PERSOONIA Published by Rijksherbarium/ Hortus Botanicus, Leiden Volume Part 491-512 16, 4, pp. (1998) Multivariateanalysis of the Scutellinia umbrorum complex (Pezizales, Ascomycetes) from five ecotopes in the Netherlands 2 Jaanus Paal Bellis Kullman & Henk+A. Huijser The multivariate structure of the Scutellinia umbrorum complex (Pezizales, Ascomycetes), based on the morphometrical parameters of 81 specimens from five ecotopes in the Nether- five lands, was analysed. According to conventional expert estimation, putative taxa resp. Svrček, S. umbro- species were established: S. patagonica (Rehm) Gamundi, S. aff. subhirtella form rum (Fr.) Lambotte, S. parvispora J. Moravec and S. subhirtella s. Kullman. These taxa a taxonomic continuum hardly separable by traditional taxonomy. Five clusters obtained by UPGMA (with the generalized J-distance for mixed data asa measure of resemblance) are more distinct and in good accordance with ecological factors; some of them, however, are statistically not well separated. The revision of the clusters’ structure by k-means approach yields highly discontinuous clusters. The morphometric characters of specimens differ when going from habitats the forest. Differences also revealed in the open to are phenology: growing season starts in the forest later than in open habitats. The data are divided into two subsets according ornamentation and the variation of either subset is caused to spore spore width, withingroup mainly by the length of marginal hairs. On the basis of several statistical methods a supposi- tion was introduced that the S. umbrorum complex probably consists of two polymorphic species, S. umbrorum (Fr.) Lambotte and S. subhirtella Svrček s.l., with the mean value of 450 The clus- marginal hairs longer than 450 µm and shorter than µm, respectively. UPGMA ecodemes of ters can be interpreted as respective species. Introduction The systematics of the order Pezizales (Ascomycetes ) is disputable and rather unstable (Kimbrough & Gibson, 1989; van Brummelen, 1994; Bunyard et al., 1995). The sepa- ration of some species is based on the morphological description of one or two specimens of only. The result is a multitudeof putative taxa. When studying more abundantmaterial variation of characters closely related species in Pezizales, an extensive morphological becomes evident. Taking into account that the Pezizales have "a virtually invariant haplophase with pre- it be dominantly homomictic mating" (Burnett, 1987; Weber, 1992) may considered that these fungi exist in natural conditions as lineages, i.e. as groups of clones with the same Scutellinia Lambotte emend. Gal there DNA haplotype. In the genus (Cooke) Le are Scutellinia scutellata Lambotte & Wil- homothallic species, e.g. (Fr.) (Gwynne-Vaughan liamson, 1933). of Scutellinia follows continuous the substrate The evolutionary pattern divergence, being the most important natural selection factor for these saprotrophic fungi (Kullman, ') Institute of Botany and Ecology, University ofTartu, Lai St. 40, EE-2400 Tartu, Estonia. 2) Institute ofZoology and Botany, Riia St. 181, EE-2400 Tartu, Estonia. 3) Frederikstraat 6, 5671 XH Nuenen, The Netherlands. 492 PERSOONIA Vol. 16, Part 4, 1998 1979, 1982, 1986; Kullman & Rahi, 1988a, 1988b, 1989, 1990; Schumacher, 1990). Sometimes response to the substrate and other environmental conditions is expressed also by local ecotypical variability which is revealed in pronounced morphological difference (Stebbins, 1950; Kullman, 1977, 1995). aims to the structure of dataset of The present paper (i) analyse a comparatively large the Scutellinia umbrorum complex, hardly distinguishable morphologically, (ii) test the statistical significance of clusters obtained by different phenetic approaches of classifi- cation of these data, and (iii) analyse the dependence of data structure on substrate and habitat conditions. MATERIAL AND METHODS Study sites The material was collectedin the Netherlands during 1979-89.Five localities, consti- tuting a trophic gradient from ecotopes poor in nutrients to richer ones were studied: of brick-work. The habitat (i) Best-Son (denoted as B). Former loam-pit represents after the a deposition of loam or loamy sand being permanently wet or drying slowly re- dominated Salix moval of loam. Only pioneer vegetation has formed, by Equisetum spp., Alnus 16 collected. spp., glutinosa, mosses, etc. specimens (ii) Nuenen (N). Abandoned loam-pit. Habitat and vegetation quite similar to that of Best Son. 16 specimens collected. (iii) Eindhoven, Urkhoven (E). Paludifiedmeadow with a sparse field layer on sandy- loamy soil. Traversed by car-tracks. Transition zone between wet heathland and reed The main vascular are: Pedicularis Gentianapneumonanthe, Dac- swamp. species spp., tylorhiza maculata, Platantherabifolia, Carex spp., Phragmites australis, Sphagnum spp. 7 specimens collected. Rather moist meadow (iv) Helmond, De Schouw (H). open moderately eutrophic (hay- Dominant vasculars Carex land) on sandy-loamy soil. Traversed by car-tracks. are: spp., collected. Equisetum spp., and Dactylorhiza majalis. 25 specimens (v) Elsloo-Geulle, Bunderbos (L). Eutrophic forest slope (with calcareous springs) on brooklet. Abundant vasculars slowly drying black mud along a regularly overflowing are: Equisetum telmateia, Impatiens noli-tangere, Chrysosplenium sp., and Allium ursinum. 16 specimens collected. Measurements Thecharacteristics offungal fruit-bodies were measured with themicroscope 'Olympus observation medium. For BH-2', magnification up to 1000 x. Tap water was used as an measured: hairs all 81 specimens, 4 morphological parameters were length of marginal on width of hairs the the apothecium (PILL), marginal on apothecium (PILW), spore length (SPOL), spore width (SPOW). For every specimen all morphological parameters were measured in not less than ten replications. The mean values were used as initial data for multivariate analysis. A subtractedparameter, the ratioof spore length to spore width (Q), ornamentation was also included in the data matrix. Additionally, the type of spore (OR) was identified as (i) tuberculate, (ii) verrucose, and treated as a binary variable. The type blue solutionin ofornamentationwas studied by staining the spore wall in cotton lactophenol electron 1 and as well as from photographs of the scanning microscope (Figs. 2). Paal, Kullmann & Huijser: Multivariate analysis of the Scutellinia umbrorum complex 493 Fig. 1. Spores with a tuberculate type of ornamentation. Specimen of S. parvispora according to the ex- 5 — 2. with of pert estimation. Magnification 3000 x (15 mm = μm). Fig. Spores a verrucose type orna- mentation. Specimen of S. subhirtella s. Kullman according to the expert estimation. Magnification 3000 x (15 mm = 5 μm). 494 PERSOONIA Vol. 16, Part 4, 1998 Two nominal characteristics were used as environmental parameters. One describes from the loam to the type of the ecotope according to its trophicity and openness pit the eutrophic forest slope (see study sites: B, N, E, H, and L). The other corresponds to the O = humous soil W = wood. substrate: S = mineral soil, or decaying wood, Data At first the specimens (OTUs) were classified traditionally (see diagnoses below). The results ofthis were further taken as a basis for comparison and search for an optimal solu- tion by numerical methods. 1. Scutelliniapatagonica (Rehm) Gamundi (denoted as Pt) Diagnosis: Apothecia 3—12(—20) mm. Marginal hairs 200-1160 x 18—40(—45) |im, sample means between 535-695 x 25-32 pm, average 600 x 28.8 pm, 3-28 septate; often walls (2-)3-6 pm thick; base with 1-3 roots, the longest hairs more complex. Ascospores (17.5—)18—22 x 15-18.5 pm, sample means between 19.4-20.7 x 15.7— 17.5 pm, average 20.1 x 16.7 pm; Q = 1.1-1.3, means between 1.14-1.27, average of different size confluent 0.5—1 1.20; ornamentationtuberculate, warts rarely -2(—1.5) pm high and 0.3-1.7(—2.5) pm wide. Habitat H. in with S. described Gamundi These characteristics are good agreement patagonica as by (1975) and Schumacher (1990), but also correspond with S. arenosa (Velen.) Le Gal (Le Gal, 1966b and Moravec, 1974). 2. Scutellinia aff. subhirtellaSvrček (denoted as Sb) 12-35 Diagnosis: Apothecia 1.5—10(—15) mm. Marginal hairs 110-650(-720) x pm, sample means between 255-465 x 19-27.4 pm, average 360 x 23.6 pm, (0-)2-14 sep- tate; walls 1-5 pm thick; base usually with 1-3 roots, exceptionally more complex. Asco- between 18.9-22.5 spores (17—)18—23(—24) x (13—)14—16.5(—17.5) pm, sample means 14.3-16.3 20.5 x 15.3 = means between 1.26— x pm, average pm; Q 1.2—1.5(—1.65), variable warts 1.46, average 1.34; ornamentationheterogeneous tuberculate(-verrucose), 0.3-1.0 and 0.3-2.0 often isolated but of different size and shape pm high pm wide, sometimes confluent, forming short ridges and irregular complexes 1-1.5 pm high and and 2-3(-4) pm wide. Habitats B N. The marginal hairs fit well Schumacher's (1990) description of S. subhirtellaSvrcek, and ornamentation deviate too much and look like his S. umbrorum. but spore shape more S. umbro- Because of this resemblance such collections have certainly been conceived as the real rum (cf. Le Gal, 1947;Maas Geesteranus, 1969; they might even represent Peziza umbrorumFries, 1823). 3. Scutellinia umbrorum(Fr.) Lambotte (denoted as Um) 16-43 Diagnosis: Apothecia 3-16(-20) mm. Marginal hairs 200-1120 x pm, sample 23-30.4 568 25.9 2-29 walls 2- means between 430-745 x pm, average x pm, septate; roots but hairs also multifurcate. Asco- 7 pm thick; base with 1-3 conspicuous longest 18.5-24 13.5—17.5 between 20.4-22.5 x 14.1-16.4 spores x (13—) pm, sample means 21.3 15.5 = means between 1.30-1.47, pm, average x pm; Q 1.25—1.55(—1.7), average 1.38; ornamentationtuberculate, mostly isolated semiglobose or slightly angular warts sometimes crateriform. of different size 0.5—1.5(—2.5) pm high and 0.5-2(-3) pm wide, Paal, Kullman & Huijser: Multivariate analysis of the Scutellinia umbrorum complex 495 Habitats H and E.