«THE BRYOLOGIST [Volume 83 228 Horton,D. G. & W. B. Schofield. 1977. Cololejeuneamacounii, a second locality in North America. 80: THE BRYOLOGIST ...»
THE BRYOLOGIST [Volume 83
Horton,D. G. & W. B. Schofield. 1977. Cololejeuneamacounii, a second locality in North America.
THE BRYOLOGIST 647-650.
Mizutani,M. 1961. A revision of the JapaneseLejeuneaceae.J. HattoriBot. Lab. 24: 115-302.
Miiller,K. 1956. Die LebermooseEuropas. 3rd ed. Leipzig.
Schofield,W. B. 1968. Bryophytesof BritishColumbia.II. Hepaticaeof particularinterest.J. Hattori
Bot. Lab. 31: 265-282.
Schuster,R. M. 1956. North AmericanLejeuneaceaeIII. Paradoxae:Cololejeunea(Concl.), Dipla- siolejeunea.Jour. Elisha MitchellSci. Soc. 72: 87-125.
1. 1969. TheHepaticae and Anthocerotaeof North AmericaEast of the Hundredth Meridian.
Vol. II. ColumbiaUniv. Press, New York.
1977. A checklistof the liverwortsandhornwortsof NorthAmerica.
Stotler,R. & B. Crandall-Stotler.
THE BRYOLOGIST 405-428.
Underwood, M. 1890. A new North AmericanLejeunea.Bull. TorreyBot. Club 17: 258-259.
Vailia,J. 1976. Studien fiber die Jungermannioideae(Hepaticae). 10. Nardia. Folia Geobot. Phy- totax., Praha, 11: 367-425.
The Bryologist 83(2), 1980, pp. 228-233 Copyright @ 1980 by the American Bryological and Lichenological Society, Inc.
Acid-Base Color Reactions: The Status of Triquetrellaferruginea, Barbula inaequalifolia and B. calcarea
RICHARD H. ZANDER
Encouraged by recent reports (Hill 1976, Lane 1978) of the utility of color reactions to acids and bases in Sphagnum taxonomy, I made a survey of the response of the leaves of various species of Pottiaceae to a number of histochemical and chromatographic spray reagents given by Haas and Hill (1928), Hais and Macek (1968), Jensen (1962), Mueller (1973) and Seikel (1964). The only reactions that seemed to be commonly specific to taxa and therefore potentially useful in classification were simply changes in leaf color on application of strong mineral acids and bases. Such color reactions are usually reversible.
Leaves of Pottiaceae species readily blacken in 10% ferric chloride solution and
of the presence of various anthocyanins or flavonoids, which are phenolics commonly exhibiting changes in coloration with changes in pH (Geissman 1955, Robinson 1967).
Flavonoids are apparently common and diverse in acrocarpous mosses (McClure & Miller
1967) and have proven to be of considerable value in vascular plant taxonomy because of their chemical stability and general lack of variation in populations, even in environmentally modified plants. Martensson and Nilsson's (1974) review of the chemical bases of morphological color in bryophytes emphasized that to this date few color reactions to chemical reagents have been shown to be potentially important in bryophyte taxonomy.
Stone (1976) used differing reactions to staining by Toluidine Blue followed by a lactic acid rinse to discriminate, in part, two new species of Acaulon. Koponen (1968, 1974) reviewed color responses of Mniaceae species to ferric chloride and to potassium hydroxide solutions. Crundwell (1979) found color changes in moss rhizoids after application of acids and bases to be often strong, distinctive and valuable as taxonomic characters for some species and groups. Several papers compiled by Suire (1978) emphasize the utility of phenolic analyses in taxonomy of certain bryophyte groups, but these involve comparatively lengthy chromatographic methods, not simple color reactions of the plants themselves.
I now routinely test herbariumspecimens with concentratedhydrochloricacid ("Cl"), a 10% solution of potassium hydroxide ("K"), concentrated nitric acid ("N"), and a 2 : 1 mixture of concentratedsulphuricacid and ethanol ("SE"). The alcohol in the last reagent(add acid to alcohol slowly in a cold water bath) retardsthe dissolutionof the leaf cell walls by the acid-dilution of the acid by water weakens or stops the color reactions of the moss plants, which is the reason blotting of wet plants is importantbefore testing. The pink partialreactionproductof the acid and alcohol has no evident effect on the color reactions. The four reagentsare corrosive but are safely kept in small, squeezable, plastic dropperbottles or in glass dropperbottles with rubberbulbs replaced by plastic bulbs when attackedby acids. Mixturesof both young and matureleaves are preparedfrom cleaned plants that have been soaked in hot water for 1-5 seconds, then lightly blotted. Portionsof these mixtures are placed in the slots of depression slides and saturatedwith 1-3 drops of each reagent. Extra acid reagentis added whenever calcareoussoil is adherentto the leaves. Whenchlorophyll appearsto mask the color response, as is commonlythe case in fresh plants, a lactic acid (concentrated)presoak of 15-30 seconds, followed by a hot water rinse, "clears" the plants and allows easy recognitionof color response in K but often not in other reagents for which mature, achlorophylloseleaves must be examined.The lactic acid apparently does not affect the subsequent response to reagent.The colors of the leaves (not the costae or the stems) are then recordedagainst under a dissecting microscope(up to two minutesare allowed to elapse if there a white background is no immediatereaction), and the slides are then cleaned of the reagentsby rinsingwith tapwater from a washbottleinto a large, glass canister. This technique is fast and, with some care, safe to clothes and person. A small fan may be used to disperseacridfumes.
Color reactions do not appear to lessen in intensity with aging of the herbarium specimen; indeed, the fading of chlorophyll allows better color discernment. Young plants or plants that grew in unusually moist conditions often exhibit a less strong response than do older plants or older portions of plants, or plants from more exposed stations. But sometimes young leaves have brighter color responses than do mature leaves. Some variation in color response in herbarium specimens may be due to leaching by preservatives after collection or degradation of chemicals by heat (see Coradin & Giannasi 1980, Culberson et al. 1977). But, in spite of some variation, many taxa appear to have color responses that may be used to distinguish them from other taxa. This can be of importance in separating taxa with morphologically similar gametophytes that are easily confused when sterile.
For example, sterile Anoectangium aestivum (Hedw.) Mitt. is easily distinguished from Hymenostylium recurvirostrum (Hedw.) Dix. by the N deep red reaction-the latter species is N light to medium orange-brown, seldom light to medium reddish-brown. Also, [Volume 83 230 THE BRYOLOGIST small plants of Didymodon acutus (Brid.) K. Saito and D. rigidulus Hedw. are readily distinguishable from morphologically similar small specimens of species of Didymodon sect. Vineales (Steere) Zander by the K light red-brown or light to strong yellow- or redorange reaction of the first two species versus the K medium to deep red response of sect.
Vineales. Of course, similar color reactions may be produced by different organic substrates, but acid-base color reactions are here suggested only as characters additional to those of morphology.
An instance in which acid-base color testing has proved of particular importance is in a review of the genus Triquetrella in Mexico. I have examined all reported (Bescherelle 1872, Crum 1951, Theriot 1931) Mexican collections (NY, PC,TENN) of this genus, including the type of T. ferruginea (Schimp.) Ther., and these proved to be "Barbula reflexa (Brid.) Brid." This weak segregate of D. fallax (Hedw.) Zander is treated as D. rigidicaulis (C.
Muell.) K. Saito by Saito (1975) and Zander (1978a). Although morphological intergradation with typical D. fallax is not uncommon, "B. reflexa "-characterized by small plants with strongly papillose, recurved leaves that reach 2.0 mm in length-may be better treated
Didymodon fallax var. reflexus (Brid.) Zander, comb. nov.
Tortulareflexa Brid., Musc. Recent. Suppl. 1: 255. 1806, basionym.- Barbula reflexa (Brid.) Brid., Mant. Musc. 93. 1819.- Barbulafallax var. reflexa(Brid.)Brid., Bryol. Univ. 1: 558.
BarbulaferrugineaSchimp.ex Besch., Mem. Soc. Nat. Sci. Nat. Cherb. 16: 181. 1872,syn. nov.
Type: Mexico, San Cristobal,Miiller s.n. (Pc-holotype; BM,NY-isotypes). - Triquetrella ferruginea (Schimp. ex Besch.) Th6r., SmithsonianMisc. Coll. 85(4):9. 1931.
Additionalsynonyms,includingthree other combinations Triquetrella given by Saito (1975, in are p. 502); however, Saito's citation of "Tortulafallax var. recurvifoliaWils., Musc. Hib.: 48 (1804)" is incorrect(see van der Wijket al. 1969).
Judging from the two specimens (including the type) at NY, Triquetrella californica (Lesq.) Grout is a good species of Triquetrella, similar to other species of the genus in that the stem transverse section is triangular and shows a central strand; the leaves are trifarious and have strongly recurved lower margins; the laminal papillae are sharp, single over the cell lumina; and the costa lacks an adaxial epidermis and is abaxially bulging and sharply papillose. It has not yet been collected in Mexico and is apparently restricted to California (but note that California: Bolander 61-cu, distributed by the U.S. National Herbarium under the basionym Anomodon californicus Lesq., is actually Leskeella arizonae (Williams) Flow.). Didymodon fallax var. reflexus is quite similar in general appearance but differs by the stem pentagonal in transverse section, the leaves in 3-5 rows and having weakly recurved lower margins, the laminal cells less strongly papillose, and the costa not bulging abaxially and only weakly papillose. It may well be that only specialists with access to authentic material for comparison purposes can readily distinguish these two taxa on morphological bases alone. However, the acid-base color reactions of
the two taxa are quite different. Triquetrella californica responds strongly to all reagents:
Cl deep yellow, K deep orange, N deep red, SE deep yellow-brown. These same strong colors are also found in the related species Leptodontium flexifolium (Dicks. ex With.) Hampe in Lindb. and L. viticulosoides (P. Beauv.) Wijk & Marg. But Didymodon fallax var. reflexus (and also the var. fallax) has the following color reactions: C1 negative or pale yellow-brown to light orange, K light to deep red-brown, N light tan to light red, SE light to medium red-brown.
Another difficult taxonomic case "solved" with the aid of acid-base color reactions involves the delimitation of the genera Barbula and Bryoerythrophyllum. In a recent synTABLE1. Comparison of acid-base color reactions of New World species of Barbula and Bryoerythrophyllum after G = green, O = orange, R = red; 1. = light, med. = medium, brt. = bright, dk. = dark, dp. = deep; neg. = negati
opsis of Barbula in North America (Zander 1979), I noted that Barbula inaequalifolia Tayl. and Barbula calcarea Ther. had the red coloration in nature (in some collections) and hollow laminal papillae typical of Bryoerythrophyllum species, but had the long, twisted peristome teeth characteristic of Barbula species. All Bryoerythrophyllum species have rudimentary or short, straight peristome teeth or are, rarely, eperistomate (Zander 1978b).
A survey of acid-base reactions of nine Barbula and seven Bryoerythrophyllum species was conducted (Table 1). When available, several specimens of each were tested. The color reactions of these two Barbula species are the same as those typical of Bryoerythrophyllum species. Salient differences between the two genera follow: Barbula is Cl green to bright green usually present and predominating, K light to deep yellow- to orangebrown, SE green usually present plus light to medium yellow- to orange-brown in mature leaves; Bryoerythrophyllum is Cl green seldom present or other colors predominating, K light to deep red or red-brown, SE green usually absent or weak, when present usually blackish, plus medium to deep red or red-brown in mature leaves. With this, it is apparent that some Bryoerythrophyllum species have long, twisted peristome teeth, just as a few Barbula species at least occasionally have short, straight peristome teeth. In both Bryoerythrophyllum and Barbula, peristome length and degree of torsion are less conservative characters than are acid-base color reactions.
Bryoerythrophyllum inaequalifolium (Tayl.) Zander, comb. nov.
Barbula inaequalifolia Tayl., London J. Bot. 5: 49. 1846, basionym. - Tortula inaequalifolia (Tayl.) Mitt., J. Linn. Soc. 12: 153. 1869. For additionalsynonyms, see Zander(1968).
Bryoerythrophyllum calcareum (Ther.) Zander, comb. nov.
Barbula calcarea Ther., Smiths. Misc. Coll. 85(4): 20. 1931, basionym. - Barbula linguaefolia Bartr., THEBRYOLOGIST 204. 1947.
Crundwell (1979) found that there appeared to be two main groups of pigments in moss rhizoids: red to purple or violet pigments that become red in acid but bluer in alkali, and orange to orange-red or red-brown pigments that become more yellow in acid and more red in alkali. The presence of various amounts of these pigment groups in different taxa might also account for the differential color changes in moss leaves discussed above; of course, chemical isolation and analysis is needed to demonstrate this conclusively. In any event, acid-base color reactions do have utility as additional taxonomic characters in dealing with some difficult moss groups.
I thank the curatorsof BM,CU, NY and TENN loan of specimens, and M. O. Hill, R. Magill, for G. Pierce and W. C. Steere for helpfulsuggestionsand information.
Bescherelle, E. 1872. Prodromus bryologiae mexicanae. Mem. Soc. Nat. Sci. Nat. Cherbourg 16:
Coradin, L. & D. E. Giannasi. 1980. The effects of chemical preservatives on plant collections to be used in chemotaxonomic surveys. Taxon 29: 33-40.