Heredity 59 (1987) 337—343 The Genetical Society of Great Britain Received 17 December 1986 Electrophoretic evidence of reproductive isolation between two varieties of the moss, Climacium americanum Jonathan Shaw, Department of Botany, Duke University, Durham, Thomas R. Meagher and North Carolina 27706, U.S.A. Pamela Harley Twohundred and twenty individuals of the moss, Climacium americanum, from three localities in the Piedmont of North Carolina were sampled for a study of electrophoretically detectable genetic variation. Eight enzymes (ACP, ADH, ESTfl, GOT, PER, PGI, PGM, and SOD) were assayed and only two (GOT, SOD) were monomorphic at all three localities, in spite of the extreme rarity of sexual reproduction in this species. Although the var. kindbergii has generally been considered a form of C. americanum induced by submersion in water, there was a strong correlation between enzyme phenotypes and morphological characters distinguishing the two varieties of C. americanum. These data suggest that there is linkage disequilibrium among the loci coding for ACP, PGM, and PGI, and between these loci and those controlling several morphological characters that distinguish the two varieties of C. americanum. INTRODUCTiON However, the ecological correlation is far from absolute and both forms sometimes occur in what TheClimaciaceae is a small family of mosses con- appears to be the same habitat. sisting of four species in a single genus, Climacium. The dimorphism in growth habit in Climacium In North America, the genus is represented by two americanum has resulted in various systematic species, C. americanum Brid. and C. dendroides treatments of the species. Renauld and Cardot (Hedw.) Web. and Mohr. Climacium dendroides (1890) described C. americanum var. kindbergii has a circumboreal range and is widespread in based on plants from Louisiana and Massachusetts north-temperate North America, whereas C. having more distant leaves and shorter, oblong leaf americanum is endemic to the eastern United cells as compared to the var. americanum. Although States. Species of Climacium are among the most Renauld and Cardot did not discuss growth form robust and recognizable of temperate mosses, when they described the var. kindbergii, the varietal characterised by below-ground rhizomatous stems epithet has since become associated with forms of and erect, branched, dendroid secondary branches. C. americanum characterized by irregular, non- The common name, tree moss, is descriptive of the dendroid branching. Grout (1901) raised the var. dendroid growth form typical of species in the kindbergii to specific rank, arguing that C. kindber- genus. The production of sporophytes is very rare gii difiers in geographic distribution as well as in both species of Climacium, apparently a result morphology. In 1904, Cardot and Theriot, of most natural populations being unisexual, pro- however, described C. americanum var. pseudo- ducing only female gametangia (Bedford, 1938). kindbergii from Missouri, remarking that it has the Climacium americanum varies in growth form growth form of C. kindbergii but the areolation of from erect and strongly dendroid to elongate and C. americanum, and argued against considering trailing with an irregular branching pattern (fig. them two species. On the basis of additional 1). The variation in growth form of C. americanum observations, Cardot and Theriot (1906) argued has been observed to be dimorphic and is corre- that C. americanum and C. kindbergii intergrade lated with habitat: plants are generally dendroid to a significant extent. in mesic forests and are trailing with irregular More recently, C. kindbergii has been con- branching on rocks in small streams or in swamps. sidered to be an environmentally induced variant 338 J. SHAW, T. R. MEAGHER AND P. HARLEY exhibit the dendroid growth form. However, the species also occurs on rocks in a small stream in a narrow ravine that drains into the Eno River. All plants observed in the latter habitat are of the non-dendroid, or "kindbergii", form. The small stream varies seasonally in flow rate but some water is apparently always present and many of the clumps of Climacium were at least partially sub- merged. Sixty plants were collected along a transect in the floodplain parallel to the Eno River and sixty were collected along a transect in the LI seasonal stream. Only a single plant was collected kind berg ii from each clump encountered along the transects. americanum At the remaining two localities, plants con- Figure 1 Morphologicaldifferences between Climacium forming to the vars. americanum and kindbergii americanum var. americanum (left) and var. kindbergii grew in closer proximity to one another although (right). any particular clump exhibited either dendroid or nondenroid growth, i.e., the two forms did not of C. americanum (e.g., Crum and Anderson, grow intermixed in the same discreet clump. Forty 1981). Horton and Vitt (1976) and Crum and plants were collected in the floodplain of New Anderson (1981) interpreted C. kindbergii as a Hope Creek, a small river in the Duke Forest about phenotypic expression of C. americanum induced 3 miles southeast of Durham in Durham County. by submersion in water during all or part of the Again, collections were made at least one meter growing season. Shaw (in press) grew forms refer- apart to avoid multiple collections from the same able to C. americanum and C. kindbergii under clump. Plants characterised by dendroid versus controlled environmental conditions and found nondendroid growth were sampled as they were only weak evidence for discontinuity between encountered along the transect. Sixty plants were them. The degree of tolerance to desiccation varied collected from a similar floodplain habitat along among populations but did not correlate with habi- the Flat River, approximately 18 miles north of tat or morphology. Shaw presented experimental Durham in Person Co. Plants of the two contrasting evidence suggesting that the degree of desiccation growth forms occurred as discreet clumps and were tolerance was more closely related to environ- sampled as encountered along the transect. At both mental pretreatment than to genetic differences New Hope Creek and along the Flat River, plants among plants. were subjectively classified as var. kindbergii or This study of electrophoretically detectable var. americanum as they were collected. Intermedi- variation was initiated in order to assess the level ate forms were noted as such. of genetic variation in C. americanum, a species in Plants were refrigerated in a moistened state which sexual reproduction appears to be extremely for 2-3 days prior to enzyme extraction. Several rare, and to determine whether variation in enzyme extraction buffers were tried in a preliminary study, phenotypes correlates with growth form variation including those described by Kelley and Adams corresponding to the vars. kindbergii and (1977) for sequestering phenolic compounds americanum. released by Juniperus leaves, and by Soltis et a!. (1983) for extracting enzymes from gametophytes of ferns and fern allies. These extraction buffers MATERIALSAND METHODS did not improve the results obtained by extraction in a simpler 01 M tris-HC1 (pH 7.2) buffer with Collectionswere made at three localities in the 05 per cent mercaptoethanol so that the latter was Piedmont of North Carolina near the city of used exclusively in this study. Extractions were Durham. The first locality was along the Eno River tried with and without freezing the plant material in Durham County where the river crosses U.S. in liquid nitrogen prior to grinding; better results Route 70, about 12 miles northwest of Durham. were obtained from frozen material, probably due Climacium americanum is extremely common in to more effective fractionation of the tissue. After the floodplain between two and 15 feet from the the enzymes were extracted and absorbed onto river bank, and forms discreet clumps of irregular paper wicks, the latter were stored in a deep-freeze size and shape. Floodplain plants invariably at —70°C until use. Electrophoretic separation of REPRODUCTIVE ISOLATION IN A MOSS 339 enzyme variants was accomplished on 12 per cent TabJe 1 Summary of electrophoretic variability in C. horizontal starch gels in either a discontinuous americanumby enzyme system and locality. M = monomorphic;P =polymorphic.Numbers in parentheses 02 M tris-citrate/lithium borate buffer system at indicate the number of different bands present among pH 83 (ADH, ACP, GOT, MOH, PGI) or a con- individuals in populations for a given enzyme system tinuous 0065 M histidine buffer atpH 57 (ESTfl, PER, PGM, SOD) (Cardy et a!., 1983). Eno River New Hope Creek Flat River Approximately 20 enzyme systems were tested ADH P(2) M P(2) for activity, and scorably distinct bands were ACP M P(2) P(2) obtained for 8: acid phosphatase (ACP), alcohol GOT M M M dehydrogenase (ADH), fluorescent esterase PGI P(8) P(8) P(8) (ESTfl), glutamate oxaloacetate transaminase Esifi M P(2) P(3) (GOT), peroxidase (PER), phosphoglucosiso- PER M P(2) M merase (PGI), phosphoglucomutase (PGM), and PGM P(3) P(4) P(3) superoxidedismutase (SOD). Scorable bands were SOD M M M obtained from some individuals for malate dehy- drogenase (MDH) but the amount of activity was The single ESTfl band present at Eno River was too inconsistent to provide scorable reSults. Stain- also present at the other two localities. A second ing of enzymes was according to recipes slightly ESTfl band was present at both Flat River and modified from Shaw and Prasad (1970) except for New Hope Creek localities, and a third, much ESTfl, for which the staining schedule of Soltis et faster, band was expressed in three individuals al. (1983) was followed. (out of 60) from the Flat River. ADH was monomorphic at New Hope Creek but was poly- morphic at the Eno and Flat River localities. Plants RESULTS at the latter two localities expressed the same band as was present at New Hope Creek, plus a second Sixout of the eight enzyme systems studied were band in common between the two localities where polymorphic in at least one of the three collection ADH was polymorphic.