The Cladonia pyxidata-fimbriata complex in the Netherlands , with description of a new variety

are recognized: Cladonia fimbriata, Cl. conistea, Cl. conista, Cl. pyxidata. Cl. chlorophaea. Cl. cryptochlorophaea, Cl. merochlorophaea and Cl. grayi. A strain with novochlorophaeic acid is described as a new variety; Cl. merochlorophaeaAsah. var. novochlorophaea (colour reactions: P orange-red or often negative; K -> negative; C -> negative or yellow; KC -> negative). In morphological and ecological respect a close relationship has been found between 1. Cladonia conistea and Cl. conista, and 2. Cladonia cryptochlorophaea and Cl. merochlorophaea. For comparison also some material from other European countries has been studied.


INTRODUCTION
Since the taxonomic value of these new species, often considered as chemical strains, was questioned by some authors (Dahl 1950), a more extensive study of the differences between them was necessary. For this an examination of the morphology, chemistry, and ecology of the taxa is required. To eliminate a good deal of the variation caused by geographical isolation or large climatical differences, a study of larger sets of material from limited areas seemed to be The group of lichens with which this paper is concerned, conveniently called the Cladonia pyxidata-fimbriata complex, comprises Cladonia pyxidata ( L.) Hoffm., Cl. fimbriata (L.) Fr., and a number of closely related species which in this century have been separated from them, mainly on the basis of their chemistry.
The occurrence in Europe of these species was studied mainly by Asahina (1940Asahina ( , 1941, Ahti (1966), and Leuckert et al. (1972), the second author working especially with Finnish, the third with Central European material.
No Dutch material was included in these studies. Reports of species belonging to this complex from the Netherlands by Maas Geesteranus (1954) and Hennipman (1969) comprise Cladonia fimbriata. Cl. pyxidata var. chlorophaea Flk., and Cl. pyxidata var. grayi Merr., the last-named variety being distinguished by its negative P-reaction after Sandstede (1938 (Ahti 1966) and the U.S.A. (Kristinsson 1971). The large amounts of material collected in the Netherlands offered another, excellent opportunity; the results of the study of this material are described in the following. For the chemical analysis, microcrystal-tests with acetone-extraction were used (Thomson 1968). Besides, colour reactions were checked on filter-paper impregnated with part of the extract. For the C reaction a bleach for domestic use was applied. This occasionally produces a colour reaction different from that of the usual calcium-hypochlorite solution, in particular the C -> yellow reaction in Cladonia merochlorophaea var. novochlorophaea (see below). After some experience, observation of the dried acetone extract together with the colour reactions in most cases proved to be reliable for identification.
All measurements have been taken from dry material. The dimensions of the soredia and granulae generally were determined on the surface of well-developed cups with the aid of a microscope with ocular micrometer. The smallest measurements given are from young stages of soredia or granulae, the larger ones from fully developed stages. The soredia and granulae were distinguished from scales by their rounded shape and the absence of lobes.
An insight into the ecology of the species was obtained by study of the accompanying mosses and lichens and of remnants of the substrate, as present in the collections, in connection with data on habitat and vegetation from the labels.
It is remarkable that the dimensions of the soredia and granulae found by the author are smaller than those given by some other authors. In Cladonia cryptochlorophaea, for example, the diameter of the soredia was found to be 20-80 jxm, contrasting with 30-130 urn in Kristinsson (1971). It is true that the measurements of Kristinsson were based on wet, thus swollen material, whereas the author worked with dried material. However, it is the author's impression that this does not satisfactorily explain the differences. Climatical differences probably are responsible.
As to the apothecia in the Cladonia pyxidata-fimbriata complex, two types could be distinguished. Colour reactions'. P-> orange-red; K->yellow, often difficult to distinguish from the reaction K->negative (weakly orange, after a few minutes brownish); C->negative; KC->negative.
In exposed sites, for instance on the Wadden Islands, the species is conspicuous by its strongly developed thallus leaves curled up and exposing their white lower side when dry; its podetia are small and whitish. Inland forms generally have a smaller thallus and larger podetia with longer stalks. Proliferating podetia with cups on three levels were seen in a collection from Bretagne (de Smidts.n. 6.V11.1963, U).
Distribution and ecology, occurring all over the Netherlands, on weakly acid, sandy or loamy soil, frequently in weakly calcareous sand-dunes and often associated with Ceratodon purpureus and Cladonia fimbriata, rarely epiphytic.
A find of the species on a dam near the harbour of Terschelling suggests that it may be salt tolerant.
Very close to Cladonia conistea from which it differs in chemistry only; instead of atranorin Cladonia conista contains "substance H", which may be seen in the dried acetone extract as very long (often over 100 |im) colourless needles arranged in a criss-cross pattern. The same result is obtained with the GAW test. The K reaction is negative.
Distribution and ecology: common all over the Netherlands, on weakly acid, sandy or loamy soil, often together with Ceratodon purpureas but also often with Pohlia nutans and Polytrichum piliferum, sometimes epiphytic.
In the Netherlands the species seems to prefer less acid substrates than the next species. The same is observed in Finland (Ahti 1966).
Colour reactions: K-» orange, after a few minutes claret; C->rosy for one second; KC-> strongly rosy for one second.
Because of the positive P-reaction all material belongs to f. cryptochlorophaea Asah.
Dried acetone extract: on the edge a more or less complete gummy ring, better developed than in the last two taxa, is formed. Locally accumulations of short, prismatic, colourless crystals are present. Sometimes oblong, brownish crystals arranged in radiate groups are also present.
With GAW thick, prismatic, colourless crystals are obtained which originate very slowly from oil droplets; often it is better to make the observations after an overnight's wait. They may appear as small, rhombic double prisms, often crossing in pairs. Sometimes they form groups of rather large, single prisms.
Various other types of colourless crystals may be present: oblong, thick ones, oblong and flat ones, and large or small needle-shaped ones. These aberrant crystals may dominate the prismatic ones.
The prismatic crystals fit the description of novochlorophaeic acid as given by Dahl (1950) and Thomson (1968). Culberson & Kristinsson (1969) made chromatograms of the novochlorophaeic acid strain and found a mixture of sekikaik (dominant) and homosekikaik acid. Ahti (1966) reported the occurrence of merochlorophaeic acid in the novochlorophaeic acid strain. In the course of the present study crystals were found resembling those of merochlorophaeic acid, but absence of the characteristic colour reactions indicated that they belong to some other substance. Distribution and ecology: in general as in the two previous taxa, with which it often grows intermingled. P-negative specimens are most common in the northern part of the country and are absent from the South.
According to Thomson (1968) the K reaction should be negative. The P-positive material is called f. aberrans Asah.
Morphology: In the scanty material from the Netherlands no clear morphological differences with the two foregoing species could be found. According to Ahti (1966) the apothecial groups and the soredia are smaller than those of Cladonia merochlorophaea. Kristinsson (1971) found a considerable overlap in the size of the soredia of Cl. grayi and Cl. cryptochlorophaea.
A more distant relationship between Cladonia grayi and Cl. cryptochlorophaea with Cl. merochlorophaea was suggested by Shibata & Chiang (1965) on account of the chemical structure of their lichen acids.
Distribution and ecology: an overall resemblance with the two foregoing species was found, but they were not found growing together.
The distribution in the Netherlands is restricted to a small area in the East ("subcentreuroop district"), probably an outpost of a more continental range.
These data together suggest a close relationship between the following taxa: novochlorophaea. The taxa of each of these groups may better be united into single species. The chemical variants within them could be interpreted as chemical strains of lower than specific rank. To confirm this conclusion, it is needed to check the existence outside the Netherlands of correlations between morphological, chemical and ecological characters.
These conclusions largely agree with those drawn for Central Europe by Leuckert et al. (1971), who found no distinction between Cladonia pyxidata and Cl. chlorophaea, and no morphological differences between Cladonia cryptochlorophaea, Cl. merochlorophaea, and the novochlorophaeic acid strain.

EXPLICATION OF THE ABBREVIATIONS
The abbreviations employed in describing the chemistry are the usual ones in the lichenological literature. They include the following colour reactions: P: adding a droplet of c. 10% paraphenylenediamine solution in ethanol.
The wetted spot may turn yellow, orange or red. Instead of this highly instable solution (to be replaced after 5 hours) the 'Steiner's solution is used: 1 % paraphenylenediamine and 10% sodium hyposulfite in distilled water with a trace of a detergent. In the microcrystal tests the following abbreviations are used GAW: a drop of glycerine-ethanol-distilled water mixture (1:1:1) is added to the dried lichen extract. After some gentle heating (until boiling) the extracted products may recrystallize and produce much better developped crystals.
GAoT; a drop of glycerine-ethanol-orthotoluidine mixture (2:2:1) is added to the dried lichen extract. After gentle heating (until boiling) a reaction between the orthotoluidine and some extracted lichen product may give rise to a substance with characteristic crystals.
The lichen extracts are prepared on an object slide, and for recrystallizing a cover slide is put on the mixture.
For more extensive descriptions of these methods see Thomson (1968