD1 Foul1 Ex-type Isotype
claroideum claroideum claroideum claroideum claroideum claroideum claroideum claroideum claroideum (n ¯ 34) fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum (n ¯ 32) fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum (n ¯ 121) fistulosum fistulosum fistulosum (n ¯ 122) fistulosum (n ¯ 114) fistulosum fistulosum fistulosum fistulosum fistulosum lamellosum maculosum
Spore width (µm)
Voucher number
Min
Max
Mean
Min
Max
Mean
W2507 W2845 W2417 W2503 W2846 W2839 W2847 W2595 W947 W1839 W1842 W1840 W1841 W2844 W2837 W2840 W1872 W2902 W2114 W1843 W2020 W1853 W2871 W2849 W2850 W2851 W2852 W2853 W2111 W2370 W2374 W2868 W505
80 95 72 75 65 85 79 63 95 93 87 88 80 90 99 107 83 99 99 60 84 97 103 88 103 68 111 92 80 94 90 84 102
160 181 192 140 154 197 199 181 175 190 169 179 197 167 201 176 165 155 176 165 153 199 168 153 180 168 180 164 164 181 192 219 178
124 128 129 110 108 144 138 129 130 137 122 131 135 127 137 136 124 132 136 124 124 140 136 123 129 125 144 124 124 124 141 136 138
84 95 75 70 70 81 81 51 92 85 87 80 80 93 111 99 93 95 99 60 80 97 103 84 96 79 107 92 84 92 95 81 102
222 178 192 139 165 224 226 188 205 190 175 158 243 160 160 191 179 165 183 165 160 210 180 153 172 168 180 168 164 165 183 174 178
126 128 129 108 109 147 139 130 133 138 124 131 135 126 136 138 125 132 138 124 126 141 138 123 130 126 145 126 125 125 142 135 139
Width rank
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 39 30
Voucher
W2852 W2839 W2374 W1853 W2847 W505 W1839 W2871 W2840 W2114 W2868 W1841 W2902 W1840 W2850 W2595 W2845 W2851 W2844 W2507 W2112 W2853 W2111 W1872 W2370 W1843 W1842 W2849 W2846 W2503
1 2 3 4 5 6 7 9 10 8 12 11 13 14 15 16 17 18 19 24 22 20 23 25 21 26 28 27 30 29
Length rank A B A B A B AC B AC BDA C BDA C BDA C BDA C BDA C BDE C BDE C F DE C F DE C F DE C F DE F E F E F E F E F E F F F F F F F G G
Tukey Grouping for width 145±3 141±9 141±5 139±3 138±8 138±7 137±9 137±9 137±9 137±8 135±2 134±8 132±0 131±1 130±4 129±9 128±4 128±3 126±4 126±0 126±0 125±6 125±3 125±0 124±6 124±1 124±1 123±3 109±5 108±1
Mean width (µm) A B A B A B A C B A C B A C BDA C EBDA C EBDA C EBD C EBDA C EBDA C E D CF E D GC F E D G F E G F E G F E G F G F G F G F G F G F G G F G G G H H
Tukey Grouping for length 144±2 141±9 141±0 138±6 138±5 138±1 136±8 135±9 135±7 135±5 136±0 135±7 132±3 130±6 129±3 129±1 128±0 127±8 126±6 123±9 124±3 124±4 124±0 123±6 124±4 123±5 122±2 123±3 107±9 109±7
Mean length (µm) 634 251 43 74 251 491 271 269 632 422 244 5 313 5 435 622 598 480 5 598 420 635 223 335 564 79 271 383 597 597
Attempt number 3 1 1 3 1 0 7 3 3 1 6 8 2 6 3 3 6 3 19 6 1 3 3 3 3 3 5 3 6 5
Culture number fistulosum claroideum claroideum fistulosum fistulosum maculosum fistulosum fistulosum fistulosum fistulosum lamellosum fistulosum fistulosum fistulosum fistulosum claroideum claroideum fistulosum fistulosum claroideum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum fistulosum claroideum claroideum
Original identification
V187 Foul2—Sonchus (JM) Foul2—Raspberry (CW) V14b Foul1—Sonchus (JM) ISOTYPE Gryndler 23A V151 V12 V127 Ex-type Ex-type V112a Ex-type V174 HOLOTYPE BR147A (INVAM) Dried V184 Ex-type BR147A (INVAM) Fresh (UY) V138 V198 V92 V13 Irish. UCD1 V128 Gryndler 23A V170 SC186 (INVAM) Fresh (UY) SC186 (INVAM) Dried
Other identity and notes
T 5. Analysis of lengths and widths (µm) of 100 spores sampled from pot cultures in the comparatie study of Glomus claroideum and similar arbuscular mycorrhizal fungi
614 Walker and Vestberg—Glomus claroideum Emended
Walker and Vestberg—Glomus claroideum Emended specimens and may be an artefact. The evanescent component is relatively thick (appearing unitary) on some spores, but has disappeared completely on others. Although it is assumed that this culture consists of just one species, it was produced only from a multi-spore attempt, originally in open pot culture. Consequently, there remains a possibility that more species were present in the pot, either from an original error in selecting spores, or from later contamination. Two spores examined had pustules on the outer wall, reminiscent of those on G. pustulatum. The other extype material of this species came from three different subcultures of the ex-type. Each of these was started with pot culture substrate from its predecessor. The specimens from Attempt 244–4 were from a sealed pot culture on Plantago lanceolata. In the first voucher, W2117, the wall structure was observed on mature spores as A(EL)B(F). Some specimens had a luteous haloed effect due to the thick coloured wall. Many of these spores were parasitized, apparently resulting in failure to develop the innermost flexible component. Specimens such as this appear to have a muronym of A(EL). Samples from the second voucher, W2432 correspond with the illustrations in the protologue, but lack the flexible wall component. These spores are indistinguishable from those of G. clarum. Later, the culture was lost through a build up of parasites, but not before a sample had been sent to Dr Dodd in Canterbury. A substrate sample from this culture from Kent (Attempt 244–5) was retrieved, and a voucher (W2392) taken before the remainder was used to establish Attempt 244–6, in sealed bags, with Plantago lanceolata. The W2392 collection has evanescent, laminated and flexible components typical of the species examined here, though the flexible one is very thin and difficult to see, perhaps because the culture was not fully mature. Voucher W2712 from Attempt 244–6 consisted only of spores corresponding with the descriptions of G. clarum and G. claroideum. A few spores possessed macular structures yet showed no signs of attack by micro-organisms. The wall structure of the other sample from Attempt 244–6, W2868, is A(JL)B(F) where J indicates a mucilaginous or jelly-like component. The J component is embedded with bacteria and fine particles and appears to be exuded by the fungus after the structural wall components have formed. In some spores there is an additional structure that could be interpreted as an evanescent component. Such spores have a muronym of A(JEL)B(F). The spores of G. pustulatum have a wall structure of A(UoL)B(F). The ornamentation (o) consists of rounded pustules or collicles on the outer wall surface. Although the wall structure description of G. multisubstenum is given as having only ‘ two inseparable layers ’, illustrations in the protologue show evidence of both an outer, evanescent component [remnants attached to the spore in Mukerji et al., 1983 their Figs 1, 2 and 4] and a flexible inner component [Mukerji et al., 1983 their Fig. 4], corresponding to the wall-structure of G. claroideum. The wall structure of G. albidum, interpreted from the protologue, consists of an evanescent component surrounding a laminated component. However, re-examination of the type material and of preserved specimens from ex-type pot cultures showed that it also may possess a very thin, flexible innermost wall
615
component. For the type specimens of G. przelewicense, there are two possible interpretations. Some spores have an evanescent component attached to a laminated component [A(EL)], whilst in a few specimens there could be an extremely thin flexible innermost component that can only be detected by wrinkling in the paler spores [A(ELF)]. There is no evanescent layer on the spores of G. diaphanum which seems to have a wall structure of A(UF) or A(U)B(F), but which probably has a laminated outer component rather than a unitary one [A(LF) or A(L)B(F)].
Fistulae and pustules Specimens from the type of G. claroideum have the same overall wall structure, and in one spore (Fig. 14) similar maculae (Fig. 13) as G. maculosum. These occur on the inner wall component only, and like those of G. maculosum, they show no clear evidence of invasion by parasitic microorganisms. Re-examination of G. maculosum provided no evidence of an external agent that might have stimulated formation of the maculae. On some spores, however, there are unusual structures in the cytoplasm. Possibly these are invasive parasites which have caused the fungus to react by localized deposition of wall material. Some spores in the culture of G. manihotis had developed maculae indistinguishable from those which were used to separate G. maculosum from other species in the genus. Evidence from two other fungi examined during this study, one of G. claroideum and one of an undetermined Glomus sp., shows that these swellings are not confined to one species. The ex-type material of G. lamellosum and the similar fungus found contaminating a culture of G. mosseae proved to be confusing. Spores produced from these cultures divided into three groups. One group had the characteristics as described for the species, but another appeared to be G. claroideum and the third had the characteristics of G. clarum. In addition, spores similar to G. pustulatum were found in one of the cultures of G. lamellosum. Glomus pustulatum was originally described from field collections. It has therefore not been proven that the production of pustules on the surface of the spores is a consistent, genetically controlled character that can be used for species delineation. Although described from both field and pot-cultured specimens, no samples of G. multisubstensum remain in the herbarium at New Delhi, and none could be found (Tewari, Canada, pers. comm. ; Mukerji, India, pers. comm.). It can therefore be argued that the species was not validly published. Nevertheless, it is illustrated sufficiently well in the protologue to allow conclusions to be drawn about its identity, and we consider it worthwhile to make comparisons with other species with similar descriptions, rather than pursuing complex legalistic arguments. We examined all 20 of the type specimens of G. przelewicense preserved in PVLG. These were field specimens, and it was not easy to observe all the necessary characteristics. Nevertheless, with the exception possibility of G. albidum, they did not fit well with any of the other species. The difference between specimens of G. diaphanum
616
Walker and Vestberg—Glomus claroideum Emended
and the other species examined were distinctive, and the original species description is substantially correct. Reaction to PVLG}Melzer’s In the experimental evaluation, there was no reaction in fresh spores of G. fistulosum to PVLG}Melezer’s, but in the dry spores a very weak pink reaction was noticed infrequently. Although this may seem insignificant, it is recorded for completeness. Apart from this, the reaction to Melzer’s reagent was tested in 31 different samples from Finland, and except for one instance where a slight yellowing of laminae was recorded, there was no reaction. Similarly, there was only one occasion when a reaction to PVLG} Melzer’s was noted (other than a slight yellowing of the laminated wall component) in any other specimen among all cultures examined. Specimens of G. claroideum (W1404) displayed a rather slow, patchy orange reaction, probably from the remnants of the evanescent wall component. The reaction to Melzer’s reagent or PVLG}Melzer’s of spores of G. multisubstensum and G. przelewicense is unknown. Spores of the species G. lamellosum are recorded in its protologue as reacting only by an increase in contrast of the laminations in the main structural wall component. Neither of the G. diaphanum spore wall components has a reaction to Melzer’s reagent (from observations of W949, ex-type material), although it was not assessed for the isotype, W952. Subtending hypha The subtending hypha was usually more or less straight (Fig. 14) or slightly curved (Figs 15 and 23). On some specimens, however, it was sharply recurved to the point of following the outline of the spore. In such specimens, it was often difficult to detect. Except for G. manihotis and G. clarum, the subtending hyphae of all cultures examined in this study develop in the same manner. In immature spores, it is two-layered and continuous with the layers of the supporting mycelium. The point where the somatic hypha changes into the spore’s subtending hypha is not always easy to determine, but there is usually a gradual change in colour of the innermost component, where it becomes pigmented and evidently laminated. As the spore matures, the outermost wall components usually begin to disintegrate. The evanescent wall and any gelatinous or mucigel-like structures disappear, leaving only the rather thin laminated component which is normally short and rapidly tapering. Consequently, in such specimens, the subtending hypha is quite difficult or impossible to locate (Fig. 16). Spores of G. diaphanum and G. manihotis normally retain a distinct and persistent subtending hypha formed by a relatively much longer and sturdier laminated wall component. Due to the poor condition of the type specimens of G. claroideum, only the laminated component of the subtending hypha can be seen, though in some specimens it is split along the laminae, appearing then to consist of two components (Fig. 9). Of all the G. claroideum-like species studied, 2 to 7 % of spores had at least two subtending hyphae. These were usually fairly close together (Fig. 7), but on occasions they were at opposite ends of the spore (Fig. 8).
Germination characteristics Glomus fistulosum was the only species tested experimentally for germination characteristics. Results from the frozen spores were 17, 39, 43, 50, 51 and 90 % (six trials). For spores not given the freezing treatment, germination was 0, 6 and 61 % (three trials only). Spores germinated in two ways. Some produced regrowth directly through the remnants of the subtending hypha at the spore base (Fig. 23). This is considered to be the ‘ normal ’ type of germination for Glomus spp. However, in other spores, up to five germ tubes emerged from each spore by direct penetration of the main structural spore wall (Figs 22 and 24). Spores could have one or both of these germ tube emergence patterns. A few spores in the ex-type and type material of G. claroideum were germinating at the time of preservation. Germination is both through the subtending hypha and by direct growth through the spore wall. The spores of G. maculosum are capable of germinating by emergence of a germ tube through the subtending hypha (Miller and Walker 1986), but no experimental approach was taken in assessing germination. There is no discussion of germination in the protologue of G. lamellosum, but a few spores of the G. clarum morphotype in the collection W2868 have germinated by direct regrowth through the subtending hypha. No germination characteristics are recorded for G. pustulatum and nothing is known of the germination characteristics of G. multisubstensum. The spores of G. albidum are described and illustrated in the protologue as germinating directly through the spore wall, but for G. przelewicense no description of the germinating characteristics is given in the original species description, and there are no germinating spores in the type material. The germination characteristics of G. diaphanum are not described, and we have not been able to locate germinating spores in specimens available to us.
Mycorrhizas Glomus fistulosum forms mycorrhizas with abundant arbuscules and sparse, thin-walled vesicles (Fig. 26). However, Gryndler (pers. comm.) indicates that mycorrhizas formed by the species (attempt 271) with Hedera helix (and occasionally in maize) are atypical, consisting mainly of hyphal coils and atypical arbuscule-like structures formed by production of fine lateral hyphae branching directly from the coils. The same organism forms typical vesiculararbuscular mycorrhizas with other hosts. The species also produces spores in roots (Fig. 6), but these are sparse and not always present in a sample. Glomus claroideum is known to form vesicular arbuscular mycorrhizas, but no description is available other than that they are ‘ typical ’, and that spores were rarely produced in roots (Schenck and Smith, 1982). However, the holotype includes a microscope slide of stained mycorrhizas. These fall into two distinct categories (Figs 25–27), a fairly typical Glomus vesicular arbuscular mycorrhiza (Figs 25 and 26) (Abbott, 1982), and one with distinctly lobed vesicles (Fig. 27). The cultures studied in the description of G. maculosum
Walker and Vestberg—Glomus claroideum Emended did not form typical arbuscular mycorrhizas. The mycorrhizas that were formed were seen only to produce hyphal coils, and neither vesicles nor arbuscules were noted. However, the fungus proved impossible to stain in the hosts used, and it is probable that arbuscules were present but were not visualized. According to the description in the protologue, G. lamellosum forms typical arbuscular mycorrhizas with vesicles, although Fig. 7 of that publication shows rather stunted arbuscules. G. pustulatum is known to form arbuscular mycorrhizas with vesicles but there is no published detailed description, and although G. multisubstensum is said to form mycorrhizas, they are not illustrated. Glomus albidum forms vesicular arbuscular mycorrhizas but they are not illustrated in the description, whilst the mycorrhizal status of G. przelewicense is unknown, the only known specimens consisting of spores collected in the field. G. diaphanum forms both vesicular arbuscular mycorrhizas and abundant spores in roots. D I S C U S S I ON To avoid doubt, we must stress that our interpretation of developmental events is based on observations made by light microscopy only, and the assumption (not necessarily true) that the spore wall structure advances from simple to complex. Our conclusions must inevitably, therefore, be subject to doubts that can probably only be solved by ultrastructural observations. We believe that Stu$ rmer and Morton (1997) were in the same situation, and suggest that their phylogenetic and ontogenetic analysis must therefore be subject to the same caveats, and may be premature and flawed. Stu$ rmer and Morton (1977) erroneously interpret earlier work (Walker, 1992), claiming that it suggests the innermost layer in this group of organisms is evidence of polyphyly. No such statement is made or implied in that work, which was written with the intention of pointing out unclear areas that were ripe for further research. With regard to the flexible innermost wall, its ontogeny remains unclarified, and until further studies are carried out, speculation about its analogies or homologies remains a matter of conjecture. Doubt remains about the precise wall structure of this species. Our interpretation of light microscopy observations is that the inner wall of G. claroideum spores forms a complete and separate entity developing only after the completion of the growth of the original spore. Therefore it might not be correct to consider it to have the same kind of blastosporic ‘ chlamydospore ’ as is found in most other species of the genus Glomus. Our interpretation is more consistent with an ‘ ectospore and endospore ’ type of development similar to that proposed for members of the genus Scutellospora (Ferrer and Herrera, 1981). Stu$ rmer and Morton (1997) draw different conclusions, clearly stating, though not adequately illustrating, that the component is contiguous with the innermost lamina of the main structural wall component. Thus, there are two different interpretations possible from the same evidence. The apparent endospore-like nature of the innermost wall component may be an artefact of specimen preparation.
617
Ultrastructural developmental studies may be needed to ascertain its true nature. We take a more cautious view than Stu$ rmer and Morton (1997) in proposing that there is evidence for an endospore, but accepting that the matter remains unresolved. We therefore sound a note of caution in warning that any new phylogenetic analysis must be based on sound, repeatable and unambiguous evidence. The fact that germination can take place by direct penetration of multiple germination tubes through the spore wall is also different from the supposedly normal form of germination found in the genus Glomus (regrowth of the subtending hypha). Such germination was discussed by Walker and Rhodes (1981), when the possibility of using it as a criterion for separating a new genus from Glomus was discussed. The evidence is increasing, but more information will be required before a separation can be confidently made. Ideally, such studies would gather information from many different sources. Suitable methods might include ultrastructural studies, DNA sequences of many different genes, isozyme profiles with several enzymes, and fatty acid profiles. Colour Colour is a continuous variable that is rather difficult to assess, especially for the untrained observer. It is unreasonable to expect to obtain a high degree of exactitude in defining and matching specimens with these characteristics, particularly when matched against charts made up by inexpensive printing processes. This is particularly true of colour charts made by the half-tone printing process (Gonnet, 1995). In this study, colour differences exist both within and among isolates, but could only be used to separate G. albidum and G. diaphanum. Spores of the former are generally very much paler than those of most species examined here, and the latter does not have pigmented spores. Even these fall completely within a subset of the full range recorded for the other fungi which generally ranged from hyaline (only in very young, immature specimens) through ivory (2) and very pale creams (3 and 4) when immature, to a range of colours between yellowish cream (5) to ochraceous (8) for mature spores. More infrequently, older cultures have a small proportion of pale apricot (47) spores. Comparisons with colours quoted, from the halftone chart used for G. claroideum (Stu$ rmer and Morton 1997), show mostly the same range, depending on stage of spore development, from white, through pale cream to dark cream which equates roughly to pale yellowish cream (3) to pale ochraceous to ochraceous (3–6–8). The range of colours we detected is broader than this, both paler and much darker, possibly as a result of different storage and culturing conditions, but we could find the full range in mature specimens with their innermost wall component developed. Unfortunately, many species of the Glomales have spores in this colour range, though few appear to have quite such a broad spectrum. Glomus multisubstensum spores are described as ‘ light brown ’, and the wall layers as ‘ brown ’ or ‘ pale yellow brown ’ in the species description, both of which rather imprecise terms can be applied to mature and field-collected specimens of G. fistulosum and G. claroideum.
618
Walker and Vestberg—Glomus claroideum Emended
Spore colour varies considerably within and among cultures, perhaps depending on such factors as age of the culture, soil conditions or host (Table 3). It is impossible from the data we have to determine if these are due to differences in perception among observers, differences caused by age of culture, differences induced by physical factors such as pot culture substrate, or differences among spores grown on different hosts. This variability can be viewed with some concern, since spore colour is often considered to be of considerable importance in species descriptions among the members of the Glomales. Taxonomic characteristics must be consistent if they are to be used to separate organisms at any given taxonomic level.
Spore dimensions Like colour, the dimension of spores is also a continuous variable, although making measurements is a mechanical process and thus is inherently more accurate than judging colour by eye. Nevertheless, accuracy differs depending on the minimum graticule size used and the magnification, and it is also possible that size may vary with the environmental conditions experienced by the fungi. The assumption has always been that spore size is a stable taxonomic characteristic in the Glomales. An analysis of variance showed that significant differences existed among mean lengths and breadths of the different species examined. Generally, the bulk of the isolates formed a large group that were not distinguishable when an overall view was taken (Tables 4 and 5), but the culture of G. claroideum from South Carolina could clearly be separated, being significantly smaller in both dimensions (P % 0±05). Although slight differences in ranking occurred (Table 5), the results were substantially the same for both length and breadth of spores. A few individual comparisons could be made where the same culture had been sampled at different times. The analyses showed that with only one attempt, samples taken at different times from different cultures produced spores with significantly different dimensions (Tables 4 and 5). There is a significant difference (P % 0±05) for both spore length and width between samples from Attempts 271–5, produced in the Czech Republic and Attempt 271–7, grown in Scotland. However, the former measurements were made from dried spores, whereas fresh spores were used for the latter. At first, we thought that the differences might be the result of shrinkage during drying, but comparisons of dried s. fresh spores in two other cultures did not show such a difference, and additional measurements of spores taken at different times from these two samples always showed the same difference. If spore dimensions are valid as a taxonomic characteristic, they must be consistent and must remain unaffected by external influences. If external factors influence their size, spores produced from a symbiosis with one plant, or with a different substrate, might produce an apparently different species when grown with another. The result for the Czech culture (Table 5, Fig. 29) suggests such factors may influence spore dimensions, though a more detailed experimental
approach will be required to ensure that this is not a Type I error, accepting significance where it does not really exist (Snedecor and Cochran, 1967). The South Carolina G. claroideum (Attempt 597) used by Schenck and Smith (1982) to support the species description had significantly smaller spores than any of the others. Intriguingly, the same culture apparently produced much larger spores in the study by Stu$ rmer and Morton (1997), though they provide no explanation of the method or criteria used to select spores for measurement. Certainly, these results raise some element of doubt about the validity of spore dimensions as a stable taxonomic characteristic, and indicate the need for a more detailed, experimental examination of the matter. Subtending hypha The possession of more than one subtending hypha (Figs 7 and 8) on Glomus spores is not an uncommon occurrence and thus must be viewed with some scepticism as a major specific character. It is useful as part of the overall description of a species, but there are species in the genus Glomus which may have spores with more than one subtending hypha for which no mention of such a character has been made in the literature. This highlights the desirability of as detailed a knowledge as possible of the entire group when erecting new species. Melzer’s reaction Reaction to Melzer’s reagent was somewhat unpredictable. In some dried spores there was a weak reaction of wall component 1. In most dried spores and all fresh material, there was no reaction. Thus, for this species, the reaction to Melzer’s reagent, commonly used to support descriptions of arbuscular mycorrhizal fungi, is of doubtful taxonomic value. Stu$ rmer and Morton (1997) are convinced not only that such a reaction exists, but that it is of considerable phylogenetic and ontogenetic significance. It would be better if features used to draw such conclusions were consistent, repeatable, not subject to environmental or sampling differences, and if they were readily observed. Spore wall thickness Spore wall thickness is of little taxonomic value in this group of organisms, although it is traditionally quoted in descriptions of species. The laminated component, which constitutes the main structural component of the spore, is very variable, but always increases in thickness over time as layers are added. The evanescent component becomes thinner with time as it disintegrates. The mucilaginous layer seems to be deposited in response to some external stimulus, and also may be consumed by soil micro-organisms. Consequently, it can become thicker or thinner. The innermost, flexible component is difficult or impossible to see (and hence to measure) in immature spores, but thickens with maturity. It never becomes as thick as the laminated component. The nature and order of wall components, however, remain consistent and useful characteristics.
Walker and Vestberg—Glomus claroideum Emended
619
Spore length, µ m
250 200 150 100 50
G. claroideum
G. fistulosum
G. lamellosum G. maculosum
BR
1 BR 47 14 SC 7 -0 SC 9 18 SC 6 18 Fo 6 ul Fo 2 Ho ul2 lo t y Iso pe ty p G2 e 3a G2 Ex 3a -t Ex ype -t Ex ype -t Ex ype -ty pe V1 2 V1 V1 3 12 a V1 27 V1 28 V1 38 V1 4b V1 51 V1 70 V1 74 V1 84 V1 87 V1 98 V9 2 Iri sh Fo Ex ul1 -ty Iso pe ty pe
0
Spore width, µ m
250 200 150 100 50
G. claroideum
G. fistulosum
G. lamellosum G. maculosum
BR
1 BR 47 14 SC 7 -0 SC 9 18 SC 6 18 Fo 6 ul Fo 2 Ho ul2 lot y Iso pe ty p G2 e 3a G2 Ex 3a -t Ex ype -t Ex ype -t Ex ype -ty pe V1 2 V1 V1 3 12 a V1 27 V1 28 V1 38 V1 4b V1 51 V1 70 V1 74 V1 84 V1 87 V1 98 V9 2 Iri sh Fo Ex ul1 -ty Iso pe ty pe
0
Culture identity F. 29. Graphical comparison, with their original species identifications, of mean, maximum and minimum spore lengths and breadths (µm) for the samples herein synonymized as Glomus claroideum.
Wall structure and deelopment Glomus lamellosum is described as having a readily evident mucilaginous layer, though on some spores we examined, it had either not been formed, or had been shed. The laminated nature of the first main structural component of G. lamellosum (component 1) is not easy to see and requires further investigation. Although it is mentioned in the species description, it is not well illustrated. It may be important to establish if there are different kinds of laminated wall components in spores of members of the Glomales so that proper phylogenetic analyses can be made. Glomus geosporum may also have an inner wall layer that appears discontinuous with the subtending hypha (Walker, 1982), but this does not seem to be flexible and is not easily detached from the laminated spore wall component. It probably is not homologous with the inner flexible component of G. claroideum. The protologue of G. claroideum describes the spores as having ‘… 1 or 2 walls with the outer wall laminate and usually thicker than the inner wall …’, but our examination of the type material shows a different structure consisting of three components in two groups. The wall structure of mature spores seems to be stable in the species examined, as does the nature of the subtending hypha. Nevertheless, we offer a word or two of caution. The difficulties in studying spores of Glomus spp. by light microscopy were a problem in this study. For some spores
viewed at relatively low power, the outer component was seen as a single layer ; increasing magnification gave the appearance of two layers, whilst observation at maximum power shows an apparent third layer. Some small element of doubt must therefore remain, pending studies by electron microscopy, although we examined a very large number of spores and believe we have interpreted our observations correctly. From the present studies, the development sequence of the spore wall components has been hypothesized. The spore develops firstly by blastic swelling of a hyphal tip, or sometimes by a similar intercalary swelling producing a spore with two subtending hyphae. The components (components 1 and 2, Fig. 12) of the wall (group 1) are continuous with those of the subtending hypha. A thin septum may then be produced (Fig. 21), continuous with the inner laminated components of the main structural wall group, near the spore base to occlude the cytoplasm. At this stage, the subtending hypha is persistent and prominent (Fig. 15) and there is no evidence of any other wall group. The outer components form the main structural spore wall group, reaching full development before further wall structures begin to grow. The cytoplasm next begins to form a thin, diffuse line (Fig. 10) around the perimeter which then becomes a flexible component (group 2). This seems not to be continuous with the components of the subtending hypha (Figs 20 and 21), but apparently forms a complete ‘ endospore ’ (Fig. 19). The flexible component then thickens
620
Walker and Vestberg—Glomus claroideum Emended
(Figs 11 and 21), but no laminae can be seen under the light microscope. As development proceeds, the outer component disintegrates and disappears (Figs 11 and 21), and the subtending hypha usually collapses and becomes detached rather close to the spore (Fig. 16). Consequently, on mature spores, the subtending hypha may be difficult or impossible to see, except as a small scar or a much reduced remnant. Coupled with the flexible innermost wall component, this, on some specimens, can give an erroneous impression that the spore is of an Acaulospora sp. On the specimens studied, we only rarely observed anything, even on very young spores, that could be interpreted as an additional mucigel-like deposit forming externally, and even then we were never certain it existed. Yet this was defined as a major characteristic of the species by Stu$ rmer and Morton (1997). Having studied some of the same cultures produced under the same conditions we are unable to explain this difference, but would merely draw it to the attention of the reader. Inevitably there are differences among laboratories in techniques, and it is possible that the ones we used differed sufficiently from theirs to render this feature unobservable in our system. In view of this, we suggest that others might also find it difficult to see, though it is included as a possible wall component in the new species re-description.
Spore deelopment Stu$ rmer and Morton (1977) have made much of the development of spores in the Glomales, but caution must be exercised when making comparisons. Firstly, ontogeny is not always a good guide to phylogeny as it is possible that developmental aspects may be lost through evolution. Secondly, we have already drawn attention to the difficulties inherent in studying spores of members of the Glomales, and we are somewhat doubtful that sufficiently detailed and accurate information can be obtained through the light microscope alone. We have found it possible, for example, to suggest a different conclusion about the nature of the innermost flexible wall component in G. claroideum, though we are careful to point out that we have doubts about its veracity.
Germination characteristics In those instances where we have been able to observe germination of G. claroideum and G. fistulosum, we have established that germination can take place both through the subtending hypha and by direct production of germ tubes through the spore wall, more akin to the mode of germination seen in Gigaspora species. However, we do not feel able to draw any conclusion about analogy or homology of such germination in widely separated genera. The proportion of species in the Glomales for which anything is known about germination characteristics is very low. Though it is often assumed that the normal mode of germination of species of Glomus is by direct regrowth through the subtending hypha, G. claroideum germ tubes may emerge directly through the main structural spore wall
components. Germination apparently cannot occur until the inner flexible component has fully developed. Consequently, its completion must be considered to be a pregermination event such as those in Gigaspora (Spain et al., 1989) and Scutellospora (perhaps also Acaulospora and Entrophospora), casting doubt on the assumption that these propagules are simple chlamydospores. When the species G. albidum was first described, Walker and Rhodes (1981) discussed the possibility of using germination characteristics to separate genera, though they concluded that insufficient evidence existed to do so. Both G. albidum and G. claroideum germinate in this manner and both possess an inner, flexible wall component that must develop before germination. Evidence is gathering, therefore, to support the splitting of such fungi from Glomus sensu stricta, but with only one of the species available in culture, and other species with similar wall structure for which no details of germination characteristics are known, it is still too early to do so.
Mycorrhizas Glomus fistulosum forms fairly typical arbuscular mycorrhizas, though the vesicles are very sparse, thin walled, and can be difficult to stain (Fig. 26). In the type material of G. maculosum, mycorrhizas failed to stain with a variety of stains, and consisted of sparse hyphal coils that could be seen only by sophisticated contrast enhancement techniques (Miller and Walker, 1986). Glomus claroideum is known to form vesicular arbuscular mycorrhizas, but no description is available other than that they are ‘ typical ’ and that spores were rarely produced in roots (Schenck and Smith, 1982). However, the holotype includes a microscope slide of stained mycorrhizas. At first, we thought the mycorrhizas were unusually variable, but more careful examination showed them to fall into two distinct categories. One (Fig. 25), is a fairly typical but weakly staining Glomus vesicular arbuscular mycorrhiza (Abbott, 1982). The second has distinctly lobed vesicles (Fig. 27). At first, this was confusing, but when more preserved spores from the holotype were examined, a specimen of Acaulospora laeis Gerd. & Trappe was discovered (Fig. 28). It is assumed, therefore, that the lobed vesicles belonged to this species rather than to G. claroideum, and this reinforces the need for carefully produced pure cultures which are maintained so as to preclude contamination by other arbuscular mycorrhizal fungi. This is not to imply that lobed vesicles are confined to members of the genus Acaulospora, as has been assumed by some, but A. laeis is known to produce such structures (Gerdemann & Trappe, 1974).
GENERAL DISCUSSION The type of G. fistulosum seems to have been made from spores that were past maturity and which had been attacked or decayed by soil micro-organisms. The spores in the type material exhibited the ‘ fistules ’ used to characterize the species. These are not taxonomic characters, but are the
Walker and Vestberg—Glomus claroideum Emended ‘ fine radial canals ’ caused during the decay of moribund spores (Lee and Koske, 1994). This species cannot be distinguished from G. claroideum, and must be synonymized. The cultures of G. claroideum from South Carolina and Brazil can be separated only by spore dimensions. Their wall structures are identical to that of the type material of G. claroideum. Two samples of each of these isolates were examined, one received directly from INVAM as dried pot culture substrate and the other from a living pot culture on Plantago lanceolata. The appearance of the dried and fresh spores under the dissecting microscope differed somewhat. In the South Carolina isolate, dried spores (W2503) were matt and generally whiter than freshly extracted specimens from an active culture (W2504). The Brazilian isolate displayed a similar matt appearance when dried, though in this isolate dried spores (W2506) were more pigmented than fresh ones (W2507). The morphological characteristics of the Danish G. claroideum agree well with those of both the type specimen (preserved material) and the South Carolina isolate (living material), and the isolate can confidently be assigned morphologically to the species G. claroideum. The maculae used to separate G. maculosum from other similar species are most likely not genetically controlled consistent developmental features. This is reinforced by their appearance on other species in the Glomales, both within and outside the group of species with flexible innermost wall components at maturity. Consequently, the species cannot be maintained with confidence and it is thus reduced to synonymy with G. claroideum, which it resembles in all other respects. The developmental stages of G. maculosum spores are the same as in the isolates of G. fistulosum and G. claroideum. In the protologue of G. maculosum, Miller and Walker (1986) wrote ‘ Some spores of G. maculosum have multiple hyphal attachments … , a characteristic feature of G. multisubstensum … Nevertheless, the description of the latter indicates that these species have little else in common, size range, color (sic), and wall structure all being different ’. Examination of the illustrations in the protologue of G. multisubstensum leads us to consider that this opinion should now be revised, as the wall structure evidently includes a flexible innermost layer. The multiple subtending hypha used to furnish the epithet is not a particularly uncommon characteristic in members of the genus Glomus. The feature is particularly common on spores of G. maculosum, G. claroideum and G. fistulosum, but it also occurs in G. etunicatum, G. geosporum and G. occultum (Walker, unpubl. res.). The species description and illustrations in the protologue do not, therefore, give sufficient information to allow G. multisubstensum to be distinguished from G. fistulosum or G. maculosum, and other than in colour, from G. claroideum. It is safe, therefore, either to reject or synonymize the names. Glomus claroideum has precedence, being published the year before G. multisubstensum, and thus, even if validly published, the latter is subsumed within the former and no further confusion should arise. The wall structure of G. lamellosum is quoted in its protologue as A(LLM), with an additional ‘ foliated lamellate surface that sloughs away with age …’. This is a
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mucilagenous outermost component, perhaps exuded by the fungus after the structural wall components have formed, extending the muronym to A(JLLF), where J indicates a mucilaginous or jelly-like component. Some of the spores also had an apparent evanescent outermost component, in which case its muronym would be A(JEL)B(F). The variability of spore wall structure in G. lamellosum is difficult to understand. It is possible that this is an extremely variable fungus which produces more than one spore morphology. There is, however, a possibility that the culture examined is mixed, either from contamination at some time or as a result of its isolation by multi-spore culture. Single spore culturing will be required to clarify it. At first it was thought that it might be possible to determine its status in this study, but complications caused by the uncertainty of its purity preclude this. Consequently, we suggest that the species is in need of more detailed study and possibly requires revision. In relation to G. pustulatum, Błaszkowski (1994) observed spores with pustules in his studies of the Polish Glomales. We did not examine spores from these collections, but it appears from the descriptions that, other than a slight size difference [(85–) 100 (–110) µm] and the presence of pustules, the spore description fits that of G. claroideum. Several of the Finnish isolates of Glomus spp. also produce pustules on their outer surface but are otherwise clearly G. claroideum, and we observed them on spores in the Canadian culture supposedly of G. lamellosum. As with G. lamellosum, this species has some characteristics of G. claroideum, but because we have not been able to examine living material we are unable to draw a firm conclusion. The ranges of colour and size quoted in the protologue of this species are consistent with those of the G. claroideum. The validity of this species should be confirmed by further investigation possibly leading to a revised description. In particular, the pustules, like the maculae in G. maculosum may be environmentally induced and therefore perhaps might not be useful to circumscribe a species. Unless obtained in pure and consistent pot culture and the characteristics verified for consistency, G. pustulatum should be considered a doubtful species. Glomus albidum remains a distinctive and valid species, but it would be beneficial if a new, living culture could be found so it could be re-examined in detail. Its spores may have an internal flexible wall component, but it is difficult to observe in preserved material and we have been unable to determine its origin. Glomus przelewicense is not a very distinctive taxon and, because it was field collected, there can be no certainty that all 21 spores in the collection are of the same species. Detailed observations of some of the specimens shows that there is possibly a very thin, flexible inner wall component. Regardless of whether or not only one species is present, it is clear that G. przelewicense is not synonymous with G. claroideum. From the protologue, the wall structure of G. przelwicense is described as two laminated components in a single group [A(LL)], but examination of the type specimens leads us to draw a different conclusion. This species was examined only because of its innermost flexible wall component, which superficially places it in a grouping with
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Walker and Vestberg—Glomus claroideum Emended
G. claroideum and similar species. To clarify its validity, careful study is needed to see how the flexible component is formed and to establish its germination characteristics which have not been determined. The culture of G. manihotis was included only because some of its spores developed maculae indistinguishable from those which were used to separate G. maculosum from other species in the genus. Their development on an otherwise clearly different species supports the opinion that they have no validity as a taxonomic characteristic. The spores of G. diaphanum, (39)–74 (–121) µm, are much smaller than those of even the smallest G. claroideum, though there is a slight overlap at the upper range of the former and the lower of the latter. Although G. diaphanum differs substantially from G. claroideum, it does produce very pale-coloured spores with a flexible innermost wall component, so we have considered it in comparison with the other fungi discussed here. All the specimens that had been determined as G. fistulosum, G. claroideum or G. maculosum along with G. multisubstensum had such similar morphological characteristics that they can be considered as conspecific. Glomus claroideum is validly described but its description requires some emendation. Stu$ rmer and Morton (1997) made an informal redescription that did not completely agree with our observations. Of the group of species examined in this study, it is the earliest published and therefore takes precedence over all others that are considered synonymous. Among the fungi studied there are a few minor differences in spore morphology, but with the possible exception of the culture from South Carolina, their spores cannot be reliably separated on size, colour, occlusion, wall structure, subtending hypha size and shape, development, and (as far as can be established) germination characters. Accordingly, we synonymize these species and offer a re-description and formal emendation of G. claroideum. Perhaps further study using molecular or other non-morphological techniques (e.g. DNA, isozyme or immunofluorescence methods) will allow species or sub-species to be distinguished with confidence, but these are dependent on obtaining living organisms or specimens that have not been damaged too much by fixatives and preservatives. The G. claroideum isolate of Skipper was used informally in the original species description but is not typical because of its spore dimensions. In considering an appropriate culture to represent the species, we thought of the ex-type of G. fistulosum. However it is not a single spore culture, and thus is not verifiably a pure isolate. We therefore consider that the best representative of G. claroideum sensu Walker & Vestberg is the Irish culture of G. fistulosum. The suitability of this as an epitype [… a specimen selected to serve as an interpretative type when the holotype, lectotype, or previously designated neotype, or all original material associated with a validity published name is demonstrably ambiguous and cannot critically be identified for the purpose of the precise application of the name of a taxon … (Greuter et al., 1994)] is reinforced by the fact that it is available as a pot culture that has been permanently maintained in a closed pot culture, and therefore has not been placed in peril of contamination.
REDESCRIPTION Glomus claroideum Schenck & Smith emend Walker & Vestberg. Figs 1–29. 3 Glomus claroideum Schenk & Smith. Mycologia 74 : 84 (1982). ¯ Glomus maculosum Miller & Walker. Mycotaxon 25 : 218 (1986). ¯ Glomus fistulosum Skou & Jakobsen. Mycotaxon 36 : 274 (1989). ¯ Glomus multisubstensum Mukerji, Bhattacharjee & Tewari. Transactions of the British Mycological Society 81 : 3 (1983). Sporocarps unknown. Spores formed singly or in loose clusters in soil. Fresh spores white (1) to iory (2) to occasionally cream (3) when young becoming yellowish cream (5) or, rarely, pale apricot (47) when mature, with the walls sometimes haing a pale fuscous black haloed appearance under reflected light : after drying in soil, cream to ochraceous, the haloed effect becoming less eident. Spores globose to subglobose, (59)–70–199¬(51)–70–226 (®243) µm. Spore wall structure in young spores of two components in one group [muronym (Walker, 1986) A(EL)] or, in mature spores, of three components in two groups [A(EL)B(F )]. Wall component 1 in group 1, a hyaline eanescent wall, 1–2 µm thick in younger spores, partly disintegrating in older spores. Wall component 2 in group 1, the main structural component, finely laminated, 6–8±5 µm thick at maturity. Wall component 3, in group 2, flexible (more or less membranous), up to 0±5 µm thick, absent in ery young spores. A mucigel-like outermost component or deposit perhaps occurring on some specimens. Sporophore (Figs 17 and 18) consisting of a subtending hypha continuous with walls 1 and 2 of the spore, straight or recured, hyaline, sometimes with cured septa. Subtending hypha 5–16 µm in diameter at the spore base. Length ery ariable in young spores, 19–306 µm or more, 0–137 µm in old spores. Some spores with two, rarely more, subtending hyphae. Many older spores appearing sessile due to the loss of integrity of the subtending hypha, mainly due to the disintegration of wall component 1. Spore contents sometimes occluded by a septum formed by ingrowth of wall component 2, 10–73 µm from the spore base. Subtending hypha 3–6 µm diameter at spore base tapering distally to 1 µm diameter or less. Wall component 1 sometimes reacting pale pink in Melzer’s reagent, but normally not reacting. Germinating, after completion of deelopment of wall component 3, by emergence of a germ tube through the subtending hypha, or directly through wall group 1, to produce abundantly-branched coenocytic or sparsely septate mycelium with occasional intercalary swellings. On mature spores, subtending hypha usually not eident, except on some specimens as a small remnant. When eident, sometimes occluded by a thin septum formed from the innermost lamina of wall component 2. Forming esicular arbuscular mycorrhizas with sparse esicles, sometimes difficult or impossible to stain. In some
Walker and Vestberg—Glomus claroideum Emended circumstances, forming atypical mycorrhizas lacking esicles, or possessing an unusual type of arbuscule. Type material Holotype. OSC No. 40252 (W2595 in the private herbarium of C. Walker). Isotypes (W947) FH ; FLAS No. F52578 (Schenck and Smith, 1982). Epitype. A sample (W2370) from the Irish isolate (Attempt 564) is designated as the epitype. Part of this has been lodged at the herbarium of CAB International (IMI). To comply with the eminently desirable Recommendation 8B.1 of the Botanical Code (Greuter et al., 1994), an ex-epitype culture has been lodged at IMI and in La Banque Europe! enne des Glomales (BEG 62). Distribution Widely distributed in arable land in Finland and apparently in Northern Europe and the USA. Not so far known from tropical localities but one isolate identified from the southern hemisphere (temperate Brazil). Known also from a field collection made in temperate India. Mycorrhizal associations Under its various names, the species has been recorded as forming arbuscular or vesicular arbuscular mycorrhizas with Allium porrum, A. cepa, Alstroemeria sp., Chrysanthemum superbum, Coleus¬hybridus, Echinacea purpurea, Fragaria¬ananassa, F. esca, Fraxinus excelsior, Glycene max, Hedera helix, Hordeum ulgare, Hyssopus officinalis, Lycopersicum esculentum, Malus baccata, M. domestica, Origanum ulgare, Paspalum notatum (Fig. 25), Phlox paniculata, Plantago lanceolata (Fig. 26), P. major, Prunus domestica, Ricinus communis, Solanum tuberosum, Sorbus aucuparia, Sorghum sudanense, Tagetes sp., Trifolium pratense, Trifolium repens, Trifolium subterraneum and Zea mays. A C K N O W L E D G E M E N TS We thank J. Błaszkowski (Szczecin), A. Camprubi (Cabrils), Y. Dalpe! (Ottawa), M. Gryndler (Prague), D. Mitchell and J. O’Neill (Dublin), I. Jakobsen (Roskilde), M. Daft (Dundee), A. Fitter & J. Merryweather (York), and F. Sanders (Leeds) who supplied cultures or assisted with collection of soil samples, and K. G. Mukerji & J. P. Tewari for help with establishing the facts about the missing type specimens of G. multisubstensum. Alice Broome, David Clark, Anna-Maija Kartano-Ylen and Prikko Jalkanen provided expert technical assistance. We also thank the herbarium curators at the University of Copenhagen and Oregon State University and an anonymous referee who offered useful suggestions. Cultures of arbuscular mycorrhizal fungi from outside the European Community were imported into Great Britain under Scottish Agricultural Science Agency soil importation licence number IMP} SOIL}14}1996 and IMP}SOIL}16}1996.
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L I T E R A T U R E C I T ED Abbott LK. 1982. Comparative anatomy of vesicular-arbuscular mycorrhizas formed on subterranean clover. Australian Journal of Botany 30 : 485–499. Anon. 1969. Royal Botanic Garden Edinburgh. Flora of British fungi. Colour identification chart. Edinburgh : Her Majesty’s Stationery Office. Bentivenga SP, Morton JB. 1995. A monograph of the genus Gigaspora, incorporating developmental patterns of morphological characters. Mycologica 87 : 719–731. Berch SM. 1986. Endogonaceae : taxonomy, specificity, fossil record, phylogeny. In : Mukerji KG, Singh VP. Frontiers in applied microbiology Vol. 2. Lucknow, India : Print House, 161–188. Berch SM, Koske RE. 1986. Glomus pansihalos, a new species in the Endogonaceae, Zygomycetes. Mycologia 78 : 832–836. Błaszkowski J. 1994. Polish Glomales. XI. Glomus pustulatum. Mycorrhiza 4 : 201–207. Błaszkowski J. 1988. Three new vesicular-arbuscular mycorrhizal fungi (Endogonaceae) from Poland. Bulletin of the Polish Academy of Sciences. Biological Sciences 36 : 271–275. Dalpe! Y, Koske RE, Tews LL. 1992. Glomus lamellosum sp. nov. : a new Glomaceae associated with beach grass. Mycotaxon 43 : 289–293. Ferrer RL, Herrera RA. 1981. El genero Gigaspora Gerdemann et Trappe (Endogonaceae) en Cuba. Re. Jardin Botanico Nacional, Habana 1 : 43–66. Gerdemann JW, Nicolson TH. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Transactions of the British Mycological Society 46 : 235–244. Gerdemann JW, Trappe JM. 1974. The Endogonaceae in the Pacific Northwest. Mycologica Memoir 5 : 1–76. Gilmore AE. 1968. Phycomycetous mycorrhizal organisms collected by open-pot culture methods. Hilgardia 39 : 87–105. Gonnet J-F. 1995. A colorimetric look at the RHS chart—perspectives for an instrumental determination of colour codes. Journal of Horticultural Science 70 : 191–206. Greuter W, Barrie FR, Burdet HM, Chaloner WG, Demoulin V, Hawksworth DL, Joergensen PM, Nicolson DH, Silvia P, Trehane P, McNeill J. International Code of Botanical Nomenclature (Tokyo Code) Regnum Vetetabile 131. Koeltz Scientific Books. Gryndler M. 1995. Arbuscular mycorrhizal fungi in artificial cultivation systems. In : Turnau K, ed. COST 821. Arbuscular mycorrhizas in sustainable soil-plant systems. Arbuscular mycorrhizas as a link between East and West European countries. Proceedings of a conference held at Kazimierz Pułaski Polonia Collegium, Jagellonian Uniersity, KrakoU w, Poland, 2 to 5 June 1994. European Commission Luxembourg, 15–19. Koske RE, Friese C, Walker C, Dalpe! Y. 1986. Glomus pustulatum : a new species in the Endogonaceae. Mycotaxon 26 : 143–149. Lee P-J, Koske RE. 1994. Gigaspora gigantea : parasitism of spores by fungi and actinomycetes. Mycological Research 98 : 458–466. Miller DD, Walker C. 1986. Glomus maculosum sp. nov. (Endogonaceae) : an endomycorrhizal fungus. Mycotaxon 25 : 217–227. Morton JB. 1986. Three new species of Acaulospora (Endogonaceae) from high aluminium, low pH soils in West Virginia. Mycologia 78 : 641–648. Morton JB. 1995. Taxonomic and phylogenetic divergence among five Scutellospora species based on comparative development sequences. Mycologia 87 : 127–137. Morton JB, Walker C. 1984. Glomus diaphanum : a new species in the Endogonaceae common in West Virginia. Mycotaxon 21 : 431–440. Mukerji KG, Bhattacharjee M, Tewari JP. 1983. New species of vesicular-arbuscular mycorrhizal fungi. Transactions of the British Mycological Society 81 : 641–643. Omar MB, Bolland L, Heather WA. 1979. A permanent mounting medium for fungi. Bulletin of the British Mycological Society 13 : 31–32. Schenck NC, Smith GS. 1982. Additional new and unreported species of mycorrhizal fungi (Endogonaceae)from Florida. Mycologia 74 : 77–92. Schenck NC, Spain JL, Sieverding E, Howeler RH. 1984. Several new
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Walker C. 1986. Taxonomic concepts in the Endogonaceae : II. A fifth morphological wall type in endogonaceous spores. Mycotaxon 25 : 95–99. Walker C. 1992. Systematics and taxonomy of the arbuscular endomycorrhizal fungi (Glomales)—a possible way forward. Agronomie 12 : 887–897. Walker C, Gianinazzi-Pearson V, Marion-Espinasse H. 1993. Scutellospora castanea, a newly described arbuscular mycorrhizal species. Cryptogamie, Mycologie 14 : 279–286. Walker C, Giovannetti M, Avio L, Citernesi AS, Nicolson TH. 1995. A new fungal species forming arbuscular mycorrhizas : Glomus iscosum. Mycological Research 99 : 1500–1506. Walker C, Mize CW, McNabb HS Jr. 1982. Populations of endogonaceous fungi at two locations in central Iowa. Canadian Journal of Botany 60 : 2518–2529. Walker C, Rhodes LH. 1981. Glomus albidus : a new species in the Endogonaceae. Mycotaxon 12 : 509–514. Walker C, Vestberg M. 1994. A simple and inexpensive method for producing and maintaining closed pot cultures of arbuscular mycorrhizal fungi. Agricultural Science in Finland : 3 : 233–240.
