Journ. Hattori Bot. Lab. No. 63: 395-410 (Dec. 1987)

LIMBELLA FRYEI (WILLIAMS) OCHYRA DISTINCT FROM L. TRICOSTATA (SULL.) CM. (MUSCI: )

JOHN A. CHRISTyl

ABSTRACT. Investigation of branching patterns, morphology, karyotype, isozymes and shoot growth confirmed specific differences between tricostata (Sull .) C. M. and L. /ryei (Williams) Ochyra. Significant differences were observed in branch bud frequency, leaf insertion angle, leaf areola­ tion, chromosome length, electrophoretic mobility of superoxide dismutase, and shoot regeneration. Chromosome numbers of both taxa are n = l1. A key and descriptions segregate the taxa.

INTRODUCTION In Flora of the Pacific Northwest, Lawton (1971) reduced the pleurocarpous North American moss Limbellafryei - then known as Sciaromiumfryei - to a synonym of the Hawaiian L. tricostata, because she could find no significant morphological dif­ ferences between from the two areas. Morphology of plants from the two regions is superficially similar, but closer inspection indicated to me that differences might exist in branching pattern and leaf insertion angle. The Hawaiian L. tricostata exhibits branching at more or less regular intervals up to I cm apart, giving the plants a pinnate or frondose habit, especially in aquatic forms (Fig. I). In contrast, branches of the North American L. fryei arise in more closely-spaced groups, giving the plants a penicillate, brush-like appearance. Plants are therefore nearly always dendroid (Fig. I). In both dried and living material, leaves of Hawaiian plants are appressed, whereas those of North American plants are erect to wide-spreading (Fig. 3). Also, habitat conditions for Limbella in Hawaii and Oregon are in most respects strikingly dissimilar (Christy 1980). These differences in morphology and habitat, coupled with a disjunctive distri­ bution of 4,600 km of open ocean and 25 ° of latitude, indicated that the Hawaiian and North American populations might not be conspecific, as had been proposed by Lawton (1971). Christy (1985) investigated the differences in order to determine whether or not plants from the two areas were conspecific.

MATERIALS AND METHODS

1. Quantification of Branching Patterns. Branching pattern in Limbella was quantified by calculating branch bud frequency per merophyte interval. Bud frequencies were deter­ mined by (1) counting the number of merophytes along the stem, following the spiral growth

1 Botany Section, Milwaukee Public Museum, 800 West Wells Street, Milwaukee, Wisconsin 53233, U.S.A. 396 Joum. Hattori Bot. Lab. No. 63 198 7

FIG. 1. Limbella fricosfata (left of scale) and L. fryei (right of scale). Scale = 5 cm.

pattern from the lowermost bud found, toward the distal end of the shoot, until a bud or branch was encountered, (2) recording that merophyte number, (3) resuming the count at one with the next merophyte, following the spiral pattern again until the next bud or branch was encountered, (4) recording that merophyte number and (5) repeating the sequence until the shoot apex was reached. This count gave a measure of the number of times a bud or branch was encountered per merophyte interval. The numbers of buds occurring at each merophyte interval were totaled, the percent occurrence at each merophyte interval (= bud frequency) calculated, and the frequencies plotted against the corresponding merophyte in­ tervals. Twenty shoots (1, J 34 merophytes) of L. tricOSfafa and 17 shoots (689 merophytes) of L. fryei were counted. Differences in frequency were analyzed by an R x C test of inde­ pendence (Sokal & Rohlf 1981). 2. Phenetic Analysis. Twelve specimens of L. tricostafa and 12 of L. fryei, including types, were scored for 28 morphological characters (Table 1). The data were analyzed by one­ way analysis of variance (ANOY A) using the Statistical Analysis System package (SAS Institute 1985). The data for two variables (shortest basal leaf cell length, leaf tooth length) were log transformed prior to analysis to normalize their distributions. Sporophytes were not scored because those of L. fryei are unknown. Leaves and stem sections were measured 1.0- 1.5 cm below the tips of main shoots or largest branches with intact apices. Leaf apical angle and marginal tooth angle were measured using an ocular protractor (Wagner 1951; Christy 1987). J. A. CHRISTY: Limbellafryei distinct from L. tricostata 397

TABLE 1. Morphological characters measured in Limbella tricostata and L. fryei.

1 . Shoot length (cm) 2. Branch length, longest (cm) 3. Leaf insertion angle (degrees) 4. Leaf length, shortest (mm) 5. Leaf length, longest (mm) 6. Leaf width, at widest part (mm) 7. Leaf apical angle, excluding mucro (degrees) 8. Leaf tooth length, longest (jtm) 9. Upper leaf cell length , shortest (jtm) 10. Upper leaf cell length, longest (,Im) 11 . Upper leaf cell width, shortest (jtm) 12 . Upper leaf cell width, longest (jtm) 13. Upper leaf cell length: width 14. Basal leaf cell length, shortest (jtm) 15. Basal leaf cell length, longest (jtm) 16 . Basal leaf cell width, shortest (,lm) 17 . Basal leaf cell width, longest (jtm) 18. Basal leaf cell length : width 19. Costa width at insertion (jtm) 20 . Limbidial width at insertion (,Im) 21. Number of cell layers in costa 22 . Costa thickness (Ilm) 23 . Number of cell layers in limbidia 24. Limbidial thickness (pm) 25 . Stem diameter, longest dimension (Ilm) 26 . Number of cells in stem diameter 27 . Width of stem cortical layer (1/m ) 28 . Number of cells in stem cortical layer

3. Growth Chamber Studies. Sods of living L. tricostata and L. fryei were maintained 2 1 for nine months in a growth chamber under fluorescent lighting of 600--800 pE m- s- , 16 hr light: 8 hr dark at 18°C and 100 % RH, conditions thought to approximate those in the fie ld. Specimens of L. tricostata were grown immersed in tap water and subjected to circula­ tion and aeration. Specimens of L. fryei were placed in closed, transparent plastic boxes and misted with tap water 3- 5 times weekly. Portions of collections of one taxon were also grown in the same container with the other taxon for ten weeks, simultaneously exposing each to the environmental conditions experienced by the other. Growth responses of L. tricostata and L. fryei were observed by examining new shoots produced by Limbella fragments in vitro. Shoots of both taxa were stripped of existing branches, cut into 1 cm segments, and equal numbers from each taxon placed together on moist filter paper in petri plates. The segments were firmly attached to the filter paper with monofilament fishing line. The petri plates were kept in a growth chamber for ten weeks, during which time new shoots developed from buds. Twelve replicate plates were prepared in this manner, containing 120 shoot segments, 60 of each taxon. 4. Isozyme Banding Patterns. Isozyme banding patterns of Limbella were obtained by means of horizontal starch gel electrophoresis, following the methods of Helenurm (1983) and 398 J oum. Hattori Bot. Lab. No. 63 1 9 8 7

Lay ton and Ganders (1984). Living plants were procured from field-collected material that had been maintained in a growth chamber for 1-4 months. Whole plants of 0.2- 3.0 g wet weight were immersed in liquid nitrogen and ground to a powder in a chilled mortar (Fahselt 1980). A few drops of distilled water (Szweykowski et al. 1981 ) were added to the thawing powder to make a paste which was transferred to chilled 1.5 ml polypropylene micro test tubes and centrifuged under refrigeration for 15 min at 20,000 g. The extracts were obtained from (1) field-collected plants that had been maintained in a growth chamber (53 individual Hawaiian plants from four different populations on one island, and 49 Oregon plants from one population), (2) sporelings grown from spores from two different populations on two different islands and (3) reciprocally-cultivated growth chamber material. Extracts were run simultane­ ously on two gels, one with morpholine citrate buffer (Clayton & Tretiak 1972) and one with Ridgway buffer (Ridgway et al. 1970). The buffer system used in the trays was the same as that used in the particular gel being run. Gels were run at ca. 50 mAmps for 3-4 hours, and were stained for 12 enzymes. After initial trials, five enzyme assays were selected because they stained most clearly : malic dehydrogenase (MDH), phosphoglucose isomerase (PG I), phos­ phoglucomutase (PG M), 6-phosphogluconate dehydrogenase (6PGDH) and superoxide dis­ mutase (SOD). 5. Karyotype Morphology. About 150 squash proparations were made from shoot and branch apices, using 14 plants from four different Hawaiian populations and I1 plants from one North American population. Attempts to obtain chromosome figures from L. tricostata sporelings (sensu Nehira 1984) were unsuccessful. Material was obtained from field-collected plants that had been maintained in a growth chamber 4- 5 months. Steel's (1978) procedures were followed with some modifications adopted from Beeks (1955) and Przywara and Kuta (1983). Differences in relative lengths (relative length = absolute length : total length of chro­ mosome complement) were analyzed by one-way analysis of variance (Sokal & Rohlf 1981). 6. Phenolic Spot Patterns. Dried plants were treated following the methods of Gornall and Bohm (1980). Two samples of L. tricosfafa were tested (37.3 g and 69.6 g, respectively) and one of L. fryei (46.0 g) . One-gram quantities of the taxa were also tested separately. Both two-dimensional and sequentially-spotted one-dimensional chromatograms were run .

RESULTS 1. Quantification of Branching Patterns. The results indicated that the frequency of branch bud patterns was dependent on moss species (P < .000 I, G = 206; R x C test of independence). In Hawaiian plants, buds occurred most often every fourth mer­ ophyte (26.4 %), with lesser peaks every first, ninth and eleventh merophtye (Fig. 2). A few buds were separated by up to 20 merophytes. In contrast, buds of plants from Oregon most often occurred every first merophyte (30.0 %), with lesser peaks every fourth, sixth and ninth merophytes. A few buds were separated by no more than 13 merophytes. In a very few cases, individual plants exhibited atypical bud frequencies, but populations as a whole appeared to be uniform when all bud frequencies were averaged. 2. Phenetic Analysis. Analysis of phenetic measurements showed that 5 char­ acters varied significantly with moss species (Table 2). Leaf insertion angle was greater in L.fryei than in L. tricostata (P < .OOOI, F= 54.5). Leaf tooth length was also signifi- J. A. CHRISTY: Limbella /ryei distinct from L. tricostata 399

3D

-;;- 20 ..,... ..,c ...= ~ '" ...'"= 10

11 13 15 Me rophyte number FIG. 2. Branch bud frequencies of L. tricostata and L. /ryei, per merophyte interval. Shaded=L. tricostata, unshaded =L. /ryei. Sample size for L. tricostata= 20 (1134 mero­ phytes) and L. /ryei= I 7 (689 merophytes).

TABLE 2. Measurements of morphological characters (mean + standard error of mean) of L. tricostata and L./ryei. Sample size for each taxon = 12. L. tricostata L./ryei Leaf insertion angle (degrees) 38 .3+ 1.7 54.6+ 1.4 Leaf tooth length ("m) 14 .0 + 1.0 18 .5+ 1.2 Basal leaf cell length, shortest ("m) 16.7+ 1.5 8.7+ 0.6 Basal leaf cell length, longest ("m) 58.6+ 4.0 37 .3+ 2 .9 Costa width (pm) 91.5 + 4 .3 78 .3+ 3.9 cantly greater in L. fryei (P< .OI, F = 7.9). Both longest and shortest basal leaf cell lengths (P <.0003, F = 18.4 and P <.OOOI, F = 27.5, respectively) were greater in L. tricostata than in L.fryei. Costa width was greater in L. tricostala than in L.fryei (P< .04, F = 5.2). Of the five characters identified as being statistically significant, leaf insertion angle and longest basal leaf cell length were most conveniently measurable. 3. Growth Chamber Studies. After ten weeks, new growth in plants of L. tricostata grown under culture conditions of L. fryei developed leaf insertion angles and basal leaf cell lengths typical of L. tricostata, but growth rates slowed. Similarly, after ten week's exposure to immersion and current, new growth in L. fryei developed leaf insertion angles and basal leaf cell lengths typical of L. fryei. In a few cases the leaves of L. fryei became squarrose. Growth response exhibited by the shoot segments secured with monofilament line in petri dishes were the same as those of unsecured segments. Limbella tricostata developed plagiotropic shoots ranging from horizontal 400 Journ. Hattori Bot. Lab. No. 63 198 7

FIG. 3. Regenerated shoots, showing differences in orientation and leaf insertion. L. tricostata on left, L . [ryei on right. to about 60° orientation, whereas L. fryei exhibited in all cases orthotropic shoots of nearly vertical orientation (Fig. 3). Leaf insertion angle and basal leaf cell length remained the same as in typical forms. 4. Iso zyme Banding Patterns. Banding appeared to be monomorphic in each taxon (Fig. 4A & B). Bands of SOD proved to be the best-resolved of the five enzymes tested. Limbella tricostata and L. fryei each displayed its own invariant monomorphic pattern of SOD banding, both consisting of two zones of activity (Fig. 4C). There was a consistent difference in the mobility of SO D between the two taxa: L. tricostata showed bands of greater mobility and usually more intense negative staining than those of L. fryei. Populations of L. tricostata from two different islands (both mature plants and sporelings) showed identical band patterns. Reciprocally-cultivated growth chamber material of both taxa showed band patterns identical to those of their parent stocks, indicating that patterns were not influenced by different environmental regimes. 5. Karyotype Morphology. The chromosome number of both L. tricostata and L. fryei is n = 11 (Fig. 5). Absolute chromosome lengths ranged from 0.7-3.5 ,urn. The mean absolute length of each chromosome in each complement (1.6 ,urn), and the total mean absolute length of all chromosomes in each complement (17.5 pm) were congruent. Calculation of mean relative lengths of each chromosome in each complement revealed differences between the two taxa (Fig. 6, Table 3). Chromosomes 1 and 2 were signifi­ cantly longer in L.fryei (P < .OO2, F= 13.1 and P <.03, F = 5.6, respectively). In con­ trast, chromosomes 6, 7, 8 and 9 were longer in L. tricostata (P <.03, F=5.5; P < .OO3, I. A. CHRISTY: Limbella [ryei distinct from L. tricostata 401

FIG. 4. Zymograms of crude tissue extracts of L. tricostata and L. [ryei. White bands = SOD activity, dark bands=PGM activity in A and B, and MDH in C. (A) Left of gap = L. tricostata (4 different populations), right of gap = L.jryei. Morpholine citrate buffer sys­ tem. (B) Left= L,fryei, right = L. tricostata (2 different populations). Ridgway buffer system. (C) L. tricostata= l- Kauai, Hawaii; 3-Kauai, Hawaii; 5-reciprocally cultivated growth chamber material, originally from Kauai, Hawaii ; 7-sporelings, grown from spores from Maui, Hawaii; 8-sporelings, grown from spores from Kauai, Hawaii ; 9-Kauai, Hawaii. L. [ryei= 2-reciprocally cultivated growth chamber material, originally from Sutton Lake, Oregon; 4, 6-Sutton Lake, Oregon. Ridgway buffer system.

F = 11.4; P<.008, F = 8.5; and P <.005, F = lO.O, respectively). When mean absolute lengths were plotted against mean relative lengths, the chromosomes of L. fryei ex­ hibited a broader range of size classes than did those of L. tricostata (Fig. 7). 6. Phenolic Spot Patterns. Both two-dimensional and four-dimensional chro­ matograms and sequentially-spotted fractions of Limbella extracts exhibited phenolic activity. Blue, yellow and orange spots were visible under long-wave ultraviolet light after spraying with 0.1 % b-aminoethyl diphenylborinate, but were colorless in visible light. No purplish UV-absorbing spots typical of fiavonoids were detected. There was as much variation in spot pattern among different samples of each taxon as there was between the patterns of both taxa. This variation obscured any recognizable configura­ tions of possible taxonomic significance between the two taxa as far as phenolics are concerned. 402 Journ. Hattori Bot. Lab. No. 63 1 987

A D #~ ,#", ~ # I • ...... C • " ~~'.. , B E

~ ~ I' '.f~ • ' ... ,.~ .., " . C F FIG. 5. Chromosome figures of (A-C) L. fricosfafa (Christy 5368) •and (D-F) L. fryei (Chrisry 5505). 1 15 .~ l/ 1 ~ bIl = 10 ... ~ ...> ~ -;;; U ... ~ a: ~ ~ ~ JlL 5 ~ ~ ~ ~ ~

~ ~ . ~ o 3 5 7 9 11 Chromosome number FIG. 6. Mean relative lengths of chromosome complements of L. fricostata and L. /ryei. Shaded = L. tricostafa, unshaded= L.fryei. Sample size for L. tricostata= 14 and L. /ryei= 11. J. A. CHRISTY: Limbellafryei distinct from L. tricostata 403

TABLE 3. Relative chromosome lengths (mean+ standard error of mean) of L. tri­ costata and L. fryei. Sample size for L. tricostata = 14 and L. fryei = l1. Chromosome number L. tricostata L.fryei 13.72+ 0.41 15.99+ 0.47 2 12.07 + 0 .26 13 .29 + 0.48 6 8.68+ 0 . 15 8.15 + 0 . 17 7 8 . 30+ 0 . 14 7.60+ 0 .1 5 8 7 .74 + 0.14 7.10+ 0.18 9 7 . 50+ 0 .1 8 6.66+ 0.19

FIG . 7. Mean absolute chromosome lengths plotted against mean relative lengths. Triangle= L. tricostata, closed circle= L./ryei. Sample size for L. tricostata= 14 and L./ryei = 11.

DISCUSSION Reexamination of Hawaiian and North American populations of Limbella reveal­ ed significant differences overlooked by earlier investigators. Consistent and statistically significant differences in bud frequencies of plants from Hawaii and Oregon confirmed the existence of quantifiable differences in branch­ ing patterns. When such buds produce branches, the branches of L. tricostata are by necessity more widely spaced than those of L. fryei. One-way analysis of variance identified five morphological characters as being statistically significant. Two of these, leaf insertion angle and basal leaf cell lengths, were shown by cultivation experiments to be stable under a variety of environmental conditions and presumably more strongly controlled by genetic than by environmental factors. 404 Journ. Hattori Bot. Lab. No. 63 1 9 8 7

N• 250m

FIG . 8. Distribution of four populations of L. Iricoslala exhibiting monomorphic elec­ trophoretic profiles, Na Pali-Kona Forest Reserve, Kauai, Hawaiian Islands. Modified from Haena, Hawaii 7.5 topographic map, U. S. Geological Survey.

Both taxa of Limbella have the same chromosome number ofn= 11, but differences in relative chromosome length were shown to be statistically significant. The apparent monomorphic banding patterns observed in the enzyme systems tested are incongruent with recent data that indicate a high degree of genetic variation in sexually-reproducing populations of bryophytes (Wyatt & Stoneburner 1984; Wyatt 1985; Wyatt et al. 1987). In L. tricostata, it is possible that continual fragmentation of shoots in the stream environment (Conboy & Glime 1971; Glime et al. 1979), coupled with rapid transport and regeneration of shoot fragments (Christy 1985), led to the development of clonal populations in four localities in two different watersheds (Fig. 8), despite ample production of sporophytes. In the single known population of L. fryei, sporophytes are absent and dispersal is limited to vegetative J. A. CHRISTV: Limbel/a/rye; distinct from L. tr;costafa 405 fragmentation. Genetic uniformity would be expected in such a population, as was observed in Racopilum by Vries et al. (1983). SOD is usually monomorphic in and animal tissues and only rarely has been reported to be polymorphic (Baur & Schorr 1969; El-Kassaby 1980; Yeh & O'Malley 1980; Yamazaki 1981). A consistent dif­ ference in the usually monomorphic banding pattern of SOD, especially in the pat­ terns observed in the reciprocally-cultivated material, indicated a stable genetic dif­ ference between the taxa and demonstrated that the environmentally-caused variation in banding patterns cited by Gottlieb (1981) did not occur. The lack of any consistent phenolic spot patterns between the Limbella specimens tested rendered these chromatograms of little taxonomic use. The results, using up to seven times more plant material than that used by McClure and Miller (1967), cor­ roborated their conclusion that L. tricostata contains no flavonoids. Results of the reciprocal cultivation and shoot regeneration experiments provided evidence that two key morphological characters identified by phenetic analysis, angle of leaf insertion and basal leaf cell lengths, remained stable under a variety of environmental conditions and could be used reliably to distinguish the taxa. Con­ sistent differences between the species in shoot orientation, as shown by the regene­ ration experiments, were used as an additional character to distinguish the taxa. Identification of these differences between L. tricostata and L. fryei confirms the observations of Vitt (1982) and Wyatt (1985) that populations of some continentally "disjunct" bryophyte "species" may be found, upon closer examination, to consist of one or more narrowly-distributed taxa.

TAXONOMY la. Mature shoots 7-20(50) cm long, lower branches 3- 10 cm long ; leaves erect-spreading 35-45°, basal leaf cells to (32)60-77 p m long; inner perichaetial bracts usually ecostate; on rock, rarely on soil or wood, aquatic or emergent in perennial or intermittent streams; Hawaiian Islands ...... Limbella tricostata (Sull.) C. M. lb. Mature shoots 4-8(13) cm long, lower branches 1.5-4 cm long; leaves spreading 50-60°, basal leaf cells to 25- 50(60) !tm long; inner perichaetial bracts costate; on wood, peat, or bark, rarely aquatic, in dense swampy shrub-carr; coastal northwestern North America (Oregon) ...... Limbella !ryei (Williams) Ochyra

Limbella (C.M.) C.M. Flora 82 : 466. 1896. Hypnum sect. Limbelfa C. M., Forschungsr. "Gazelle" Bot. 4: 36. 1889. Sciaromium (Mitt.) Mitt., previously applied to taxa of Limbella, is a synonym of Echinodium Jur. (Churchill 1986). Plants trailing or dendroid, forming yellow-green to blackish sods or mats. Pseudo par­ aphyllia foliose. Leaves limbate, somewhat contorted when dry, ovate-oblong or ovate­ lanceolate, acuminate or cuspidate, keeled. Margins plane, unistratose, 1(2) cells wide, entire or serrate in upper third, sometimes serrulate to base. Limbidia submarginal, disappearing just below acumen, of (2)4 layers of stereids. Costa strong, percurrent to shortly excurrent. 406 Journ. Hattori Bot. Lab. No. 63 1 987

Upper laminal cells subquadrate to rhomboidal. Basal cells mostly oblong, walls at insertion incrassate, sparingly pitted, often yellow. Dioicous; perichaetial bracts lanceolate, long­ acuminate, squarrose; perigonial bracts short-acuminate, concave. Setae elongate, smooth, straight or f1exuose, often tortuose in aquatic plants. Capsules horizontal to cernuous, ob­ long-cylindric, asymmetrical, sometimes arcuate, usually shrunken below the mouth when dry; annulus of 2-3 rows of cells, deciduous; operculum conic-apiculate to rostrate; ex­ othecial cells quadrate to rounded-rectangular; stomata superficial. Peristome teeth 16, yel­ low-brown, lanceolate, sometimes cribose at base, cross-striolate below, papillose above, trabeculate at back; endostome hyaline or pale yellow, with high basal membrane, cilia ap­ pendiculate. Calyptra cucullate, naked, cylindric, early-deciduous.

Limbella tricostata (Sull.) C.M. Flora 82 : 466. 1876. Neckera tricostata Sull., Proc. Amer. Acad. Arts Sci. 3 : 81. 1854; Hypnum tricostatum (Sull.) Sull., U. S. Expl. Exped. Wilkes Musci 13. 1859; Sciaromium tricostatum (Sull.) Mitt. in Seem., Fl. Vit. 400. 1873; Hypnodendron tricostatum (Sull.) Jaeg. ex Jaeg. & Sauerb., Ber. That. St. Gall. Naturw. Ges. 1877-78: 360. 1879. HYPllum subtricostatum C. M., Forschungsr. "Gazelle" Bot. 4: 37. 1889, nom. nud; Sciaromium sub­ tricostatum (c. M.) Par., lnd. Bryol. 1156. 1898, nom. nudo Limbella intralimbata Card., Annuair. Cons. Jard. Bot. Geneve 15-16: 176. 1912; Hypnodendron in­ tralimbatum (Card.) Broth., Nat. Pfl. ed. 2, 11: 531. 1925. Limbella leptolomacea C. M., Flora 82 : 467. 1896 ; Sciaromillm leptolomaceum (c. M.) Par., Ind. Bryol. 1155.1898; Hypnodendron leptolomaceum (c. M.) Broth., Nat. PI'!. 1 (3): 1170. 1909. Limbella limbatula C. M., Flora 82 : 467. 1896; Sciaromium limbatulum (c. M.) Par., Ind. Bryol. 1155. 1898; Hypnodelldron Iimbatulum (C. M.) Broth., Nat. Pfl. 1 (3): 1170. 1909. Sciaromillm /lagellare Broth. in Lev., Bull. Bot. Soc. Ital. 1904: 23. 1904, nom. nudo Sciaromium /lexicaule Broth. in Lev., Bull. Bot. Soc. Ital. 1904 : 23. 1904, nom. nudo Sciaromillm porotrichoides Broth. in Lev., Bull. Bot. Soc. Ital. 1904: 23. 1904, nom. nudo Lectotypus nov.: "U. S. Ex. Wilkes 1838-42," (FH; isolectotypes: BM, NY) . Variously labeled "12" or "W20". "Hab. at forest at eastern base of Mauna Kea, Hawaii, Sandwich Islands" (Sullivant 1854). The specimen at FH was chosen as the lectotype because it is clearly the same specimen as that depicted by Sullivant (1859) and is from Sullivant's personal herbarium. Plants coarse and robust, 7- 20(50) cm long. Branches 3- 10 cm long, when present usually J. A. CHRISTY: distinct from L. tricosrara 40 7 arising every fourth merophyte. Leaves 2.2-3.5 x 0.5-1.4 mm, sometimes homomallous, erect­ spreading 30-45°, serrate in upper third, teeth 7- 20 !lm long, serrulate to base; upper laminal cells 5-36 x 3-18 !lm, basal cells 7- 77 x 3- 18 ,urn. Perichaetial bracts 18- 25, costa weak or absent, limbidia absent; perigonial bracts 12-20, costa weak or absent, limbidia absent. Seta 1.5- 3.5 cm long. Capsules 1.5- 3 mm long; operculum 0.5-1.5 mm long; calyptra 4-5 !lm. Spores minutely papillose, 14- 18!lm in diameter, green. Chromosome number : n = l1. Illustrations: Figs. 1, 9, 10. Bartram 1933 : Fig. 96 a-e. Brotherus 1909: Fig. 824 a-e; 1924 : Fig. 385 a-e. Christy 1980 : Figs. 1-6. Christy 1985 : Figs. 25- 28 . Sullivant 1859 : PI. 9B, Figs. 1-13. Specimens examined : BISH (49), BM (13), CAS (3), COLO (5), FH (25), H-BR (16), M (1), MO (5), NY (30), S (12), UBC (9), uc (5), us (6), WTU (3), YU (I), Herb. J. A. Christy (34). See Christy (1980) for partial citation of specimens examined. Sporophytes ('f L. tricostata were collected by David Dwight Baldwin as early as 1875 (Christy 1980). During the intervening \05 years, four specimens with sporophytes had been distributed, three to NY and one to BISH. The specimens at NY bear the label "Plantae Hawaiienses. Duplicates from herb. D. C. Eaton, purchased from J. K.

FIG . 10. Exostome of L. tricosrata. (A) Outer face, (B) inner face, (C) detail, distal por­ tion of outer face, (D) detail, basal portion of outer face (Christy 5208). 408 10urn. Hattori Bot. Lab. No. 63 1 987

Small, 1920.", and were probably sent to Eaton by Baldwin (Miller 1956). The spe­ cimen at BlSH was obtained from Baldwin's granddaughter in 1960. I found sporophytes of L. tricostata to be rather common in the field, contrary to the reports of Sullivant (1854), Ba rtram (1933), Miller (1954), Crosby (1965) and Hoe (1974). My collections of specimens with sporophytes have been distributed to the herbaria listed above. Limbella tricostata was collected first by James Macrae in 1825 (St. John 1978 ; Hoe 1979), 29 years before it was described by Sullivant (1854). Although Macrae's collections were reportedly sent to Hooker and De Candolle, among others (Mann 1866), it is surprising that L. tricostata was not described first by European bryologists. Macrae's specimens at BM and NY were probably identified by Mitten, who cited them for the first time in Seeman's Flora Vitiensis (Mitten 1873). Traces of glue on the sheets in the Hooker herbarium at BM match - in reverse - the plants in the Mitten herbarium at Y, indicating that Mitten obtained the specimens from Hooker, presumably after L. tricostata had been described by Sullivant in 1854.

Limbella fryei (Williams) Ochyra J. Hattori Bot. Lab. 61: 313. 1986. Sciaromium [ryei Williams, Bryologist 35: 52. 1933. Lectotypus nov.: "Cape Arago, Oregon," Frye s.n., 8. viii. 1922 (NY; isolectotypes : usc, WTU). "Oregon, Coos County, Cape Arago, 1-1.5 miles northeast of Charleston on east side of highway. On ground in more or less wet pasture" (UBC, WTU). The specimen at NY was chosen as the lectotype because it is presumably the same specimen upon which Williams (1933) based his description and illustra­ tion. Plants coarse, 4- 8(13) cm long. Branches 1.5--4 cm long, when present usually arising in clusters from adjacent merophytes. Leaves 1.5-3.8 x O.l-1 mm, spreading 50-60°, serrate in upper third, teeth 14- 25,um, serrulate to base; upper laminal cells 3-32 x 3-14 ,um; basal cells 7- 50(60) x 3- 14 [tm. Perichaetial bracts 14-30, costate, limbidia absent; perigonia un­ known. Sporophytes unknown. Chromosome number: n = 11. Illustrations: Fig. I. Christy 1985 : Figs. 29-32. Grout 1934: PI. 80, Figs. 1-8. Kawai 1968: Fig. H, 1. Lawton 1971: PI. 158, Figs. 11-17. Williams 1933: Figs. 1-8. Specimens examined: BISH (5), NY (6), usc (7), us (5), WTU (7), Herb. 1. A. Christy (17). See Christy (1980) for partial citation of specimens examined.

ACKNOWLEDGMENTS Grants awarded to W. B. Schofield by the National Research Council of Canada, and to J. A. Christy by the Northwest Scientific Association, are gratefully acknowleged. I thank curators from BISH, BM, CAS, COLO, FH, H-BR, JE, M, MO, NY, S, UBC, UC, us, WTU and YU for loans of specimens. I am indebted to W. J. Hoe and the botany staff of the B. P. Bishop Mu­ seum, Honolulu, for assistance in Hawaii. Many thanks are also due to T. Noeske for the statistical analyses, and to W. R. Buck, M. J. Dibben, W. B. Schofield, W. C. Taylor and R. Wyatt for critical reviews of the manuscript.

LITERA TURE CITED Bartram, E. B. 1933. Manual of Hawaiian . Bull. B. P. Bishop Mus. 101 : 1-275. J. A. CHRISTY: Limbella Iryei distinct from L. Iricoslala 409

Baur, E. W. & R . T. Schorr. 1969. Genetic polymorphism of tetrazolium oxidase in dogs. Science 166 : 1524-1525. Beeks, R . M. 1955. Improvements in the squash technique for plant chromosomes. Aliso 3: 131- 133. Brotherus, V. F. 1909. Musci. In A. Engler & K . Prantl, Die naturlichen Pflanzenfamilien, Ed. 1, Teil. 1, Abteil 3, Halfte 11 , p. 701-1246. - - . 1924. Musci. In A. Engler & K . Prantl, Die naturlichen Pflanzenfamilen, Ed. 2, Band 10, 478 pp. Christy, J. A. 1980. Rediscovery of Sciaromium Iricosratum (SuB.) Mitt. ( = Limbella tricostata (SuB.) Bartr.) in North America. Bryologist 83: 521-523. - - . 1985. Identity and li mits of Limbella tricoslala (Musci: Amblystegiaceae), 216 pp. M . Sc. thesis, Univ. of British Columbia, Vancouver, --. 1987. K. A. Wagner's ocular protractor, for measurement of angles with a compound micro­ scope. Bryol. Times 41: 4. Churchill, S. P. 1986. A revision of Echinodium Jur. (Echinodiaceae: Hypnobryales). J. Bryol. 14: 117-133. Clayton, J. W. & D. N. Tretiak. 1972. Amine-citrate buffers for pH control in starch gel electrophore­ sis. J. Fisheries Res. Board Canada 29 : 1169-1172. Conboy, D. A. & J. M. Glime. 1971. Effects of drift abrasives on Fontinalis novae-allgliae Sui!. Cast­ anea 36: 111-114. Crosby. M. R. 1965. New records for Hawaiian Island mosses. Bryologist 68: 457-462. EI-Kassaby, Y. A. 1980. Isozyme patterns of a selected Pseudotsuga menziesii (Mirb.) Franco popu­ lation, 137 pp. Ph. D. dissertation, Univ. of British Columbia, Vancouver Fahselt, D . 1980. Alternative method for analyzing protein characters in lichens. Bryologis t 83 : 340-343. Glime, J. M., P. C. Nissila, S. E. Trynoski & M. C. Fornwal!. 1979. A model for attachment of aquatic mosses. J . Bryo!. 10 : 313- 320. Gornall, R . J. & B. A. Bohm. 1980. The use of flavonoids in the of Boykinia and allies (Saxifragaceae). Can. J. Bot. 58: 1768- 1779. Gottlieb, L. D. 1981. Electrophoretic evidence and plant populations. Progress Phytochem. 7 : 1-46. Grout, A. J. 1934. Moss Flora of North America North of Mexico, Vo!. 3, part 4, 98 pp. Published by the author, Newfane, Vermont. Helenurm, K. 1983. Genetic differentiation of Hawaiian Bidells, 107 pp. M . Sc. thesis, Univ. of British Columbia, Vancouver. Hoe, W. J. 1974. Annotated checklist of Hawaiian mosses. Lyonia 1 : 1- 45. --. 1979. The phytogeographical relationships of Hawaiian mosses, 357 pp. Ph. D . disserta­ tion, Univ. of Hawaii, Honolulu. Kawai, I. 1968. Taxonomic studies on the midrib in Musci. I. Significance of the midrib in systematic botany. Sci. Rep. Kanazawa Univ. 13 : 127-157. Lawton, E.1971. Moss Flora of the Pacific Northwest, 362 pp. Hattori Botanical Laboratory, Nichi­ nan, Japan. Layton, C. R. & F . R. Ganders. 1984. The genetic consequences of contrasting breeding systems in Plectritis (Valerianaceae). Evolution 38: 1308-1325. Mann, H . 1866. Enumeration of Hawaiian plants. Proc. Amer. Acad. Arts Sci. 7 : 143-235. McClure, J. W. & H. A . Miller. 1967. Moss chemotaxonomy. A survey for flavonoids and the taxono­ mic implications. Nova Hedwigia 14: 111-125. Miller, H . A. 1954. Field observations on associations of Hawaiian mosses. Bryologist 57: 167-172. --. 1956. A phytogeographical study of Hawaiian Hepaticae, 123 pp. Ph. D. dissertation, Stanford Univ. 410 Journ. Hattori Bot. Lab. No. 63 1 9 8 7

Mitten, W. 1873. Musci, Jungermanniae, Marchantiae, p. 378-419. In B. Seeman, 1865-1873, Flora Vitiensis, 453 pp. L. Reeve & Co., London. Nehira, K. 1984. Spore germination, protonema development and sporeling development. In R. M. Schuster (ed.), New Manual of Bryology, p. 343-385. Hattori Botanical Laboratory, Nichinan. Przywara, L. & E. Kuta. 1983. An acetic-hematoxylin method for cytological investigations of Bryophyta. Bryologist 86 : 141-143. Ridgway, G. J., S. W. Sherburne & R. D. Lewis. 1970. Polymorphisms in the esterases of Atlantic herring. Trans. Am. Fisheries Soc. 99 : 147-15 1. SAS Institute. 1985. SAS User's Guide: Statistics. Version 5 ed. SAS Institute, Inc., Cary, North Carolina. Sokal, R. & F. J. Rohlf. 1981. Biometry, 2nd ed. W. H. Freeman & Co., San Francisco. St. John, H. 1978. Plants of the Sandwich Islands collected by James Macrae. Phytologia 39 : 307- 319. Steel, D. T. 1978. The taxonomy of Lophocolea bidentata (L.) Dum. and L. cuspidata (Nees) Limpr. J. Bryol. 10: 49-59. Sullivant, W. S. 1854. Notices of new species of mosses from the Pacific Islands. Proc. Amer. Acad. Arts Sci. 3 : 73-81. --. 1859. Botany, Musci. United States Exploring Expedition During the Years 1838, 1839, 1840,1841,1842 Under the Command of Charles Wilkes, U. S. N ., 32 pp., 26 pI., imperial folio. e. Sherman & Son, Philadelphia. Szweykowski, J., W. Prus-Glowacki & M. Mendelak. 1981. Serological variability in the central European liverwort genus Pellia - a preliminary report. J. Hattori Bot. Lab. 50: 269- 276. Vitt, D. H. 1982. Populational variation and speciation in austral mosses. J. Hattori Bot. Lab. 52 : 153-159. Vries, A. de, B. O. van Zanten & H. van Dijk. 1983. Genetic variability within and between popula­ tions of two species of Racopi/um (Racopilaceae, ). Lindbergia 9 : 73-80. Wagner, K. A. 1951. The Neckeraceae of North America, 195 pp. Ph. D. dissertation, Univ. of Michigan, Ann Arbor. Williams, R. S. 1933. Sciaromium fryei sp. novo Bryologist 35 : 52-53. Wyatt, R. 1985. Species concepts in bryophytes : input from population biology. Bryologist 88: 182-189. -- & A. Stoneburner. 1984. Biosystematics of bryophytes : an overview. In W. F . Grant (ed.), Plant Biosystematics, p. 519-542. Academic Press, Toronto. --, I. J. Odrzykoski & A. Stoneburner. 1987. Genetic variability in natural populations of Plagi­ omnium ciliare (Mniaceae). [Abstract]. Amer. J. Bot. 74 : 605. Yamazaki, T. 1981. Genetic variabilities in natural population of haploid plant, Conocepha/u/n conicum. I. The amount of heterozygosity. Jap. J. Genet. 56: 373-383. Yeh, F. e.-H. & D. O'Malley. 1980. Enzyme variations in natural populations of Douglas-fir, Pseu­ dotsuga menziesii (Mirb.) Franco from British Columbia. 1. Genetic variation patterns in coastal populations. Silvae Genet. 29: 83- 92.