Tuatara: Volume 19, Issue 1, November 1971
Chemical Aids to Species Recognition in the Lichen Genus Cladonia
Chemical Aids to Species Recognition in the Lichen Genus Cladonia
Over Much of the Earth the genus Cladonia is represented on heaths, bogs, and sterile soils, or on decaying logs, at elevations ranging from sea-level to alpine habitats. The majority of some seventy species indigenous to New Zealand occur at elevations under 1,200 metres. Endemic species are few; some are cosmopolitan, and most are wide-ranging. Some species are easy to recognise; but as Bruce Fink has pointed out when referring to the lichen flora of North America. ‘Cladonias are the most variable of all lichens, and the most difficult to describe accurately. Nothing but the most careful observation will enable anyone to determine a Cladonia with any degree of certainty even with the best descriptions.’
Since these observations were recorded, however, a great deal of research has been devoted to the genus Cladonia by Asahina, Evans, Ahti, Sandstede, Des Abbrayes, Culberson, and others, all of whom have studied the chemical as well as the morphological attributes of the various species. A majority of lichens produce ‘lichen acids’, most of which are found only in lichens, and for the recognition of many of which simple microchemical tests have been devised. Usnic acid and atronorine are located in the cortex but most of the others form an incrustation on the hyphae of the medulla. The most commonly used reagents are (1) an aqueous solution of potassium hydrate (KOH); (2) a saturated aqueous solution of calcium hypo-chlorite (CaOCl2) or alternatively one of the commercial bleaching fluids; and (3) an alcoholic solution of para-phenyline-diamine freshly prepared. Each of these produces a distinctive colour change when applied to the cortex or medulla when certain acids are present, and none in their absence.
Many species of Cladonia constantly produce the same acid or acids which in consequence are termed ‘diagnostic’ for that species. Other accompanying acids not invariably present are termed ‘accessory’. If therefore a specimen believed to be a particular species is found not to contain an acid diagnostic for that species then the determination is wrong and must be revised. The above reagents are indicated as a rule by the letters K, C, and P or Pd. KC indicates a drop of K is immediately followed by a drop of C on the same area. A + sign following indicates a colour change results and this is usually followed by the first letter of the colour resulting. Thus K + y indicates a colour change to yellow, while K — symbolises no colour change. The identification of acids that produce no colour reaction can best be determined by the form of their crystals or crystals of their salts or by means of paper chromatography. page 7 The procedure for these tests and the form of the crystals may be consulted in the Lichen Handbook by Mason E. Hale — a Smithsonian Institute publication.
Every lichen represents a symbiotic association of a fungus, and a colony of algal cells on which the fungus is dependent for food and enzymes, as all fungi are devoid of chlorophyll. The relationship is more than simple symbiosis, however, as the lichen acquires its own distinctive form and often produces structures such as soredia, isidia, cephalodia, or cyphellae unknown save on lichens. The fruiting bodies (apothecia) are those of the fungus only and the contained spores are of course fungus spores, and can only reproduce a new generation of lichens when on germination they fortuitously make contact with the appropriate algal cells living free in the area. It is believed, however, that propagation in Cladonia is rarely from spores but from such diaspores’ as soredia, isidia, or thallus fragments in which both symbionts are already associated.
The germinating diaspore develops into a primary thallus either squamulose or crustose in form. The crustose form is characteristic of sections Unciales and Cladinae and the squamulose form of most other species. The largest squamules 2-3cm. long are found in Sections Foliosae and Podostelides. After a time a secondary fruticose thallus may form on the surface or margin of the primary thallus, known as a podetium. The apothecia in Cladonia are always borne on the tips of the podetia or of their branches. A cross-section of a typical podetium displays a central hollow cylinder of compact chondroid tissue surrounded by a medulla of fungal hyphae, then by an algal zone, and commonly by an outer cortex which may be more or less continuous, or areolate, and often sorediate.
Weise has demonstrated experimentally that only the chondroid cylinder arises from the tissue of the primary thallus, the outer layers being derived from windborne soredia that lodge on its surface. In the absence of an algal layer in the podetial wall, the central cylinder often atrophies for lack of the supplementary nourishment these algae are able to provide.
The podetia of the more primitive species are short and simple or few branched, and the pycnidia and apothecia may occur on the primary thallus. In the Cladinae the podetia are often densely branched and both pycnidia and apothecia are confined to the branch tips of fertile plants. The primary thallus may be persistent or ephemeral, and soon disappears when crustose. Some species have podetia that die at the base but continue growth at the apex. Some species rarely or never produce apothecia or spores.
A problem not yet finally resolved relates to the so-called ‘chemical species’. Do chemotypes have true taxonomic status? Most botanists recognise them, but not all. It has been suggested that when two or more lichens morphologically alike but each with a different acid content occupy different regions, they should be page 8 regarded as autonomous species, but when they range over the same area they should be deemed chemical strains of a single species. Thus some botanists consider Cladonia chlorophaea, Cladonia grayi, Cladonia merochlorophaea, and Cladonia cryptochlorophaea as four chemical strains of Cladonia chlorophaea. Three additional species closely resembling them, viz. Cl. fimbriata, Cl. conista, and Cl. pyxidata, can be distinguished by morphological features and are deemed valid species by all.
A number of species supposedly indigenous have been reported so seldom or once only that further corroboration is desirable. Even expert specialists can and do make wrong determinations. Thus New Zealand members of Section Cladinae were long regarded as Cl. rangiferina, Cl. sylvestris, Cl. alpestris, and Cl. pycnoclada — not one of which was correctly determined. This section today comprises Cl. mitis, Cl. alpestroides, and Cl. leptoclada, all P —, though three of the former species are P +. Species collected only once and listed as indigenous include C. chondrotypa, Cl. Krempelhuber, Cl. metalepla, Cl. oceanica, and Cl. solida. Other doubtful indigens include Cl. decorticata, Cl. medusina, and Cl. ventricosa. The supposed endemics are Cl. neozelandica, Cl. southlandica, Cl. murrayi, and Cl. enantia. The first two seem amply distinct but Cl. enantia scarcely differs from Cl. cariosa, and, though morphologically distinct, its chemistry has suggested the possibility that Cl. murrayi may be a monstrous form of Cl. deformis.
A key to the indigenous Cladoniae may be consulted in Trans. Roy. Soc. N.Z., vol. 85, part 4, pp. 603-22 (1958). The diagnostic lichen acids accompanying each Cladonia species together with their chemical reactions to K, KC, and P are listed below. It may be noted that all New Zealand species in Sections Unciales, Cladinae, and Foliosae contain usnic acid, and Podostelides, Thallostelides and Chasmaria species are devoid of usnic acid. All Thallostelides species other than Cl. grayi and Cl. nernoxyna contain fumarprotocetraric acid which is absent from all New Zealand Cladinae and Unciales. The lichen acids bellidiflorine, didymic acid, and zeorine are confined to the Cocciferae.
Commoner Lichen Acids and Their Reactions to K, KC, and P
|K + yellow||Atronorine, stictic, and thamnolic acid|
|K + blood red||Norstictic acid, salazinic acid|
|K + wine red||Cryptochlorophaeic acid, merochlorophaeic acid|
|KC + yellow||Usnic acid|
|KC + red||Gyrophoric, electoronic, and lecanoric acids|
|P + light yellow||Psoromic acidpage 9|
|P + deep yellow||Norstictic acid, salazinic acid|
|P + dull red||Fumarprotocetraric acid|
|P + orange red||Stictic acid|
|P + bright red||Homosekikaic acid|
The following lichen acids give no colour reactions with K, KC, or P — viz. Barbatic, Cervicornic, Didymic, Fimbriatic, Rangiformic, Grayanic, Perlatolic, and Squamatic acids — but can be identified from the form of crystals of the acid or of its salts. For illustrations of these crystals and the technique of obtaining them see Hale's Lichen Handbook, chapter 6, pp. 66-75.
Chemistry of Indigenous Species of Cladonia
|Cladonia alcicornis||−||+fy||+r||Fumarprotocetraric, Usnic|
|" amaurocraea||−||+||−||Usnic, Barbatic|
|" borbonica||±f||−||+||Fumarprotocetraric, Grayanic|
|" boryi||−||+y||α||Usnic, Zeorin|
|" carneola||+y||+||−||Usnic, Barbatic|
|" cenotea (?)||−||−||−||Squamatic|
|" coccifera||−||+||−||Usnic, Barbatic|
|" cylindrica||+r||Fumarprotocetraric, Grayanic|
|" deformis||−||±||−||Usnic, Zeorine|
|" didyma||−||−||−||Barbatic, Didymic|
|" enantia||±f||−||+?||Atronorine (?)|
|" fimbriata||±f||−||+r||Fumarprotocetraric, Fimbriaticpage 10|
|" floerkeana||−||±f||−||Barbatic, Didymic, Usnic,|
|" leptoclada||−||+||−||Usnic, Perlatolic (sometimes)|
|" mitis||−||+||±||Usnic, Rangiformic|
|" pleurota||−||±f||−||Usnic, Zeorine|
|" polydactyla||+or-v||+y||+y||Thamnolic (= C. flabelliformis)|
|" subcariosa||Sq+y-r||−||+y||Atronorine, Norstictic|
|" subulata||−||−||+r||Fumarprotocetraric (Syn. = Cl. cornutoradiata)|
|" uncialis||−||+||−||Usnic, Squamatic|
|" vulcanica||+y||−||+y||Thamnolic (Barbatic, Didymic accessory)|
(Note: f = faintly; ± = sometimes +, sometimes -; br = brown, r = red, y = yellow; sq = squamules)
To identify a species of Cladonia first consult the key to the indigenous species in Vol. 85, part 4, of the Trans. Roy. Soc. of N.Z., and make a tentative determination both of the species and of the section of Cladonia to which it belongs. Now apply the reagents K, KC, and P to the cortex or medulla as the case may require, and note the reactions. Compare these with those of the presumed species in the above table. If they differ your tentative determination is probably incorrect, but if they agree you are probably but not necessarily correct. Final corroboration requires comparison of the specimen with a full description of the species; but if this is not available, compare the plant with authentic specimens in some herbarium, page 11 always making allowance for a possible wide range of variation. Most species should present no great difficulty, though it should be noted that many plants are, or at least resemble, hybrids between more clearly defined species, and are well-nigh unclassifiable.
Some species show their reactions best when the contained acids are extracted by placing a portion of the plant in the bottom of a test-tube and covering with acetone. The specimen is now removed and the solution evaporated to a small quantity which is then transferred to a microscopic slide to complete the evaporation. The solid residue is now divided into separate portions to which the reagents are separately applied, and any colour changes noted. Many botanists apply this technique to all specimens.