AMERICAN we FERN — JOURNAL January—March 2009 QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY Megalastrum (Dryopteridaceae) in Sere Paraguay, and Urugu Robbin C. Moran, Jefferson Pik and Paulo H. Labiak Local Knowledge and Management of the Royal Fern (Osmunda regalis L.) in Northern pain: Implications for Biodiversity Conservation Maria Molina, Victoria Reyes-Garcia, Manuel Pardo-de-Santayana SHORTER Notes Salvinia molesta in Mexico Arturo Mora-Olivo and George Yatskievych Type Specimens of Dracoglossum sinuatum Uncovered in the Rio de Janeiro Herbarium Maarten J. M. Christenhusz Review Illustrated Flora of Ferns and Fern Allies of South Pacific Islands Barbara Joe Hoshizaki 45 58 The American Fern Society Council for 2009 WARREN D. HAUK, Dept. of Biological Sciences, Denison University, Granville, OH 43023. President MICHAEL WINDHAM, Dept. of Biology, Duke University, PO. Box 90338, Durham, NC 27708. President-Elect W. CARL TAYLOR, National Science Foundation, 4201 Wilson Blvd., Arlington, VA 22230. Secretary JAMES D. CAPONETTI, Div. of Biology, University of Tennessee, Knoxville, TN 37996-0830. Treasurer GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. Membership Secretary JAMES D. MONTGOMERY, Ecology III, 804 Salem Blvd., Berwick, PA 18603-9801. Curator of Publications JENNIFER M. O. GEIGER, Dept. of Natural Sci Carroll College, Helena, MT 59625 Journal Editor R. JAMES HICKEY, Dept. of Botany, Miami University, Oxford, OH 45056. Memoir Editor JOAN N. E. HUDSON, Dept. of Biological Science, Sam Houston State University, Huntsville, TX 77341-2116. DAVID SCHWARTZ, 9715 Christey Way, Bakersfield, CA 93312-5617. Bulletin Editors American Fern Journal EDITOR JENNIFER M. O. GEIGER Dept. of Natural Sciences, Carroll College, Helena, MT 5 ph. (406) 447-4461, e-mail: a eo MANAGING EDITOR JILL ANNE DIP ee Dept. of Natural ep Carroll College, Helena, MT 59625, . (406) 447-5176, e-mail: jdill@carroll. edu ASSOCIATE EDITORS GERALD fF GASIONY: (os. Dept. of Biology, Indiana University, Bloomington, IN 47405-6801 GARY Ky GREER ois ecw Biology Dept., Grand Valley State University, Allendale, MI 49401 CHRISTOPHER H. HAUFLER ................--- Dept. of Ecology and Evolutionary eS ieee rsity of Kansas, e, KS 66045-2106 R. JAMES HICKEY Dept. of Botany, Miami ee pasty ‘Oxford, OH 45056 ROBBIN C. MORAN New York Botanical Garden, aan NY 10458-5126 JAMES E. WATKINS, JR. mbridge, MA 02138 The “American Fern Journal” CISSN 0002-8444) is an —. nga devoted to bys general opens of ferns. It is owned by the American Fern Society, and published ociety, Yo Mi Botanical Garden, P. O. Box 299, § St. Louis, MO 63166-0299. ca sats ug at sec Louis, MO, 2 a additional entry. Claims for missing issues, made 6 months (domestic) to 1 for back issues should be addressed to Dr. James D. spetoseaes orien Ill, 804 Salem Blvd., fascioey PA ~ 3 a = f. Ll } (4 wh 1. LA. |= + R c +, dues, oC rer (3 r - _ Back volumes are avalible for most years as ue issues or on microfiche. Please contact the Back Issues Curator for prices and availability. Sihectoinne: Society Membership — USA, Canada Mex (inelnead T 1 4 Cl AAlal ic ) $25 WiC DCTSHIP i includ T j CiAdist ic 2 Society t ifm RZ, t Le 2M) fon CLAN “ae g L 2 £ outside USA, Canada, Mexico) Regular Membership -USA, ‘Canada, Nh (ack Aue Widlichead ) $1 ar Member (4 4 BAA ee $19 sin ark Messen < $35 to USA, Canada, Mexico; $45 elsewhere (-$2 agency fee) Postmaster: Send address changes to American Fern Journal, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299. American Fern Journal 99(1):1-44 (2009) Megalastrum (Dryopteridaceae) in Brazil, Paraguay, and Uruguay Rossin C. Moran The New York Botanical Garden, Bronx, NY 10458-5126, USA, email: rmoran@nybg.org JEFFERSON PRADO Instituto de Botanica, C.P. 3005, CEP 01061-970, Sao Paulo, SP, Brazil, email: jprado.01@uol.com.br PauLo H. LasBiak Universidade Federal do Parana, Depto. de Botanica, C.P. 19031, 81531-980, Curitiba, PR, Brazil, email: plabiak@ufpr.br Asstract.—We provide keys, descriptions, illustrations, full synonymies, maps, and a list of specimens examined for species of Megalastrum found in Brazil, Paraguay, and Uruguay. Eighteen littorale, M. organense, M. retrorsum, and M. ubstrigosum. The species occur primarily in the coastal mountains; none occur in Amazonia, The mountains of coastal Brazil are a center of endemism and diversity for the genus. Key Worps.—Megalastrum, Dryopteridaceae, Brazil, floristics, taxonomy, systematics, ferns surprising given that the Atlantic rainforest of coastal Brazil is isolated from the Andes, the main region where the genus occurs in South America. The genus is absent from Amazonian Brazil (F ig. 1). Although Christensen (1913, 1920) treated 10 Brazilian species of Mega- lastrum, his treatment was based on the relatively few specimens then available and is now badly out-of-date. Brade (1972) published a helpful study of the Brazilian species, also including species now placed in Dryopteris and Ctenitis, but it too needs updating because recent collections have extended the known ranges of many Brazilian species and new species have been discovered. Moran et al. (2008) have shown that one of the species assigned to the group by Christensen, Brade, and others (M. lasiernos (Spreng.) A. R. Sm. & subincisa’”’ of subgen. Ctenitis. He assigned about 30 species to the group. Holttum (1986) elevated the subincisa group to generic rank, as Megalastrum, and made combinations for the Jamaican type of the genus (M. villosum (L.) 2 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) f q j { 0 500 Km 0° ie HR ‘al f ag XS { sae, y, ( Aes 3 ( \ ® Fad \ 5 - l ( pa, ap ee ney | y 10°S } 7 5 f ( \ % \ ( . f af J s J f : = 9 ff —/ ©@ 20°S 4 : e y . - : e i ® be { : | aan e.8 e 60°W 50°W 40°W lL L aN Fic. 1. Distribution of Megalastrum in Brazil, Paraguay, and Uruguay. Holttum) and the African and Madagascan species. Combinations for 39 Neotropical species were subsequently made by Smith and Moran (1987). Since then, two new species have been described from Costa Rica (Rojas- Alvarado, 2001), one from Peru (Smith, 2006), and six from Bolivia (Kessler and Smith, 2006). Megalastrum differs from other dryopteroid genera by lamina cutting, venation, and type of hairs on the axes adaxially. As one goes distally along the pinna, the basal basiscopic pinnules gradually become decurrent and broadly adnate to the pinna rachis. Correspondingly, the vein that supplies the broadly adnate segment or lobe arises from the costa, not the costule. This is unique among dryopteroid ferns. Another helpful characteristic is the form of the hairs on the adaxial surfaces of pinna rachises. Generally, these are coarse, whitish, septate, sharp-tipped, and antrorsely strigose or spreading (Smith and Moran, 1987). Often they remain terete after drying, the cells not collapsing and twisting at right angles to each other. The veins in most species of Megalastrum end before the lamina margins in enlarged clavate tips (hydathodes). This type of vein termination is uncommon among dryopteroid ferns, which typically have the vein tips slender and reaching the margin. The dryopteroid genera that have hydathodes are Didymochlaena (pers. obs.), Elaphoglossum sect. Setosa (Mickel and Atehor- ttia, 1980; Moran et al., 2007), and Stigmatopteris (Moran, 1991). Judging from the dryopteroid portion of the cladogram presented by Schuettpelz and Pryer MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 3 (2007), the presence of hydathodes in these genera represents independent evolutionary developments. The spores of Megalastrum are of two types: 1) densely echinate, and 2) cristate. The echinate type resembles that found occasionally in other dryopteroid ferns (e.g., Elaphoglossum papillosum, E. pygmaeum, E. oblan- ceolatum; Moran et al., 2007). The cristate type, however, seems unique among dryopteroid ferns. Most dryopteroid ferns have broadly folded perispores, but the cristate spores of Megalastrum have sharp thin crests, and they are often parallel (see images at www. plantsystematics.org). Previously, Megalastrum was thought to be closely related to Ctenitis, which it resembles in lamina cutting (Christensen, 1920). Recent DNA-based phylogenetic studies, however, reveal that Rumohra is the sister genus to Megalastrum, and that these two genera are sister to Lastreopsis (Schuettpelz and Pryer, 2007). This clade is sister to the “former lomariopsids” (Bolbitis, Elaphoglossum, Lomagramma, and Teratophyllum). Megalastrum comprises medium- to large-sized ferns, often with finely divided laminae. This imparts challenges to working with herbarium specimens, which are often fragmentary, consisting only of a few pinnae or an apex. In decompound ferns, the laminae will vary from less divided juvenile leaves to highly divided large ones. Thus, two species that differ greatly in the division of their laminae might appear to overlap in keys and descriptions, even though their dissection is, on the whole, quite different. For this reason we give in the descriptions the cutting for the basal pinnae, where the laminae are most finely divided. Given the problems with using lamina division, we emphasize the more consistent characteristics of the hairs and scales in our keys and comparative discussion. Hairs and scales in Megalastrum are readily distinct. The hairs are often whitish, uniseriate, and multicellular, whereas scales are usually broad, and flat or bullate, with reddish walls and denticulate margins. Towards the lamina margins, the scales become gradually reduced to uniseriate structures that resemble hairs. If these are interpreted as hairs, it will cause confusion in using the keys and descriptions. These reduced hairs, or “proscales,’’ are called uniseriate scales in this paper. They are usually appressed, reddish, and several-celled. That they are reduced scales is shown by their complete transition with large, broader scales; i.e., proscales are serially homologous with typical scales. In some species, the hairs are modified as glands. The glandular cell is spherical and typically yellowish (less commonly reddish). Glandular hairs may be capitate, that is, each with a glandular cell at its apex (Fig. 10R, W), or they may be sessile (Fig. 10E). The glands are only 0.05-0.1 mm long and are best seen with at least 30 times magnification. Megalastrum is mostly Neotropical, occurring from Mexico and Cuba to southern Chile. Three species occur in the Old World (Africa, Comores, Réunion, Madagascar). Brazil shares four species with Paraguay and Uruguay (Table 1). 4 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) TasLe 1. Distribution of the Megalastrum in Brazil, Paraguay, and Uruguay. Brazil Alagoas (1): eugenii Bahia (6): canescens, connexum, eugenii, grande, indusiatum, umbrinum Ceara (1): eugenii Espirito Santo (4): connexum, grande, substrigosum, wacketii Minas Gerais (4): abundans, connexum, crenulans, umbrinum Parana (6): abundans, albidum, brevipubens, connexum, crenulans, umbrinum Pernambuco (1): eugenii Rio de Janeiro (10): abundans, canescens, connexum, crenulans, grande, inaequale, littorale, organense, retrorsum, umbrinum Rio Grande do Sul (6): abundans, adenopteris, connexum, crenulans, oreocharis, umbrinum Santa Catarina (5): abundans, adenopteris, connexum, oreocharis, umbrinum Sado Paulo (11): albidum, brevipubens, canescens, connexum, crenulans, grande, inaequale, littorale, organense, umbrinum, wacketii Paraguay Alto Parana (1): connexum Amambay (2): connexum, umbrinum um Pp) e p = p oo > a ea 3 S = is wn San Bernardino (1): connexum Uruguay Rivera (1): connexum Tucuaremb6 (2): connexum, oreocharis METHODS Herbarium specimens were borrowed from 23 herbaria (see Acknowledge- ments). Living plants were studied in the field by two of us (Prado and Labiak). To show the variation in lamina cutting, silhouettes were prepared from herbarium specimens for all species. Digital images were taken of basal pinna, and the images were then adjusted to provide a white background and a black lamina. To produce the distribution maps, the geographic coordinates were estimated for many specimens because this information was not provided on the labels. Our estimates of geographic coordinates are given in brackets in the Specimens Examined section below. For the Specimens Examined, only two or three specimens were cited per state. The dot distribution maps based on all specimens were generated in the GIS laboratory at the New York Botanical Gard RESULTS Geography.—Two-thirds of the species of Megalastrum treated here are restricted to the coastal mountains of southeastern Brazil (Table 1). Four of these species (M. littorale, M. organense, M. retrorsum, and M. wacketii) are narrow endemics, occurring collectively only in the mountains in the states of MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 2 Sao Paulo and Rio de Janeiro. Species that occur beyond the coastal mountains are M. adenopteris, M. brevipubens, M. connexum, M. crenulans, M. oreocharis, and M. umbrinum. Of these, M. connexum has the widest distribution, occurring from Bahia, Brazil, to Tucaremb6, Uruguay. Given these distributions, the coastal mountains of Brazil are a center of endemism and diversity for the genus (Fig. 1). Taxonomic treatment.—Eighteen species are here recognized for the region, seven of which are new. No infraspecific taxa are recognized. The species may be separated by the key below. Megalastrum Holttum, Gard. Bull. Straits Settlem., ser. 3, 39: 161. 1986. PE.—Megalastrum villosum (L.) Holttum [basionym: Polypodium villo- sum L.] Ctenitis subsect. Subincisae (C. Chr.) Tindale, Contr. New South Wales Natl. Herb. 3: 252. 1965. Dryopteris sect. Subincisae C. Chr., Index F ilic., Suppl. 3: 7. 1934, j Plants terrestrial; rhizomes erect to decumbent: petioles scaly toward the base, with 4-10 vascular bundles, the two adaxial bundles enlarged; laminae 1- pinnate-pinnatifid to 4-pinnate-pinnatifid, catadromic above the basal pinnae; basal pinnae inequilateral and more developed on the basiscopic side or (less commonly) equilateral; rachises, costae, and costules not grooved or only shallowly so adaxially, scaly and pubescent abaxially, densely pubescent on the adaxial surfaces, the hairs whitish, spreading to antrorsely strigose, multicellular, if glands present, these ca. 0.1 mm wide, spherical, shiny, yellowish to orangish, sessile to stalked: basal basiscopic segment of more distal pinnules becoming decurrent and adnate to the pinna rachises, the vein supplying the segment springing from the pinna rachis instead of the costule; hydathodes (enlarged vein ends) present adaxially; indusia absent or (less commonly) present, circular, brown, firm, in some species minute and fugacious; x=41. KEY TO THE SPECIES OF MEGALASTRUM IN Brazi_, PARAGUAY, AND UruGUAY 1. Indusia present, persistent 2. Indusia completely g tissue between the veins adaxially glandular....................... 7. M. crenulans 2. Indusia partially covering the sori, about the size of a single sporangium capsule; scales non-bullate on the pinna rachises abaxially; lamina tissue between the veins adaxially setidet nag CO ne ee 11. M. indusiatum ‘ } = 1 has tias +h : yar 1 ea lamina 4. Hairs on the abaxial surfaces ca. 1-2 mm long, 2-8-celled; glands on the lamina tissue abaxially stalked, never sessile AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) 4. Hairs on the abaxial surfaces ca. 0.1—0.2 mm long, 1- or 2-celled; glands on the lamina tissue abaxially sessile or short-stalked 6. Hairs on the abaxial surfaces of the costules more than 1 mm long .. 6. Hairs on the abaxial surfaces of the costules 0.2-0.3 mm 7. Laminae adaxially densely and evenly pubescent between veins; rachis scales appressed, inconspicuous; minute fugacious indusia present. . . . 2. M. adenopteris 7. Laminae adaxially glabrous between veins or sparsely sibeniceat with a few hairs near the margins; rachis scales spreading, conspicuous; minute ere indusia 7. M. .18. M. wacketii BOGGUL se 7. M. umbrinum 3. Laminae eglandular abaxiall 8. Scales of the petioles and rachises retrorsely denticulate Ses 15. M, retrorsum 9, Laminar tissue between the veins glabrous on both surfaces ...... 14. M. organense 8. Scales of the petioles and rachises entire, or if denticulate, not retrorsely so 10. Lamina tissue pubescent between the veins abaxially, the hairs erect or spreading, often acicular 11. Hairs on the abaxial surfaces of the lamina between the veins 0.4—0.6 mm long Oe ek ae oa Ft res Cee Py haa Sage iene 3. M. albidum 11. Hairs on the abaxial surfaces of the lamina between the veins Ca. 0. neat Ue a kes Peed es ee ea be ete ee eee beerinubens 10. Lamina tissue glabrous between the veins abaxially (sometimes with appressed reddish uniseriate scales, but no hairs 12. Scales on the abaxial surfaces of the costae and costules sub-bullate to bullate 13. Laminae 3-pinnate-pinnatifid at base, subcoriaceous, lustrous gerpiens . abun 12. Scales on the abaxial surfaces of the costae and costules flat tanita) 14. Costae glabrous adaxially alg Cas Oe aries are a eo 9. M. grande 14. o — adaxially Hai n the pinna rachises, costules, and veins abaxially 0.5—-0.7 mm heey Ha our Rae ae ee ane ae ee eae 13. M. oreocharis 15. Hairs on the pinna rachises, costules, and veins abaxially 0.1-0.3 mm lon 16. Pinna rachises abaxially densely pubescent, hairs substrigose .. . SEG aS Oe Ee Pe ee ee ee 6. M. substri sirigondsi 16. Pinna rachises abaxially glabrous or glabrescent, the hairs (when present) not substrigose 17. Laminae 1-pinnate-pinnatisect at the middle; petiole scales dark OWE i es eee ug 17. Laminae 2-pinnate-pinnatifid or more divided at the ‘sdinldies M. petiole scales yellowish brown .......-.-.--- 6. M. connexum 1. Megalastrum abundans (Rosenst.) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Dryopteris abundans Rosenst., Hedwigia 46: 133. 1906. Ctenitis abundans (Rosenst.) Copel., Ann. Crypt. Phytopath. [Gen. Fil.] 5: 124. 1947. LECTOTYPE (here designated).—BRAZIL. Rio Grande do Sul: Mun. te Cruz, [29°43’S, 52°52'W], Sette Lagoas do Herval do Paredao, 2 Mar 1905, Jiirgens 195 [Rosenstock Filices Austrobrasilienses no. 367] (NY; duplicates: B, NY, P, R-n.v., S-n.v.). Figs. 2A, 5A, 8A-H. MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY t ' T / 0 $00Km oJ L / 0 500Km go { l j 5 l j He ? | 10°sa Fe } 10°S + / - 20°s4 20°s Se : 7 a 4 © / a ped e 30°S4] F 30°S ‘ e@ M. aAcBipum @ M. ABUNDANS m@ M. ADENOPTERIS @ M. CANESCENS A 4 MV. BREVIPUBENS B 60°W ow 40°W 60°W 50°W 40° i 1 i yeh Fea t / 0 500Km 4. 0 500Km — oo] Seaman ee 2 j » - 5 = 10°S4 10°S 4 - e 20°S4 e 20°S = e * 6 ° a) wee 30°S4] F 30°S 4 r e Ds CG D 60°W s0°w 40°W 60°W 50°W 40°w i i i ic. 2, Distribution of six species of Megalastrum. A. M. abundans and M. canescens. lans F albidum, M. adenopteris, and M. brevipubens. C. M. connexum. D. M. crenu ans. 8 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Dryopteris martiana Rosenst., Hedwigia 46: 132. 1907. LECTOTYPE (here designated).—Brazil. Parana: Villa Nova, 1906, Annies 71 (Rosenstock Filices Austrobrasilienses no. 117) (S-1691; duplicates: B-n.v., BM, GH, MICH, NY, P, R-n.v., S, U, UC, US). Leaves to 2.5 m long; scales of petiole bases 1-3 X 0.2—0.35 cm, linear, sparsely denticulate, brown; laminae 1-2 m long, 4-pinnate-pinnatifid at base, 3-pinnate-pinnatifid medially; proximal pinnae ca. 0.7 m long, strongly inequilateral, pinnules acroscopically reduced toward bases of pinnae; pinna rachises abaxially non-glandular, sparsely pubescent, hairs 0.5 mm, 4- or 5- celled, adaxially non-glandular, densely pubescent, hairs 0.3-0.4 mm long, 5- to 7-celled, scales 0.8-1.0 mm long, bullate, brown, ovate-lanceolate; costules on abaxial surfaces non-glandular, pubescent, hairs 0.1-0.5 mm, 1—4-celled, sparsely scaly, scales 0.8-1.0 mm long, bullate, brown, ovate-lanceolate; laminar tissue between veins abaxially non-glandular, glabrous to sparsely pubescent, hairs ca. 0.1 mm long, 2-celled, erect, adaxially glabrous (sparse hairs only on veins); veins visible on both surfaces, abaxially non-glandular, pubescent and minutely scaly, hairs 0.2—0.3 mm long, 1- or 2-celled, scales ca. 0.3 mm long, uniseriate, appressed, reddish, adaxially non-glandular, sparsely pubescent, hairs 0.3-0.5 mm long, 1—3-celled; Jamina margins ciliate, hairs ca. 0.1 mm long, 1-celled, glandular hairs absent; indusia absent. Distribution and ecology.—Endemic. to coastal Brazil; 600-1200 m. ADDITIONAL SPECIMENS ExAMINED.—BRAZIL. Minas Gerais: Juiz de Fora, Pogo Danta, [21°45’S, 43°21'W], Jul 1902, Schwacke 14801 (P). Parana: Pitanga, Borboleta, 24°45'S, 51°45’W, 600 m, 13 Dec 1973, Hatschbach 33505 (C, UC); Piraquara, Mananciais da Serra, Morro do Canal, 25°30'58"S, 48°48'20"W, 1200 m, 20 Oct 2003, Labiak et al. 3002 (NY, UC, UPCB). Rio de Janeiro: Serra da Estrela, [22°53’S, 43°13’W], Luetzelburg s.n. (S). Santa Catarina: Blumenau, 27°56'S, 49°03'W, 850-900 m, 3 Apr 2008, Schwartsburd & Ceolin 1631 (NY, SP); Lages, [27°48’S, 50°18’W], Mar 1912, Spannagel 324 (HBR, S, SP). Megalastrum abundans is characterized by bullate scales on the pinna rachis and costules abaxially, lamina tissue glabrous adaxially between veins, veins pubescent adaxially, and non-indusiate sori (Fig. 8A—H). It and M. crenulans are the most finely divided species in the area, with laminae 4-pinnate (or more) at their bases (Fig. 5A). Megalastrum abundans differs from M. crenulans by lack of indusia. 2. Megalastrum adenopteris (C. Chr.) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Dryopteris adenopteris C. Chr., Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. Afd., ser. 8, 6: 85. 1920. Ctenitis adenopteris (C. Chr.) Ching, Sunyatsenia 5: 250. 1940. LECTOTYPE (here designated).—BRAZIL. Rio Grande do Sul: Silveira Martins, Val Veneta, ad terram silvae primaevae, in 1893, Lindman s.n. (Regnell A 1313) (BM- 00907710; duplicates: BM, C-n.v., L-n.v., S-n.v., U-n.v., US; fragm. MO; photo MICH ex BM). Figs. 2B, 6F, 10G-O. MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 9 Dryopteris villosa (L.) Kuntze var. tomentosa Rosenst., Hedwigia 46: 130. 1916. LECTOTYPE (here designated).—BRAZIL. Rio Grande do Sul: Mun. Rio Pardo, Fazenda Soledade, [29°59'23’S, 52°22’41"W], 1906, Jiirgens s.n. (Rosenstock Filices Austrobrasilienses no. 207) (MICH; duplicate: S-n.v.). Dryopteris oreocharis Sehnem var. canescens Sehnem, FI. Ilustr. Catarinense ASPI 1: 177. 1979. LECTOTYPE (here designated).—BRAZIL. Santa Catarina: Lauro Miiller, Novo Horizonte, 400 m, 24 Oct 1958, Reitz & Klein 7516 (PACA; duplicate: HBR). Leaves to 4 m long; scales of petiole bases 1-2 X ca. 0.1 cm, linear, sparingly denticulate (nearly entire), light brown; Jaminae 1—2 m long, 4-pinnate at base, 3-pinnate-pinnatifid medially; basal pinnae ca. 1m long, strongly inequi- lateral, pinnules acroscopically reduced toward bases of pinnae; pinna rachises abaxially glandular with sessile to short-stalked glandular hairs, adaxially pubescent and glandular, hairs 0.1-0.3 mm long, 1-3 celled; costules on abaxial surfaces sparsely scaly, scales 0.50.7 mm long, light brown, ovate- lanceolate, non-bullate, pubescent, hairs ca. 0.1-0.2 mm long, with many gland-tipped hairs (more so than on tissue between veins), glandular cell yellowish pubescent, hairs on adaxial surfaces 0.3-0.4 mm long, 3- or 4-celled; laminar tissue between veins densely and evenly puberulent on both surfaces; hairs on abaxial surfaces ca. 0.1 mm long, erect, 1-celled, some of the hairs gland-tipped, glandular cell yellowish, spherical, sometimes sessile or nearly so; hairs adaxially on tissue between veins, ca. 0.2 mm long, 1-celled, glandular hairs sparse; veins visible, pubescent on both surfaces, hairs abaxially ca. 0.3 mm long, 2-celled, hairs adaxially sparser, ca. 0.5 mm long, 3- or 4-celled; lamina margins ciliate, hairs 0.2—-0.3 mm long, 1-celled, glandular hairs sparse to absent: indusia < 0.3 mm, fugacious and usually seemingly absent, glandular and pubescent, hairs sometimes appearing mixed among the sporangia. Distribution and ecology.—Brazil, Argentina; 500-850 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Rio Grande do Sul: Val Veneta, 1893, Lindman s.n. (BM, MO, US): Rio Pardo, Fazenda Soledade, [29°59’22’S, 52°52’41”W], 1906, Jiirgens s.n. [Rosenstock Filices Austrobrasilienses no. 207] (MICH). Megalastrum adenopteris is characterized by dense even uniform, erect hairs on the abaxial surfaces of the laminae, glands on both surfaces of the laminae, pinna rachises, and costules, and minute fugacious indusia (Fig. 10G—O). Glands are usually most evident on the pinna rachises and costules abaxially, either sessile or with a one-celled stalk. Unlike most Megalastrum in the region, the rachis scales are widely spreading. The indusia are often apparently absent, or they appear as a cluster of several minute (ca. 0.05 mm long) scales. Whether this condition is homologous with true indusia is uncertain. The most similar species is Megalastum umbrinum, which differs by laminae glabrous adaxially between the veins (or with a few hairs near the 10 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) margin), rachis scales appressed, and lack of minute fugacious indusia (Fig. 10A-F). Megalastrum abundans differs by numerous bullate scales on the pinna rachises and costules abaxially, sparser pubescence of the laminae abaxially, absence of glandular hairs, and lack of indusia (Fig. 8A-H). Megalastrum crenulans differs by persistence large indusia, the hairs between the veins abaxially all short (ca. 0.1 mm long) glandular, and hairs on the adaxial surfaces of the costules 0.5—-0.7 mm long, 5- or 6-celled (Fig. 10P—AA). 3. Megalastrum albidum R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.— BRAZIL. Parana: Mun. Morretes, Serra da Graciosa, Caminho dos Jesuitas, 29°59'23"S, 52°22’41”W, 1000 m, 20 Oct 2003, Labiak & Gold- enberg 3011 (holotype: UPCB; isotypes: NY, SP). Figs. 2B, 7A, 9E-H. AM. canescenti laminis ad basim 3-pinnato-pinnatisectis, ad medium 2- pinnato-pinnatisectis, utrinque pubescentibus atque rachidibus pinnarum abaxialiter squamis linearibus et pilis 0.3-0.6 mm longis albidis vestita differt. Leaves to 2m long; scales of the petiole bases ca. 1.5-2 Xx 0.04—0.1 cm, linear, sparsely denticulate, yellowish to golden brown, flat (not twisted), en masse not forming a woolly tuft; Jaminae 1.5 m long, to 3-pinnate-pinnatisect at base, 2-pinnate-pinnatifid medially; basal pinnae to 45 cm long, stalks to 2 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward the bases of pinnae; pinna rachises abaxially non-glandular, sparsely pubescent, very sparsely scaly, hairs 0.3-0.6 mm long, 3—5-celled, scales 1— 2 mm long, linear, brown, denticulate, flat (not bullate), slightly spreading, adaxially non-glandular, densely pubescent, hairs ca. 0.3-0.6 mm long, 3-5- celled, patent (not strigose); costules abaxially non-glandular, pubescent, hairs of relatively uniform length, ca. 0.8-1.0 mm long, 5-8-celled, acicular, whitish, scaly, scales small (ca. 0.5—1 mm long), subentire to denticulate, linear, subappressed, adaxially non-glandular, pubescent, hairs ascending to antrorsely strigose, ca. 0.5 mm long, 3-5-celled; laminar tissue between veins abaxially non-glandular, densely pubescent, sparsely scaly, hairs ca. 1—1.5 mm long, 6—9-celled, erect, scales ca. 0.1 mm long, uniseriate, linear, appressed, reddish, inconspicuous, adaxially pubescent, hairs 0.4-0.7 mm long, 4-7- celled, spreading to erect, very sparse scaly, scales ca. 0.2-0.4 mm long, uniseriate, appressed, light reddish, both surfaces dull; veins visible on both surfaces, non-glandular, pubescent and scaly, hairs ca. 1-1.5 mm long, 6—9- celled, adaxially densely pubescent, hairs 0.4-0.7 mm long, 4-7-celled; lamina margins ciliate, non-glandular, hairs ca. 0.2-0.3 mm long, 2-3-celled; indusia absent. Distribution and ecology.—SE Brazil (Parana and Sido Paulo); 0—-1000 m. ApDpITIONAL SPECIMENS EXAMINED.—BRAZIL. Parana: Trés Barras, 27 Jan 1916, Dusén 17563 (S). SAo Pauto: Iguape, Serra de Itatins, [24°42'28"S, 47°47'18’W], 600 m, Mar 1924, Brade s.n. (US); Santos, [23°57'S, 46°20’W], 15 Nov 1874, Mosén 3091 (S). MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 11 Megalastrum albidum has linear scales on the pinna rachises abaxially, these mixed with whitish hairs 0.4-0.6 mm long (Fig. 9E-H). Also character- istic are the laminae pubescent on both surfaces between the veins. It resembles M. canescens (Fig. 12A-F), a species that differs by glandular, stalked hairs on the abaxial lamina surfaces, hairs of mixed length on the costules, and generally shorter hairs on the laminar tissue between the veins (shorter than those along the veins). Furthermore, the laminae of M. canescens are 2-pinnate-pinnatisect at base, 2-pinnate-pinnatifid medially (Fig. 5B), whereas the laminae of M. albidum are 3-pinnate-pinnatisect at base, 2- pinnate-pinnatifid medially (Fig. 7A). Megalastrum albidum is named for the dull whitish hairs on both surfaces of the laminae. 4. Megalastrum brevipubens R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.— PARAGUAY. Amambay: Sierra de Amambay [ca. 23°00’S, 58°00’W, 300 m], Sep 1907, Hassler 10802 (holotype: NY; isotypes: BM, K, MICH). Figs. 2B, 7F, 11F-K. A M. connexo lamina abaxialiter pilis acicularibus 0.1-0.2 mm longis erectis inter venas vestita differt. Leaves ca. 1.0 m long; scales of petiole bases ca. 2 X 0.15 cm, linear to lanceolate, sparsely denticulate, light brown to yellowish or golden, twisted or crispate, en masse forming a dense wool: laminae ca. 75 cm long, to 3- pinnate-pinnatifid at base, 2-pinnate medially; basal pinnae ca. 30-50 cm long, stalks to 2 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward the base of pinna; pinna rachises abaxially non- glandular, glabrous or nearly so, with a few (usually at pinna base) scales, these 2.5 mm long, linear, denticulate, adaxially densely pubescent, non- glandular, hairs 0.1-0.3 mm long, 1-4-celled; costules abaxially non-glandu- lar, pubescent to lacking hairs, sparsely scaly, hairs 0.2-0.4 mm long, 2—4- celled, scales 1-2 mm long, filiform to narrowly lanceolate, non-bullate, adaxially pubescent, hairs 0.1-0.6 mm long, 3-5-celled; laminar tissue between veins abaxially non-glandular, puberulent, hairs ca. 1 mm, 1-celled, erect to substrigose, sometimes with sparse uniseriate scales, these ca. 0.2 mm long, appressed, brown, glabrous adaxially; veins adaxially pubescent, visible, abaxially pubescent, hairs 0.2—0.3 mm long, 1-3-celled, with sparse uniseriate, filiform scales, these ca. 0.2 mm long, appressed, brown; lamina margins non- glandular, sparsely ciliate, hairs ca. 0.1-0.2 mm long, 1(2)-celled; indusia absent. Distribution and ecology.—Brazil, E Paraguay; [200-300 ml]. ADITIONAL SPECIMENS EXAMINED.—BRAZIL. Sao Paulo: Sete Barras, Fazenda Intervales, Base de Saibadela, 20 Jul 1994, Salino 1987 (AAU). Unknown: Burchell 3160 (K); 30 Mar 1883, Urban s.n. (MICH). P GUAY. Guaira: Tororo, Arroyo Polilla, 25°55'S, 56°56’W, 14 Dec 1988, Soria 2848 (MO). 12 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Megalastrum brevipubens is distinctive by the short (ca. 0.1 mm long), erect, acicular hairs between the veins on the abaxial surfaces of the laminae (Fig. 11F—K). It resembles M. connexum, a species that is glabrous between the veins abaxially (Fig. 11L—P). 5. Megalastrum canescens (Kunze ex Mett.) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Phegopteris canescens Kunze ex Mett., Abh. Senckenberg. Naturf. Ges. 2: 314. 1858. Polypodium villosum L. var. canescens Baker, Flora Brasil 1(2): 483. 1870. Ctenitis canescens (Kunze ex Mett.) C. V. Morton, Contr. U.S. Natl. Herb. 38: 54. 1967. LECTOTYPE (here designated) BRAZIL. Bahia: Castelnovo, 1836, Blanchet 2454 (NY; B-n.v.; isotypes: GH, K, P). Figs. 2A, 5B, 12A-F. Phegopteris cana Mett., Ann. Mus. Bot. Lugduno-Batavi 1: 223, 1864. TYPE.— BRAZIL. s.d., collector unknown (B-n.v.). [Placed here in synonymy following Christensen, 1920]. Phegopteris mollivillosa Fée, Mém. Soc. Sci. Nat. Strasbourg [Mém. Foug. 10] 32. 1865. LECTOTYPE (here designated).—BRAZIL. State unknown: s.d., Martius 320 (P-00600562, 00600563; duplicate: BM). Leaves to 2 m long; scales of petiole bases ca. 1 X ca. 0.1 cm, lanceolate, sparsely denticulate, brown to dark brown, sometimes with blackish denticulate margins (this color often absent on scales on the distal portion of petiole); Jaminae 1-2 m long, 2-pinnate-pinnatisect at base, 2-pinnate medially; basal pinnae ca. 0.4m long, stalks to 3.5 cm long, strongly inequilateral, pinnules acroscopically reduced toward pinna bases; pinna rachises abaxially glandular, pubescent and sparsely scaly (scales sometimes apparently absent), glands ca. 0.1 mm long, hairs ca. 1 mm long, 5—7-celled, scales ca. 1.5mm, non-bullate, ovate-lanceolate, subentire, apices long- acuminate; adaxially apparently non-glandular, densely pubescent, hairs ca. 1.2 mm long, 5- or 8-celled; costules glandular, glands 0.1 mm long, pubescent abaxially with two sizes of hairs, longer ones ca. 1 mm long, 3—5-celled, shorter ones ca. 0.2 mm long, 1- or 2-celled, sparsely scaly, scales to 1 mm long, uniseriate, appressed, brown; Jaminar tissue between veins abaxially glandular and pubescent, glands ca. 0.1 mm long, 1- or 2-celled, yellowish, abundant to nearly absent, hairs 0.3-0.5 mm long, 2- or 3-celled, adaxial surfaces sparsely pubescent, hairs 0.2-0.4 mm long, 1- or 2-celled, appressed; veins visible and pubescent on both surfaces, hairs on abaxial surfaces 0.4— 0.8 mm long, 1—3-celled, adaxial surfaces with hairs 0.5-0.8 mm long, 3-6- celled, glandular hairs absent; Jamina margins ciliate, hairs ca. 0.4—0.5 mm long, 1- or 2-celled, glandular hairs apparently absent; indusia absent. Distribution and ecology.—SE Brazil; 600-1200 m. AppITIONAL SPECIMENS EXAMINED.—BRAZIL. Bahia: Camacan, Ramal para a Torre da Embratel na Serra Boa, N de Sao Joao da Panelinha, [15°25’8"S, 39°39'45"W], 6 Apr 1979, Mori & dos Santos 11703 (K, NY, US); Camacan, MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY ais: RPPN Serra Bonita, 9.7 km de Camacan na estrada para Jacareci, dai 6 km SW na estrada para a RPPN e torre da Embratel, 15°23'30"S, 39°33’55”W, 835 m, 3 Mar 2006, Matos et al. 1094 (CEPEC, UPCB). Rio de Janeiro: Bico do Papagaio, 600 m, 4 Jun 1929, Brade s.n. (US); Parati, Parque Nacional da Serra da Bocaina, 23°11'22’S, 44°50'15”"W, 1200 m, 7 Jan 2008, Labiak et al. 4372 (NY, SP, UPCB). Sao Paulo: Iguape, Morro das Pedras, [24°42'28"S, 47°47'18’W], Oct 1920, Brade 7713 (GH, HB, NY, S, UC, US); Rio Ipiranga confluéncia com Rio Juquid, [24°20'29"S, 47°47'44”W], Oct 1925, Brade 21315 (HB, GH). Unknown: Riedell 51 (GH). Megalastrum canescens is characterized by hairs ca. 1 mm long and 5-8- celled on the abaxial surfaces of the laminae, intermixed with shorter ones, up to 0.2 mm long, non-bullate scales, and lack of indusia (Fig. 12A—-F). The petiole base scales of this species tend to have blackish margins. Superficially, this species resembles M. albidum, which see for comparison. Phegopteris brevinervis Fée, Mém. Foug. 10: 32. 1965. TYPE.—BRAZIL. State nknown: s.d., Claussen s.n. (holotype: P). Phegopteris eriopoda Fée, Crypt. Vasc. Brésil 1: 102, t. 31, fig. 1. 1869. LECTOTYPE (here designated).—BRAZIL. Rio de Janeiro: Rio de Janeiro, Gavia sur les bords du Chemin, [22°56’S, 43°17'W], 12 Mar 1868, Glaziou 2397 (P-00610813; duplicates: C, S, photos MICH, MO ex C). Phegopteris adnata Fée, Crypt. Vasc. Brésil 1: 103. 1869. LECTOTYPE (here designated).—BRAZIL. Rio de Janeiro: Rio de Janeiro, Alto Macaé, [22°56’S, 43°17'W], 21 May 1868, A. Glaziou 2398 (P-00610860; duplicates: C; photo MICH ex C). Phegopteris propinqua Fée, Crypt. Vasc. Brésil 1: 103, t. 32, fig. 3. 1869. TYPE.—BRAZIL. Rio de Janiero: Rio de Janeiro, Fazenda do Ariro [22°56’S, 43°17'W], 28 Jun 1868, Glaziou 2399 (Holotype: P). Polypodium willsii Baker, Ann. Bot. 5: 458. 1891. Dryopteris willsii (Baker) C. Chr., Index Filic. 301. 1905. TYPE.—BRAZIL. Rio de Janeiro: Rio de Janeiro, [22°56’S, 43°17’W], Jun 1881, Wills s.n. (holotype: K). Phegopteris lateadnata H. Christ, Ann. Cons. Jard. Bot. Genéve 3: 36 1899. Dryopteris lateadnata (H. Christ) C. Chr., Index Filic. 274. 1905 [as ‘“D. latealata,” a typographical error]. Dryopteris connexa var. lateadnata (H. Christ) C. Chr., Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. 14 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Afd., ser. 8, 6: 80. 1920. LECTOTYPE (here designated)—PARAGUAY. Caaguazu: 1 Apr 1885 s.d., Balansa 313a (holotype: P-00610679; duplicates: B-o.v., K. Lavv., P). Leaves to 1.5-2.5 m long; scales of the petiole bases ca. 2 X 0.15 cm, linear to lanceolate, sparsely denticulate, light brown to yellowish or golden, twisted or crispate, en masse forming a dense wool; laminae 1-2 m long, to 3-pinnate at base, 2-pinnate medially; basal pinnae ca. 30-50 cm long, stalks to 2 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward pinna bases; pinna rachises abaxially non-glandular, glabrous or nearly so, with a few (usually at pinma base) scales, these 2.5 mm long, linear, denticulate, adaxially densely pubescent, non-glandular, hairs 0.1— 0.3mm long, 1-4-celled; costules abaxially non-glandular, pubescent or lacking hairs, sparsely scaly, hairs 0.2-0.3 mm long, 2—4-celled, scales 1— 2 mm long, filiform to narrowly lanceolate, non-bullate, adaxially pubescent, hairs 0.1-0.6 mm long, 3—5-celled; laminar tissue between veins abaxially non- glandular, glabrous, sometimes with sparse uniseriate scales, these ca. 0.2 mm long, appressed, brown, adaxially glabrous; veins adaxially glabrous or with scattered hairs, visible, abaxially glabrous or pubescent, hairs ca. 0.2 mm long, 1-3-celled, with sparse uniseriate, filiform scales, these ca. 0.2 mm long, appressed, brown; Jamina margins non-glandular, sparsely ciliate or appar- ently eciliate, hairs ca. 0.1 mm long, 1(2)-celled; indusia absent. Distribution and ecology.—Brazil, Argentina, Paraguay, and Uruguay; 0— 1000 m ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Bahia: Camacan, RPPN Serra Bonita, 9.7 km de Camacan, Fazenda Serra Bonita, 9.7 km W de Camacan na estrada para Jacareci, dai 6 km SW na estrada para a RPPN e torre da embratel, 15°23'30’S, 39°33’55’"W, 835 m, 13 Feb 2005, Matos et al. 439 (UPCB); Ilhéus, [14°47’S, 39°39’W], Moricand 2469 (K). Espirito Santo: Concordia, Concordia a Cachoeira, 1889, Bello 533 (R). Minas Gerais: Serra de Caldas, [17°44’S, 48°37'W], 10 Sep 1873, Mosén 2184 (P, S, U). Serra de Ouro Preto, 07 Jan 1894, Schwacke 10230 (P). Parana: Vila Nova, [24°42'50"S, 53°53'34”W], Mar 1905, Annies s.n. [Rosenstock Filicies Austrobraslienses 100](MICH, S); Campo Mourao, Mata ao lado da Usina Mourao, 24°07'06"S, 52°19'13”"W, 24 Dec 2007, Labiak et al. 4260, 4262 (NY, SP, UPCB). Rio de Janeiro: Gavla sur les bords du chemin, 12 Mar 1868, Glaziou 2397 (C, P, S); Alto Macaé, 21 May 1868, Glaziou 2398 (C, P). Rio Grande do Sul: Sao Leopoldo, [29°45'36"S, 51°51'49"W], 23 Apr 1905, Eugénio 39 (NY); Pareci Novo. Montenegro, 100 m, 13 Oct 1945, Sehnem 1343 (US). Santa Catarina: Florianépolis, Sertaéo da Lagoa, [27°35'49’S, 48°48'56’W], 26 Jun 1948, Rohr 1071 (HB, HBR, NY, US); Chapec6, Seminario Diocesiano, W of Chapecé, 27°06’S, 52°37’W, 16 Dec 1964, Smith & Klein 14041 (US). Sao Paulo: Sado Paulo, Serra da Cantareira, [23°22'S, 46°46’W], 11 Jan 1914, Brade & de Toledo 6634 (GH, HB, NY, Ease Iguape, Morro das Pedras, [24°42'28"S, 47°47'18"W], Aug 1916, Brade 7714 (GH, HB, K, NY, S, UC). Unknown: Burchell 1915 (GH, K, P); Glaziou 1780 (C, P); Glaziou 7948 (C, P). MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 15 PARAGUAY. Alto Parana: Reserva Bioldégica de Itab6, 35 km W Rio Parand, 25°5'S, 54°54’W, 10 Oct 1990, Schinini & Marmori 27051 (GH); Estancia Rio Bonito, Forest I, 25°38'50"S, 54°54’45”W, 240 m, 29 Aug 1994, Zardini & Vera 40653 (MO). Amambay: In altapanitie et declivibus, Sierra de Amambay, [22°56’S, 55°55’W], 1907-1908, Hassler 10278 (BM, MICH, P); In altapanitie et declivibus, Sierra de Amambay, [22°56’S, 55°55’W], 1907-1908, Hassler 10802 (B, K, MICH, NY). Caazapa: Tavai Pacuri, Comunidad Mbya, 26°10'S, 55°55’W, 22 Dec 1988, Basualdo 2127 (MO); Parque Nacional de Caaguazu, Bosque de hasta 20 m de altura, Linea del Parque hacia ao Ité y ao Jaku’y Ruta Prov. 101; camino al aeropuerto, sobre el desvio a las Cataratas del Iguazu, [28°6'60"S, 56°56’60"W], 22 Jul 1986, Molas 823 (MO). Central: in regione Lacus Ypacaray, [25°23’S, 57°16’W], Hassler 12944 (P). Paraguari: prope Sapucay, [26°3’S, 56°56’W], Aug 1913, Hassler 12203 (C, K, MO, P, S, US); Parque Nacional Ybycu’{, Sendero dentro del bosque ribereno, [26°3’S, 56°56’W], 31 May 1987, Zardini & Pérez 2881 (MO). San Bernardino: [25°16’S, 57°19’W], 2 Sep 1915, Osten 8435 (S). Unknown: Apr 1881, Balansa 2910(BM,C,P, S);1885-— 1895, Hassler 1841 (BM, K, NY, P); 1885-1895, Hassler 660b (BM, K, NY, P, S). URUGUAY. Rivera: Tranqueras, [31°0’S, 46°0'W], 7 May 1945, Legrand 3994 (US). Tacuarembé: Sierra del Tacuaremb6, gruta de los helechos, [33°00’S, 56°00’W], 300 m, 24-28 Aug 1907, Herter 3534a (P). Megalastrum connexum is characterized by glabrous pinna rachises abaxially and filiform scales on the costules and veins abaxially (Fig. 11L— P). Both surfaces of the lamina between the veins are glabrous. The costular indument is variable. In all specimens there are hairs and scales on the costules abaxially, but some specimens have more scales than hairs, and vice- versa. Judging from the number of specimens, this is one of the most common species in Brazil. Megalastrum brevipubens has similar laminar cutting but differs by minute (ca. 0.1 mm long), erect, acicular hairs on the laminar tissue between the veins (Fig. 11F-K). Two specimens from Paraguay (Hassler 12203, 12942a) are unusual in lamina cutting. The pinnae are extremely large, with ultimate segments widely spaced and slightly acute and falcate; however, we find no differences in the indument with typical plants, and no other differences correlate with lamina division. Therefore, we consider the specimens part of the variation within M. connexum. Both Christensen (1920) and we were unable to find Kaulfuss’s type specimen of Polypodium connexum (Brazil, Sta. Catarina, s.d., Chamisso s.n.), which should be housed at B or LE. For this reason, we are neotypifying this long-used name. The neotype chosen is from the island of Santa Catarina where the Chamisso specimen was collected. 7. Megalastrum crenulans (Fée) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Aspidium crenulans Fée, Crypt. Vasc. Brésil 1: 139, t. 47, fig. 1. 1869. Dryopteris crenulans (Fée) C. Chr., Kongel. Danske Vidensk. Selsk. 16 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Skr., Naturvidensk. Math. Afd., ser. 8, 6: 90. 1920. Ctenitis crenulata (Fée) Ching, Sunyatsenia 5: 250. 1940. LECTOTYPE (designated by Christen- sen, 1920): BRAZIL. Rio de Janiero: Rio de Janeiro, [22°56’S, 43°17’W], Glaziou 1781 (C; duplicates K, P, RB-n.v.; photos MICH, MO ex GC). Figs. 2D, 5D, 10P-AA. Dryopteris villosa var. glandulosa Rosenst., Hedwigia 46: 129. 1907. Dryopteris crenulans forma glandulosa (Rosenst.) C. Chr., Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. Afd., ser. 8, 6: 91. 1920. LECTOTYPE (here designated).—BRAZIL. Rio Grande do Sul: Mun. Rio Pardo, banks of Rio Cyriaco, 1906, Jiirgens s.n. [Rosenstock Filices Austrobrasilienses no. 206] (BM-000907729; duplicates: GH, HB, K, MICH, MO, NY, P, S-n.v., UC). Leaves to 2.5 m long; scales of the petiole bases ca. 2 X ca. 0.1 cm, linear, sparsely denticulate, light brown, twisted, en masse forming a dense woolly tuft; Jaminae 1m long, to 4-pinnate at base, 2-pinnate-pinnatifid medially; basal pinnae ca. 30-50 cm long, stalks to 2.5 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward the pinna bases; pinna rachises abaxially glandular, pubescent, hairs 0.8—-1.2 mm long, 4- or 5-celled, densely glandular, glands ca. 0.1 mm long, 2-celled, scaly, scales ca. 1 mm long, lanceolate, subentire, flat (non-bullate), adaxially densely pubescent, hairs ca. 1 mm long, 3—6-celled, non-glandular; costules abaxially glandular, pubescent, hairs 0.3-0.4 mm long, 1-3-celled, glands ca. 0.1 mm, 2-celled, scaly, scales ca. 1 mm long, bullate, subentire, adaxially pubescent, hairs 0.4— 0.8 mm long, 1—-3-celled, sparsely glandular, glands like those on the abaxial surfaces: laminar tissue between veins abaxially densely glandular and pubescent, hairs ca. 0.1 mm long, 1-celled, adaxially glandular but slightly less so than abaxially, hairs absent; veins visible on both surfaces, very sparsely glandular abaxially, pubescent abaxially, hairs 0.2—-0.3 mm long, 1- or 2-celled, adaxially sparsely pubescent, hairs ca. 0.4—0.5 mm long, 1—3-celled; lamina margins densely ciliate, hairs ca. 0.3-0.4 mm long, 1- or 2-celled; indusia present, circular, dark brown, glandular, pubescent, or both, hairs 0.3— 0.4 mm long, 1- or 2-celled. Distribution and ecology.—Venezuela, Brazil, and Paraguay; 725-1760 m. AppITIONAL SPECIMENS EXAMINED.—BRAZIL. Minas Gerais: Vicosa, Fazenda de Aguada, [20°47'45"S, 50°50’W], 725 m, 17 Sep 1930, Mexia 5060 (NY, §, UG, US). Parana: Apucarana, Pirap6, [23°33’S, 51°26’W], Jun 1951, Tessmann 593 (HB). Rio de Janeiro: Teres6polis, Quebra frascos, [22°24'44"S, 42°42'56”W], 17 Oct 1929, Brade 9695 (GH, NY, UC); Fazenda da Santa Anna, [22°54'S, 43°43’W], Glaziou 2351 (C, K, MO, P [syntypes]). Rio Grande do Sul: 1865, Page s.n. (US). Sao Paulo: Sao Paulo, Agua Funda, nativa no Jardim Botanico, [23°32'52"S, 46°46’9”"W], May 1973, Handro 2224 (GH, HB, US); Campos do Jordao, [22°46’S, 45°31'W], Jul 1945, Leite 3567 (UC). PARAGUAY. Central: Cordillera Central, [25°22’60"S, 57°57'60”W], Dec 1900, Hassler 6898 (GH, MICH, NY, P). MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 27 Megalastrum crenulans is nearly unique in the region by having a large persistent indusium (Fig. 10P—AA). It is further distinctive by glands on both surfaces of the laminae and bullate scales on the abaxial surfaces of the costules. The indusia are variable in the presence of hairs and glands, but no other character apparently correlates with this. The only other species with a well-defined indusium is M. indusiatum, but the indusium in that species is much smaller, only about the size of a single sporangial capsule, and are easily overlooked. Fée (1869) applied the name Aspidium consobrinum Fée (=Ctenitis), type from Guadeloupe, to the Brazilian specimens Glaziou 2350, 979, and Gaulthier s.n. In our opinion, these are typical M. crenulans. 8. Megalastrum eugenii (Brade) A. R. Sm. & R. C. Moran, Amer. Fern 1. 773 127. 1987. Dryopteris eugenii Brade, Rodriguésia 4(13): 298, t. 2. 1940. Ctenitis eugenii (Brade) Brade, Bradea 1(22): 209. 1972. LECTOTYPE (here designated).—BRAZIL. Ceara: Serra de Baturité, Sitio Santa Clara, [4°19'44"S, 38°53'06"W], 9 Dec 1937, Eugénio s.n. (US: duplicates: HBR, RB-n.v.). Figs. 3A, 7C, 11Q-V. Leaves to 1.01.5 m long; scales of the petiole bases ca. 1.5 X 0.05-0.07 cm long, linear, sparsely denticulate, brown, slightly twisted, en masse forming a woolly tuft; Jaminae 1m long, to 2-pinnate-pinnatifid at base (rarely 1- pinnate-pinnatisect), 1-pinnate-pinnatisect medially; basal pinnae 20-30 cm long, stalks to 1 cm long, inequilateral, pinnules acroscopically not or only slightly reduced toward pinna bases; pinna rachises abaxially non-glandular, glabrous to puberulent, sparsely scaly, hairs 0.1-0.2 mm long, 1—3-celled, scales 1-2 mm long, narrowly lanceolate, brown, denticulate, flat (non- bullate), adaxially non-glandular, pubescent, hairs 0.3-0.4 mm long, 2- or 3- celled, strigose; costules abaxially non-glandular, glabrous to puberulent, hairs 0.1—0.2 mm long, 1—3-celled, scaly, scales ca. 1 mm long, linear to narrowly lanceolate, non-bullate, sparsely denticulate, adaxially puberulous through- out, hairs 0.4—0.5 mm long, 1—3-celled; laminar tissue between veins abaxially non-glandular, glabrous to subglabrous, hairs (when present) ca. 0.2 mm long, 1- or 2-celled, uniseriate scales often present, these appressed, reddish, inconspicuous, adaxially non-glandular, glabrous to sparsely pubescent (often near margins), hairs 0.3-0.4 mm long, 2- or 3-celled; veins visible or obscure on both surfaces, non-glandular, glabrous to sparsely pubescent; lamina margins sparsely to densely ciliate, hairs 0.2-0.4 mm long, 2—3-celled; indusia absent. Distribution and ecology.—Endemic to coastal NE Brazil (Alagoas, Bahia, Ceara, and Pernambuco); 600-700 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Alagoas: Ibateguara, Engenho Coim- bra, 9°00’S, 35°51’W, 380-400 m, 19 Dec 2000, Pietrobom 4698 (HB, SP). Bahia: Arataca, Serra do Peito de Moga, estrada que liga Arataca a Una, ramal ca. 22,4km de Arataca com entrada no Assentamento Santo Antonio, 18 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) 20°S 7] 30°S 5 e@ M. INAEQUALE m WM. inDusiatuM B OW 40°W 60°W i i i t 1 / 0 500Km go oe re { = : f Es 7 - 10°S a Pi < J 20°S 44 — { 30°S 3 @ M. OREOCHARIS eo @ M. ORGANENSE mw M. LiTTORALE ; m M. suBsTRIGOSUM D 60°W 50°W f 60°W 50°W 40°W i i i i i Fic. 3. Distribution of eight species of Megalastrum. A. M. grande and M. eugenii. B. M. inaequale and M. indusiatum. C. M. oreocharis and M. littorale. D. M. organense and M. substrigosum. 15°10'25"S, 39°20'30"W, 650 m, 6 Aug 2006, Labiak et al. 3673, 3678 (CEPEC, NY, UPCB); prope Sado Pedro de Alcantara ad Vila dos Ilheos, [14°47'20"S, 39°39'56”W], 1839, Luschnath Mart. Herb. FI. Bras. 327 (BM, K, NY, P). Pernambuco: Timbatiba, Complexo do Mascarenhas, Mata do Estado, 7°37’S, MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 19 35°23'W, 304-394 m, 28 Apr 2001, Pietrobom et al. 5225 (HB, SP); Sao Vicente Ferrer, Complexo do Mascarenhas, Mata do Estado, 7°35'0"S, 35°35’0"W, 600— 650 m, 17 Aug 1998, Pietrobom 4394 (NY). Megalastrum eugenii resembles M. grande but differs by the presence of hairs on the costae adaxially (cf. Fig. 11Q-V and Fig. 9J—M). This is the only species that occurs in the northern part of northeastern Brazil. 9. Megalastrum grande (C. Presl) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Polypodium grande C. Presl, Deliciae Pragenses 1: 171. 1822. Dryopteris grandis (C. Presl) C. Chr., Index Filic. 268. 1905. Ctenitis grande (C. Presl) Copel., Ann. Crypt. Phytopath. [Gen. Fil.] 5: 124. 1947. TYPE.—BRAZIL. Rio de Janeiro: collector unknown, Christensen (1920) suspected J. E. Pohl s.n. (holotype: PR-n.v.; isotype: W?-n.v.). Figs. 3A, 5E, 9J-M. Polypodium auriculatum Raddi, Syn. Fil. Bras. 10 (no. 74). 1819 [seors. Prae- impr. ex Opusc. Sci. Bol. 3(5): 288. 1819], nom. illeg., non L. 1753. Polypodium formosum Raddi, Pl. Bras. Nov. Gen. 1: 25, tab. 38. 1825, nom. nov. for P. auriculatum Raddi, non L. 1753. TYPE.—BRAZIL. Rio de Janeiro: Mt. Corcovado, [22°56’S, 43°17'W], s.d., Raddi s.n. [specimen no. 1 with “Polypodium formosum nob.” written by Raddi on the cover], (Lectotype: designated by Pichi Sermolli & Bizzarri, 2005): Pl-n.v.; duplicates: FI-n.v., P, UC). Polypodium macropterum Kaulf., Enum. Fil. 111. 1824. Phegopteris macro- ptera (Kaulf.) Fée, Gen. Filic. 243. 1852. Polypodium splendidum Kaulf., Enum. Fil. 112. 1824. Phegopteris splendida (Kaulf.) Fée, Gen. Filic. 243. 1852. Polypodium macropterum Kaulf. var. splendida Baker, Fl. Bras. 1(2): 502. 1870. LECTOTYPE (here designated).—BRAZIL. Rio de Janeiro, [22°56’S, 43°17'W],s.d., Mertens s.n. (C; duplicates: B-n.v.; photos MICH, MO, NY ex C). Polypodium repandum Vell., Flora Flumin. 11, tab. 73. 1827, nom. illeg., non Lour. 1790. TYPE.—unknown. Polypodium pohlianum C. Presl, Tent. Pterid. 180. 1836, nom. nud. Alsophila fischeriana Regel, Index Seminum Hort. Petrop. 1855, nom. nud. Phegopteris scrobiculata Fée, Crypt. Vasc. Brésil 1: 102, t. 31, fig. 2. 1869. LECTOTYPE (here designated): BRAZIL. Rio de Janeiro: Tijuca, Bico do Papagaio, [22°56’'S, 43°17’W], 12 Mar 1867, Glaziou 2066, pro parte (P- 00610859; duplicates: C, K, MO, P, U; photos MICH, NY ex C). Leaves to 1.0—-1.5 m long; scales of the petiole bases ca. 1.5 X 0.05—0.07 cm long, linear, sparsely denticulate, brown, slightly twisted, en masse forming a woolly tuft; Jaminae 1m long, to 2-pinnate at base (rarely 1-pinnate- pinnatisect), 1-pinnate-pinnatifid medially; basal pinnae 20-40 cm long, stalks to 1 cm long, inequilateral, pinnules acroscopically not or only slightly _ reduced toward pinna bases; pinna rachises abaxially non-glandular, glabrous to puberulent, sparsely scaly, hairs 0.1-0.2 mm long, 1-3-celled, scales 1— 20 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) 2mm long, narrowly lanceolate, brown, denticulate, flat (non-bullate), adaxially non-glandular, glabrous; costules abaxially non-glandular, glabrous to puberulent, hairs 0.1-0.2 mm long, 1—3-celled, scaly, scales ca. 1 mm long, linear to narrowly lanceolate, non-bullate, sparsely denticulate, adaxially glabrous; Jaminar tissue between veins abaxially non-glandular, glabrous to subglabrous, hairs (when present) ca. 0.2 mm long, 1- or 2-celled, uniseriate scales often present, these appressed, reddish, inconspicuous, adaxially non-glandular, glabrous to sparsely pubescent (often near margins), hairs 0.3-0.4 mm long, 2- or 3-celled; veins visible or obscure on both surfaces, non-glandular, glabrous to sparsely pubescent (similar to lamina tissue between veins); Jamina margins sparsely to densely ciliate, hairs 0.2-0.4 mm long, 1—3-celled; indusia absent. Distribution and ecology.—Brazil, primarily Atlantic rainforest from Bahia to Sao Paulo; 300-1000 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Bahia: Castelnovo, 1836, Blanchet 2494 (P). Espirito Santo: Santa Teresa, Alto de Santo Anténio, terreno do Bozza, 19°54'31"S, 40°40’28”W, 760 m, 12 Jul 2007, Labiak et al. 4041, 4047 (NY, UPCB); Santa Teresa, trilha que sobe a encosta ao lado da entrada do Country Club, 25 Feb 1996, Salino 2636 (BHCB, UC). Rio de Janeiro: Tijuca, [22°53’S, 43°13’W], 8 Oct 1867, Glaziou 1676 (C, K, P, S); [22°56’S, 43°17’W], s.d., Glaziou 1780 (C, P, RB-n.v.). Teresépolis, Parque Nacional da Serra dos Orgaos, 22°26'56"S, 42°59'06”W, 950 m, 13 Jan 2008, Labiak et al. 4475 (NY, SP, UPCB). Sao Paulo: Ubatuba, Parque Estadual da Ilha Anchieta, [23°32'S, 45°03’W], 4-11 Jan 1993, Salino 1649 (BHCB, UC). Unknown: Claussen 2111 (NY, P); 1901, Glaziou s.n. (K). Megalastrum grande is unique in the genus by having the pinna rachises glabrous adaxially. It is the least divided species in coastal Brazil, with laminae to 2-pinnate at the base, and usually broadly adnate segments that are slightly falcate (Fig. 5E). The hairs (when present abaxially) are generally inconspicuous, and the costal scales are sparse and linear to linear- lanceolate (Fig. 9J, M). Glandular hairs are absent from all parts of the plant. Although Christensen (1920) included Phegopteris scrobiculata as a synonym of Megalastrum connexum, the type at K and MO seem to us to be typical M. grande. 10. Megalastrum inaequale (Kaulf. ex Link) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 127. 1987. Polypodium inaequale Kaulf. ex Link, Hort. Berol. 2: 107. 1833. Ctenitis inaequale (Kaulf. ex Link) Copel., Ann. Crypt. Phytopath. [Gen. Fil.] 5: 124. 1947. LECTOTYPE (here designated).— collected in Brazil in 1839 and cultivated in the Berlin Botanical Garden, “Hb Link,” collector unknown s.n. (B-200059182; duplicates: BM, P). Figs. 3B, 5F, 9A, B. Phegopteris marginans Fée, Crypt. Vasc. Brésil 105, t. 61, fig. 1. 1869. LECTOTYPE (here designated)—BRAZIL. Rio de Janeiro: Rio de Janeiro, MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 21 Tijuca, [22°56’S, 43°17’W], 18 Oct 1867, A. Glaziou 1681 9631") (P- 00600397; duplicates: C, P). Phegopteris fulgens Mett. ex Schenck, Hedwigia 35: 166. 1896, nom. nud. Leaves to 3 m long; scales of the petiole bases ca. 2 X 0.15 cm, narrowly lanceolate, denticulate, brown, flat (not twisted), en masse not forming a woolly tuft; Jaminae 2 m long, to 3-pinnate-pinnatifid at base, 2-pinnate- pinnatifid medially, shiny on both surfaces, paler abaxially; basal pinnae 20- 40 cm long, stalks to 1 cm long, inequilateral, pinnules acroscopically slightly reduced toward the pinna bases; pinna rachises abaxially non-glandular, pubescent, scaly, hairs 0.1-0.3 mm long, 1-3-celled, antrorsely substrigose (slightly curved toward apex), scales ca. 2.5 mm long, ovate-lanceolate to (less commonly) linear, light brown, subentire, flat to sub-bullate, adaxially non- glandular, densely pubescent, hairs ca. 0.5 mm long, 3- or 4-celled, strigose; costules abaxially non-glandular, pubescent, hairs ca. 0.1-0.2 mm long, 1-3- celled, scaly, scales like those of pinna rachises but more bullate, adaxially with hairs like those of costae; laminar tissue between veins non-glandular and glabrous on both surfaces; veins visible on both surfaces, non-glandular, abaxially sparsely pubescent and inconspicuously scaly, hairs ca. 0.1 mm long, 1-celled, slightly strigose, scales 0.3-0.4 mm long, uniseriate, appressed, reddish, adaxially sparsely puberulent, hairs 0.2-0.3 mm long, 1-3-celled; Jamina margins thick, glabrous to sparsely ciliate, hairs ca. 0.1 mm long, 2-celled, strigose; indusia absent or appearing absent, if present easily overlooked, fugacious. Distribution and ecology.—Endemic to coastal Brazil (Rio de Janeiro and Sao Paulo); 300-1200 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Rio de Janeiro: Itatiaia, Lote 17, [22°29'46"S, 44°44’48”W], 1934, Brade 14017 (MO, NY); Rio de Janeiro, Corcovado, 15 Jul 1866, Glaziou 967 (C, K, P, S). Sao Paulo: Iguape, Serra do Itatins, [24°42’28’S, 47°47'18"W], Mar 1921, Brade 8276 (C, HB, NY,R, S, UC, US); Raiz da Serra, [23°53’42”S, 46°46’30’W], 20 Jan 1907, Wacket 21761 (GH, NY, UC). Megalastrum inaequale is characterized by slightly antrorsely strigose hairs on the pinna rachises and costules abaxially and moderately dense, subbullate scales on the axes (Fig. 9A, B). The laminae are thick, slightly shiny adaxially, and paler abaxially. 11. Megalastrum indusiatum R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.—BRAZIL. Bahia: Camacan, RPPN Serra Bonita, 10 km W de Camacan na estrada para Jacareci, 6 km Sw na estrada para a RPPN e Torres de Transmissao, 15°23'35"S, 39°33'53”W, 750 m, 14 Apr 2007, Matos et al. 1365 (holotype: UPCB; isotypes: CEPEC, NY). Figs. 3B, 5C, 8j-O. A M. crenulanti indusiis minoribus fugacibus, laminis abaxialiter squamis non bullatis secus axin, adaxialiter glabris inter venulas atque rhachidibus costis costulisque abaxialiter eglandulosis differt. 22 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Leaves to 2.0m long; scales of the petiole bases ca. 2 cm long, linear, sparsely denticulate, light brown, twisted, en masse forming a dense woolly tuft; Jaminae 1 m long, to 4-pinnate-pinnatifid at base, 2-pinnate-pinnatisect medially; basal pinnae ca. 30-50 cm long, stalks to 2.5 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward pinna bases; pinna rachises abaxially non-glandular, slightly pubescent, hairs 0.8-1.0 mm long, 4- or 5-celled, scaly, scales ca. 5 mm long, lanceolate, subentire, flat (non- bullate), adaxially densely pubescent, hairs ca. 1 mm long, 3—6-celled, non- glandular; costules abaxially non-glandular, pubescent, hairs 0.3-0.4 mm long, 1—3-celled, scaly, scales ca. 1mm long, non-bullate, subentire, adaxially pubescent, hairs 0.4—0.8 mm long, 1-3-celled, non-glandular; /aminar tissue between veins abaxially non-glandular, pubescent, hairs ca. 0.1 mm long, 1- celled, adaxially non-glandular, hairs absent; veins visible on both surfaces, non-glandular abaxially, minutely scaly and pubescent abaxially, hairs 0.2— 0.3 mm long, 1- or 2-celled, scales ca. 0.1-0.3 mm long, uniseriate, reddish, appressed; adaxially sparsely scaly and pubescent, hairs ca. 0.4—0.5 mm long, 1—3-celled, scales like those abaxially; Jamina margins ciliate, hairs ca. 0.3— 0.4 mm long, 1- or 2-celled; indusia present, circular, dark brown, pubescent, hairs 0.3-0.4 mm long, 1- or 2-celled. Distribution and ecology.—Endemic to Bahia, Brazil; 100-800 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Bahia: Almadina, Serra do Corcov- ado, 9,8 km ao SW de Coaraci na estrada para Almadina, dai N até a Fazenda Sao José, 14°42'21’S, 39°36/12”W, 650-750 m, 19 Jul 2005, Matos et ol 717 (UPCB); Ilhéus, Estrada entre Sururti e Vila Brasil, a 6-14 km de Sururi, a 12— 20 km ao SE de Buerarema, [14°47'20’S, 39°39'56”W], 100 m, 10 Nov 1979, Mori & Benton 12991 (NY). Megalastrum indusiatum is characterized by small indusia (about the size of a single sporangial capsule), non-bullate scales on the axes, glabrous tissue adaxially between the veins, and non-glandular axes abaxially (Fig. 8J—O). The laminae typically dry dark brown adaxially. The indusia in M. crenulans are much larger and conspicuous, covering the sori. 12. Megalastrum littorale R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.— BRAZIL. Rio de Janeiro: Parati, Parque Nacional da Serra da Bocaina, floresta Atlantica, 23°12'03”S, 44°49'43”W, 800 m, 7 Jan 2008, Labiak et al. 4378 (holotype: UPCB; isotypes: NY, SP). Figs. 3C, 7D, 12P-W. AM. canescenti Jamina abaxialiter pilis ca. 2 mm longis albidis et glandulis capitatis bi- vel tricellularis dense vestitae differt. Leaves to 2m long; scales of the petiole bases ca. 1 cm, lanceolate, sparsely denticulate but not retrorsely so, brown to dark brown, without blackish denticulate margins; Jaminae 1-2 m long, 3-pinnate-pinnatifid at base, 2-pinnate-pinnatisect medially; basal pinnae ca. 50 cm long, stalks to 5 cm long, strongly inequilateral, pinnules acroscopically reduced toward MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 23 pinna bases; pinna rachises abaxially sparsely glandular, pubescent and sparsely scaly (sometimes apparently absent), glands ca. 0.1 mm long, hairs 1— 2mm long, 5—8-celled, scales ca. 1.5 mm, non-bullate, linear, subentire, adaxially apparently non-glandular, densely pubescent, hairs ca. 2 mm long, 7- or 8-celled; costules sparsely glandular abaxially, glands 0.1 mm long, pubescent abaxially, hairs 1-2 mm long, longer ones ca. 2 mm long, 5-8- celled, shorter ones ca. 1 mm long, 5- to 8-celled, sparsely scaly, scales to 1mm long, uniseriate, appressed, brown; laminar tissue between veins abaxially glandular and pubescent, glands 0.1—-0.2 mm long, 1- or 3-celled, capitate, whitish, abundant, hairs 1-2 mm long, 5- to 8-celled; adaxial surfaces densely pubescent, hairs 0.5—1 mm long, 4- to 6-celled, erect: veins visible and pubescent on both surfaces, hairs on abaxial surfaces 1-2 mm long, 5—8-celled, on adaxial surfaces hairs 0.5-1 mm long, 4—6-celled, glandular hairs sparse; lamina margins ciliate, hairs ca. 1-2 mm long, 5—8-celled, glandular hairs apparently absent; indusia absent. Distribution and ecology.—SE Brazil (Rio de Janeiro and Sao Paulo); 0— 800 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Rio de Janeiro: Parati, Parque Nacional da Serra da Bocaina, 23°12'03”S, 44°49’43”W, 800 m, 7 Jan 2008, Labiak et al. 4377 (NY, SP, UPCB). Sao Paulo: Ubatuba, pr6ximo a Base Norte (Instituto Oceanografico), Jul 1960, Vdlio 112 (SP, SPF 1 Megalastrum littorale is distinctive by being densely pubescent throughout with whitish hairs ca. 2 mm long (Fig. 12P—W). Also distinctive are its stalked glands with one or two basal cells below the capitate apical cell (Fig. 12T, V). It resembles M. canescens and can be distinguished from that species by couplet 7 of the key. This species grows near the coast, thus the specific epithet littorale. 13. Megalastrum oreocharis (Sehnem) Salino & Ponce, Darwiniana 45: 237. 2007. Dryopteris oreocharis Sehnem, FI. Ilustr. Catarinense ASPI 1: 177. 1979. Ctenitis oreocharis (Sehnem) R. M. Bueno & R. M. Senna, Caderno Pesquisa, ser. Bot. (Sta. Cruz do Sul), 4: 11. 1992. TYPE.—BRAZIL. Santa Catarina: Lages, [27°49'0"S, 50°19'34”W], 950 m, 10 Jan 1951, Sehnem 5508 (holotype: PACA; isotype: PACA). Figs. 3C, 7B, 11A-E. Dryopteris connexa (Kaulf.) C. Chr. var. minor Legrand, Com. Bot. Mus. Historia Nat. Montevideo 2(21): 19. 1952. SYNTYPES: PARAGUAY. Cerro Largo: Isla Zapata, Arechavaleta 2048 (P-n.v.). PARAGUAY. Tacuarembo: Gruta de los Cuervos, D. Legrand 3325 (P-n.v.). Leaves to 1 m long; scales of the petiole bases 1-2 X 0.15 cm, linear, sparsely denticulate, light brown to brown, twisted, en masse forming a dense wool; laminae 80 cm long, to 2-pinnate-pinnatisect at base, 2-pinnate-pinnatifid medially; basal pinnae ca. 15-20 cm long, stalks to 1 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward the pinna bases; 24 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) pinna rachises abaxially non-glandular, very sparsely pubescent, hairs 0.5— 1.5 mm long, 4-9 celled, with a few (usually at pinna base) scales, these 2.5 mm long, linear, denticulate; adaxially pubescent, non-glandular, hairs 0.1-0.3 mm long, 1—4-celled; costules abaxially non-glandular, pubescent, sparsely scaly, hairs 0.7-1.5 mm long, 6—11-celled, scales 1-2 mm long, filiform to narrowly lanceolate, non-bullate, adaxially pubescent, hairs 0.6— 1 mm long, 4—5-celled; Jaminar tissue between veins abaxially non-glandular, glabrous, sometimes with sparse uniseriate scales, these ca. 0.2 mm long, appressed, brown, adaxially glabrous; veins adaxially sparsely pubescent, visible, abaxially pubescent, hairs to 1 mm long, 4—10-celled, with sparse uniseriate, filiform scales, these ca. 0.2 mm long, appressed, brown, veins adaxially pubescent, hairs 1.5-2.0 mm long, 8—10-celled; Jamina margins non- glandular, sparsely ciliate or apparently eciliate, hairs 0.1-0.2 mm long, 1(2)- celled; indusia absent. Distribution and ecology.—S. Brazil (Paranda, Santa Catarina, Rio Grande do Sul), Paraguay, and Uraguay. 0—1000 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Parana: 19 Oct 1963, Hatschbach 10745 (U). Rio Grande do Sul: Piratiny, [31°26’S, 53°06’W], 1892, Lindman 869A (S); Ex colonia Santo Angelo, [28°17'S, 54°15’W], 1893, Lindman 985A (K, S). Santa Catarina: 6 May 1896, Reineck s.n. (P); Lages, [27°49’S, 50°19’W], 950 m, 10 Jan 1951, Sehnem 5508 (PACA). URUGUAY. Tacuarembé: Cerro Largo, Isla Zapata, [33°00’S, 56°00'W], 1877, Arechavaleta s.n. (P, S); Gruta de los Cuervos, [31°36’S, 53°06’W], 19 Mar 1913, Osten 6619 (S, US). Megalastrum oreocharis resembles a small version of M. connexum but differs by longer hairs (0.7-1.7 mm) on the pinna rachises, costules, and veins abaxially (Fig. 11A—E). The plants are generally thinner textured than M. connexum. From M. wacketii, it differs by the glabrous laminar tissue between the veins. Geographically, M. oreocharis and M. wacketii do not overlap (cf. Fig. 3C and Fig. 4A). In our region, M. oreocharis has the southernmost distribution of any species. 14. Megalastrum organense R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.— BRAZIL. Rio de Janeiro: Mun. Teres6polis, Parque Nacional da Serra dos Orgaos, Floresta Atlantica, 22°26'56"S, 42°59'06”W, 1700 m, 13 Jan 2008, Labiak et al. 4485 (holotype: UPCB; isotypes: NY, SP). Figs. 3D, 7E, 8T-W. AM. retrorso Jaminis utrinque inter venulas glabris, differt. Leaves to 1.5 m long; scales of the petiole bases ca. 1.5 X 0.15 cm, narrowly lanceolate, retrorsely denticulate, brown, flat (not twisted), en masse not forming a woolly tuft; Jaminae to 1 m long, to 3-pinnate-pinnatifid at base, 2- pinnate-pinnatisect medially, dull on both surfaces, paler abaxially; basal pinnae 35-45 cm long, stalks to 1.5cm long, inequilateral, pinnules MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 25 Foe { ' ' f 0 500 Km — 40] 0 500 Km go. { on Ease ae ye! f 4 a bap Fe eas ris Sad ct \ rd ¥ i 2 ; | ome 10°S4] F- } rey. | oe 10°S { \ } Lee 2 oo f 20°S44 A a 20°s 4 ; 3 f } Poot 30°S4 CI 30°S @ M. RETRORSUM ‘ @ M. Wacketi A B 60°W 50°W 40°W 60°W 50°W 40°W i i i I Fic. 4. Distribution of three species of Megalastrum in Brazil and Paraguay. A. M. retrorsum and M. wacketii. B. M. umbrinum. acroscopically slightly reduced toward the pinna bases; pinna rachises abaxially non-glandular, pubescent to glabrescent, scaly, hairs 0.2-0.3 mm long, 3- or 4-celled, patent, scales ca. 3-7 mm long, narrowly lanceolate to linear, brown, retrorsely denticulate, flat (not bullate), adaxially non- glandular, pubescent, hairs ca. 0.5 mm long, 3—5-celled, spreading to antrorsely strigose; costules abaxially non-glandular, puberulent, hairs ca. 0.2—0.3 mm long, 3-celled, scaly, scales like those of pinna rachises but smaller and lanceolate, entire to retrorsely denticulate so, shiny and brown, loosely appressed, ca. 3 mm long, adaxially puberulent, hairs ca. 0.1 mm long, 2- or 3- celled, spreading; laminar tissue between veins abaxially non-glandular, glabrous with some inconspicuous, uniseriate, appressed, reddish scales, adaxially glabrous; veins visible on both surfaces, non-glandular, abaxially glabrous or sparsely pubescent, hairs ca. 0.1-0.2 mm long, 1- or 2-celled, adaxially pubescent, hairs ca. 0.3-0.5 mm long, 1—-3-celled, spreading to antrorsely strigose; lamina margins sparsely ciliate, hairs ca. 0.1-0.2 mm long, 2-celled, spreading to appressed; indusia absent. Distribution and ecology.—Endemic to Rio de Janeiro, Brazil; 1500-1750 m. ADDITIONAL SPECIMENS ExAMINED.—BRAZIL. Rio de Janeiro: Teresépolis, Rio Roncador, [22°24’44"S, 42°42'56”W], 1750 m, 3 Nov 1959, Brade 9851 (BM, GH, NY, US); Teresdpolis, Parque Nacional da Serra dos Orgaios, 22°26’56"S; 42°59’06"W, 1700 m, 13 Jan 2008, Labiak et al. 4483, 4502 (NY, SP, UPCB). 26 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Fic. 5. Basal pinna dissection in six species of Megalastrum from Brazil. A. M. abundans (Jiirgens 195, NY). B. M. canescens (Mori & Santos 11703, NY). C. M. indusiatum (Mori & Benton 12991, D. M. crenulans (Handro 2224, US). E. M. grande (Labiak et al. 4047, NY). F. M. inaequale (Luetzelburg 6911, NY). MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 27 Fic. 6. Basal pinna dissection in six species of Megalastrum sip Brazil. A. M. umbrinum (Matos & Gomes 1196, NY). B. M. retrorsum (Brade 12712, Yi t: substrigosum (Labiak et al. gee NY). D. M. connexum (Matos et al. 1104, NY). E. M. ee (Labiak et al. 4000, NY). M. adenopteris (Venturi 886, US). 28 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) . Proximal pinnae dissection in six species of Meqilasiran from Brazil and eeepc A. M. usc (Labiak et al. 841, NY). B. M. oreocharis (Lindman 985, K). C. M. eugenii (Matos 9 D. M. littorale (Labiak et al. 4378, NY). E. M. organense (Labiak et al. 4485, NY). F. M. romana Hassler 10802, B) _— MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 29 Megalastrum organense is named for the Organ Mountains of Rio de Janeiro. Like M. retrorsum, it has retrorsely denticulate scales (Fig. 8W). See comments under that species for comparison. 15. Megalastrum retrorsum R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.— BRAZIL. Rio de Janeiro: Itatiaia, Rio Bonito, [23°10'15”"S, 44°50’08’"W], 900m, Sep 1933, Brade 12712 (holotype: NY; isotypes: MO, RB). Figs. 4A, 6B, 8P-S. A M. organensi Jaminis utrinque inter venulas dense et aequaliter pubescentibus differt. Leaves to 1.5 m long [estimate]; scales of the petiole bases ca. 1.5 X 0.2 cm, narrowly lanceolate, retrorsely denticulate, brown, flat (not twisted), en masse not forming a woolly tuft; Jaminae to 1 m long, to 3-pinnate-pinnatifid at base, 2-pinnate-pinnatifid medially, dull on both surfaces, paler abaxially; basal pinnae 30-40 cm long, stalks to 2 cm long, inequilateral, pinnules acroscopi- cally slightly reduced toward the pinna bases; pinna rachises abaxially non- glandular, puberulous, scaly, hairs 0.1-0.2 mm long, 2- or 3-celled, patent, scales ca. 5 mm long, narrowly lanceolate, brown, retrorsely denticulate, flat (not bullate), adaxially non-glandular, puberulent, hairs ca. 0.4—0.7 mm long, 3—5-celled, patent; costules abaxially non-glandular, puberulent, hairs ca. 0.1— 0.2 mm long, 1—3-celled, scaly, scales like those of pinna rachises but smaller, ca. 3 mm long, adaxially puberulent, hairs ca. 0.3-0.5 mm long, 3- or 4-celled; laminar tissue between veins abaxially non-glandular, densely to sparsely puberulent, hairs ca. 0.1, 1- or 2-celled, mixed with some inconspicuous, uniseriate, appressed, reddish scales, adaxially densely puberulent, hairs 0.2— 0.3 mm long, 1—-3-celled, erect; veins visible or obscure on both surfaces, non- glandular, abaxially densely puberulent and inconspicuously scaly, hairs ca. 0.1 mm long, 1- or 2-celled, adaxially densely puberulent, hairs ca. 0.3 mm, 1— 3-celled; Jamina margins sparsely puberulent, hairs ca. 0.1-0.2 mm long, 1- or 2-celled, substrigose; indusia absent. Distribution and ecology.—Endemic to Rio de Janeiro, Brazil: wet forests, 900-1450 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Rio de Janeiro: Parati, Parque Nacional da Serra da Bocaina, 23°10'15"S, 44°50’08”W, 1450 m, 7 Jan 2008, Labiak et al. 4352 (NY, SP, UPCB). Megalastrum retrorsum is named for its retrorsely denticulate scales (Fig. 8R), a character it shares with M. organense. It can be distinguished from M. organense by the abaxial surfaces of laminae densely and evenly puberulent between the veins (Fig. 8S), whereas M. organense is glabrous between the veins (Fig. 8V). 16. Megalastrum substrigosum R. C. Moran, J. Prado & Labiak, sp. nov. TYPE.—BRAZIL. Espirito Santo: Mun. Castelo, Parque Estadual do 30 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) C ase vi Pat Fic. 8. Indument of four species of Megalastrum. A-H. M. abundans (Annies 117, MICH). A. ent apex detail. C. Hairs from adaxial surface of costule. D. > EE — iz) c Ry Oo o ° a) ae] = D i=) a es) pi llate scal indusiatum (Mori & Benton 12991, NY). J. Adaxial surface of pinnule. K. Scale from pinnae rachis. MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 31 Forno Grande, afloramentos rochosos, com matas timidas nos vales, 20°31'16'S, 41°05'50’W, 1300 m, 18 Jul 2007, Labiak et al. 4222 (holotype: UPCB; isotype: NY). Figs. 3D, 6C, 9C, D. A M. inaequali Jaminis crassis non lucidis, utrinque inter venulas puberulis, ad marginem ciliatis differt. Leaves to 1 m long [estimate]; scales of the petiole bases ca. 1.5 X 0.07— 0.08 cm, linear, denticulate, light brown to yellowish, flat (not twisted), en masse forming a wool-like tuft; laminae 0.5 m long [our estimate], to 3- pinnate-pinnatifid at base, 2-pinnate-pinnatifid medially, drying light green on both surfaces; basal pinnae to 35 cm long, stalks to 2.cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward pinna bases; pinna rachises abaxially non-glandular, pubescent, scaly, hairs 0.2-0.5 mm long, 1-3-celled, scales ca. 1.3-2 mm long, narrowly lanceolate, brown, sparsely denticulate, flat (non-bullate), adaxially non-glandular, densely pubescent, hairs ca. 0.4—0.5 mm long, 2—4-celled, strigose; costules abaxially non-glandular, pubescent, scaly, hairs ca. 0.2—0.4 mm long, 2—3-celled, scales like those of the pinna rachises, also with reduced, uniseriate, reddish appressed scales, adaxially densely pubescent, not scaly, hairs 0.3-0.4 mm long, 2—4-celled; laminar tissue between veins abaxially non-glandular, sparsely puberulent, very sparsely scaly, hairs ca. 0.2 mm long, 1- or 2-celled, spreading, scales ca. 0.3 mm long, uniseriate, linear, appressed, adaxially appearing glabrous but actually very sparsely pubescent, hairs ca. 0.2 mm long, substrigose, whitish; veins visible on both surfaces, non-glandular, abaxially sparsely pubescent and inconspicuously scaly, hairs 0.2-0.3 mm long, 1- or 2-celled, erect, scales 0.3—0.4 mm long, uniseriate, appressed, reddish, adaxially nearly glabrous to pubescent, hairs ca. 0.2—0.3(-0.5 mm, 1— 3-celled, substrigose, scales ca. 0.3-0.4 mm long, uniseriate, appressed, reddish; Jamina margins ciliate, hairs ca. 0.2 mm long, 1- or 2-celled, substrigose; indusia absent. Distribution and ecology.—Endemic to Espirito Santo, known only from the type; 1300 m. Megalastrum substrigosum can be recognized by substrigose hairs (thus the meaning of the specific epithet) on the abaxial surfaces of the pinna rachises, costules, and veins (Fig. 9D). Also characteristic are the puberulent abaxial lamina surfaces between the veins (Fig. 9D). The substrigose hairs along the _ Abaxial surface of pinnule. T—-W. M. organense (Labiak et al. 4483, NY). T. Adaxial surface of innule. U. Hair from adaxial surface of lamina. V. Abaxial surface of pinnule. W. Scale from abaxial surface of pinnae rachis. Scale bars = 1 mm. a2 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) Fic. 9. Indument of four species of Megalastrum. A-B. M. inaequale (Pereira 353, MO). A. Adaxial surface of pinnule. B. Abaxial surface of pinnule. C—D. M. substrigosum (Labiak et al. 4222, NY). C. Adaxial surface of pinnule. D. Abaxial surface of pinnule. E-H. M. albidum (Dusén 14693, S). E. Adaxial surface of pinnule. F. Detail of scale. G. Abaxial surface of pinnule. H. Hair from pinna rachis. J—M. M. grande (Labiak et al. 4047, NY). J. Abaxial surface of pinnule. K. Scale from pinna rachis. L, M. Adaxial surface of pinnule. Scale bars = 1 mm MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 33 axes are exactly like those of M. inaequale. That species, however, differs by laminae glabrous between the veins on both surfaces, thick shiny laminae, and glabrous to sparsely ciliate lamina margins (Fig. 9A, B). 17. Megalastrum umbrinum (C. Chr.) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 129. 1987. Dryopteris umbrina C. Chr., Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. Afd., ser. 8, 6: 81. 1920. Ctenitis umbrina (C. Chr.) Ching, Sunyatsenia 5: 250. 1940. LECTOTYPE (here designated).—Brazil. Sao Paulo: Cantareira [ca. 23°20'S, 46°41'W], Jun 1913, Brade 6532 (NY-00149429; duplicates: BM-n.v., R-n.v.: photo MICH ex BM). Figs. 4B, 6A, 10A-F. Leaves to 1.5 m long; scales of the petiole bases 1.5—2 X 0.07-0.1 cm, linear, sparsely denticulate (nearly entire), brown; laminae ca. 1 m long, 3-pinnate- pinnatisect at base, 2-pinnate-pinnatisect medially; basal pinnae ca. 30 cm long, strongly inequilateral, pinnules acroscopically reduced toward pinna ases; pinna rachises abaxially glandular, pubescent, scaly, glands ca. 0.05 mm long, sessile, yellowish, hairs of two sizes, from 0.1—-0.4 mm long, 2—6-celled, acicular, scales to 3.5 mm long, lanceolate, non-bullate, entire to subentire, brown, spreading, adaxially inconspicuously glandular, pubescent, glands like those abaxially, hairs 0.4-0.7 mm long, 2- to 5-celled; costules abaxially with indument like that of the pinna rachises, adaxially pubescent, not scaly, hairs 0.4-0.7 mm long, 2-5-celled, erect; laminar tissue between veins abaxially sparsely glandular, not scaly, hairs ca. 0.1-0.2 mm long, 1- or 2- celled, erect to (rarely) appressed, inconspicuous, adaxially glabrous or rarely with a few hairs near margins; veins visible on both surfaces, pubescent on both surfaces, abaxially hairs 0.2-0.3 mm long, 1- or 2-celled, adaxially the hairs denser, 0.1-0.5 mm long, 2-5-celled, spreading to appressed; Jamina margins ciliate, non-glandular, hairs 0.2-0.3 mm ong, 2- or 3-celled, appressed; indusia absent. Distribution and ecology.—Southeastern Brazil, Paraguay; 100-1200 m. ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Bahia: Camacan: RPPN Serra Bonita, 9.7 km de Camacan, estrada para Jacareci, dai 6 km SW na estrada para a RPPN e torre, 15°23’S, 39°33’W, 835 m, Matos 1076 (UPCB). Minas Gerais: Caldas Novas, [17°44’S, 48°37'W], Dec 1854, Lindberg 547 (K): Juiz de Fora, Poco Danta, [21°45'S; 43°21’W], [700] m, Jul 1902, Schwacke 14955 (P). Parana: Morretes, Estrada da graciosa, Grota Funda, [25°14’S, 48°48'’W], 19 Oct 1963, Hatschbach 10745 (HB); Antonina, Reseva Natural do Rio Cachoeira - SPVS, Trilha do meio, Floresta Ombréfila Densa, 25°18’S, 48°48'W, 200 m, 28 May 2006, Matos & Gomes 1193 (NY, UPCB). Rio de Janeiro: Serra do Itatiaia, [22°29'S; 44°33’W], 900 m, 16 Jun 1930, Brade 10056 (BM, C); Parati, Parque Nacional da Serra da Bocaina, 23°11'22"S, 44°50'15”W, 1200 m, 7 Jan 2008, Labiak et al. 4371 (NY, SP, UPCB). Santa Catarina: Hansa, Blumenau, [26°56’S; 49°03’W], 1906, Liiderwaldt s.n. (S, US); Joinville, [26°19'S, 48°50'W], 4 Jan 1906, Miiller 162 (UC, S). Sao Paulo: Sado José do Barreiro, oa ~ tp at mops i as ee re :é Sak * ‘ bP me.” ange ee tf Ie, a air Filices Anaprohenstliesies no. 207], S). G. t. J. midrib o GoD. A “a ty he aes Ai c OLA) - ae AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) (faba retin tt paket Peg H Rel 4 X rs ‘ SEN 4. tas as ¥ Ines Hee * ‘at A % a + eb ei il Abaxial aes of pinnule. K. Scale from abaxial surface of costule. MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 35 Estrada para o Parque Nacional da Serra da Bocaina, Rodovia SP 221, 1000 m, 6 Jan 2008, Labiak et al. 4316 (NY, SP). PARAGUAY. Amambay: In altapanitie et declivibus, Sierra de Amambay, [22°56’S, 55°55’W], 1907-1908, Hassler 10421 (BM, K, MICH). Megalastrum umbrinum is distinctive by small but conspicuous, spreading scales along the rachises and pinna rachises—a characteristic that will help distinguish this species from many others in Brazil (Fig. 10E). Also helpful are the abaxial surfaces of the axes that are densely puberulent with short glandular hairs, and acicular (non-glandular) hairs of mixed sizes (Fig. 10E). The most similar species is Megalastrum adenopteris, which differs by the adaxial surfaces between the veins densely and evenly pubescent (Fig. 10G). It also has minute fugacious indusia. 18. Megalastrum wacketii (Rosenst. ex C. Chr.) A. R. Sm. & R. C. Moran, Amer. Fern J. 77: 129. 1987. Dryopteris wacketii Rosenst. ex C. Chr., Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. Afd., ser. 8, 6: 84. 1920. Ctenitis wacketii (Rosenst. ex C. Chr.) Ching, Sunyatsenia 5: 250. 1940. TYPE.—Brazil. Sao Paulo: Pilar, Wacket 223 (holotype: BM). Fig. 4A, 6E, 12G-O. Leaves to 1.5 m long [estimate]; scales of the petiole bases ca. 2 X 0.3— 0.35 cm, narrowly lanceolate to linear, sparsely denticulate, brown, flat (not twisted), en masse not forming a woolly tuft; Jaminae 1 m long [our estimate], to 3-pinnate-pinnatisect at base, 2-pinnate-pinnatifid medially; basal pinnae to 35 cm long, stalks to 1 cm long, strongly inequilateral, pinnules acroscopically slightly reduced toward pinna bases; pinna rachises abaxially non-glandular, moderately to densely pubescent, very sparsely scaly, hairs 0.3-1.0 mm long, 2-7-celled, scales 0.5—1 mm long, narrowly lanceolate to linear, brown, entire to sparsely denticulate, flat (not bullate) adaxially non-glandular, densely pubescent, hairs ca. 1 mm long, 5- or 6-celled, patent, not strigose; costules abaxially non-glandular, pubescent, hairs generally of two lengths, long ones ca. 0.8-1.0 mm long, 4- or 5-celled, and shorter ones ca. 0.2 mm long, 1- or 2- celled, scaly, scales like those of pinna rachises, adaxially non-glandular, pubescent, hairs of two sizes, longer ones ca. 1 mm, 3- or 4-celled, and shorter ones ca. 0.3mm, 1- or 2-celled; Jaminar tissue between veins abaxially glandular, densely pubescent, and sparsely scaly, glands 0.1-0.2 mm long, 2- Gun L. Detail of costule showing hairs and glands. M. Hair from pinna rachis. N. Hairs and stalked glands on rachis. O. Glandular hair. P-AA. M. crenulans (Jiirgens 206, UC). P. Adaxial surface of pinnule. Q. Hair from adaxial surface of vein. R. Detail of pi his showing hai d glandul hairs. S. Glandular hair. T. Hair from adaxial surface of pinna rachis. U. Abaxial surface of pinnule. V. Hair from laminar tissue between veins. W. Pinna rachis with hairs and glands. X. Hair. Y. Glandular hair. Z. Sorus showing indusium with glandular hairs. AA. Bullate scale. Scale bars = 1 mm. AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) eeramat W. Aufnte~ Fic. 11. Indument of four species of Megalastrum. A-E. M. oreocharis (Lindman A869, S). A. Adaxial surface of pinnule. B. Scale from pinna rachis. C. Hairs on abaxial surface of pinna rachis, costule, and veins. D. Hair from pinna rachis. E. Abaxial surface of pinnule. F—-K. M. brevipubens (Salino 1987, AAU). F. Adaxial surface of pinnule. G. Scale from pinna rachis. H. Hairs on abaxial surface of lamina. J. Abaxial surface of pinnule. K. Detail of indument. L—-P. M. connexum (Rohr MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 37 celled, erect, capitate, hairs ca. 0.2 mm long, 1-celled, erect, scales ca. 0.3 mm long, uniseriate, linear, appressed, reddish, inconspicuous, adaxially sparsely pubescent, hairs 0.2-0.3 mm long, 1- or 2-celled, spreading, sparsely scaly, scales ca. 0.2-0.4 mm long, uniseriate, appressed, light reddish, surfaces shiny and darker than abaxial surfaces; veins visible on both surfaces, non-glandular, pubescent and scaly, hairs of two sizes, longer ones ca. 0.6 mm, 1-3-celled, and shorter ones ca. 0.2 mm, 1- or 2-celled, adaxially densely pubescent, hairs 0.6—0.7 mm long, 3—4(—5) celled, smaller ones ca. 0.2 mm long, 1- or 2-celled; lamina margins ciliate, non-glandular, hairs ca. 0.3 mm long, 1- or 2-celled; indusia absent. Distribution and ecology.—SE Brazil (Espirito Santo and Sao Paulo; 500— 1000 m ADDITIONAL SPECIMENS EXAMINED.—BRAZIL. Espirito Santo: Jatiboca, [19°52’15’S, 40°40'15"W], 10 May 1946, Brade & Apparicio 18071 (BM, GH, NY, U); Santa Teresa, Localidade de Julido, Floresta de encosta Semidecidua, abaixo de inselberg, 19°44'50"S, 40°40'16”W, 500 m, 10 Jul 2007, Labiak et al. 4000, 4002 (NY, UPCB). Sao Paulo: Bosque da Satide, [23°32’S, 46°38’W], 11 Jan 1914, Brade 6635 (S). Megalastrum wacketii has laminae that dry dark greenish or blackish. The short, capitate-glandular hairs are distinctive on the laminar surfaces abaxially (Fig. 12K—O). It most resembles M. abundans by the division of the laminae and the short (ca. 0.1 mm long) hairs on the laminar surfaces between the veins, but the latter species lacks glandular hairs on the abaxial surfaces of the laminae and has conspicuous bullate scales on the costae abaxially (Fig. 8A—H). ACKNOWLEDGMENTS Alison Paul (BM) for assistance with information about type specimens, Alejandra Vasco (NY) for help generating the lists of specimens examined and index to collectors’ names and numbers, and Hannah Stevens (GIS lab at the New York Botanical Garden) for help with the distribution maps. The drawings of indument were made by Mr. Haruto Fukuda. We also thank an anonymous reviewer for careful comments on the manuscript. ec 1071, US). L. Adaxial surface of pinnule. M. Detail of hai adaxial surface. N. Abaxial surface of innule. O. Scale from costule. P. Detail of costule showing scale and hairs. Q-V. M. eugenii (Labiak et al. 3678, NY). Scale bars = 1 mm. 38 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) sy ~ aay > a2) ~~ se T H i Fic. 12. Indument on four species of Megalastrum. A—F. M. canescens (Brade 7713, US). A. Adaxial surface of pinnules. B. Scale from rachis. C. Adaxial surface of pinna rachis showing strigose hairs and stalked glandular hairs. D. Abaxial surface of pinnule. E. Pinna rachis showing long hairs and stalked glandular hairs. F. Glandular hair. G-O. M. wacketii (Labiak et al. 4002, NY). G. Adaxial surface of pinnule. H. Hair from vein. K. Abaxial surface of pinnules. L. Glandular hair. MORAN ET AL.: MEGALASTRUM IN BRAZIL, PARAGUAY, AND URUGUAY 39 LITERATURE CITED BRADE, oA ? 1972. O género ‘‘Dryopteris’’ (Pteridophyta) no Brasil e sua divisao taxonémica. Bradea 1:191—261. Ss c. - A monograph of the genus Dryopteris, Part I. The tropical American pinnatifid- PH species. Kongel. Danske Vidensk. Selsk. Skr., Naturvidensk. Math. Afd., ser. 7 10:55-282. CHRISTENSEN, C. 1920. A monograph of the genus Dryopteris, Part II. The tropical American fn sien — [eani-Aemerey [eini-Aqieou = ueqin = ayRuIey = OPM salioseyeo jusUIESeURYy dnos a8y UISIIO [eInd-ueqiE) Jepuas) ‘dnois o8e pue ‘urst10 ‘1apues 0} 8utpio90e soli080}e9 JUSUIESeURUT UO SJUPWIOJUT JO UOT]NGLYSIG ‘% ATAV I, 5Z AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) # informants >70 % informants 0% 20% 40% 60% 80% 100% Fic. 4. Informant’s distribution by sex, age group, and origin among ‘non collector-consumers’. Urb, Nea, Far: see Fig. 3 sellers’ suggests that ethnobotanical knowledge of antojil is not threatened. Our finding meshes with previous studies in the region that describe the marketing of other medicinal plants (i.e., Chamaemelum nobile (L) All. and Sideritis hyssopifolia L.) locally preferred to pharmaceutical medicines (Pardo- de-Santayana, 2004). The results also agree with research among indigenous populations that shows that market economy is not necessarily linked to the loss of local knowledge of wild plants (e.g., Zarger and Stepp, 2004; Reyes- Garcia et al., 2007). We found that most collectors were people over 50 years of age who lived in remote villages in direct contact with the resource (Fig. 5). Besides collectors, only four informants had the knowledge for harvesting the rhizome and preparing antojil wine. Our data also highlight that most sellers were young men and women living in towns and cities who lacked ecological knowledge of antojil (Fig. 7). Consumers were from urban and nearby-rural origins and generally also lacked the ecological knowledge of the plant. Our data suggest # informants <40 41-50 S100 61-70 535 Age group % informants 0% 20% 40% 60% 80% 100% Fic. 5. Informant’s distribution by sex, age group, and origin among ‘collector-consumers’. Urb, Nea, Far: see Fig. MOLINA ET AL.: OSMUNDA REGALIS IN NORTHERN SPAIN 53 a bo] ts z 12 # informan <40 41-50 51-60 61.79 Age grou >70 gender origin % informants 9% 20% 40% 60% 80% 100% Fic. 6. Informant’s distribution by sex, age group, and origin among ‘collector-sellers’. Urb, Nea, Far: see Fig. 3. that urban consumption of antojil might increase over the next decades due to the development of a wide urban customer network and to the presence of an aging population. The dissociation between the consumption of a remedy ade Irom an unprotected wild plant and the ecological knowledge of the plant might have important implications for the conservation of the species. Unaware of harvesting practices, urban consumers and sellers do not understand the risk of overexploitation associated with the rising demand. We found that all collectors expressed concern about the effects of destructive harvesting generated by a rising demand. Moreover, three ‘collector consumers’ expressed concern over the fact that some collectors and sellers are only driven by economic incentives and do not care about the sustainability of the resource. Informants perceive economic interests as a risk for antojil’s conservation and for the quality of the marketed medicinal products because some traders are diluting antojil wine which might destroy the efficacy of the # informants <40 41-50 51-60 61-79 = grou % informants 9% 20% 40% 60% 80% 100% Fic. 7. Informant’s distribution by sex, age group, and origin among ‘non collector-sellers’. Urb, Nea, Far: see Fig. 54 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) remedy. The concerns expressed by informants are similar to those reported in other case studies, when the commercialization of a medicinal plant has lead to its overexploitation (Botha et al., 2004; Pardo-de-Santayana et al., 2005). Results from this research suggest that local ecological knowledge and practices are still alive in rural areas of developed countries, and that local harvesters are interested in the sustainable use of wild resources. This offers an opportunity to design management programs where local people participate actively encouraging the acceptance and internalization of environmental norms (Pardo-de-Santayana and Morales 2001). ACKNOWLEDGMENTS We are eens to all the people who kindly shared their knowledge and time. We thank J. Tardio, R. Morales, S. Gonzdlez, L. Aceituno and two anonymous reviewers for revising and improving the manuscript. LITERATURE CITED fi J. B. 1993. tig cng peoples and conservation. Conserv. Biol. 7:424—426. TIN, D. F., ed. 2004. Florida Ethnobotany. CRC Press, Florida. eit F. ge Sacpaian community-based conservation. Conserv. Biol. 18:621—630 BERNARD, H. R. 2006. Research methods in Anthropology. Qualitative and quantitative approaches. Fourth edition. Altamira Press, Walnut Creek. Boom, B. M. 1985. Ethnopteridology of the Chacobo Indians in Amazonian Bolivia. Amer. Fern J. 75:19—21. Bora, J., E. T. E. hhirciasome G. Sys heoemenaiees = ne hin K. i slownaieags 2004. Socio-economic differentiation in Lowveld, South Africa: 8 esos for resource management initiatives. Int. J. Sust. Dev. World Ecol. 11:280—29 Cuane, H. C., o l Huanc, D. C. Acrawat, C. L. Kuo, C. R. Wu and H. S. Tsay. 2007. Antioxidant pohcebacg ~ polyphenol contents of six folk medicinal ferns used as ‘‘Gusuibu”’. Bot. Stud. 48:3 CULPEPER, see (1995). sy ond s complete herbal. Wordsworth, Ware, Hertfordshire. ih . OrtEGA. 2004. Especies vegetales protegidas en Espafia: plantas vasculares. Suis de Medio pero Madrid. ESCOP (European SCIENTIFIC COOPERATIVE ON PHYTOTHERAPY), eds. 2003. ESCOP Monographs. The scientific foundation for herbal medicinal products. ESCOP, Exeter. Thieme, Stuttgart, New York. GosiERNO DE CANTABRIA. 2005. Plan Forestal de Cantabria. Plan vanes Regional sobre el Medio Natural. Documento operativo. Direccién General de Montes y Conservacién de la Natu- raleza. Available from http://www.dgmontes. orp/acrobat/ PFC. PARLAMENTO_completo.pdf (accessed April 2008) Lancz, D. 1998. Europe’s medicinal and aromatic plants: their use, trade and conservation. Traffic International, Cambridge, United Kingdo Larsen, H. O. and C. ~ OLSEN. apgr. Unsustainable collection and unfair trade? Uncovering and malayan medicinal plant conservation. Biodivers. Conserv. 16:1679-1697. LoriENnTE, E. 1999. Ecologia y corologia ke las plantas espontdneas de Cantabria I (Pteridophyta- Gimnospermae). Tantin, Santande Macta, M. J. 2004. A comparison of siete ose tp de ote two Amerindian groups from Amazonian Bolivia and Ecuador. Amer. Fern J. 94 MOLINA ET AL.: OSMUNDA REGALIS IN NORTHERN SPAIN 55 oe M. 2006. Conocimiento y manejo de Osmunda regalis L. en la medicina popular de antabria. abe ia sis. Department of Biology. Meagher cuss Auténoma de Madrid, Madrid. ae He, » P. O. Lyver and M. Kistatiociu. 2004. Combining science and traditional ancl knowledge: monitoring populations oe co-managem ment. Ecol. Soc. 9(3):2. Available from htt t2/ (accessed April 2008). Pace, C. N. 1996. The fons af Britain and Ireland. 2nd edition. Cambridge, University Press, Cambridge, era eh PARDO-DE-SANTAYANA, M. Cole de las plantas medicinales de Cantabria. Salud y tradicién popular. Estvdio, iO PARDO-DE-SANTAYANA, M. and R. Moraes. 2001. Patrimonio natural, usos tradicionales y conservacion. Quercus 189:64—-65. PARDO-DE-SANTAYANA, M. and R. Morates. 2005. Fi itoterapia popular. Plantas medicinales de uso tradicional en la Peninsula Ibérica. Rev. Fitoterapia 5(1):227—228. PARDO-DE-SANTAYANA, M., J. TaRDio and R. Moraes. 2005. The gathering sa Se nie of wild edible a in a (Cantabria, Spain). Int. J. Food Sci. Nutr. 56:529-542. Reyes-Garcia, V., V. Vapez, T. Huanca, W. R. Leonarp and T. McDane, 2007. peas development and oa colagcl knowledge: A deadlock? Quantitative research from a native Amazonian ane ar col. 35:371-377. Ricat, M., A. Bonet, S. ae » T. Garnatye and J. Vatiés. 2007. Studies on pharmaceutical a. in the high he Ter valley (Pyrenees, Catalonia, Iberian Peninsula). J. Ethnopharmacol. 113:267—27 Puabe. B. and S. CaANiGuERAL, is 2003. Fitoterapia, vademécum de prescripcion. 4th edition. evier - Masson, Barcelon ZarcgR, R. and J. R. Stepp. 2004. Persistence of botanical knowledge among Tzeltal Maya children. Curr. Anthropol. 45:413—-4 American Fern Journal 99(1):56—58 (2009) SHORTER NOTES Salvinia molesta in Mexico.—The genus Salvinia Ség. comprises ten species of mostly tropical ferns that are floating aquatics or less commonly stranded on receding shorelines. Among these, perhaps the best known is S. molesta D.S. Mitchell (Kariba weed, giant salvinia, giant water spangles), which is notorious as an extremely aggressive invasive exotic in both the New and Old Worlds. This species is extremely fast growing and has the capacity to cover the surface of even large bodies of standing and slow-moving water, forming so dense a continuous mat that oxygen exchange is inhibited and light passage is precluded, to the detriment of other aquatic organisms. Because it is a sterile pentaploid (n=45; Loyal and Grewal, Cytologia 31: 330-338. 1966), S. molesta reproduces only vegetatively by fragmentation and regrowth; thus humans, waterfowl, and surface drainage are the main dispersal agents. The taxon first came to the world’s attention in the 1930s, when plants inadvertently released into a lake in Sri Lanka quickly grew into a major infestation. Subsequently, it caused similar problems in portions of Australia, India, southeastern Asia, and Africa (Moran, Fiddlehead Forum 19[4+5]: 26—28. 1992). It was not until 1972 that the taxon was correctly determined to represent an unnamed species and was described as new to science, based on plants infesting Lake Kariba, along the border between Zambia and Zimbabwe (Mitchell, Brit. Fern Gaz. 10: 251— 252. 1972). In the United States, S. molesta and its relatives are considered noxious weeds under the U.S. Department of Agriculture’s Animal and Plant Health Inspection Service (USDA-APHIS) and thus are prohibited by law from international import or interstate shipment. Salvinia molesta is a member of the S. auriculata Aubl. complex, which consists of four taxa, all native to South America and Trinidad (Forno, Aquatic Bot. 17: 71-83. 1983). All of these taxa (but in particular S. molesta) are considered potentially severe aquatic weeds outside of their native ranges. Forno and Harley (Aquatic Bot. 6: 185-187. 1979) were the first to discover native populations of S. molesta growing at relatively low elevations in temperate southeastern Brazil. Curiously, although the species has proliferated in the Old World tropics, it apparently has not spread significantly thus far in the neotropics (Moran, Fl. Mesoamer. 1: 396-397. 1995). The introduction of S. molesta into temperate North America is very recent. Nauman (Flora of North America 2: 336-337. 1993) saw no wild-collected material, but mentioned that it was a candidate for future escape based on its cultivation in Florida. The species was first reported in the wild from a small pond in South Carolina (Johnson, Aquatics 17[4]: 22, 1985), soon thereafter from a reservoir in eastern Texas and adjacent Louisiana (Jacono, Sida 18: 927— 928. 1999), and subsequently from several other southeastern states (Jacono et al., Castanea 66: 214—226. 2001). At about the same time, infestations were first noted in agricultural canals in southeastern California, and subsequently in the Colorado River. Salvinia molesta was first reported as present on the SHORTER NOTES ny Arizona side of the Colorado River by Tellman (Plant Press [Tucson] 23[3]: 4, 14. 1999) and has been documented from both La Paz and Yuma Counties (Yatskievych and Windham, Canotia 4: 46649. 2008). In California, it was first reported by Jacono and Pitman (Aquatic Nuisance Species Digest 4: 13-16. 2001). Outside the Colorado River drainage and adjacent agricultural canals in Imperial and Riverside Counties, the Consortium of California Herbaria website (http://ucjeps.berkeley.edu/ tium) now records sporadic occur- rences west to San Diego County and northward to San Luis Obispo and Mendocino Counties (we have not verified these specimens). In the recent, comprehensive account of Mexican pteridophytes (Mickel and Smith, Mem. New York Bot. Gard. 88: 1B1054. 2004), only two species of Salvinia were treated (S. auriculata and S. minima Baker); S. molesta was not mentioned. We recently were made aware of the existence of an apparent 2002 collection from the Villahermosa area in Tabasco (C. Jacono, U.S. Geological Survey, pers. comm.), which may be the earliest documented infestation of the species in the country. Given its presence in the Lower Colorado River drainage of Arizona and California, it also is not surprising that S. molesta should eventually become established in adjacent portions of Mexico. Anecdotal reports place the time of its first discovery in northwestern Mexico during 2003. An online document issued by the Lower Colorado River Giant Salvinia Taskforce (http://www.|crsalvinia.org/ plisl ts/Mexi PRES% 20SM% 20BLYTHE% 2029-JUN-05.pdf) provided photographic evi- dence of the presence of the species in Baja California and maps its presence nearly throughout the Mexicali Water District. Until recently, we had not seen a voucher specimen in support of these reports, but our colleague, Richard Felger, kindly collected one at our request: MEXICO, Baja California, Mpio. Mexicali, ca. 0.3 km S of Presa Morelos, wetland with stagnant water pools and sandy-silty river soils, floating aquatic, forming 100% cover on some small, stagnant pools and common at edge of large pools, 32°42'16.0"N, 114°43'44.4"W Long., elev. ca. 108 ft, 16 Mar. 2006, R. S. Felger et al. 06-5 (BCMEX, MO, SD, UC). Despite all of the attention given to the presence of S. molesta in Baja California, the species apparently has not been reported officially from the state of Sonora. However, a recently located voucher specimen was collected by the eminent Mexican aquatic plant researcher, Alejandro Novelo R:: MEXICO, Sonora, Mpio. San Luis Rio Colorado, Canal de riego El Barrote, 16 km SO de San Luis Rio Colorado, vegetaci6n acuatica, herbacea, hidréfita libremente flotadora de 0.10 m, asociada a Myriophyllum y Potamogeton, 7 Oct. 2004, A. Novelo R. 4566 (MEXU). Water that enters Mexico in the Colorado River (much of it pumped from deep wells in the desert south of Yuma, Arizona) is impounded behind the Presa Morelos, forming a reservoir that provides water for irrigation of crop fields and other human uses through a complex series of ditches and canals. The stretch of the river from below the dam to its mouth at the Gulf of California has additional water diversions. Salvinia can be spread by water flow through the system, as well as by human activities and waterfowl that 58 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) utilize the water and transport pieces of plant in mud attached to their feet and feathers. It seems likely that in coming years the species will continue to become increasingly common in both northeastern Baja California and northwestern Sonora. The Lower Colorado River Giant Salvinia Taskforce has completed experimental releases of the biocontrol agent for the weed, a weevil native to southern Brazil called Cryptobagous salviniae Calder & Sands (Coleoptera: Curculionidae), which has been used successfully in some tropical countries of the Old World (Thomas and Room, Nature 320:581— 584. 1986; http://www.Icrsalvinia.org/weevil.htm). Some stands also have been sprayed seasonally with herbicides, but thus far these efforts have not succeeded in stopping the spread of S. molesta in the Lower Colorado River.— Arturo Mora-Ouvo, Instituto de Ecologia Aplicada, Universidad Aut6noma de Tamaulipas, 13 Blvd. Adolfo Lé6pez Mateos No. 928, 87040 Cd. Victoria, Tam., México, and GeorcE YaTskiEvycH, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166 U.S.A. Type Specimens of Dracoglossum sinuatum Uncovered in the Rio de Janeiro Herbarium.—During a recent survey of the A. L. A. Fée collections in the Jardim Botanico herbarium, Rio de Janeiro (RB), some specimens belonging to the newly published fern genus Dracoglossum Christenhusz (Thaiszia J. Bot. 17:1-10. 2007) were uncovered. These specimens are original material cited in the descriptions of Bathmium macrocarpon and B. sinuatum by Fée (Mém. Foug. 5. Gen. Filic.:288. 1852), but previously were not recognized as types. These specimens both belong to the same species: Dracoglossum sinuatum (Fée) Christenhusz. Bathmium macrocarpon Fée is an illegitimate name, as stated by Christenhusz (2007), but Morton (Amer. Fern J. 56:123. 1966) erroneously took the name as a legitimate basionym and thus unintentionally established a new name: Tectaria plantaginea (Jacq.) Maxon var. macrocarpa C.V.Morton, with Fée’s specimen (French Guiana, Cayenne, Poiteau s.n., anno 1825) as the type. Morton assumed that this specimen was in Paris, without consulting P, but the isotypes found there are originally from the herbarium of Caen (CN), and had nothing to do with Fée. The specimen in RB is from Fée’s herbarium and annotated by him and is therefore the holotype. The specimen of Bathmium sinuatum was cited by Fée (1852) as ‘‘Habitat in Guyana, Leprieur s.n. in Herb. Moug.’’. The Mougeot herbarium was deposited in the herbarium of Montpellier (MPU) but this specimen is not present here. Since the specimen was considered lost, Christenhusz (2007) designated a neotype. Nevertheless the specimen in RB is original material from the Fée collection and thus it is the holotype, superseding the designated neotype.— Maarten J. M. CuristeNHusz, Department of Biology, University of Turku, 20014 Turun yliopisto, Finland. American Fern Journal 99(1):59-60 (2009) REVIEW Illustrated Flora of Ferns and Fern Allies of South Pacific Islands, produced by the South Pacific Fern Studies Group of the Nippon Fernist Club, directed by Takehisa Nakamura and Sadamu Matsumoto. Published by the National Museum of Nature and Science as series No.8. Printed by Tokai University Press, 2008. Front matter xxxi pp., text 295 pp. Hard cover. Price: ¥ 8200 (about $80-$90 depending on exchange rate) ISBN 978-4-486-0179-2, 16 color plates; 110 black and white plates. Book 7.5 by 10.5 inches. http://www. press. tokai.ac.jp; tupcustomer@press.tokai,ac.jp; Fax: 048-432-0381. The South Pacific islands included in this generously illustrated book are New Caledonia, Vanuatu, Fiji, Samoa, and other nearby Pacific Islands. The book is useful to botanists, students, amateurs, naturalists, and environmental workers who are interested in the flora and nature of this area. The South Pacific Fern Studies Group of the Nippon Fernist Club produced this book under the leadership of Dr. Takehisa Nakamura, former President of the Nippon Fernist Club, and Dr. Sadamu Matsumoto of the Tsukuba Botanical Garden. The Nippon Fernist Club is comprised of botanists, knowledgeable fern enthusiasts and naturalists. The fern families and their genera are treated by various well-known Japanese fern botanists. Fortunately for those of us who do not read Japanese there are English translations throughout the book, though in some areas they are shortened versions from the Japanese text. The front matter includes maps of the area followed by colored photographs and comments on the geography of the major islands. The photographs show major island terrains and habits of some of their ferns. Following this section are several pages discussing the new classification of ferns adopted in the book. The classification system is mainly adapted from Smith et al. (2006), A classification for extant ferns. Taxon 55: 705-731. Most of the book consists of botanical keys reaching the species level and black and white illustrations. The fern and fern allies of the area are treated family by family with each family and its contents being authored by one or more pteridologist. The family entry is followed by a brief description of the family, its possible diversification pathway, number of species, and comments on its distribution in the area. A botanical key to the genera in the family follows. Each genus is very briefly discussed and followed by a botanical key to the species of the area. Each species is then listed with reference to its diagram and coded information on the specimens collected, studied, diagramed, and the institution where vouchers are deposited. Though one might wish for lengthier descriptions, one is grateful that this work is not a mere checklist without any descriptions, botanical keys, or illustrations. The botanical keys are well constructed, clear, short and easy to use. The botanical terms used in the keys are well known and do not require a very erudite botanist to decipher. Most of the species are illustrated by excellent line 60 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 1 (2009) drawings, which will serve as a great help in identifying the ferns. Many of the line drawings are selected to show the diagnostic features of the species and have scale bars as well. For English readers this book will be a valuable field guide to have when visiting these South Pacific Islands. Additionally the botanist who needs to work on ferns from this area will find careful documentation of specimens used in producing this work, a brief botanical history of the area and an extensive bibliography on South Pacific pteridophytes. As with most bilingual books, particularly where the written languages are very different, there are typographical and other errors. However, these are relatively small distractions considering the value of having such a fern treatment on this area. We are indebt to the organizers of this team effort, Dr. Takehisa Nakamura and Dr. Sadamu Matsumoto, in producing this book and making it available to the English reading public as well.—Barsara Joe Hosuizaki, 557 N. Westmoreland Ave., Los Angeles, CA 90004-2210. INFORMATION FOR AUTHORS Authors should adhere to the following guidelines; manuscripts not so prepared may be returned for revision prior to review. Electronic submission is st gly ged; how- ever if it is necessary to submit hard copy, please submit one copy of the manuscript and include a review copy of illustrations and originals of illustrations. After acceptance, please space manuscripts throughout, including title, author’s names and full addresses, a short, informative abstract, key words, text (including heads and keys), literature cited, tables (separate from text), and figure captions ( grouped as consecutive paragraphs separate from figures). Arrange parts of manuscript in order just given. Include author’s name and page number in the upper right corner of every sheet, and provide an abbreviated running title. Provide margins of at least one inch (25 mm) all around on typed pages. Do not submit right-justified text, avoid footnotes, and do not break words at end of lines. Make table headings and figure captions self-explanatory. Use S.I. (metric) units for all measures (e.g., distance, elevation, weight) unless quoted or cited from another source (e.g., specimen citations). For nomenclatural matters (i.e., synonymy and typification), use one paragraph per basionym (see Regnum Veg. 58:39-40. 1968). Abbreviate titles according to Botanico- Periodicum-Huntianum (Lawrence et al., 1968, Hunt Botanical Library, Pittsburgh) and its parenthetically in text. Use Index Herbariorum (Regnum Veg. 120:1-693. 1990; or http:// websun.nybg.org/bsci/ih/) for designations of herbaria. For more detailed instructions on manuscript preparation, see http://amerfernsoc.org/. Ff 1 fo aon £ d ling el ic f print ord Ss are sent to authors by the printer. Authors should send proof corrections of corrected proofs to the editor and reprint orders to the printer. Authors will t 1 charges for ive alterations made after type has been set. ; For other matters of form or style, consult recent issues of the American Fern Jour- nal and The Chicago Manual of Style, 14" ed. (1993, Univ. of Chicago Press, Chicago). Occasionally, departure from these guidelines may be justified. Authors are encouraged to . consult the editor for assistance with any aspect of manuscript preparation Papers of longer than 32 printed pages may t ¢ io the Editor of Preridoipoin ( Editor, see journal cover page 2). : o He eG PTERIDOLOGIA ISSUES IN PRINT The following issues of Preridologia, the memoir series of the American Fern Society, are available for purchase: 1. Wagner, David H. 1979. Systematics of Polystichum in Western North America North of Mexico. 64 pp. $10.00 plus postage and handling. : 2A. Lellinger, David B. 1989. The Ferns and Fern-allies of Costa Rica, Panama, and the Choc6 (Part 1: Psilotaceae through Dicksoniaceae). 364 pp. $32.00 plus postage and handling. 3. Lellinger, David B. 2002. A Modern Multilingual Glossary for Taxonomic Pteri- dology. 263 pp. $28.00 plus postage and handling. For orders and more information, please contact our authorized agent for sales at: Missouri Botanical Garden Press, P.O. Box 299, St. Louis, MO 63166-0299, tel. 314-577- 9534 or 877-271-1930 (toll free). For online orders, visit: http://www.mbgpress.org. AMERICAN FERN JOURNAL ON MICROFICHE Volumes 1-61 of the American Fern Journal are available as archival quality, silver positive microfiches. Single volumes or the entire run may be purchased. The fiches are easily read with 10x or greater magnification (using a dissecting microscope and transmit- ted illumination or a fiche reader). Silver negative microfiches of vols. 1-50 are also avail- able. The price is $4.00 per volume or $244.00 per set of 61 volumes, postpaid. Send your inquiry or order with a check or money order to: American Fern Society, Inc., % Ecology III, 804 Salem Blvd., Berwick, PA, 18603-9801. FIDDLEHEAD FORUM The editor of the Bulletin of the American Fern Society welcomes contributions from members and non-members, including miscellaneous notes, offers to exchange or purchase materials, personalia, horticultural notes, and reviews of non-technical books on ferns. SPORE EXCHANGE Ms. Denia Mandt, 12616 Ibbetson Ave., Downey, CA 90242-5050, is Director. Spores exchanged and lists of available spores sent on request. http://amerfernsoc.org/sporexy. html GIFTS AND BEQUESTS ft 4. tt tn tthe < ude ee le +t ft, Ait ambherc At therc interested in ferns. Back issues of the Journal and cash or other gifts are always welcomed and are tax-deductible. Inquiries should be addressed to the Membership Secretary. VISIT THE AMERICAN FERN SOCIETY’S WORLD WIDE WEB HOMEPAGE: http://amerfernsoc.org/ AMERICAN = FERN nae JOURNAL pe QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY AAAI 1 Peta fc. nr ees os Jak on Oe St ee Py, Wee. BO | fA Lb Ek, £7. o 7 ei a: v ipinna from Taiwan Ho-Ming Chang, Wen-Liang Chiou, and Jenn-Che Wang 61 Molecular Cloning and Sequence Analysis of Cyanovirin-N Homology Gene in Ceratop-teris thalictroides Xiaogiong Qi, Yongxia Yang, Yingjuan Su, and Ting Wang 78 A New Species of Adiantum from Cuba Manuel G. Caluff 93 New B ili Sp i f the G Asplenium e fA ] Fernando B. Matos, Paulo H. Labiak, and Lana S. Sylvestre 101 New nr 3s. 2 = DI Tes mD.!] 3; [e c ah . Brazil Al, rt; Cisks 106 A Hybrid Phlebodium (Polypodiaceae, Polypodiophyta) and Its Influence on the Circumscrip- ion of the Genus J. Daniel Tejero-Diez, John T. Mickel, and Alan R. Smith 109 2008 AFS Symposium Summary of the 2008 AFS Symposium: From Gels to Genomics: The Evolving Landscape of Pteridology. A Celebration of Gerald Gastony’s Contributions to Fern Evolutionary ology. Michael S. Barker and George Yatskievych 117 A Brief History of Gerald J. Gastony’s Botanical Career Michael S. Barker and George Yatskievych 117 Gels and Genetics: The Historical Impact of Isozymes on Paradigm Shifts in Hypotheses about Fern Evolutionary Biology Christopher H. Haufler 125 Using — and Nuclear DNA Sequences to Redraw Generic Boundaries and Demystify Spe- es Complexes in Cheilanthoid Ferns Michael D. Windham, Layne Huiet, Eric Schuettpelz, Amanda L. Grusz, Carl Rothfels, James Beck, George Yatskievych, and Kathleen M. Pryer 128 Di.) << 2 £T . ee Ohl ~ ‘Paul G. Wolf, Aaron M. Duffy, and Jessie M. Roper 132 Fern Genome Structure and Evolution Takuya Nakazato 134 Ss cr, Dp 1 sh uU:..k Ch AT 4. RE Enea 7 Evolutionary Genomic A Ir, Dp 5 PD. sa a ee Pee +. Anviosperms ; : Michael S. Barker = 136 Flora de Nicaragua. Tomo 4. Helechos Alan. Smith 142 —~ The American Fern Society Council for 2009 WARREN D. HAUK, Dept. of Biological Sciences, Denison University, Granville, OH 43023. President MICHAEL WINDHAM, Dept. of Biology, Duke University, P.O. Box 90338, Durham, NC 27708. President-Elect W. CARL TAYLOR, National Science Foundation, 4201 Wilson Blvd., Arlington, VA 22230. Secretary JAMES D. CAPONETTI, Div. of Biology, University of Tennessee, Knoxville, TN 37996-0830. Treasurer GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. M, bh pee JAMES D. MONTGOMERY, Ecology III, 804 Salem Blvd., Berwick, PA 18603-9801. : Curator of Publications JENNIFER M- O. GEIGER, Dept. of Natural Sciences, Carroll College, Helena, MT 59625 Journal Editor R. JAMES HICKEY, Dept. of Botany, Miami University, Oxford, OH 45056. Memoir Editor so N. E. HUDSON, nore f Biol i Sam H University, Huntsville, TX 77341-2116. AVID SCHWA 9715 Christey =a Bakersfield, CA 93312-5617. Bulletin Editors American Fern Journal EDITOR JENNIFER M. O. GEIGER Dept. of Natural Sciences, pete College, Helena, MT 59625, ph. (406) 44 1, e-mail: jgeiger@carroll.edu MANAGING EDITOR JILL ANNE DILI Dept. of Natural Sciences, Carroll College, Helena, MT 59625, ph. (406) 447-5176, e-mail: jdill @carroll.edu ASSOCIATE EDITORS GERALD J. GASTONY Dept. of Biology, Indiana University, Bloomington, IN 47405-6801 GARY K. GREER Biology Dept., Grand Valley State University, Allendale, MI 49401 -POPHER 71. HAUPLER ....::..... Dept. of Ecology and Evolutionary Biology, University of Kansas, wrence, KS 66045-2106 R. JAMES HICKEY Dept. of Botany, Miami University, Oxford, OH 45056 ROBBIN C. MORAN New York Botanic al Garden, Bronx, NY 10458-5126 JAMES E. WATKINS, JR. H y, Cambridge, MA 02138 The “American Fern Journal” (ISSN 0002-8444) is an — quarterly devote to the general oma of ferns. It is owned by the American Fern Society, and published at The American Fern Society, % Misso Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. hella postage eg at St. Louis, MO, a additional entry. L 7m for back ay should be addressed to Dr. James D. Rice en, Fale Ill, 804 Salem Blvd., same PA 18603-980 dunes 4 75 4 y = bh 34 kh 4 AA } | aed iw + it rr =: r Z Back volumes are avalible for most years as printed issues or on microfiche. Please contact the Back Issues Curator for prices and availability. Subscriptions: Society Membership USA, Canada, Mexico (includes Journal and Fiddlehead Forum) = Society Membership (includes Journal and Fiddlehead F _ itgaantnecrs pepe pvp iling harge for outside USA, Canada, Mexico) Saga Ueoeniag USA, Canada Mexico (includes Fiddlehead F Bi he Oe ee ae $19 Insti Rienioeel Membership — $35 to USA, Canada, Mexico; $45 elsewhere (-$2 agency fee) PosTMA hanges to American Fern Journal, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299. American Fern Journal 99(2):61—77 (2009) Molecular Evidence for Genetic Heterogeneity and the Hybrid Origin of Acrorumohra subreflexipinna from Taiwan Ho-Minc CHANG Department of Life Science, National Taiwan Normal feicarsets No.88, Ting-Chow Rd., Sec 4, 116, Tai Division of Botany, Endemic Species pater aie. No.1, Ming-Sheng E. Rd., Chi-Chi, Nantou 552, Taiwan Wen-LIANG Cutou Division of Forest Biology, Taiwan Forestry Research Institute No. 53, Nanhai Rd., Taipei 100, Taiwan JENN-CHE Wanc* Department of Life Science, National maedey Normal oe No.88, Ting-Chow Rd., Sec 4, aipei 116, Taiw: Asstract.—Acrorumohra subreflexipinna, an endemic fern of Taiwan, has been suspected to be a hybrid species. The aims of this study were to detect possible multiple origins of this species, determine the genetic variation in different populations, and clarify their lineages. One nuclear and three organellar DNA fragments were sequenced to determin and to examine genetic differentiation among populations. Sequenc ce data support ‘the conclusion that A. subreflexipinna arose from the hybridization of A. hasseltii and A. diffracta, and the hybridization was uni-directional, i.e., based on the assumption of maternal inheritance in organellar DNA, the former was its maternal species while the latter was its paternal source. A convincing coli Shecwraat is that the female gametes of A. hasseltii gametophyte could be fertilized by the male gametes from apogamous A. diffracta. Unique nuclear alleles present in different han A. hassel at sympatric sites. Our pi ts show that any phd variation of A. Gleb came its parents and that it maintains this significant genetic variability because of recurrent ph beta. Key Worps.—Acrorumohra diffracta, Acrorumohra hasseltii, Acrorumohra subreflexipinna, hybridization, monilophytes, multiple origins Hybridization followed by polyploidization is an important mechanism driving the formation of new lineages of ferns and other plants (Paun et al., 2007). By means of diploidization processes, such as chromosomal rearrange- ments, intergenome recombination, and gene silencing, the genomic constitu- tion of many extant taxa might be the outcome of ancient hybridization and polyploidy (Bowers et al., 2003; De Bodt et al., 2005; Haufler, 1987; Paun et al., 2007). Hybridization events often begin these cycles and high chromosomal base number in ferns was achieved as the result of repeated cycles of * corresponding author: e-mail: biofv017@ntnu.edu.tw 62 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) polyploidization (Haufler, 1987; Klekowski and Baker, 1966; Nakazato et al., 2006). Accessing the parentage of hybrids or allopolyploids is essential for understanding relationships within taxonomically complex groups. Although allozyme studies could provide tenable evidence to indicate the possible origin of hybrid-originated taxa, they have rarely been utilized to distinguish maternal lines from paternal ones. However, direct DNA evidence, such as nucleotide sequences and DNA fingerprints, can provide more informative insights into these evolutionary processes than enzymes. In most plants, organelle genomes are maternally inherited via female gametes while nuclear DNA is biparentally inherited (Soltis et al., 1992). Comparing organellar DNA of hybrid taxa and their possible parents therefore could reveal the maternal origin (Gastony and Yatskievich, 1992; Vogel et al., 1998) while comparing nuclear DNA of those taxa could show both putative parentages (Small et al., 2004). In addition, any evolutionary trace, theoretically, would be deposited in nucleotide sequences and could be detected by DNA-based molecular technology. Unique local variation would be detectable if applicable DNA markers were chosen (Soltis et al., 1992). DNA markers containing non-coding regions have been shown to be the best choice to reconstruct eperisnat of hybrid and parental populations (Small et al., 2004; Xiang et al., Studies of north temperate ferns have clearly indicated the -citeeiden of hybridization and polyploidization to fern evolution (Barrington et al,. 1989; Bennert et al., 2005; Pintér et al., 2002; Wagner, 1973; Werth et al., 1985). Out of the 420 species of lycophytes and ferns that grow in North America, nearly 20% are of hybrid origin (Flora of North America Editorial Committee, 1993), and reticulate networks and ploidy levels of most taxonomically complex groups have been well studied (Barrington, 1986; Stein and Barrington, 1990; Wagner, 1954, 1962, 1973; Xiang et al., 2000). However, only a few ferns from other regions have received taxonomic attention like those in Europe and North America (e.g., Barrington, 1990; Ebihara et al., 2005; Takamiya et al., 2001; Terada and Takamiya, 2006). Some hybrid ferns have been recorded from Taiwan (Holttum and Edwards, 1986; Kuo, 1988, 1990; Miyamoto and Nakamura, 1983), but until now, no direct evidence has been reported to test and verify their parentage. Acrorumohra is a small genus with about seven species distributed in Eastern and Southeastern Asia. This genus has an intermediate morphology between Dryopteris and Arachniodes; therefore, species of Acrorumohra were once treated in these genera. However, Acrorumohra was treated as an independent genus in the Flora of Taiwan (Shieh et al., 1994) and Flora Reipublicae Popularis Sinicae (Hsieh, 2000) based on the presence of the zigzag rachis and anadromous pinnules of pinnae. Acrorumohra subreflex- ipinna (M. Ogata) H. Ito, an endemic species of Taiwan, produces shriveled and abortive spores and has an intermediate morphology between A. hasseltii (Blume) Ching and A. diffracta (Baker) H. Ito. Given its morphological characteristics, A. subreflexipinna has been suspected as a hybrid of these two species (Moore, 2000). Moreover, the fact that A. subreflexipinna always grows CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA 63 Le Population site of A. subreflexipinna in Taiwan: * A: Mt. Kentuerhshan Ms teen Fic. 1. Distribution of Acrorumohra subreflexipi A. hasseltii, and A. diffracta, and collection sites in this study. sympatrically with the later two species reinforces the reasonable hypothesis of its hybrid origin. The narrowly defined genus ‘Acrorumohra’ was followed and the scientific name ‘A. subreflexipinna’ is used throughout the study, although palynological and unpublished breeding data indicates it a sterile F1 hybrid. In this study, chloroplast, mitochondria and nucleus DNA markers were used to identify the parentage of this suspected hybrid. Furthermore, the hypothesis that hybrid populations in Taiwan each originated independently was tested. In addition to haplotype comparison, genetic variation in different populations was determined to clarify lineage relationships. MATERIALS AND METHODS Plants of Acrorumohra subreflexipinna were sampled from three sites in Taiwan: Mt. Howeishan, Lake Chunglingchih and Mt. Kentuerhshan (Fig. 1). Leaf tissue of four to 11 individuals per population was collected for molecular analyses. Ten individuals of the two putative parent species, A. hasseltii and A. diffracta, were also sampled in each sympatric site (Table 1). Two plants of Dryopteris polita Rosenst. were also sampled to detect any possible parental relationship because based on phylogenetic analysis of a chloroplast trnS-rps4 data set, D. polita and A. hasseltii are sister species (Li and Lu, 2006). Two chloroplast intergenic spacers (trnL-trnF and trnS-rps4 IGS) and one mitochondrial intron (nad5 intron 2), which have been frequently used for AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) SLLZ6ZNA/SI-FL UoNUy 918d ILZZ6ZN4/SI-F1 Uonu jI8d 60ZZ6ZNA/SI-FL UoQut 718d 80L26ZNA/SI-FL uonU 918d 189462) A/qU-Tuy AIV.L/SZ99 Supyy AIV.L/PZ99 Sunyy AIV.L/€Z99 dupyy AIV.L/OFS9 Suvyy AIV.L/FFS9 supyy AIV.L/6ES9 Supyy ON.L/0ze9 supyy ON.L/6LE9 supyD aIV.L/Z999 Sunyy FIV.L/96s9 Ssupy’) ANL/9LEg supyy €¢/V 62/S ST/E ST/€ ST/E SL/€ OL/ (D) UeYstomoy “IW “se OL/ (@) YrysurpsunyD eye] *Z OL/ (V) UeYsyionjuey “WA ‘TL OL/ (9) UeYystamoyH “I ‘€ OL/ (a) YryosurpsunyD eye *z OL/(V) UeYysyIonjuey "WY TL IjassDYy DIYOUTNIOIOY pooiffip pryournso1gy ‘OU UOISsa00R yurgue/uolsel YNG Un eqiat paysodap/reyono, ‘ou/‘ou atdures pauoy’) saUuoyo jo ‘ou e[dures/(apoo uoyerndod) Aytye00"] uOXe |, ‘Apnys sty} Jo sisAyeue se[Noe[OUI UI pasn exe} JOJ siequINU UOIssaD0R YURGUAy pue o[dutes Jo AJQUeNb ‘uoyeUTIOJUT JaYONOA “| TIAVY, LEXIPINNA ? SZZZ6ZNA/SI-PL uoNu 718d P892Z6ZNA/qUG-Tuy aIV.L/e069 Sunyy OL/Z z/yrpoenyuery pyyod st1ajdoAiq $2ZL6ZN4/SI-F1 UoQut 718d 22LZ6£NA/SI-FL uo! DIsd AIV.L/2Z99 Suvyy 929262NA/AUay-Tuy] AIV.L/1Z99 8uvyy 16/11 11/{>) weustamony AW ‘c LIZLZ6ZNA/SI-FL out 718d aIV.L/ZFS9 Suny 02ZZ6ZNA/SI-FL UonUT 918d ONL/2ze9 Buvyy 869Z62N4/z UoNUI gppu 88926£Na/Psdi-guy puutdrxayfaiqns 84946204 /qUd}-TU] ONL/ZLe9 suvyD ZS/S c/(V) UeYysyrenquey “yA ‘T piyourniolgy ‘OU UOIssaD0R wintieqiey sauo[o Jo ‘ou a[dures/(apoo uOXB], yuegues/uodel VNC peysodapsiayono, = ‘ous‘ou aydures peuoy) uonejndod) Aytyeo0"] CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREF. ‘ponunuoy “T a1avy, 66 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) phylogenetic analysis at lower taxonomic levels, were employed to reveal maternal history, while introns of the single-copy nuclear gene pgiC (including introns 14 and 15, and exon 15) were used to observe bi-parental inheritance. These sequences were chosen because of their significant phylogenetic information relative to other fragments and the availability of usable primers (Ishikawa et al., 2002; Nadot et al., 1995; Smith and Cranfill, 2002; Vangerow et al., 1999). Dry or fresh tissues of young leaves were homogenized with liquid nitrogen. Genomic DNA was extracted from ca. 100 mg of leaf tissue by using a Plant Genomic DNA Mini Kit (Viogene, USA). The PCR amplification of all segments was performed in an ABI thermocycler (9700). Primers for trnL-trnF IGS, trnLF-11 5’- GGG CAA GTT GCG GTA GAA CGA-3’' and trnLF-12 5’-CTG CTC TAC CGA CTG AG CTA-3’, were modifications of those utilized by Taberlet et al. (1991). The primers tsr4-f/tsr4-r 5’-CCC GCA AAG CTT AGT GAT CA-3’/ 5’-CCG AGG GTT CGA ATC CCT C-3’, nadh2-f/nadh2-r 5’-GGG GCT ATA TCG CCA TCC-3’/5’-CCG CAC GTG CAA GTT TCC-3’, and pgiC-14fA/pgiC-16rA 5’- GTG CTT CTG GGT CTT TTG AG-3’/5'’-GTT GTC CAT TAG TTC CAG GT-3’ were developed for this study referring to Smith and Cranfill (2002), Vangerow et al. (1999) and Ishikawa et al. (2002), respectively. PCR reactions were carried out in 20 uL reactions containing 2 pL unstandardized template DNA, 0.2 mmol/L of each dNTP, 0.8 units of Taq polymerase (ABgene, USA) and 6.25 pmol each of the forward and reverse primers, and programmed for 5 min at 95°C, 35 cycles of 1 min at 95°C, 1 min at annealing temperature and 2 min at 72°C, followed by a 8 min extension at 72°C. The annealing temperature was 59°C in amplifying the chloroplast trnL-trnF IGS and the mitochondrial nad5 intron 2, and 52°C in amplifying the chloroplast trnS-rps4 fragment. When amplifying nuclear pgiC intron 14-15 segment, annealing was performed at 57°C for the first 3 cycles, at 55°C for the next 3 and at 54°C for the final 29. PCR products were directly sequenced, using one amplification primer, on an ABI 373A automated sequencer (Applied Biosystems, USA) with the Taq Dye Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems). For the electrophoresed bands with lengths greater than 500 bp, sequences were determined in both directions. Additionally, pgiC intron 14-15 segments of all A. subreflexipinna samples and 3-5 samples in each population of A. hasseltiiand A. diffracta were cloned. The PCR products of the nuclear segment were purified by electrophoresis using 1X TAE buffer on a 1.2% agarose gel. Electrophoresed bands were cut and eluted using the Gel-M gel extraction system (Viogene). Purified nuclear DNA was cloned with the yT&A cloning kit (Yeastern Biotech, Taiwan) following the manufacturer’s protocol. Five to eight colonies were chosen to perform colony PCR using TA-F forward and TA-R reverse primers (Yeastern Biotech). Purified nuclear DNA was sequenced with M13 universal and reverse primers which are located on the DH5« vector termination site. When any different haplotype was detected, repeated PCR reactions using a different Taq polymerase (Genomics, Taiwan) or using DNA from another three colonies were chosen to check whether it was a real variant or not. All sequences were deposited in the GenBank nucleotide CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA 67 sequence database, and accession numbers and their corresponding DNA regions are listed in Table 1. The sequences were aligned by BioEdit 7.0 and manual correction, and compared with nucleotide sequences available through GenBank to determine their boundaries of coding region. Haplotypes were named after the first letter of the specific epithet, and followed by a lowercase letter and number to designate different, minor haplotypes (those differing from their correspond- ing major haplotype at only one base) of A. hasseltii. Genetic diversity at population and species levels was estimated with the software package DNA Sequence Polymorphism (DnaSP 4.20.2, Rozas et al., 2003). The haplotype diversity (h) and nucleotide diversity (x) of these three populations were calculated separately and totally. Genetic differentiation (ys7, Nei, 1982) among these three populations and between pairs of populations was also calculated by this package. y,;, but not Fs; or Nsy, was used because the three sampled populations were the only ones of interest (Lynch and Crease, 1990). Because no variation was detected in the nuclear sequences of A. diffracta, only the A. hasseltii haplotypes cloned from A. subreflexipinna were used when analyzing genetic diversity and differentiation among the populations of A. subreflexipinna. Haplotypes of A. hasseltii and A. subreflexipinna were identified and coded by direct sequence comparison, and unrooted haplotype networks were constructed with the program TCS 1.21 (Clement et al., 2000). RESULTS Total aligned length and GC content of the sequences of nuclear pgiC intron 14-15, chloroplast trnL-trnF IGS and trnS-rps4 IGS, and mitochondrial nad5 intron 2 were 725 bp/37.8%, 268 bp/34.9%, 374 bp/36.5%, and 728 bp/ 52.6%, respectively. Low GC content of chloroplast segments agreed with the AT-rich property of most non-coding spacers (Graur and Li, 2000). In the chloroplast and mitochondria segments, all 50 individuals of A. subreflexipinna and A. hasseltii had the same nucleotide sequences but were different from those of A. diffracta and D. polita (Tables 2—4). In the nuclear pgiC intron 14-15 sequences, A. subreflexipinna possessed both the A. hasseltii and the A. diffracta haplotypes (Table 5) but not that of D. polita (data not shown). pgiC intron 14-15 sequences of A. diffracta from the three populations were all the same (haplotype ‘D’), but those of A. hasseltii and A. subreflexipinna in each population had two to three haplotypes (Tables 5 and 6). There were two major (Ha and Hb) and another three minor haplotypes (Ha1, Hb1 and Hb2) found in A. hasseltii (Table 5). These minor haplotypes differ from their corresponding major haplotypes at only one base, and were found in the three populations respectively. In total, five and four haplotypes were found in A. hasseltii and A. subreflexipinna, respectively. When calculating haplotype diversity (h) and nucleotide diversity (r) (Table 6), the haplotype ‘‘D” was, a priori, removed from the genetic pool of A. subreflexipinna to avoid interference in comparison with that of A. hasseltii. In A. hasseltii, haplotype diversity (h) among these three populations ranged 68 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) TaBLE 2. The variable nucleotide sites (indel & base substitution) of chloroplast trnL-trnF intergenic spacer sequences. Columns shaded are sites identical to the hybrid sequences. Variable sites Species A. diffracta A. subreflexipinna A. hasseltii D. polita from 0.533 to 0.689, and it was 0.724 at the species level. In A. subreflexipinna, haplotype diversity among populations ranged from 0.400 to 0.667, and it was 0.674 at the species level. Nucleotide diversity (x) among the three populations of A. hasseltii ranged from 0.00074 to 0.00446, and it was 0.00485 at the species level. In A. subreflexipinna, nucleotide diversity among populations ranged from 0.00055 to 0.00553, and it was 0.00466 at the species level. For the nuclear pgiC segment of A. hasseltii and A. subreflexipinna, the Ha haplotype could be clearly distinguished from Hb by six sites with different base pairs and two indel sites (Table 5; Fig. 2). The Ha haplotype has a minor type (Ha1) with a single base difference. This minor haplotype is found only in the Mt. Kentuerhshan population of A. hasseltii and A. subreflexipinna (Fig. 2). On the other hand, the Hb haplotype has two single base change minors (Hb1 and Hb2) occurring respectively in Lake Chunglingchih and Mt. Howeishan populations of A. hasseltii (Fig. 2(i)). In A. subreflexipinna, genetic variation among different individuals and/or populations directly came from different haplotypes of A. hasseltii. For example, in A. subreflexipinna of Mt. Kentuerhshan, except for the haplotype that was identical to A. diffracta, there were two haplotypes (Ha and Ha1) that were also found in A. hasseltii of the sympatric site. Nuclear haplotypes of A. hasseltii in Mt. Howeishan and Lake Chunglingchih were identical except for the two minors (Hb1 and Hb2). However, only one major haplotype (Ha) was found in A. hasseltii and A. subreflexipinna of Mt. Kentuerhshan. The Hb and derivatively minor haplotypes were found neither in A. hasseltii nor A. subreflexipinna of Mt. Kentuerhshan. CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA 69 TaBLeE 3. The variable nucleotide sites (indel & base substitution) of chloroplast trnS-rps4 intergenic spacer sequences. Columns shaded are sites identical to the hybrid sequences. Variable sites OO. 80. 44 8 1 ae 2S ee a ee 8 Species SPT eae eee OO ae Se eS ee 2 62 0 1 ee e 2 7 eee ee ee A. diffracta y AT A —.c FA A. subreflexipinna A. hasseltii D. polita The level of divergence among the three populations could not be revealed by the organellar fragments because only one haplotype was detected in each species (Tables 2-4). For nuclear pgiC intron 14-15 sequences, however, DnaSP analysis revealed high levels of genetic differentiation among three populations of A. hasseltii and A. subreflexipinna (ygr = 0.44377 and Yst = 0.26399; Table 7). Additionally, higher levels of genetic differentiation were also detected between northern and southeastern populations (A—B and A-C; Table 7) of these two species while little differentiation was found between those northern two (B-C; Table 7). For this same fragment, on the other hand, A. diffracta had only one haplotype and indicated no pattern of population structure. DIscUSSION Hybridization and parentage.—Similar to the traditional circumscription of species, hybrid species and hybrid parentage are usually postulated initially based on morphological characters and degree of spore/pollen abortion (Barrington, 1989, 1990). Acrorumohra subreflexipinna is suspected as a natural hybrid between A. hasseltii and A. diffracta (Moore, 2000) because A. subreflexipinna has abortive spores and intermediate morphology between A. hasseltii and A. diffracta, and occurs sympatrically with these two species. In addition, A. subreflexipinna’s spores show no germination, but those of A. hasseltii and A. diffracta germinate at a rate of more than 80% (unpublished data). Therefore, A. subreflexipinna appears to be a sterile F1 hybrid. 70 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Taste 4. The variable nucleotide sites (indel & base substitution) of mitochondrion nad5 intron 2 sequences. Columns shaded are sites identical to the hybrid sequences. Variable sites Species A. diffracta A. subreflexipinna A. hasseltii D. polita Acrorumohra subreflexipinna with its perennial habit, however, could occupy an original habitat for a long time despite of all spores being sterile. Repeated hybridization where the putative parents sympatrically exist might also replenish the stock of this hybrid. Organelle genomes are generally maternally inherited in monilophytes (Gastony and Yatskievich, 1992; Vogel et al., 1998). The assumption that chloroplast and mitochondria are maternally inherited is adopted through this study. All organellar sequence data indicated that A. hasseltii was the maternal parent of A. subreflexipinna. Nuclear pgiC sequences indicated that A. diffracta was the other genome donor of this hybrid. In addition to the three taxa of Acrorumohra discussed here, another species, A. yoroii (Seriz.) Shieh, was reported in the second edition of Flora of Taiwan (Shieh et al., 1994). In Taiwan, it grows in high montane regions and never sympatrically with other three taxa of Acrorumohra. Samples of that species were also collected from Taiwan and sequenced. It has organellar and nuclear sequences different from those of A. subreflexipinna, and phylogenetic analysis indicates a distant relationship between them (data not shown). There are three other species of this narrowly defined genus. Acrorumohra dissecta Ching ex Hsieh is distributed in a few locations of southwestern China, and A. obtusissima (Mett. ex Kuhn) Ching and A. undulata (Bedd.) Ching are distributed throughout Sri Lanka. Though we cannot reject the hypothesis, the possibility of these species contributing to the formation of this hybrid is extremely low because of their restricted habitats and disjunct distribution from this hybrid. CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA 7 ABLE 5. The haplotype and variable nucleotide sites (indel & base substitution) of nuclear pgiC intron 14-15 sequences. Columns shaded are sites different from those of other populations. Variable sites Havlotyoe cole Fiiuency G0. 0110.98 2 8 a ae gg eG ee ge ee ge Site* iS 16 8 2 AOS A OSCR OR eee pac og Te eae Sk 26 8 TAS SEO a ae eg oO ae A. diffracta D 10 CG A G COC T ANT. 6 CTA GA CA Gabo. Ae eas D 5 Ol kage oan ee ee A, subreflexipinna Ha 1 tO POC PRC G6) ae AA ee ee ee he Hal 4 rererreectPMcanc--cccrosrece 6 Bene Co ee a Hal 4 reterrecetPMcanc--ccctostace A. diffracta D 10 CCR Go oe A Er Oo eee ee GA A ae ea ee Aor oe D 4 C G10 GC Cie A TGC PAO: Ae Aer a AG CA EF A. subreflexipinna Ha 2 TG Ge ee Se eee OMe Cay Cee ees Bg < Dec ea B Hb 2 Be Qccccc BR Be Ha ee ee eee A. hasseltii Hb 5 t Pe TO 8 eo be ac Hb1 3 T TS 1826 Boye qc A. diffracta D 10 CoG ACE CC TAF Gc a bo a eee eee ee A. subreflexipinna Ha 5 TG TCT 6.6 €.F C0 AA O22 6 C8 T6°A TO eG c Hb 6 7 Ha gee eee Hb 5 a Te Hb2 1 T @ Tc ree “ A: Mt. Kentuerhshan, B: ry c: Mt. Howeishan. * First letter feaenorvege tense erent haplot 2 ies; D: A. diffracta, H: A. hasseltii. L | i A. hasseltii. 72 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Tas_e 6. Number of haplotypes, estimates of i diversity (h) and nucleotide diversity (x) of A. hasseltii and A. subreflexipinna. The diffracta haplotype ‘D’ cloned from A subreflexipinna were ada prior to this ae of of aplotype Nucleo Populations Species Bie eae eee (h) ay i Total (A+B+C) A. hasseltii 30 5 0.724 0.00485 A, reli eon 20 3 0.674 0.00466 Mt. Kentuerhshan (A) _ A. hasseltii 10 2 0.533 0.00074 A.s ae 5 Zz 0.400 0.00055 Lake Chunglingchih (B) A. hasseltii 10 3 0.689 0.00360 A. subreflexipinna : 2 0.667 0.00553 Mt. Howeishan (C) A. hasseltii 10 3 0.644 0.00446 A. subreflexipinna 11 2 0.545 0.00453 Although the phylogenetic analysis based on chloroplast trnS-rps4 IGS sequence show a sister-group relationship between D. polita and A. hasseltii (Li and Lu, 2006; our unpublished data), this study reveals that A. subreflexipinna has sequences different from those of D. polita in both organellar (Tables 2-4) and nuclear (data not shown) genomes. Therefore, Dryopteris polita did not contribute to the formation of this hybrid. These molecular data plus morphological and ecological information explicitly suggest that A. subreflexipinna arose through hybridization of A. hasseltii and A. diffracta, and that the former was its putative maternal parent while the latter was its paternal source. Acrorumohra hasseltii is distributed in tropical Asia, including Java, Borneo, Thailand, Nepal, East Himalayas, Vietnam, Hainan, Taiwan and southern Japan (Fig. 1). The range of A. diffracta overlaps with A hasseltii in the northern portion of the range of A. hasseltii, i.e., East Himalayas, northern Thailand, Vietnam, Hainan and Taiwan (Fig. 1). Except for southwestern China, the geographic range of A. diffracta almost completely overlaps with that of A. hasseltii. However, A. subreflexipinna has only been reported from Taiwan. It is suspected that A. subreflexipinna might be established at some sites across this widely overlapping range of these two putative parents but misidentified as A. diffracta because of their similar zigzag rachis. Careful recognition is needed to identify this hybrid in future field investigation where the range of these two species overlaps. Gender bias in hybridization events has been demonstrated many times in plants (Emms et al., 1996; Vogel et al., 1998; Weiblen and Brehm, 1996; Xiang et al., 2000). For reasons not entirely clear, the hybridization of A. hasseltii and A. diffracta in our study was absolutely biased, i.e., A. hasseltii always was the supplier of egg while A. diffracta was that of sperm. This phenomenon has also been found in other hybrid species (e.g., Arnold and Bennett, 1993; Peng and Chiang, 2000; Smith and Sytsma, 1990; Wendel et al., 1991). In ferns, mating systems usually correlate with ploidy levels and could be a decisive factor in the nuclear-organellar combination pattern of parental genotypes in hybrid- ization. In fact, A. diffracta was reported as a tetraploid (Tsai and Shieh, 1975) CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA ne (i) (ii) A: Mt. Kentuerhshan -----—- B: Lake Chunglingchih C: Mt. Howeishan Fic. 2. pgiC intron 14-15 network for Acrorumohra h tii (i) and A. subreflexipinna (ii). Letters and number in the boxes or circles are haplotyp . Dashed boxes indicate site(s) in which the haplotype is detected. The little rhombuses indicate the hypothetical haplotypes. Each line between haplotypes represents a mutational step. and A. hasseltii a diploid (Iwatsuki, 1995), and our observation showed that A. hasseltii has 64 spores per sporangium but A. diffracta has 32 spores (unpublished data). These agree with the general rule that a sexual species usually has 64 spores per sporangium whereas there are typically 32 spores in an apogamous species. According to the Dopp-Manton Scheme and Braithwaite modes of reproduction (Raghavan, 1989), Acrorumohra diffracta 74 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) TABLE 7. Genetic ge uidies between pairwise and among all populations of A. hasseltii and A. subreflexipinn Genetic differentiation between pairwise populations (ysr)* Genetic differentiation among all Species populations (ys) A-B A-C B-C A. hasseltii 0.44377 0.60815 0.40303 0.06301 A. subreflexi- pinna 0.26399 0.40000 0.31460 0.00162 “A: Mt. Kentuerhshan, B: Lake Chunglingchih, C: Mt. Howeishan. might be an obligate apogamous species with functional antheridia. These observations and molecular evidence lead to a convincing hypothesis for uni- directional hybridization that sexual A. hasseltii could adopt sperm from the apogamous tetraploid, A. diffracta. Additionally, a lack of heterozygosity of the A. hasseltii genotypes in this hybrid further supports that A. hasseltii was the haploid female gamete donor in these hybridization events. In this case, because fertile sperm were liberated from an apogamous, tetraploid gameto- phyte, A. subreflexipinna should be a pentaploid. C-value results based on flow cytometry show that A. subreflexipinna has the highest ploidy level among these taxa (unpublished data), which confirms this hypothesis. Further observations on chromosome counts and the mating system of A. subreflex- ipinna and both parents in the future would provide more direct evidence. Multiple independent origins.—Although A. subreflexipinna has a larger body size than both putative parental species, it produces no fertile spores, and its small population size and rare occurrence strongly suggests it is a product of occasional hybridization event(s). Both cpDNA and mtDNA fragments of populations of A. hasseltii and A. subreflexipinna were identical in sequence. Single direction hybridization and allopolyploidization may produce cyto- plasmically uniform hybrids or allopolyploids despite distinct origins (Soltis et al., 1992). The variation in cpDNA and mtDNA from the taxa of this study was uninformative regarding multiple origins of A. subreflexipinna. Nuclear pgiC sequences, however, revealed different haplotypes and genetic differen- tiation among populations of A. hasseltii and A. subreflexipinna. Southern and northern populations of A. hasseltii had distinct and unique genotypes, and the genetic uniqueness was transmitted to their hybrid offspring (Table 5; Fig. 2). In both A. hasseltii and A. subreflexipinna, there were nine variable sites that had different bases in southern and northern populations (Table 5). Haplotype Ha1 was found in A. hasseltii of Mt. Kentuerhshan and was present in A. subreflexipinna in the same location. On the other hand, major haplotype Hb was only found in the northern populations of both species but not in the southern ones (Fig. 2). These are direct indicators of multiple independent origins of A. subreflexipinna. There was no variation of organellar DNA fragments in populations of A. hasseltii and A. subreflexipinna, but high nucleotide diversity was found in the nuclear pgiC intron 14-15 of the same species. The fact that, usually, CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA %5 evolutionary rates of nuclear DNA are faster than those of chloroplast and mitochondria in plants (Graur and Li, 2000) may provide a reasonable interpretation of this significant difference. This also indicates that nuclear DNA markers may be a better choice when analyzing population variation due to relatively short evolutionary time. In this case, geographical barriers might effectively hinder gene flow, causing geographical subdivision in nuclear NA, but the time of isolation might not have been long enough to accumulate new mutations in organellar DNA. Several studies indicate that multiple origins of hybrid and polyploid species are common, if not the rule, in plants (see Soltis et al., 1992 and references therein). Genetic variation of the parental species could be incorporated into and preserved in hybrids and their derivative taxa by these processes (Arft and Ranker, 1998; Paun et al., 2007; Peng and Chiang, 2000). This phenomenon was also found in this study. Unlike a small population usually having fixed alleles for most loci, populations of A. subreflexipinna, even when only four individuals were found in a population, have high haplotype diversity. Moreover, the results agree with our anticipation that in each collecting site there were fewer haplotypes and lower haplotype diversity of A. subreflexipinna than those of A. hasseltii. Because A. subreflexipinna is a sterile hybrid, any genetic variation should come from its parents. It is the recurrent hybridization of this hybrid that might maintain its significant genetic variability and provide operative materials for future evolution, i.e., polyploidization and subsequent fertilization. ACKNOWLEDGMENTS The authors thank James R. Shevock for advice and revision of English. We are thankful to Mrs. Lu, Pi-Fong and Dr. Liu, Yea-Chen for the information of distribution sites and to Miss Chao, Yi- Shan for help in experiment. We also extend appreciation to Dr. Geiger and two anonymous reviewers for their critical reviews of the manuscript. This study was supported by Council of Agriculture (grant number 96AS-11.2.3-EI-W2), Taiwan. LITERATURE CITED Arrt, A. M. and T. A. Ranker. 1998. Allopolyploid origin and population genetics of the rare orchid Spiranthes diluvialis. Am. J. Bot. 85:110-122 Arno_p, M. L. and B. D. Bennett. 1993. Natural hybridization in Louisiana irises: genetic variation and ecological determinants. In R. G. Harrison (ed.), Hybrid zones and the evolutionary process, pp. 115-139. Oxford University Press, New York. Barrincton, D. S. 1986. The morphology and cytology of Polystichum x potteri hybr. nov. (=P. acrostichoides X P. braunii). Rhodora 88:297—313. Barrincton, D. S. 1989. New species and combination in tropical American Polystichum (Dryopteridaceae). Ann. Miss. Bot. Gard. 76:365—373. Barrincton, D, S. 1990. Hybridization and allopolyploidy in central American polystichum: cytological and isozyme documentation. Ann. Miss. Bot. Gard. 77:297—305. BarrincTon, D. S., C. H. Haurter and C. R. Wertx. 1989. Hybridization, reticulation, and species concepts in the ferns. Am. Fern J. 79(2):55-64. BENNERT, W., M. LusiENski, S. KOrNER and M. STEINBERG. 2005. Triploidy in Equisetum subgenus Hippochaete (Equisetace, Pteridophyta). Ann. Bot. 95:807-815. 76 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Bowers, J. E., B. A. CHAPMAN, J. Ronc and A. H. Paterson. 2003. Unravelling angiosperm genome evolution by phylogenetic vag ae of chromosome duplication events. Nature 422:433-438. CLEMENT, M., D. Posapa and K. A. CRranpa.L. 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9:1657—1660. De Bopr . Magre and Y. VANDE Pegr. 2005. Genome duplication and the origin of angiosperms. Toads Ecol. Evol. 20:591-597. EsrHara, A., H. IsHikawa, S. Matsumoto, S. Lin, K. Iwatsuxi, M. Takamiya, Y. WaTaNo and M. Ito 2005. Nuclear DNA, dhigeiias DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex i aehyliaes in Japan and adjacent areas. Am. J. Bot. 92:1535-1547. Ems, S. K., S. A. Hopces and M. L. ArNoLp. 1996. Pollen-tube competition, siring success, and consistent asymmetric hybridization in pees irises. Evolution 50:2201—2206 FLorA oF NortH AMERICA EpiToriAL Committee. 1993. Flora of North America. Vol. 2, Pteridophytes and Gymnosperms. Oxford University fet New York. Gastony, G. J. and G. YATsKIEVICH. 1992. Mat ea inheritance of the chloroplast and mitochondrial genomes in a ferns. Am. J. Bot. 79:716—722. Gravr, D. and W.-H. Li. 2000. Searle. sP molecular evolution, 2"¢ ed. Sinauer Associates, napa d. UFLER, C. H. 1987. pope neepvies is modifying our concepts of evolution in homosporous siege 74:953—966. Ho trum, R. E ee j. Sovtanh 1986. Studies in the fern genera allied to Tectaria II. Dryopsis, a gigs ea Gs ll. 41:171-204 HsIEH, wey -T. 0. Acrorumohra (H. Ito) H. Ito. In S.-G. Wu (ed.), Flora Reipublicae Popularis Sinicae ee Science Press, oe (in Chinese) IsHikawa, H., Y. WaTANO, K. Kano, M. Ito and S. Kurita. 2002. pamiereeiene of primer sets for PCR amplification of the PgiC gene in elas J. Plant Res. 115:6 Iwatsuxk!, K. 1995. Dryopteridaceae. In K. akg et al. (eds.), ans of Japan I, Pteridophyta and Gymno: Sane pp. 120-173. Kodansha, Tokyo. KLEKowskI, E. ve H. G. Baker. 1966. msoeneel ts significance of polyploidy in the Pteridophyta. Science ae ser Kuo, C.-M. 1988. A new Asplenium hybrid from Taiwan. Bot. Bull. Acad. Sin. 29:109—-11 Kuo, C.-M. 1990. Mateduale for the Lomariopsidaceae of Taiwan. Bot. Bull. Acad. Sin. 31: abet Li, C.-X. and S.-G. Lu. 2006. Phylogenetics of Chinese Dryopteris (Dryopteridaceae) based on the chloroplast rps4-trnS SS e data. J. Plant Res. 119:589-598. Lyncu, M. and T. J. Crea pe The analysis of population survey data on DNA sequence colin Mol. Biol. are 377-394. Mryamoro, F. and T. Nakamura. 1983. Three new hybrids of Polystichum from Taiwan. J. Jap. Bot. 58:146—-150. Moors, S.-J. 2000. Acrorumohra subreflexipinna. In S.-Y. Lu, et al. (eds.), Rare and en- dangered plants in Taiwan, pp. 1-2. Council of Agriculture Executive Yuan, Taipei. (in inese Napot, S., G. Brrrar, L. Carter, R. Lacrorx and B. Lejeune. 1995. A phylogenetic analysis of sicicweyiacidi based on st chloroplast gene rps4, using parsimony and a new numerical phenetics method. Mol. Phylogen. Evol. 4:257—282. Nakazato, T., M.-K. Junc, E. A. Houswortu, L. H. Rieseserc and G. J. Gastony. 2006. Genetic map- based mo ite of genome structure in the homosporous fern Ceratopteris richardii. Genetics 173:1585— Ne!, M. 1982. olution of human races at the gene level. In B. BonNE-Tam (ed.), H Genetics, \ e Unfolding Genome, pp. 167-181. Alan R. Liss, New York. Paun, O., M. : Fay, D. E. Sottis and M. W. Cuase. 2007. Genetic and epigenetic alterations after hybridization and genome doubling. Taxon 56:649-656 Penc, C.-I. and T.-Y. Cxtanc. 2000. Molecular centicweina of unidirectional hybridization in Bewonia x taipeiensis Peng (Begoniaceae) from Taiwan. Ann. Miss. Bot. Gard. 87:273—285. CHANG ET AL.: HYBRID ORIGIN OF ACRORUMOHRA SUBREFLEXIPINNA 77 PINTER, I., F. BAKKER, J. BARRETT, C. Cox, M. Guay, S. HENDERSON, M. Morcan-Ricuarps, F. RUMSEY USSELL, S. Trewick, H. ScHNemer and J. VoceL. 2002. Phylogenetic and biosystematic e 229 RaAGHAVAN, V. . Developmental biology of fen gametophytes. Cambridge University Press, Cambridge. Rozas, J., J. C. SANCHEz-DetBarrio, X. Messecyer and R. Rozas. 2003. DnaSP, ae polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496— SHIEH, W.-C., C. E. Devon and C.-M. Kuo. 1994. Dryopteridaceae. In T.-C. oat fe a (eds.), Flora of Taiwan 24 ed, 1:303-351. Editorial Committee of the Flora of Taiwan, Second Edition, Taipei SMALL, R. iE R. C. CRonn and J. F. WENDEL. es bs yas @ Johnson Review No. 2, Use of nuclear genes for phylogeny severe in plan t. Syst. Bot. 17:145-170 SmitH, A. R. and R. B. Cranriit. 2002. Lene relationships of the thalyptascid ferns elyperdacoae Am. Fern J. 92(2):131-149. SmitH, R. L. and K. J. Syrsma. 1990. Evolution of Populus nigra (sect. Aigeiros): uphatpes hybridization and the chloroplast contribution of Populus alba (sect. Populus). Am. J. B 77:1176—1187. eae P.S., J. J. Doyie and D. E. Soxtis. 1992. Molecular data and polyploidy evolution in plants. In P. S. Soxtis, et al. (eds.), Molecular systematics of plants, pp. 177-201. Chapman & Hall, New ork. Stein, D. B. and D. S. Barrincton. 1990. Recurring hybrid formation in a population of Polystichum X potteri: stionia from chloroplast DNA comparisons. Ann. Miss. Bot. Gard. 77:334—339. Taser.et, P., L. Getty, G. Pautou and J. Bouvet. 1991. Universal primers for amplification of three non-coding 2. of chloroplast DNA. Plant Mol. Biol. 17:1105-1109. Takamiya, M., N. Onta, Y. Yarase and N. Murakami. 2001. Cytological, morphological, genetic, and molecular phylogenetic studies on Abtraspectfic differentiations within Diplazium doederlei- nii (Woodsiaceae: Pteridophyta). Int. J. Plant Sci. 162:625—-636. Terapa, Y. and M. Takamrya. 2006. Cytological and genetic study of two putative hybrids and their parents of Athyrium scencony isos in Yakushima Island, southwestern Japan. Acta Phytotax. Geobot. wes i ):9 Tas, J.-L. and W.-C. SHE. 1 Sg leiae ag of the fern family Aspidiaceae (sensu Copeland) in Taiwan. J. fe Engin. 12:321— Vancerow, S., T. TEERKORN and V. Knoop. 1999. SRE a in the mitochondrial nad5 gene of pteridophytes: RNA editing and intron sequences. Plant Biol. 1:235-243 VocEL, J. C., S. J. Russe, F. J. Rumsey, J. A. Barrett and M. pati 1998. Evidence maternal transmission of nia: DNA in the genus Asplenium (Aspleniaceae, Pteridophyta). Bot. Acta 111:247— Wacner, W. H. 1954. aie evolution in the Appalachian aspleniums. Evolution 8:103-118. Wacner, W. H. 1962. Irregular morphological development in hybrid ferns. Phytomorphology 12:87-100. Wacner, W. H. 1973. Reticulation of aes pee (Polystichum) in the western United States and adjacent Canada. fee Fern (3):99— Wers.en, G. D. and . BREHM. 1996. Recrhiciive strategies and. barriers to egpisbtiaorgoe hetveoans Tellima pons and Tolmeia menziesii (Saxifragaceae). Am. J. Bot. 83:910-918. WencEL, J. F., J. M. Stewart and J. H. Retric. 1991. Molecular evidence for homoploi reticulate evolution hare Australian species of Gossypium. Evolution 45:6 Werth, C. R., S. I. Gurrman and W. H. EsupaucH. 1985. Electrophoretic ieee of reticulate pane ae in the Appalachian Asplenium complex. Syst. Bot. 10:184—192. X1anc, L., C. R. Wert, S. N. Emery and D. E. McCauzey. 2000. Population- -specific gender-biased hybridization between patie intermedia and D. carthusiana: evidence from chloroplast A. Am. J. Bot. 87:1175-11 American Fern Journal 99(2):78—92 (2009) Molecular Cloning and Sequence Analysis of Cyanovirin-N Homology Gene in Ceratopteris thalictroides XIAOQIONG QI Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei 430074, China YONGXIA YANG Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei 430074, China YINGJUAN Su* School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China TinG WaNG* Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei 430074, China Asstract.—A new full-length genomic DNA, encoding a member of the cyanovirin-N (CV-N) homologous protein family, has been cloned from the fern species Ceratopteris thalictroides by chromosome walking. It is 1993 bp ca eeegreon a 723 bp open ame (ORF) that encodes a deduced protein (named CtCVNH) w 150 amino acid residues. ~CtCVNH has a predicted isoelectric point (PI) of 4.47 and a peoree molecular mass 15.9556 kDa. It possesses the conserved anti-HIV (human immunodeficiency virus) CV-N domain, which is the same as the cyanovirin-N homology (CVNH) members that were isolated from filamentous ascomycetes and C. richardii. Modeling of the tertiary structure indicated that CtCVNH is an elongated, largely B-sheet protein that displays internal two-fold pseudosymmetry. Comparative structure analysis of the predicted CtCVNH with native CV-N nope: that the mee iiprecareonds changes os during the evolution of plant CVNHs were: 1 and C a loop to helix transition at the helical-turn regions. Phylogenetic analysis showed that CtCVNH was grouped together with the two CVNHs from C. richardii. Worps.—Ceratopteris thalictroides, chromosome walking, single oligonucleotide nested PCR, inverse PCR, thermal asymmetric interlaced PCR, CVNH, bioinformatic analysis Cyanovirin-N (CV-N) is an 11 kDa anti-HIV (human immunodeficiency virus) protein originally isolated from the extract of the cyanobacterium Nostoc ellipsosporum (Des.) Rabenh. (Boyd et al., 1997). It consists of a single chain with 101 amino acids, exhibits significant internal sequence duplication between residues 1-50 and 51-101, and contains two intramolecular disulfide bonds (Gustafson et al., 1997). CV-N is largely comprised of fB-sheets with a two-fold pseudosymmetry (Bewley et a/., 1998). Its antiviral activity depends on the high-affinity binding to the HIV surface envelope glycoprotein, gp120 (Boyd et al., 1997; Mori et al., 1997). CV-N can specifically interact with high mannose groups (Bolmstedt et al., 2001; Botos et al., 2002), thereby blocking the interaction between gp120 and the receptor CD4 on target cells (O’Keefe et * corresponding authors: Yingjuan Su, Tel: +86-20-84035090, Fax: +86-20-84036215, E-mail: suyj@mail.sysu.edu.cn; Ting Wang, Tel: +86-27-87510677, Fax: +86-27-87510251, E-mail: tingwang@wbgcas.cn QUIET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 79 al., 1997). Besides HIV strains (Boyd et al., 1997), CV-N is also able to inactivate simian immunodeficiency virus (SIV), Ebola virus (EBO), herpes simplex virus-1 (HSV-1), and hepatitis C virus as well (Barrientos et al., 2003; O’Keefe et al., 2003; Barrientos and Gronenborn, 2005; Helle et al., 2006). The potent inactivation of HIV plus unique biophysical properties make CV-N a candidate for a topical anti-HIV microbicide. The CV-N preclinical develop- ment is underway (Colleluoria et al., 2005). Recently, a family of CVNH (cyanovirin-N homology) has been identified. All CVNH proteins share a common fold that matches the one previously thought to be unique in CV-N (Percudani et al., 2005). Current research on CVNHs is mainly focused on structural information, antiviral activity, carbohydrate-binding specificities or structure-function relationships (Percu- dani et al., 2005; Koharudin et al., 2008). For example, solution structures of three CVNHs from Tuber borchii Vittad., Ceratopteris richardii Brongn., and Neurospora crassa Shear et Dodge have been determined (Koharudin et al., 2008) and may be helpful in elucidating the roles that these proteins play in the organs and during evolution. CVNHs show a patchy organism distribution regarding the anti-HIV domain. They are present in organisms as diverse as cyanobacteria, filamentous ascomycetes and seedless plants (Percudani et al., 2005). However, among plants, CVNHs have only been identified in the fern C. richardii until now. To provide useful information for understanding the evolution of CVNHs and developing antiviral polypeptides, here we report the cloning and sequence analysis of the full-length CVNH genomic DNA in Ceratopteris thalictroides (L.) Brongn. together with an analysis of CVNHs phylogeny and modeling of the protein tertiary structure. MATERIALS AND METHODS Plant materials.—Ceratopteris thalictroides was collected from Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, Hubei, China. Young and healthy leaves were sampled, immediately frozen in liquid N>, and stored at —70°C until used. Genomic DNA extraction.—Total genomic DNA was extracted from fresh leaves following the modified CTAB protocols (Su et al., 1998). DNA concentration and purity were determined by measuring UV absorption using a Pharmacia 2000 UV/Visible spectrophotometer. DNA intactness was checked by 1.0% agarose gel electrophoresis. Molecular cloning of the full-length genomic DNA.—Based on the C. richardii EST sequence (Accession No. BQ087187), specific primers were designed to amplify the internal region of CVNH in C. thalictroides. The forward primer CVNH-F was 5'-GTGGGCGTCTAGCGATTTCCTTT-3’, and the reverse primer CVNH-R was 5’-ATCATCCGCTGCTTGCTTCTTCG-3’. The reaction mixture (20 yL) contained 50 ng template DNA, 40 pmol each primer, 1 pmol each dNTP, 1.0 U Tag DNA polymerase and 1 x Taq polymerase buffer. PCR was performed using the following protocol: the template was 80 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) TaBLe 1. The primers used in chromosome walking. Primer name Primer sequence 5S IPCR 5’-GGTGATATTGCCCGTCGGTGCCTTT-3’ 5'SON-1 5'-ATCACTGTTGAGGCAATCTGCGGCT-3’ 5'SON-2 5’-GCTGCGATCAAGACGATGAGAAAAC-3’ 5'SON-3 5'-CCATCGCTTCTAGGAGTAAACAGAC-3’ 3’TAIL-1 5’-GTGCAAAGGCACCGACGGGCAATAT-3’ 3’TAIL-2 5’-GGGGTGTTGGATTTCTGTGGCTATG-3’' 3'TAIL-3 5’-AAGCGAAGAAGCAAGCAGCGGATGA-3’' denatured at 94°C for 5 min followed by 36 cycles of amplification (94°C for 50 s, 61°C for 50 s, 72°C for 90 s) and a final extension of 10 min at 72°C Based on the sequence obtained from the internal DNA region, two sets of nested primers for 5’ single oligonucleotide nested PCR (SON-PCR) (Antal et al., 2004) and 3’ inverse PCR (IPCR) (Triglia et al/., 1988) combined thermal asymmetric interlaced PCR (TAIL-PCR) (Liu and Whittier, 1995) were designed to amplify the 5’ and 3’ flanking sequences. These primers included 5’IPCR, 5’SON-1, 5’SON-2, 5’SON-3, 3’TAIL-1, 3’TAIL-2, and 3’TAIL-3 (Table 1, Fig. 1). They were of high annealing temperatures and synthesized by Invitrogen (Shanghai). The 5’ flanking sequence was amplified by SON-PCR. The primary PCR was carried out in a 20 ul volume containing 50 ng genomic DNA, 50 pmol single primer (5’SON-1), 50 mol/L each dNTP, 2.0 U Taq DNA polymerase and 1 Taq polymerase buffer. For the secondary PCR, two single primers (5’SON-2 and 5’SON-3) were separately used. The reaction solution was the same as that of primary PCR except that 1 ul of a 1:50 dilution of the primary PCR products was used as the template. The 3’ flanking sequence was obtained using IPCR combined TAIL-PCR. Ceratopteris thalictroides genomic DNA was digested with Pac I (NEB, BSA 5 Uug * of DNA) at 37°C for 3 h, and then heated at 65°C for 20 min. The digested DNA was self-ligated overnight at 15°C with a concentration of 0.3— 0.5 ug/ml in the presence of 3 U/ml T4 DNA ligase (Promega). PCR was carried out in a 20 ul volume with 1 ul ligated product, 1 pmol each dNTP, 40 pmol each primer (5’IPCR and 3’TAIL-1), 1.0 U Taq DNA polymerase and 1 X Taq polymerase buffer. The primary PCR of TAIL-PCR was performed using primer 3TAIL-1 3TAIL-2 3TAIL-3 5’ 3’ AA. id tf oF +— ee eee Fic. 1. Schematic view es sieges aoe eleriemcsot of = —— used in this study and of their relative positions t The rectangle frame indicates the sequence obtained by specific PCR, whereas the line | represents regions determined by further chromosome walking. QI ET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 81 Taste 2. Cycling conditions used for SON-PCR, IPCR, and TAIL-PCR. Name Reaction Cycle no. Thermal condition 5’SON-PCR Primary 4 94 °C (5 min) 5 94 °C (30 s), 65 °C (1 min), 72 °C (2.5 min) 1 94°C(30 s), 29°C(3 min), ramping to 72°C over 3 min, 72°C(2.5 min) 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 72°C(7 min) 94°C(5 min) 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 72°C(7 min) io2) So Secondary w wo POR RP WHER OR RB SIPCR IPCR 94°C(1 min), 65°C(1 min), 72°C(2 min) 72°C(10 min) 93°C(1 min), 95°C(1 min 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 94°C(30 s), 25°C(3 min), ramping to 72°C over 3 min, 72°C(2.5 min) 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 94°C(30 s), 44°C(1 min), 72°C(2.5 min) 1 72°C(5 min) Secondary 1 94°C(1 min) 15 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 94°C(30 s), 65°C(1 min), 72°C(2.5 min) 94°C(30 s), 44°C(1 min), 72°C(2.5 min) 1 72°C(5 min) 3’TAIL-PCR Primary ee ol 3’TAIL-1 as the gene-specific primer and primer AD (5’-TC(G/C)TICGNA- CIT(A/T)GGA-3’) (Liu and Whittier, 1995) as the arbitrary degenerate primer in a total 20 ul volume that contained 1 ul of a 1:50 dilution of the IPCR products, 2 pmol each dNTP, 40 pmol primer 3’TAIL-1, 500 pmol primer AD, 2.0 U Taq DNA polymerase and 1 X Taq polymerase buffer. For the secondary PCR, two gene-specific primers (3’TAIL-2 and 3’TAIL-3) were separately used with the same arbitrary primer as used in the primary one. The reaction solution was the same as that used for the primary PCR except that 1 ul of a 1:50 dilution of the primary PCR products was used as the template. Thermocycling profiles used for SON-PCR, IPCR, and TAIL-PCR are listed in Table 2. Recovery of PCR products.—PCR products were purified by running them through a 1.0% low melting agarose gel. The desired DNA band was cut out and recovered using the DNA rapid purification kit (Omega). DNA cloning and sequencing.—A purified PCR product was ligated into a pMD 19-T (TaKaRa) vector and then used to transform competent Escherichia coli cells DH-5x. A positive clone was identified by blue/white selection and ascertained by PCR. Purified plasmid DNA was sequenced in both directions by standard methods on an ABI 3730 automated sequencer at Invitrogen (Shanghai). Primers M13F and M13R located on pMD19-T vector were utilized for sequence determination. 82 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) In silico analysis and molecular modeling.—ORF finder was used to predict coding sequence, and promoter analysis was performed online (http://www. fruitfly.org/cgi-bin/seq_tools/f ter.pl). Sequence analysis was conducted using the BLAST program (Altschul et al., 1997) and other programs available at the ExPASy server (Gasteiger et al., 2003). Multiple sequence alignment was carried out using the ClustalX software (Thompson et al., 1997). Figures of multiple sequence alignment adorned with secondary structure elements were generated with ESPript (Gouet et al., 1999). Primary structure analysis of the deduced CtCVNH (CVNH protein from C. thalictroides) was conducted with ProtParam (Gasteiger et al., 2005) by using the ExPASy server online (http:// www.expasy.ch/tools/proty html). Secondary structure was predicted with SOPMA program ystange and Deleage, 1995) online (http://npsa-pbil. ibcp.fr/cgi-bin/npsa_automat.| ge=npsa_sopma.html). Phylogenetic anal- ysis was carried out using “programs from the PHYLIP package; genetic distances were estimated with PROTDIST using the Jones-Taylor-Thornton model of amino acid substitutions. Neighbor-joining trees (Saitou and Nei, 1987) were constructed using the NEIGHBOR program; 1000 random replications were utilized for bootstrap analysis, which was performed with the SEQBOOT and CONSENSE programs. Phylogenetic trees were rendered with the TREEVIEW program (Page, 1996). The three-dimensional (3D) structural models of CtCVNH were built by the homology-based method using the SWISS-MODEL program (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006). The template used for modeling was C. richardii CVNH (PDB code 2jzjA) (Koharudin et al., 2008). Models were displayed with the PyMol program (Delano, 2002). RESULTS AND DISCUSSION Molecular cloning of the full-length genomic DNA.—Using a pair of specific primers (CVNH-F and CVNH-R), a single fragment of 775 bp was amplified from the C. thalictroides DNA [Fig. 2(a)]. Compared with the C. richardii cDNA sequence (Accession No. BQ087187), the sequence from C. thalictroides has two additional fragments that do not exist in C. richardii cDNA and the remaining parts of the sequence are identical to the C. richardii cDNA (Fig. 3). The CtCVNH intron—exon boundaries were thus deduced; it is composed of three exons and two introns. Based on the amplified sequence of the specific PCR, two sets of nested primers were further designed to obtain the 5’ and 3’ flanking sequences, respectively. A clear single band ~ 800 bp of the 5’ flanking sequence was generated in the secondary reaction [Fig. 2(b)] using SON-PCR, while a ~ 750 bp 3’flanking sequence was amplified through IPCR combined TAIL-PCR [Fig. 2(c)]. Sequence analysis of the CtCVNH gene.—The cloned full-length CtCVNH gene is 1993 bp in length, including a 818 and 452 bp 5’ and 3’ untranslated region (UTR) respectively, and a 723 bp coding region. The 5’'UTR has a TATA box in the predicted promoter elements. The ATG start codon, which is numbered +1 to +3, is flanked by G in both positions -3 (3 nucleotides before QI ET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 83 2000 1000 750 500 250 SOH-PCR 100 a b C Fic. 2. Agarose gel electrophoresis of the specific PCR(a), SON-PCR(b), and IPCR+TAIL-PCR(c) products. M is molecular weight marker (DL2000). a 1: about 750 bp fragment generated by specific PCR with primer CVNH-F and CVNH-R. b 1: smear bands produced by the first reaction of SON- PCR with primer 5’SON-1. 2: no clear band produced by the secondary reaction of SON-PCR with primer 5’'SON-2. 3: a single band obtained by the secondary reaction of SON-PCR with primer 5’SON-3. c 1: the amplified DNA fragment of secondary reaction of TAIL-PCR with primer 3'TAIL-2 and AD. the ATG codon) and +4 (1 nucleotide after the ATG codon), indicating that it is located in a sequence context for strong translational initiation (Kozak, 1999). The 3’UTR has a polyadenylation signal (AATAAA) and six ATTT domains (Fig. 4). These ATTT domains may be important for mRNA destabilization (Shaw and Kamen, 1986). The CtCVNH gene encodes a deduced protein of 150 amino acid residues with a predicted isoelectric point (pI) of 4.47 and a calculated molecular mass of 15.9556 kDa. Regarding its amino acid composition, the most abundant is Ser (13.3% by frequency), followed by Gly (9.3%), Leu (9.3%), Ala (7.3%), Asn (7.3%), Asp (7.3%), and Val (6.7%). Acidic and basic amino acids constitute 10.0% and 5.3% of the protein, respectively. Moreover, 15.3% of the amino acids are charged, and the percentages of polar and hydrophobic amino acids are 64% and 25.3%, respectively (Table 3). With regard to the predicted secondary structure, the CtCVNH protein consists of 16.00% alpha helices, 28.67% extended strands, 12.67% f turns, and 42.67% random coils. The extended strands and random coils constitute the interlaced domain of the main part of the secondary structure. CtCVNH on os CrCVNH Fic. 3. Schematic view of the exon and intron positions deduced from C. richardii CDNA. The exon and intron are indicated by rectangle frame and line, respectively. 84 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) CCATCGCTTCTAGGAGTAAACAGACATACTATAACTGC = -781 -721 ~661 -601 -541 ~481 -421 -361 -301 -241 -181 STTTATCAGTATATHTACKEACCTAATICGCACATTETEANTANE -121 ae CT ea GA -61 =i 60 REY FT PRY LL iQ 6 Po ry eT a t 120 ASAA 8 £8 ES . = 180 tcegttctcet 240 COPS PSC ED YT YY 7 Se 8 300 PRAA? CLES RGA YO 8 8.5 Lad 360 NDR TGR S ECR L VPP Gt 8 Fee 420 SaOELS YETTA SE COT TAS CEG 480 D6 .@. 7 HP FS LD LAS Cy UY NAD 540 Tiertce 7 € ¥ CE Ss tk ty gS Ss Ft OGTgtaatg g tagt 600 V ge 660 t 720 SEERA S SG 780 * ATTT 840 900 ATTT ATTT! 960 1020 ATIT 1080 ATTT. 1140 TATTTTGGATTIGTTAGTITATTACGTCTATTTATT 1175 Fic. 4. Nucleotide and deduced protein sequences of CtCVNH gene. The predicted amino acid sequence is shown below its open reading frame. The predicted promoter sequence is shown in shaded box (t iption start site shown in larger font). The TATA box is boxed with solid — The polyadenylation signal is underlined and boldface. The ATTT regions of the 3’UTR ar underlined. The introns are present in lowercase letters. QUIET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 85 TaBLe 3. Component analysis of amino acid sequence of CtCVNH. Amino acid Number Frequency(%) Hydrophobic amino acid 38 25:3 Charged amino acid 2a 15.3 Polar amino acid 96 64.0 Acidic amino acid 15 10.0 Basic amino acid 8 5.3 Ala(A) 11 75 Gly(G) 14 9.3 Met(M) 3 2.0 Ser(S) 20 13.3 Cys(C) 7 4.7 His(H) 2 1.3 Asn(N) 11 7.3 Thr(T) 10 6.7 Asp(D) 11 79 Dle(D 4 27 Pro(P) 3 2.0 Val(V) 10 6.7 Glu(E) 4 27 Lys(K) 4 27 Gln(Q) 3 2.0 Trp(W) 0 0.0 Phe(F) 7 4.7 Leu(L) 14 9.3 Tyr(Y) 8 a0 Arg(R) 4 27 Amino acid sequence alignment and phylogenetic analysis.—Initial homol- ogy searches were conducted with the deduced CtCVNH amino acid sequence in the non-human, non-mouse EST database at the NCBI (National Center for Biotechnology Information, NIH, Bethesda) by using the tblastn program (Altschul et al., 1997). A new member of CVNHs was uncovered from the plant Selaginella moellendorffii Hieron. by conducting these searches. The results (Table 4) showed that the CVNH members were present in fungi and plants (E value < 0.01). Above 70% of the members occurred in fungi. A comparison of the deduced CtCVNH against other CVNHs revealed that CtCVNH shares a high degree of similarity with the two CVNHs from C. richardii (99% and 53% identity, respectively), and a reduced level of similarity with the CVNHs from fungi (26-33%). Multiple sequence alignment indicated that the anti-HIV domain is conserved [Fig. 5(a)]. The most conservative sites were F4, L18, G27, L36, G41, N42, G45, F54, L69, G78, L87, N93, and G96 (the numbering is in line with the N. ellipsosporum CV-N). These residues are predominantly located in the hydrophobic core region, which are involved in hydrophobic interactions between the B-hairpin and the underlying triple-stranded B-sheet of each repeat (Percudani et al., 2005). Also conserved are hydrophilic amino acids involved in the formation of the hydrogen-bonded bridges that connect AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) ‘aseqejep [Sy 9SnOow-UOU ‘UeUINY-UWoOU oy} Jo ezIs ay} UO paseq (XIeUT ZQUINSOTq) suOsteduIOD astmited s0j sontea poayoedx7y,, ‘HNADID peonpep 94} 0} yedser YLM AyQUEp! JUedIEg, ‘poyioda st souenbas [gq eayejueseidas & Jo JequINU UO[ssa90e ay} ‘BLP WNYUI Woy poonpaep seouenbes utejoid 10,4, £00'0 ee sayaoAWOLIepIo0sS sualiA pa1s0dAyy POL VNU L9ETBEDA £00'0 ee Sa}BDAUIOLIePIOS IIx1] pal20dAp] SOL VNwuL ZOLSL6[V Z00'0 9Z saad AUOlep10s aDITyop UINIIIy.1a A €PL VNwu O€ZOLLO Aresqi] LSA poxtut F0-9L Pe - umnJopou Dilapydsoapyg / UNAYsSaD UMNITILL], 90L VNMUL 9c6rrONd G0-96 o£ so] AUIOLIep10g UMOITDUIOIS DULI@POYIIL J, LOL VNwut ZEZZ16[V 0-92 €E sajaoAuoapryyoq SINS umniopou bilapydsoapydg 901 VNU TeLS6€HH CO-8L of sooo ATO NOINY apzAio snjjisiadsy 90L VNU PL68ZPA GO-eL o£ sajoo AO nOIN snapjf snyisiadsy 90L VNU Z668ELOD Z00'0 Le saTe][autseas iffiopuajjaout pjjautspjag 901 VNwu OOosesetaa b2-9£ €S Sayer papyoud si1aydojyo.19a-) Zbl VNU 218280048 18-92 66 SO[PROTL lpapyaid siiajdojp1a‘) OSL VNU Z81Z80048 od ql WeoI0q WOT}LOYISse[D UISTUPSIO ptoe ourury 9010S ,»UOIssaD0Y ‘saseqejep ploe Ofe[INU UI palyyUapt SHNAD 9} Jo slequiayy “fF ATAV, QIET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 87 Ceratopteris thalictroides MEYFTPRYLL IVLIAASNASAQCDB Ceratopteris richardii 718 natrieariinos. FLEVLiAREwAGAnd poccom ty aetna 751 MRALAPSFRLLAGLLLLIVLSLSSANAQC Aspergilius ee Phaatepheete nodonan G15 OE CR Ree ha wa New bee ee womans M Triticum aestivum /Phaeosphaeria nodorum1 ...... 2-262. ee ee M WO a ee VLPAPPFEAPLSPXPNTSIM ee ee es ey ie SKMS OR aie akipce Mie wear nog aig: Mung U sai giuta ang geuegiene Eee QMs| Trichoderma stromaticum ; A er ena arate neds meee ote MS Nostoc ellipsosporum boca te actin ae Eee eet 10 al re aie: : —- QQQQ 69 DGRT vyis LDFCGYGVGKSTAYVKSSTVS EEAS 69 reDaHT vive 3 LDPCGYGVGRSTAYVESSTVSEEASSG 70 LNDGHT VIGIN S| MEPCR..ASNADHVLKSS..SE..... 42 FGFEGGAHVP RVE NINH FEF QY 42 PGPEGGAHVP RVIFINNINIH PEF QY 43 FSIEGDGEVP RVIS|DN PRO ce as as bo ke ee 43 FSIEGDGEVP seek oj oben wo eghabe 3 EP ee BN TG Hp ee 62 FELEGDDG La NDIJAFSFDGHHEEEQQEEEAEPEEEE..... 44 RLDGT Md INE LVF 43 WRLDG iN LVF 43 »QLEGT RiI\s QLVV 43 LAGSS ‘AD LKYE 52 [aaa eae YiFIGo YLEF first half MEYFTPRYLLLQGFLIVI GNFLAADCLNSDGA second half Q a 2 Se he ee ee first half *, RK, sR REE Fic. 5(a). Multiple ali t of the CVNHs. - Sequence conservation is visualized ding to the are boxed in gray. The deduced secondary structure of CtCVNH is shown above the alignment. The predicted N-termini of the mature fern polypeptides are indicated by a black triangle. Fig. 5(b). Comparison of the amino acid sequences of domains 1-76 and 77-150 of CtCVNH. Sequence homology of the domains was maximized by insertion of gaps (-). Identical amino acids (-) and conserved amino acids (*) are indicated. B-strands 1-9 and 4—6 (Bewley et al., 1998). These suggest a critical structural role or their involvement in carbohydrate binding. Sequence similarity was also examined between the first (residues 1-50, according to the numbering in the N. ellipsosporum CV-N) and the second half (residues 51-101) of the CVNHs. Like CV-N, all CVNHs comprise two tandem sequence repeats with identities ranging from 24.0% to 41.1% (data not symmetrically interconnected CVNH fold (Percudani et al., 200 A neighbor-joining tree (Fig. 6) was constructed to analyze nes phylogenetic relationships of CtCVNH with other CVNHs (Table 4). It shows that CtC closely related to the member from C. richardii (BQ087187), and Cun 88 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Ceratopteris thalictroides Ceratopteris richardii (BQ087187 Selaginella moellendorffii Nostoc ellipsosporum Aspergillus oryzae Ceratopteris richardii (BQ087517) Aspergillus flavus Verticillium dahliae Hypocrea lixii Triticum aestivum / Trichoderma stromaticum Phaeosphaeria nodorum Hypocrea virens Phaeosphaeria nodorum SN15 _|OPAM Fic. 6. Phylogeny of the CVNH proteins. The unrooted tree was constructed by neighbor-joining analysis (Saitou and Nei, 1987) of genetic distances estimated with the Jones-Taylor-Thornton model. Branch lengths are proportional to genetic distances as indicated by the scale bar representing 10 PAMs (point-accepted mutations). belonging to different phyla form monophyletic groups. The CVNH domains may have common origin; however, Percudani et al. (2005) suggested that in fungi and seedless plants the domain has been separately amplified with different copy numbers following the separation of these two lineages. Predicted CtCVNH tertiary structure and the structural evolution of CVNHs/ CV-N.—Understanding the structural properties of CtCVNH is important for clarifying the conservation and variation of CVNHs as well as the roles they play in plants. In silico methods exist to predict with high reliability the tertiary structure of proteins from template structures (Saenz-Rivera et al., 2004; Gopalasubramaniam et al., 2008). Predicting a structure can yield insights into potential evolutionary patterns for CVNHs. Because CtCVNH and QI ET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 89 Fic. 7. Panel (a) overlay of predicted CtCVNH (gray) and native CV-N (black) tertiary structures. Panel (b) overlay of CrCVNH (gray) and native CV-N (black) tertiary structures. f-strands are indicated with 61-10 and helical turns with «1-4. Arrow shows the two sugar binding pockets C. richardii CVNH (CrCVNH) are approximately 50% identical, we predicted the tertiary structure of CtCVNH using CrCVNH as a template. Fig. 7a further shows that the predicted CtCVNH comprises two tandem sequence repeats. They form equivalent, elongated structures via the combination of a triple- stranded f-sheet and a f-hairpin. Thus two symmetrically related fold- domains are created, each containing a sugar-binding site. Fig. 7a indicates that CtCVNH structure is quite similar to that of native CV-N, including the positions of triple-stranded antiparallel B-sheet (the first sequence repeat: £1, 62, and $3; the second: £6, 67, and £8), B-hairpin (formed by B4 and 65, B9 and B10, respectively), and «-helical turn («1—4). However, the structures differ in that the N- and C-terminal regions are longer in CtCVNH than in CV-N, the helical turn (#3) folds differently, and an (3/4 turn) «-helix exists within the C- terminal region of predicted CtCVNH. Moreover, the B1 and £6 strands are 90 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) shorter in CtCVNH than in CV-N. To further understand the CVNH evolution in plants, we also compared the tertiary structure of CrCVNH with CV-N. Fig. 7b shows that the native CrCVNH structure is more similar to that of native CV-N, and most differences exist in the helical turn regions (a2, «3, «4) rather than in the B-strand ones. It is worthwhile to note that these differences are located in the sugar binding pockets of the proteins, which imply that CrCVNH and CV-N may have different affinities for mannose disaccharide ligands (Percudani et al., 2005) In conclusion, molecular cloning and characterization of CtCVNH showed that CtCVNH is very similar to other CVNHs from ascomycete fungi and the fern C. richardii, having a typical anti-HIV domain [F ig. 5(a), 7], indicating that CtCVNH belongs to CVNH family. This is the first time a full-length genomic DNA of CVNH in plants has been cloned. Our results provide a basis for a deeper understanding of CVNH function and evolution. ACKNOWLEDGMENTS This project was supported by the “100 Talent Project” of Chinese Academy of Sciences Msi No.: 0729281F02), the National Sean Science Foundation of China (Grant No.: 30771763, 30170101), and the ‘Outstanding Young Scientist Project’”’ of the Natural Science nde of Hubei Province, China (Grant No.: 0631061H01). LITERATURE CITED ALTSCHUL, S. F., T. L. MADDEN, A. gs eased epee is cougieeai Z. Prime es pops and me : Lipman. 1997. Gapped BLAST and PSI-BLA Pp rograms. Nucl. Oo AntAL, Z., C. Rascie, M. Fevre and C. Brue.. 2004. Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags. C 46. ARNOLD, K., L. Borpoui, J. Kopp and T. Scuwepe. 2006. The SWISS-MODEL cio oa a aa based environment for protein structure homology modeling. Bioinformatics 22:1 Barrientos, L. G. and A. M. Gronensorn. 2005. The highly specific carbohydrate- nono protein peer Piiganps anti-HIV/Ebola activity and possibilities for therapy. Mini Rev. Med. Chem. 5 BARRIENTOS, ee a R O’Keere, M. Bray, A. SANCHEZ, A. M. GRoNENBORN and M. R. Boyp. 2003. Cyanovirin-N binds to oe viral surface glycoprotein, GP1,2 and inhibits infectivity of Ebola virus. Antivir. Res. 58:47-56. Bew ey, C. A., K. R GUSTAFSON, ws R. Bop, D. G. Covet, A. Bax, G. M. Core and A. M. GRONENBORN. 1998. Solution struct yanovirin-N, a potent HIV-inactivating protein. Nat. Struct. Biol. Boimstept, A. J., B. R. O’Keere, S. R. SHENoy, J. B. McMauon and M. R. Boyp. 2001. Cyanovirin-N defines a new class of antiviral agent targeting serps - -mannose glycans in an ee manner. Mol. Pharmacol. 59 Boros, I., T. Mori, L. K. CARTNER, M. R. Boyp and A. pane so ‘Toda ol structure of a mutant oF etaionin. Biochem. Biophys. Res. Commun. 294:184—190 Boyp, M. R., K. R. Gustarson, J. B. McMauon, R. H. SHoemaxer, B. R. O’Keere, a ue sees sags Wu, M. I. Rr NS, J. H. Carpetuina, R. W. UCKHEIT, JR., P. L. Nara, L. K. PANNELL, R. C. Sowper and L. E. HENDERSON. 1997. pes dc of QIET AL.: MOLECULAR CLONING OF CYANOVIRIN-N IN CERATOPTERIS THALICTROIDES 91 cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope aeoahee gp120: potential applications to microbicide development. Antimicrob. Agents Chemother. 41:1521-1530. Co.teLuoria, D. M., D. ieee F. Kanca, T. Pacueta, R. Kussa, T. McCormicka, K. Watsons, K. McFappenc, I. CHAIKENC, R. W. Buckuerr and J. W. RoMANOA. ee: Expression, purification, and characterization of cebacat suaiiwode -N for vaginal anti-HIV microbicide development. Protein Express, Purif. 39:229-236. DELANO, W. hi 2002. The PyMOL molecular graphics system. DeLano Scientific, Palo Alto, CA org. GasTEIGER, E., Qo Genes, C. Hooc.anp, I. Ivanyi, R. D. Appe, and A. Bairocu. 2003. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucl. Acids Res. 31:3784—-3788. GasTEIcgR, E., C. HoocLanp, A. Gatriker, S. Duvaup, M. R. Winkins, R. D. Appet and A. Bairocu. 2005. Protein (denliicaticn and analysis tools on the ExPASy server. The Proteomics Protocols Handbook. Humana Press, Totow GrourjON, C, and G. DELEacr. 1995. SOPMA: significant improvements in protein eprint: Seem prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 11:681-684. GoPALASUBRAMANIAM, S. K., bee GA bdcsnunee G. B es, N. Pastor and R. ARREDONDO-PETER. 2008. Use ili lyze the tertiary structure of plant hemoglobins Math Ensymalogy 436: 393-41 0. Gouet, P., E. Courcet.e, D. I. Sruarr and F. Meroz. 1999. ESPript: analysis of multiple sequence alignments in rr hcnee Hipinfornaticn 15:305-—308. Guex, N. and M. C. Perrscu. 1997. SWISS-MODEL and the geet ait! an environment for comparative protein modeling. Bectochecie: 18:2714—2723 Gustarson, K. R., R. C. Sowner, L. E. HENperSON, J. H. CARDELLINA, J. B. McManon, U. RajAMANI Lt and M. R. Boyp. 1997. Isolation, primary sequence determination, and disulbide bond structure of cyanovirin-N, an anti-HIV (human immunodeficiency virus) protein from the cyanobacterium Nostoc EEE aes Biochem. Biophys. Res. Commun. 238:223-228. HELLE, F., C. Wycuows, N. Vu-Dac, K. R. Gustarson, C. Voisset and J. Dusuisson. 2006. Cyanovirin-N inhibits hepatitis C virus gow by binding to envelope protein glycans. J. Biol. Chem. 281:25177-25183 Konarupin, L. M., A. R. Viscomi, J. G. Jee, S. OrroneLto and A. M. GRoNENBORN. 2008. T evolutionarily conserved family of evans -N homologs: structures and carbohydrate specificity. Structure 16:570—-584 Kozak, M. 1999. Initiation of translation in prokaryotes and eukaryotes. Gene 234:187—208. Liu, Y. G. and R. F. Wurrrier. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674-681 Mok, T., R. H. SHoeMaker, R. J. GuLakowski, B. L. Krepps, J. B. McMauon, K. R. Gustarson, L. K. PANNELL and M. R. Boyp. 1997. Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV were immunodeficiency virus)-inactivating protein. Biochem Biophys. Res — 238:218—222. O’Keert, B. R., J. A. B R, J. H. Carpeuuina, R. J. Gutakowski, B. L. Krepps, J. B. MCMaAuon, R. C. Sowner, L. E. Festa: SON, L. K. PANNELL, S. A. Paro and M. R. Boyp. 1997. Isolation and became ETH of niphatevirin, a human irus-inhibitory glycoprotein m the marine sponge Niphates erecta. Eur. J. Biochem. 245: 47 —53. ae B. R., D. F. Smee, J. A. Turpin, C. J. Saucepo, K. ae idee j pipe 1): BLARESLEE, R. Buckuerr and M. R. Boyp. 2003. Potent anti-infl with viral hemagglutinin. Antimicrob. Agents Chemother. ‘47: 2518-2525. Pack, R. D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. par Biosci. 12:357-358. PERCUDANI, R., NTANINI and S. OrroneLto. 2005. The anti-HIV cyanovirin-N domain is ci: casmeried and occurs as a protein module in eukaryotes. Proteins 60:670-678. 92 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) SAENZ-RIvERA, J., G. SARATH and R. ARREDONDO-PETER. 2004. Modeling the t f (Zea mays ssp. mays) non- sagan enamine Plant Physiol. Biscay 42:891-897. Sarrou, N. and M. Net. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic iresa, Mol. Biol. Evol. 4:406—425. ScHWEDE, T., J. Kopp, N. Guex and M. C. Perrscu. 2003. ioe MODEL: an automated protein ee server. Nucl. Acids Res. 31:3381-3 Suaw, G. and R. Kamen. 1986. A conserved AU sequence from we 3! untranslated region of GM-CSF mRNA mediates come mRNA iy Say Cell 46:659-667. Su, Y. J., T. Wane, W. D. Yanc, C. Hanc and G. K. Fan. 1998. DNA extraction and RAPD analysis of P odo THompson, J. D., T. J. Gipson, F, PLEwN1AK, F. JEANMOUGIN and D. G. Hiccins. 1997. The CLUSTAL_X windows interface: flexible aie for Sia ae sequence alignment aided by quality analysis tools. Nucl. ary Res. 876-4882. Tricia, T., M. G. Pererson and D, J. Kemp. 1988. A procedure for in vitro amplification of DNA segments that lie se the boundaries of known sequences. Nucl. Acids Res. 16:8186. American Fern Journal 99(2):93—100 (2009) A New Species of Adiantum from Cuba MANUEL G. CALUFF Jardin de los Helechos de Santiago de Cuba, Centro Oriental de Ecosistemas y Biodiversidad (BIOECO), Carretera del Caney No. 129, La Caridad, Santiago de Cuba, Cédigo Postal 90400, Cuba TRACT.—Adiantum alomae is described from eastern Cuba. It is characterized by all oa of the leaves and its small size. Its habitat is also distinctive, occurring on limestone cliffs and walls, usually facing and very near the sea, receiving salt spray. A key is given to differentiate t from the related Cuban endemic Adiantum sericeum, and illustrations of the distinctive characteristics of both species are presented. Key Worps.—Adiantum alomae, Cuba, new species Adiantum is represented in Cuba by 23 species (Duek, 1971). These species grow mainly in gallery forest and secondary forest, from sea level to ca. 700 m in elevation or in coffee, cacao and citrus plantations. The genus is nearly absent in rain and cloud forests at high elevations. Two species of Adiantum grow in eastern Cuba in coastal shrub vegetation or on limestone cliffs near or facing the sea. The first is Adiantum alomae, described below, which occurs mainly along the southern coast. The second is A. deltoideum Sw. occurring along the northern coast. Eaton (1869) described Adiantum sericeum and pointed out that it differed from many other species of Adiantum by the pubescence on all parts of the leaf. Since then, many collections of densely pubescent Adiantum have been gathered in Cuba and identified as A. sericeum. Recent herbarium and field observations, however, suggest that they represent not one but two species: A. sericeum, growing in central and western Cuba, and a new one confined to eastern Cuba, which is here described. Adiantum alomae Caluff, sp. nov. TYPE. Pemiece Santiago de Cuba. Castillo del Morro y sus alrededores, en rocas y paredones calizos, localmente abundante, 0-50 m, matorral costero, 1 se hes 2007, Caluff, Shelton, & M. Serguera 6356 (Holotype: BSC!; isotypes: HAC!, HAJB!). Fig. 1 A-I. Ad A. sericeum D.C. Eaton affinis differt, frondium magnitudine (12-51 x 4— 10cm in A. sericeo autem 5-28 X 1.3-2cm in A. alomae), laminae architectura (2-3 pinnata in A. sericeo autem 1-2 pinnata in A. alomae), rhizomatis squamarum longitudine (4-20 mm in A. sericeo autem 1.3—5 mm in A. alomae), atque rachium trichomatibus (trichomata densissima, rigida, patentia obscuro-rubescentiaque in A. sericeo, autem sparsa, flexuosa, albescentia usque claro-fusca in A. alomae). Plants epipetric or rarely terrestrial. Rhizome ascending, ramified, cylindri- cal, blackish, 2-3 mm in diam., scaly toward the apex, the scales (Fig. 1E) ovate to ovate-lanceolate, brown, 1.3-5 X 0.8—-1.2 mm, nearly clathrate, the 94 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) 1mm Scene Fic. 1. Adiantum alomae Caluff. Caluff & M. Serguera 6226 (BSC). A. Silhouette; B. Sterile a pinnulae; C. Partially fertile apical pinnulae; D. Fertile apical pinnulae; E. Rhizome scales; Rhizome scale mong, tions; G. Rachis and stalks pubescence; H. Rachis unicellular hair; : Rachis pluricellular hai CALUFF: ADIANTUM ALOMAE IN CUBA 95 cells in the basal and medial portion roundish, distally enlarged, basifixed, the base truncate to more or less cordate, denticulate, the teeth (Fig. 1F) recurved or incurved, concolorous, usually conformed by two cells, the apex filiform: fronds (Fig. 1A) numerous, fasciculate, 5-30 cm long; Jaminae linear-lanceo- late, 1-pinnate or occasionally 2-pinnate in the largest medial pinnae, 4-24 x 1—3.2 cm, with an apical, the biggest, conform pinna (Fig. 1B—D), usually hairy on both surfaces; stipes cylindrical, 1-6 cm long and 0.2—1.0 mm diam., reddish brown when young, eventually blackish, lustrous, scaly at the very base, hairy throughout like the rachis and pinnae stalks (Fig. 1G), the hairs weak and deciduous, of two types: some numerous, unicellular (Fig. 11), whitish to clear brown, usually flexuous 0.8-0.9 (1.3) mm long, cylindrical, sometimes paler and flattish, with an enlarged base, others, occasional, found near the pinnae insertion and in the stalks, pluricellular (Fig. 1H), translucent, flattish, flexuous, with some cateniform cells, the septae reddish, up to 3 mm long, scales also present, these, deltate-enlarged, clear brown, lustrous, the base truncate, the cells 2-4 times longer than wide; pinnae 8-16 pairs, stalked 1-3 mm, 0.5-1.7 X 0.4—1.3 cm, alternate, with 3(-5) lobules, the margins entire to shallowly dentate, the base cuneate, lightly oblique, articulate, deciduous, the stalk and its blackish color stopping abruptly in a dilatate, discoid joint, the simple ones never overlapping the rachis, the sterile herbaceous, rounded or with blunt lobes, the fertile ones somewhat contracted, chartaceous, saggitate, the apex and lobules acute; pinnules (if any) 1 or 2, similar to the pinnae but smaller; veins free, flabellate-dichotomous, clear brown, lightly raised over the laminar tissue and hairy on both surfaces; laminar hairs abundant, unicellular, whitish, flexuous, 0.6—0.9 mm long. Sori linear, usually continuous and curved, avoiding the lobe apex; indusia brown to dark brown, entire, with numerous rigid, whitish to clear brown hairs, 0.4— 0.5 mm long; sporangia glabrous; spores tan to yellowish, globose-tetrahedral, retate, 40—45 SPECIMENS EXAMINED.—CUBA. Granma: municipio Pilédn, Boca de Toro, desembocadura del rio Boca de Toro, suelo calizo, 0-10 m, 19 Mar 1988, Gabriel Brull s/n (HAC; HAJB). Santiago de Cuba: farall6n costero, Sardinero, Santiago., 8 Jul 1949, Alain 815 (HAC); Oriente, Florida Blanca, sobre rocas, 10 Jan 1960, Hno. Alain, Acufia, Lépez-Figueiras & Ramos s/n (HAC); playa Sardinero, Justici, Santiago de Cuba, entrando a la playa, sobre paredones calizos, 5 m, matorral xeromorfo costero, 13 Jul 1980, Caluff 920 (BSC); Castillo del Morro de Santiago de Cuba, comtn en paredes y resquicios calizos, 50 msm, vegetaci6n costera, 20 Aug 1979, Caluff & Couso 1 (BSC); playa Sardinero, Reserva Siboney-Jutici, Santiago de Cuba, en rocas y paredones a 1 km de la playa, 10 m, bosque semideciduo micréfilo, 29 Feb 2007, Caluff & M. Serguera 6226 (BSC); paredones a la entrada de la playa Sardinero, Santiago de Cuba, 5 m, matorral xeromorfo costero, 19 Mar 1990, Caluff & Shelton 2942 (BSC); alrededores de Santiago de Cuba, Jul 1920, Clemente 131 (HAC); alrededores del Morro, Santiago, 18 Nov. 1937, Clemente 2248 (HAC); sobre una roca en los farallones que rodean a la Playa de Sardinero, Santiago de 96 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Cuba, 25 Nov 1951, Lopez Figueiras 315 (HAC); provincia de Oriente, cercanias de la desembocadura del rio San Juan, en la playa de Aguadores, 26 Oct 1952, Lopez Figueiras 1716 (HAC, 3 sheets; HAJB); provincia de Oriente, en farallones del Monte Picote, cercanias del central Miranda, 12 Mar 1955, Lopez Figueiras 2001 (HAC; HAJB); Santiago de Cuba, Morro Castle, 24 7. 1909, Voisard & Boelloz 1556 (HAC). Without locality: 27 Jul 1952, F. G. C. s.n. (HAC); Garcia Canizares s.n (HAJB); Cuba orientali, 1859-1860, Wright 1078 (3 sheets) (HAC). DisTRIBUTION.—Endemic to the coasts of southeastern Cuba, Santiago de Cuba and Granma Provinces, with a single small population found inland, growing in the karstic belt of the southern side of Sierra de Nipe Mountains. Hasirat.—Terrestrial or epipetric in dry coastal shrubs vegetation and in semi deciduous microphillous forest, on limestone, in cave entrances, on big rocks, cliffs and old walls, exposed or partially shaded, usually in crevices, facing the sea and receiving the salt spray, 0-50 m elevation; the population inland, on limestone cliffs, usually in open and sunny places, 80-150 m elevation. Adiantum alomae grows in dry habitats near and commonly facing the sea, receiving the salt spray. The largest population was found in the Morro Castle of Santiago de Cuba and nearby areas, consisting of hundreds of individuals. They grows on old walls, big rocks, around a cave entrance, in the ground. The stone masonry associated with the castle was built using calcareous rocks and terracotta bricks cemented with lime mortar. Adiantum alomae and A. sericeum are the only two completely hairy species of this genus in Cuba. The hairs cover the stipes, rachises, indusia, and laminar tissue on both surfaces. These species can be distinguished by the following key: 1. Leaves up to 30 X 2.3 cm; lamina 1-pinnate to occasionally 2-pinnate; the simple pinnae not sla tne ~ rachis; unicellular hairs of the non laminar axes whitish to clear brown, Oak, HOMUOUS 6s eee la he rt es A. alomae ; behead up ee ~ x 10cm; lamina 2-pinnate to occasionally 3-pinnate; basal acroscopic pinnule of each pinna elise the rachis; unicellular hairs of the non laminar axes dar reddish, acicular, rigid, peteht. 0 A. sericeum =) Adiantum alomae and A. sericeum resemble A. tricholepis Fée from the United States, Mexico, and Mesoamerica. They all have denticulate rhizome scales and pubescent laminar tissue on both surfaces. Adiantum tricholepis differs from the other two species by laminae ovate to deltate and 3-4 pinnate, the acroscopic basal pinnules not overlapping the main rachis, the rachis and costae glabrous, and the apex of the stalks not or lightly enlarged (Moran et al., 1995) Adiantum alomae is similar to A. deltoideum in habitat, small size, and pinna shape. Adiantum deltoideum differs basically in being glabrous and in its distribution, confined to the northern coast of eastern Cuba, growing likewise, on the northern and east coasts of Jamaica, and in Hispaniola (Proctor, 1985). EponyMy.—This species is dedicated to Omar Alomé Moreno, Director of the Macradenia Orchid Garden, in Palmira, Cienfuegos Province, central Cuba, CALUFF: ADIANTUM ALOMAE IN CUBA 97 who first called my attention to the differences between A. sericeum and the new species. Because the protologue for Adiantum sericeum is very simple and this rare Cuban endemic is poorly known, a complete description of this species is given. Adiantum sericeum D. C. Eaton, Botanisch Zeitung. Berlin. 27. 361. 1869. TYPE.—prope Trinidad, Wright 3950 (isotypes: MBG!; HAC!; NY! {5 sheets}). Fig. 2 A- Plants epipetric or r terrestrial. Rhizome ascending, branched, cylindrical, blackish, 2.5-9 mm in diam., densely scaly at the apex, the scales (Fig. 2E) lustrous, concolorous, light brown, deltate-attenuate to deltate-lanceolate, 4— 10 X 0.3—1.5 mm, non clathrate, basifixed, the base cordate or lightly so, the margins aie (Fig. 2F) with spaced and usually straight, minute, concolorous, one or two celled teeth, the cells in the basal portion rounded to quadrangular, toward the medial and distal portion gradually more enlarged, 2-5 times longer than wide; fronds numerous (Fig. 2A), fasciculate, ca. 51 cm long; lamina 8-38 x 4—10 cm, lanceolate to oblanceolate, 2-pinnate throughout or occasionally 3-pinnate at the base and in the base of the medial largest pinnae, gradually tapered to an apical, conform, simple pinna similar to the apical pinnules of the largest pinnae (Fig. 2. B—D), herbaceous to papyraceous, densely hairy on both surfaces with unicellular, acicular, patent, whitish to deep reddish hairs 0.6—0.9 mm long; stipes 4-13 cm long and 0.6— 1.8 mm diam., cylindrical, reddish black, lustrous, scaly at the very base with scales similar to thouse of the rhizome, densely hairy throughout like the rachises and stalks (Fig. 2G), the hairs deciduous, of two types, the commonest acicular (Fig. 2H), unicellular, patent, dark reddish, with a pustular, persistent, enlarged base, ca. 0.7mm long, and very occasional hairs pluricellular, flexuous, some flattish, translucent with reddish septae, sometimes with some cateniform cells; pinnae 10—17 pairs, alternate, 2-7 x 1—2.7 cm, oblong-attenuate, stalked 2-5 mm, lightly oblique, with a terminal conform, biggest pinnule; pinnules alternate, oblique, articulate, stalked 1—- mm, the black color of the stalk suddenly stopping at the pinnule base, leaving a discoid black joint when it falls off, the sterile ones larger, the base cuneate, distally rounded or with the apex and lobules blunt, the outer margins crenate and dentate to lobulate, the fertile ones contracted, saggitate, the apex and lobules acute, lobulate, dentate toward the lobules apex; pinnules of the first order 2—7 pairs, 0.5—1.7 X 0.5—1.7 cm, the basal acroscopic in each pinna overlapping the primary rachis, the apical one always the largest, ca. 2.1 2.3 cm; pinnules of the second order similar to those of the first order but more smaller; veins free, flabellate dichotomous, ending in teeth, light brown, lightly raised over the laminar tissue and hairy on both surfaces. Sori oblong, usually discontinuous, curved to straight, avoiding the lobe apex; indusia brown to dark brown, the margin entire, densely hairy with dark reddish, 0.4— 0.5 mm long hairs; spores tan, globose-tetrahedral, lightly tuberculate. 98 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) 0.1 mm i }. Cas H 1cm 1cm B Cc D Fic. 2. Adiantum sericeum D. C. Eaton. Caluff, Shelton & O. Alomd 6224 (BSC). A. Silhouette; B. Sterile apical pinnulae; C. Partially fertile apical pinnulae; D. Fertile apical pinnulae; E. Rhizome scale; F. Rhizome scale denticulations; G. Rachis and stalks pubescence; H. Rachis unicellular hair. CALUFF: ADIANTUM ALOMAE IN CUBA 99 SPECIMENS EXAMINED.—CUBA. Sancti Spiritus: Farallé6n del Charco de Oro, rio Higuanojo, Area Protegida El Naranjal, Alturas de Sancti Spiritus, en farallones calizos, 300 m, 26 Aug 1994, E. Bécger & E. Martinez 3444 (BSC); alrededores del Hoyo del Naranjal, mdrgenes del rio Higuanojo, Alturas de Sancti Spiritus, prov. Sancti Spiritus, en farallones rocosos, 280 m, bosque siempreverde secundario, 30 Nov 1994, Caluff & Shelton 3854, 3855, 3856 A/B (BSC): cascada Las Cortinas, arroyo La Yaba, finca La Vega, km 40 de la carretera desde Cienfuegos a Trinidad, a unos 200 m de la carretera, en un pedregal de rocas micdceas carbonatadas esquistosas, subiendo por el lado derecho de la cascada, 60-80 m, en bosque semideciduo mes6filo, 3 Feb 2007, Caluff, Shelton & O. Alomd 6224 (BSC, 16 sheets); Cienfuegos: arroyo Navarro, Mina Carlota, SE de Cumanayagua, Sierra de San Juan, 330 m., 22 Mar 1957, Proctor 29409 (HAC). EASTERN CUBA. Pinar del Rio: paredones del Pan de Aziicar, Vifiales, del Rio, 5 Feb 1956, Acufia & Morton 20106 (HAC); sobre las rocas, base del mogote Pan de Azticar, Vifiales, 9 Oct 1955, Alain 4425 (HAC); La Guira, 7 km de Punta de La Sierra, Pinar del Rio, exploracién sur del mogote, 12 Nov 1972, Bobrov & Cdrdenas 29409 (HAC); exploracion sur del mogote La Guira, 12 Nov. 1972, Bobrov & Cardenas 29811 (HAC); cercanias de Sumidero, Pinar del Rio, Jul 1012, J. A. Shafer & Leén 3171, (Shaffer 13407) (HAC). DisTRIBUTION.—Endemic to central Cuba, Sancti Spiritus, and Cienfuegos Provinces, Trinidad and Sancti Spiritus Heights, and western Cuba, Pinar del Rio Province, Sierra de los Organos. Hasirat.—Semi deciduous and evergreen secondary forest and in karstic (mogote) vegetation, on limestone, in well drained and inclined stony soil, on big rocks and cliffs, usually in crevices, in filtered sun, 60-300 m alt., locally common. Adiantum sericeum grows inland in moderately humid places, typically with pteridophytes such as Adiantopsis rupicola Maxon, Adiantum fragile Sw., Adiantum tenerum Sw., Anemia adiantifolia (L.) Sw., Anemia cuneata Poepp. ex Spreng, Blechnum occidentale L., Lygodium venustum Sw., Pteris longifolia L., Selaginella eatonii Hieron. ex Small, Selaginella spp., Thelypteris dissimulans (Maxon & C. Chr.) C. F. Reed, T. kunthii (Desv.) C. V. Morton, and T. scolopendrioides (L.) Proctor. ACKNOWLEDGMENTS I thank Dr. Robbin C. Moran and Dr. Victor Fuentes Fiallo, and the two anonymous referees for revising the manuscript; MSc. Susana Carreras G. for the Latin diagnosis, and Ing. Maité Serguera N. for spore determination. LITERATURE CITED Duek, J. J. 1971-1972. Lista de las especies cubanas de Pyoopenianny Psilotophyta, Equisetophyta y Polypodiophyta (Pteridophyta). Adansonia, ser. 2, 11:559-578, 717-731 Eaton, D. C. 1989. Ein neues Adiantum von Cuba. Botanisch Zeitung. Berlin. 27. 361. 100 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) and A. C. Jermy. 1995. Adiantum. In Moran, R.C. & R. Riba. (eds. ge ae es tastes , G. M., M. Sousa S. and S. Knapp (eds. pa Flora Mesoamericana. Vol. 1 Pullotacen a Salviniaceae. Univ. Nac. Aut6noma de México, Inst. de Biologia, México, Proctor, G. R. 1985. esas of ee British Museum (Nat. Hist.) London. American Fern Journal 99(2):101—105 (2009) A New Brazilian Species of the Genus Asplenium L. (Aspleniaceae) FERNANDO B. Matos Universidade Federal do Parana, Depto. de Botanica, C.P. 19031, 81531-980, Curitiba, PR, Brazil. fbttms@yahoo.com.br Pauto H. Lasiak Universidade Federal do Parana, Depto. de Botanica, C.P. 19031, 81531-980, Curitiba, PR, Brazil. plabiak@ufpr.br LANA S. SYLVESTRE Universidade Federal Rural do Rio de Janeiro, Depto. de Botanica, BR-465, Km 7, CEP 23890-000, Seropédica, RJ, Brazil. lana@ufrrj.br Asstract.—Asplenium truncorum, a new asplenioid fern from the Brazilian Atlantic Rain Forest, is described, illustrated and compared to the most similar species. So far, it seems to be restricted to the montane moist forests of southern Bahia and Espirito Santo States, at elevations of 750 to 950 m. Field observations suggest that this species grows exclusively as an epiphyte on the trunks of tree ferns, especially Alsophila setosa Kaulf. (Cyatheaceae). Key Worps.—Asplenium truncorum, Atlantic Rain Forest, Bahia, Espirito Santo, ferns, taxonomy The asplenioid ferns, including the genus Asplenium L. and its putative segregates, make up one of the most species-rich groups among leptospo- rangiate ferns, comprising approximately 700 species, mainly with tropical distribution (Schneider et al., 2004; Smith et al., 2006). According to Sylvestre and Windisch (2003), Brazil harbors about 70 species of Asplenium, representing nearly half of the diversity found in the Neotropics (close to 150 species, according to Tryon and Tryon, 1982). As is the case with many other fern genera (e.g., Moran, 1981; Moran et al., in press), the Serra do Mar mountains along the coast of southeastern Brazil play a very important role in the diversification of this group, presenting a high level of endemism. Recent botanical expeditions to these mountains, in the States of Bahia and Espirito Santo, have revealed a new species of the genus Asplenium, which we describe as follows: Asplenium truncorum F. B. Matos, oie & L. Sylvestre, sp. nov. TYPE.— BRAZIL. Bahia: — RPPN Serra Bonita, 15°23'25”S, 39°34'05”"W, 920 m, 29 Jul 2008, F. B. Matos et oa 1537 (holotype: UPCB; isotypes: CEPEC, NY, RB, po Figs. 1, 2A-C, F-G. Species Asplenio martiano C. Chr. similaris, differt laminis minus divisis, ad basin 1-pinnatis, petiolis laminis dimidio brevioribus et habitu epiphytico. Plants epiphytic. Rhizomes erect; scales 1.5—-2 < 0.3—0.5 mm, lanceolate, atrocastaneous, clathrate, tips twisted and long attenuate, margins with 102 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) rs ee 7 ~—— a ee — ae ry oo ee cage - r _ At SPO ~ * Ke Fic. 1. A-—D. Asplenium truncorum (Matos 1537, UPCB). A. Habit. B. Medial portion of the lamina. C. Rachis and sori detail. D. Rhizome scales. irregular projections; roots thin and wiry, not proliferous. Fronds (5)10—16(30) cm long, arcuate, clustered; indument abaxially of scattered, linear, clathrate scales, 0.2-1 mm long, also with inconspicuous clavate hairs, especially on leaf axes. Stipes 2—5(8) cm long, 0.3-0.7 mm diam., ca. 1/3—1/2 of the lamina length, brownish at base and greenish to stramineous distally, dull, with narrow green wings less than 0.4 mm wide. Blades 4—15(21) cm long X 1—5(13) MATOS ET AL.: A NEW BRAZILIAN SPECIES OF ASPLENIUM 103 Fic. 2. A-—C. Silhouettes illustrating the morphological variation in Asplenium truncorum. A. (Matos 1537, MBM). B. (Matos et al. 806, CEPEC). C. (Matos 1537, UPCB). D-E. Asplenium martianum (Handro 2664, NY), spores with alate folds, echinulate wings. D. Lateral view of the spore. E. Distal view of the spore. F—G. A. truncorum (Thomas et al. 13796, CEPEC), spores with low folds and large areolas. F. Lateral view of the spore. G. Proximal and lateral view of the spores. cm wide, membranaceous, 1-pinnate proximally, with long attenuate pinnat- ifid apices; rachises greenish to stramineous, dull, with narrow green wings up to 0.5 mm wide; pinnae 1-11 cm long, less than 1 cm wide, flabellate to linear- lanceate, subfalcate, 2—5 pairs, the bases cuneate, non-auriculate, apices obtuse 104 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) to long-attenuate, margins dentate; veins mostly simple except for the proximal ones, which are forked, readily visible on both sides, vein ends expanded adaxially. Sori 1-5(12) pairs per pinna, occasionally diplazioid; indusia 5 mm long < 0.3 mm wide, linear, firmly membranaceous, margins entire; spores reniform, monolete, with a few large and broad anastomosing ridges. DisTRIBUTION AND EcoLocy.—Asplenium truncorum is known only from the montane moist forests of coastal Brazil, in the States of Bahia and Espirito Santo, at 750-950 m above sea level. This species seems to grow exclusively as a low-trunk epiphyte on tree ferns (Fig. 1, A), especially Alsophila setosa Kaulf. (Cyatheaceae). CoNsERVATION.—Despite of its remarkable species richness and exceptional concentration of endemics, the devastation of the Brazilian Atlantic Forest continues at a very alarming rate. Nowadays it is considered one of the most threatened biomes on Earth, with a very fragmented distribution along the Brazilian coast. Because it has a narrow extent of occurrence in this scenario, Asplenium truncorum meets the IUCN criteria (IUCN, 2001) of vulnerable species (VU: B1 a + b iii). Etymo.ocy.—The specific epithet ‘‘truncorum’’ was chosen due to its habitat preference for tree fern trunks. PaRATYPES.—BRAZIL. Bahia: Almadina, Serra do Corcovado, 9,8 km ao SW de Coaraci na estrada para Almadina, dai N até a Fazenda Sao José, 14°42’21’S, 39°36'12”W, 750 m, 19 Apr 2007, Matos et al. 1408 (CEPEC, UPCB); Camacan, RPPN Serra Bonita, 15°23'30"S, 39°33’55”W, 835 m, 1 Feb 2004, Thomas et al. 13796 (CEPEC); Camacan, RPPN Serra Bonita, 15°23’30’S, 39°33'55”W, 835 m, 3 Feb 2005, Matos et al. 305 (CEPEC, UPCB); Camacan, RPPN Serra Bonita, 15°23'30"S, 39°33'55”"W, 835 m, 13 Feb 2005, Matos et al. 446 (CEPEC, UPCB); Jussari, RPPN Serra do Teimoso [750 m], 27 Jul 2005, Matos et al. 806 (CEPEC, UPCB). Espirito Santo: Santa Teresa, Alto Sao Lourenco, Sitio da Cachoeira, 25 Oct 2000, Demuner et al. 1477 (BHCB, MBML); Santa Teresa, Nova Lombardia, Reserva Biolégica Augusto Ruschi [800 m], 27 Jul 2002, Vervloet et al. 559 (BHCB, MBML); Santa Teresa, Nova Lombardia, Reserva Biolégica Augusto Ruschi [800 m], 18 Dec 2002, Rose & Pereira 20 (BHCB, MBML). Asplenium truncorum can be recognized by its erect rhizome, stipes with ca. 1/3 to 1/2 of the lamina length, 1-pinnate or less divided lamina, non-conform apical pinnae, and membranaceous to chartaceous leaf texture. Superficially, it resembles Asplenium auriculatum Sw. in habit, leaf dissection and color. However, the latter can be easily recognized by the presence of prominent auricles in the acroscopic base of the pinnae that often overlap the rachis. Asplenium martianum C. Chr. is probably one of the most closely related species in Brazil, being distinct by its longer stipes (the same length as the lamina or longer), blades usually 2-pinnate at base (or at least deeply 1- pinnate-pinatifid), and preferentially terrestrial habitat. Besides that, their spores are quite distinct, with those of Asplenium martianum showing alate MATOS ET AL.: A NEW BRAZILIAN SPECIES OF ASPLENIUM 105 folds and echinulate wings (Fig. 2, D-E). Asplenium austrobrasiliense (Christ) Maxon also seems to be related morphologically, differing mainly by its chartaceous to coriaceous blades with conform apical pinnae, and longer stipes with approximately the same length as the laminae. Asplenium cariocanum Brade, which is ecologically similar in habitat, differs in having fringed stem scales with pronounced dark teeth, pinnae with lobately serrate margins and nearly symmetric pinnae bases that are usually auriculate. ACKNOWLEDGMENTS The authors thank André M. Amorim (UESC) and Wm. Wayt Thomas (NYBG) for supporting field work in southern Bahia and studies at the NYBG, as part of the project “Flora of the montane forests in Southern Bahia, Brazil’’ (Beneficia Foundation, NSF 9972116, NGS 7785-05, and CNPq 474648-4), and CAPES for providing the Master’s scholarship to the first author. This contribution was also partially funded by CNPq (Proc. n. 306878/2007-0 and 309415/2008-0) and NSF (DEB 0717056, in name of Dr. Robbin C. Moran). We also thank Dr. William A. Rodrigues for the Latin diagnosis, Diana Carneiro for preparing the illustrations, and Judith Garrison Hanks for preparing the SEM images of the spores. LITERATURE CITED IUCN. 2001. IUCN Red list categories and criteria: Version 3.1. IUCN Species Survival Comission. IUCN, Gland, Switzerland and Cambri Moran, R. C. 1987. meen S - - Neotrenica hrs genus Polybotrya (Dryopteridaceae). Illinois Nat. Hist. Surv. Bull. 34:1 Moran, R. C., J. Prapo and P. pos Megalastrum (Dryopteridaceae) in Brazil, Paraguay and Uruguay. aes Fern J. (in press). penn = S. J. Russet, C. J. Cox, F. BAKKER, S. HANDERSON, F. Rumsey, J. Barrett, M. Gipsy and J. C. VoceL. 2004. Chloroplast phylogeny of asplenioid ferns based on rbcL and trnL-F spacer squncos Piles alata Aspleniaceae) and its implications for a sear Syst. Bot. 29:260— Situ, A. R., fs M. Pryer, E. ScHuETTPELZ, P. Koratt, H. ScHNemper and P. G. Wo r. 2008. Fern classification. Pp. 417-467. In: Ranker, T. A. and C. H. Haurter. (eds.). ‘Daloay and evolution of ferns and lycophytes. Cambridge University Press, United Kingdom. Sytvestre, L. S. and P. G. Winpiscu. 2003. Diversity and distribution patterns of cagogreangel in Brazil. Pp. 107-120. In: CHanpra, S. and M. Srivastava. (eds.). Pteridology in the N Millennium. Kluwer Academic posoncpuit Dordac ht. Tryon, R. M. and A. F. Tryon. 1982. Ferns and allied plants, with special reference to tropical America. Springer-Verlag, New York American Fern Journal 99(2):106—108 (2009) New Combinations in Pleopeltis (Polypodiaceae) from Southeastern Brazil ALEXANDRE SALINO Departamento de Botanica, Instituto de Ciéncias Biolégicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 30123-970, Belo Horizonte, MG, Brasil, email: salino@icb.ufmg.br ABsTRACT.—From taxonomic studies of Pleopeltis from southeastern Brazil, some new combina- tions are made: Pleopeltis alborufula (Brade) Salino, P. bradei (de la Sota) Salino, P. desvauxii (Klotzsch) Salino, P. minarum (Weath.) Salino, P. monoides (Weath.) Salino, and P. trindadensis (Brade) Salino. Key Worps.—Ferns, pteridophytes, Polypodiaceae, Pleopeltis, Southeastern Brazil The generic limits of Pleopeltis are under active revision (Andrews and Windham, 1993; Windham, 1993; Sota, 2003; Schneider et al., 2004; Schneider et al., unpubl. ms.). The most recent definition of Pleopeltis based on molecular phylogeny (Schneider et al., 2004) includes the genera Dicranoglossum and Neurodium, as well as the Polypodium species with scaly leaf blades. The distribution of scales in th q te species varies widely, but at least some are always present between the veins on the abaxial side of the blade (Kessler and Smith, 2005). In this definition, Pleopeltis comprises about 75 Neotropical and a few African species (Kessler and Smith, 2005). Some of the necessary combinations in Pleopeltis for Brazilian squamate Polypodium have been made by Sota (2003), Kessler and Smith (2005) and Sota etal. in Zuloaga et al. (2007), but other Polypodium and Dicranoglossum species need to be transferred. The necessary new combinations are proposed here to allow their use in regional floras and modern taxonomic treatments. The Polypodium species combined here were studied by Weatherby (1947) and Sota (1965, 1966) and clearly belong to the squamate clade of Schneider et al. (2004). With these additions, Pleopeltis is represented in Brazil by 14 species, with seven endemic to th theastern region New ComsBINATIONS Pleopeltis alborufula (Brade) Salino, comb. nov. Polypodium alborufulum Brade, Arq. Jard. Bot. Rio de Janeiro 11: 29. 1951. TYPE.—BRAZIL, Espirito Santo, Castelo, Forno Grande, 12 May 1949, A.C. Brade 19791 (RB !). DisTRIBUTION.—Endemic to Brazil (only in Espirito Santo state). Pleopeltis bradei (de la Sota) Salino, comb. nov. Polypodium bradei de La Sota, Revista Mus. La Plata, Secc. Bot. 9 (42): 266. 1965. TYPE.—BRAZIL, SALINO: NEW COMBINATIONS IN PLEOPELTIS 107 Espirito Santo, Castelo, Forno Grande, 12 May 1949, A.C. Brade 19791 B (RB!) DistRIBUTION.—Endemic to Brazil (only in Espirito Santo state). Pleopeltis desvauxii (Klotzsch) Salino, comb. nov. Taenitis desvauxii Klotzsch, Linnaea 20: 431. 1847. LECTOTYPE.—(designated by Proctor, Flora Lesser Antiles 348. 1977): Hooker & Greville, Icon. Fil. 1, t.7. 1827, based on a Guilding specimen from St. Vincent (not seen). DisTRiBUTION.—Neotropical. Pleopeltis minarum (Weath.) Salino, comb. nov. Polypodium minarum Weath., Contr. Gray Herb. 165: 78. 1947. TYPE.—BRAZIL, Minas Gerais, Serra da Piedade, 1843. Claussen 78 (P, not seen). DistrIBUTION.—Endemic to Espinhago range and Iron Quadrangule, in Minas Gerais state, Brazil. Pleopeltis monoides (Weath.) Salino, comb. nov. Polypodium monoides Weath., Contr. Gray Herb. 165: 78. 1947. TYPE.—BRAZIL, Bahia, forests of the rio Gongogi basin, 100-300 m, 10/30 Nov. 1915 (US!). DisTRIBUTION.—Endemic to Brazil (Bahia, Espirito Santo, and Minas Gerais states) Pleopeltis parenenoe (Brade) eee comb. nov. Polypodium trindadense Brade, Arq. Inst. Biol. Veg. 3: 4, t. 3, 4, 6. 1936. TYPE.—-BRAZIL, Ilha da Trindade, 14 i 1937, C, sate 585 (RB!). DIsTRIBUTION.—Endemic to Trindade Island, Brazil. LITERATURE CITED Anprews, E. G., M. D. WinpHaM. 1993. Pleopeltis Humbold & Bonpland ex Willdenow, in Fl. North Amer. Ed. Comm. (ed.). Flora of North America, North of Mexico, 2:324—327. New York, Oxford: pone University Press. Kessier, M. and A. R. SmirH. 2005. Seven new species, ae new combinations, and one new name of Polypodaceae from Bolivia. Candollea emu 271- ScHNEIDER, H.., Situ, R. Cranriti, T. J. H ies H. Haur_er and T Unraveling we phylogeny of poly eenadd grand (Polypodiaceae sad es ammitidaceae): exploring aspects of the aedsice of epiphytic plants. Molecular Phylogenetics and Evolution 31:1041-1063. Sora, E. R. DELA. 1965. Las species escamosas del género “Polypodium” L. (s. str.) en Brasil. Revista Museu de la Plata, Secc. Botanica 9:243-271 Sora, E. R. DELA. 1966. Revision de las species americanas del grupo “Polypodium squamatum’”’ L. Polypodiaceae (s. str.). Revista Museu de la Plata, Secc. Botanica 10:69-186. 108 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Sora, E. R. pDELA. 2003. Nueva combinacién Itis (P ). Hickenia 3(47):195-197. Sota, E. R. peLa., A. Saino and F. C. Assis. sei ie ke ea In: F. O. Zutoaca, O. ‘Morr NE and M. J. Betcrano, (eds.). Novedades taxonémicas y nomenclaturales para la flora pani del cono sur de Sudamérica. Darwiniana 45(2):236—241. Weatuersy, C. A. 1947. Polypodium lepidopteris and its relatives in Brazil. Contribution from the Gray Herbarium 1 WinpuaM, M. D. 1993. New taxa and nomenclatural changes in the a American fern flora. Contributions from the University Michigan Herbarium 19:31-6 American Fern Journal 99(2):109—116 (2009) A Hybrid Phlebodium (Polypodiaceae, Polypodiophyta) and Its Influence on the Circumscription of the Genus J. DANtEL TEyERO-DfEz Universidad Nacional Auténoma de México, Facultad de Estudios Superiores Iztacala, arrera de Biologia, Apartado Postal 314, Tlalnepantla 54090, México, México. JOHN T. MICKEL The New York Botanical Garden, Bronx, NY 10458-5126, U.S.A. ALAN R. SMITH University Herbarium, University of California, Berkeley, CA 94720-2465, U.S.A. TRACT.—The fern genus Phlebodium is traditionally described as having a row of costal areoles lacking included veins, with the sori located in extra-costal areoles and each sorus served by two veinlets. The discovery of a hybrid between Phlebodium pseudoaureum and Polypodium pleurosorum raises questions about the limits of Phlebodium and necessitates a revised taxonomic circumscription of the genus Key Worps.—ferns, hybrid, Mexico, Phlebodium The fern genus Phlebodium has a Spun aay distribution and has been thought to comprise three species: P. aureum (L.) J. Sm., P. decumanum (Willd.) J. Sm., and P. pseudoaureum (Cav) Lellinger isyn, P. areolatum (Humb. & Bonpl. ex Willd.) J. Sm.] (see e.g., Proctor, 1989; Nauman, 1993; Mickel and Smith, 2004). When first recognized at generic rank, Phlebodium (R. Br.) J. Sm., based on Polypodium sect. Phlebodium R. Br., was a superfluous name because it included sect. Pleopeltis Humb. & Bonpl. ex Willd., an older name that should have been adopted under current rules (see Smith, 1981). Article 52.3 (McNeill et al., 2005; see also its Ex. 15) is applicable to this matter. Since Phlebodium is based on a name-bringing synonym (in other words, it has a basionym, i.e., Polypodium sect. Phlebodium R. Br., that is legitimate), Phlebodium is not illegitimate. Because Smith’s genus was a stat. nov., Art. 7.4 dictates that the type of R. Brown’s section must also be the type of Phlebodium. Art. 10.2 establishes that the type must be either P. aureum or P. decumanum, given that these were the only two species included in sect. Phlebodium by Brown. Phlebodium was lectotypified by Phlebodium aureum (L.) J. Sm. (Smith, 1875), and this choice has been reaffirmed by several authorities (e.g., Copeland, 1947; Tryon and Tryon, 1982). Phlebodium has usually been characterized by venation that is highly reticulate (but free near margins), with 1 to 4 rows of fertile costal polygonal areoles and two or three rows of alternate marginal sterile areoles (without free included veinlets) (Fig. 1J). The costal areoles include one secondary areole that extends laterally from secondary vein to secondary vein, with two 110 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) included excurrent veinlets meeting at apices. The genus is further characterized by having pinnatifid to pinnatisect blades (Fig. 1G). Often, Phlebodium aureum has been treated in a broad sense (e.g., by Tryon and Stolze, 1993), to include also Ph. pseudoaureum and segregates of that species. Tryon and Tryon (1982) placed Phlebodium aureum s.]. and Polypodium lowei C. Chr. [= Po. pleurosorum] in with a group of Mexican and Mesoamerican species related to Polypodium plesiosorum Kunze, P. subpetiolatum Hook., and several other species. The Po. plesiosorum group is now thought to be closely related to true Polypodium (type: Po. vulgare L.), and less closely related to Phlebodium (Schneider et al., 2006; Tejero-Diez, 2005). In 2002, the first author (JDTD) discovered in Chiapas, Mexico, a specimen (Fig. 1 A-C) that appears to be a hybrid between the most common species of Phlebodium in Mexico, Ph. pseudoaureum (Figs. 1G—J), and a simply pinnate species of Polypodium, Po. pleurosorum Kunze ex Mett. (Figs. 1D-F). The plant has blades that are pinnate proximally and pinnatifid distally, a mixture of sori each served by a single vein or by two veins, and differential development of secondary costal sterile areoles (Figs. 1A and C). Its sori have abundant sporangia and mostly malformed spores (Fig. 2H). Some authorities have considered Phlebodium and Polypodium as only distantly related (e.g., Copeland, 1947, who thought Phlebodium to be derived from Pleopeltis), while others have thought them to be more intimately related (e.g., Tryon and Tryon, 1982, p. 691). Closer examination was made to see if Polypodium pleurosorum might in fact belong to Phlebodium. Moore (1855), in his description of Polypodium pleurosorum (under the name Phlebodium inaequale T. Moore) wrote: “The sori are large, round, situated in a single series near the midrib; sometimes seated on the apex of a veinlet within a costal areole, which is characteristic of Goniophlebium; sometimes on a veinlet exterior to the costal areole, sometimes at the point where two or more veins unite, which is the normal condition of Phlebodium. It is consequently an osculating species between the genus Goniophlebium and Phlebodium.” He also noted that it resembles Phlebodium aureum but has truly pinnate fronds. Examination of herbarium specimens of Polypodium pleurosorum shows that although most of the sori are located in costal areoles and served by a single vein, there are occasional sori, especially distally, that are served by two veins. Recent phylogenetic studies based on DNA molecular characters (Schneider et al., 2004; Schuettpelz and Pryer, 2007) show that Phlebodium pseudoaur- eum and P. decumanum are sister to a clade comprising species of Pecluma. Sampled are Pe. alfredii (Rosenst.) M. G. Price, Pe. eurybasis (C. Chr.) M. G. Price, and Pe. ptilodon (Kunze) M. G. Price and two Mexican/Mesoamerican species of Polypodium, Po. hartwegianum Hook. and Po. longepinnulatum E. Fourn. the last two species, as well as some others, are probably better referred to Pecluma, but these transfers await more comprehensive sampling in the Pecluma clade. The Phlebodium + Pecluma clade is in turn sister to a large group (75+ spp.) of scaly polypods, the Pleopeltis clade (Otto et al., in press), including scaly species usually included in Polypodium s.]. The true Polypodium clade, comprising Po. vulgare L. and allies (Haufler and Ranker, TEJERO-DIEZ ET AL.: A HYBRID PHLEBODIUM 111 Fic. 1. A—-C. Phlebodium X hemipinnatum (Tejero-Diez 4362, NY). A. Habit. B. Rhizome scale. C. Pinna detail. D-F. Phlebodium inaequale (Mickel 1099, NY). D. Rhizome and pinnae. E. Rhizome ale. F. Pinna detail. G-J. Phlebodium pseudoaureum (King & Soderstrom 4757, MICH). G. Rhizome and blade. H. Rhizome scale. J. Pinna detail. 112 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Fic. 2. Spores of Phlebodium. A-C. Phlebodium inaequale (Tejero-Diez 4931, IZTA). A. Distal view. B. Medial view. C. Proximal view. D. Phlebodium pseudoaureum (Tejero-Diez 4195, IZTA). Medial view (bar 20 um). E-H. Phlebodium Soares 0° eaegleat (Tejero-Diez 4362, MEXU). E. Medial view. F, G. Proximo-medial view. H. Malformed spor 1995), is yet more distantly related to Phlebodium. Phlebodium inaequale has now also been sampled for DNA (Schneider, unpubl. data), and nucleotide sequence data show that Phlebodium, as redefined here and including the newly transferred P. inaequale, is monophyletic, with strong bootstrap and Bayesian support, sister to the Pecluma alliance (Schneider, pers. comm Proctor (1989) reported that in Puerto Rico, where Phlebodium aureum, Ph. pseudoaureum, and Ph. decumanum occur together, both P. pseudoaureum and P. decumanum appear to be diploid, Phlebodium aureum s.s. is t fertile, allotetraploid hybrid, and at least one sterile, triploid backcross hybrid was reported. Chromosome counts for Phlebodium include three counts of 2n = 74 (diploid, based on x = 37) for Ph. decumanum from Trinidad (Walker, TEJERO-DIEZ ET AL.: A HYBRID PHLEBODIUM 113 1985), three counts of n = 74, 2n = 147 for Ph. aureum from Trinidad and Tobago (Walker, 1985), and four counts n = 37, 2n = 74 of Ph. aureums.1. from Jamaica and Mexico (Walker, 1966; Mickel and Smith, 1977, reported as Po. araneosum M. Martens & Galeotti, now considered a synonym of Ph. pseudoaureum). These last diploid counts likely pertain to the species now called Ph. pseudoaureum, and not the true Ph. aureum, which appears to be tetraploid. Walker (1985) reported spontaneous, sterile, triploid hybrids between what he called Po. aureum s.l. and Po. decumanum in Trinidad. There is also an early report of a hybrid called Phlebodium Xschneideri, reputed to be the hybrid between Po. aureum s.l. and Po. vulgare L. (Schneider, 1894). The parentage of this hybrid now seems in doubt, because of the relatively distant relationship between Phlebodium and Polypodium, as currently defined. In an attempt to verify hypothesized relationships among species of Phlebodium, Caruso (1985) studied living plants of Phlebodium aureum, Ph. pseudoaureum, and Ph. decumanum growing in the greenhouses of the New York Botanical Garden. Although cytological studies were unsuccessful, measurements of spores and stomatal guard cells showed significant differences, with the tetraploid, Ph. aureum having the larger measurements. The rarity of the Tejero-Diez collection (4362) and its morphological intermediacy suggest that it is a hybrid, and with its significant bearing on the circumscription of the genus Phlebodium, we hereby give it a hybrid name. Phlebodium Xhemipinnatum Tejero, Mickel and A. R. Smith, hyb. nov. TYPE.—MEXICO: Chiapas, Mpio. San Cristébal de las Casas, Km 67 de la carretera federal 190, Tuxtla Gutiérrez a San Cristébal de las Casas (16° 42’ 23” N, 92° 46’ W), bosque de Pinus-Quercus, 2440 m, 6 Ago 2002, Tejero- Diez 4362 (Holotype: MEXU; isotypes: IEB, IZTA, NY, UAMIZ). Figs. 1A-C. Phlebodio pseudoaureo atque Polypodio pleurosoro proxima, sed laminis hemipinnatis, id est basis pinnatis apiceque pinnatifidis, plane differt. Rhizomes long-creeping, 4-6 mm diam. (excluding scales), pruinose, densely scaly; rhizome scales 8-12 X 2—4 mm, ovate, long-attenuate, yellowish brown, each with enlarged, round, peltate base, darker at point of attachment, margins denticulate to short-ciliate and erose throughout, with short to long, flexuous, contorted, hairlike tips; fronds (55) 60-70 cm long; stipes 1/3—1/2 the frond length, brown, glabrous; blades ovate-deltate to broadly-oblong, 26— 35 cm wide, 1-pinnate at middle basal part, becoming pinnatifid above the middle, terminal segment subconform, 5-16 cm long; pinnae (segments) 8—12 pairs, 12-30 mm wide, linear-oblong to linear-lanceolate, some falcate, acuminate, glabrous, green-yellowish, margins entire to repand; veins netted, free near margins, with 1 row of fertile costal polygonal areoles, each with a single simple or bifurcate, excurrent included veinlet or 2 veinlets that form a secondary areole and meet at their tips, 2-3 rows of similar areoles closer to pinna margins, these mostly without included veinlets; sori round, 2-3 mm diam., submedial, one row on each side of the costa; spores mostly malformed, 114 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) bilateral, monolete, (33)39(45) x es et um, tuberculate, tubercles dome- shaped, somewhat overlapping, am ParaTyPE.—MEXICO: Chiapas, Mpio. Tenejapa, a 3.5 km al NE del paraje Balum Canal (16° 48’ 05” N, 92° 31’ 50” W), Acahual derivado de bosque de Pinus-Quercus, 2200 m, 8 Mar 1995, Ramirez-Marcial & Herndndez-Rojas 654 (MEXU!, ECOSUR - herbarium of the Colegio de la Frontera Sur, Chetmul, Quintana Roo, Mexico). Hasirat.—Epiphytic in pine-oak forests and adjacent disturbed areas; 2200— 2500 m. DisTRIBUTION.—Mexico, Chiapas, montane areas. The existence of this new hybrid, with characters intermediate between Phlebodium pseudoaureum and Polypodium pleurosorum, causes us to conclude that the latter species can once again be included in the genus Phlebodium, with the earliest available name, Ph. inaequale T. Moore. Impetus for the recircumscription of polypod genera has been given by several other recent phylogenetic studies on Polypodiaceae, most importantly the one by Schneider et al. (2004), outlining a global phylogeny for the family. Subsequently, several other papers directed toward the placement of problematic Neotropical polypods have appeared (e.g., Krier et al., 2007; Schneider et al., 2006; Tejero-Diez, 2005), are in press (Krier et al., 2008), or have been submitted for publication (Otto et al., in press). The redefinition of Phlebodium also recalls the recent recircumscription of the polypod genus Microgramma, necessitated by the finding of a new and radically different species of the genus in coastal Brazil (Salino et al., in press). Cladistic analysis of morphological characters in species of Polypodium and related taxa (Tejero-Diez, 2005) suggests that the critical characters separating Phlebodium from its sister group (Pecluma, and Mexican/Mesoamerican species allied to Pecluma but still placed in Polypodium; Schneider et al., 2004; Schuettpelz and Pryer, 2007) are: a) spores with tuberculate ornamen- tation (Fig. 2A—H; b) small size of spore body (33) 38 (45) um; and c) the presence of several rows of marginal sterile polygonal areoles. Of the aforementioned characters, the spore ornamentation in Phlebodium and the smaller spore size are unique in Polypodiaceae, but the ornamentation is somewhat similar to spores of Polypodium arcanum Maxon and some species of Serpocaulon (Tryon and Lugardon, 1991; Tejero-Diez, 2005). It is clear that the taxonomic limits of Phlebodium cannot be governed by the way in which the internal veinlets of the main costal areoles are organized. The species of Phlebodium and the newly described hybrid can be separated by the following key: 1. Blades 1-pinnate, at least proximally; sori each at the end of a simple or bifurcate veinlet; secondary costal areoles absent or irregularly so. 2. Blades pinnate throughout their length... ... . 2... oe oe ee ee we P. inaequale 2. Blades pinnate proximally but pinnatisect or pinnatifid distally....... P. Xhemipinnatum 1. Blades pinnatifid or pinnatisect; sori each at the end of two veinlets; secondary costal areoles regularly present. TEJERO-DIEZ ET AL.: A HYBRID PHLEBODIUM 115 3. Sori in 1 row on each side of costae; (170-)550-2500 m.............. P. pseudoaureum 3. Sori in 2 or more rows on each side of costae; 0-500 m. 4, Sori on 3 or more rows on each side of costae, 2. 2... P. decumanum 4. Sori on 2 (infrequently 1) rows on each side of costae.................2-. P. aureum The use of the name Phlebodium inaequale T. Moore for what has been called Polypodium pleurosorum Kunze ex Mett. requires a brief explanation. The former name was published first by Moore (1855), but when treated as belonging in Polypodium cannot be used because of the existence of an earlier homonym, Polypodium inaequale Link, published in 1833 (Mickel and Smith, 2004) ACKNOWLEDGMENTS e thank Harald Schneider for permission to use unpublished information on the phylogenetic gicunes of Phlebodium inaequale. We also thank John Wiersema for onary ante advice on the legitimacy of Phlebodium. Spore images were obtained by Rafael Emiliano Quin anar-Zuihiga, using a scanning microscope Jeol 6380 LW at the Facultad de Estudios Superiores ee of the Universidad Nacional Auténoma de México. Haruto Fukuda prepared the line drawings in Fig. 1. LITERATURE CITED Caruso, L. 1985. Cytological relationships in the Phlebodium aureum-decumanum species complex, unpublished. Library of the New York Botanical Garden. aisle E. B. 1947. Genera Filicum: the Genera of Ferns. ae mesa Phytopathol. 5:] xvi + 7 pp. + 10 pls. Chronica Botanica, Waltham, Massachuse Evans, M. 1963. New chromosome observations in the sone ane and Grammitidaceae. Caryologia 16:671-677. Haur er, C. H. and T. A. Ranker. 1995. rbcL sequences provide dota insights among sister sa of the fern genus ad eon Amer, Fern KRIER, , M. Rex, K. Welsinc, M. Kesster, A. R. Smiro and H. an 2008. Inferring the Apher aa of the eens fern genus Serpocaulon (Polypodiaceae) in South America Psa — last sequences and amplified fragment length polymorphisms. Pl. Syst. Evol. (in p Krier, H. P, ri Rojas-ALvarapo, A. R. SmirH and H. pein 2007. Hyalotrichopteris is indeed a Campyloneurum (Polypodiaceae). Amer. Fern J. 97: MCNEIL, J. et al. 2006. International Code of a plata (Vienna Code). [Regnum Veg. 146]. A. R. G. Ganter Verlag, —— Liechtenst MickeEL, J. T. and A. R. Smrru. 1977. Chromosome counts ice ferns. Brittonia 29:391—398. MicxeL, J. T. and A. R. Smit. 2004. The Pteridophytes of Mexico. Mem. New York Bot. Gard. 88:1—1054. Moorz, T. 1855. New fi o. IV. 8. a ete Gard. Chron. 1855:660, t. 58, f. 4. Moran, R. C. 1995. ee Pp. 333-366 R. C. Moran y R. Riba (eds.), Flora ee Vol. LE _Psilotaceae a ee Universidad Nacional Auténoma de i F mm.]. Flora of North America North of Mexico, Vol. 2, Pteridophytes and Gymnosperms. Oxford University Press, Oxford. Orto, E., M. T. JANssEN, H.-P. Kreter and H. Scuneter. In Press. New insights into the phylogeny of Polypodiani. Pleopeltis, and related Neotropical sail (Polypodiaceae, Polypodiopsida). Molec. Phylogenet. Evol. Proctor, G. R. 1989. Ferns of Puerto Rico and the Virgin Islands. Mem. New York Bot. Gard. 53:1—389. 116 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Sa.ino, A., T. E, ALMempa, A. R. SmirH, A. NAavarro-Gomez, H.-P. Krerer and H. ScuHNetper. 2008. A new species of Microgramma (Polypodiaceae) from Brazil and recircumscription of the genus 0-635. ScHNEIDER, H., H.-P. KRErER, R. Witson and A. R. Smitu. 2 hee The Synammia enigma: evidence for a yao fro Seaseas of polygrammoid ferns (Polypodiaceae, Polypodiidae) in southern South Syst. B See os os R ae R. Cranritt, T. J. Hitpepranp, C. H. HaurLer and T. A. Ranker. 2004. Unraveling the phylogeny of soly ygrammoid ferns (Polypodiaceae and Gre mumitidaceae): exploring aspects of the diversification of epiphytic plants. Molec. Phylogenet. Evol. 31:1041—1063. SCHUETTPELZ, E. and K. M. Pryer. 2007. Fern en inferred from 400 leptosporangiate species and three plastid genes. Taxon 56:1037— Smit, A. R. 1981. Pteridophytes. In: D. E. ae. (ed.). Flora of Chiapas, Vol. 2. California Academy of Sciences, San Francisc Situ, A. R., H.-P. Kreter, C. H. HAuFier, . A. RANKER and H. ScHNEWwER. 2006. Serpocaulon, a new genus segregated from Polypodium. Taxon 55:919—930. Situ, J. 1875. capi Filicum. Macmillan, London TEJERO-DfEz, J. . Revisi6n taxonémica del complejo Polypodium plesiosorum Kun Sener tlo pispadieade Tesis (doctorado en ciencias biolégicas), Uutivanaided Auténoma Metropolitana, México Tryon, A. F. and B. Lucarpon. 1991. Spores of the Jayne sss Li ctielirs dors New York. Tryon, R. M. and R. G. Sroize. 1993. bea phyta of Peru . V. 18. Aspleniaceae — 21. Polypodiaceae. Fieldiana, Bot. 32:1— Tryon, R. M. and A. F. Tryon. 1982. Ferns ne Allied Plants, with Special Reference to Tropical merica. Springer-Verlag, New Water, T. G. 1966. A cytotaxonomic eens of the pteridophytes of Jamaica. Trans. Roy. Soc. ol: Wa ker, T. G. 1985. Cytotaxonomic studies of the ferns of Trinidad 2. The cytology and taxonomic nls Bull. Brit. Mus. (Nat. Hist.), Bot. 13:149-249. American Fern Journal 99(2):117—141 (2009) 2008 AFS Symposium SUMMARY Summary of the 2008 AFS Symposium: From Gels to Genomics: The Evolving Landscape of Pteridology. A Celebration of Gerald Gastony’s Contributions to Fern Evolutionary Biology.—The study of pteridophyte evolutionary biology has undergone remarkable developments during the past 40 years. Central to these developments have been the efforts of Gerald J. Gastony and his academic offspring to advance our understanding of these plants. Accordingly, on 29 July, 2008, during the Botany 2008 Conference in Vancouver, British Columbia, former students, colleagues, and friends gathered to celebrate Jerry Gastony’s productive career at the forefront of pteridology. The symposium highlighted some of the major methodological and philosophical advances that have evolved during his exemplary career. Trained as a classical taxonomist, Prof. Gastony has continually reinvented himself since arriving at Indiana University in 1970. His initial forays into enzyme electrophoresis shed light on such diverse topics as the breeding system of ferns, the role of cryptic taxa in reticulate lineages, and the contributions of paleo- and neopolyploidy to fern systematics and evolution. These questions have been persistent throughout Jerry’s career and have been influential in shaping the field of pteridophyte evolutionary biology. The 2008 AFS symposium revisited these questions and showed how new tools are building on the foundation that Prof. Gastony helped lay over the last 40 years. In lieu of a formal Proceedings, the present text presents a brief summary of each of the presentations from the symposium, credited individually to each speaker and his co-authors.—Edited by Micuac. S. Barker, Department of Botany, University of British Columbia, 3529-6270 University Blvd, Vancouver, BC V6T 1Z4, CANADA, and Department of Biology, Indiana University Jordan Hall 142, 1001 E Third St., Bloomington, IN 46405-3700 and Gerorce YarskievycH, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299. A Brief History of Gerald J. Gastony’s Botanical Career.—After graduating from St. Ignatius High School in Cleveland, Ohio, Gerald J. Gastony (1940~ ) attended St. Louis University for his undergraduate training. His initial focus was on the humanities and in 1964 he received his Bachelor’s Degree in the College of Philosophy and Letters. Through this focus, he became fluent in Latin and comfortable in Greek, skills that aided his future career as a plant systematist. Jerry also became interested in botany through a course from the distinguished taxonomist and floristician, John Dwyer, and he wound up taking the equivalent of a major’s worth of classes in biology and supporting sciences in addition to those in his major. Dwyer subsequently encouraged Jerry to apply to Tulane University, where eventually he was advised by the noted naturalist and botanical historian, Joseph Ewan while supported by a predoctoral fellowship from NASA. It was during his work at Tulane that Jerry 118 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) became interested in ferns, which would be the focus of his doctoral work and future career. Ewan and Walter Hodge (then at NSF) were among those who encouraged Jerry to accept a Master’s Degree (in 1966) from Tulane and to apply to the doctoral program at Harvard University (although this meant abandoning his NASA fellowship for support through a grant from NSF). There, he completed his Ph.D. in 1971 under Rolla Tryon, one of the preeminent classical fern systematists of his time. Jerry’s doctoral work on the taxonomy of the tree fern genus Nephelea (Gastony, 1973) not only prepared him for a career in systematics, but it also stimulated his interest in related topics, such as the comparative morphology of fern spores, variation in the fern life cycle, and speciation. Jerry accepted a faculty position at Indiana University in 1970, straight from graduate school. His initial research in Bloomington focused primarily on the spore morphology of tree ferns (Gastony, 1974, 1979, 1981, 1982; Gastony and Tryon, 1976). However, several years into his position, Jerry became aware that in order to lead a successful career in a department that emphasized evolutionary studies beyond the organismal level, he would have to expand the focus of his research to address basic questions in evolutionary biology. In order to gain technical skills that would allow him to broaden his research program. Jerry sat in on several courses at Indiana University on biochemistry and genetics. He then applied this knowledge to a new effort to adapt the developing field of isozyme electrophoresis to ferns. He also spent his first sabbatical in Leslie Gottlieb’s lab at the University of California at Davis, where he perfected his isozyme techniques and began to apply them to evolutionary and population genetic studies in ferns. At the time, existing protocols to extract, resolve, and genetically interpret the banding patterns of common enzyme systems mostly did not work with ferns (Soltis et a/., 1983), and Jerry was challenged to prove himself in the Gottlieb lab. Ferns in the genus Pellaea are abundant and cytologically diverse in California, and these became Jerry’s model system for many future studies involving taxonomic relationships, population genetics, formation of polyploids, and the contributions of apogamous taxa to fern evolution (Gastony and Gottlieb, 1982, 1985; Gastony, 1988, 1990, 1991, Gastony and Windham, 1989). The coupling of classical and molecular techniques led to Jerry’s pioneering work on fern isozymes, and his lab (known as ‘‘Sky Lab” because of its location on the top floor of Jordan Hall) became a popular destination and invaluable resource for graduate and postdoctoral students interested in plant systematics and evolution. In the mid-1980s, Jerry and his students and collaborators further expanded the lab’s repertoire to include restriction-site variation of DNA. Jerry’s lab was one of the first to use variation in fern chloroplast DNA to understand historical relationships among fern species and genera (Yatskie- vych et al., 1988, Stein et al., 1989; Gastony et al., 1992). A few years later, Jerry began studying DNA sequence data for phylogenetic analyses of ferns, which eventually led to the first comprehensive phylogeny for ferns (Hasebe et al., 1995). Most recently, his lab generated the first genetic linkage map for a 2008 AFS SYMPOSIUM SUMMARY 119 Fic. 1. Gerald J. Gastony working in the greenhouse at Indiana University in 2008. fern, which will provide an important and permanent resource for fern genetics (Nakazato et al., 2006). Because of the great diversity of Jerry’s contributions to fern systematics and evolution, it is difficult to summarize all of them here. For example, his early work on spore morphology of the Cyatheaceae (Gastony, 1974, 1979; Gastony and Tryon, 1976) provided some of the initial evidence that the prevailing generic classification was unnatural. He was the first to count the chromo- somes of the sporophyte-less taxon, Vittaria appalachiana Farrar & Mickel, which required adapting existing cytological protocols to the special demands of mitotic cells in gametophytic tissue (Gastony, 1977). He also demonstrated that ferns have diploid isozyme expression patterns despite their high chromosome numbers and that, contrary to prevailing wisdom at the time, homosporous ferns are highly heterozygous rather than homozygous (Gastony and Gottlieb, 1982, 1985). He later showed that fern genes can become silenced following genome doubling (Gastony, 1991). His work on cheilanthoid ferns provided the first robust phylogeny of that large and taxonomically difficult group (Gastony and Rollo, 1995, 1998), but he also has made substantial contributions to the understanding of other fern groups, in such families as Apleniaceae (Gastony, 1971; Gastony, 1986; Gastony and Johnson, 2001), Onocleaceae (Gastony and Ungerer, 1997), and other subfamilies of Pterida- ceae (Gastony and Baroutsis, 1975; Baroutsis and Gastony, 1978; Gastony and Johnson, 2001; Nakazato and Gastony, 2003). 120 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) 1) Charles A. Weatherby L2) Rolla M. Tryon, Jr. 3) Gerald oo oe 4) J L-5) ¢ 6) Douglas E E. (Baroutsis) Gordon mont an H. Haufler 40) C ne. — Scag 45) Jennifer M. Ramp Neale a Ghaano D. Fehiberg 47) Jonathan — 12) 1 apne Windham 48) Loreen Allphin 13) ies Brooks — 14) rans Andrews Hooper 15) J inwei Li — 16) aa — 17) Terri Hildebrand Soltis 18) Loren H. Rieseberg ' 49) Aaron Liston — 67) ping K. Blackman 7) James E. Vogelmann -— 8) ee Yatskievych 9) 7 — 10) akuya Nakazato ae Barker 19) pete at Brunsfeld 20) Pau —24) Qiu-Yun Xia 73) Chuanzhu Fan 74) Wenhang Zhang ings +-31) Matthew Gitzendanner —38 Ge. poene An academic genealogy of Gerald J. Gastony, his botanical een and his academic pprhee ie See Table 1 for further information on each person name In 1995, Jerry Gastony (Fig. 1) received the Edgar T. Wherry Award from the Botanical Society of America (Anonymous, 1995). In 2006, he was one of the honorees for a Centennial Medallion Award from the Botanical Society of America. He was chairman of the Pteridological Section of the Botanical 2008 AFS SYMPOSIUM SUMMARY 121 TABLE 1. Biographical summary of individuals in the Academic Genealogy of Gerald J. Gastony. See Fig. 2 for chronology and context. — Ls Se as hy o Ree & © = 9 = i = ed i] = harles A. Weatherby. Academic grandfather. See American Fern Journal 40(1) for information. a M. Tryon, Jr. Academic father. Ph.D. Harvard University, 1941. See American Fern Journal 92(1): 1-9, 2002 for further information. Gerald J. Gastony. Ph.D. 1971, Harvard University. Currently Professor of Biology Emeritus, Indiana Bap Metts Bloomington Judith E. (Baroutsis) Gordon. Ph. D. 1976 (as Judith G. Baroutsis), Indiana University, Bloomin ngton. Currently Professor of Biology Emerita, Department of Biology, Augusta Sate University, Augusta, Christopher H. Haufler. Ph.D. 1977, Indiana University, Bloomington. Currently scaneivel and Chair, Department of rea and Evolutionary Biology, University of Kansas, Lawre Douglas E. Soltis. Ph.D. 1980, Indiana University, Bloomington. Currently Sialeagie ‘ad Chair, Department of eg arctan of Florida, Gainesville. James E. Vogelmann. Ph.D ; iana University, Bloomington. Currently Research Ecologist, U.S. Geological Saver Barth Resources Observation 8 Science Center, Sioux Falls, SD and Adjunct Gegares South Dakota State University, Brookings. George Yatskievych. Ph.D. 1990, Indiana University, Bloom Bees Currently Curator and Director of the Flora of pyreet Project, Missouri Botanical Garden, St. Louis and Research Associate Professor and Adjunct Graduate Faculty, University of Missouri—-St. Louis and Research Associate, Arizona-Sonora Desert Museum, Tucson. Takuya Nakazato. Ph.D. 2005, Indiana University, Bloomington. Co-advised by Loren H. Rieseberg. Currently Assistant ena Department of Biology, University of Memphis, Memphis, Tennessee. cae see number Michael S. Barker. Ph.D. 2009, Indiana Sale Bloomington. Co-advised by Loren H. Rieseberg. resend: Postdoctoral Associate, Department of Botany, University of British Thomas A. pone "Ph. D. 1987, University of Kansas. erscnseed Professor and Chair, Department of Botany, University of Hawaii at Manoa, Honolulu, H Michael Windham. Ph.D. 1988, University of Kansas. Currently coe Scientist and Curator of Vascular iri Department of Biology, Duke Uuircais, Durham Ralph Brooks. Ph.D. 1989, University of Kansas. Currently Senior Hicircamentel Scientist Black & Veatch, Eke Oswego, nage Elizabeth Andrews Hooper. Ph.D. 1994, University of Kansas. Currently Associate Professor of Biology, Truman State ss ae Kirksville, MO. Jianwei Li. Ph.D. 1996, University of Kansas. Currently Bioinformatics Engineer III, J. Craig Venter Institute, Rockville, MD. Jay Therrien. Ph.D. 2003, University of Kansas. Currently Director of Sales, Asia Pacific and Japan, Illumina, Inc., orients VIC, Australia Terri Hildebrand. Ph.D. 2005, University of Kaien. Currently Assistant Professor of Botany, Department of Hinlosy, Southern Utah University, Cedar City, UT. Loren H. Rieseberg. Ph.D. 1987, Mhectng oe State University. Currently Professor and Canada Research Chair, Department of Botany, University of British Columbia, Vancouver, British appeia! —o and Biaienubed Professor, Department of Biology, Indiana University, Bloom Seven, Tao feld. Ph.D. 1990, Washington State University. Professor, Department of Forest Resources, University of Idaho, Moscow. Deceased, 2007 (http://www.cnrhome.uidaho.edu/ default.aspx?pid=96887). Paul Wolf. Ph.D. 1990, Washington State University. Advised by Pamela Soltis, co-advised by Douglas ae Currently Professor, Department of Biology, Utah State University, Logan. ryan Ness. Ph.D. 1992, Washington State ao Prices Associate Professor, De ee of Biology, Pacific Union College, Angwin AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) TABLE 1. Continued. to tS N wo NS ao S) i i) N ~S i) ad oo) ry w ~ w - w oi wo sd cs o ney wr Michael S. Mayer. Ph.D. 1993, Washington State University. Advised by Pamela Soltis, co- advised by Douglas Soltis. Currently Associate Professor, Department of Biology, University of San Diego, San Diego, Gregory Plunkett. Ph.D. 1994, Washington State University. Currently Curator and Director, is an Program in Molecular Systematic Studies, The New York Botanical Garden, Bronx, ae Xiang. Ph.D. 1995, Washington State University. Currently Associate Professor, NC. Joanna Schultz. Ph.D. 1996, Washington State University. Advised by Pamela Soltis, co- advised by Douglas Soltis. Currently Senior Consultant, Earth Informations Systems, Houston, Linda Cook. Ph.D. 1998, Washington State University. Advised by Pamela Soltis, co-advised by Douglas Soltis. Currently Lecturer part time, Washington State University, Pullman T. Michael Hardig. Ph.D. 1998, Washington State University. Advised by Pamela Soltis, co- advised by Douglas Soltis. Currently Associate lorena pees of Biology, Chemistry, and Mathematics, University of Montevallo, Montevallo, A Robert K. Kuzoff. Ph.D. 1998, Washington State Nae Co-advised by Larry Hufford. phen — Professor, Department of Biological Sciences, University of Wisconsin, heneage ark E. oven Ph.D. 1999, Washington State University. Currently Associate Professor, ecm a of Ecology and Evolutionary Biology and Associate Curator of the McGregor Matthew Gitzendanner. Ph.D. 2000, Washington Sete pene Advised bel Pamela Soltis, co-advised by Douglas Soltis. Currently , University of Florida, Gainesville. Jason Koontz. Ph.D. 2000, Washington State University. Advised by Pamela Soltis, co-advised by Douglas Soltis. Currently Assistant Professor, Department of Biology, Augustana College, Rock Island, Michael Zanis. Ph.D. 2002, Washington State University. Currently Assistant Professor, Department of Botany eo Plant Pathology, Purdue University, West Lafayette, IN. Pablo Speranza. Ph.D. 2005, University of Florida. Advised by Pamela Soltis, co-advised by Douglas Soltis. Currently Profesor Adjunto he Fitotecnia, Departamento de Biologia Vegetal, Universidad de la iene Montevideo Ashley B. Morris. Ph.D. 2006, University of eo Aabieod by Pamela Soltis, co-advised by Douglas Soltis. Currently Assistant Professor, Department of Biology, University of South Alabama, Mobile Christine E. Edwar ds. Ph.D. 2007, University of Florida. Co-advised by Douglas Soltis and Pamela Soltis. irbigiaes Postdoctoral Research Scientist, Department of Botany, University of Wyoming, Lar Monica Asahi. oh D. 2008, University of Florida. Currently Postdoctoral Fellow, RI. Chrissen E. C. Gemmill. Ph.D. 1996, University of Colorado, Boulder. Curre ntly Senior Lecturer ie Associate seria in U.S.), Department of Biological Sciences, University of Waikato, seer New Zealand. Robin A. Bin Ph.D. 1997, University of Colorado, Boulder. Currently Professor, Department of Nato and Environmental Sciences, Western State College, Gunnison, CO. 2008 AFS SYMPOSIUM SUMMARY 123 TABLE 1. Continued. 42. Carla A. Wise. Ph.D. 1997, University of Colorado, Boulder. Yan Linhart, co-advisor. Currently independent pager al and Science Writer, Corvallis, OR. Jennifer M. O. Geiger. Ph.D. 2003, University of Colorado, Boulder. Currently Associate > ow 44. Laura Mujica. Ph.D. 2004, University of Colorado, Boulder (as Laura RSE See Patrick Bourgeron co-advisor. nay ag Term Assistant Professor, Chemistry Department, University of Alaska, Anchorage, A Jennifer M. Ramp Neale. Ph.D. 2005, University of Colorado, Boulder. Sharon Collinge, co- advisor. Currently Associate Director of Research and Director of the Conservation Genetics Program, Denver Botanic — Denver, oe 46. Shannon D. Fehlberg. Ph.D. 2006, University of Colorado, Boulder. Currently Conservation > ol 47. Jonathan Krieger. Ph.D. 2007, University of Colorado, Boulder. Robert P. Guralnick co advisor. Currently Postdoctoral Research Associate, Department of Palaeontology, The Natural History Museum, London. 48. Loreen Allphin. Ph.D. 1996, pondapnas of Utah, Salt Lake City. Advised by Delbert Wiens, co- advised by Michael Windham. Currently cae Professor, Department of Plant and Wildlife Sciences, Brigham Young University: # 49. Aaron Liston. Ph.D. 1990, Claremont Graduate Univesiin: Claremont, CA. Nominally advised by Thomas S. Elias, co-advised by Loren H. Rieseberg. Currently Professor, Department of Botany and Plant Pathology and Director of the OSU Herbarium, Oregon State University, Corvallis, OR. 50. Oscar Dorado. Ph.D. 1992, Claremont Graduate University. Currently Professor, Universidad Autonoma del Estado de aia Cuernavaca, Mexico 51. Michael Hanson. Ph.D. , Claremont Graduate University. Currently tenured botany Instructor, ste ag Cage, i em 52. DulceM. Arias. Ph.D. 1 5 Clonee Graduate aang Currently Professor, Universidad Auténoma del Estado ve eke Cuernavaca, ico. 53. Peter Morrell. Ph.D. 1997, Claremont ae University. Currently Senior Research Geneticist, Monsanto Co., St Louis, MO. 54. Stanley Spencer. Ph.D. 1997, Claremont Graduate University. spate! Senior Biologist at wa ray, 56. Mark Ungerer. Ph.D. 2000, Indiana University, Bloomington. Currently Assistant Professor, Division of Biology, Kansas State University, Manhattan, KS. 57. Diana Wolf. Ph.D. 2000, Indiana University, Bloom mington. amen! Assistant Professor, Institute of Arctic Biology, University of Alaska, Fairbanks 58. Mark Welch. Ph.D. 2002, Indiana University, Bloomi ee ea urrently Assistant Professor, Department of Biological Presi Mississippi State University, Mississippi State, M 59. Eva Sanders Allen. Ph.D. 2002, Indiana University, Bloomington. Ellen Ketterson, co-advisor. Currently Grants Specialist, Department of Biology, Indiana University, Bloomingto 60. Keith Gardner. Ph.D. 2004, Indiana University, Bloomington. Currently ecedceecal | Fellow, Royal Botanic Garden, meget ci UK. 61. Takuya Nakazato. Ph.D. 2005, Indiana University, Bloomington. Co-advised by Gerald J. Gastony. Currently Assistant Professor. Department of Biology, University of Memphis, Memphis, Tennessee. Also see number 9. Cécile Edelist. Ph.D. 2007, Université Paris-Sud 11, Orsay, France. Advised by Christine Dillmann and Delphine Sicard, co-advised by Loren H. Rieseberg. Currently research engineer, Conservation des ns Sestaierticn et Suivi des Populations, National Museum of Natural History, Paris, France sor) tS AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) TABLE 1. Continued. fo>] oo > - Briana Gross. Ph.D. 2007, Indiana University, Bloomington. Co-advised by Elizabeth Kellogg, einai of Missouri, St. Louis. Curren ly Postdoctoral Research Fellow, Department of Bi O. Nolan Kane. Ph.D. 2007, Indiana University, Bloomington. Currently Postdoctoral Research Associate, Fated ot Botany, University of British Columbia, Vancouver, British Columbia, Canada. Abigail Harter. Ph.D. 2008, Indiana University, Bloomington. Currently Postdoctoral Fellow, University of Edinburgh, UK. Troy Wood. Ph.D. April 2009, Indiana University, Bloomington. Benjamin K. Blackman. Ph.D. May 2009, Indiana University, Bloomington. Co-advised by Scott Michaels. Sedonia Sipes. Ph.D. 2001, Utah State University. ates aed a Professor, Department of Plant Biology, Southern Illinois University, Carbondale, Roy Murray. Ph.D. 1997, Utah State University. Currently oa hacker, IEM. Mark W. Ellis. Ph.D. May, 2009, Utah State University, Logan, Antoine N. Nicolas, Ph.D. May 2009, Virginia Commonwealth University, Richmond, VA Pedro Fiaschi Ph.D. anticipated, August 2009, Virginia Commonwealth Linivocsity. Richmond, VA. Chuanzhu Fan. Ph.D. 2003, North Carolina State University. Currently Assistant Research Scientist, Arizona egeuged apron University of Arizona, Tucson, AZ Wenhang Zhang. Ph.D. , North Carolina State University. Michael Purugganan, co- advisor. Currently Peicual Fellow, SRa HOE of Organismic and Evolutionary Biology, Harvard University, Cambridge, M Alexander Krings. Ph.D. 2007, North Carolina stor University. Jon M. Stucky, co-advisor. Currently Extension Acdetant Professor and rR cella the Herbarium, Department of Plant Biology, North Carolina sem University, Ralei A. Jennifer Floyd. Ph.D. 2000, North Carolina ea Gaivesity. Nina oat co- poadns Most gaan 2 Assistant Professor, Biology Program, University of Guam, Mangilao B. Terri L. Weese. Ph.D. 2004, Brigham Young University. Currently Editor, Hae Plant Name Sodas (APND, eee Plant Industry, Canberra, Australia Nicholas Levsen. Ph.D. 2008, University of Kansas. Currently Postdoctoral Fellow, Institute of AK. Francisco J. Camacho. Ph.D. 1999, Oregon - University. James M. Trappe co-advisor. Currently homemaker, San juan Capistrano, John Wheeler. Ph.D. 1998, Oregon State pe Currently Associate Professor, Department of Biology, penne of Wisconsin, River Falls Barbara Wilson. Ph.D. 1999, Oregon State University. Currently Partner, Carex Working Group LLC [botanical consuling firm], Eugene, OR. John odes Ph.D. 2006, Oregon State University. Co-advised by Richard C. pene tases Forest Service PNW. Gosule Assistant Professor, Linfield College, McMinnville, Jason Alexander. Ph.D. 2007, Oregon State University. Currently Herbarium pe ‘Utah Valley University, Orem, a Ann Willyard. Ph.D. 2007, Oregon State University. Currently Post Doctoral Fellow, Department of Biology, pile of South Dakota, Vermillion, SD. Assistant Professor, Brian Knaus. Ph.D. 2008, Oregon State University. Co-advised by Richard C. Cronn, USDA Forest Service iets Currently Postdoctoral Research Geneticist, USDA Forest Service PNW, Corvallis, OR. 2008 AFS SYMPOSIUM SUMMARY 125 Society of America (1979-1980) and also served as vice president (1994-1996) and president (1996-1998) of the American Fern Society. He has been an Associate Editor of the American Fern Journal since 1973 and was editor-in- chief of Systematic Botany from 1992 through 1995. Thus far, three species of plants new to science have been named in his honor: a Caribbean moss, Macrocoma gastonyi Norris & Vitt (1973); a Mexican polystichoid fern Phanerophlebia gastonyi Yatskievch (1992), and the uncommon allopolyploid Pellaea gastonyi Windham (1993). In addition to his contributions to scientific research and service to several scientific societies, Jerry Gastony has been a caring and skilled teacher of both undergraduate and graduate students. His Vascular Plants course was widely recognized as one of the best courses in the Department of Biology at Indiana University, and in 2001 he was honored with the Department of Biology Senior Class Award for Teaching Excellence in Biology and Dedication to Under- graduates. He has also been a much loved and respected mentor to a small dynasty of graduate students, several of whom have gone on to become eminent plant systematists in their own right (Fig. 2, Table 1). During his tenure as director of the Evolution, Ecology, and Behavior Graduate Program in the IU Department of Biology from 1991 to 2002, this program developed into one the strongest of its kind in the country. Even after Gerald Gastony’s retirement in 2006, he has continued to be a major force in pteridology and to interact with many researchers and students in the field.—MicnagL S. BARKER, Department of Botany, University of British Columbia, 3529-6270 University Blvd, Vancouver, BC V6T 1Z4, CANADA, and Department of Biology, Indiana University Jordan Hall 142, 1001 E Third St., Bloomington, IN 46405-3700 and GEORGE YATSKIEVYCH, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299 Gels and Genetics: The Historical Impact of Isozymes on Paradigm Shifts in Hypotheses about Fern Evolutionary Biology.—Although it is comforting when new discoveries confirm established hypotheses, it is positively exciting when novel techniques and observations demand rejection of reigning textbook concepts. The history of genetics for homosporous ferns is an exemplar of how technical innovations and discoveries lead to significant modifications of our working models in biology. Homosporous ferns were originally placed in the mysterious group called the “cryptogams” because, unlike their “‘phanerogamic’”’ cousins, their manner of breeding was hidden from obvious observation and investigation. Once botanists began culturing the gametophytes of ferns, their reproductive biology was revealed, and a method for conducting genetic experiments (crosses and progeny rearing) became available. The earliest studies of fern genetics were those of Lang (1923) and Anderson-Kott6 (1931), who demonstrated that most ferns showed simple Mendelian inheritance of traits. In 1950, Irene Manton published her magnum opus, ushering in a new era of genetic and biosystematic research on seed-free plants. Manton’s extensive survey demonstrated that most ferns had 126 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) extraordinarily high chromosome numbers and that often what appeared to be polymorphic species were actually reticulate complexes of diploid species and their allopolyploid derivatives. This research helped to demonstrate the importance of including genetic aspects of species in understanding their origins and their population dynamics. In the 1970s, Edward Klekowski (1979) brought a renewed focus to fern genetics by developing logically consistent and compelling correlations and hypotheses about the evolutionary biology of homosporous vascular plants. Klekowski observed that because homosporous ferns had _poten- tially bisexual gametophytes they should be highly inbred, and because these plants have high chromosome numbers, they should be polyploid. Klekowski further hypothesized that this polyploidy could represent an adaptive response that would buffer the homozygotizing effects of consistent inbreeding. Genetic variation stored among the several to many homoeologous genomes contained in polyploids could be released by non-homologous pairing mistakes during meiosis. Indeed, Hickok (e.g., 1978) provided evidence consistent with pairing between homoeologs. Klekowski’s hypoth- eses were intriguing because if accurate they provided a different genetic system and different evolutionary trajectory for homosporous vascular lants 1 * Population variation would be reduced and polymorphism constrained. Polysomic inheritance ld require diff t algorith fi i i dynamics of homosporous vascular plants. If single spores could germinate to become bisexual gametophytes that generated sporophyte offspring, wind dispersal and migration would surmount most geographic barriers and lead to large species ranges. population genetic rs 4 o U. oO Although some breeding experiments and chromosomal studies proved to be consistent with Klekowski’s hypotheses, central implications of them could not be addressed until enzyme electrophoresis provided a window on molecular genetics. Whereas the hypotheses predicted that ferns should have numerous duplicated genetic loci and be predominantly homozygous, isozymes demonstrated that species with generically basal chromosome numbers were genetically diploid and possessed numerous heterozygous loci (Gastony and Gottlieb, 1982; Haufler and Soltis, 1986). These discoveries required revised hypotheses and forced a revolution in modeling population- level phenomena for ferns. * Mechanisms promoting outcrossing were explored and verified through coordination of laboratory and field studies (e.g., Haufler and Soltis, 1984). Given a new (higher base numbers) starting point, polyploidy levels in homosporous vascular plants actually approximated those of other plant groups (Vida, 1976). No longer constrained by lethargic rates of change because of polygenic systems, it was reasonable to posit that diploid ferns could adapt and diversify along with their seed plant descendants (Schneider et al., 2004). At the species level, migration via single spores became a specialized rather than a standard capacity for ferns (Haufler, 2002). Fern biogeographers were required to consider a new variety of possible outcomes from dispersal and vicariance (e.g., Wolf et al., 2001). 2008 AFS SYMPOSIUM SUMMARY 127 ¢ Within populations, standard diploid-based models of population genetics obtain. Most diploids have random-mating breeding systems with inbreeding restricted to specialist species, those with subterranean gametophytes, and (of course) polyploids (Ranker and Geiger, 2008). Dismissing ferns as stagnant evolutionary dead-ends ceased to be an option, and with exciting new evidence from DNA and genomic studies, new vistas are opening all the time. Discovering the paradox that ferns had high chromosome numbers but were genetically diploid necessarily led researchers to ask how this unusual condition could have evolved. One hypothesis was that ferns differed (once again) from other organisms and the lineage started with a larger number of chromosomes (Soltis and Soltis, 1987). A second hypothesis stated that ferns (and other homosporous vascular plants) accumulated chromosomes through cycles of polyploidy events, followed by a return to genetic diploidy through gene silencing (Haufler, 1987). Why ferns retain chromosomes after silencing half their genes remains unclear (and does suggest they differ from other organisms), although it may be related to strong genetic control of bivalent formation (multivalents—that can result in chromosome losses—are rare in ferns having a balanced number of chromosome sets). Experiments and observations aimed at testing these hypotheses (Pichersky et al., 1990; Gastony, 1991; McGrath and Hickok, 1999; Nakazato et al., 2008) have all demonstrated that ferns having chromosome numbers that are basic within genera appear to have experienced ancient polyploidy followed by gene silencing. Support for the polyploidy plus silencing hypothesis is also consistent with new evidence that plant genomes are remarkably volatile and fluid (e.g., Adams and Wendel, 2005). Resolving these genetic mysteries of vascular cryptogams leads to a whole new set of open questions: ¢ We know little or nothing about the actual processes mnyniver Dobos | ages silencing in ferns. What is the mechanism that results in the paradoxical g n of the homosporous vascular plants? The majority of studies on fern genetics have focused on temperate groups. With most diversity in the tropics, and the origin of temperate groups tied to tropical ancestors, we need to know more about how tropical populations work and whether the conclusions drawn from studies of temperate populations apply to tropical ones We still know surprisingly little about the actual mechanisms that control breeding systems in ferns. More studies that coordinate laboratory analyses of gametophyte biology with surveys of natural populations may help to link mechanis sms with observed patterns of ares variation. * ° S » 17 2) n = 5 uo} oC. ° =] D re) a ° c nal =a ® =] Q ive) 5 n — ® re) —e ® ” - . 9 ty =A ° =| o> rt) = ® > ® © 5 as] ° a] ° wn ® > canal = is] 3 ° a re) n ny So) n wh ores arrive in a new location? What limitations are imposed on species migration by outcrossing fhe iap systems? Again, coordination of lab and field studies may help to resolve these open questions. ¢ Perhap sae bigg t g mystery involves the crigin « of abel — With demonstrations tion tak f ngiosperms diversify, it may that f be seule to study early stages in the oan process of ferns. These and other vistas await future generations of scientists interested in understanding the fascinating world of homosporous vascular plants and revealing the cryptic nature of their biology and genetics.—CurisTopHer H. 128 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Haurier, Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045. Using Plastid and Nuclear DNA Sequences to Redraw Generic Boundaries and Demystify Species Complexes in Cheilanthoid Ferns.—Cheilanthoid ferns constitute a monophyletic group of 400-500 species within the Pteridaceae (Smith et al., 2006; Schuettpelz and Pryer, 2007; Schuettpelz et al., 2007). They are noteworthy for their ability to colonize xeric and semi-xeric habitats, niches that are rarely exploited by other ferns (Tryon and Tryon, 1979, 1982). Relationships within this lineage are highly problematic, and cheilanthoids have been called “the most contentious group of ferns with respect to a practical and natural generic classification” (Tryon and Tryon, 1982: 248). It is not surprising, then, that molecular phylogenetic analyses to date have revealed that most of the larger cheilanthoid genera are polyphyletic (Gastony and Rollo, 1998; Kirkpatrick, 2007; Prado et al., 2007; Schuettpelz et al., 2007; Zhang et al., 2007; Rothfels et al., 2008). Cheilanthoid ferns have long been a topic of interest for Dr. Gerald Gastony, the honoree of this collection of papers. His contributions run the gamut from studies of chromosome numbers and apomixis in Bommeria E. Fourn. (Gastony and Haufler, 1976), through genetic analyses of various species groups (Gastony, 1988; Gastony et al., 1992), to documenting tetrasomic inheritance and gene silencing in polyploids (Gastony, 1990, 1991), and maternal inheritance of plastids in Pellaea Link (Gastony and Yatskievych, 1992). His phylogenetic studies of cheilanthoids (Gastony and Rollo, 1995, 1998) were the first to demonstrate that rbcL sequences could provide a valuable, independent tool for circumscribing genera in this taxonomically controversial group of ferns. We are now poised to take the “next step” toward redefining generic boundaries among the cheilanthoids. It is clear that the number of genes and taxa analyzed must be significantly increased if we hope to obtain a robust phylogeny of the group. To this end, we have initiated a large-scale phylogenetic study using DNA sequences derived from three plastid regions (rbcL, atpA, trnG-R). To date, we have sequenced all three plastid regions (representing nearly 4000 base pairs) for 157 species. Maximum likelihood analyses of these data identify seven, well-supported subclades of chei- lanthoid ferns (Fig. 3). Ludens clade.—Previously published analyses (Schuettpelz et al., 2007; Zhang et al., 2007) revealed that Doryopteris ludens (Wall. ex Hook.) J. Sm. is not closely related to most taxa traditionally placed in this genus, including the type species, D. palmata (Willd.) J. Sm. Whereas Doryopteris J. Sm. in the strict sense is strongly supported as a member of the hemionitid clade (Fig. 3), D. ludens and its close allies appear to represent a rather isolated lineage within the Pteridaceae. Analyses by Schuettpelz et al. (2007) resolved D. ludens as sister to all other cheilanthoid ferns while those of Zhang et al. (2007) suggested a possible affinity to other pteroid lineages. Though the placement of this species varies depending on taxon sampling, it is clear that it 2008 AFS SYMPOSIUM SUMMARY 129 hemionitids a - 2 notholaenids a pellaeids Yj myriopterids a! i skinneri clade Py bommeriids 4 * /udens clade — — Fic. 3. Summary of phylogenetic relationships within cheilanthoid ferns. Topology results from maximum likelihood analyses of atpA, rbcL, and trnG-R sequence data for 157 species; tree rooted with Doryopteris ludens. Thumbnails identify seven, well-supported cheilanthoid clades. Triangles indicate proportion of named species belonging to each clade; darker portion of each triangle represents the proportion of species included in the current analysis 130 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) is more closely related to cheilanthoid ferns than to any other potential outgroup sampled to date. For this reason, we have used it in our analyses to root the remaining cheilanthoid tree. The D. Judens clade encompasses a total of four species (only one of which is included in our sample) whose combined range extends from continental Asia to New Guinea. Because its phylogenetic divergence and geographic isolation from Doryopteris s.s. are substantial, this lineage is in the process of being transferred to a new genus: “Calciphilopteris”’ (Yesilyurt and Schneider, in press). Bommeriids.—As shown in earlier studies (Gastony and Rollo, 1995, 1998), species of Bommeria sensu lato (including B. elegans (Davenp.) Ranker & Haufler; see Ranker and Haufler, 1990) are sister to all cheilanthoids other than the D. Judens clade. Our data confirm Ray Cranfill’s (unpubl. data) assignment of Cheilanthes brandegeei D. C. Eaton to this clade, suggesting that the circumscription of Bommeria may need to be expanded yet again. Some species that Tryon and Tryon (1982) considered close relatives of C. brandegeei are strongly supported as members of the notholaenid clade in our analyses (Rothfels et al., 2008), and these already have been transferred to Notholaena (Yatskievych and Arbelaez, 2008). The remaining members of the “C. brandegeei group” (sensu Tryon and Tryon, 1982) need to be sampled before the bommeriid clade can be accurately delimited. Based on available data, we estimate that this lineage ultimately will encompass about 2% of cheilanthoid species, half of which have now been included in our analyses. Skinneri clade.—In a recent parsimony analysis of rps4, rps4-trnS, and trnL-F sequences by Kirkpatrick (2007), Cheilanthes skinneri (Hook.) R.M. Tryon & A.F. Tryon was weakly supported as sister to all cheilanthoids other than Bommeria (the Judens clade was not included in her sampling). In our studies, this taxon is strongly supported as sister to the myriopterid + pellaeid clade; together, these three clades are sister to the notholaenid + hemionitid clade (Fig. 3). Our molecular data also support a close relationship between C. skinneri and C. lozanoi (Maxon) R.M. Tryon & A.F. Tryon, an association previously proposed based on morphology (Mickel, 1987). Although these species have been transferred back and forth between Pellaea (in the pellaeid clade) and Cheilanthes (hemionitid clade) in the past, our data indicate that neither generic placement is tenable. Mickel (1987) identified several other taxa that may be related to C. skinneri, and these must be sampled before we can adequately circumscribe the clade and determine the correct generic name for it. Based on the available data, we estimate that this primarily North American lineage will include 4-5 species (about 1% of cheilanthoid diversity), two of which were included in the current analysis. Myriopterids.—This clade encompasses a group of primarily North Amer- ican species traditionally placed in Cheilanthes. A similar assemblage, also sister to the pellaeid clade, was recovered by both Gastony and Rollo (1998) and Kirkpatrick (2007). Our analyses indicate that this group is only distantly related to the type species of Cheilanthes (C. micropteris Sw., a member of the hemionitid clade) and, as such, all included taxa will need to be transferred to another genus (Grusz et al., in prep.). The type species of Myriopteris Fée 2008 AFS SYMPOSIUM SUMMARY 131 (1852), Cheilosoria Trev. (1877), and Pomatophytum M.E. Jones (1930) all belong to this clade, so there is no shortage of potential names. The challenge will be to identify morphological features that consistently separate this group from Cheilanthes sensu stricto. We estimate that this lineage comprises approximately 10% of cheilanthoid diversity; 75% of recognized species have been sampled to date. Pellaeids.—In addition to Pellaea s.s. (described by Link in 1841), this clade includes four genera named within the last 70 years: Argyrochosma (J. Sm.) Windham, Astrolepis D.M. Benham & Windham, Paraceterach Copel., and Paragymnopteris K.H. Shing. As revealed by earlier molecular analyses (Gastony and Rollo, 1998; Kirkpatrick, 2007), Argyrochosma (with ca. 30 species) is sister to all other pellaeids and can continue to be recognized as a distinct genus as proposed by Windham (1987). The other three genera, although morphologically more divergent than Argyrochosma, are nested within the traditional circumscription of Pellaea section Pellaea. It appears that members of this clade have switched from a typical Pellaea morphology (highly divided, nearly glabrous leaves) to an Astrolepis-Paraceterach- Paragymnopteris morphology (usually simply pinnate, densely scaly or hairy leaves) on no less than three occasions on three different continents. The taxonomic problems posed by this situation are not easily resolved; Kirkpatrick (2007) provided a good discussion of the potential synapomor- phies of each pellaeid subclade and the various nomenclatural options. The pellaeid clade comprises about 12% of cheilanthoid diversity; 65% of the species are represented in our analyses and additional representatives were sampled by Kirkpatrick (2007). Notholaenids.—This primarily North American lineage, the subject of a recent study by Rothfels et al. (2008), is sister to the large, cosmopolitan hemionitid clade. Most of the species included in the notholaenids are farinose, with abaxial leaf surfaces covered by ‘powdery’ (predominantly flavonoid) deposits produced by underlying glandular trichomes. This feature has often been considered a synapomorphy for the genus Notholaena R. Br. (sensu Yatskievych and Smith, 2003), but our data place two nonfarinose taxa deep within the clade and a strongly glandular, but non-farinose, species as the earliest diverging branch. Additional morphological studies are underway (Rothfels et al., in prep.) to identify characters that can be used to circumscribe an expanded Notholaena. This lineage comprises roughly 8% of cheilanthoid diversity; 60% of recognized species have been sampled to date. Hemionitids.—This is, by far, the largest and most diverse clade of cheilanthoids; its members are found on every continent except Antarctica and the geographic ranges of two species, Cheilanthes farinosa (Forssk.) Kaulf. and C. concolor (Langsd. & Fisch.) R.M. Tryon & A.F. Tryon, cover most of the subtropics (Tryon and Tryon, 1973). The lineage includes the type species of more than a dozen genera named between 1753 (Hemionitis L.) and 1991 (Pentagramma Yatsk., Windham & E. Wollenw.). Nearly all of these generic names are associated with well-supported subclades in our analyses, but relationships among these groups are largely unresolved in the plastid tree. 132 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) The hemionitid lineage appears to have undergone a rapid radiation (possibly associated with its colonization of new habitats and continents), and much additional data will be needed to clarify generic boundaries in this group. We estimate that this lineage comprises about 67% of cheilanthoid diversity; only 20% of known species are represented in the current analysis. Future directions.—Ultimately, we hope to include more than 60% of cheilanthoid species in our studies, with a special emphasis on under-sampled diversity hotspots in South America and Africa. The type species of all validly named genera will be sampled, as well as the majority of species of uncertain or disputed relationship. Phylogenetic analyses of these plastid DNA sequences will be used to identify well-supported monophyletic lineages. These clades can then be evaluated for morphological synapomorphies that will provide the foundation for a revised generic classification.—MIcHag. D. WinpHAM, Layne Hurer, Eric Scuuerrpetz, AMANDA L. Grusz, CarL ROTHFELS, and JaMes Beck, Department of Biology, Duke University, Durham, NC 27708-0339, Grorce YATSKiEvycH, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299, and KaTHLEEN M. Pryer, Department of Biology, Duke University, Durham, NC 27708-0339. Phylogenetic Use of Inversions in Fern Chloroplast Genomes.—Evolutionary studies at the genome level are nothing new, even in ferns, for which the earliest approaches can be attributed to the cytogenetic investigations of Irene Manton (Manton, 1950). Yet, within a decade of the development of recombinant DNA techniques, researchers were examining genomes through the study of DNA rather than chromosomes. This began with the pioneering work of Jeffrey Palmer and Diana Stein who demonstrated the utility of variation in the chloroplast genome for evolutionary studies in land plants, including ferns (Palmer, 1987; Palmer and Stein, 1982; Stein et al., 1986). Although the chloroplast genome (hereafter plastome) is generally conserved in structure (Palmer and Stein, 1986), it contains sufficient variation to be used at a wide range of phylogenetic scales. Two general approaches were used to study structural variation in plastomes, both involving restriction site analysis. The first entailed mapping via heterologous probes. This provided data on structural changes which can be informative especially at deep phylogenetic levels (Raubeson and Jansen, 1992). Hasebe (1992) compared the plastome structure of the fern Adiantum capillus-veneris to that of tobacco and found that the gene order in Adiantum was reversed throughout much of the inverted repeat region. A series of inversions was necessary to explain the difference. Later Stein et al. (1992) attempted to examine this aspect of plastome structure across ferns. The study found that Osmunda has the tobacco gene order, whereas the remaining taxa studied (a tree fern and several polypods) all had the Adiantum gene order, with no additional changes in structure detected. The second approach to comparing plastomes used variation at the sequence level, detected by presence or absence of restriction sites. This approach was used for more 2008 AFS SYMPOSIUM SUMMARY 133 phylogenetically focused studies including polystichoid ferns (Stein et al., 1989), Cyatheaceae (Conant et al., 1994), and the genus Pellaea (Gastony et al., 1992). Furthermore, maternal inheritance of the plastome was demonstrated in ferns (Gastony and Yatskievych, 1992). By the 1990s, DNA sequencing had become feasible for systematists, such that it replaced restriction site analysis as the method of choice. This had several effects. One was that now researchers were more focused on variation in one or a few genes, those for which PCR and sequencing primers were first developed. However, the genome scale approach had been lost. Yet nucleotide variation was so useful that much of the overall framework of fern phylogeny was established (Hasebe et al., 1994, 1995) using the gene rbcL, alone at first, but later adding data from additional genes (Pryer et al., 2004) We posit that evolutionary studies are now moving back to a genome scale perspective. This latest shift is again driven by technological advances, mostly those associated with high-throughput genomics, and the concomitant reduction in cost. Several researchers are starting to examine the highly complex nuclear genomes of ferns, and some of that work was included in this symposium. Our research group is focused on the plastome, of which two complete sequences are available for ferns: Adiantum (Wolf et al., 2003) and Angiopteris (Roper et al., 2007). Complete genome sequences provide advantages over the earlier mapping approaches: it is much easier to add taxa to a study and there is no need for additional cross probing. Also, the data provide both nucleotide data and genome structure data, deduced from gene order in the genome annotation. Although we do not yet have additional complete fern plastome sequences, we can use the information from Angiopteris and Adiantum to focus on a few key areas of the plastome. Now that a more robust phylogenetic framework is available for ferns, we can screen appropriate taxa to examine genome reorganization in more detail. Our research asks two main questions: how phylogenetically informative is gene order, and what are the evolutionary dynamics of genome structure? Gene order can be phylogenetically informative if the individual events that make up a genome reorganization each fall on a different branch of the tree. Alternatively, if there are temporal destabilization events, then a series of rearrangements can occur on the same branch, reducing the number of informative characters, and in some cases preventing the interpretation of actual events (but still providing strong support for one branch). Furthermore, if physical hotspots for rearrangements are common then it is possible that characters of genome structure might be susceptible to homoplasy. We used the plastome sequences of Adiantum and Angiopteris to design primers and used PCR and DNA sequencing to determine gene order in representatives of all major lineages of ferns. Here we focus only on a few regions that we know to vary, based on the two complete plastome sequences available. Details of the technique will be published elsewhere. We found that the complex reorganization of the inverted repeat in ferns (Stein et al., 1992) occurred via two main events. Angiopteris, Osmunda, filmy ferns, and gleichenioid ferns all possess the ‘tobacco’ (ancestral) gene order. The 134 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) schizaeoid ferns appear to have undergone one large (approximately 18 kb) inversion. The remaining lineages have a second large inversion which occurred after the first, and the result is the Adiantum gene order, with the rRNA genes occurring in the reverse order, as seen in all other land plant lineages studied to date. Thus, this structural reorganization appears to be comprised of two separate events that map consistently onto the fern phylogenetic framework of Pryer et al. (2004). However, other smaller rearrangements are composed of several inversions on the same branch, reducing their phylogenetic utility. Despite major strides in our understanding of fern phylogeny, several key branches remain poorly resolved. One clade that seems to be well-supported is the monilophytes, which include the Ophioglossales/Psilotales, leptospo- rangiate ferns, marattioid ferns, and the horsetails (Pryer et al., 2001), and we have found a 3 kb inversion that unites this clade. However, resolution among the four constituent lineages remains unclear. Another problematic area is the filmy ferns and gleichenioid ferns, which may be sister taxa, although the support for this is weak (Pryer et al., 2004). As more ferns plastomes are sequenced it should be possible to discover more phylogenetically informative rearrangements that may help address such unresolved issues. Moreover, genome scale data can be used for more than just phylogenetic studies. For example, several plastomes contain nucleotide repeats that may be variable at the population level. Although shifts in the type of data collected may have been driven by advances in techniques, the trend seems to be an increased ability to generate large amounts of data. Thus, future developments will likely depend on the ability to manage and analyze large data sets.—Pau. G. WoLr, Aaron M. Durry, and Jessi M. Roper, Department of Biology, Utah State University, Logan, UT 84322-5305. Fern Genome Structure and Evolution.—We now know that genome structure is a dynamic entity, and understanding how it evolves is of fundamental importance in biology. Ferns and seed plants are sister groups, and yet they show interesting differences in their genome structure. Hence, comparative analyses of their genome structure provide insights into what is unique in each group and how the genome structure differences evolve. One major difference between the fern and seed plant genome is their chromosome numbers. Chromosome numbers of ferns, particularly homospor- ous ferns, are much higher than those of seed plants (Klekowski and Baker 1966), and the underlying cause of this phenomenon has long been of a great interest to biologists. It is traditionally thought that ferns have high chromosome numbers because they are polyploids (Wagner and Wagner, 1980; Grant, 1981). However, Gastony and Gottlieb (1982) showed that, despite their high chromosome numbers, ferns with the lowest chromosome numbers in their genus show isozyme expression patterns typical of diploid organisms. To resolve the paradox of high chromosome numbers and diploid gene expression in ferns, Haufler (1987) hypothesized that they have acquired their 2008 AFS SYMPOSIUM SUMMARY 135 high chromosome numbers through repeated cycles of polyploidization and genome diploidization via gene silencing. Consistent with the Haufler’s hypothesis, Gastony (1991) showed that duplicated genes in a recent tetraploid species have been progressively silenced since the polyploidization event. More recently, Nakazato et al. (2006) looked for evidence of past polyploidiza- tion event(s) in a ‘diploid’ fern at the DNA level, by constructing a linkage map of Ceratopteris richardii Brongn.. They detected a large number of duplicated genes, one of the highest proportions among past mapping studies in plants, supporting the hypothesis that ferns are polyploids. The distribution of gene duplicates in the genome, however, revealed no apparent homoeologous chromosomes, evidenced by clustering of sets of gene duplicates in different chromosomes. Nonetheless, statistical tests for clustering of gene duplicates at the genome level were highly significant, suggesting that C. richardii has a polyploid-like genome structure. Therefore, it appears that C. richardii and perhaps other ‘diploid’ ferns have experienced ancient polyploidization(s), but homeologous chromosomes have been broken up by subsequent gradual chromosomal rearrangements. Furthermore, mapping the distribution of chromosome numbers on the known fern phylogeny revealed an apparent increase in the base chromosome numbers at the divergence between the water fern lineage and its sister, ca. 200 MYA (Nakazato et al., unpubl.), although many exceptions to the pattern make it premature to draw a firm conclusion. Together with the results from the linkage mapping study (Nakazato et al., 2006) and EST sequence analyses (Barker et al., unpubl.), it can be concluded that ferns probably have experienced ancient polyploidization event(s). Therefore, results from the past studies have largely support the Haufler hypothesis of repeated cycles of polyploidization and diploidization, and this phenomenon seems to explain the high chromosome numbers in ferns. However, it has become increasingly clear that polyploidization events are ubiquitous not only among ferns, but also among angiosperms (reviewed in Lockton and Gaut, 2005). Therefore, polyploidization events in ferns alone do not seem to explain the higher chromosome numbers in ferns than in seed plants, unless ferns experience more polyploidizations and extinctions of diploids. Interestingly, the modes of chromosome structural evolution seem to be substantially different between ferns and seed plants, and this may help us to understand why ferns have higher chromosome numbers. Genome size and chromosome number are significantly positively correlated in ferns (Nakazato et al., 2008), which is expected if no significant structural changes occur to chromosomes. However, no such correlation exists in angiosperms or gymnosperms, suggesting that chromosomal structure is highly dynamic in seed plants, but not in ferns. Also, the distributions of genome size and chromosome number are highly skewed toward low values in angiosperms, so there appears to be selection for small genome size and low chromosome number, but not among ferns. 136 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) So why are seed plant genomes more dynamic than fern genomes? Although we do not have good answers yet, we can speculate several alternatives. First, because most ferns are homosporous, and seed plants are heterosporous, this difference in reproductive system may induce selection on genome size and chromosome numbers, although the exact nature of this selection is not known. In support of this hypothesis, heterosporous ferns generally have low base chromosome numbers. Alternatively, chromosomal inheritance patterns may be fundamentally different between ferns and seed plants. Although multivalent formation is common among seed plants, in ferns multivalents that may start to form early in meiosis rarely survive to the late prophase stage. Finally, it is possible that ferns and seed plants have some differences in their genome composition. Transposable elements, in particular, are known to have a substantial contribution to genome size, especially in grasses (Bennetzen, 2002). Although highly speculative, it is possible that fern chromosome structure is highly stable because transposable element activity is lower relative to seed plants. Answers to the question of why ferns and seed plants have different genome structure will come only from detailed empirical studies. It is highly desirable in future studies to investigate what makes up the large fern genomes and how they are different from those of seed plants. Also, we need to conduct hypothesis-driven studies to establish causal links between the genome structure differences and biological differences between ferns and seed plants, such as reproductive systems and chromosomal inheritance.—Takuya NAKA- zATO, Dept. of Biology, The University of Memphis, 3700 Walker Ave., Memphis, TN 38152 Evolutionary Genomic Analyses of Ferns Reveal that High Chromosome Numbers are a Product of High Retention and Fewer Rounds of Polyploidy Relative to Angiosperms.—Ever since the first chromosome counts of homosporous pteridophytes revealed that they possess astonishingly high numbers of chromosomes, botanists have recognized the unique genomic composition of these plants. Basal chromosome counts for fern genera are significantly higher than similar values from angiosperms (homosporous ferns n = 57.05, angiosperms n = 16; Klekowski and Baker, 1966), a result that led early workers to assume that as many as 95% of ferns are polyploids. Numerous hypotheses have been proposed throughout the years to explain the origin and maintenance of these chromosome numbers, but Klekowski and Baker’s (1966) hypothesis of homoeologous heterozygosity received the most attention as it was supported by early studies. However, this hypothesis was refuted through a series of convincing isozyme investigations of fern genetics by Gastony and colleagues (Gastony and Gottlieb, 1982, 1985; Haufler and Soltis, 1986; Gastony, 1991). These studies demonstrated that homosporous fern species with the lowest numbers in their genera possess diploid gene expression patterns, and led to a 2008 AFS SYMPOSIUM SUMMARY 137 hypothesis that fern chromosome numbers are the product of numerous rounds of paleopolyploidy. To test these hypotheses, I analyzed Sanger and 454-sequenced ESTs from four polypod fern species for evidence of ancient genome duplication. My analyses demonstrate that a single genome duplication occurred near the base of the polypod ferns, a lineage that comprises >80% of extant fern diversity. Combined with available fossil data, I also provide the first estimate of fern nuclear genome evolutionary rates with polypodiaceous nuclear genomes evolving at approximately 4.79 X 10 ° subst. fees site/year and places the ancient genome duplication at 178 +/— 32 MYA Assuming that rates of chromosomal loss in ferns are comparable to angiosperms, this is fewer genome duplications than expected, as many angiosperms with much lower chromosome numbers have experienced numerous rounds of genome duplications (Cui et al., 2006). To further elucidate this pattern, I calculated a rate of paleopolyploidzation for angiosperms and ferns from genomic data sets of 192 species (Barker et al., in prep). This rate comparison reveals that, on average, ferns experience approximately half as many paleopolyploidizations as angiosperms. o, why then do homosporous ferns possess so many more chromosomes than angiosperms? It appears that pteridophyte genomes are simply less dynamic than angiosperm genomes and maintain their chromosomes with higher fidelity. Consistent with this hypothesis of gene silencing with little loss of physical genetic material is the observation of significantly lower gene density in the Ceratopteris genome relative to seed plants (Rabinowicz et al., 2005). Additionally, pteridophytes are the only lineage of vascular land plants that have a strong, positive correlation between genome size and chromosome number (Nakazato et al., 2008). Possibly involved in the maintenance of these chromosomes is another peculiar pteridophyte trait, the strong bivalent pairing of chromosomes (Wagner and Wagner, 1980). Further research is needed to identify the forces and mechanisms driving the striking differences in genome evolution and organization between seed plants and monilophytes. Perhaps the ultimate tool for addressing this question will be whole-genome sequences of homosporous and heterosporous ferns. Considering innovations in sequencing technology and the declining cost of sequencing, we are likely only a few years away from having such data and further elucidating this most outstanding pteridological mystery.—Micna S. Barker, Department of Botany, University of British Columbia, 3529-6270 University Blvd, Vancouver, BC V6T 1Z4, CANADA, and Department of Biology, Indiana University Jordan Hall 142, 1001 E Third St., Bloomington, IN 46405-3700 ComBINED LITERATURE CITED Apas, K. L. and J. F. WenpEL. 2005. Polyploidy and genome evolution in plants. Curr. Opin. PI. Biol. 8:135-141. Anpersson-KorT90, I. 1931. The genetics of ferns. Biblio. Genet. 8:269-294. 138 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Anonymous 1995. Awards and prizes at BSA Annual Meeting. Pl. Sci. Bull. 41:45—-46. — J. G. and - : Gastony. 1978. Chromosome numbers in the fern genus Anogramma: 2. r. Fern J. 6 Bawa, J. L. 200 eg Fe and rates of genome expansion and contraction in flowering 2 eee 115:29-—36. Conant, a fs D. B. Stem, A. E. C. Vauinskt, P. SuDARSANAM and M. E. AHEARN. 1994. sei implications of chionnslast DNA variation in the ope! 1. Syst. Bot. 19:6 Cur, L. Y., P. K. Watt, J. H. Leepens-Mack, B. G. Linpsay, D. E. S JJ Doms P. 8. 80 Bo sy Carson, K AN, A. . ALBERT, H. “ve dC. W. eas. 2006. Widespread genome duplications Sevughout the history of foweelnn plants. Genome Res. 38-749. Gastony, G. a 1971. Asplenium pinnatifidum X trichomanes - a new 1 for Indi Amer. Fern 2-34 Gasrony, G. iF 1973, A revision of the fern genus Nephelea. Contr. Gray Herb. 203:81—-148. Gastony, G. J. 1974. Spore morphology in the Cyatheaceae: 1. The perine and sporangial capacity: general considerations. Amer. J. Bot. 61:672—680. Gastony, G. J. 1 Chromosomes of the eicienngnes tiie Appalachian gametophyte: a new source a taxonomic evidence. Syst. B GasToNy, pa J. 1979. Spore morphology in the sa nce a: The genus Trichipteris. Amer. J. Bot. 66:1238—1260. cae. G. J. 1981. Spore morphology in the Dicksoniaceae: 1. The genera Cystodium, hyrsopteris, and Culcita. Amer. J. Bot. 68:808—819. Gastony, G. J. 1982. Spore morphology in the Dicksoniaceae: 2. The genus Cibotium. Canad. J. Bot. 55— 1955-9 So Og F 1986. Electrophoretic evidence for the origin of fern species by unreduced spores. r. J. Bot. 73:1563-1569. Cancer, C J. 1988. The Pellaea glabella ha ei cope poe eee for the derivations of 44-67. the a goa ne taxa and a en aie my. an Gastony, G. J. 1 El Hotei heterozygosity in South Ain Pellaea rufa A.F.Tryon Srearn Ann. ican Bot. Gard. 77:306-313. Gastony, G. J. 1991. nen ngwenag ig in a polyploid homosporous fern—paleopolyploidy revisited. Proc. Natl. oe U.S.A. 88: 1602-100>, G. J. GasTony and J. G. eet 1975 bers in the fern genus Anogramma. Amer Gastony, G. J. and D. A Darrow. 1983. Chloroplastic and cytosolic isozymes of the homosporous fern Athyrium filix-femina L. Amer. J. Bot. 70:1409-1415. Gastony, G. J. and L. D. Gorriies. 1982. Evidence for genetic heterozygosity in a homosporous fern. Am Gastony, G. J. and L. D. Gorrus. 1985, Genetic variation in the homosporous fern Pellaea andromedifolia. Amer. J. Bot. seat 67. Gastony, G, J. and C. H. Haurter. 1976. Chromosome numbers and apomixis in the fern genus Bommeria (Gymnogrammaceae). Biotropica 8:1-11. Gastony, G. J. and W. P. Jounson. 2001. Phylogenetic placements of Loxoscaphe thecifera (Aspleniaceae) and dominoes radiata (Pteridaceae) based on analysis of rbcL nucleotide s ee 7-213 Gastony, G. J. and D. R. Roto. fee . Ph dea en i ipti f cheilanthoid ferns Pride Cheilanthoideae) fast from rbcL nucleotide ‘sequences. Amer. Fern J. ae Cues ee J. and D. R. Roto. 1998. Cheilanthoid ferns (Pteridaceae: Cheilanthoideae) in the eh ae United States and adjacent Mexico—a molecular phylogenetic reassessment of generic lines. Aliso 17:131-144. Gastony, G. J. and R. M. Tryon. 1976. Spore morphology in the Cyatheaceae. II. biog genera Lophosoria, Metaxya, sey eg Alsophila, and Nephelea. Amer. J. Bot. 63:738—758. Gastony, G. J. and M. C. UNcerer. 1997. Molecular systematics and a revised taxonomy of the onecleoid ferns fisupenitansae Onocleeae). Amer. J. Bot. 84:840—-849. 2008 AFS SYMPOSIUM SUMMARY 139 Gastony, G. J. and G. YATsKlEvycH. 1992. sbiipeiee maproapninasis of the chloroplast and mitochondrial genomes in cheilanthoid ferns. Amer. J. B 9:716-722. Gastony, G. J., G. YATSKIEVycH and C. K. Dixon. as Chloroplast DNA restriction site variation in the fern genus Pellaea - Phylogenetic relationships of the Pellaea glabella complex. Amer Bot. 79:1072—1080. Grant, V. 1981. Plant Speciation. peeieen University Press, New York. Hasse, M. and K. Iwatsuki. 1992. Gene localization on the poh ots DNA of the maiden hair fern: Adiantum capillis-veneris. Sip Mag. (Tokyo) 105:413-419 Hasese, M., P. G. Wotr, W. D. Lah J. R. Manuart, C. H. HAuFier, R. SANo, G. J. Gastony, E. H. Crane, K. M. Pryer, N. Murakamt, J. Pes and M. Iro. 1994. A global analysis of fern sparen ie based on rbcL ie sequences. Amer. J. Bot. 81(6, supplement):120-121 [Abstract]. HaseseE, M., 7 G. Wor, K. M. Pryer, K. Uepa, M. Ito, R. Sano, G. J. Gastony, J. YOKOYAMA, J. R. Manuart, N. Murakami, E. H. Crane, C. H. Haurer and W. D. Haux. 1995. Fern phylogeny based on rbcL nucleotide sequences. Amer. Fern J. 85:134—181. igen C. H. 1987. Electrophoresis is modifying our concepts of evolution in homosporous eben ier J. Bot. 74:953-966. one. G, 2. Homospory 2002: An odyssey of progress in pteridophyte genetics and evolutionary ‘oe tee rane 52:1081—-1093. Haurier, C. H. and D. E, Soxtis. 1984. Obligate outcrossing in a homosporous fern: field te of a evaded prediction. Amer Haur er, C. H. and D. E. Soxtis. 1986. Genetic evidence suggests that agora oe ferns with high chromosome e numbe ers are paren Proc. Natl. Acad. Sci. U.S.A. 83:4389-4393 es L. G. 8. Homoeologous chromosome pairing and ose EE, in the fern mes Amer. ot. 74:1173—1183. SE a: R. E. B. 2007. eventing the monophyly e — (Pteridaceae) in the context of a phylogeny vs eeu aspiat Syst. Bot. ne ac KLEKOWSKI, E. ba gy of ferns. Pp. 133-170 in Dyer, A. Fi, ed. The Experimental Biology a Ferns. Pigg Press, London. KiEKowskl, E. and H AKER. 6. Evolutionary significance of polyploidy in the J. scar end pn i 8. Lane, W. H. 1923. vie ba genetic analysis of a heterozygote plant of Scolopendrium vulgare. J. Genet. 13:167—-1 LocxkTon, S. and B. S. 2005. Plant conserved non-coding sequences and paralogue evolution. Trends Genet. 21:60-65. Manton, I. 1950. Problems of eateed and Evolution in the Pteridophyta. Cambridge University Press, Cambridge, Englan McGrath, J. M. and L. G. tates 1999. eens aren RNA gene a in the genome of the homosporous fern Ceratopteris richardii. Canad. J. Bot. 77:1199-12 L, J. T. 1987. A new fern from western eos and its sis on the taxonomy of cheilanthoid ferns. Amer. Fern. J. 77:109— Nakazato, T. and G, J. Gastony. 2003. lies -ehieiapeciis = Anogramma species and related genera (Pteridaceae: Tacatidenaasa’ Syst. - 9 Nakazato, T., M. S. Barker, L. H. RIESEBERG an dG. re ae 2008. Evolution of the nuclear genome of ferns and lycophytes. Pp. 175-198 in T. A. Ranker and C. H. Havr er, eds. Biology and Evolution of Ferns and Lycophytes. Cambridge University Press, Cambridge, England. Nakazato, T., M.-K. Junc, E. A. Houswortn, L. H. Rieseperc and G. J. Gastony. 2006. Genetic map- based analysis of genome structure in the homosporous fern Ceratopteris richardii. Cac 173:1585—1597. Norris, D. H., D. H. Virr. Macrocoma gastonyi Pp. 209, f. 1-7, 20-23 in: et H. Virr. A revisionary study of the genus Macrocoma. Rev. Bryol. Lichénol., n.s. 39:205-2 Pater, J. D. 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA variation. Amer. Naturalist 130:6—-2 140 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) Patmer, J. D. and D. B. Stem. 1982. Chloroplast DNA from the fern Osmunda ci : physical oo gene localization and comparison to angiosperm chloroplast DNA. Curr. Genet. 170. Bees I D and D. B. Stein. 1986. Conservation of chloroplast genome structure among vascular s. Curr. Genet. 10:823-833. Sauce . D. E. Soutis and P. S. Souris. 1990. Defective chl ll a/b- ae Lalas genes in the genome of a homosporous fern. Proc. — Acad. Sci. U. S. A. 8 Prapo, J., C. D. N. Ropricugs, A. SaLatino and M. L. F. SALaTINoO. 2007. itachi relationships among Pteridaceae, including Brazilian | species, inferred from rbcL sequences. Taxon 56:355—368. PRYER, = M., . — dh G. Wate, hie , SCHNEIER, A. R. SmirH and R. Cranrit. 2004. Phylogeny f fern ith a focus on the early leptosporangiate divergences. Amer. J. Bot. 91:1582—1598. Pryer, K. M., H. Scunewer, A. R. SmirH, R. Cranritt, P. G. Wotr, J. S. Hunt and S. D. Sipss. 2001. Horsetails and ferns are a monophyletic group and the closest living relatives to seed plants. Nature 409:618-622. Rasinowicz, P. D., R. Crrex, M. A. Bupiman, A. Nunserc, J. A. Bepett, N. Lakey, A. L. O’SHAUGHNESSY, L. U. Nascimento, W. R. McCompire and R. A. MarrTIENSSEN. 2005. Differential methylation of genes and repeats in lead plants. Genome Res. 15: ee Ranker, T. A. and C. H. Haurter. 1990. A new combination in (Adiant ). Amer. Fern 0:1-—3 Ranker, T. A. and J. M. O. Geicer. 2008. Population genetics. Pp. 107-133 in T. A. RANKER and C. H Havr ter, eds. Biology and Evolution of Ferns and Lycophytes. Cambridge University Press, Cambridge, Englan Rauseson, L. A. and R. K. ecu 1992. Chloroplast on evidence on the ancient evolutionary split in vascular land plants. Science 255:1697—269 Roper, ws = S. K. Hansen, P. G. Wotr, K. G. Karot, D. : Manpout, K. D. E. Everett, J. Kuen and J. L. 2007. The complete plastid genome sequence of Angiopteris evecta (G. Forst.) Hoffm. patens Fern J. 97:95— RorHFes, C. J., M. D. Wieeae . L. Grusz, G. J. GaAstony and K. M. Pryer. 2008. Toward a monophyletic Notholaena eetaen resolving patterns of eviludanary convergence in xeric- gigs ferns. Taxon 57:712-724 SCHNEIDER, H., S eeu K. M. Pryer, R. Cranritt, R. MaGaLton and R. Luria. 2004. Ferns diversified i in the shadow of angiosperms. Nature 428:553-557. ScHUETTPELZ, E. and K. M. Pryer. 2007. Fern ap ia ad inferred from 400 leptosporangiate species and three plastid amas aia 56:1037. TTPELZ, E., H. ScHNEweER, L. Hurt, M. “ ole and K. M. Pryer. 2007. A molecular phylogeny of the fogs family Pteridaceae: assessing overall relationships and the affinities of prev pages unsampled genera. Molec. Phylogen. ers 44:1172-1185. SmirH, A. R., K. M. Pryer, E. Scuuerrpetz, P. Korat, H. ScHnewer and P. G. Worr. 2006. A classification of extant ferns. bog 55:705-731. Soxtis, D. E., C. H. Haurer, D. A. Darrow and G. J. Gastony. 1983. Starch gel electrophoresis of fern: a laa grant of paced Suiiees gel and electrode buffers, and staining schedules. Amer. m 3:9-27. Soxtis, D. E. sae S. Soxtis. 1987. P 1k ling syst in homosporous Pteridophyta: a reevaluation. Amer. Naturalist : 130: :219-232. Stein, D. B., J. D. Patmer and W. F. Tompson. 1986. picasa lee cee ogi flip-flop sgn of chloroplast DNA in the fern genus Osmunda. 10:835-841 Sten, D. B., G. Yatskevycu and G. J. Gastony. 1989. eee DNA pu and is of some polystichoid ferns. Biochem. Syst. Ecol. 17:93—1 Sten, D. B., rae Pape M. y AHEARN, E. T. JORDAN, S. A. ni , M. Hasse, K. Iwatsuxi, M. K. TAN d Structural rearrangements of the chloroplast genome provide an important caren ak in ferns. Proc. Natl. Acad. Sci. U.S.A. 89:1856—1860. 2008 AFS SYMPOSIUM SUMMARY 141 Tryon, R. M. and A. F. Tryon. 1973. Geography, spores, and rue! relations in the Seer fornia. Pp. 145-153 in A. C. Jermy, J. A. CRABBE she B. A. Tuomas, eds. The Phylogeny and genase of Ferns. Academic Press, New York. Tryon, R. M. and A. F. Tryon. 1982. Ferns and Allied Plants, with fate Reference to Tropical America. Scvisis Vorlan, New York. Vina, G. 1976. The role of polyploidy i in evolution. Pp. 267-294 in V. J. A. NovAk and B. Pac.tovA, Wacner, W. H. and F. S. Wacner. 1980. Polyploidy in pteridophytes. Pp. 199-214 in W. H. Lew ed, Polyploidy, Biological Relevance: Proceedings i ne sahara Canteraics on Polyploidy, Biological Relevance, Plenum Press, New WinpuaM, M. D. 1987. Argyrochosma, a new genus of Saiae aoad ferns. Amer. Fern J. 77:37—41. WinpuaM, M. D. 1993. New taxa and eet changes in the North American fern flora. Contr. Univ. Michigan Herb. 19:3 WinpuaM, M. D. and G. YatTskIEVYCH. has Faas studies of cheilanthoid ferns Pioigesroms Cheilanthoideae) from the western United States and Mexico. Amer. J. Bot. 90:1788-1800. Wotr, P. G., C. A. Rowe, R. B. SincLa and M. Hasse. 2003. Complete nucleotide sequence of the chloroplast genome from a leptosporangiate fern, Adiantum capillus-veneris L. DNA Res 10:59-65. Wor, P. G., H. Scuneter and T. A. Ranker. 2001. Geographic distributions of homosporous ferns: does onl aps obscure evidence of vicariance? J. Biogeogr 28:263-270. YATSKIEVYCH, G. 1 ng aleass in the fern genus Phanerophlebia. Novon. 2:445—446. YATSKIEVYCH, a " Stein and G., J. Gastony. 1988. Chloroplast DNA evolution and systematics of camronilehic and See fern oy Proc. Natl. Acad. Sci. U.S.A. 85:25 89-2593 ices G. and A. L. A. ARBELAEZ. new species and three generic transfers to the fern genus Notholaena sh haandizate seat 18:120-124. YATSKIEVYCH, G. an . SMITH. 2003. Typification of Notholaena R. Br. (Pteridaceae). Taxon 52:331—336. YESILYURT, J. C. ~ H. Scuneier. In press. oe a new genus of Pteridaceae. Blumea. ZHANG, G., X. ZHANG, Z. CHEN, H. Liu and W. Yanc. 2007. First insights in the phylogeny of Asian cicilenihold raat based on sequences - a chloroplast markers. Taxon 56:369—37 American Fern Journal 99(2):142—144 (2009) REVIEW Flora de Nicaragua. Tomo 4. Helechos, the flora edited by W. D. Stevens, O. M. Montiel, and A. Pool, the volume authored by L. D. Gémez and A. L. Arbeldez. Monographs in Systematic Botany 116: 1-348, + i-xvii. 2009. ISBN 0161-1542. Published by the Missouri Botanical Garden, St. Louis. Hard cover. Price: $109.00. ISBN 978-1-930723-87-0, 151 figures (full page plates), illustrations by A. L. Arbeldez. In Spanish. orders@mbgpress.info; web: http://mbgpress.info This fourth and concluding volume of the Flora of Nicaragua (previous volumes, all seed plants, published in 2001, ISBN 9780915279951) contains coverage of the ferns and so-called “fern allies’, and treats, in alphabetical order, 102 genera and 551 species known from the country. Twelve additional genera and 82 species are also given full treatment, and included in the keys, in expectation that many of these will eventually be found in Nicaragua, since the known distribution is in countries immediately to the north and/or south of Nicaragua. For each species, we are given the accepted name, citation of publication, basionym, salient synonyms, description, habitat, representative specimens (collector and number), range, occasional brief taxonomic discus- sion, an endangerment code, original line drawings (habit or diagnostic details), and a dot distribution map. Keys to species, but not to families or genera, are included. Introductory sections include a discussion and maps for concentration of both pteridophyte and vascular plant diversity in Nicaragua, and for density of collections within the country (by Stevens), discussion of conservation issues (by Montiel), placement of genera within families, and a general bibliography. This volume presents an updated and more focused version, for Nicaragua only, of the earlier general flora for the region, Flora Mesoamericana, Vol. 1 (Davidse et al., eds., 1995). There are, indeed, many first literature reports of species for Nicaragua, contained within this new work. The authors have generally adopted the most recent classification/taxonomy available for a given genus, with only minor exceptions: filmy ferns are presented in the traditional two genera system, rather than the recently published 9-genus classification by Ebihara et al. (2006); and Cnemidaria is Arete hate from Cyathea. I noticed only a few questionable taxonomic decisions, the Committee for Pteridophyta has declared that the saiot incon of Acrostichum ebeneum L., by Tryon, must stand (Taxon 54:831. 2005), the effect being that that name is regarded as a synonym of Pityrogramma tartarea (Cav.) Maxon); Pityrogramma ebenea (L.) Proctor was used for this species by the authors. Dryopteris rossii is included in the flora on the basis of Gémez 6160, but I think it likely that this specimen is either mislocalized or misidentified. Nephrolepis cordifolia is said to be naturalized in the Neotropics, but the type, from the Dominican Republic, is conserved (McNeill et al., eds.,Vienna Code, 2006), and the species generally considered to be native to at least parts of the New REVIEW 143 World. Also, Nephrolepis multiflora is listed as a synonym of N. hirsutula, even though Hovenkamp and Miyamoto (Blumea 50: 279-322) included the former as a synonym of N. brownii (Desv.) Hovenkamp & Miyam., a species also accepted in the present flora; most likely, specimens assigned to N. hirsutula by Gémez and Arbeldez are really N. brownii, and specimens determined as the former are misidentifications. One somewhat confusing aspect of this flora is that a substantial number of species (e.g., Psilotum nudum, Botrychium schaffneri, B. virginianum, Ophioglossum crotalophor- oides, Hymenophyllum pulchellum, H. trapezoidale, H. undulatum, and Asplenium salicifolium, to name a few) listed by G6mez (1976; Brenesia 8:41— 57) in his enumeration of ferns of Nicaragua are included in the present flora on the expectation of their possible occurrence in Nicaragua—this, despite the statement by Gomez (1976, p. 41) that the earlier list was compiled from ferns “conocidos hasta la fecha como resultado de una revisién de literatura y el examen de varios miles de ejemplares colectado por mi y depositado en el Herbario Nacional de Costa Rica y mi herbario personal.’’ One would have preferred an unambiguous statement to the effect that the current authors were now unable to verify the existence of the species in question in Nicaragua. This underscores the inadvisability of accepting range statements for floras on the basis of literature citations. I myself have been guilty of this (Smith, 1981, Flora of Chiapas), accepting, uncritically, range statements for species said to be in Nicaragua by Gémez (1976); in turn, my range statements (for Nicaragua) were taken up in the Flora Mesoamericana (Moran & Riba, 1995). In this way, the cycle of misinformation continues. The largest pteridophyte genera for Nicaragua are Thelypteris s.l. (51 spp.), Asplenium (39 spp.), Elaphoglossum (28 spp.); Trichomanes s.l. (28 spp.), Adiantum (26 spp.), Diplazium (23 spp.), and Selaginella (21 spp). In fact, the 10 largest genera comprise nearly half of the species known from the country. Only two species are considered to be endemic: an unnamed Anemia and Thelypteris mombachensis. From the distributions maps, one can readily discern the most common (often weedy) ferns in Nicaragua: Adiantum concinnum, Blechnum occidentale, Lygodium venustum, Microgramma percussa, Pityrogramma calomelanos, Tectaria heracleifolia and T. panamen- sis, Thelypteris dentata (naturalized) and T. nicaraguensis. These, and a few others, are represented by more than 30 collections. All species are estimated to fall into one of several categories depending on abundance/rarity of collections: in order of greatest endangerment these categories are CR, in critical danger; EN, in danger; VU, vulnerable; NT, somewhat threatened; LC, of lesser concern. Given the intrinsic uncertainties of assessing species vulnerability in any tropical area, approximately 35 spp. are considered as CR (usually only one collection known from the country); 160 spp. are EN (generally 1—2, up to ca. 7, collections known); and 156 spp. are VU (generally 4—10 collections known). By these estimates, more than 60% of the pteridophytes of Nicaragua are vulnerable, if not greatly threatened, a staggering percentage. Even though nearly all species of ferns have wider distributions outside the country, these statistics should cause concern. That 144 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 2 (2009) so many Nicaraguan ferns are known from only one or two collections also suggests that there are likely many species not yet collected in the country. The illustrations are helpful, well executed, and pleasingly arranged by Alba Arbeldez, a co-author of the book. Kudos to her for her artistry, and for citing vouchers for the drawings! Also, the editing process is superb, the book is about as error-free as a flora can be. I enthusiastically recommend this book to anyone wanting to know about, or identify, pteridophytes from Nicaragua. ALAN R. Smit, University Herbarium, University of California, Berkeley, CA 94720- 2465. INFORMATION FOR AUTHORS Authors are encouraged to submit manuscripts pertinent to pteridology for publica- tion in the American Fern Journal. Manuscripts should be sent to the managing editor at amerfernj@hotmail.com. Acceptance of papers for publication depends on merit as judged by two or more referees. Authors are encouraged to contribute toward publishing costs; however, the payment or non-payment of page charges will affect neither the accept- ability of manuscripts nor the date of publication. Authors should adhere to the following guidelines, manuscripts not so prepared set = returned for revision prior to review. El ever if it is necessary to submit hard copy, please submit one copy of the sino pss include a review copy of illustrations and originals of illustrations. After acceptance, please submit final versions of manuscripts via FTP (contact the managing editor for instructions), email, or on diskette or CD ROM (see below for figure formatting). If submitting hard copies, use standard 8.5 by 11 inch paper of good quality, not “erasable” paper. Double- space manuscripts throughout, including title, author’s names and full addresses, a short, informative abstract, key words, text (including heads and keys), literature cited, tables (separate from text), and figure captions (grouped as consecutive paragraphs separate from figures). Arrange parts of manuscript in order just given. Include author’s name and page number in the upper right corner of every sheet, and provide an abbreviated running title. Provide margins of at least one inch (25 mm) all around on typed pages. Do not submit right-justified text, avoid footnotes, and do not break words at end of lines. Make table headings and figure captions self-explanatory. Use S.I. (metric) units for all measures (e.g., distance, elevation, weight) unless quoted or cited from another source (e.g., specimen citations). For nomenclatural matters (i.e., synonymy and typification), use one paragraph per basionym (see Regnum Veg. 58:39-40. 1968). Abbreviate titles according to Botanico- Periodicum-Huntianum (Lawrence et al., 1968, Hunt Botanical Library, Pittsburgh) and its supplement (1991). References cited only as part of nomenclatural matter are not included in literature cited. For shorter notes and reviews, omit the abstract and put all references parenthetically in text. Use Index Herbariorum (Regnum Veg. 120:1-693. 1990; or http:// websun.nybg.org/bsci/ih/) for designations of herbaria. For more detailed instructions on manuscript preparation, see http://amerfernsoc.org/. Illustrations should be proportioned to fit page width (5 inches or 12.5 cm) with caption ultimately to be included on the same page. Halftone and color images should be scanned at a minimum of 300 pixels per inch (ppi). Line art should be scanned at 1200 ppi when- ever possible. Please note that nearly all images that are downloaded from the Internet or that are in JPEG or GIF format will be 72 dpi and not acceptable for the printing process. Indicate the file format of the graphics. Please submit image files in TIFF (preferable) or EPS format. Provide margins of at least 25 mm on all illustrations. For continuous-tone illustrations, design originals for reproduction without reduction or by uniform amount. In composite blocks, abut edges of adjacent photographs. Avoid combining continuous- tone and line-copy in single illustrations or blocks. Coordinate sequence and numbering of figures (and tables), with order of citation in text. Explain scales and symbols in figures themselves, not in captions. Include a scale and reference to latitude and longitude in each map. Pig j - » | is £; roe by ‘te printer. Authors should s send proof corrections of corrected Proofs to the editor and reprint orders to the printer. Authors de after type has been set. For other matters of form or style, consult recent issues of the American Fern Jour- nal and The Chicago Manual of Style, 14" ed. (1993, Univ. of Chicago Press, Chicago). Occasionally, departure from these guidelines may be justified. Authors are encouraged to consult the editor for assistance with any aspect of manuscript preparation. P: 7 UN OEE ON EES LARIAT UE LS apers Editor, see = cover page ae PTERIDOLOGIA ISSUES IN PRINT The following issues of Pteridologia, the memoir series of the American Fern Society, are available for purchase: 1. Wagner, David H. 1979. Systematics of Polystichum in Western North America North of Mexico. 64 pp. $10.00 plus postage and handling. 2A. Lellinger, David B. 1989. The Ferns and Fern-allies of Costa Rica, Panama, and the Choc6 (Part 1: Psilotaceae through Dicksoniaceae). 364 pp. $32.00 plus postage and handling. 3. Lellinger, David B. 2002. A Modern Multilingual Glossary for Taxonomic Pteri- dology. 263 pp. $28.00 plus postage and handling. For orders and more information, please contact our authorized agent for sales at: Missouri Botanical Garden Press, P.O. Box 299, St. Louis, MO 63166-0299, tel. 314-577- 9534 or 877-271-1930 (toll free). For online orders, visit: http://ww I org or AMERICAN FERN JOURNAL ON MICROFICHE Volumes 1-61 of the American Fern Journal are available as archival quality, silver positive microfiches. Single volumes or the entire run may be purchased. The fiches are ~~ read with 10x or greater magnification (using a dissecting microscope and transmit- ted illumination or a fiche reader). Silver negative microfiches of vols. 1—50 are also avail- able. The price is $4.00 per volume or $244.00 per set of 61 volumes, postpaid. Send your inquiry or order with a check or money order to: American Fern Society, Inc., % Ecology Ill, 804 Salem Blvd., Berwick, PA, 18603-9801. FIDDLEHEAD FORUM The editor of the Bulletin of the American Fern Society welcomes contributions from members and non-members, including miscellaneous notes, offers to exchange or purchase materials, personalia, horticultural notes, and reviews of non-technical books on ferns. SPORE EXCHANGE Ms. Denia Mandt, 12616 Ibbetson Ave., Downey, CA 90242-5050, is Director. Spores exchanged and lists of available spores sent on request. http://amerfernsoc.org/sporexy. html GIFTS AND BEQUEST S Cutt dh ta the & it to ex nal it } At, tHe OOC ciety cu able i interested in ferns. Back issues of the Journal and cash or other ots are always welcomed and are tax-deductible. Inquiries should be addressed to the Membership Secretary. VISIT THE AMERICAN FERN SOCIETY’S WORLD WIDE WEB HOMEPAGE: http://amerfernsoc.org/ AMERICAN se FERN Number 3 JOURNAL oe QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY Comparative siesipniamaey Capacity of Abaxial and Adaxial Leaf Sides as Related to Exposure in Two Epiphytic Ferns in a Subtropical Rainforest in Northeastern Craig E. Martin, Rebecca (Chia-Chun) Hsu, and Teng-Chiu Lin 145 Selected Physiological Responses of Salvinia minima to Various Temperatures and Light Safaa H. Al-Hamdani and Jamil J. Ghazal 155 Habitat Differentiation of Ferns in a Lowland Tropical Rain Forest James E. Watkins, Jr. and Catherine Cardelts 162 — Microbial Communities Associated with the Rhizosphere of the Temperate rm Thelypteris noveboracensis (L.) Nieuwl. O. Roger Anderson 176 f the Rhi Vascular System of Four Polypodium S Archana Srivastava and Subhash Chandra 182 Isoetes maxima, a New Species from Brazil R. James Hickey, C. Cecilia Macluf, and Melanie Link-Pérez 194 New Records of Polyphlebium borbonicum, an African Filmy Fern, in the New World and Polynesia Atsushi Ebihara, Joel H. Nitta, David Lorence, and Jean-Yves Dubuisson 200 ap sea Tree Fern ee to South and Central Ameri Claudia Cristina L. Fiori, Marisa Santos, ned Aurea M. Randi 207 In vilt Oo Stud hyte D t of Epiphytic Fern, Arthromeris himalay- ensis spent ) Ching, of South S Sikkim. India am Ganguly, Kaushik Sarkar, and Radhanath Mukhopadhyay —_217 a. Of: Pee Se ee elo ya 0 Ctarikiesah ig Wu Hua, Chen Ping-Ting, Yuan Li-Ping and Chen Long-Qing 226 The American Fern Society Council for 2009 WARREN D. HAUK, Dept. of Biological Sciences, Denison University, Granville, OH 43023. President MICHAEL WINDHAM, Dept. of Biology, Duke University, P.O. Box 90338, Durham, NC 27708. President-Elect W. CARL TAYLOR, National Science Foundation, 4201 Wilson Blvd., Arlington, VA 22230. Secretary JAMES D. CAPONETTI, Div. of Biology, University of Tennessee, Knoxville, TN 37996-0830. Treasurer GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. JAMES D. MONTGOMERY, Ecology III, 804 Salem Blivd., Berwick, PA 18603-9801. Curator of Publications JENNIFER M. O. GEIGER, Dept. of Natural Sciences, Carroll College, Helena, MT 59625 Journal Editor R. JAMES HICKEY, Dept. of Botany, Miami University, Oxford, OH 45056. Memoir Editor apd N. E. HUDSON, gor ps of Biological Science, Sam Houston State caine Huntsville, TX 77341 -2116. AVID Christey Way, Bakersfield, CA 93312-5617 Bulletin Editors American Fern Journal EDITOR JENNIFER M. O. GEIGER Dept. of Natural Sciences, Carroll College, Helena, MT 59625 ph. 447-4461, e-mail: jgeiger@carroll. edu MANAGING EDITOR JILL ANNE DILI Dept. of Natural Sciences, Carroll College, Helena, MT 59625, ph. (406) 447-5176, e-mail: jdill @carroll.edu ASSOCIATE EDITORS GERALD J GASTONY Dept. of Biology, Indiana University, Bloomington, IN 47405-6801 GARY © GREER ae Biology Dept., Grand Valley State University, Allendale, MI 49401 CHRISTOPHER H. HAUFLER ................... sr of Ecology and Evolutionary apo ihe) of Kansas, e, KS 66045-2106 R. JAMES HICKEY Dept. of Botany, Miami mie Oke OH 45056 ROBBIN C. MORAN New York Botanical Garden, pte NY 10458 fib JAMES E. WATKINS, JR. dge, MA 0213 The “American Fern Journal” (ISSN 0002-8444) is an illustrated apne devoed to the apa cs of ferns. It is owned by the American Fern Society, and published at The Am m Society, % Misso Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. Periodicals came Sas at St. Louis, MO, a additional entry. for back issues should be addressed to Dr. James D. iy dente nase Il, 804 Salem Blvd., slang PA 18603- 9801. dd , dues, 1 1 ad rr r Z nf 1 ~e £ et wh 7 prs ee q 4a 1: PS £, .. 1lAt + L RA . Pape Back volumes are avalible for most years as printed issues or on microfiche. Please contact the Back Issues Curator for prices and availability. Subscri Poi Membership - USA, (Canada, Mexico (includes J ] and Fiddlehead F ) $25 Society M mbership 1er ¢s. ‘$0 fi , 3 1 30: AA . 4 Memb $300 (add $140 ili ig h s' fe tside USA , Canada, Mexico) oe Mea USA, ‘Canada, b Mexico (includes Fiddlehead F $12 i Cr AA 1. $19 mien Membership — $35 to USA, han Mexico; $45 elsewhere eee agency fee) STER: Send address changes to American Fern Journal, Missouri Botanical Garden, P.O. Box 299, St. ites MO 63166-0299. American Fern Journal 99(3):145—-154 (2009) Comparative Photosynthetic Capacity of Abaxial and Adaxial Leaf Sides as Related to Exposure in Two Epiphytic Ferns in a Subtropical Rainforest in Northeastern Taiwan Craic E. MARTIN aiwan Forestry Research Institute, 53 Nanhai Rd., Taipei 10066, Republic of China (Taiwan) cee eet of Ecology & Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA Repecca (CH1A-CHun) Hsu and Tenc-Curu Lin* Taiwan Forestry Research Institute, 53 Nanhai Rd., Taipei 10066, Republic of China (Taiwan) Asstract.—Photosynthetic gas exchange was measured in situ with either the adaxial or abaxial leaf surface illuminated on vertical, horizontal, and angled leaves of Asplenium nidus and vertical leaves of Ophioderma pendula, two epiphytic ferns in a subtropical rain forest in northeastern Taiwan. Leaves for gas exchange measurements were selected to ensure a diversity of different exposures of the two leaf surfaces to direct sunlight. For most leaves of both species, photosynthetic rates were higher when the side of the leaf that typically received more direct insolation was illuminated during the gas exchange measurement. ee | pave of net CO, uptake hen one side of the leaf was illuminated, relative to rates when th was illuminated, were attributable to a greater biochemical capacity hed photosynthesis, a 2 ome stomatal con cthoorsbas seeps) on — results of this study, the 5 fthe a leaves for the most part, reflects the degree of exposure a each side of the leaf to direct sunlight, as ee om found in similar studies of terrestrial taxa. Y Worps.—abaxial leaf surface, adaxial leaf surface, Asplenium, epiphytes, illumination, leaf angle, Ophid oa. photosynthesis, subtropical forest, Taiwan Most leaves are green and, thus, presumably capable of some level of photosynthetic activity, even if just recycling respiratory CO, on both their adaxial and abaxial surfaces (Moore et al., 1998; Terashima, 1986). Work with terrestrial taxa has shown that the capacity for photosynthesis is equal, o nearly so, when either leaf surface of vertically oriented leaves is illuminated, as long as both surfaces intercept similar amounts of solar radiation during leaf development (Syvertsen and Cunningham, 1979; DeLucia et al., 1991; Poulson and DeLucia, 1993). In contrast, if one side of a vertically oriented lea typically receives more insolation than the opposite side, the photosynthetic capacity of the leaf is greater when the normally sunlit surface is irradiated during photosynthetic measurements, relative to photosynthesis when the shaded side is irradiated (Poulson and DeLucia, 1993; but see Vaclavik, 1984) *Corresponding author. T.-C. Lin, Department of Life Science, Taiwan National Normal University, 88 Ting-Chou Road, Section 4, Taipei, 116 Republic of China (Taiwan). tel.: +886-2- 29336875. E-mail, tclin@ntnu.edu.tw 146 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Likewise, the photosynthetic activity of horizontally oriented leaves is greater when their adaxial surface is illuminated than when their abaxial surface is illuminated (Syvertson and Cunningham, 1979; Terashima, 1986; DeLucia et al., 1991) The latter applies only to the sun leaves, not the shade leaves, of Sitka spruce (Leverenz and Jarvis, 1979). Epiphytic vascular plants appear to have been excluded from such studies, yet are ideal subjects for such investigations. Epiphytic vascular plants often exhibit a great diversity of leaf orientations and exposures (Benzing, 1990). For example, epiphytes with a rosette growth form often have leaves ranging from vertical to horizontal, and many have intermediate angles. Furthermore, a number of epiphytic taxa have vertically oriented leaves that are, unlike their terrestrial counterparts, positively geotropic. Most epiphytes also live in a complex light environment, being shaded by the host tree stem and canopy, as well as surrounding trees, depending on the location of the sun at any point in time. Given their leaf angles and the complexity of the light environment in which epiphytes grow, it is difficult to predict how the photosynthetic capacity of the two sides of the leaves of such plants compare and whether or not findings based on terrestrial taxa might apply to epiphytes. Therefore, the goal of this study was to determine if photosynthesis in epiphytes, particularly ferns, responds to leaf surface illumination in a similar manner as has been found in terrestrial plants. MATERIALS AND METHODS Study site and species.—Leaf photosynthetic parameters were measured for six individuals of Asplenium nidus L. and five individuals of Ophioderma pendula (L.) Presl in situ at the Fushan Experimental Forest, a comparatively pristine tract of subtropical rainforest (121°34’E, 24°46’N) at an elevation of ~600 m located 40 km southeast of Taipei in northeastern Taiwan. For general climatic conditions at the Fushan site, see Martin et al. (2004). Environmental conditions during the week of measurements (11-15 July 2005) were: 25.1° C average daily air temperature (29.8° C average daily maximum; 21.3° C average daily minimum), 4.2 mbar average daily vapor pressure deficit (vpd); and 20.0 mol m * day’ average daily Photosynthetic Photon Flux Density (PPFD). Asplenium nidus and O. pendula were chosen for this investigation to ensure a diversity of different exposures of the two leaf surfaces to direct sunlight. Plants were selected in a partially disturbed section of the forest to allow easy access for in situ measurements of photosynthesis. The study site included several walking trails and was tens of meters from a laboratory building. Species of dominant trees at this site were numerous, primarily in the families Fagaceae and Lauraceae; examples include Litsea acuminata (B1.) Kurata (Lauraceae), Machilus zuihoensis Hayata (Lauraceae), Castanopsis cuspidata (Thunb. ex Murray) Schottky var. carlesii (Hemsl.) Yamazaki (Fagaceae), and Pasania hancei (Benth.) Schottky (Fagaceae). MARTIN ET AL.: PHOTOSYNTHETIC CAPACITY OF TWO EPIPHYTIC FERNS 147 All plants were large (plant diameter for A. nidus = 0.5m and leaf length for O. pendula = 0.5m) growing epiphytically on a variety of host trees, including those listed above. Most plants had sporangia on some leaves at the time of this study (sporangia-bearing portions of the leaves were avoided in all measure- ments to avoid effects of sporangia on the measurements (Chiou et al. 2004). All leaves were measured no higher than three to four meters from the ground, i.e., within arm’s reach while standing, with or without a ladder. Only mature, non-senescent leaves lacking substantial insect damage were sampled; very young and very old leaves were avoided. Leaves were selected without regard to host tree species, height from the ground (except as noted), and degree of canopy shade at the time of measurements. Photosynthesis ts.—Photosy is was measured on three different leaves for each of six plants of A. nidus; the three leaves were selected for measurements based primarily on the likelihood of exposure of each leaf surface to direct sunlight. Horizontal leaves were older (based on size, presence of sporangia, weathering, and phyllotaxy of the epiphyte) than the other two leaves selected for measurements and grew perpendicular to and away from the host tree trunk. Such leaves should intercept very little direct sunlight on their abaxial surface, whereas their adaxial surface should intercept direct sunlight during much of a sunny day. Angled leaves grew at . about a 45 degree angle from the tree trunk, so should occasionally intercept direct sunlight on both surfaces of the leaf. Vertical leaves grew close to the trunk of the host tree, and, thus, were shaded by the trunk much of the day. These leaves should intercept little light on their adaxial surface most of the day, but occasionally direct sunlight on their abaxial surface, depending on the location of the sun. All leaves of Ophioderma pendula grew with similar exposure to light on their two surfaces as in the ‘‘vertical” leaves of A. nidus, but, in contrast to leaves of A. nidus, the growth of O. pendula leaves was positively geotropic. Another important difference between the “vertical” leaves of these two epiphytic ferns is that the leaf sides are reversed in the two taxa, i.e., in A. nidus, the abaxial side of the “vertical ‘“‘ leaves faces outward, and is thus more exposed to solar irradiation, whereas, the adaxial surfaces of the O. pendula leaves face outward and are thus more exposed to direct sunlight. Although intercepted sunlight on all leaves and their two surfaces was not measured, field observations during this study confirmed the above statements. Photosynthesis was measured with a LI-COR (Lincoln, NE) LI-6400 Portable Photosynthesis System. Because all leaves measured were large, the area of leaf for which gas exchange was measured matched the maximum area possible (6 cm’) in the gas exchange chamber. Photosynthetic parameters were measured two different ways at the central portion of each leaf: once with the adaxial surface illuminated and again adjacent to the same leaf location with the abaxial surface illuminated. The exact same location on the leaf was not used for both measurements to ensure that manipulation by inserting the leaf into the chamber and clamping the chamber on the leaf for the first measurement did not influence the second measurement. Although gas 4} 148 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) exchange was always measured for both sides of the leaf simultaneously, the chamber was oriented such that only the adaxial or abaxial surface received light from the blue and red diodes in the top half of the chamber. Very little ambient light reached the opposing leaf surface during the measurements as a result of shading by parts of the gas exchange chamber, the investigators, and nearby vegetation. For both species, photosynthesis was measured three times with illumination on one surface of a leaf at a low PPFD (100 nmol m * s_‘), then three times at a high PPFD (1000 umol m~* s_'). Using the same leaf, the chamber was then reversed to measure gas exchange with illumination (both PPFD levels) on the opposite leaf surface. Net CO, uptake in A. nidus saturated at approximately 500 umol m * s_' (determined with preliminary gas ex- change measurements). Other environmental conditions during all measure- ments were maintained by the LI-6400 system at the following values: air COz concentration of 370 pmol mol’, chamber (‘‘block’’) temperature of 30°C (leaf temperatures were typically 0.5° C higher), vapor pressure deficit (vpd) of 0.9 mbar, and flow rate of 200 umols ‘. Lower vpd values resulted in exceedingly low transpiration rates, which led to unrealistic values for C;; any such data were discarded. For each gas exchange measurement, data were recorded only when gas exchange rates were stable (Coefficient of Variation of exchange rates of both gases and flow rates not varying by more than 0.2% among successive measurements every 2—3 seconds), typically within 10 sec- onds of inserting the leaf in the gas exchange chamber or after the previous measurement (for a total of three repeated measurements). The gas exchange chamber remained clamped to a leaf for approximately five minutes at each light level, allowing for stable readings, as well as steps taken to ensure instrument accuracy (e.g., using the ‘‘match”’ function of the LI-6400 prior to each measurement). Statistical analyses.—For both species, means (N=5 or 6 plants; the value for each plant being a mean of three repeat measurements; see above) of abaxial and adaxial gas exchange parameters at each light level were compared with a paired Student’s t-test when the data met the assumptions of parametric statistics (Sokal and Rohlf, 1981) or with a Mann-Whitney U-test otherwise. RESULTS AND DISCUSSION The adaxial side of the vertical leaves growing out of the rosettes of A. nidus is unlikely to receive direct radiation due to shading by the host tree trunk, whereas the exposed abaxial side should at least occasionally intercept direct solar radiation. Thus, based on results with terrestrial plants (Syvertson and Cunningham, 1979; Terashima, 1989; DeLucia et al. 1991; Poulson and DeLucia 1993), it was predicted that the illumination of the abaxial side of the vertical leaves of A. nidus would result in higher photosynthetic rates than when the adaxial side of the same leaf is illuminated. Measurements of photosynthesis at both high and low PPFD of plants in northeastern Taiwan did not, however, support this prediction (Fig. 1). In contrast, although not statistically significant (high PPFD P= 0.28; low PPFD P = 0.17), the trend in MARTIN ET AL.: PHOTOSYNTHETIC CAPACITY OF TWO EPIPHYTIC FERNS 14 © |S ,.u jouurl ‘aBueyoxg “O95 39N Net CO, Exchange, pmol m” s™ VRAD VRAB HZAD HZAB ANAD ANAB VRAD VRAB HZAD HZAB ANAD ANAB Leaf Type & Side Leaf Type & Side Fic. 1. Mean (lines projecting from bars are standard deviations; n = 6 plants, three repeated measurements/leaf/plant) rates of net CO, exchange (positive values indicate CO, uptake) for different leaves and with illumination at two light levels on either side of the leaves of the epiphytic fern Asplenium nidus measured in situ in a subtropical rain forest in northeastern Taiwan). Abbreviations for type and side of leaf are: ““VR” = vertical, “HZ” = horizontal, “AN” = angled (45° from vertical); and ‘‘AD’ indicates illumination (A, 100 umol m~* s7?; B, 1000 pmol m~* s_*) provided to the adaxial side of the leaf during gas exchange measurements; “AB” indicates illumination (low and high PPFD as in AD) provided to the abaxial side of the leaf during measurements. The abaxial and adaxial means for two leaves at low PPFD are significantly different at P < 0.05 or P < 0.01 indicated by “*” or ‘‘**”’, respectively, above each pair of means, while the other pairs of means are not significantly different (P > 0.05, indicated by ‘‘ns’”’ above each pair of means). the data indicated the opposite of expectations, i.e., photosynthetic rates at either PPFD appeared higher when the adaxial surface was illuminated. According to the statistical analyses, however, photosynthetic rates at both light levels were equal regardless of which side of the leaf was illuminated (Fig. 1). Light interception of the two surfaces of the horizontal leaves of the epiphytic fern A. nidus is quite different from that of the vertical leaves, and the prediction of comparative photosynthetic capacities when the two sides of this leaf are illuminated is the opposite of that of the vertical leaves of this fern. Because the adaxial surfaces of these leaves intercept more direct solar radiation than do the abaxial surfaces, photosynthetic rates when the adaxial surface of the horizontal leaves of this epiphyte are illuminated should be higher than those of the leaf when the abaxial surface of the same leaf is illuminated. Measurements of photosynthetic rates confirmed this prediction, although the higher photosynthetic rates when the adaxial side of the leaves was illuminated were statistically significant only when measurements were made at the lower PPFD (Fig. 1). These higher net CO, uptake rates were accompanied by equal transpiration rates (Fig. 2) and stomatal conductances (Fig. 3), while internal CO, concentrations were significantly lower (Fig. 4) These gas exchange results indicate that the higher photosynthetic rate was most likely the result of a greater biochemical capacity for photosynthesis and not the result of greater stomatal opening and, hence, easier gas diffusion into = 1) i=) AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) VRAD VRAB HZAD HZAB ANAD ANAB VRAD VRAB HZAD HZAB ANAD ANAB Leaf Type & Side Leaf Type & Side j-8 zt jouw ‘aBueyoxy O2y JON Net H9O0 Exchange, mmol m”? 5"! Fic. 2. Mean (lines projecting from bars are standard deviations; n = 6 plants, three repeated poe peor ee rates of net HO exchange (positive values indicate water vapor loss) for different leaves and with illumination at two light levels on either side of the leaves of the euak fern Asplenium nidus measured in situ in a subtropical rain forest in northeastern Taiwan). Abbreviations for type and side of leaf are: ‘“VR”’ = vertical, “HZ” = ~ horizontal, Dior yale = angled (45° from vertical); and ‘‘AD’ indicates illumination ne 100 umol m*s?; B, 1000 umol m * s~*) provided to the adaxial side of the leaf during gas exchange measurements; “AB” indicates illumination (low and high PPFD as in AD) provided to the abaxial side of the leaf during measurements. None of the abaxial and adaxial means at any leaf location are significantly different (P > 0.05, indicated by ‘‘ns” above each pair of means) “ g % 2 3 : E re) 7s o o a 2° 4 =| 5 2 2 2 fee » i) 7) oO 4 VRAD VRAB HZAD HZAB ANAD ANAB VRAD VRAB HZAD HZAB ANAD ANAB Leaf Type & Side Leaf Type & Side Fic. 3. Mean (lines projecting from bars are standard deviations; N = 6 plants, three agai measurements/leaf/plant) stomatal conductances for different leaves and with illumination at tw light levels on either side of the leaves of the epiphytic fern Asplenium nidus measured in situ in a subtropical rain forest in northeastern Taiwan). Abbreviations for type and side of leaf are: “VR” = vertical, ““HZ’’ = horizonta 1, “AN” = angled (45° from vertical); and “AD” indicates illumination (A, 100 pmol m~* s~*; B, 1000 i m * s_') provided to the adaxial side of the leaf during gas exchange measurements; “AB” indicates luminaton (low and high PPFD as in AD) provided to the sbadal side of the leaf during measurements. The abaxial and adaxial means at two leaf locations at high PPFD are significantly eet at tP < 0.05, indicated by ‘**” above each pair of means, while the other pairs of means are not significantly different (P > 0.05, indicated by ‘“‘ns” above each pair of means). MARTIN ET AL.: PHOTOSYNTHETIC CAPACITY OF TWO EPIPHYTIC FERNS 15 pu eno — 500 — oa 3 E 3 800 400 Fs ° A = —_ = 300 300 g '~ 9 - O, 200 200 . g o 3 100 100 3 » ° = ee VRAD VRAB HZAD HZAB ANAD ANAB VRAD VRAB HZAD HZAB ANAD ANAB Leaf Type & Side Leaf Type & Side Fic. 4. Mean (lines projecting from bars are standard deviations; N = 6 plants, three repeated measurements/leaf/plant) leaf internal CO, concentrations (external CO. concentration was 370 umol mol“ ) for different leaves and with illumination at two light levels on either side of the loaves of the epiphytic fern Asplenium nidus measured in situ in a subtropical rain forest in northeastern Taiwan). Abbreviations for type and side of leaf are: “VR” = vertical, “HZ” = horizontal, ‘‘AN’’ = angled (45° from vertical); and ‘‘AD” indicates illumination (A 100 umol m~’ s_ '; B, 1000 umol m * s') provided to the adaxial side of the leaf during pes exchange measurements; “AB” an isin (low and high PPFD as in AD) provided to the abaxial side of the deve during measurements. The abaxial and adaxial means at esi leaf locations fi y different at P < 0.05 or P < 0.01, indicated by “*” or ““**”, ectively, above each po r of means, while the other pairs of means are not significantly different e > 0.05, indicated by ‘“‘ns” above each pair of means). the leaf (Farquhar and Sharkey, 1982; Sharkey, 1985). In agreement with the latter interpretation, it is possible, especially for the measurements made at high light, that illumination of the abaxial surface resulted in photoinhibition in this lateral half of the section of leaf being measured. This possibility is supported by previous findings that the side of a leaf that is typically less exposed to sunlight has pacthniors and photosynthetic features typical of shade-adapted leaves (Schreiber et al., 1977; Kulandaivelu et al., 1983; Terashima and Inoue, 1984; Terashima et al., 1986). Differences in photosyn- thetic capacity depending on which side of the leaf is illuminated might also reflect other anatomical or optical (e.g., absorptance) features of the two sides of the leaf (Terashima 1986; DeLucia et al., 1991). Such differences would also be interpreted as non-stomatal and non-diffusional mechanisms responsible for differences in photosynthesis between the two sides of the leaf, as was found in this study. Both the adaxial and abaxial surfaces of the ‘‘angled” leaves of A. nidus should intercept direct sunlight, at least for brief periods, throughout a day. Thus, one might predict that the photosynthetic capacity of these leaves is comparable, regardless which surface is illuminated (Syvertsen and Cunning- ham, 1979; Vaclavik, 1984; DeLucia et al., 1991; Poulson and DeLucia, 1993). Based on measurements made in situ with this epiphytic fern in northeastern Taiwan, this prediction was supported when gas exchange was measured at high PPFD (Fig. 1), but the photosynthetic rate when the adaxial leaf surface — a NS AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Net CO Exchange, jxmol m=? 571 Net H,O Exchange, mmol m” s* ADLL ABLL ADHL ABHL ADLL ABLL ADHL~ ABHL Leaf Side & PFD Leaf Side & PFD Fic. 5. Mean (lines projecting from bars are standard deviations; n = 5 plants, three repeated measurements/leaf/plant) rates of net CO, exchange (A; positive values indicate CO, uptake) and rates of net water vapor exchange (B; positive values indicate water vapor loss) with illumination at two light levels on either side of the leaves of the epiphytic fern Ophioglossum pendula measured in situ in a subtropical rain forest in northeastern Taiwan). fu oneao for side of leaf and light level are: “AD” indicates illumination (LL = 100 pmol m?s~ L= 1000 pmol m * provided to the adaxial side of the leaf during gas exchange Ae es “AB” indicates illumination (low and high PPFD as in AD) provided to the abaxial side of the leaf during measurements. The abaxial and adaxial means at high PPFD are significantly different at P < 0.01, indicated by “*” above that pair of means, while the other pairs of means are not significantly different (P > 0.05, indicated by ‘‘ns” above that pair of means). was illuminated exceeded that when the abaxial surface of the leaf was illuminated at low PPFD (Fig. 1). As was the case with the horizontal leaves, the higher net CO, exchange rate of the angled leaves was apparently the result of a greater biochemical capacity for photosynthesis, generating a lower leaf internal CO, concentration (Fig. 4), and not due to a greater stomatal conductance (Fig. 3; Farquhar and Sharkey, 1982; Sharkey, 1985). These findings contrast directly with those for Sitka spruce by Leverenz and Jarvis (1979), who found that differences in photosynthetic capacity of the leaves, depending on which side of the leaf was illuminated could be ascribed to differences in stomatal conductance, not to the biochemical capacity of the leaf. The leaves of Ophioderma pendula are positively geotropic, hanging vertically from the main body of this epiphytic fern and remaining close to the main stem of the host tree. As a result of shading by the immediately adjacent host tree trunk, the abaxial surfaces of these leaves seldom receive direct solar radiation. Thus, it was predicted that photosynthetic rates, at least at the higher PPFD, when the adaxial surface of these leaves is illuminated, would exceed those when the abaxial surfaces of these leaves are illuminated for plants measured in situ in this subtropical rain forest. This was indeed the case for net CO2 exchange measurements at both high and low PPFD (Fig. 5). Because rates of transpiration and stomatal conductances were equal when either side of the leaves was illuminated during gas exchange measurements MARTIN ET AL.: PHOTOSYNTHETIC CAPACITY OF TWO EPIPHYTIC FERNS 153 0.08 500 = w ae i 400 5 E 5 ro) — € 300 pes o x o <= 00 © o : 3 S o es 100 3 c ° fo} = oO —_ ADLL ABLL ADHL- ABHL ADLL ABLL ADHL ABHL Leaf Side & PFD Leaf Side & PFD Fic. 6. Mean (lines projecting from bars are standard deviations; n = 5 plants, three repeated measurements/leaf/plant) stomatal conductances (A) and leaf internal CO, concentrations (B;: external CO, concentration was 370 umol mol ') with illumination at two light levels on either side of the leaves of the epiphytic fern Ophioglossum pendula measured in situ in a subtropical rain forest i 5 ey rcp ). Abbreviations for side of beat and light level are: ““AD” indicates ieee? tion (LL 00 pmol m~* s_'; HL= 1000 upmol m Fae provided to the adaxial side of the leaf during gas Bes measurements; ‘“AB”’ cas Hianination (low and high PPFD as in AD) provided to the abaxial side of the leaf during measurements. Neither pair of abaxial and adaxial means is significantly different (P > 0.05, indicated by “ns” above the pairs of means). (Figs. 5, 6), and because internal CO, concentrations were lower (although not statistically significantly so at high PPFD; Fig. 6) when the adaxial surface was illuminated, the mechanism underlying the higher rate of net CO, uptake when the adaxial surface was illuminated appears, as was the case in several instances with A. nidus, to reflect a greater biochemical capacity for photosynthesis, not a greater stomatal conductance allowing easier CO, diffusion into the leaf (Farquhar and Sharkey, 1982; Sharkey, 1985). Overall, the results of in situ gas exchange measurements with two epiphytic ferns in a subtropical rain forest in northeastern Taiwan lend considerable, but not complete, support to past findings with terrestrial taxa (Syvertsen and Cunningham, 1979; Terashima, 1989; DeLucia et al., 1991; Poulson and DeLucia, 1993). In most, but not all, cases, if a leaf is oriented such that one side receives more direct solar radiation than the other, the leaf has a higher photosynthetic capacity when the more exposed surface is illuminated. In addition, this higher capacity reflects a greater biochemical capacity for photosynthesis and not easier diffusion of CO, into the leaf (Farquhar and Sharkey 1982; Sharkey, 1985) ACKNOWLEDGMENTS Financial support from National Science Council (Taiwan) grant #93-2621-B-018-001 to T.-C. Lin is gratefully acknowledged. We greatly appreciate C.-T. Chang, H.-S. Wang, Y.-H. Tseng, Elizabeth Forsyth, and M.-S. Huang for their assistance in the field. Special thanks are extended to Zhih-Hong Zhuang and Gene-Sheng Tung for their help with several aspects of this study and to Yue-Joe Hsia for sharing his meteorological data 154 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) LITERATURE CITED BenzinG, D. H. 1990. Vascular epiphytes. General biology and related biota. Cambridge Univ Press, GC idge. Cuiou, W.-L., C. E. Martin, T.-C. Lin and S.-H. Lin. 2004. Ecophysiological differences betwee sterile and nia koads by “al ne subtropical a: fern Pyrrosia lingua fBolypodiacene\ in in Taiwan. Amer. Fern J. 9 De.ucia, E. H., H. D. ‘Ses o1, S. eB ae ar A. Day. sha os symmetry of sun and shade leaves of different a Oecologia Farquuar, G. D. and T. D. SHARKEY. 1982. Stomatal gah ll and photosynthesis. Annu. Rev. Plant Physiol. 33:317—345. KULANDAIVELU, G., A. M. NoorupeeN, P. SAMpaTH, S. PerryANAN and K. RAMAN. 1983. Assessment of the phatosynthotic electron transport properties of upper and lower leaf sides in vivo by fluorometric method. Photosynthetica 17:204—209. LEverENz, J. W. and P. G. Jarvis. 1979. Photosynthesis in sitka spruce VIII. The effects of light flux density and direction on the rate of net photosynthesis and the stomatal conductance of needles. J. i a Ecol. 16:919-932. Martin, C. E., T.-C. Lin, C.-C. Hsu, S.-H. Lin, K.-C. Lin, Y.-J. Hsia and W.-L. Cuiov. 2004. Ecophysiology and plant size in a tropical epiplytic fern, Aplenium nidus, in Taiwan. Int. J. ant Sci. 165:65—72 . CLark and D. S. bie 1996. i Hotany.. bere culscrgianl Hill, New York. Pouison, M. E, and E. H. Dezucia. 19 ructural acclimation to light direction in vertical leaves of Silphium suave ata ial a 95:393—400. ScureiBer, U., R. FINK and W. Vipaver. 1977. Fluorescence induction in whole leaves: differentiation between ns two leaf sides and adaptation to different light regimes. Planta 133:121-129. Suarkey, T. D. 1985. Photosynthesis in intact leaves of C3 plants: physics, physiology and rate Tole by Rev. 51:53-105. SoxaL, R. R. and F. J. Rouir. 1981. Biometry. The principles and practice of statistics in biological research. 2nd Ed. WH Freeman & Co, New York. SYVERTSEN, J. P. and G. L. Cunnincuam. 1979. The effects of irradiating adaxial or abaxial leaf surface on 98: rate of net photosynthesis of Perezia nana and Helianthus annuus. Photosynthetica 7203. tase I. 1986. Dorsiventrality in photosynthetic light response curves of a leaf. J. Exp. Bot. 7:399—405. TERASHIMA, I. and Y. INOUE. eis: Carpatenye photosynthetic Be gsoee of gues a rian aicatans and spongy functional adj ihe FE rocachpaaae apparatus to light environment within a leaf. Plant Cell Physiol. fA ig ig ‘y . as and N. reste) 1986. Intra-leaf and intracellular gradients in chloroplast ultrastructure of dorsiventral lea ll om the adaxial or abaxial side during their d VActavik, J. 1984. Photosynthetic CO, uptake by Zea mays leaves as influenced by unilateral irradiation of adaxial and abaxial leaf surfaces. Biol. Plant. 26:206—214. American Fern Journal 99(3):155—161 (2009) Selected Physiological Responses of Salvinia minima to Various Temperatures and Light Intensities SaFAA H. AL-Hampani* and Jami. J. GHAZAL Department of Biology, Jacksonville State University, Jacksonville, AL 36265, USA BSTRACT.—Two separate experiments were conducted to determine the influence of temperature (15, 23 and 35°C) and various light intensities, ranging from 80 to near 700 umol/m?/s on selected experiment was carried out for 14 days under controlled environments, with a light intensity of 120 umol/m?/s and 14 h photoperiod. Plant growth was the highest at 23 and 35°C, in comparison to those grown at 15°C. The chlorophyll concentration was less influenced by the temperature than by the growth; however, carotenoid concentration at 35°C was significantly higher than those obtained from the plant grown at 15°C. Salvinia acclimation to cold temperature possibly included an increase in athocyanin and soluble sugar concentrations. The second experiment was carried out under greenhouse conditions, 25—27°C and various light intensities ranging between 80 to near 700 umol/m7/s in order to determine the light saturation curve. Salvinia was shown to have a wide range of acclimation ability to various light intensities ranging from 80 to near 700 umol/m2/s. This study should be helpful for determining the ecological distribution of salvinia. Key Worps.—Salvinia minima, CO, assimilation, photosynthetic pigments, light intensities The genus Salvinia, from the family Salviniaceae, is comprised of 12 known species (Nauman, 1993). Salvinia minima Baker is a small, free-floating freshwater fern found in tropical and temperate regions (DeBusk and Reddy, 1987) and in areas such as North, South, and Central America, the West Indies, and Africa (Nauman, 1993). This species is commonly referred to as water fern and South American pond fern (Nauman, 1993). In the United States, salvinia was first discovered in the St. John’s River, Florida in 1928 (Long and Lakely, 1976) and was speculated to have been introduced by accident through its discharge from contaminated boat ballasts from South American ships or from accidental release from aquarium sources (Schmitz et al., 1991). This plant can be found floating near the edges of slow moving streams and in nutrient enriched ponds. Salvinia forms a ‘“‘mat”’ that covers very large portions of the body of water where it grows. Salvinia reproduces exponentially by vegetative fragments, with a leaf doubling time of 3.5 days (Nichols et al., 2000). The rapid growth of salvinia has been recognized as an ecological problem in many southern coastal regions of the United States. This is due to the impact on suppressing the growth of native vegetation and the degradation of water quality; this includes reducing oxygen concentration, reduces the light *Corresponding author: S. H. Al-Hamdani, Department of Biology, Jacksonville State University, 700 Pelham Road, Jacksonville, AL 36265. Tel: (256) 782-5801, E-mail: sah@jsu.edu 156 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) penetration, and other ecological impacts (McFarland et al., 2004). However, The unique characteristics of salvinia, which include rapid growth, the high acclimation capacity of the plant to wide range of temperatures, and relative tolerance to a wide range of contaminants make it a prime candidate for phytoremediation (Olguin et al., 2003; Olguin et al., 2007). For instance, Salvinia minima demonstrated the ability to withstand aluminum (Al) concentrations of 20 mg/l through the manipulation of the media pH from 3.9 to near 7 within 24 hours (Gardner and Al-Hamdani, 1997). In addition, salvinia has the ability to survive and grow under highly eutrophic environments unsuitable for other species found in similar environments such as azolla (Azolla caroliniana Willd.) and duckweed (Lemna minor L.) (Reddy and DeBusk, 1985). Whether salvinia is perceived as either a noxious or beneficial weed, it is essential to determine the environmental requirements for its growth. Temperature and light intensity are among major environmental factors determining the ecological distribution of any plant (Larcher, 2003). There is a lack of general information regarding these environmental requirements for salvinia growth. Therefore, in this study, the influence of selected tempera- tures (15, 25, 35°C) on salvinia growth was determined. To examine the ability of salvinia to combat the impact of low temperature, the accumulation of soluble sugar was examined. The accumulation of soluble sugar is one of the major defense mechanisms of plants to cold temperatures (Larcher, 2003). In addition, the photosynthetic light response curve was determined to evaluate the photosynthetic response of salvinia to different light intensities. The photosynthetic pigment concentrations at different temperatures were evalu- ated and anthocyanin concentration was also determined. Anthocyanin is a major flavonoid pigment which is considered a free radical scavenger reducing the damages resulting from oxidative stress such as photooxidation (de Pascual-Teresa and Sanchez-Ballesta, 2007). MATERIALS AND METHODS To accomplish the objective of this study, several separate experiments were carried out under controlled environments of temperature and light intensity. To determine the effect of temperature (15, 23, 35°C) on salvinia growth, a total of 20 fronds were placed into 250 ml Erlenmeyer flasks containing 125 ml of 10% Hoagland’s solution (Hoagland and Arnon, 1938). The plants utilized in this study were taken from stock material growing under greenhouse conditions. The plants of all treatments were grown for fourteen days in a growth chamber at 23°C, 120 wmol/m*/s photon flux density and 14h photoperiod. Twelve flasks (samples) per treatment were used. The samples of each treatment were placed in separate containers. Water was added to each container, and the flasks were partially submerged in a water bath. The individual temperatures were controlled by passing water with the desirable temperature from a circular water bath unit through cooper coils located inside the individual water containers. AL-HAMDANI AND GHAZAL: PHYSIOLOGICAL RESPONSES OF SALVINIA MINIMA 157 The plants of six samples (flasks) from each treatment were used for growth determination. The remaining six samples of each treatment were used to determine the soluble sugar content. Salvinia growth was determined using fresh weight and frond number doubling time. Frond number of each sample was counted at the initial day of the experiment (day1) and on day 7 and 14. The frond numbers were used to evaluate the growth rate using the following doubling time formula: DT = t log 2 [log (wyw, *)]' (Moretti and Gigliano, 1988) where DT is the doubling time (days), t is the experiment duration (days), w; is the final number of frond, and w, is the initial frond number. Approximately 0.1 g fresh weight of each sample was used for measuring chlorophyll a and b, and carotenoid concentration. The plants were placed into 5 ml of N,N-Dimethylformamide (DMF) solution. The samples were incubated in the dark for 36 hrs at 4°C. Chlorophyll a and b was determined spectrophotometrically at wavelengths of 647 and 664.5 nm (Inskeep and Bloom, 1985). The anthocyanin was determined by homogenizing 0.1 g fresh tissues in 5ml methanol containing 1% HCl (v/v) for 2 min on ice. The homogenate was filtered and absorbance of the extract was determined spectrophotometrically by the method of Mancinelli (1990). Soluble sugar analysis was conducted following a procedure slightly modified from Chatterton et al. (1987). Six randomly selected samples of each treatment were oven dried at 65°C for 48 h. The dry samples were ground into a fine powder, and a 100-500 mg portion was placed in a sealed vial and used to measure soluble sugars as reported in detail by Wilson and Al-Hamdani (1997). This experiment was repeated twice each and statistically analyzed as a randomized complete block design (Steel and Torrie, 1980). This design ensured that observed differences in plant performances were largely due to treatments rather than variation among the four blocks (experiments conducted at different times). Mean separations for the treatments with significant F values (P = 0.05) of ANOVA analysis were based on the least significant difference (LSD) test (Steel and Torrie, 1980). The second experiment was carried out to determine the photosynthetic light response curve. The salvinia was grown in Hoagland’s solution, as in the first experiment. Six flasks containing 20 fronds of salvinia were grown for 7 days under greenhouse conditions of 25—27°C and various light intensities, depending on the time of day, ranging from 80 to near 700 pmol/m?/s. Carbon dioxide assimilation of the six samples was measured starting four hours after the onset of the light period at day seven. The selected plants of each sample were placed on wet filter paper and enclosed in a flow-through plexiglass assimilation chamber (4.5 by 11.8 by 7.3 cm) of a Li-Cor 6200 photosynthesis system (Lincoln, NE, USA) as described by McDermitt et al. (1989). Standard measurement conditions were 27°C, various light intensity with ranges from 80 to near 700 pmol umol/m’/s photon flux density and 75% relative humidity. To establish a light saturation curve, photosynthesis measurements were taken at various times of the day to obtain the desired range of light intensities. This experiment was repeated three times. 158 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) TasLe 1. The effect of different temperatures on salvinia growth and soluble sugar accumulation. Doubling time (days) Length of exposure (days) Soluble sugar* mg/g y weigh Temperature (°C) 7 14 15 —33.559a 53.364a 78.130a 23 3.865b 4.668bb 33.830b 39 3.468b 7.741b 14.953b Mean within the column followed by the same lower case letter are not significant based on LSD (P -05). * The soluble sugar was determined after fourteen days of exposure at the selected temperatures. RESULTS AND DISCUSSION Assessment of salvinia growth at the three selected temperatures clearly showed that exposure to colder temperatures (15°C) resulted in significant growth decline in contrast to samples exposed to higher temperatures (Table 1). After seven days of growth at 15°C, salvinia doubling time showed a negative value, indicating no determinable change in the growth status during that period. This result was expected since salvinia is a tropical plant and is susceptible to cold temperatures (Gaudet, 1973). Debusk and Reddy, 1987 reported that the growth rate of Salvinia rotundifolia was significantly lower at 10°C, compared to samples grown at 25°C. The optimum gro temperature for most tropical plant species was reported between 23 to 32°C. (Lee et al., 2007). Salvinia grown at 23 and 35°C showed similar rapid growth, as indicated by the doubling time values, which was less than four days (Table 1). The growth results after 14 days were comparable to those at seven days. The exception were the plants grown at 15°C, which demonstrated very slow growth rate, with a positive value for the doubling time (53.36 days). This result indicates that salvinia can survive at low temperature of 15°C. Chlorophyll a and b concentrations were similarly influenced by the three temperature treatments (Table 2). The exception was plants grown at 35°C which showed significantly higher chlorophyll a concentration than the other temperatures. Similar findings were reported by McWilliam and Naylor (1967). They reported a reduction in chlorophyll content in corn (Zea mays L.) associated with lowering the growth temperature from 28 to 16°C. The commonly observed reduction in chlorophyll concentration in tropical plants at low temperature was attributed in part to an aberrant development of the thylakoid membranes (Hodgins and van Huystee, 1986). In addition, low temperature was found to induce a reduction in several enzymes associated with chlorophyll synthesis in the plant (Tewari and Tripathy, 1998). In this study, the temperature treatments of 35°C induced significant increases in carotenoid concentration in comparison to those grown at 15°C (Table 2). However, temperature effect of 23°C on carotenoid concentration was not significantly different to those grown at 15 and 35°C. Lefsrud and Kopsell (2005) reported an increase in B-Carotene concentration in kale (Brassica AL-HAMDANI AND GHAZAL: PHYSIOLOGICAL RESPONSES OF SALVINIA MINIMA 159 Tasie 2. The influence of selected temperatures on chlorophyll a (chl a), chlorophyll b (ch! b), carotenoid and anthocyanin concentrations in salvinia after fourteen days of growth. Chl a Chl b Carotenoid Anthocyanin Temp. (°C) (mgg fr.wet.) (mgg' fr.wet.) (ugg * fr.wgt) (ugg * fr.wgt) a Fo) 6.163a 3.747a 515.075a 31.480a 23 6.762a 4.235a 686.625ab 10.478b oo 8.903b 4.963a 1252.452b 21.698c } eer 4 Mg efe he j An LSD (P = Mean within the column followed by the same lower 0.05). oleracea L.) in response to gradual temperature increases from 15, 20, 25 to 30°C. Similarly, Leipner et al. (1997) found a decline in chlorophyll a and chlorophyll b and carotenoid concentrations in corn associated with lowering the temperature from 25 to 15°C. Anthocyanin concentration was significantly different among the plants grown at different temperatures (Table 2). The highest anthocyanin concen- tration was obtained from the plant grown at 15°C, followed in decreasing order by those grown at 35 and 23°C. The increase in anthocyanin concentration in response to the relatively low (15°C) and high (35°C) temperatures might be considered an acclimation response to stress. This conclusion was also supported by Doong et al. (1993), who reported that anthocyanins are produced by most aquatic plants in response to stress factors such as high light intensity, high temperature, or nutritional limitations, and can be used as a stress indicator. Increased anthocyanin concentrations were also found to be induced in azolla by Al stress (Ayala-Silva and Al-Hamdani, 1997) and by Cr (VI) (Wilson and Al-Hamdani, 1997). Salvinia accumulation of soluble sugar was significantly increased with each decrease in temperature from 35 to 23 and 15°C (Table 1). This increase in soluble sugar might represent an acclimation response to low temperature. Sugar accumulation could play an important role in combating the influence of low temperature (Levitt, 1980; Hurry et al., 1995). Similar findings were reported by Al-Hamdani and Thomas (2000). Strand et al. (1997) suggested that an increase in soluble sugar accumulation is an essential response to combat the cold temperature stresses. The advantage of increasing soluble sugar is associated with the reduction in freezing point and increase plant tolerance to cold lemperaturs aes and Orcutt, 1996). Th between light inte y d h 1 that the light- limiting portion of the light pudeeion curve = cttceniol to near 300 umol/m?/s (Fig. 1). This can be used as an indicator that photosynthesis was operating linearly with the light intensity during this portion of the curve. The CO, limiting portion of the curve extended from 350 to near 700 umol/m’*/s, Similar findings were reported for Floating Pennywort (Hydrocotyle ranunculoides L.f.) with a light saturation of CO, gas exchange between near 350 to near 800 umol/m?/s (Hussner and Losch, 2007). In conclusion, salvinia growth was the highest at 23 and 35°C and lowest at 15°C. However, the reduction in plant growth was not severe enough to totally 160 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) 0.5 ~~, nt MO 04 - a «£ ay aa ng = ah A aA => a a . He = 03 | a 4 ane . 2) A ® S 02, — a ” A g s oO 214 5 ow a 0.0 T T T q T T 0 100 200 300 400 500 600 700 . . -2 -2 Light Intensity (uEm‘s °) Fic. 1. The relationship between photosynthesis and light intensity for Salvinia minima. inhibit plant reproduction. Salvinia acclimation to cold temperature possibly included the increase in anthocyanin and soluble sugar concentrations. The light saturation curve indicated that salvinia has a wide range of acclimation to various light intensities ranging from 80 to near 700 umol/m’/s and should be helpful for determining the ecological distribution of salvinia. Since one of the major criteria in selecting plants for ecological restoration is the acclimation capacity to the range of temperature and light intensity (Salt et al., 1998), salvinia’s capability to withstand a diverse range of environmental variables make it a prime candidate for phytoremediation. ACKNOWLEDGMENTS The authors wish to acknowledge Ms. Kristin Schwarzauer for her technical assistance in advancing the manuscript and Jacksonville State University for supporting the projec LITERATURE CITED Au-Hampanl, S. H. and T. S. Tuomas. 2000. Influence of root chilling on winter and spring wheat growth and carbon dioxide assimilation. Acta Agriculturae Scandinavica, Sect B, Soil and Plant Sci. 50:149-154. Aya.a-SiLvA, T. and S. H. At-Hampani. 1997. interactive effects of polylactic acid with different aluminum concentrations on growth, Laces concentrations, and carbohydrate accumula- tion of Azolla. Am. Fern J. 87:120—12 AL-HAMDANI AND GHAZAL: PHYSIOLOGICAL RESPONSES OF SALVINIA MINIMA 161 De Pascuau-Teresa, S. and M. T. SaNncHez-BaLtesta. 2007. Anthocyanins: from plant to health. Phytochem. Rev. 7(2):281-29 oi and Desusk, W. F. K. R. Reppy. 1987. Growth and nutrient uptake potential of Azolla spa baigaat Willd. and nips ne Willd as a function of temperature. J. Exp.Bot. 27(2):215 Doone, R. L., G. : MacDona_p and D. G. Suiinc. 1993. Effect of fluridone on orci ale carotenoid and anthocyanin content of sien Be dees Plant. — 31: 55— ais - L. and S. H. At-Hampani. 19) bis t nia. ° Aquat. Plant. Manage. 35:30-34. GAUDET, a J. 1973. Growth of Pay peor weed, Salvinia, under standard conditions. a cle 41:107—112. Hopes, R. and R. B. van Huysteg. 1986. Porphyrin metabolism in chill stressed maize (Zea mays L.). J. Plant. Physiol. 125:325-336. Hurry, V. M., O. Keersurc, T. PARNIK, P. Garpstrom and G. Oquist. 1995. Cold hardening results in increased activity of ee en in carbon metabolism in leaves of winter rye (Secale cereale L.). Planta. 195:554—-562. Hussner, A. and R. Loscu. 2007. Growth and Photosynthesis of Hydrocotyle ranunculoides L. fil in Central Europe. Flora. 202:653-6: LarcHER, W. 2003. Physiological shoe t Ecology 4'" ed. Springer, Germany. Lee, K. S., S. R. Park and Y. K. Kim. 2007. Effects of irradiance, neon peng and nutrients on growth amics of seagrass: a review. a Exp. Mar. Biol. Ecol. 350:144—175. LEIPNER, J., Y. FRACHERBOUD and P. Stamp. 1997. Acelimetion by ical Growth Temperatures Diminishes Photooxidative Damage in Maize Leaves. Plant. Cell. Environ. 20(3):366—372 Levitt, J. 1980. Responses of plants to environmental stresses. Physiolo. Ecol. Series. 1(2): wna Lone, R. W. and O. Laxey. 1976. Flora of Tropical Florida. 2"¢ ed. Banyan = Miam McFartanp, D. G., L. S. Netson, M. J. Gropowrtz, R. M. SMart and C. S. Owens. 2004. has molesta D. S. Mitchell (Giant Salvinia) in the United States: A Review of ike Ecology an Approaches to Management. Special Rep. U.S. Army Corps Eng., Eng. Res. Devolp. Cent., : Ni Nauman, C. E. 1993. Flora of North Aiea: Volume 2. Oxford Press, N.Y., N.Y. Nicuots, P. B., J. D. Coucu and S. H. At-Hampant. 2000. Selected hope seam responses of Salvinia minima to different chromium concentrations. Aquat Bot. 68:313-319 Nitsen, E. T. and D. M. Orcutt. 1996. Physiology of Plants ee Stress. john Wiley and Sons, Inc., WY NY. ’ Oxcum, E. J., D. Ropricuez, G. SANCHEZ, E. HERNANDEZ and M. E. Ramirez. 2003. Productivity, protein content and nutrient removal from anaerobic efflu uents of coffee wastewater in Salvinia ):259-270 Otcun, E. J., G. SANcHEz-GaLvaN and T. Prrez-PerEz. 2007. Assessment of the ee eee ee Salvinia minima Baker to sarge polyrrhiza in high strength organ wastewater. Water, Air and Soil Pollut. 181:1 Reppy, K. R. and W. R. Desusk. 1985. Growth oda of aquatic macrophytes cultured in nutrient enriched water: Azolla, Duckweed and Salvinia. Econ. Bot. 39:200—208. Satt, D. E., R. . SmitH and I. Raskin. 1998. Annu. si Plant Physiol. Plant Mol. Biol. 49:643-68. Scumitz, D. C., B. V. NELson, L. E. Natt and J. D. ScHarpr. 1991. Exotic Aquatic Plants in Florida: A Historical Perspective and Review of oe Present Aquatic Plant Regulation Program. Proceedings of the Symposium on Exotic fa Plants. National age Publication Office. National Park Service, Denver, CO. NPS/NREVER/NRTRR-91/06 STRAND, A., V. Hurry, P. Gustarsson and P. seach 1997, santepealeny of Gmabbdopale thaliana leaves at low temperatures releases the suppression of photosynthesis and oe gene expression rg a the prompts of soluble carbohydrates. Plant J. 12(3):605 B. Tewari, A. K. and B. C. Trirpatuy. 1998. Temperature-stress-induced impairment of chicebell bi avatlens reactions in cucumber phe wheat. Plan. Physiol. 117:851—858. Witson, G. and S. At-Hampani. 1997. Effects of chromium (vi) and pape substances on selected physiological responses of Azolla caroliniana. Am. Fern. J. 87: American Fern Journal 99(3):162-175 (2009) Habitat Differentiation of Ferns in a Lowland Tropical Rain Forest James E. Warkins, JR. and CATHERINE CARDELUS Colgate University, Department of Biology, 13 Oak Drive, Hamilton, NY 13346 Asstract.—Fern species and growth form diversity peak in tropical rainforests. In such forests, ferns often play important ecological roles. However the distribution and diversity patterns of different growth forms (i.e., epiphytic vs. terrestrial ferns) have not been broadly quantified. We 21 species of epiphytic and 20 terrestrial ferns was recorded, with only one species found as an epiphyte and as a terrestrial species. Epiphytic species also exhibited increasing species diversity with i ing trunk height. Epiphytic species exhibited predictable patt f distribution along the trunk and were easily grouped into high-trunk, low trunk, or bimodal categories. In terms of percent cover and number of species, simple-leaved f dominated the epiphytic growth form, 13 of 21 species, whereas ferns with compound or dissected leaves dominated the hemi-epiphytic and terrestrial floras with 20 of 20 species. These results indicate that there are significant functional differences in the ecology of epiphytic and terrestrial ferns and that reciprocal establishment is difficult and extremely rare. Key Worps.—Pteridophyte, epiphyte, canopy, microclimate, gametophyte Pteridophytes, especially the ferns, make up an important component of tropical and temperate floras and serve important functions in ecosystem processes in both the canopy (Hietz, 1997) and forest floor habitats (Hill and Silander, 2001). Epiphytic ferns make up an especially conspicuous component of tropical wet forest regions around the world. For example, in Costa Rica, 70% of the entire pteridoflora is epiphytic, while at La Selva Biological Station in northeastern Costa Rica epiphytic ferns comprise 42% of this lowland forest flora (Grayum and Churchill, 1987). Surprisingly, our understanding of the ecology of epiphytic taxa is especially limited. Recent studies on the comparative biology of epiphytic and terrestrial fern species have revealed significant differences in the gametophyte ecology of these functional types (Watkins et al., 2007a, b, c). Some epiphytic taxa have evolved fantastic degrees of desiccation tolerance in the gametophyte generation that likely contributes to establishment potential (Watkins et al., 2007b) and ultimately controls species distributions. The gametophytes of epiphytic taxa also exhibit significant demographic differences from terrestrial species. Epiphytic gametophytes may live for years and perhaps decades and beyond, while their terrestrial counterparts have significantly reduced longevities (e.g. 1-2 months; Watkins et al., 2007a). Such significant ecological differences do not disappear in the sporophyte generation and epiphytic taxa WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 163 exhibit divergent patterns of leaf-level nutrient and carbon relations relative to terrestrial taxa (Watkins et al., 2007c). What factors combine to influence the distribution of epiphytic and terrestrial species with such radical ecological differences? Most attempts to answer this question for epiphytes have focused on sporophyte ecology. Within canopy habitats, studies have demonstrated that water use efficiency and drought tolerance of sporophytes can affect epiphytic species distributions (Andrade and Nobel, 1997; Hietz and Briones, 1998). Epiphytic fern sporophytes also seem to be invested in biochemical (Hietz and Briones, 2001) and, to a more limited extent, morphological (Watkins et al., 2006b) photoprotective measures which likely influence their distributions. Substrate preference also seems to play a significant role in structuring some species distributions (Moran et al., 2003, Moran and Russell, 2004). Still others have attempted to quantify the effects of microclimate (Freiburg, 1998; Cardeltis 2002; Cardeltis and Chazdon, 2005), tree characteristics (Ter Steege and Cornelissen, 1989; Cardeltis, 2002, 2007, Cardeltis and Chazdon, 2005), and individual plant adaptations (Benzing, 1986, 1987) on overall epiphytic plant distributions. In comparison to epiphytic species, our understanding of terrestrial species ecology is broader but remains limited for tropical species. Pioneering studies on the distributions of terrestrial ferns in the tropics have revealed the importance that edaphic specialization has on species distribution (Jones et al., 2007, 2008; Tuomisto and Ruokolainen, 1994; Tuomisto and Dalberg, 1996; Tuomisto et al., 1998; Tuomisto et al., 2002). Sporophyte stress tolerance has also been demonstrated to influence the distribution of terrestrial tree fern species (Durand and Goldstein, 2001) and at the community level, both environmental and neighborhood effects have been shown to influence habitat specialization and act as an important determinant of the distribution of tree ferns (Jones et al., 2007). While these studies have elucidated important aspects of fern ecology, few comparative studies on epiphytic and terrestrial species exist. In a recent study comparing the distribution of epiphytic and terrestrial species along an elevational gradient, Watkins et al. (2006) found that out of 264 species only one grew as both a canopy epiphyte and a terrestrial species, and this from only a single site. A similar finding was reported by Kluge and Kessler (2006) along the same gradient in Costa Rica. Such a result is perhaps not surprising given the apparent ecological differences between these two groups. To understand better the patterns of fern habitat differentiation, we examined the vertical distribution of ferns on the trunk of an emergent canopy tree and compared this to species distribution in terrestrial plots. We asked the following questions: 1) How does overall species richness change and is there variation in the vertical distribution of epiphytic fern species along the trunks of an emergent tree species, 2) How does trunk fern richness and terrestrial fern richness compare and is there species overlap between habitats? 3) Are there differences in functional morphology between epiphytic and terrestrial species? 164 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) MATERIALS AND METHODS Species composition and distribution —This study was conducted at La Selva Biological Station in Heredia Province, Costa Rica. The site is a 1400 ha tropical wet forest with an average rainfall of 4000 mm per year (McDade et al., 1994). The trunks of six Hyeronima alchorneoides Allemao (Euphorbiaceae) trees were sampled for ferns using single rope climbing techniques (Perry, 1978). Hyeronima alchorneoides was chosen as this evergreen species maintains relatively high epiphyte species richness and has a well studied canopy habitat (Cardeltis, 2002, 2005; Cardeltis and Chazdon, 2005). All trees sampled were greater than 1m in diameter above the buttresses with an average diameter among trees of 1.5 m. A 26 m transect, from the forest floor to the main bifurcation was established along the trunk of each of six trees and broken into contiguous 2 m X by 2 m plots. We found that 26 m was an ideal length that put the upper bound of the transect just below the first trunk bifurcation of all trees sampled. This small size 2m x 2 m plots established along the trunk allowed for a finer level analysis of richness and cover. The average trunk area sampled per tree was 122 m’. Identity, abundance (estimated by measurements of percent cover) frond morphology, and life form (e.g., primarily epiphytic, primarily hemi-epiphytic, and primarily terrestrial) were documented for each fern species in each plot. While the actual number of individuals in a given plot may be a better measure of species richness, accurate counts of individuals from six trees was difficult. Species such as Hymenophyllum brevifrons Kunze (Hymenophyllaceae) form mats of potentially hundreds of individuals; thus, we utilized percent cover as a proxy for dominance and ignored the actual number of individuals. Percent cover was estimated by determination of the total area covered in each 2m X 2m plot by a given species. As leaves can overlap, it was possible to a plot to have a total percent cover of >100% when summed across species. For comparison of epiphytic species with hemi-epiphytic and terrestrial species, a circular plot with a radius of 26 m (total sampled area of 2122 m?) was established terrestrially around the base of each sample tree. The 26 m radius plot was established to mimic the total tree height sampled. Each terrestrial plot completely encircled each sampled tree. The number of terrestrial individuals in these plots was often too low (4—5 individuals) to accurately allow for determination of percent cover; therefore, only presence/ absence data was noted. Voucher specimens were collected from within each terrestrial plot and deposited in the National Herbarium of Costa Rica. For species identification we used the taxonomic concepts of Flora Mesoamer- icana (Moran et al., 1995). In addition to richness data, we quantified variation in leaf morphology among the different habitats sampled. Each species encountered was recorded as having compound or simple leaves. A chi square was run to determine if species from either habitat were associated with a given leaf morphology. We also evaluated specific leaf weight of species from three sections on the trunk. Data were collected from terrestrial plots, the buttress zone (0-2 m), and the WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 165 bifurcation zone (22-24 m). Due to limited time we choose these location subsets rather than sampling species along the entire trunk. The buttress zone and the bifurcation zone data also correspond to areas where we measured microclimate (see below). Microclimate measurements.—Measurements of temperature and relative humidity were recorded on June 26, 2005. Three Hobo Pro Temperature/RH (Onset Corp., Bourne, ME, USA) data loggers were placed at three different locations on a single tree. One sensor was placed at 1.5 m above the forest floor to measure microclimate of the buttress zone, another sensor was suspended at 11 m above the forest floor to measure the mid-bole zone, and a final sensor at 23 m above the forest floor to represent the highest level or bifurcation zone. Temperature and humidity were recorded every 5 min for 12 hours. Water potential of the air was calculated using the formula: ‘¥ = RT In e/e®, where R is the gas constant, T is the absolute temperature and e/e® is relative humidity expressed as a fraction (i.e. 50% r.h. = 0.5). This value was divided by the partial molal volume of water to convert to pressure units. Light levels were measured with a Licor quantum sensor (Li-190, Lincoln, NE, USA) connected to a Licor data logger (Li-1400, Lincoln, NE, USA) at the same levels as temperature and humidity. Measurements were taken on June 27, 2005 and percent light transmittance was calculated by comparing sensor data to a control sensor measuring at the same time in an open field. RESULTS Species composition and distribution.—A total of 40 fern species was found: 21 epiphytic species (plus gametophytes of Vittariaceae), 15 terrestrial species and 4 hemi-epiphytic species (Table 1). Olfersia cervina (Dryopteridaceae), was the only species found in both habitats (Table 1). The average number of epiphytic species found per tree was 11 (+/—2 species) with the sporophytes of Vittaria stipitata Kunze (Vittariaceae), Elaphoglossum herminieri (Bory & Fée) T.Moore (Elaphoglossaceae), Oleandra articulata (Sw.) C.Presl (Dryopterida- ceae), and Vittariaceae gametophytes occurring on all individual trees (Table 1). Examination of presence/absence data suggests that terrestrial species are less abundant relative to the epiphytic species (Fig. 1). In addition, no single terrestrial species was represented in all 6 terrestrial plots. Only a single hemi-epiphytic species, Polybotrya osmundacea Humb. & Bonpl. ex Willd., was found in all six terrestrial transects. Even though a significantly lower total trunk area was surveyed in the epiphytic when compared to terrestrial habitats, we encountered a higher diversity of epiphytic relative to terrestrial and hemi-epiphytic species (Table 1, Fig. 1). The number of epiphytic species remained relatively constant along the trunk up to 16m, when diversity quickly increased (Fig. 1). Abundance (% cover) of epiphytes along the trunk did not follow this trend, but showed a strongly bimodal distribution (Fig. 2 Microclimatic variation and extremes were differentially distributed over the trunk (Fig. 3). The buttress zone was consistently darker and exhibited — . 166 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) TaBLE 1. Trunk and terrestrial plots where epiphytic, terrestrial, and hemiepiphytic fern aaron p cu Hemiepiphytic species were all recorded from these terrestrial plots. Data from this study were gathered from La Selva Biological Station in Costa Rica. | Plot Height on Tree Trunk (m) 0-2 2-4 4-6: 6-8 8-10 10-12 12-14 14-16 16-18 18-20 20-22 22-24 epipnyitc Species Colchlidium serrulatum (Sw.) Bishop Grammatid sp. 1 lycopodioides (L.) Copel. Phliebodium pseudoaureum (Cav.) Lellinger stipit Hecistopteris pumila (Spreng). ) J. Sm. Asplenium serra opal dae Hymenophyllum sp. 1 Elaphoglossum latifolium (Sw.) J. Sm. Vittariaceae Gametophytes Trichomanes ini Hook. Trichomanes e Wess. Boer pecan (L. mo Terrestrial Species Dennstaedtia aoe ge ) T. Moore diantum obliqum Wil Alsophila pc (knee D.S.Conant Cyathea mul s Mett. Salpichiaena volubilis (Kaulf.) J. Sm. Tectaria dracontifolia (D.C. Eaton) Copel. Tectaria incisa Cav. oe ee Christ) C.F. Reed —— yensis (E. Fourn.) C.V. Morton 4 5 6 iumb. & Bonpl. ex Willd. Ta ae ae Polybotrya osmundacea Hi Olfersia cervina arg Kunze Lomariopsis vestita E. Fourn. caudata Kunze significantly wetter air and less variation than the mid-trunk or bifurcation zone. Variation increased along the trunk with the most extreme and variable microclimate occurring in the bifurcation zone (Fig. 2 There were also several species specific distribution. patterns. For example, Elaphoglossum sp.1 is a high light, high canopy species, whereas its congener laphoglossum latifolium (Sw.) J.Sm. seems to tolerate more variable microhabitats often occurring in the dark, wet buttress zone (Fig. 4). A similar pattern exists between Hymenophyllum brevifrons, a high canopy species, and the related H. hirsutum (L.) Sw. which follows a bimodal pattern similar to E. latifolium. In contrast, a pair of filmy ferns, Trichomanes godmanii Hook. ex Baker and T. ekmanii Wess.Boer are present at high densities on the low trunk and buttresses but neither occur in high canopy locations. WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 167 16 Total Number of Species Per Plot o * 4 q T Roun T T T T t T T q , ea 0-2 2-4 46 6-8 8-10 10-12 12-14 14-16 16-18 18-20 20-22 22-24 Plot Height From Forest Floor Fic. 1. Epiphytic, terrestrial, and hemiepiphytic fern species area curve sampled at La Selva Biological Station, Costa Rica. Epiphytic species were recorded on the trunks of six emergent canopy trees (Hyeronima alchorneoides) whereas terrestrial and hemiepiphytic species were collected in ground transects under each sampled tree. Another intriguing distribution pattern unfolds when members of the Vittariaceae are examined. Due to their unique gemmae production (asexual propagules), we were able to identify the gametophytes of this family (Farrar, 1974). The gametophytes exhibit an interesting bimodal distribution occurring at both the buttress and the crown, while sporophytes of Vittaria were only encountered on high trunks (Fig. 5). Frond Morphology.—When frond morphology was examined in epiphytic, terrestrial and hemi-epiphytic species, epiphytic ferns had significantly more species with simple leaves than terrestrial and hemi-epiphytic species (y? = 18.13; p = 0.0001) Thirteen of the 21 epiphytic species had simple leaves and there were no terrestrial or hemi-epiphytic species exhibiting this leaf morphology. Specific leaf weight also increased from terrestrial to bifurcation zone species (Fig. 6 DISCUSSION Species distributions.—In the first part of this study, our goal was to describe and compare the abundance (in terms of percent cover) and distribution patterns of epiphytic ferns in canopy habitats to determine if there is predictable vertical distribution of epiphytic fern species along the trunks of an emergent tree species. When the total number of species per plot was 168 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) First Bifurcation (sensors at 23 m) 100 oo 8 = £ 80 oa = 22-24 < ~ 2 60 ‘5 -20 4 g 3 30 = 40 20-22 2 49 = 20 a = 5 -50 8 0 os oO 18-20 ae o. - 0099 4.00% ,.00%..00 5.00 90:09, .909 7.90:00 16-18 Time Time — Mid Trunk Sag is at 11 m) S 14-16 @ 1 AS we s” _ ee § 80 wo = ® = -10 4 E 5 12-14 re 2 60 ms ‘5 -20 5 © 3 B 39 = 40 2 s > ic 10-12 © 49 - + 20 = e 5) RAR > & -50 a” ro Do EF 810 = -60 a xe) 600% ,09:09 00:09 500.99, 00509 5:00:09 9,00. ,4.0:09 7.90.00 a 6-8 Time Time Buttress (sensors at 1.5 m) 46 100 e : a er aes £ 80 < E 2-4 = 2 60 ‘5 -20 + e w F 40 ra S 0-2 $ 4p 4 = 20 ao = = 5 0 - ET = 60 gl gee eng 0 10 20 30 40 50 60 70 OO MRpc0M oop coO$,0009 5.000 gg09, 0000700: Mean Percent Cover Time Time Fic. 2. The bimodal carves: of mean percent cover of all epiphytic fern species along ce ee tree trunks. Humidity and light sensors were placed at three separate locations along the trunk: 1 , 11 mi, ne 23 m. Microclimatic variables and light were measured at 5 min intervals at these loutoae examined, there was a predictable pattern of increasing species diversity with plot/tree height above 12 m (Fig. 3). The lower buttress zone (0-2 m) of any given tree was less diverse than the top bifurcation zone (22-24 m, Fig. 3). While diversity increased, percent cover exhibited a highly bimodal distribution (Fig. 2). Thus, while the number species increase with plot height, the total percent cover was similar between buttress and bifurcation zones. The buttress zone is homogenously dark and wet whereas the mid- an upper-trunk are brighter and drier and exhibit greater environmental WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 169 7 6 Elaphoglossum sp. 1 5 2 6 (o) O 4 S 3 5 2 1 0 Hymenophyllum brevifrons oe ® > (o) oO e ® os o ou 60 Trichomanes godmanii ih s o) O 40 S 30 o a 20 10 0 r ¢) 5 10 15 20 25 0 ss. 10 15 20 25 Plot Height From Forest Floor (m) Plot Height From Forest Floor (m) Fic. 3. The relationship of trunk height above ground on Hyeronima alchorneoides with the richness of san be fern species. Highest richness was consistently within 2-3 m of the first branching of the trun heterogeneity (Fig 2). These factors likely contribute to differences in diversity and abundance along the trunk. Indeed, whereas edaphic characters have been shown to influence terrestrial species diversity, light and water likely play an important role in shaping epiphytic species distribution (Hietz and Briones, 1998). Our data suggests that microenvironmental heterogeneity, rather than absolute values, is particularity important for epiphytic ferns. Along with the zone specific microclimatic variation, we also found broad species specific patterns of distribution. The filmy ferns Trichomanes ekmanii and T. godmanii, dominated the dark buttress zone. The group, ‘‘filmy ferns,” get their name from fronds that lack stomata, are one cell layer thick, and thus 170 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) EB Vittaria stipitata Sporophytes "7 [9 Vittariaceae Gametophytes Percent Cover 0-2 2-4 46 6-8 8-10 10-12 12-14 14-16 16-18 18-20 20-22 22-24 Plot Height From Forest Floor (m) . Examples of a subset of epiphytic species distribution along the trunks of Hyeronima ie. Most species fell out into primarily buttress species or bifurcation species with few occurring across the trunk. prone to desiccation. It is not uncommon to find large scale mortality of these two species following tree falls that expose them to brighter drier environ- ments (pers. obs). Hymenophyllum brevifrons is similar in size to T. ekmanii and T. godmanii yet is completely absent from the dark buttress areas and quite abundant in high trunk habitats. Hymenophyllum hirsutum had a much broader range, occurring in most plots along the trunk (Fig. 4). The upper trunk had both a high percent cover and high species diversity which may reflect a more heterogeneous microenvironment to which different ferns are adapted. This variation may be important in maintaining high levels of fern diversity in tropical forests, especially on small local scales employed in this study. An additional pattern to emerge from this study is the differential distribution of the gametophytes and sporophytes of the Vittariaceae. While the area remains poorly studied, it is thought that gametophytes may exhibit broader ecological distributions that their sporophyte olen Sete (Sato and Sakai, 1980; Sato and Sakai, 1981; Peck et al., 1990). Vittariod gametophytes are easily identifiable give there unusual morphology and gemmae production. We observed that Vittariod gametophytes exhibited a distinctly bimodal distribution relative to sporophytes which were confined to plots higher along the trunk (Fig. 5). There was not a single Vittariod sporophyte below 4 m on any of the trees sampled. We encountered hundreds of gametophytes from potentially several different non-Vittariod species which suggests that the WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 171 a0 —#*— Epiphytic Fern Species - Terrestrial Fern Species —-¥— Hemiepiphytic Fern Species oe oO uo om Oo q o 15 ° = Co = aD, 5 104 ao a Oo 5 ae 2 .| oe eel oe ee ¥ ¥ ¥ — e+ 0 T Pies qT T T T 1 2 3 4 5 6 Tree Number Fic. 5. Distribution of sporophytes and gametophytes of the Vittariaceae. Sporophytes are only found on the upper portions of the trunk, whereas gametophytes were found in both the upper portions and lower portions of the trunk. gametophytes of other species may exhibit similar patterns of distribution. Gametophytes may be more highly adapted to growth in dark environments as the carbon budget of an individual, and thus growth rates, are small compared to the needs of the sporophyte (Farrar, 1998). Whereas gametophytes can establish in a broader range of environments, sporophytes may be more restricted to more stable niches. Greater ecological plasticity in the gameto- hyte generation may be important in “habitat exploration’”’ for species as it is the first living stage to encounter new environments. Data from temperate species has shown that gametophyte plasticity is important to establishment and sporophyte distributions (Greer et al., 1997). Species diversity.—The second part of this study examined how trunk fern diversity and terrestrial fern diversity compare and asked if there is species overlap between habitats. The area sampled on all six trees was less than the area sampled in the first terrestrial plot, yet the number of epiphytic species is much higher than terrestrial species (Fig. 1). As with any study on tropical species diversity, our species area curve indicates that we under-sampled poison species. We ceased to discover any additional epiphytic species, yet we from an earlier floristic survey of La Selva (Grayum and Churchill, 1987) on this too represents an underestimation of epiphytic and hemi-epiphytic species. i272 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) 100 C (F=5.01, p=0.01) 80 - av £ Roe = 60 - a ® = ow a o = .o) ® Qa ” 20 - 0 Terrestrial Buttress (0-2m) Bifurcation (22-24m) Location Page art See bea & and bifurcation iphvt G 6 pecific leaf ig ine hyt yee 7 ~ Oo Oo Hyeronima alchorneoides at La Selva Biological Station, Costa Rica. Nevertheless, in this study, epiphytic species were more diverse than terrestrial and hemi-epiphytic species. While it is has been difficult to show that host specificity influences epiphyte composition (Zotz and Vollrath, 2003) it is known that certain tree species harbor greater diversity and numbers of epiphytes (Cardeltis, 2002). The tree chosen for this study has a diverse and abundant epiphyte flora relative to other emergent tree species in La Selva. The epiphytic fern flora of Hyeronima alchorneoides makes for interesting comparisons with the terrestrial fern flora as the species grows on both alluvial bottoms and upland terraces. We were thus able to sample a diversity of soil types and found that alluvial bottoms were areas of particularly high terrestrial fern diversity. For example, the final terrestrial transect sampled happened to occur along a small stream on one of the alluvial bottoms at La Selva. The number of terrestrial species in this particular plot was almost double that of the most diverse terrestrial plot in the sample. While other factors may be involved, this observation further supports the importance of edaphic factors to patterns of terrestrial fern distribution (Tuomisto and Ruokolainen, 1994; Tuomisto and Poulsen, 1996; Tuomisto et al., 1998). When we examined the species overlap between habitats, we found that Olfersia cervina was the only species that was found growing in epiphytic and terrestrial habitats. This species has been described as a low climber (Moran, 1995) and as a hemi-epiphyte which could exclude it from both the epiphytic WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 173 and terrestrial groupings. The species was only observed in pockets of deep soil that form on buttresses and never above 2 m from the forest floor. This is an unusual species in that it is most frequently encountered growing on large fallen trees in advanced stages of decay (pers. obs). Hyeronima often forms buttresses that can collect large amounts of detritus and thus provides an important habitat for Olfersia. Perhaps of greater interest is that there was not a single species that grew on terrestrial soil and on the upper trunk further corroborating the reports by Watkins et al. (2006). One question that has often plagued pteridologists is how the evolution of epiphytisim actually came about. In spite of the fern’s dispersal syndrome, such intensive local sampling effort combined with regional studies (Watkins et al., 2006) suggest that reciprocal establishment of epiphytic and terrestrial species is rare. In the ferns, epiphytisim likely arose through some intermediate form, most likely passing through some hemi-epiphytic form before radiation into a completely epiphytic condition. Leaf morphology.—There are striking differences in leaf morphology between the epiphytic and terrestrial species studied here. The majority of epiphytic species (13 of 21) have simple leaves whereas terrestrial and hemi- epiphytic species have compound morphologies. Interestingly, of the epiphytic species with compound leaves, only two species in the Hymeno- phyllaceae had leaves that were more than once pinnate. In an opposite pattern, 11 of the 15 terrestrial species exhibited leaves that were more than once divided; the other four species had once pinnate leaves. These patterns are repeated throughout the Costa Rican pteridoflora (pers. obs) and this convergence of leaf form in the canopy, in several divergent lineages, suggests that these traits are adaptive and are under direct selective pressure. Epiphytic species from the bifurcation zone also had significantly increased specific leaf weight compared to terrestrial and buttress epiphytes. Canopy habitats tend to be hotter, drier (Fig. 2), and experience more wind than terrestrial habitats in most tropical forests. Thus, it is likely that the combination of both energy and mechanical aspects have influenced the evolution of leaf morphology and leaf thickness in epiphytes. ACKNOWLEDGMENTS This study was completed during the Tropical Plant aye course (98-9) taught under the auspices of the Organization for Tropical Studies. The course was supported by a grant to OTS m the Andrew W. Mellon Foundation. ihe Bret author received support from the pegeoialy = at the University of Connecticut, and Marjorie Pohl ua: at lowe State Mestieiew We aa thank Dr. Robbin Moran and Dr. ahi Farrar for help wit d Dr. Robin Chazdon, Joanne Sharpe, and an unknown reviewer for ee commentary on an earlier version of the manuscript. LITERATURE CITED Anprabe, J. L. and P. S. Noset. 1997. Microhabitats and water relations of epiphyti ti and ferns in a lowland neotropical forest. Biotropica 29:261-270. 174 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) BenziNG, D. H. 1986. The vegetative basis of vascular epiphytism. Selbyana 9:23-43. Benzinc, D. H. 1987. Vascular epiphytism: taxonomic participation and adaptive diversity. Annals of the Missouri Botanical Garden 74:183—204 CarbeLUs, C. 2002. Distribution and abundance of vascular epiphytes in tropical wet forests. Dissertation, University of Connecticut, Sto Carpe.us, C. 2007. Vascular epiphyte c ae de in thet inner-crown of Hyeronima sgt gpae aa and she ampla at La Selva Biological Station, Costa Rica. Biotropica 39(2):171-176. Carve ts, C. and R. L. CHazpon. 2005. Inner-crown microenvironments of two jects tree species in a lowland wet be Biotropica 37:238-244. Duran, L. Z. and G. GotpsTEIn. 2001. Photosynthesis, iBone and nitrogen use efficiency in native and invasive tree ferns in Hawaii. Oecolog 7345-354 FARRAR, es R. 1974. Gemmiferous Fern Gametophytes: Silariacoss inicdieen Journal of Botany 6-15 FARRAR, as R. 1998. The tropical flora of — a formations in the eastern United States. i Freipurc, M. 1998. The influence of canopy ip lcs on temperature in a tropical rain forest of French Guiana. Second International Forest Canopy Conference, Forest Canopies 1998: Global Perspectives, steuinicn Florida: 3 GrayuM, M. H. and H. W. Cxurc 1987. an ensue to the Pteridophyte flora of Finca La Selva, Costa Rica. Anas Ferm Journal 77:73-89. GREER, G. = R. M. Lioyp and B. C. Mc nel 1997. Factors influencing the distribution of pteridop ee in a aha Ohio hardwood forest. Journal of the Torrey Botanical Society. 124 Hietz, P. 1997. sehen dynamics of epiphytes in a Mexican humid montane forest. Journal of Ecology 85:767-775. Hitz, P. and O. Briones. 1998. Correlation between water relations and within-canopy distribution of — ferns in a Mexican cloud forest. Oecologia 114:305—-316. Hierz, P. and O. Briones. 2001. spate any chlorophyll fluorescence and ieee -canopy distribution of ee ferns in a Mexican cloud forest. Plant Biology 3:279-28 Hit, J. D. and J. A. Sitanper. 2001. capri and dynamics of two ferns: ee taedtia ecetaapisiaas (Denna and Thelypteris noveboracensis Tholyptrdacoo) ina Northeast mixed hardwoods-hemlock forest. America Journal of Botany 88:894—902. Jones, M. M., P. 0. Rojas, H Poon Misto and D. B. Ciark. 2007. Environmental aa neighbourhood effects on tree fern distributions in a neotropical lowland rain forest. J. Veg. Sci. 18(1):13-24. Jones, M. M., H. Tuomisto, D. Borcarp, P. Lecenpre, D. B. CLark and P. C. Ouivas. 2008. pear variation in tropical plant c community composition: influence of environmental and spati data quality. regan 155(3):593 Kuuce, J. and M. Kesser. 2006. Fern ete and its apace Se from an elevational transect in Cats Rica. Diversity and Distributions 12(5):535-545. McDape, L. A., K. S. Bawa, H. A. HESPENHEIDE and G. S. ati 1994. La Selva: Ecology and natural history of a neotropical rain forest. Chicago University Press, Chicago. Moran, R. C., S. Kumas and M. Cartsen. 2003. a epiphytic ferns on tree ferns versus angiosperms in Costa Rica. Nepean 35:4 Moran, R. C. and A. R. SmitH. 2001. Phytog eS relationships between neotropical and African-Madagascan pteridophytes. Brittonia 53:304-351. — R. C. and R. Ria. 1995. Psilotaceae a Salvinaceae. In: G. Davidse, M. S. Sousa and S. Knapp, eds. Flora Mesoamericana. Universidad Nacional Autonoma de Mexico, Mexico City. Moran, R. C. and R. V. Russet. 2004. The occurrence of Trichomanes godman sh algpa fod ceae) on Welfia georgii (Arecaceae) at the La Selva Biological Station, Costa Rica. Ameri 0-76 Peck, J. H., C. J. Peck and D. R. Farrar. 1990. Influences of life history attributes on formation of local and distant fern populations. American Fern Journal 80:126—-142 Perry, D. R. 1978. A method of access into the crowns of emergent and canopy trees. Biotropica 0:155—157 WATKINS AND CARDELUS: HABITAT DIFFERENTIATION OF FERNS 175 Sato, T. and A. Sakai. 1980. Freezing resistance of gametophytes of the temperate fern, bias wie retroso-paleaceum. Can. J. Bot. 58:1144—1148 ill. 1. 1981. Cold tolerance of gametophytes of some cool temperate ferns native to kkaido. on J. Bot. 59:604—608. J. H. C. Corne.issen. 1989. Distribution and ecology of vascular epiphytes in land rain forest of Guyana. Biotropica 21:331-339. Tuomisto, H. and K. RuoKOLAINEN. 1994. Distribution of pteridophyta and Melast t ] edaphic gradient in an Amazonian rain forest. Journal of Vegetation Science 5:25-34. Tuomisto, H. and A. Datserc. 1996. Influence of edaphic specialization on pteridophyte distributions in neotropical rain forests. Pets of Biogeography 23:283-293. Tuomisto, H. and A. D. Poutsen. 1996. Influence of edaphic specialization on pteridophyte distribution in neotropical rain forests. denier of biogeography VRE nial 293 Tuomisto, H. A., D. PouLsen and R. C. Moran. 1998. E genus Adiantum in Western Amazonia. Biotropica 30:392-—399. Tuomisto, H., K. RUOKOLAINEN, A. D. Pou.sen, R. C. UINTANA, G. Canas and J. CEL. Distribution and diversity of laser da iad ioc, one edaphic ie He in Yasuni National Park, Ecuadorian Amazonia. Biotropica 34:516-53 Warkins, Jr., J. E., C. Carpets, R. K. CoLweLt and R. C. Moran. ist Species richness and distribution of ferns along an elevational gradient in Costa Rica. American Journal of Botany 93:73—83. fat... £, rr Warkins, Jr., J. E., A. Y. Kawanara, S. A. Leicur, J. R. AutD, A. J. BicKsLer and K. Kaiser. 2006b. Fern laminar scales protect against photoinhibition from excess light. American Fern Journal. 96(3):83-92. . Mack and S. Mu key. 2007a. Gametophyte ecology and oe of epip hytic and lane tropical ferns. American Journal of Botany. 94(4):701 Watkins, Jr., J. E., M. Mack, T. Sinctai and S. Mutkey. 2007b. Ecological a Ae pnsequences of desiccation tolerance in tropical fern gametophytes. New Phytologist. pes vee WarkIns, Jr., J. E S Rene and C. L. Carpe.ts. 2007c. The influence of life form on carbon and nitrogen relationships i in tropical rainforest ferns. Oecologia. 153(2):225-232. Zotz, G. and B. VottratH. 2003. The epiphyte vegetation of the palm Socratea exorrhiza correlations with tree size, tree age and bryophyte cover. Journal of Tropical Ecology American Fern Journal 99(3):176—181 (2009) Eukaryotic Microbial Communities Associated with the Rhizosphere of the Temperate Fern Thelypteris noveboracensis (L.) Nieuwl. O. RocER ANDERSON Biology, Lamont-Doherty Earth Observatory of Columbia University, Torrey Cliff, Palisades, Y 10964, U.S.A. ora@LDEO.columbia.edu RACT.—Microbial communities, associated with terrestrial mosses (Bryopsida) and the rhizosphere of agricultural and natural occurring seed plants, have been rather extensively examined; but less is known about associations with seedless vascular plants, including ferns. The New York fern (Thelypteris noveboracensis), typically found within deciduous forests, occurs in locally extensive stands in North America extending from northeastern Canada to southeastern U.S.A. Soil samples were obtained in autumn (2007) and early summer (2008) within a plot of T. negate in the understory of deciduous trees in the forest reserve at Torrey Cliff, NY to document the rhizosphere (root-associated) density of commonly occurring ace: pe microbes (protozoa), including microflagellates, naked amoebae and testate amoe 10°), naked amoebae (1.8 X 10°—4.0 < 10°) and testate amoebae (ca. 400). Very few active ciliates were observed. This is the first report of guaien cguansrapes iter = with the rhizosphere of ferns and etidd ee a step t of protozoa associated with plant communities. S parative data of protozoa associated with mosses and seed plants are also presented. RDs.—microbial diversity, microflagellates, naked amoebae, New York fern, soil biology, terrestrial ecology, testate amoebae In recent decades, considerable ecological research has focused on understanding the coupling of aboveground and belowground processes; that is, how primary production of plant organic matter, including exudates from capi affects the belowground soil microbial communities. Bacteria supported n part by organic matter from plants, and stimulated by the presence of suk pee microbes (protozoa) and secondarily by bactivorous nematodes, mineralize and recycle soil nutrients, thus enhancing plant growth. Therefore, understanding the functioning of land plants must include knowledge of the protozoan community in soils and the rhizospheres surrounding roots. Soil microbial communities associated with terrestrial mosses (e.g., Anderson, 2006, 2008) and the rhizosphere of agricultural and naturally occurring seed plants, have been extensively studied in a wide range of terrestrial locations (Bamforth, 1984; Clarholm, 1985, 1989; Foissner, 1987; Griffiths 1990; Cowling, 1994; Anderson, 2000; Adl, 2003; Li et al., 2005). However, less is known about microbial communities associated with the rhizosphere of seedless vascular plants, including ferns; although, they are widely distributed geographically from arctic to tropical habitats (e.g., Moran, 2004). Fern rhizomes and roots, in addition to decaying shed fronds, are substantial ANDERSON: FERN RHIZOSPHERE MICROBIOTA Ves sources of organic matter that may support rich microbial communities. Fern root systems also may secrete possible allelopathic and antimicrobial substances (e.g., Horsley, 1977; Stetsenko et al., 1984; Hill and Silander, 2001). Hence, a better understanding of microbial communities associated with fern rhizospheres is warranted to more fully document the microbial communities associated with a wide range of plant groups, and eventually to more completely understand the dynamics of the interactions of ferns with associated terrestrial eukaryotic microbes. The purpose of this research was to contribute to our knowledge of fern rhizosphere microbial communities by examining the eukaryotic microbes associated with the rhizosphere of Thelypteris noveboracensis (L.) Nieuwl., a widely distributed, temperate fern in eastern Canada and U.S.A. MATERIALS AND METHODS Sample site.—Soil samples, using a LaMotte model EP corer, were obtained from the rhizosphere of a well-established plot of T. noveboracensis in the Torrey Cliff Forest Reserve at Palisades, NY (40°59’ 58” N; 73° 54’ 30” W). Three separate soil cores approximately 2 cm long were taken on each sampling date and combined to obtain a more representative soil sample. The mixed sample was placed in a sealed plastic bag and immediately returned to the Lamont-Doherty Observatory laboratory for analysis. Samples were taken in October and November of 2007 and early June of 2008 to provide some evidence of seasonal differences. Thelypteris noveboracensis grows by a spreading rhizome and each plant produces an extensive patch of growth. For example, at Black Rock Forest (Cornwall, NY), an expansive patch occupying ca. 0.1 km? (10 ha) developed in a forest clearing within several seasons after opening of the canopy (Schuster, pers. comm., 2008). Soil analyses.—Moisture content (expressed as % w/w) was determined gravimetrically by difference in weight between the fresh sample and after drying to constant weight at 109°C. The pH of the soil samples in aqueous suspension (5 g per 50 ml distilled water) was obtained using an Accumet'™ model 15 pH meter (Fisher Scientific, Fairlawn, NJ). Percent (w/w) organic content was determined by difference in weight between the fresh sample and after combustion at 375°C for 12-16 hours. Microbiota.—Densities of bacteria that typically serve as prey for the eukaryotic microbiota were estimated by direct fluorescent microscopic counting (Anderson et al., 2001). Microflagellate densities in aqueous extracts of the soil samples, fixed in 2% TEM grade glutaraldehyde, were determined using an acridine orange fluorescence method (Anderson et al., 2001) adapted from Hobbie et al., 1977. Testate amoebae and ciliates were enumerated by exhaustive examination of 3 ml (50 ul subsamples per observation) of aqueous- suspended soil samples fixed with 2% TEM grade glutaraldehyde and stained with Lugol’s iodine (Anderson, 2008). Densities of naked amoebae were estimated using a standard culture observation method (COM), and cyst densities were estimated using the dried aliquot culture observation method 178 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) (DCOM) as published previously (Anderson, 2000). For the COM, a freshly collected sample of soil was suspended in micropore-filtered pond water (MFPW) in a ratio of 1 g in 50 to 80 ml total, and thoroughly dispersed. A 10 ul aliquot was dispensed per well of a 24-well Falcon™ tissue culture dish containing 2 ml of MFPW and a small cube of malt yeast agar (MYA) serving as a nutrient source for prey bacteria. Triplicate plates were prepared for each sample. After 10 to 14 days incubation at 25°C, each well was examined to determine the presence or absence of a given amoeba morphospecies indicating, if present, that at least one individual of that morphospecies was in the 10 ul aliquot. The total tally of each morphospecies was obtained and converted to number per ml of the original suspension. The number of amoebae per g dry weight of soil was calculated based on the weight of soil used to make the original suspension (Anderson, 2000). For the DCOM method, a 10 ul aliquot of the soil suspension was deposited in each of the dry wells of the Falcon culture dish and rapidly dried by flowing air before the MYA and 2 ml of MFPW were added. Thus, only the encysted amoebae survive the drying; and their count, based on observations of emergent morphospecies in the wells of the tissue culture dish, indicates the density of encysted amoebae in the original soil sample. Diversity of naked amoebae morphospecies was determined using the Shannon-Wiener formula (H = 1 — 2 pj * log. pj.) where p; is the proportion of each morphospecies relative to the total. All microscopic observations were made with a Nikon Diaphot™ inverted compound microscope using phase contrast optics. RESULTS Fern soil moisture at the sampling site ranged from 30 to 34%, pH was 4.4, and the organic content of the soil was 20%. Densities (number/g dry weight) of bacteria ranged from 2.5 to 7.1 x 10° comparable to published data for other terrestrial sites. The densities of protozoa in the autumn and June samples are presented in Table 1, including the percentages (in parentheses) of naked amoebae that were encysted and of testate amoebae that were atrophied or present as empty shells. No ciliates were observed in the Lugol’s iodine preserved samples, which is consistent with other reports that a significant number of soil ciliates are usually encysted under typical field conditions, except when soil water is elevated following heavy precipitation (e.g., Foissner, 1987). However, as observed with terrestrial moss samples (Anderson, 2008), occasional ciliates were observed in the COM culture wells, confirming that encysted ciliates were probably present and became active when more fully hydrated. The diversity of naked amoebae was relatively high (H = ca. 3.0). The major genera of naked amoebae identified included Acanthamoeba, Arachnula, Cochliopodium, Hartmannella, Mayorella, Sacca- moeba, Thecamoeba, Vahlkampfia, Vexillifera and Vannella, further indicat- ing relatively rich species diversity. The major genera of testate amoebae were Trinema, Euglypha, Tracheleuglypha, Corythion and occasionally cryptodif- ANDERSON: FERN RHIZOSPHERE MICROBIOTA 179 TaBLE 1. Densities of microbiota in fern soil samples during autumn 2007 and June 2008. Sample date Micro-flagellates Naked amoebae Testate amoebae Oct. 24 6.5 X 10° 4.0 X 10° (53%) 400 (66%) Nov. 8 9.8 x 10° 2.1 X 10° (48%) 444 (80%) June 7 1.3 x 10° 1.8 X 10° (50%) 400 (20%) Densities are number/g dry weight of soil. All microflagellates counted appeared to be trophic stages; the percentages of encysted naked amoebae and of testate amoebae with empty shells or atrophied cytoplasm are presented in parentheses. flugia. A globose testate amoeba, possibly Geopyxella, was occasionally abundant. Interestingly in this study, the density of naked amoebae in the June sample (ca. 2 X 10°) was less than in the October and November samples (ca. 4.0 and 2.0 x 10°, respectively). However, the microflagellate density was substan- tially higher in June (ca. 1 x 10%) compared to the October and November samples (6.5 and 9.8 < 10°, respectively). While the total density of testate amoebae was relatively constant for the three sampling dates (ca. 400), the percent inactive was lowest in June (20%). Comparative data with other plant groups obtained from some representative published sources, including terrestrial mosses and the rhizosphere of seed plants, are presented in Table 2. In general, the densities of flagellates and naked amoebae were within the broad range found in other plant communities, but the testate amoeba densities were somewhat lower. DISCUSSION This research presents some of the first data on eukaryotic microbial organisms associated with the rhizosphere of a temperate fern in a northeastern U.S.A. hardwood forest. The robust growth of the fern’s creeping rhizome, and production of organic matter due to secretory products, exfoliation of rhizome scales, deposition of shed fronds, etc., may contribute to the relative high organic content of the rhizosphere soil (20%) and resulting relatively high moisture content (ca. 30%), thus supporting a robust microbial community. Overall, the densities of fern-associated protozoa are reasonably similar to that found in other plant communities, but as discussed below in some cases they exceeded the densities associated with seed plants in organically enriched soils. The published data on seed plant rhizosphere microbiota encompasses a broad range of habitats including deserts, cultivated soils, grasslands, temperate forests, and tropical rain forests (Table 2). A more informed analysis is possible when the data from the current study are compared to some illustrative published results from organically rich soils. For example, in organically rich soils, the reported density of flagellates was in the range of 10°, and naked amoebae 1.4 to 2.6 x 10* (Griffiths, 1990), or as much as 2 X 10° in tropical soil litter (Bamforth, 2006). Clarholm (1994) reported peak flagellate densities of ca. 4 X 10° in litter of a pine forest following rain 180 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Taste 2. Densities of microbiota in the fern rhizosphere compared to some published data for other plant communities. Plant group Terrestrial Mosses' Fern T. noveboracensis Seed plants* Flagellates 1 x 10°—4 x 10” 1 X 10'-1 x 10° 1.3 x 10°-4 x 10° (Griffiths, (Anderson, 2008) 1990; Clarholm, 1994) Naked amoebae 3.5 X 10°-3.6 x 10° 2 x 10°-4 x 10° 2 X 10°-2 x 10° (Clarholm, (Anderson, 2006, 2008) 1989, 1994; Griffiths, 1990; Bamforth, 2006 Testate amoebae 3 X 10°-6 X 10° 90-3007 1.3 X 10*-3.7 x 10° (Lousier, (Anderson, 2008) 1982; Wanner and Xylander, 2005; Bamforth, 2006) * Data are from published sources on terrestrial mosses in Torrey Cliff, NY and Toolik, AK. * Adjusted data for living individuals, excluding those atrophied or with empty shells. * Data for seed plants are largely from cultivated soils and tree-bearing sites. during September 1977, but the lowest values were on the order of 2 X 10°. Both the flagellates and naked amoebae in the fern soil examined in this study reached densities substantially higher, including published data for terrestrial mosses (Anderson, 2006, 2008). The densities and diversity of testate amoebae are less than those reported in other terrestrial sites, although the genera detected are not particularly unusual for natural soils (Foissner, 1987; Cowling, 1994). verall, this study indicates that T. noveboracensis, at least at the Torrey Cliff site, sustains a rich and substantial community of soil microbiota. Additional research is needed to replicate this work at other geographic locations, including other fern species, to more fully document fern rhizosphere microbiota. However, these data provide at least the first evidence of protozoan densities in a fern rhizosphere at a temperate woodland site. In general, there is limited biogeographic data on terrestrial microbial commu- nities associated with diverse plants and much additional systematic research is needed to more fully understand the population structure and ecological dynamics of plant-associated microbial communities on a global scale. ACKNOWLEDGMENT This is Lamont-Doherty Earth Observatory Contribution Number 7257. LITERATURE CITED AbL, S. 2003. The Ecology of Soil Decomposition. CAB International, Oxford, United Kingdom. ANDERSON, O. R. 2000. Abundance of terrestrial gymnamoebae at a northeastern U. S. Site: A four- ANDERSON, O. R. 2006. The density and diversity of gymnamoebae associated with terrestrial moss communities (Bryophyta: Bryopsida) in a northeastern U.S. forest. J. Eukaryot. Microbiol. 53:275—279. ANDERSON, QO. R. 2008. The role of amoeboid protists and the microbial community in moss-rich terrestrial ecosystems: Biogeochemical implications for the carbon budget and carbon cycle, especially at higher latitudes. J. Eukaryot. Microbiol. 55:145—150. ANDERSON: FERN RHIZOSPHERE MICROBIOTA 181 ANDERSON, O. R., T. GorRELL, A. BERGEN, R. Kruzansxky and M. Levanpowsky. 2001. Naked amoebae and bacteria in an oil-impacted salt marsh community. Microb. Ecol. 42:474—481. BamrortH, S. S. 1984. Microbial distributions in Arizona deserts and woodlands. Soil Biol. Bamrortu, S. S. 2006. Protozoa from alysis and ground soils of a tropical rain forest in Puerto at eaten 50:515—5 CLaRHOLM, M. 1985. Interactions of nee protozoa and plants leading to mineralization of soil nitrogen. sel Biol. Biochem. 17:181-188. CLarHoLM, M. 1989. Effects of plant-becterial- -amoebal interactions on plant uptake of nitrogen under ald conditions. Biol. Fert. Soils 8:373-378. CLaRHOLM, M. 1994. The microbial loop. Pp. 221-230. In: K. Rrrz, J. Dicuton and K. E. Giuter. (eds.). Beyond i pelt New York.Wile Cow .inc, A. J. 1994. Protozoan distribution aA adaptation. Pp. 5-42. In: J. F. Darsysuire. (ed.). Soil rotozoa. CAB International, Oxford, United Kingdom FoissNeR, W. 1987. Soil protozoa: fundamental alata ecologinal peceaarranits ser daa in ciliates and testaceans, bioindicatos and guide to the literature. Prog. P A GrirFitHs, B. S. 1990. comparison of microbial Pitti nematodes ee proioue in the rhizosphere of different oa Biol. Fert. Soils 9:83-88. Hu, J. D. and . Jk. SILANDER. 2001. Distribution pa dynamics of two ferns: Dennstaedtia punctilobula (Donusinediiabeae) and Thelypteris eayeniaego (Thelypteridaceae) in a northeast mixed hardwoods-hemlock forest. Am. J. B 8:894—902 Hossir, J. E., R. J. DaALey and S. Jaspers. 1977. Use of naclenpor filters he counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:1 28. Horsey, S. B. 1977. Allelopathic inhibition of black cherry, er 2. Inhibition by woodland grass erns and eae Can. J. Forest Res. 7:515—519. Li, Q., E. Mayziisn, I. SHamir, S. PEN-Mouratov, M. STERNBERG and Y. STEINBERGER. 2005. i of grazing on soil biota in a Mediterranean grassland. Land Degrad. Develop. 16:581— Lousirr, J. D. 1982. Colonization of decomposing deciduous leaf litter ef Petes Pe 88. Rhizopoda): fees cies succession, abundance and biomass. Oecologia Moran, R. gee) A Natural History of Ferns. Timber Press, Portland, Oregon. StetsEeNko, N. M., N. D. cgiestidt fet and GevepzE. 1984. Antimicrobial properties of eae oe ferns. Rastitel’ oe Resursy 20:1 100-106. Wanner, M. an E. R. XYLANDER. 2005. Biodiversity ace oa ft trial testate amoebae: is there any succession at all? Biol. Fertil. Soils 41:428—438 American Fern Journal 99(3):182—193 (2009) Structure and Organization of the Rhizome Vascular System of Four Polypodium Species ARCHANA SRIVASTAVA Career Convent Girls Degree College, Vikas Nagar, Lucknow- 226026 (India) SUBHASH CHANDRA* D-15 sector ‘G’, Aliganj, Lucknow-226 020 (India) Asstract.—The present investigation is a detailed study of the vasculature of the rhizome of four species of Polypodium (P. cambricum, P. fauriei, P. interjectum, and P. sibricum). The vascular architecture of the rhizomes of the Polypodium species studied denotes a line of reduction and simplification of characters. The characteristic nature of the association of branches with leaves in Polypodium does not support a close relationship with Goniophlebium as has previously been hypothesized. However, more extensive study of Polypodium is needed to arrive at any definite conclusion. Key Worps.—Polypodiaceae, rhizome, vascular system, Polypodium The genus Polypodium is typified by P. vulgare Linn., a fern of the north temperate zone of the world. Goniophlebium is regarded by some taxonomists as congeneric with Polypodium while many prefer to regard it as separate genus (Rod|]-Linder, 1990; Schneider et al., 2004; Srivastava and Khare, 2005). Though predominantly a neotropical genus, Polypodium includes a few species in Africa and eastern Asia and one, P. vulgare Linn., the type species, in Europe. Christensen (1938) redefined the genus Polypodium, excluding hundreds of species treated in it by earlier authors. At the same time he included Goniophlebium in the genus Polypodium, which he considered a natural genus of about 50 species in tropical and subtropical America, Europe and Asia to Polynesia. Ching (1940) merged Goniophlebium into Polypodium which he placed in tribe Polypodieae of the subfamily Polypodioideae. Holttum (1949), who recognized five groups within the Polypodiaceae, included Goniophlebium in Polypodium, which he placed in Phymatodes group. Copeland (1947) and de la Sota (1973) preferred to separate the palaeotropic species to constitute the genus Goniophlebium, which they regarded as closely related to Polypodium s.s. Nayar (1970, 1974) and Crabbe et al. (1975) included Polypodium and Goniophlebium in their subfamily Polypodioideae of the family Polypodiaceae. Pichi-Sermolli (1977) recognized 14 groups within the Polypodiaceae, one of them included the genera *Author for Correspondence: Former Head, Pteridology Laboratory, National Botanical Research Institute, Lucknow, India (chandrasum@rediffmail.com). SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES 183 Polypodium and Goniophlebium. Tryon and Tryon (1982) and Rodl-Linder (1990) considered Goniophlebium an Old World genus with articulate pinnae and not closely related to Polypodium s.s., which is predominantly a New World genus. Hennipman et al. (1990) provisionally included Goniophlebium in Polypodium sensu lato, tribe Polypodieae of the subfamily Polypodioideae. Recently, Smith et al. (2006) included both in Polypodiaceae of the order Polypdiales under Polypodiopsida. In the genus Polypodium, rhizomes are short to long creeping, the rhizome scales are peltate or pseudopeltate rarely basifixed and clathrate or opaque with a broad central band. Fronds are uniform, monomorphic and simple with the lamina usually pinnatifid or pinnate and with no laminar hairs or scales. Vascular morphology of the rhizome of ferns is currently well accepted as a conserved feature, which is minimally affected by the environment and is a highly reliable comparative criterion thought to be of significance in taxonomic and phyletic studies of homosporous ferns. Hence, the structure and organization of the vascular system in the fern shoot have been useful in comparative studies (Tansley, 1907-08; Bower, 1910, 1914, 1915, 1917, and 1918; Hayata, 1927, 1928; Tardieu-Blot, 1932; Ogura, 1972; Ching, 1940; Holttum, 1964; Nayar and Chandra, 1967; Nayar et al., 1968). In recent years, pteridologists have also demonstrated the importance of the vasculature of the rhizome in phylogeny (Kato, 1972; Lucansky and White, 1974; Chandra and Nayar, 1975; Chandra and Kaur, 1976; Chandra, 1982; Chandra et al., 2003; Hovenkamp, 1990; and Srivastava et al., 2007). Even with a long history of vasculature research, our knowledge of the vascular system of the rhizome of the genus Polypodium is meager, and, to date the only detailed description of the vasculature of the rhizome of Polypodium vulgare was provided by Srivastava and Khare (2005). The structure and organization of vascular system in the rhizome of most species of Polypodium remain almost unknown, except for few anatomical observations on the rhizome of P. microrhizoma Clarke ex Baker and petiole of P. amoenum Mett. (Bir and Trikha, 1980), and gross morphological details of Polypodium formosanum Baker and P. glaucophyllum Kunze (Hovenkamp, 1990). The present investigation is a detailed study of the vasculature of the rhizome of four species of Polypodium (P. cambricum L., P. fauriei Christ, P. interjectum Shivas and P. sibricum Sipliy) with more emphasis on the arrangement of leaf gaps, number and departure of the leaf trace strands, association between leaf gap/branch gap/leaf trace/branch trace, and associ- ation of mechanical tissue. Our goal is to assess the potential value of these characteristics in taxonomic and phyletic considerations. MATERIALS AND METHODS For the present study, four species of Polypodium were obtained from Russia through the courtesy of Dr. Nina M. Derzhavina, Herbarium, Orel State University, Russia. They are P. cambricum L. (N.M.Derzhavina 87, OHH), P. 184 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) fauriei Christ (N.M.Derzhavina 10, OHHI), P. interjectum Shivas (N.M.Derz- havina 94, OHHI) and P. sibricum Sip]. (N.M.Derzhavina 15, OHHI). Vasculature morphology was studied mainly from serial sections from hand as well as microtome sections (cut at 100-130 um) of adult rhizomes fixed in F.A.A. and stored in 70% ethyl alcohol. Anatomical observations recorded here are based on microtome sections stained with safranin and fast green. Stelar organization was studied from three-dimensional reconstructions based on camera lucida tracings of the outline of the vascular strands in serial sections. Because of the significance of vascular organization in the rhizome of ferns in taxonomic and phyletic considerations, particular attention was paid to the general form and shape of the vascular cylinder, leaf gap, leaf trace, branch gap, branch trace and the association of the branch with the leaf. RESULTS Rhizomes are moderately stout (P. cambricum and P. interjectum) to soft and slender (P. fauriei, and P. sibricum) bearing leaves restricted to two alternating, dorsal rows. The diameters of the rhizome ranges from ca. 2 mm to ca. 8 mm. Branches are usually associated with leaves on the abaxial side away from the dorsal median plane of the rhizome. Transverse section of the rhizome in any plane usually shows 9-12 cylindrical to sub-cylindrical vascular bundles, which are distributed roughly in a circle in the ground tissue. The rhizome is soft, parenchymatous with the ground tissue uniform (not differentiated into cortex and pith) and having dense starch deposits. Sclerenchyma strands, as found in other polypodiac- eous ferns, are absent in the species of Polypodium studied here. The epidermis is single layered and consists of regularly arranged small, thin- walled, rectangular cells. Form of the Vascular Cylinder The vascular cylinder of the rhizome is basically similar to the common types in the Polypodiaceae and is a highly perforated dictyostele with much elongated lacunae forming a conspicuous loose reticulum (Figs. 1-4). It is pierced with two alternating, large overlapping leaf gaps on the dorsal side and many large lacunae elsewhere so that the vascular strands are slender and cylindrical. In all four species the vascular cylinder is dorsiventral with leaf gaps closely placed at the dorsal surface so that the successive ones of the two rows overlap conspicuously. The area of the vascular cylinder between the two rows of the leaf gaps is slightly thicker than others constituting a distinct dorsal median vascular strand. Root traces are restricted to the ventral half of the vascular cylinder and originate as superficial, solitary vascular strands from the outer surface of the stelar cylinder. As the root traces passes through the cortex of the rhizome it acquires a thick sheath of sclerenchyma. SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES 185 Lear GAPp.—Leaf gaps are usually oblanceolate in P. fauriei (Fig. 3, LG) and P. interjectum (Fig. 4, LG) with bluntly rounded anterior ends, while in P. cambricum (Fig. 1-2, LG) they are broad and ovate to oblanceolate with broadly rounded to bluntly rounded anterior ends. Leaf gaps of successive leaves overlap conspicuously and successive leaf gaps of the same side also overlap, though only slightly in P. fauriei and P. interjectum. In these two species the posterior end of each leaf gap extends downwards a little on either side of the anterior end of the next leaf gap so that the region between the two leaf gaps appears as a narrow, arched vascular strand. In Polypodium cambricum and P. sibricum the leaf gaps extend only a short distance beyond the region where leaf trace separates (i.e., the leaf trace strands are given off from the margins close to the anterior end of the leaf gap), while in P. fauriei and P. interjectum the leaf gaps are longer, extending markedly beyond the region of separation of the leaf trace (i.e., the leaf trace strands are given off from towards posterior margin of the leaf gap) as also reported in P. vulgare (Srivastava and Khare, 2005). However, in P. interjectum some of the leaf gap extends only a short distance beyond the region where the leaf trace separates. Lear Trace.—The leaf trace is highly dissected by profuse, short to elongated lacunae which are regularly placed towards the basal end of the leaf trace so that the basal region of the leaf trace often forms a closed-meshed reticulum. Each leaf trace is often composed of 4—8 slender vascular strands in the species of Polypodium studied. The leaf trace strands usually branch off from the margins close to the anterior ends of the leaf gaps in P. cambricum (Figs. 1-2, L) and P. sibricum while in P. fauriei, and P. interjectum leaf trace strands usually branch off from the posterior margins of the leaf gaps (Fig. 3-4, L) making the vascular strands of the leaf trace appear as independent strands traversing the cortex of the rhizome. As in other Polypodiaceae described, the adaxial margins of the leaf trace are slightly thickened (so that the last pair of leaf trace strands is thicker than the others). BrancH Gap.—Each branch trace has a conspicuous branch gap, placed next to the ventral posterior end of the leaf gap of the associated leaf (P. cambricum and P. fauriei ) and conspicuously overlapping with the associated leaf gap though distinctly separated from it by a slender vascular strand (Figs. 1-3, BG). In P. interjectum (Fig. 4, BG) and P. sibricum the branch gaps are short, and intimately associated with the base of the leaf trace so that it appears to be a part of the reticulated base of the leaf trace (i.e., the branch gap is merged with the leaf gap becoming inconspicuous). However, in P. sibricum the branch trace is often solitary, becoming inconspicuous and appears to originate from the leaf trace strands themselves. Successive branch gaps on the same side do not overlap. BRANCH TRACE.—As is characteristic of all Polypodiaceae, a branch is associated with a leaf at its abaxial side. Each branch trace is a simple structure, often composed of 2—4 slender, cylindrical vascular strands which do not form a 186 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) ty = " =f § 5 fer wy » ‘i j, Fics. 1-2. Vascular cylinder of a portion of the adult rhizome as seen from the dorsal surface. 1. Polypodium cambricum; 2. P. cambricum (B, branch trace; BG, branch gap; L, leaf trace; LG, leaf gap). SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES Fics. 1-2. Continued. 188 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) 4 Fics. 3-4. Vascular cylinder of a portion of the adult rhizome as seen from the dorsal surface, 3. Polypodium fauriei; 4. P. interjectum (B, branch trace; BG, branch gap; L, leaf trace; LG, leaf gap). reticulate vascular cylinder (Figs. 1-4, B). There is further reduction of the branch trace strands in P. sibricum where the branch trace is often solitary. Also, in some of the leaves there is no branch associated with the leaves (Figs. 1, 3, and 4). Structure of the Vascular Cylinder The vascular cylinder of the rhizome is composed of usually 9-12 slender and mostly cylindrical vascular strands. Xylem tissue of each vascular strand is ribbon-like and tracheidal, 1-5 cells thick in P. cambricum (Fig. 5), 2—5 cells thick in P. fauriei (Fig. 6), 1-5 cells thick in P. interjectum (Fig. 7), 1-3 cells SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES 189 thick in P. sibricum (Fig. 8). The tracheids are interspersed with thin-walled individual cells or bands of xylem parenchyma in between in P. cambricum, P. fauriei and P. interjectum (Figs. 5-7) while in P. sibricum (Fig. 8), as in P. vulgare (Srivastava and Khare, 2005), the tracheids are not intermixed with xylem parenchyma. A thin sheath of 1-2 irregular layers of small parenchyma cells envelops the xylem tissue except at the free ends. The protoxylem is generally exarch in position, being present at the ends of the xylem tissue. The phloem is not continuous and is interrupted at the ends of the xylem tissue. Phloem is restricted to either surface of the xylem tissue and is interrupted at either end. It is massive, composed of 1-8 layers (P. cambricum and P. fauriei, 2-8 layers; P. interjectum, 2—4 layers; P. sibricum, 1-3 layers) of narrow, small, thin-walled parenchyma cells intermingled with few sieve cells. The pericycle is prominent, continuous around the vascular tissue, and consists usually of 1—2 layers of thin-walled, small regularly arranged polygonal parenchymatous cells. However, in P. cambricum and P. fauriei the pericycle is single layered consisting of broad, polygonal cells. The endodermis is well developed and composed of a single layer of rectangular, small, thin-walled cells with casparian thickenings on their radial walls. However, in P. cambricum the inner walls of the cortical parenchymatous cells abutting on the endodermis are usually thickened. Some of the endomermal cells have dark brown phlobaphene contents in their cells but there is no continuation in all the cells. The endodermis of some of the vascular strands are devoid of phlobaphene contents. DISCUSSION Polypodium is a fern genus of the north temperate regions of the world with a few species in Africa and eastern Asia. Holttum (1947) regards the Polypodieae as derived by reduction from Phymatodes, while Polypodium is regarded as an ancestral genus evolved independently of Microsorieae and Pleopeltideae by Copeland (1947). Copeland (1947) kept Polypodium as separate genus, most numerous in American tropics. In general, open venation (simple, free) represents an ancestral condition but in ferns, especially Polypodiaceae, the most ancestral members have a complex netted venation, the few species with free veins being derived. Thus, according to Holttum (1947, 1964) reversion has taken place in Polypodiaceae. Mickel (1982) also considers the veins in the more recently derived groups ee. The restricted distribution of free venation, found only in the Polypodium- group, suggests strongly that reticulate venation is an ancestral character in the Polypodiaceae. The north-temperate Polypodium does not represent the ancestral condition, but the late offshoot of a tropical stock. Polypodium species have free veins but are connected by intermediates with the species which have anastomizing veins (more ancestral members in Polypodium have anatomizing veins). 190 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) faye a Se = ea ge | : = a Pee | Fics. 5-8. T ti organization of a vascular strand. 5. Polypodium cambricum; 6. P. fauriei; 7. P. interjectum; 8. P. sibricum (X 400). In contrast to P. vulgare (Srivastava and Khare, 2005) the dorsal median vascular strand is distinct and slightly thicker than other vascular strands of the rhizome in all the four species studied here. Recently, Smith et al. (2006) stated that the xylem is usually mesarch in the shoot of Monilophytes (including Eu- and Leptosporangiate ferns). However, contrary to the above observations, the protoxylem is restricted to either narrowed margins (lobes) of the xylem band on the side facing the cortex of the rhizome i.e., protoxylem is exarch in all of the species studied. Each leaf trace in the Polypodium species is highly dissected and usually composed of 4—5 slender vascular strands while in P. sibricum it consists of 5— 8 vascular strands. In contrast to P. vulgare (Srivastava and Khare, 2005) the leaf trace strands usually branch off from the margins close to the anterior end of the leaf gaps in P. cambricum and P. sibricum while in P. fauriei and P. interjectum the leaf trace strands branch off from the posterior margin of the leaf gaps. However, in P. interjectum some of the leaf gap extends only a short way beyond the region where the leaf trace separates. In P. cambricum and P. sibricum, the leaf gaps extend markedly on the posterior end of the leaf trace as found in the comparatively primitive species. In other species such as P. vulgare (Srivastava and Khare, 2005) and P. interjectum the leaf gap extends only slightly on the posterior side of the leaf trace. Branch gaps are comparatively very short and often merged with the leaf gaps becoming inconspicuous (P. interjectum). There is further progressive reduction of the branch trace strands in P. sibricum. The branch trace is often solitary without any branch gap and is often merged, becomes inconspicuous, and appears to originate from the leaf trace strands themselves; thus forming an integral part of the leaf trace. Perhaps the most interesting morphological feature of the rhizome is seen in P. interjectum and P. sibricum where the characteristic association of branch trace/gap with the leaf trace/gap is observed. In some cases the branch trace/ gap is often merged with the leaf trace/gap, becoming inconspicuous and forming an integral part of the leaf trace/gap. Such type of association has also SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES 191 Fics. 5-8. Continued. been reported in P. vulgare (Srivastava and Khare, 2005) and Pleopeltis tweediana (Hook.) A. R. Smith (Srivastava and Chandra, unpublished data). The absence of associated branches with some leaves and the closer associations of leaves and branches in P. interjectum and P. sibricum seem to be significant and may indicate the evolutionary status of the taxa. Possibly the condition where the branch gap/trace extends to the leaf gap/trace is relatively more advanced than where the branch gap/trace is distinct and lateral to the leaf gap. Based on this it has been suggested that the vascular architecture of the rhizome in Polypodium species studied possibly exhibits a derived condition. However, the evolutionary significance of this association between leaf and branch needs more extensive investigation. Relationship to Goniophlebium.—Goniophlebium was regarded by some taxonomists as congeneric with Polypodium (Christensen,1938; Holttum,1949: Hennipman et al.,1990). However, many prefer to treat it as separate genus (Ching, 1940; Copeland, 1947; Holttum, 1968; Pichi-Sermolli, 1977; and Srivastava and Khare, 2005) most numerous in American tropics and maintain it as comparatively ancestral and intimately related to Polypodium. However, Tryon and Tryon (1982), considered Goniophlebium, an Old World genus with articulate pinnae, as not closely related to Polypodium s.s., which is predominantly a New World genus. Based on molecular studies, Schneider et al. (2004) showed that Goniophle- bium is more distantly related to Polypodium than has been suggested. They further indicated that Goniophlebium is part of a large Old World clade that includes various genera such as Lecanopteris, Lepisorus, and Microsorum. They also provided evidence for a monophyletic Goniophlebium, as defined by Rodl-Linder (1990). The differences in the vasculature of the rhizome observed here do not support a close association of Polypodium with paleotropic Goniophlebium. The results are consistent with the studies from molecular analyses (Schneider et al., 2004), which suggested that Goniophlebium is more distantly related to Polypodium than previously suggested. In contrast to Goniophlebium (Srivastava and Khare, 2005) Polypodium species possess 1) dorsal median vascular strands scarcely different from other vascular strands, 2) usually 192 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) obliquely placed leaf gaps, 3) Leaf trace usually with four vascular strands, 4) much reduced branch trace usually with 1-3 vascular strands, 5) narrow branch gap which is less than half as long as the leaf gap, 6) Branch trace/gap in some cases merge with the leaf trace/gap forming integral part of it, 7) in some cases no branch associated with leaves, and 8) no sclerenchyma strands in the ground tissue. Until details regarding a large number of Polypodium species become available, an evaluation of the significance of stelar architecture in this group will not be possible. ACKNOWLEDGMENTS We are greatly indebted to Prof. M. Kato (Japan) for going through the manuscript and for valuable suggestions. We are also grateful to referees and oe Jennifer Geiger (USA) for their criticism and valuable comments on the manuscript. The first author is also thankful to Dr. R. C. e and Head, Department of fae D.A-V., College, ae (India) for cooperation and encouragements. LITERATURE CITED Bir, S. S. and C. K. TrrkHa. 1980. Anatomical Observations on certain Indian Polypodiaceous Ferns. Aspects of Plant Sciences 3:139-158. Bower, F. O. 1910. Studies in the phylogeny of the Filicales- I: Plagiogyria. Ann. Bot. 24:423—450. Bower, F. 1914. Studies in the phylogeny of the Filicales IV Blechnum and allied genera. Ann. Bo eta igt Bower, F. ne 915. Studies | in the ei cassie of the Filicales V Cheiropleuria bicuspis and certain other saaesert ferns. Ann. Bot. Bower, F. O. 1917. Studies in tg isis of the Filicales-VI: hai oe Acrostichoid Sag wi special reference to Dipterid derivatives. Ann. Bot. 31:1— Bower, F. O. 1918. Studies in the phylogeny of the Filicales- VII: The se ee Bot. 32:1-68. Cuanpra, S. eon Structure and Organization of the Vascular System in the Rhizome of Drynarioid ferns. Ann. Bot. 50:585—598 CHanpra, S. and S. Kaur. 1976. Contributions to the ee of Tectaria: Vascular Organization of the Rhizome. Phytomorphology 26(1):14 Canora, S. and B. K. Nayar. 1975. Vascular fact in the Rhizome of Spleenworts. J. Indian Bot. Soc. bie cok. egies S., M. Srivastava and R. Srivastava. 2003. sige: A in the vasculature of the rhizome in me _— of lindsaeoid ferns. Indian Fern J. 2 38. Com, R C. 1940. On natural classification of the family poise Sunyatsenia 5:201—268. HING, R. C. pu The Chinese fern families and genera: systematic arrangement and historical origin. Acta Phytotax. Sin. 16:1—37. CHRISTENSEN, C. 1938. Filicineae. Pp. 522-550. In: Verpoorn, F. (ed.), Manual of Pteridology. Martinus Nijhoff. The Hague. CopeLaAnp, E. B, 1947. Genera Filicum. Chronica Boatnica Co., Waltham, Mass U: CRaBBE, J. 2 C. Jermy and J. T. Micket. 1975. A new generic sequence for eg: Pteridophyte he m. Fern Gaz. 11:141-161. Hayata, B. ne On the systematic importance of the stelar system in the Filicales-I. Bot. Mag. okyo 41:697-718 Hayata, B. 1928. On the systematic importance of the stelar system in the Filicales-II, III]. Bot. Mag. Tokyo 42:301-311; 334-348 HENNIPMAN, E., VELDHGEN and K. U. Kramer. 1990. Polypodiaceae. Pp. 203~230. In: Kusrrzixi, K. (ed.). The Families and Genera of Vascular Plants Vol. 1. Pteridophytes and Gymnosperms, Vol. Kramer K. U. and P. S. Green. (eds.). Springer-Verlag, Berlin. SRIVASTAVA AND CHANDRA: THE RHIZOME OF FOUR POLYPODIUM SPECIES 193 Hotrtrum, R. E. 1947. A revised classification of leptosporangiate ferns. J. Linn. Soc (Bot.) London 3:123-158. Ho.ttum, R. E. Aes The classification of ferns. Biol. Rev. 24:267-296 Hottrum, R. E. 1964. The evolution of the vascular system in finsin with special reference to dorsventa sare Phytomorphology 14:477-480 Hottrum, R. E. 8. A Revised Flora of Malaya II. feins of Malaya, 2"“ edition. Govt Print Off., Singapor HOVENKAMP, P. “1990. The significance of rhizome ceed pad in the systematics of the ap eeacggas ferns sensu stricto. Amer. Fern J. 80:3 Kato, M. 2. The Vascular Structure and its Taxonomic eee in the Athyriaceae. Acta ae Geobot. 25(2—3): eres Lucansky, T. W. and R. A. Wuite. 1974. Comparative studies of the nodal and vascular anatomy in re hat epages tee af Nodal and petiole patterns: Summary and conclusions. r. J. Bot. 61:818-828. ae 1. i“ 1982. sea and classification of living organisms. Mc Graw-Hill Book Co. Inc., ondo Nayar, 8B. K. 1970. A phylogenetic classification of homosporous ferns. Taxon 19:229-236. Nayak, B. K. 1974. Classification of homosporous ferns. Pp. 127-201. In: Nayar, B. K. and S. Kaur (eds.). Companion to R.H. Beddome’s Handbook to the Ferns of British India. Chronica careers New Delhi. Pp. 127-201. Nayar, B. and S. CHanpra. 1967. fee as a ae series of the genus Pyrrosia and their Oo interpretation. ae a Bot. 4 634. Nayar, B. K., N. Baypar and S. Cua Neg pie to the morphology of the genus Olean vine. J}. lann, Soc. Moule naib Ocura, Y. 1972. Comparative Anatomy of ‘ho ag oie a the Pteridophytes. Encyclopedia of Plant Anatomy. Gebruder Borntraeger, Berlin a PicH! SERMOLLI, R. E. G. 1977. eepeners seb pmese ioe aes in taxonomicum ordinem redigendi. Webbia 31:481—4 Ropt-Linper, G. 1990. Monograph ee the fern genus Goniophlebium. Blumea 34:277-423. S H., A. R. Smirn, R. Cranritt, T. E. Hitpepranp, C. H. Haurier and T. A 04. Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae and Connusitideconel: exploring aspects of the diversihea Hen of epiphytic plants. Molec. Phylog. Evol. 1063 ’ 31:1041-— SmitH, A. R., K. M. Pryer, E. ScHuETTPELz, : Korat, H. ScHNemerR and P. G. Wo tr. 2006. A classification for hee ferns. Taxon 55:705-731. Sora, E. R. pELA. 1973. On the classification aul phylogeny of the Polypodiaceae. Bot. J. Linn. Soc 67 Suppl. 1:229-244 Srivastava, A. and R. C. Kuan . 2005. Structure and “ALpuoriniar 7 fog vascular system in the rhizome of f Goniophlebium rap Polypodium. Indian Fern J. 22:168—-175. Srivastava, A., Kare and S. CHanpra. 2007. sores of si rhizome in two species of xogramme. Jo. Phytomorpholgy 87(3 * a}: eee TANSLEY, A. G. 19 ici I- X. New iota 6:25-35, a Noe 135-147, 148-155, 187-203, 219-238, 253-269; 7: 1-16, 2 TarpiEu-BLot, M. L. 1932. Les Pi arsee io Tonkin a Ouvrage couronne per i ees des sciences. IMP Basuyan & Co., France. Pp. Tryon, R. and Tryon, A. 1982. Ferns fee Allied pias with Reference to Tropical America. Springer-Verlag, Berlin. American Fern Journal 99(3):194—199 (2009) Isoetes maxima, a New Species from Brazil R. JAMES HICKEY Botany Department, Miami University, Oxford, Ohio 45056 USA C. Cecitia Mac.ur Catedra de Palinologia, sc ienig de Ciencias Naturales y Museo, pcariias Nacional de La Plata, o del Bosque s/n, 1900 La Plata, Argent MELANIE LINK-PEREZ Botany Department, Miami University, Oxford, Ohio 45056 USA Asstract.—Isoetes maxima from eastern Brazil is described as a new species. This taxon differs from other fully aquatic species in South America by a combination of its overall size, leaf coloration, finely tapering subulae, and megaspore morphology. Key Worps.—Isoetes maxima, Brazil, new species While examining Isoetes from eastern Brazil, we encountered a specimen whose large size and dark coloration are unlike any known species from that region. The plants have numerous, very narrow, densely packed, and finely tapering leaves, and its megaspores are rugulate to tuberculate. In contrast, other large Isoetes in this region of South America have broader leaves that are less densely packed, and reticulate megaspores (Fuchs-Eckert, 1986; Macluf et al., 2008). The specimens in question were collected by Aloysio Sehnem in 1970. Sehnem determined his collection to be a new species and designated it so on the label as Isoetes maxima. The label indicates the collection as the intended holotype; however, Sehnem never described or validly published the new species. We have elected to use the name that Sehnem had inscribed on the specimen label since the overall stature and robust appearance of the taxon merit the appellation. Isoetes maxima Hickey, Macluf and Link-Pérez, sp. nov. TYPE.—BRAZIL. Cambaré, Fortaleza, Aparados, in aqua rivi in campo, 1200 m, 2/5/70, A. Sehnem 10960 (PACA 74904). (holotype: PACA; isotypes: FHS). Figs, 1-14. Cormus erectus, bilobatus, 2.5-3.0 cm latus; radices succulentae, dichot- omae. Foliae 30-50, ad 45 cm longae, strictae, erectae, 1.5-2.0 mm latae ad medium, ca 8-10 mm latae basi; alae atrovirentes vel fuliginosae, 2.0 mm latae ad sporangium, 10-25 cm longae (20-50% per foliae longitudinem ascen- dentes), apice attenuato; subula atrovirens, erecta, semi-teres, apice long- iattenuato, neque nitido neque comeo; sa pee fibrosi ae ee praesentes; squamellulis carentibus. Labium triangule, 850-875 altum, 475-500 um latum. Ligula magna, auriculata bieifeate culvinse ceiduns triangularis, persistens, ca 2 cm longum et 1.5 mm latum. Velum incompletum, 30-50% HICKEY ET AL.: ISOETES MAXIMA, A NEW SPECIES FROM BRAZIL PACA 44504 fem. Glsvetacere neoten ater omen ~e Porteleza, Aper-ins, Fics. 1-2. Holotype of Isoetes maxima (Sehnem 10960, PACA). 1. Whole ee 2. Adaxial views of ae sporophylls with microsporangia, partial velum and ligule fragment J JOURNAL: VOLUME 99 NUMBER 3 (2009) AMERICAN FE Fics. 3-8. Scanning electron mic el Seta of Isoetes maxima megaspores (Sehnem 10960, PACA). 3. }- Proximal view of a megas quatorial view of a megaspore. 5. Distal view of a pe meleg . Three of the four spores of a a single tetrad. 7. High magnification of the distal surface. 8. Detail of d a weak girdle . Fused nahevck 3—5; 08 um in Fig. 6; um in ne equatorial zone, with both ihe equa le in Figs. 4, 5 and 8. Scale bar = 100 um in Figs. are visi .7 and 8 HICKEY ET AL.: ISOETES MAXIMA, A NEW SPECIES FROM BRAZIL 197 Fics. 9—14. Sc canning electron mic rographs of Isoetes maxima microspores Hp 10960, PACA). 9. Proximal view. 10. Equatorial view showing the supra-laesural 00 aspen view. 12. Distal view. 13. Several oda in different views. 14. Seige 8 echinate ornamentation. Scale bar = 5 um in Figs. 9-12 11. Equatorial High magnification of the 2, 20 um in Fig. 13, and 2.5 um in Fig. 198 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) per sporangium longitudinem descendens. Sporangium basale, ellipticum, hyalinum, 5-6 mm longum, 2-4 mm latum, non maculatum. Megasporae cretaceae, triletae, 525—-(583.8)-650 um diametro, globosae, pagina distalis verrucata vel tuberculata (vel laevis), pagina proximalis laevis, cingulum leave. Microsporae brunneae vel atrobrunneae, monoletae, 27—33 um longae, 20-23 um latae, echinatae. Plants large, corms erect, bilobed, 2.5—-3.0 cm across. Roots numerous, succulent, dichotomously branched. Leaves 30-50, to 45 cm long, straight, erect, 1.5-2.0 mm wide at mid-length, ca 8-10 mm wide at the base; alae dark green to brown, 2.0 mm wide at the sporangium, extending 10-25 cm up the leaf (20-50% of total leaf length), apex attenuate; subula dark green, erect, appearing half-terete, apex long attenuate, neither glossy nor corneous, fibrous bundles present; scales absent. Labium triangular, 850-875 ym high, 475— 500 um wide. Ligule large, massive, distinctly auriculate; cushion dark, persistent, triangular, ca 2 mm long, 1.5 mm wide. Velum extending down and covering 30-50% of the sporangium; lower velum also present and covering 5— 10% of sporangium. Sporangium basal, elliptic, hyaline, 5-6 mm long, 2- 4 mm wide, concolorous. Megaspores dull white, trilete, 525—-(583.8)-650 um in equatorial diameter, distal surface rugulate to tuberculate (to laevigate), comprised of an open reticulum of fibrils, proximal surfaces laevigate, subtriangular to globose in polar view and globose in equatorial view, laesurae 44.5 um high, equatorial ridge 30 um wide, girdle typically smoother than the distal ornamentation. Microspores brown to dark brown en masse, monolete, 27-33 um long, 20-23 um wide, elliptic in polar view, proximal face convex and distal face broadly rounded, perispore surface echinate with longer echinae distally. PaARATYPES.—BRAZIL. Itaimbezinho, Sao Francisco do Paulo, in stagno ad flumen Perdizes, alt. 900 m, Dec. 24, 1980, A. Sehnem 17148 (PACA 74905). Cambara, Fortaleza, in rivulo submersum in lectu, alt. 1000m, Jan 10, 1973. A. Sehnem 12362 (PACA 74906). Isoetes maxima is an aquatic endemic known only from three collections out of the Cambard region of Rio Grande do Sul in Brazil. It grows submerged in streams at elevations of 900 to 1200 m. Species of Isoetes from Rio Grande do Sul have either reticulate or tuberculate-rugulate to rugulate megaspores. The reticulate-spored species of South America are in desperate need of revision. Those species of eastern and southern South America (from Minas Gerais to Buenos Aires) constitute an exceptionally difficult assemblage, having had less attention paid to them than the reticulate-spored species of the northern Andes. Fuchs-Eckert (1986) treated the reticulate-spored species of this region, focusing primarily on the State of Santa Catarina. Although most non-Andean reticulate species were covered, these taxa are still imperfectly differentiated. The only two non-reticulate species in southeastern Brazil are Isoetes weberi Weber and Isoetes maxima. Megaspore ornamentation in both species varies from tuberculate to rugulate, but the species differ dramatically in megaspore HICKEY ET AL.: ISOETES MAXIMA, A NEW SPECIES FROM BRAZIL 199 size: Isoetes weberi spores have a mean of 356 um and a range of 220 to 450 um; those of I. maxima range from 525 to 650 um with a mean of 583 um. At high magnifications, the megaspore surfaces of both species consist of a coarse open reticulum of structural elements. This type of surface is not seen in any of the reticulate spored species. Microspores in both species are fundamentally echinate, but in I. weberi the echinae are broader, more columnar and distally muricate (Hickey, 1985). Both species have moderately well developed vela extending about 50% down the sporangium. The species also differ markedly in the size and shape of the labium. In I. maxima the labium is narrowly triangular, with a length to width ratio of about 1.8 to 1 and reaching lengths of 0.85-0.88 mm in height. The labium of I. weberi ranges from 1.0-1.8 mm in height, is narrowly oblong and has a length to width ratio of 2.3-3.3. The labium of I. weberi is typically bifid distally, a condition otherwise known only in I. tennesseensis Luebke and Budke (Budke et al., 5 Isoetes weberi is a lowland species growing at elevations of 10-20 m whereas I. maxima is found at about 1200 m. The presence of scale leaves around the corms in I. weberi suggests that it frequents drier habitats with only seasonal inundation, and so differs from the more aquatic Isoetes maxima. The rather large megaspores seen in Isoetes maxima suggest an origin through polyploidy. Within the vicinity of I. maxima only I. weberi stands as a likely parent. In fact, the similar morphology and habit in conjunction with the common, open reticulum of the megaspores suggest an affinity between these two. Candidates for a second parent are more problematic and perhaps must be sought further north in the states of Bahia, Espirito Santo and Rio de Janeiro, perhaps to the poorly understood J. organensis Weber or I. ulei Weber. LITERATURE CITED Bunk, J. M., R. J. Hickey and K. AFNER. 2005. Analysis of morphological and anatomical haracteristics of Isoetes using Isoetes tennesseensis. Brittonia 57:167-182. Fucus-Eckert, H 1986. Isoetdceas 3—43 in R. Reitz, ed. Flora Ilustrada Catarinense, I Parte Fasciculo. Itajai, Santa Catarina, Brasil. Hickey, R. J. 1985. Revisionary Studies of Neotropical Isoetes. Ph.D. Dissertation, Univ. of Connecticut, Storrs, CT. Mactur, C. C., M. A. Morse.u and G. E. Grupice. 2008. Megaspore morphology of Isoetes species (Lycophyta) from southern Brazil. XII Simpésio Brasilero de Paleobotdnica y Palinologia (Florianépolis, Brasil, Noviembre de 2008), Boletim de Resumos, p. 129. American Fern Journal 99(3):200—206 (2009) New Records of Polyphlebium borbonicum, an African Filmy Fern, in the New World and Polynesia ATSUSHI EBIHARA* Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba 305-0005, Japan Jor. H. Nirra Graduate School of Science, The University of Tokyo, Japan Davip LORENCE National Tropical Botanical Garden, USA JEAN-Yves DUBUISSON Université Pierre et Marie Curie, France Asstract.—Polyphlebium borbonicum is newly recorded in Central and South America and Easternmost Polynesia (Marquesas = pape! Islands). It has been misidentified as P. diaphanum and as P. endlicherianum in the orld and in the Pacific, Raganaataae dy hisroaais borbonicum is distinguishable from true ei li roader b endlicherianum by the absence of a marginal elongate vo row of the lamina. Key Worps.—distribution, Hymenophyllaceae, Marquesas In the course of preparing a treatment of Hymenophyllaceae for the Vascular Flora of the Marquesas Islands project, it came to our attention that specimens identified as Trichomanes endlicherianum C. Presl (= Polyphlebium end- licherianum (C. Presl) Ebihara & K. Iwats.) comprise two quite different forms. The first form has lanceolate fronds with an obvious row of clear elongate cells along the margins (Figs. 1a, 2d), and resembles typical P. endlicherianum distributed in the South Pacific. The second form has larger and broader onds, an ovate outline, and no clear marginal cell row (Figs. 1b, 2a). So far, no species matching the characters of the second form has been recorded in the South Pacific area. From a global viewpoint, this form best matches Polyphlebium borbonicum (Bosch) Ebihara & Dubuisson, an African species. Polyphlebium borbonicum was originally described based on a specimen from Bourbon Island (La Réunion) and is widely distributed in tropical Africa (Beentje, 2008; Kornas, 1994), but has not been recorded in either the New World or in Polynesia (with the exception of a single occurrence of P. borbonicum recently noted by Nitta (2008) in Moorea, French Polynesia; this specimen has been included in the current analysis). We also noticed that some New World plants usually identified as Trichomanes diaphanum Kunth resemble both P. borbonicum and the *Corresponding author. EBIHARA ET AL.: NEW RECORDS OF POLYPHLEBIUM BORBONICUM 201 a b Fic. 1. Laminar cells of two Marquesas species. a. Polyphlebium endlicherianum (D. Lorence 9127, PTBG) with transparent marginal elongate cells; b. P. borbonicum (K. R. Wood 10500, PTBG) without marginal elongate cells. Scale: 1 mm unidentified Polynesian plant. Examination of 1077 base pairs (bp) of chloroplast rbcL sequences (methods of DNA and phylogenetic analyses followed Ebihara et al., 2005), suggests that P. borbonicum of Réunion is the species most Closely related to Polynesian P. endlicherianum. All samples examined in this study (Table 1) form a robustly supported monophyletic clade together with P. borbonicum of Réunion (Fig. 3). Based on both morphological and genetic homologies, we treat these disjunct distributed plants as a single species, Polyphlebium borbonicum. The genus Polyphlebium as redefined by Ebihara et al. (2006) is comprised of about 15 species, located mainly in the southern hemisphere, with no species widely distributed across multiple continents. Since P. borbonicum has been misidentified as P. endlicherianum in Polynesia, and was submerged under the wider morphological variation of the P. diaphanum — P. hymenophylloides complex in the New World (Table 2), with proper 202 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) . a-c. Specimens of Polyphlebium borbonicum. a. Marquesas Islands (K. R. Wood 4522, ae 24332); b. Bolivia (M. Kessler et al. 11435, UC 1621639); c. Réunion (J.Y. Dubuisson HR1999- 22, P); d. a specimen of P. endlicherianum from the Marquesas Islands (D. Lorence 9127, PTBG). Scale: 1 cm. identification, we propose that P. borbonicum is widely distributed across the paleotropics and neotropics (Fig. 4). Polyphlebium borbonicum of the New World is recognizable by its segments of unequal length and broader fronds (more than 3 cm wide) in most cases. Trichomanes debile Bosch, a name long overlooked and synonymized under P. diaphanum (e.g., Lellinger 1989), has recently been applied by A. R. Smith to specimens having flat wings and less developed pinnae (pinnules at the basiscopic side are usually unbranched) at an acute angle against the rachis (identification was made for the herbarium specimens of UC) (Fig. 1b). Our result (Fig. 3) showed that one of two samples of T. debile is nested in the clade of P. borbonicum and that the other is closely related to the clade. We here advocate a taxonomic treatment synonymizing T. debile under P. borbonicum. ABLE 1. ee, e Cp anagccm borbonicum used for phylogenetic analysis. ‘Sequences newly pore for this s Original identification Locality Accession Voucher (Herbarium) F. Réunion AY175782 Dubuisson HR1999-2 (P) “P. endlicherianum” _ Society Islands, Moorea EU122988 __ Nitta UC “P. endlicherianum” — Marquesas Islands, Ua Huka =AB445233' Wood 10501 (PTBG) “T. debile” Bolivia, Prov. Sud Yungas EU784118' Kromer 1753 (UC) “T. debile” Bolivia, Prov. Ayopaya EU784117' Jimenez 1568 (UC) EBIHARA ET AL.: NEW RECORDS OF POLYPHLEBIUM BORBONICUM 0.01 P. hymenophylloides (AB257460) 0.97/71 P. borbonicum Marquesas (Wood 10501) 0.97 P. borbonicum Moorea (Nitta 073) 0.94/ 1.00/99 P. borbonicum Réunion (AY175782) 1.00/8 P. borbonicum Bolivia (Jimenez 1568) P. diaphanum (Y09191) 0.74/56 | P . diaphanum (AB083292) P. ingae (AB257461) P. angustatum (AY175783) P. exsectum (AB257458) P. colensoi (AB257456) P. vensoum (AY175786) P. vieillardii (AB257471) P. endlicherianum (AY175787) Crepidomanes latealatum (AB083291) Fic. 3. A phylogenetic tree of Bayesian inference based on 1077bp of chloroplast rbcL sequences. Numbers at the nodes indicate support values (Bayesian posterior probability/maximum parsimony bootstrap). 204 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Tas_e 2. A comparison of morphological characters of Polyphlebium borbonicum, P. diaphanum and P. endlicherianum. Character P. borbonicum P. diaphanum P. endlicherianum Marginal elongate cells Absent Absent Present Width of ultimate segments 0.8—1.0 mm 0.4—0.8(—1.0) mm 0.5-1.0 mm Wings of rachis Flat More or less waved Flat TAXONOMIC TREATMENT Polyphlebium borbonicum (Bosch) Ebihara & Dubuisson, Blumea 51: 240, 2006. Trichomanes borbonicum Bosch, Ned. Kruidk. Arch. 5(2): 158, 1861. Vandenboschia borbonica (Bosch) G.Kunkel, Nova Hedwigia 6: 213, 1963. TYPE.—Ins. Borboniae, Boivin 908 (holotype: L? not seen; isotype: B). Fig. 2 a—c Trichomanes debile Bosch, Ned. Kruidk. Arch. 5(2): 154, 1861. TYPE.—VENEZUELA. Prov. de Carabado, 700m, May 1846, Funck & Schlim 596 (holotype: L? not seen; isotype: UC). Rhizomes long-creeping, frequently branching, filiform, less than 0.5 mm in diameter, densely covered with brown hairs, roots few and fine. Stipes (0.8—) 2-6 cm long, at a distance from the adjacent ones. Blades bipinnatifid- << \ -v 3 Ls me, Ooh Fic. 4. A distribution map of Polyphlebium borbonicum. Black circles: new distributions by present study. White circles: previous records (Tardieu-Blot 1951, Kornas 1994, Beentje, 2008). EBIHARA ET AL.: NEW RECORDS OF POLYPHLEBIUM BORBONICUM 205 bipinnate, ovate to lanceolate, to 18cm long and 5.5 cm wide, ultimate segments 0.8-1.0 mm wide, venation anadromous, elongate marginal cells absent, false veinlets absent, internal cell walls thin and straight. Sori paratactic, tubular, lips dilate, receptacle exserted. DistTRIBUTION.—Mascarene Islands, Madagascar, Continental Africa, Costa Rica, Colombia, Venezuela, Ecuador, Peru, Bolivia, Marquesas Islands, Society Islands (Moorea) (Fig. 4). SPECIMENS ExaMINED.—VENEZUELA. Estado Carabado, Limite Distrito Bejuma Distrito Montalban, 950-1100 m, 26 Dec. 2001, W. Meier 8775 (UG 1779409). Estado Miranda, Distrito Urdaneta, Cordilera de la Costa, 500-1000 m, 20 Mar. 2004, W. Meier et al. 10391 (UC 1796788). COLOMBIA. Dept. de Narino, Ricaurte, osque nublado de montafia, cerca del Sendero Natural, 1800 m, 24 Jun. 1995, P. S. Baracaldo 050 (UC 1606850). Santa Maria, 1898— 1901, H. H. Smith 2256 (UC 219628). ECUADOR. Pichincha, L. Sodiro s.n. (UC 478203). PERU. Dept. Cuzco, Prov. Urubamba, Ruinas Machu Picchu, base of Huayna Picchu, ca. 2500 m, 3 Jan. 1963, H. H. & C. M. Iltis 1065 (UC 1348750). BOLIVIA. Dept. Cochabamba, Prov. Chapare, 910 m, 6 Sept. 1996, M. Kessler et al. 8194 (UC 1617032). Dept. Cochabamba, Prov. Ayopaya, Comunidad Grande, sendero a incacasani Grande, 2430 m, 13 Sept. 2002, I. Jimenez & A, Moguel 1568 (UC 1780573). Dept. Cochabamba, Prov. Carrasco, Parque Nacional Carrasco, 560m, 23 Sept. 1997, A. Acebey 777 (UC 1736913). Dept. La Paz, Prov. Nor Yungas, 5 km de Chuspipata hacia Coroico, 2750 m, 18 Sept. 1997, M. Kessler et al. 12037 (UC 1620790). Dept. La Paz, Prov. Nor Yungas, Canton Pacdlo a 600 m de la Estacion Biologica Tunquini, 1690 m, 22 Aug. 1998, A. Portugal et al. 204 (UC 1735646). Dept. La Paz, Prov. Sud Yungas, Alto Beni, Territorio Moseten, parcela V PIAF, 1150 m, 6 Apr. 1999, T. Kromer 1753 (UC 1749657). Dept. La Paz, Prov. J. Bautista Saavedra M., Pauji-Yuyo, entre Apolo y Charasani, 1450 m, 7 Jun. 1997, M. Kessler et al. 9860 (UC 1622866). MARQUESAS ISLANDS. Nuku Hiva, Toovii, Ooumu area, top of Tapueahu Valley off new Hwy, 3500- 3700 ft, 24-26 Sept. 1995, K. R. Wood & S. Perlman 4640 (PTBG 24438). Ua Huka, Vaikivi summit region and drainage, 700 m, 16 Jun. 2004, K. R. Wood 10749 (BISH, P, PAP, PTBG 36576, UC 1797858, US); Hitikau region, via Matukuoha Ridge overlooking Hane, 750 m, 5 Dec. 2003, K. R. Wood 10539 (BISH, P, PAP, PTBG, US). Ua Pou, Anakooma River valley ESE of Oave peak, 380 m, 17 Jul. 2003, D. H. Lorence et al. 9110 (P, PAP, PTBG, US); Pou Maka, ridge from SE base of peak heading toward Teavahaakiti, 690 m, 19 Jun. 2004, D. H. Lorence 9336 (PTBG 41942, UC 1797870): Tekohepu, 2500— 300 ft, 4—5 Jul. 1997, K. R. Wood & S. Perlman 6450 (PTBG 24434). Hiva Oa, Temetiu, 3200 ft, 24 Aug. 1995, K. R. Wood 4406 (PTBG 24440). Fatu Hiva, Trail from Omoa along Punaitai ridge crest to base of Tekou peak, 550- 840 m, 23 Jul. 1988, D. H. Lorence et al. 6179 (PTBG 6271). SOCIETY ISLANDS. Moorea, Mt. Moaputa. Trail from Vaiare to summit, ca. 700 m, 11 Nov. 2006, J. H. Nitta 73 (UC 1876625). 206 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) ACKNOWLEDGMENTS We thank curators of PTBG and UC for specimen loans, Dr. Henk Beentje for providing the Flora of Tropical East Africa manuscript and two anonymous reviewers for their helpful comments for the manuscript LITERATURE CITED BEENTJE, H. 2008. Hymenophyllaceae. In Flora of Tropical East Africa. Royal Botanic Gardens, Kew. Eprara, A., J.-Y. Dusuisson, K. Iwatsuxki, S. HENNEQUIN and M. Ito. 2006. A taxonomic Revision of —280. Eprnara, A., H. IsHikawa, S. Matsumoto, S.-J. Lin, K. Iwatsuxi, M. Takamrya, Y. Watano and M. Iro 2005. Nuclear DNA, chloroplast DNA, and ploidy analysis clarified biological complexity of the Vandenboschia radicans complex (Hymenophyllaceae) in Japan and adjacent areas. Amer. J. Bot. 92:1535—-1547. EpinarA, A., K. Iwatsuxki, M. Iro, S. HENNEQuUIN and J.-Y. Dupuisson. 2007. A global molecular phylogeny of tl genus Trichomanes (Hymenophyllaceae) with special reference to stem atomy. Bot. J. Linn. Soe. 155:1-27. Kornas, J. 1994. Filmy ferns (Hymenophyllaceae) ia Central Africa eee ees Burundi). 2. richomanes ae subgen. Microgonium). Fragm. Florist. Geobot. 3 LELLINGER, D. B. 9. The Ferns and Fern-allies hp aes lee Panama, pas the Chocé (Part 1: spun hrouth Dicksoniaceae). Pteridologia 2A:1—364. Nirta, J. H. 8. Exploring the utility of three past loci for biocoding the filmy ferns ei IRN Ste of Moorea. Taxon 57:725—7 Tarpieu-Biot, M. L. 1951. 3e Famille, Cama Grea! In Flore de Madagascar et des Comores (Plantes vasculaires), Edited by H. Humbert. Paris, Museum National d’Histoire Naturelle. American Fern Journal 99(3):207—216 (2009) Aspects of Gametophyte Development of Dicksonia sellowiana Hook (Dicksoniaceae): an Endangered Tree Fern Indigenous to South and Central America CiAupia Cristina L. Fiort, Marisa SANTos, and Aurea M. Ranpi* Department of Botany, University of Santa Catarina, 88040-900, Florianépolis, Santa Catarina, Brazil Apstract.—With the purpose of providing a basis for programs of sustainable management in the conservation of this endangered species, this paper presents morphological aspects on the gametophyte development of Dicksonia sellowiana (Dicksoniaceae) by light microscopy and scanning electron microscopy. Dicksonia sellowiana spores were germinated in Morh’s nutrient solution modified by Dyer (1979) under a 16-hour photoperiod at 23 + 2°C. To determine the best substrate for gametophyte and sporophyte development, 30 days after spore sowing filamentous gametophytes were transferred to different substrates: soil rich in organic matter; coxim (coconut fiber); sterilized typic hapludult soil (distroferric red nitosoil); and sterilized typic hapludult soil (distroferric red nitosoil) with the addition of organic compost. The best system for D. sellowiana growth was the red soil with the addition of compost. Fifteen days after spore sowing in mineral solution, gametophytes were filamentous. Some had attained laminar morphology and had established an oblique cell division, giving rise to the obconic cell. Laminar gametophytes were observed 30 days after sp g and cordate gametophyt observed after 45 days. Mature cordate gametophyt b 1 after 80-90 days. After 245 days 84.67% of gametophytes had produced sporophytes in sterilized red soil with the addition of organic compost. In typic hapludult soil, without the additional termophilic compost, sporophyte formation was delayed (development after 180 days). When gametophytes were transplanted to soil rich in organic matter they did not develop and in the “‘coxim” substrate, which is a substitute for the “xaxim”’ substrate, only filamentous gametophytes were observed at the end of the study. Key Worps.—Dicksonia sellowiana, gametophyte development, substrate comparison The Atlantic Forest biome entirely occupies three Brazilian states: Espirito Santo, Rio de Janeiro and Santa Catarina, 98% of Parand and some areas of 11 other states (IBGE, 2004; Fundacdo Biodiversitas, 2006). Ferns are an important plant group of the Brazilian flora. According to Tryon (1970, 1972) and Tryon and Tryon (1982), southeastern Brazil (from Minas Gerais to Rio Grande do Sul) contains about 600 fern species. Some of the Brazilian ferns are used as ornamental plants and members of the tree fern families Cyatheaceae, Dicksoniaceae and Cibotiaceae have been indiscriminately exploited through the commercialization of pots and substrate used in the production of ornamental plants (Windisch, 2002). For that reason, under- *Author for correspondence - amrandi@ccb.ufsc.br 208 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) standing aspects of fern biology is necessary for the development of methods that may assist in their conservation and management. In Brazil, Dicksonia sellowiana Hook. (Dicksoniaceae) is considered an endangered species of the Atlantic Forest biome (IBAMA, 1997). It occurs preferentially in high humidity environments and on river banks, independent of soil conditions (Fundag4o Biodiversitas, 2006). The stem is usually massive, ranging from 12—20 cm in diameter, arborescent, 10 m tall, basally decumbent, bearing long, dense trichomes and many fibrous roots, which may occur from the base almost to the apex. It occurs at ca. 1500-2500 m, sometimes up to 3500 m, or in Brazil at lower elevations. It occurs throughout Central America and in South America from Venezuela to Colombia, south of Bolivia, Paraguay, Uruguay and southeastern Brazil (Sehnem, 1978; Tryon 1970, 1972; Tryon and Tryon, 1982). In Brazil, it is known as ‘xaxim’ or ‘xaxim bugio’ and the trunks have been indiscriminately exploited through the commercialization of vases and substrate (Sehnem, 1978). he understanding of fern germination and establishment is required for their “ex situ” conservation. The germination of a great number of fern spores is promoted by light (Millér, 1968) and nutrients, water and mild temperatures are implicated in the growth and development of the prothallus and in sporophyte formation (Fernandez et al., 1996, 1999). Several aspects of the germination of D. sellowiana have been studied. Fillipini et al. (1999) sterilized spores in a 5% solution of commercial bleach for 10 min and reported that the spores of this species reached around 88% germination at 23 + 2°C under continuous white light, seven days after sowing in liquid mineral medium. The same authors reported that the spores stored under refrigeration remained viable for more than two years and reached 81.75% germination 10 days after spore sowing, which did not differ from the germination of recently collected spores (Fillipini et al., 1999). Under 50% and 36% irradiance, the germination of ‘D. sellowiana spores was delayed after 14 and 21 days, respectively, of culture compared to 20% and 5% irradiance. Higher percentages of germination (around 90%) and lower mean germination time (34 days) were observed for spores of D. sellowiana sterilized in a 35% solution of commercial bleach for one hour, which germinated at 20% and 5% sunlight; no statistically significant differences were observed between the two light treatments (Fillipini et al., 1999; Renner and Randi, 2004). To study the possibility of long-term spore storage of Dicksonia sellowiana for the establishment of a germplasm bank, Rogge et al. (2000) stored spores in liquid nitrogen and reported that spores remained viable after being immersed in liquid nitrogen for three months. Concerning patterns of gametophyte development, Nayar and Kaur (1969) described seven different types of prothallial development in the homosporous ferns. In previous works it was reported that germination in D. sellowiana is of Vittaria type and prothallic development is of Adiantum type (Pérez-Garcia and Fraille, 1986). To obtain sporophytes of Dicksonia sellowiana cultivated from germinated spores, Borelli et al. (1990) cultivated D. sellowiana in the soil of ‘‘xaxim’’ (D. sellowiana) trunks and observed sporophytes after six months of spore sowing. FIORI ET AL.: GAMETOPHYTE DEVELOPMENT OF DICKSONIA SELLOWIANA 209 They commented that fungal contamination was very high in all the treatments. Suzuki et al. (2005) cultivated D. sellowiana from spores and observed sporophytes that had been emerged in sterilized typic hapludult soil (red soil) with the addition of termophilic organic compost; the first sporophyte frond was observed 84 days after transplantation. The aim of the present study was to observe gametophytes of Dicksonia sellowiana grown in different substrates in the laboratory to examine morphological aspects of gametophyte development using light microscopy and scanning electron microscopy in order to determine suitable conditions for their growth and development. MATERIAL AND METHODS Sporophylls of Dicksonia sellowiana were collected from living plants in August 1999 in Urupema, a fragment of the Atlantic Forest biome, situated between 27°57'25"S and 49°53'33”W, Santa Catarina state, Brazil. Sporophylls were air-dried in an oven at 30°C for three days on filter paper in order to induce dehiscence. The spores were removed and separated from debris by filtering through lens paper, and then were stored in glass jars under refrigeration at 7 + 1°C. Spores were surface-sterilized using a 20% (v/v) solution of commercial bleach (2% active chlorine), which corresponds to 0.1% of active chlorine, for a period of 30 min before filtering through sterile filter paper and washing several times with sterile distilled water. About 20 mg of sterilized spores were sown in each of 32 conical flasks containing 20 ml of Mohr’s nutrient solution as presented by Dyer (1979) with the addition of 25 mg L"! Benomyl to avoid fungal contamination. The flasks were plugged with two layers of autoclaved transparent commercial polypropylene film (7 x 7 cm) fixed with rubber bands. All the procedures were carried out in a laminar hood. The spores were incubated in a 16-hour photoperiod (30 moles quanta - m? - s') at 23 + 2°C in January 2002. In February 2002, the young gametophytes cultivated in liquid medium were transplanted to trays containing four types of substrates: commercial substrate rich in organic matter; commercial ‘“‘coxim”: substrate produced from the coconut fiber used as substitute for the soil made from the ‘“xaxim” (D. sellowiana trunks); sterilized typic hapludult soil (distroferric red nitosoil) and sterilized typic hapludult soil (3 parts) with addition of termophilic organic compost (1 part) as described in Suzuki et al. (2005). The soil analysis was carried out in Soil Laboratory of CIDASC (Company for Agricultural Development of Santa Catarina) (Table 1). The trays were covered with transparent film to avoid excessive water evaporation and plant dehydration. Substrate sterilization was carried out in a high power microwave oven for 20 minutes. The organic compost was produced from vegetable and fruit wastes at the University of Santa Catarina. Sporophyte emergence was scored once a week. The mean and standard deviation for each day of evaluation was calculated by Excel for Windows (Microsoft). 210 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) TaBLE 1. Analysis of substratum mineral composition (CIDASC-analysis number 07462/2003). Typic hapludult Soil rich in Typic soil plus termophilic organic matter Coxim hapludult soil compost pH 5.20 4.40 5.20 P (ppm) > 50.00 38.30 2.60 > 50.00 K (ppm) 340.00 1204.00 95.00 450.00 % organic matter 4.30 > 10.00 0.80 % total N 0.12 0.35 0.03 0.26 Al (cmolc/I]) traces 0.3 2.2 traces Ca (cmolc/]) 5.4 17 17 4.8 *, Al (cmolc/I) 2.48 1.89 8.79 3.90 CEC (cmolc/l) 13,92 9.37 41,32 12.88 Specimens were collected every 15 days from the mineral solution and from the trays containing sterilized typic hapludult soil and organic compost. For light microscopy (LM) and scanning electron microscopy (SEM), gametophytes were fixed in 2.5% glutaraldeyde in 0.1 M sodium phosphate buffer 7.2 pH. All samples were fixed in Eppendorf tubes for 3-hr, then were centrifuged for 3 min, dehydrated with an ethanol series and stored in ethanol. The samples were photographed with a Leika-MPS 30 light microscope. For LM, the samples were mounted on glass slides with ethanol. For SEM, the cordate gametophytes were dehydrated with graded ethanol (80%, 90%, 96% and 3 times in 100%). Subsequently, they were transferred to HMDS (hexamethyl- desilasane) to substitute the CO, critical point, avoiding cell collapse (Bozzola and Russel, 1991). Dry samples were transferred to stubs and then were gold- coated with 20 nm of gold in a Baltec-CED 030. Examination was performed with a Philips-XL 30 scanning electron microscope. RESULTS The spores of Dicksonia sellowiana are tetrahedral, globose and trilete; the surface is densely granulated and measure 44—68um (Figs. 1 and 2). Dicksonia sellowiana germination is of the Vittaria type (Pérez-Garcia and Fraille, 1986). ring germination, the spores become swollen and the spore coat opens. The first division was parallel to the equatorial axis of the spores; small hemispheric cells were produced and gave rise to a hyaline rhizoid that does not contain plastids; subsequently a spherical prothallial cell which is rich in chloroplasts appeared (Figs. 3—5) Gametophytes of D. sellowiana were not able to develop in the soil rich in organic matter. In the coxim, only filamentous gametophytes were observed after 8 months of cultivation. In sterilized typic hapludult soil, the first sporophytes were only observed after 6 months of cultivation, but in sterilized typic hapludult soil with addition of termophilic organic compost, sporo- phytes were observed after less than 3 months of cultivation. FIORI ET AL.: GAMETOPHYTE DEVELOPMENT OF DICKSONIA SELLOWIANA 211 N;-1 . al Fics. 1-2. Ss g elect icrographs of i p 2. Tetrahedral spores. Bar = 100 um. 3. Detail of the spore with densely granulated surface. Bar = 20 um. Fifteen days after sowing, a uniseriate filament was apparent, consisting of 3-7 cells as a result of parallel divisions of the original prothallial cell. The filament cells showed abundant chloroplasts and the spore coat was still attached to the basal cell. There was only one rhizoid present (F ig. 6). A wall parallel to the axis of the filament divided the terminal cell; further division followed an oblique direction, giving rise to an obconic or meristematic cell (Figs. 7 and 8). The laminar phase of D. sellowiana gametophytes was observed after 30 days of spore sowing. The beginning of the cordate or heart phase with Fics. 3-8. Initial phase of spore germination. Bars = 20 um. 6. Germinated spore with rhizoid. Bar = 20 um. 7. Filamentous gametophyte (15 days). Bar = 100 um. 8-9. Laminar gametophyte with the initial lateral divisions (15 days). Bars = 50 um. Legend: oc — obconic cell, r - rhizoid. 212 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Fics. 9-12. 9. Laminar phase (30 days) Bar = 50 um. 10. G tophy tulated (45 days). Bar = 100 um. 11. Cordate gametophyte (75 days). Bar = 200 um, 12. Meristematic region with archegonia (90 days). Bar = 100 um. ac- obconic cell , amr - apical meristematic region,— ar- archegonium, le - lateral expansion, r — rhizoid. the development of the wings was observed 45 days after spore sowing and 15 days after gametophyte transplantation to the sterilized typic hapludult soil with addition of termophilic organic compost. After 75 days of spore sowing, gametophytes presented the heart shape (Figs. 9-11) and 90 days after spore sowing archegonia were present (Figs. 12 and 13). The archegonia grew from surface cells of the apical meristem, were bottle-shaped, and presented four rows of 4—5 exposed neck cells and the neck canal inside (Figs. 13-16). Antheridia were not observed in this SEM prepared material. The gameto- phytes did not bear trichomes. DISCUSSION The surface of Dicksonia sellowiana spores are granulated or reticulate with strands of fused spheres surrounding somewhat depressed areoles as observed by Tryon and Tryon (1982). The pattern of spore germination found for Dicksonia sellowiana was the Vittaria type (Nayar and Kaur 1971), which was described by Pérez-Garcia and Fraille (1986). The prothallial development of Dicksonia sellowiana is of the Adiantum type, described by Nayar and Kaur FIORI ET AL.: GAMETOPHYTE DEVELOPMENT OF DICKSONIA SELLOWIANA 213 Fics. 13-16. Scanning electron micrographs of Dicksonia sellowiana gametophytes. 13. General gametophyte view. Bar = 500 um. 14. Aspects of inferior face bearing archegonia. Bar = 100 um. 15. Detail of an archegonia. Bar = 20 um. (1969) and Pérez-Garcia and Fraille (1986). In this type of gametophyte development, spore germination results in a uniseriate, slender germ filament, which is generally 3-7 cells long. The terminal cell and one or two cells behind it divide longitudinally to form a prothallial plate. The division of the terminal cell is often by the formation of a wall oblique to the long axis of the filamentous gametophyte, and soon a second oblique wall delimits a central obconical meristematic cell. By the activity of the obconical cell, a spatulated prothallial plate, without trichomes, is formed in which the apex gradually becomes notched. Examples of species from the Dicksoniaceae that show the Adiantum type protallial development are Lophosoria quadripinnata (J. F. Gmel) C. Chr. (Pérez-Garcia et al., 1995) and Lophosoria quadripinnata var.contracta (Mendoza et al., 1997). The laminar phase was observed after 30 days of cultivation for Dicksonia sellowiana and the heart shaped gametophytes presented archegonia 90 days after spore sowing, but they did not present antheridia. Conversely, Pérez- Garcia and Fraille (1986) observed gametophytes of D. sellowiana bearing antheridia only after 170 days cultivation, but they did not observe gametophytes bearing archegonia. The same authors analyzed gametophyte development only under light microscopy and did not analyze time to 214 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) sporophyte formation and percentage of sporophyte formation in different soils. They found filamentous amorphous gametophytes that were not observed in the present work. The gametophyte of Lophosoria quadripinnata was spatulate after 156 days of culture; antheridia were observed after 72 days, but archegonia were seen only after 270-285 days (Pérez-Garcia et al., 1995). Additional analyses are necessary to show how sexual expression of D. sellowiana is affected by spore density in laboratory conditions. In Santa Catarina State (Brazil) there is a predominance of clay soils, cambisoils, laterites and nitosoils (IBGE, 2005). In the experimental conditions carried out in this study, the typic hapludult soil (distroferric red nitosoil) with the addition of termophilic organic compost was the most suitable for Dicksonia sellowiana development, and the first sporophytes in this substrate were observed 84 days after spore sowing. Atlantic Forest soils are poor in nutrients and the accumulation of chemical elements in cells is one of the ways tropical species tolerate low-nutrient soil (IBGE 2004; Fundagao Biodiversitas, 2006). Litterfall is a fundamental component of nutrient cycling, and it is the main means of transferring organic matter and mineral elements back to the soil surface (De Franca et al., 2007; Moraes et al., 1999). The termophilic organic compost was added in order to simulate the litter in this substrate. This substrate has a low pH and high levels of N, P, K and Ca. In contrast, when Borelli et al. (1990) cultivated gametophytes of Dicksonia sellowiana in the soil made of ‘txaxim” trunks, the first sporophytes were observed only after six months of calivaoe and the authors observed 100% contamination in their cultures and around 75% of gametophytes produced sporophytes. Therefore, the time to sporophyte formation, the percentage of sporophyte formation, and the prevention of contamination were improved in the present work. Edaphic parameters were analyzed for several fern species to elucidate their habitats including nutritional requirements and the majority of them prefer acidic soils, as does D. sellowiana (Carlson, 1979; Graves and Monk, 1982; Whitter and Moyroud, 1993; Ranal, 1995). The time to sporophyte emergence is quite variable among fern species; Lophosoria quadripinnata formed sporophytes after 36 months cultivation (Pérez-Garcia et al., 1995). The typic hapludult soil with added termophilic organic compost employed in this work was also useful for sporophyte emergence, which was observed less than three months after cultivation as observed in previous work (Suzuki et al., 2005). Information provided in this paper certainly will be useful for D. sellowiana management in green houses as part of conservation strategies. Plantlets of Dicksonia sellowiana can be easily obtained following the protocol presented here. This methodology can be used for the establishment of germplasm banks with the purpose of preserving the species in botanical gardens and to maintain its genetic variability. ACKNOWLEDGMENTS Cldudia Cristina Leite Fiori thanks CAPES (Council of Improvement of University Education Staff - Brazil) for her grant. We thank Dr. Paulo Emilio Lovato (UFSC) for suggestions and for FIORI ET AL.: GAMETOPHYTE DEVELOPMENT OF DICKSONIA SELLOWIANA 215 supplying the organic compost. Aurea Maria Randi thanks a (National Council of Scientific and Technological Development-Brazil) for the research gran LITERATURE CITED ours F. P., C. E. F. Castro, L. A. F. Marttuss, A. F. C. piece and V. Nacal. 1990. In vitro and vivo ——- of fern spores. Bra agantia 49:205—21 Poceolk. J. J. and I. D. Russe... 1991. Electron Microscopy. Piatti and techniques for biologists. Jones he as Boston, USA. Carson, T. 79. The comparative shee and frequencies of interspecific hybridization of Michigen lee ferns. Mich. Bot. 18 De Franca, E. J., E. A. De Napa FERNA NDES, gry x Baccut and C. Ettas. patie: bieodeos Forest: A natural reservoir of chemical elements. J. Radi al Nucl. Chem. 276: Dyer, A. F. 1979. The iets biology of ferns. Academic, New York, is FERNANDEZ, H., A. M. BERTRAND and R. SANCHEZ-TAMES. 1996. Influence of tissue Heagcags conditions on apogamy in : Deyoptere affinis sp. affinis, Plant sin Tissue Org. Cult. 45:93-97. FERNANDEZ, H., A. M. Bertranp and R. SANCHEZ-TAMES. 1999. pier and ingriale aspects invo ved | in bigs multiplication. Plant Cell Tissue Org. Cult. 56:211-214 FILIPPINI, E. C. P., S. R. Duz and A. M. Rano. 1999. Light and storage on oe germination of spores of 2:21—26, FunpacAo Biopiversiras. 2006. Espécies ameacadas on line. Available in http://www. biodiversitas. org.br/boletim snEADAUNEO/in dex.h Granapos, B., B. Pérez-Garcfa and A A Manesk . 2003. Fase sexual de los helechos Odontosoria schlocitendai y Odontosoria meander a (Ceuiste adtdacenat. Rev. Biol. Trop. 51:675-682 Graves, J. H. and C. D. Monk. 1982. Herb-soil relationships on a lower north store over marble. Bull. Torey Bot.Club. 109:500-507. Huan, M. Y., H. M. CHouanp and W. L. Cutou. 2004. isity aff h th and ] eng of Osmunda cinnamomea HRvcedetoert Ptoridophyta) Ann. ‘Bot. 94:229-232. IBGE. 2005. Mapas interativos: mapas de solos. Available in: http://mapas.ibge.gov.br/solos/ viewer.htm IBGE. ae. Aras interativos: mapas de biomas. Available in: http://mapas.ibge.gov.br/biomas2/ viewer. IBAMA. 1997. Foster Workshop sobre Conversagdo e Manejo de Dicksonia sellowiana (Xaxim). Urubici, Santa Catarina, Brazil MeENnboza, A., B. -Garcia and I. R. JaraMILLo. 1997. Morfogénesis de la fase acon - a Lophosoria quadripinnata var contracta (Lophosoriaceae). Rev. Biol. Trop 45 Miter, J. H. 1968. Fern gametophytes as Scie material. Bot. Rev. 34:36 1-44 a Morass, R. M., W. B. C. Deurrrt and Y. Strurraupi-De Vuono. neta — and litter nutrient content in two Brazilian sags rt aloerts ak Bras. Bot. 2 Nayar, B. K. and S. Kaur 9. Types of prothallial pear iieas in homosporous ferns. Phytomorphology 19: eae Nayar, B. K. and S. Kaur. 1971. Gametophytes of homosporous ferns. Bot. Rev. 37:295-396, Pi-Cascis, B. and M. E. Fray.e. 1986. El gametofito de Dicksonia sellowiana (Presl.) Hooker. Pérez-Garcta, B., M. E. Frayte and A. MENponza. 1995. Desarrollo ee -ongeaicas de Lophosoria quadripinnata — Lophosoriaceae). Rev. Biol. Trop. 43: . A, 1995. RANAL, Estabelecimento de pteridéfitas em mata soi pa OS do Estado de Sao Paulo. 2. Natureza = Substratos. Rev. Brasil. Biol. 55:583-594. siiesien hed ss . and A AM. Ron 2004. Effects of lscaah nse osne iradiance on germination and early Acta Bot. Brac 18:375-380. Rocce, G. D., A. M. Viana and A. M. Rano. 2000. Cryopreservation of spores of Dicksonia seats An endangered tree fern indigenous to South and Central America. CryoLetters 30. 216 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Suzoxt, G. C: LF, M: T. PAULILO and A. M. Ranopi. 2005. Substrate and irradiance affect the iid owth of the g tropical tree fern Dicksonia sellowiana Hook. (Dicksoniaceae). Am Fern J. 95:115—-125. SEHNEM, A. 1978. Ciatedceas. Flora Ilustrada Catarinense. Herbario Barbosa Rodrigues, Itajaf, Brazil. Tryon, R. M. 1970. Development and evolution of ferns floras of Oceanic Islands. Biotropica Tryon, R. M. 1972. Endemic areas and geographic speciation in Tropical American ferns. omits ; 121-1 Tryon, R. M. and A. F. TRYON. 1982. Fern and allied plants with special reference to Tropical America. Springer-Verlag, New his , USA Wnirter, D. P. and R. Moyroup. 1993. The a of spore germination and gametophyte dev velopment in Ophioglossum se ee by low J. 83:41-46 Wwoiscu, P. G. 2002. Fern conservation in Brazil. Fern ats 16:295—300. American Fern Journal 99(3):217—225 (2009) In vitro Study on Gametophyte Development of an Epiphytic Fern, Arthromeris himalayensis (Hook.) Ching, of South Sikkim, India GAUTAM GANGULY, KAUSHIK SARKAR, and RADHANATH MUKHOPADHYAY* Pteridology Research Laboratory, CAS in Botany, University of Burdwan, Golapbag-713104, West Bengal, India AssTrAcT.—Gametophyte development in sip cng § himalayensis was studied and found to be of “Drynaria type’. Germination occurred 9—10 Some prothalli showed an initial archegonial phase, which persisted throughout gametophyte development and the antheridial phase developed on separate thalli a few days later and persisted throughout the life span of the gametophytes. This type of development of sex organs may be considered as a variant new type from pain 2 reported types by earlier authors. This variant type is described here a HY s type Damnit development on separate prothalli is an indication of adaptation for ou breedin: Key Worps.—Gametophyte development, Arthromeris himalayensis, Epiphyte, Type H, out- breeding Arthromeris himalayensis (Hook.) Ching belongs to the family Polypodia- ceae and is a warm temperate fern, exclusively epiphytic in nature and distributed in India throughout the Himalayan region from Eastern Himalayas to Western Himalayas. This epiphytic fern is also found in China, Nepal and Burma in mountain areas. In Southern Sikkim this fern is generally found between 2700-3600 Germination of fern spores, growth, and further development of resulting gametophytes in artificial media is a well-studied area in pteridophyte and developmental biological research (Nayar, 1962; Atkinson and Stokey, 1964; Kato, 1969; Klekowski, 1969; Nayar and Kaur, 1969; Nayar and Kaur, 1971; Masuyama, 1975a, b; Khare and Kaur, 1983; Raghavan, 1989; Chiou and Farrar, 1997; Verma et al., 2000; Verma, 2003; Ganguly and Mukhopadhyay, 2005). Nayar and Kaur (1971) and Atkinson (1973) pointed out that the sequence and plane of cell divisions, pattern of gametophyte development, as well as the direction of initial growth of the first rhizoid and germ filament with respect to the polarity of germinating spore are distinct characteristics and can be utilized effectively for drawing phylogenetic relationships among various taxonomic groups. Nayar (1962) studied the spore germination and prothallial morphology of Arthromeris wallichiana (Sprengel) Ching along with some other polypodiaceous ferns. However, no work has been done on the prothallial development of the epiphytic fern Arthromeris himalayensis. Ferns *Corresponding author: Email: rmm13351@yahoo.com 218 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) occupy a specialized habitat as epiphytes and, as such, epiphytic ferns have evolved various gametophytic generation adapations like antheridiogen systems, production of gemmae, indefinite growth of prothalli, etc. (Farrar, 1974, 2003). Among the 15 species of Arthromeris distributed throughout the world, the majority (10 species) are found in the Eastern Himalayas (Ghosh et al., 2004). Out of these 10 species, Arthromeris himalayensis grows very successfully in the highest altitudes as epiphytes and has high antimicrobial activity (Ganguly et al., 2008). These interesting attributes prompted us to study its reproductive behavior and gametophyte development. The current study was performed to understand the details of gametophyte structure and development pattern of prothalli in Arthromeris himalayensis. MATERIALS AND METHODS Mature sporophylls of Arthromeris himalayensis were collected from healthy plants from Maenum Wildlife Sanctuary (3023 m altitude), South Sikkim in the November of 2005 and 2006. Sporophylls of individual plants were kept separately within blotting paper and mature spores from sporophyll(s) of individual plants dehisced within 48 hrs were collected in separate vials for gametophyte studies. Collected spores from three individual sporophytes were sown in separate petri plates on modified Moore’s medium (Kato, 1969). Two replicates of each set were maintained. These spores were surface sterilized by 0.1% HgCl, (w/v) solution for 5-8 minutes and rinsed three times with sterilized distilled water and then dried on sterilized blotting paper. The sterilized spores were transferred to autoclaved (at 15 lb/inch? for 15 minutes) modified Moore’s culture medium (Kato, 1969) solidified by 1% (w/v) agar in an aseptic chamber and the pH of the medium was maintained at 5.8. The cultures were incubated at 22°C—25°C under cool fluorescent white light (ca 1000 lux, 16hr/d). Gametophytes from each petri plate were studied every day randomly by light microscope (Leica DMLB) after germination of spores. Time taken for spore germination and to form mature gametophytes, initiation of sex organs and formation of sporophytes were recorded. Camera Lucida drawings of different developmental stages were made on the same microscope. Records of gametophyte development patterns from individual sporophytes were main- tained separately in order to see if there were variations in the developmental patterns among the individuals. Observations were made on ten gametophytes from each petri plate at a time. RESULTS Characteristics of spore germination and gametophyte development.— Spores were bilateral, monolete; light brown in color, perisporate, perispore thin, size 38-42 X 50-53 um. Spore germination of Arthromeris himalayensis was 79.49 + 4.66%. Spores germinated even after having been stored for two months after collection. GANGULY ET AL.: GAMETOPHYTE DEVELOPMENT IN ARTHROMERIS HIMALAYENSIS 219 Spores germinated 9-10 days after sowing. In this species the rhizoidal cell formed first (Fig. 1A) followed by the chlorophyllous protonemal cell (Fig. 1B). The protonemal cell developed into a 6-celled stage by periclinal divisions (Fig. 1C-E). Spore germination resulted in a slender uniseriate germ filament. The penultimate protonemal cell underwent oblique vertical division. In Arthromeris himalayensis the establishment of an apical meristem was much delayed and the prothalli usually developed hairs on the margin and surfaces. A broad spathulate prothalli plate was formed by repeated longitudinal and transverse divisions of its anterior cells and expansion of the resultant daughter cells (Fig. 1F—K). Mature vegetative, cordate shaped gametophytes (Fig. 1M) developed 77-80 days after spore germination. The mature prothalli measured ca 350 x 300 um in size. The prothallial plate often became 15-20 cells or more wide and broadly ovate, but was devoid of any organized meristem. Later, an obconical meristematic cell was differentiated by two oblique divisions in one of the marginal cells at the anterior end of the prothallial plate (Fig. 1K). The meristematic region (Fig. 1L-M) was located under notch. The type of development was purely “Drynaria type” as discussed by Nayar and Kaur (1969, 1971). Development of sex organs (sequence, position and duration).—Mature cordate gametophytes remained vegetative for about 30 days, after which the gametophytes started to develop sex organs. Archegonia developed first in some cordate shaped prothalli 112 + 2 days after spore germination. Archegonia were situated along the midrib region and just below the meristematic region. Archegonia consisted of a projecting neck (Fig. 1M) and a lower embedded venter. This flask shaped structure was made up of two axial rows of neck canal cells, one ventral canal cell and one egg cell (Fig. 1N). Each archegonium had a single layered jacket (Fig. 1N) and was 150-200 x 65—75ym in size. Antheridia developed on separate gametophytic prothalli, which were elongated and much longer than archegonial prothalli. Initiation of antheridia started 115 + 2 days after spore germination. Antheridia were of the emergent type (Fig. 10) with a 1-cell thick jacket, measuring about 25— 30um. In Arthromeris himalayensis, the prothalli were dioecious. The cordate shaped prothalli developed archegonia after they reached maturity and remained as archegoniate prothalli throughout the reproductive phase. Antheridiate prothalli were elongated and did not form well-defined apical meristem. Antheridia developed on the lower half of the prothallus, marginal and/or superficial in position (Fig. 1L). After initiation, antheridia took about 3-5 days to mature; spermatozoids were released after this period. The time taken for development of the different gametophytic stages of Arthromeris himalayensis is shown in Table 1. Discussion From the above observations, we can conclude that the type of gametophyte development in Arthromeris himalayensis is purely ‘“Drynaria type’. In 220 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) OO Fig. I, K-M, O Kx O Fig. A-G, J,N antheridiate prothallus. M. Development of archegonia on mature cordate prothallus. N. A mature archegonium. O. A mature antheridium. NB:[h = Hair ; m = Obconical meristematic cell ; r = Rhizoid ; s = Spore coat ; a = Antheridium ; ar = archegonium]. GANGULY ET AL.: GAMETOPHYTE DEVELOPMENT IN ARTHROMERIS HIMALAYENSIS 221 TasLE 1. Time taken for gametophyte development of Arthromeris himalyensis (Hook.) Ching. Total no. of _ taken ee sowing Sl.No. Events of the gametophyte development spore + 1, Sowing of spores 02:6 2. a germination 10> 1 s rmation of mature cage prothallus 80.3 4, fnifietion of archego 112 22 5. Initiation of scahaace 1s 273 6. Maturation of antheridia 118235 7 Initiation of sporophyte 123 5 2 ‘““Drynaria type’ development, spore germination results in a slender uniseriate germ filament. A broad spathulate prothallial plate is formed by repeated longitudinal and transverse divisions of its anterior cell and expansion of the resultant daughter cells. The prothallial plate often becomes 5-10 cells or more wide and broadly ovate, but is devoid of any organized meristem. Later, an obconical meristematic cell is differentiated by two oblique divisions on one of the marginal cells at the anterior end of the prothallial TaBLe 2. Classification of gametangial sequence on meristematic prothalli of homosporous ferns (adapted from Verma 1989, 2003). Sequential bearing of gametangia on meristematic prothalli Type Initial state Final state Symbol A Antheridiate Archegoniate. M—F Archegoniate _Persists Seahout FSF B Antheridiate Antheridia and Archegonia sensi M—>H Archegoniate Antheridia and archegonia formati F+H G Antheridiate | Antheridia and archegonia ect forsome M—>H-—F time, then only archegonia formation F-+H-F Archegoniate Same D Antheridiate § Antheridia and archegonia formation forsome MoHOFOH time, alternating periodicity in the formation FOHOMeH idia and archegonia, finally hermaphrodite. Archegoniate Same E Antheridiate | Archegonia formatio M—F Archegoniate Antheridia and mereen! formation. F+H F Archegoniate Antheridia and archegonia formation F—+H-—-MorF+M (ephemeral), then antheridia formation. G Archegoniate Antheridia and archegonia formation FH simultaneously H* Archegoniate _Persists throughout. F—F Antheridiate _Persists throughout. M—M Symbols indicate the sequential state of functional sex: M = Antheridia formation = Archegonia formation, H = Hermaphrodite. Types A, B and C are according to Masuyama (1975 b). Type D, E and F are proposed by Verma (1989). Type G is proposed by a & Mukhopadhyay (2005). Type H* is a new variant type proposed here by current authors 222 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) plate. The young prothallus becomes cordate, the apical meristematic cell is replaced by a pluricellular meristem and a midrib developed. Young prothalli are naked; hairs are usually formed when the prothallial plate becomes cordate (Nayar and Kaur, 1969). The Drynaria type of development is characteristic of Cheiropleuriaceae, Dipteridaceae, Gleicheniaceae, Lomariopsidaceae, Loxo- maceae, Thelypteridaceae and the majority of the Polypodiaceae genera (Nayar and Kaur, 1969). Smith et al. (2006) did not consider Cheiropleuriaceae a separate family; they merged the genus Cheiropleuria in the family Dipteridaceae. According to Masuyama’s (1975a, b) classification of gameto- phytes, based on gametangial sequence of development on meristematic prothalli, which was further elaborated upon by Verma (1989, 2003) and Ganguly and Mukhopadhyay (2005), the gametophytes of Arthromeris himalayensis resemble ‘type A’ to some extent. In type A, the archegoniate prothalli persist throughout development but the antheridiate prothalli become archegoniate in the later stages. In Arthromeris himalayensis, the sequence of development of the sex organs is different. Here, the archegoniate prothalli remain archegoniate and the antheridiate prothalli remain anther- idiate throughout development. Based on the sequence of sex organ development in Arthromeris himalayensis, it is identified as a new type, different from the types described by Masuyama (1975a, b), Verma (2003), and Ganguly and Mukhopadhyay (2005). Thus, we propose a new type “Type H”’ in addition to the existing seven types classified by the previous authors (Table 2). Gametophyte growth habit can be classified into three basic types in regard to the effect of form on breeding system. Type I is the familiar cordate or butterfly shaped gametophytes of most terrestrial ferns. Type II gametophytes have indeterminate growth and branching and type III gametophytes combine type II growth with production of dispersible gemmae. Type II and type III gametophytes are typical of most epiphytic species (Farrar, 2003). In Arthromeris himalayensis, the archegoniate prothalli resemble type I, which is cordate shaped. The antheridiate prothallus was elongated, having indefinite growth. Some of the gametophytes showed clonal elongation. The secondary gametophytes produced antheridia on their margins. Thus, antheridiate prothalli resemble type II gametophytes partially. The gametophytes of Arthromeris himalayensis are long lived (more than 110 days), and the advantage of long-lived gametophytic generation is to promote cross-fertilization (Klekowski, 1973, 1979). Opportunities for gamete exchange between long-lived gametophytes are much higher than for short- lived, non-clonal epiphytic gametophytes. Most species of Polypodiaceae maintain an antheridiogen system through which the robustly growing female gametophytes induce production of antheridia precociously on the smaller gametophytes growing nearby, thus enhancing the probability of cross-fertilization (Chiou and Farrar, 1997). Arthromeris himalayensis may have an antheridiogen system, as antheridia grow on separate prothalli after 3-5 days of initiation of archegonia in cordate GANGULY ET AL.: GAMETOPHYTE DEVELOPMENT IN ARTHROMERIS HIMALAYENSIS 223 shaped prothalli. This suggests that antheridiogen might have some role in controlling the reproductive system of A. himalayensis. Masuyama (1975b) recognized four basic locations of antheridia on monoecious prothalli: antheridia on the lower part of gametophyte thallus (type L), on the lower half of the wings (LW), on the lower half of the margin (type LM), on the upper half of the central cushion (type UC), or antheridia located all along the margin (type M). As Arthromeris himalayensis produces dioecious prothalli, it does not resemble any type as recognized by Masuyama (1975b), though the antheridia located on the lower half of the wings (type LW) as proposed by Masuyama (1975b). It is interesting to note that the percentage of spore germination is very high; about 79.49 + 4.66% even after two months of harvesting. This figure indicates that this species produces a high proportion of viable spores, which is likely helpful in the survival of this species. This species is restricted to a certain altitudinal regions (2700-3600 m), thus specific environmental conditions like temperature, annual rainfall, relative humidity (RH), etc. are required for its survival. RH, annual rainfall and altitude have a combined effect on the distribution and reproductive success of this species (Ganguly and Mukho- padhyay, 2008). Most homosporous fern gametophytes are potentially bisexual and due to continual self-fertilization there is a risk of exposing the lethal genes in homozygous condition. The gametophytic generation has evolved some adaptations to overcome this problem that influence the change of the mating system from intragametophytic to intergametopytic. These adaptations include the gender of the gametophytes, ecology, distribution and duration of gametangia on monoecious prothalli, and longevity of gametophytes and the capacity for vegetative reproduction (Klekowski, 1969; Lloyd, 1974a, b; Masuyama 1975a, b; Soltis and Soltis, 1987). From the above discussions, it may be concluded the Arthromeris himalayensis gametophytic generation shows some derived developmental features: 1) dioecious prothalli promotes intergametophytic fertilization (may be of sibling and/or non-sibling mating); 2) archegoniate prothalli that are meristematic and cordate shaped, continu- ously producing archegonia, increase the chances of sporophyte production; and 3) long-lived gametophytes (more than 110 days) that also promote intergametophytic fertilization. ACKNOWLEDGMENTS The authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi for financial assistance and to the Govt. of Sikkim for extending facilities. The authors are grateful to Dr. Jennifer Geiger, Chief Editor of American Fern Journal for kindly reviewing the manuscript and for constructive suggestions which has helped largely to improve the paper. LITERATURE CITED Arxinson, L. R. and A. G. Stoxey. 1964. Comparative morphology of gametophyte of the homosporous ferns. Phytomorphology 14:51-70. 224 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) Arkinson, L. R. 1973. The ecieatiag Si and family relationships. Bot. J. Linn. Soc. 67:73-90. Cuiou, W. and D. R. Farrar. 19 Gas spies gametophyte morphology of selected species of family piper poe Fern J. 87:77—-86. Farrar, D. F. 1974. Gemmiferous fern gametophytes-Vittariaceae. Amer. J. Bot. 61:146—155. Farrar, D. F. AOS Gametophyte morphology and breeding systems in ferns. Pp. 447-454, in Pteridology in the New Millennium, Kluwer Acad. Publ. Netherlands. eee G. and R. Muxuopapuyay. 2005. In vitro study on a development of Hypolepis pina (Bl.) Hook. sg cient 55(3&4):179-18 cmon "s = . Mukuopapuyay. 2008. Studies on ae diversity and pattern of vertical piphytic apsngp tndamy on their host plants of Southern Sikkim, India. Proc. Natl. Acad. Sci. . 78(2):43 GANGULY, iS K. Sarkar, S. MUKHERJEE, A. BHATTACHARJEE and R. MukHopaADHyAy. 2008. Phytochemistry and Satimmiciohial activity of an epiphytic fern Arthromeris himalayensis Hook) | Ching Pp. 114-115. International Symposium on phase in Pteridophytes. a : a2 : ee A. Biswas and R. K. Guosu. 2004. The Pteridophytic Flora of Eastern India vol. I, Botanical Survey of India. Kato, Y. 1969. Physiology and Morphogenetic studies of fern gametophytes in aseptic culture II experimental modifications of dimensional growth in gametophytes of Pteris vittata L. hytomorphology 19:114—121. Kuare, P. B. and S. Kaur. 1983. Gametophyte differentiation of pentaploid Pteris vittata L. Proc. Natl. Sc ci. Acad. B. 49 740-742. KLEKowskKI, E. : Jr. 1969. Reproductive biology of the Pteridophyta, II. Theoritical considerations. Bot. J. Linn. Soc. London 6 KLEekowski, E. 1 Jr. 1973. Soca and subsexual systems in the homosporous ferns: A new hypothesis. Amer. J. Bot. 60:535-544. Kuexowskl, E. J., Jk. 1979. The cheat and reproductive biology of ferns. Pp. 133-170, in The erator penne of Ferns, Academic Press: London, New Yor Lioyp, R. a. Mating systems and genetic load in pioneer ‘att non-pioneer Hawaiian Baan othe Bot. J. Linn. Soc. London 69:23-25. seus a - _— Reproductive biology and evolution in the Pteridophyta. Ann. Mo. Bot. Gard. ae. i. wee Th formation in t fi I I pattern and their Sabi = effect on “the fertilization with ‘special reference to the ametophytes of Athyrium. Sci. Rep. Tokyo Kyoiku Daigaku Sec. B. ae 0):4 . 1975 16(241):71-8 Nayar, B. K. 1962. Morphology of spores and prothalli of peci f Polypodi Botanical Gazette 123:223-2 Nayar, B. K. and S. sh 1969. Types of prothallial development in homosporous ferns. nie epeh ph e 19:179-188. Nayar, B. K. and S. Kaur. 1971. Gametophytes of homosporous ferns. Bot. Rev. 37:295—396. RAGHAVAN, vV. 1989. fede Biology of Fern Gametophytes, Cambridge Univ. Press: Cambridge. SmitrH, A. R., K. M. Pryer, E. Scuuerrretz, P. Korat, H. Scunemer and P. G. Wotr. 2006. Classification for eciat ferns. Taxon 55(3):705-731. Soxtis, D. E. and P. S. Sottis. 1987. Nieipe system of the fern Dryopteris expansa: evidence for mixed mating. Amer. J. Bot. 7 Tryon, A. F. and B. LuGarpon. se pes - the srionuanan a Verma, S. C. 1989. Overt and covert mechanisms of intergametophytic mating in homosparou bene. Pp. 285-300, in Plant Science Research in india, Today and Tomorrow’s Printer: Publishers: New Delhi. GANGULY ET AL.: GAMETOPHYTE DEVELOPMENT IN ARTHROMERIS HIMALA YENSIS 225 Verma, S. C. 2003. Some aspects of reproductive biology of gametophyte generation of homosporous ferns. Pp. 455-484, in Pteridology in New Millennium, Kluwer Academic Publishers: Netherland. Verma, S. C., A. Kaur and P. M. Setvan. 2000. Experimental studies on the gametophyte generation of homosporous ferns-III sexuality, gametangial sequence and mating system in some species of Pteris. Indian Fern J. 17:136—174 American Fern Journal 99(3):226—230 (2009) An Efficient Method for Surface Sterilization and Sowing Fern Spores in vitro Wu Hua, CHEN Pinc-Tinc, YUAN Li- Pinc, and CHEN Lonc-Qinc* Key paeraat of Horticultural Plant Biology (Ministry of Education), College of sietacrens and estry Science, Huazhong Agricultural On penis Wuhan 430070, P.R. Chin ABSTRACT. Tag sie are A ecamsuced used to start in vitro culture of ferns. Numerous methods for nd sowing have been developed, but spore loss and contamination are still ‘problediatle To overcome these problems, an efficient method for sterilizing and sowing spores was established. Through this method, contamination and loss of spores is minimized, and can be sown in adjustable, even densities. Key Worps.—surface sterilization, in vitro culture, spore sowing Surface sterilization is the first step for aseptic culture of ferns from spores (Dyer, 1979). Since tissue culture is widely employed as a technique for fern propagation or scientific studies, spores are widely used as a starting material. In recent years, there have been numerous studies on in vitro culture of ferns from spores (Stone, 1958; Yoroi, 1972; Kiss and Kiss, 1998; Cox et al., 2003). These researchers reported successful cultures, but admitted significant losses of spores during the sterilization process, aseptic sowing, or due to contamination (Warne et al., 1986). Because of these problems, different methods of spore sterilization and sowing have been developed (Dyer, 1979). Many of these as ape are still inefficient in terms of time and spore loss (Warne et al., 1986). ecently, we established an effective method for sterilization of spores with a ie funnel. The method is successful with spores of Osmunda japonica Thunb., Aleuritopteris argentea Gmel., Adiantum flabellulatum L., Adiantum capillis-veneris L. and Cyrtomium fortunei J. Smith (data not shown). We compared our method, here called the filter method, with two other widely used methods. With the packet method, spores were sterilized in filter bags/packets (Ford and Fay, 1999), while with the centrifugation method the spores were suspended in a sterilizing solution and then harvested in sterile distilled water by centrifugation (Fernandez et al., 1993). For these comparisons, we used spores of Adiantam reniforme var. sinense Y. X. Lin, a rare and endemic species in China. MATERIALS AND METHODS Plant materials.—Sporophytes of A. reniforme var. sinense were introduced from Wanxian County along the Yangtze River in 2001 and cultivated in the *Corresponding author: chenlq0206@163.com; email address for WH: huawu_61@163.com HUA ET AL.: METHOD OF SPORE SURFACE STERILIZATION AND SOWING 227 greenhouse of Huazhong Agricultural University (Wuhan, China). Fertile fronds of sporophytes in the greenhouse were collected and wrapped in paper bags and dried at room temperature for one week to release spores. Then the spores were collected in centrifuge tubes and stored at 4°C until used. For this study, spores were stored for 10 months. Culture media.—Murashige and Skoog (1962) medium (MS) with 1/4 strength of macronutrients were used for germination of spores. The medium was supplemented with 3% (w/v) of sucrose, solidified with 0.65% (w/v) agar and adjusted to pH 5.8 before autoclaving at 121°C and 1.1 kg cm for 20 min. Spore sterilization and sowing.—The filter method was performed in a laminar flow hood. Three mg spores were suspended and wetted with 4% (v/v) Tween solution for 5 min in a 1.5 ml centrifuge tube. Suspended spores were collected through a filter funnel (made by fast filter paper), which was placed on a proper conical flask. The tube was washed three times with fresh water and the water was poured into the funnel to collect the residual spores of the tube. Seventy percent (v/v) alcohol (chemical purity) was added to immerge the spores along the funnel margin. Thirty seconds later 30 ml fresh sterile distilled water was continually added to rinse the spores. After removing the alcohol, 4% (w/v) sodium hypochlorite (NaCIO) or 0.1% (w/v) mercuric chloride (HgCl,) was added along the funnel margin. For different disinfectants, the sterilizing time was different: NaClO (5-6 min) and HgCl, (2—3min). At the end of the sterilizing time, fresh sterile distilled water was full filled into the funnel along the margin, regardless of whether the HgCl, or NaClO solution had completely drained off or not. The same operations were repeated twice when the dilute solution seeped half from the filter funnel. Then after the solution drained off, spores were thoroughly rinsed six times with sterile distilled water. After the sterile distilled water drained off, the spores were rinsed with 40 ml fresh sterile distilled water from the filter paper into a sterile container. Using a sterile pipette, the spore suspension was distributed onto culture plates (9 cm diameter Petri dishes, containing 20 ml culture medium), which were sealed with plastic film. The sterile distilled water and different solutions were transferred by transfer pipette with sterilized tips (1ml). Ten plates were made for each treatment and the experiment was conducted three times. The waste of the HgCl, solution was collected and adjusted to pH 8-10. Enough Na,S and FeSO, were then added to react with the HgCl, and produce sediments. After sediments formed thoroughly, the sediments were collected and sent to the hazardous waste disposal department for detoxification and proper disposal. All cultures were incubated in a controlled environment room that was maintained at 23+2°C under a light intensity of 25 ul m~2 s~! with 16/8 photoperiod. The spore density of one drop of spore suspension solution from a 1 ml transfer pipette in the filter method and centrifugation method were recorded and means with standard deviations (S.D.) were calculated. In the packet 228 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) method, spores were sown by directly wiping the spores on the medium, thus spore density was not scored. Contamination and spore germination were examined on the 40th day after spore sowing. For germination rates, at least 300 random spores per plate were scored. For comparison to the other two methods, the published methodologies (Fernandez et al., 1993; Ford and Fay, 1999) were followed using 3 mg of spores, and are not described in this paper. Sterilization capacities of different sterilization methods: To test the sterilization capacities of the different methods, comparisons were made between different methods using different weights of spores (0.5g, 1.5g and 3g). The spores were administered into proper centrifuge tubes or packed in proper filter papers. All spores were sown in soil after sterilization. RESULTS In the filter method, spores sterilized by NaClO were obviously bleached and it was difficult to judge whether the spores were rinsed completely from the filter paper or if spores drifted from the tubes when poured out of the disinfecting solutions and rinse water. Table 1 shows that there were no significant differences in spore density between the two disinfectants. However, the spore densities varied greatly between the different methods tested. From the centrifugation method, there were about 69—75 spores /drop, from the filter method, there were about 235—248 spores/drop, and the number of spores not sterilized and suspended in 40 ml water directly was 260-272 spores/drop (Table 1). In the packet method, sowing of spores was achieved by wiping the spores in a swirling motion over the surface of culture medium directly, so the spore density through this method was very high in the first plate and very low in the last plate. Nevertheless, the spores tended to clump together on the first plate. Thus, the spore densities changed greatly from plate to plate and across plates. Within a single plate, the highest density was more than 3000 spores cm’; the lowest density was less than 10 spores cm *. The spores from the filter method and packet method started to germinate 10 days after sowing, while the spores from the centrifugation method started to germinate 15 days after sowing. On the 40th day, the highest germination (62.6%; see Table 1) was obtained from the filter method with HgCl2, which was followed by the filter method with NaClO (60.8%, Table 1). The germination rate of the packet method varied greatly. For example, when the disinfectant was HgCl, it ranged from 5.6% to 67%. At the highest density of about 3000 cm °, germination rate was around 20%. The germination rate in the last plate was 5.6% as there were only 287 spores in the whole plate. Only plates from the packet method were contaminated (Table 1). With increasing spore weight, different methods had different problems. For the packet method, when the spores were more than 0.5 g, removing air HUA ET AL.: METHOD OF SPORE SURFACE STERILIZATION AND SOWING 229 Table 1. Effects of different sterilization methods and disinfectants on spore germination of A. reniforme var. sinense. Sterilization Sterilization Spore Contamination Germination Disinfectant method time (min) number/drop (%) (%)+S.D. Spore color NaClO PM 5-6 36.7 40.1 + 22.9 bleached NaClO FM 5-6 235 + 7.8 ) 60.8 + 8.4 bleached NaClO CM 5-6 69 + 4.6 0 27.4+5.1 bleached HgCl, PM 2-3 as 39:9 = 26.3 unchanged HgCl, FM 2-3 248 + 5.7 0 62.6 + 9.8 unchanged HgCl, CM 2-3 76255 0 23.2 + 3.2 unchanged as “ys “6 ed c Data of spore number/drop were taken after the st ; tami dg were taken after 40 days. PM: packet method; FM: filter method; CM: centrifuge method. bubbles from the packet became very difficult, and some spores could not be wetted and sterilized. For the centrifugation method, when the spores were 0.5 g, 1.5-2 ml centrifuge tubes were proper; when the spores were 1.5-3 g, 1.5—2 ml centrifuge tubes were too small; 5-7 ml centrifuge tubes and a bigger centrifuge were needed. For the filter method, the spores, regardless of density, could be completely sterilized without any modification of the methodology. DISCUSSION When sterilizing spores via the packet method, the spores were kept in the packet during the sterilizing process. However, if the bubbles were not removed completely, the spores did not all come into contact with disinfectant and this caused an increased contamination rate. In addition, the sowing methodology caused the spores to be dispersed unevenly on the culture medium; some plates had spores that were clumped together and some plates had few spores that were spread very far apart. As a result of these discrepancies, the germination rates varied greatly, and confirmed the findings of Ashcroft and Sheffile (2000) that spore germination rate of ferns is inhibited at both high and low densities; proper spore density is important to fern culture. When sterilizing spores via the centrifuge method, the spores tended to run off when pouring out the used disinfectant solution and used sterilized distilled water. Therefore, after the whole sterilization process, few spores remained, although the spores could be sown in an even density. The results of testing sterilization capacity with this method show that when the spore weight exceeded 1.5 g, larger centrifuge tubes and a larger centrifuge were needed. When sterilizing spores via the filter method, the spores were kept in the filter funnel during the whole sterilizing process. Thus, spore loss was minimal. Besides this, the sowing spore density could be adjusted evenly through adjusting the volume of the sterilized water used to rinse off the spores from the filter paper. Given these observations, we conclude that the filter method is an effective way to sterilize spores. It is not only simple and convenient, but can be used to 230 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 3 (2009) sterilize many spores at one time and it minimizes spore loss. It also allows spores to be sown in an even density The results of this study showed that both HgCl, and NaClO were effective disinfectants. Since NaClO bleached the spores, it was difficult to judge whether the spores were rinsed off from the centrifuge tubes and filter papers thoroughly or not. However, HgCl, is not only extremely toxic to spores but also to the environment. Thus, for normal in vitro culture, it is better to use NaClO. HgCl, might be used in cases where the spores are difficult to sterilize or become too bleached. Because of the toxicity of HgCl., it should be handled very carefully, and the waste of HgCl, should be detoxified or sent to a waste disposal department for detoxification. ACKNOWLEDGMENTS he smooth accomplishment and possible achievements of this research are attributable to the support from The National Natural Science Funds of China (NSFC 30771517). LITERATURE CITED ASHCROFT, C. J. and E. SHEFFIELD. 2000. The effect of spore density on germination and development Pteridium, monitored using a novel technique. American Fern Journal 90:91—99. Cox, I. °P. Buatia and N. AsHwatu. 2003. In vitro spore germination of the fern Schizaea dichotoma. Scientia Horticulturae 97:369—37 — P. 1994. In vitro culture of ornamentals. Pp. 561-573, In: Plant Cell and Tissue Culture, asil, I. K. and Thorpe, T. A., eds, Kluwer Academic Publishers, The Netherlands. Dyer, 7 F. 2003. The culture of fern gametophytes for experimental investigation. Pp. 253-305, In: Dyer, A. F. (ed.). The Expecimental Biology of Vidion pent: Press, London Foro, M. V. and M. F. Fay. 1999. Spore-d thod of propagation Pp. 159-168, In: Hall, R. D. (ed.). Plant cell culture protocols, 1st edn. Surrey, U Kiss, H. G, and J. Z. Kiss. 1998. Spore germination in populations of Schizaea pusilla from New Jersey and Nova Scotia. International Journal of Plant Science 159:848-852. Stone, I. G. 1958. The gametophyte and embryo of php ayer venosum (R. Br.) Copeland (Hymenophyllacees). Australian Journal of Botany 6:183-20 _— T. R., G. L, Waker and L. G. Hickox. 1986. . novel ee for surface-sterilizing and wing fern spores. American Fern Soumial 76:187—188. os R 1972. Studies on spore germination and BasSe of Japanese Hymenophyllaceae. Science Reports of Tokyo Kyoiku Daigaku 15:81—11 INFORMATION FOR AUTHORS Authors are encouraged to submit manuscripts pertinent to pteridology for publica- tion in the American Fern Journal. Manuscripts should be sent to the managing editor at amerfernj@hotmail.com. Acceptance of papers for publication depends on merit as judged by two or more referees. Authors are encouraged to contribute toward publishing costs; however, the payment or non-payment of page charges will affect neither the accept- ability of manuscripts nor the date of publication. Authors should adhere to the following guidelines; manuscripts not so prepared may be returned for revision prior to review. 1; how- ever if it is necessary to submit hard copy, please submit one copy of the manuscript and include a review copy of illustrations and originals of illustrations. After — please submit final versions of manuscripts via FTP (contact email, or on diskette or CD ROM (see below for figure formatting). If cidaseiting hard copies, use standard 8.5 by 11 inch paper of good quality, not “erasable” paper. Double- space manuscripts throughout, including title, author’s names and full addresses, a short, informative abstract, key words, text (including heads and keys), literature cited, tables (separate from text), and figure captions (grouped as consecutive paragraphs separate from figures). Arrange parts of manuscript in order just given. Include author’s name and page number in the upper right corner of every sheet, and provide an abbreviated running title. Provide margins of at least one inch (25 mm) all around on typed pages. Do not submit right-justified text, avoid footnotes, and do not break words at end of lines. Make table headings and figure captions self-explanatory. Use S.I. (metric) units for all measures (e.g., distance, elevation, weight) unless quoted or cited from another source (e.g., specimen citations). For nomenclatural matters (i.e., synonymy and typification), use one paragraph per basionym (see Regnum Veg. 58:39-40. 1968). Abbreviate titles according to Botanico- Periodicum-Huntianum (Lawrence ef al., 1968, Hunt Botanical Library, Pittsburgh) and its supplement (1991). References cited only as part of nomenclatural matter are not included in literature cited. For shorter notes and reviews, omit the abstract and put all references parenthetically in text. Use Index Herbariorum (Regnum Veg. 120:1-693. 1990; or http:// websun.nybg.org/bsci/ih/) for designations of herbaria. For more detailed instructions on manuscript preparation, see http://amerfernsoc.or Illustrations should be proportioned to fit page width (5 inches or 12.5 cm) with caption ultimately to be included on the same page. Halftone and color images should be scanned at a minimum of 300 pixels per inch (ppi). Line art should be scanned at 1200 ppi when- ever possible. Please note that nearly all images that are downloaded from the Internet or that are in JPEG or GIF format will be 72 dpi and not acceptable for the printing process. Indicate the file format of the graphics. Please submit image files in TIFF (preferable) or EPS format. Provide margins of at least 25 mm on all illustrations. For continuous-tone illustrations, design originals for reproduction without reduction or by uniform amount In composite blocks, abut edges of adjacent photographs. Avoid combining continuous- tone and line-copy in single illustrations or blocks. Coordinate sequence and numbering of figures (and tables), with order of citation in text. Explain scales and symbols in figures themselves, not in captions. Include a scale and reference to latitude and longitude in each ee, ford are sent to authors by the printer. Authors should ‘send proof corrections of comrected Proofs be the editor and reprint orders to the printer. Authors after type has been set. For other matters of form or style, consult recent issues of the American Fern Jour- nal and The Chicago Manual of Style, 14" ed. (1993, Univ. of Chicago Press, Chicago). Occasionally, departure from these guidelines may be justified. Authors are sisson is to consult the editor for assistance with any aspect of ae preparation. Papers of longer than 32 pri ges may it to the Editor of Preridologia(memoir Editor, see ered cover page 2). PTERIDOLOGIA ISSUES IN PRINT The following issues of Pteridologia, the memoir series of the American Fern Society, are available for purchase: 1. Wagner, David H. 1979. Systematics of Polystichum in Western North America North of Mexico. 64 pp. $10.00 plus postage and handling. 2A. Lellinger, David B. 1989. The Ferns and Fern-allies of Costa Rica, Panama, and the Choc6 (Part 1: Psilotaceae through Dicksoniaceae). 364 pp. $32.00 plus postage and handling. 3. Lellinger, David B. 2002. A Modern Multilingual Glossary for Taxonomic Pteri- dology. 263 pp. $28.00 plus postage and handling. For orders and more information, please contact our authorized agent for sales at: Missouri Botanical Garden Press, P.O. Box 299, St. Louis, MO 63166-0299, tel. 314-577- 9534 or 877-271-1930 (toll free). For online orders, visit: http://www.mbg org I AMERICAN FERN JOURNAL ON MICROFICHE Volumes 1-61 of the American Fern Journal are available as archival quality, silver positive microfiches. Single volumes or the entire run may be purchased. The fiches are easily read with 10x or greater magnification (using a dissecting microscope and transmit- ted illumination or a fiche reader). Silver negative microfiches of vols. 1-50 are also avail- able. The price is $4.00 per volume or $244.00 per set of 61 volumes, postpaid. Send your inquiry or order with a check or money order to: American Fern Society, Inc., % Ecology III, 804 Salem Blvd., Berwick, PA, 18603-9801. FIDDLEHEAD FORUM The editor of the Bulletin of the American Fern Society welcomes contributions from members and non-members, including miscellaneous notes, offers to exchange or purchase materials, personalia, horticultural notes, and reviews of non-technical books on ferns. SPORE EXCHANGE Ms. Denia Mandt, 12616 Ibbetson Ave., Downey, CA 90242-5050, is Director. Spores exchanged and lists of available spores sent on request. http://amerfernsoc.org/sporexy. GIFTS AND BEQUESTS - Pes vs c oe Bs = At th acc Gifts and bequests to the Socie iety enable it t t others interested in ferns. Back issues of the Journal and cash or ee pits are always welcomed and are tax-deductible. Inquiries should be addressed to the Membership Secretary. VISIT THE AMERICAN FERN SOCIETY’S WORLD WIDE WEB HOMEPAGE: http://amerfernsoc.org/ AMERICAN FERN — October-December 2009 JOURNAL QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY Obituary: Alice Faber Tryon (1920-2009) Gerald J. Gastony, David S. Barrington, and David S. Conant Obituary: Prem Kumar Khare (1946-2009) Rama Shankar and R. C. Srivastava Isoétes todaroana (Isoétaceae, Lycopodiophyta), a New Species from Sicily (Italy) Angelo Troia and Francesco M. Raimondo On Neolepisorus emeiensis and N. dengii (Polypodiaceae) from China jao-si Guo and Bin Li Botrychium ascendens W. H. Wagner (Ophioglossaceae) in Newfoundland and Notes on its Origin Peter F. Zika and Donald R. Farrar Spore Maturation and Release of Two a Macaronesian Ferns, Culcita acrocarpa and Woodwardia radicans, along an Altitudinal Gradient Marta L. Arosa, Luis G. Quintanilla, Jaime A. Ramos, Ricardo Ceia, and Hugo Sampaio Nutrient Levels Do Not Affect Male Gametophyte Induction by Antheridiogen in Ceratopteris richardii Asya Ayrapetov and Michael T. Ganger Transplanting Tree Ferns to Promote Their Conservation in Mexico Ana Alice Eleutério and Diego Pérez-Salicrup iati in F from South Ecuador Marcus Lehnert, Ingrid Kottke, Sabrina Setaro, Linda F. Pazmifio, Juan Pablo Sudrez, and Michael Kessler Mycorrhizal 4 Differences In Post-Emergence Growth Of Three Fern Species Could Help Explain Their Varying Local Abundance Kai Riink and Martin Zobel The Function of Trich f Amphibious Fern, M ili q di if hi Tai-Chung Wu and Wen-Yuan Kao SHORTER NOTES Isoetes duriei New to Lebanon Lytton J. Musselman and Mohammad S. Al-Zein Erratum Referees for 2009 Table of Contents for Volume 99 231 236 273 279 292 307 323 The American Fern Society Council for 2009 WARREN D. HAUK, Dept. of Biological Sciences, Denison University, Granville, OH 43023. President MICHAEL WINDHAM, Dept. of Biology, Duke University, P.O. Box 90338, Durham, NC 27708. President-Elect W. CARL TAYLOR, National Science Foundation, 4201 Wilson Blvd., Arlington, VA 22230. Secretary JAMES D. CAPONETTI, Div. of Biology, University of Tennessee, Knoxville, TN 37996-0830. Treasurer GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. Membership Secretary JAMES D. MONTGOMERY, Ecology III, 804 Salem Blvd., Berwick, PA 18603-9801. Curator of Publications JENNIFER M. O. GEIGER, Dept. of Natural Sciences, Carroll College, Helena, MT 59625. Journal Editor R. JAMES HICKEY, Dept. of Botany, Miami University, Oxford, OH 45056. Memoir Editor JOAN N. E. HUDSON, Dept. of Biological Science, Sam Houston State University, Huntsville, TX 77341-2116. DAVID SCHWARTZ, 9715 Christey Way, Bakersfield, CA 93312-5617 Bulletin Editors American Fern Journal EDITOR JENNIFER M. O. GEIGER Dept. of —- Sciences, Carroll College, Helena, MT 59625, h. (406) 447-4461, e-mail: jgeiger@carroll.edu MANAGING eon JILL ANNE DILL Dept. of Natural Sciences, Carroll College, Helena, MT 59625, 447-5176, e-mail: jdill@carroll.edu ASSOCIATE EDITORS Syeaees J. GASTONY Dept. of Biology, Indiana University, Bloomington, IN 47405-6801 GARY KE. GREER oo Biology Dept., Grand Valley State University, Allendale, MI 49401 CHRISTOPHER H. HAUFLER ................... Dept. of Ecology and Evolutionary Biology, cao of Kansas, Lawre 66045-2106 R. JAMES HICKEY Dept. of Botany, Miami Geneuics, prey OH 45056 ROBBIN C. MORAN New York Botanical Garden, Bronx, NY 10458-5126 JAMES E. WATKINS, JR. H d University, Cambridge, MA 02138 The “American Fern Journal” (ISSN 0002-8444) is an illustrated quarterly devoted to the general study of ferns. It is owned by the American Fern Society, and published at The American Fern Society, % Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. Periodicals postage paid at St. Louis, MO, and additio nal en entry. eS 1 f or back issues should be addressed to Dr. James D. “ehesunisig eine Il, 804 Salem Blvd., ear PA 18603 9801. 4 SR A ye s. Lt . \* @™ i +h AA. hk. | 7G ¢ he dues, Back volumes lible for most years as somes issues or on microfiche. Please contact the Back Issues Curator for sii and availability. ubscriptio Society Metiheaha USA, » Canada, Mexico (includes Journal and Fiddlehead Forum) $25 Society Membership other countries (includes Journal and Fidd $32 Soci ife Memt (add $140 mailing surcharge for outside USA, Canada, Mexico) Regular eos - USA, ‘Canada Mexico (includes Fiddlehead Fi $12 All other ies (i des Fiddlehead Fi $19 Institutional Membership — $35 to USA, Canada, Mexico; $45 elsewhere (-$2 agency fee) POSTMASTER: Send address changes to American Fern Journal, Missouri Botanical Garden, P.O. Box 299, St. Louis, MO 63166-0299. American Fern Journal 99(4):231—235 (2009) Obituary: Alice Faber Tryon (1920-2009) GERALD J. GASTONY Department of Biology, Indiana University, Bloomington, IN 47405-7005 Davip S. BARRINGTON Department of Plant Biology, University of Vermont, Burlington, VT 05405-1745 Davin S. CONANT Department of Natural Sciences, Lyndon State College, Lyndon, VT 05851-0919 Alice Faber Tryon became a member of the American Fern Society in 1946 and in 1978 was elected to honorary membership, a special category of membership for those who have made outstanding contributions to the study of ferns. An eminent student of ferns and their spore morphology, she was born Alice Elizabeth Faber in Milwaukee, Wisconsin on August 2, 1920 (according to her sister Jane, she celebrated her birthday on August 1, although her birth certificate reads August 2). She was the second of three children of Arthur H. and Laura Bindrich Faber, and all four of her grandparents had roots in Germany. Known in her family as an ambitious and hardworking woman, Alice was Aunt Fern to her nieces and nephews. Alice graduated from the Milwaukee State Teacher’s College, now the University of Wisconsin at Milwaukee, in 1941. After several years teaching in public schools, she went to the University of Wisconsin at Madison where she met Rolla M. Tryon Jr. and married him on March 16, 1945. This initiated a happy and enduring domestic Photograph by Gerald Gastony at Alice Tryon’s Azalea Trace apartment in 2005. 232 AMERICAN FERN JOURNAL: VOLUME 99.NUMBER 4 (2009) partnership and a research synergism whose productivity has nourished pteridologists throughout the world. Also in 1945, she completed her master’s thesis at Wisconsin, began her doctoral studies there under Rolla’s direction, and moved with him to the University of Minnesota where Rolla served briefly as an Assistant Professor. The couple moved to the Missouri Botanical Garden in St. Louis in 1947 where Alice completed her doctoral degree at Washington University in 1952. Alice’s life work was the study of fern diversity. During her career, she published nearly 50 contributions to the literature on ferns, including three full-length books. Spores have always been prominent in Alice’s work, beginning with her master’s thesis, which addressed the taxonomic utility of spore characters in the spikemoss genus Selaginella. Her doctoral dissertation analyzed the diversity and taxonomy of the New World species of Pellaea, a genus of xerically adapted ferns of the Pteridaceae, a family that remained central to her work during the first half of her career. Her time in St. Louis was followed by a year at the University of California at Berkeley where Rolla was a Research Associate during 1957. In 1958, she and Rolla joined the staff of the Gray Herbarium at Harvard University, where her next major focus was a monograph (1962) of the Andean alpine gymnogrammoid genus Jamesonia. Following this, she monographed the closely related Andean-centered gymnogrammoid genus Eriosorus (1970). To these revisions, she added papers on reproductive biology and biogeography of the Pteridaceae, notably studies of apogamy in Pellaea (1968, 1972), and of incipient heterospory in Platyzoma (1964, 1967). Fern spores were the central focus of Alice’s interests in the second half of her career, resuming an interest in spores first expressed in her master’s research on the spores of Selaginella (1945, 1949). At Harvard, she played a central role in introducing the scanning electron microscope as a research and teaching tool, pioneering its use in the study of fern spores. Prominent among her contributions on spores are her works on evolutionary and ecological trends in spore features (1964, 1973, 1986, 1990), including her study of the specialized spore surfaces of the myrmecophytic ferns (1985). Her book with Bernard Lugardon, Spores of the Pteridophyta (1991), is likely to remain the authoritative reference on spore morphology for decades to come. Alice’s professional and personal history is inextricably tied to that of her husband, Rolla M. Tryon, Jr. (1916-2001). Their jointly published work most notably includes Ferns and Allied Plants with Special Reference to Tropical America (1982), an in-depth survey of fern diversity with emphasis on the New World tropics. This monumental book, containing numerous photographs by Walter H. Hodge, continues to provide many taxonomic hypotheses that are testable by today’s molecular techniques. Together, Alice and Rolla mentored a group of graduate students who have gone on to be prominent in pteridology (see discussion in Gastony et al., 2002). They organized and taught their Fern Biology in Mexico course with Ramon Riba, one of their students, five times between 1971 and 1981. This stimulating opportunity to do science with ferns in the field was a formative experience for all participating students. GASTONY ET AL.: ALICE FABER TRYON 233 Alice and Rolla had a lifelong investment in creating venues in which scientists could interact in the kinds of informal, relaxed settings that lead to the development of new insights about the botanical world, especially the ferns, but in much broader contexts as well. Prominent among these is the Missouri Botanical Garden’s annual Systematics Symposium, initiated by Rolla and Alice during their time in St. Louis, and the New England Fern Conference, which Rolla and Alice inaugurated in 1970. Following her arrival in New England in 1958, Alice was deeply involved in the New England Botanical Club. She was elected its first woman member in 1968. After serving as recording secretary and vice president, Alice was elected the club’s first woman president in 1978. During her time as president, the club inaugurated several successful programs, including a focus on New England’s rare and endangered species in the 1979 symposium Rare and Endangered Plant Species in New England, the proceedings of which were published in 1980. Her interest in New England and long-time residence there led Alice to develop her last book, The Ferns and Allied Plants of New England (1997), coauthored with Robbin Moran. This book is notable for its images of the plants, including both the classic photographs of Robert L. Coffin and the more recent work of noted botanist and photographer Walter H. Hodge. For this book, Alice included spore images for each of the New England pteridophytes, a fortunate inclusion for students of New England Pleistocene biogeography who find this resource invaluable in their analyses of palynological cores. After their retirements from Harvard, Alice and Rolla retired to Florida in 1989. While there, they continued their pattern of supporting small venues for the discussion of scientific ideas by founding the Institute for Systematic Botany and the Tryon Lecture Series at the University of South Florida in Tampa. In 1990, Alice and Rolla were honored with a festschrift occupying pages 222-339 of Annals of the Missouri Botanical Garden volume 77. This tribute to the Tryons featured an opening photograph of them at the portrait of Daniel C. Eaton (first American pteridologist) at Harvard University, an introductory summation of their contributions to pteridology, a closing photograph of them by Walter H. Hodge, and contributed papers by the following: Cathy A. Paris and David S. Barrington; R. James Hickey; Robbin C. Moran; Alan R. Smith; Robert G. Stolze; Gillian A. Cooper-Driver; Ram6én Riba and Irma Reyes J.; David S. Conant; David S. Barrington; Gerald J. Gastony; Christopher H. Haufler, Michael D. Windham, and Thomas A. Ranker; Karl U. Kramer; and Diana B. Stein and David S. Barrington. For more than a decade, Alice’s and Rolla’s partnership in scholarly work and community outreach about the ferns of Florida were centered at the University of South Florida, ending with Rolla’s death in 2001. Following that, Alice moved to the Azalea Trace retirement community in Pensacola, Florida where the Tryons’ good friends Walter and Barbara Hodge were already in residence. Among Alice’s final acts of scientific altruism were her generous establishments of endowments for the Field Museum in Chicago, the New England Botanical Club, the Alice and Rolla Tryon Pteridophyte Library at the 234 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Pringle Herbarium of the University of Vermont, and the Rolla and Alice Tryon Scholarship Fund in support of the Woman in Science program of the Department of Botany at the University of Wisconsin, Madison. Comforted by her care-giving friends, Alice died peacefully in her garden apartment at Azalea Trace on March 29, 2009, surrounded by many mementoes of a life happily shared with Rolla and dedicated to advancing our knowledge of ferns. On September 27, 2009, the authors and their wives united Alice’s ashes with Rolla’s on a ferny hill in northern Vermont. A simple bronze plaque affixed to a boulder at the site records their passing with the following words. IN Memory OF Rotia M. Tryon Jr. (1916-2001) AND ALICE F. Tryon (1920-2009) EMINENT PTERIDOLOGISTS LITERATURE CITED Gastony, G. J., D. S. cups = S. Conant. 2002. Obituary: Rolla Milton Tryon, Jr. (1916— 2001). Amer. Fern J. 9 BIBLIOGRAPHY OF ALICE F. Tryon (1920-2009) Tryon, A. F. 1945. A taxonomic study of spores of Selaginella subgenus relies in North merica north of Mexico. Master’s Thesis. University of Wisconsin, Madis Tryon, A. F. eee Glandular prothallia of Notholaena standleyi. Amer. Fern J. ae 88-89. Tryon, A. F. 1949. Spores - oy genus Selaginella in North America, north of Mexico. Ann. Micaredt’ Bot. Gard. 36:4 TrYON, A. F. 1952. A revision - fe American species of the fern genus Pellaea. Ph.D. Dissertation. isbreo ah Medea f BE Lae ae sp abe Ameri ies of Pellaea. Proc. Eighth Int. Bot. Cong. t eh oe Pa 1955. A new Pellaea from South Africa. Ann. Missouri Bot. Gard. 42:101-103. Tryon, A. F. 1957. A revision of the fern genus Pellaea section Pellaea. Ann. Missouri Bot. Gard. 25-193. Tryon, A. F. 1957. The vegetable lamb of Tartary. Amer. Fern J. 47:1-7. Tryon, A. F, 1957. A leaf of fern. Perspective 3:12-15. —, F. and D. M. Brirron. 1958. Cytotaxonomic studies on the fern genus Pellaea. Evolution 1Z 7-145. TRYON, . F. sae nai of the Incas. Amer. Fern J. 49:10—24. Tryon, R. and A N. 1959, Observations on the epee ferns: the hardy species of tree ferns (Dicksonia es Cyatheacoae) hiswogh ron} 49:129-142 se a F. 1960. Obs j f Pell l lifolia. Contr. Gray Herb. 7:6 ce ne i F. 1961. Some new aspects of the fern Platyzoma microphyllum. Rhodora 63: ag o Tryon, A. F. 1962. A monograph of the fern genus Jamesonia. Contr tr. Gray Herb. 191:109— Tryon, A. F. 1963. Hermann Karsten, his collections and the Flora Columbiae. Taxon 12: gi ie Tryon, A. F, 1963. Notes on the fern genus Eriosorus. Rhodora 65:56—57. GASTONY ET AL.: ALICE FABER TRYON 235 TRYON, eh F. 1964. Platyzoma—a Queensland fern with incipient heterospory. Amer. J. Bot. 51:939-9472. TRYON, A ¥ 1965. Trichomes and paraphyses in ferns. Taxon 14:214-218. Tryon, A. F. 1965. A parcel of Cameroon ferns. Amer. Fern J. 55:49-57. Tryon, A. F. 1966. Origin of the fern flora of Tristan da Cunha. Brit. Fern Gaz. 9:269-276. Tryon, A. F. and G. Vina. 1967. Platyzoma: a new look at an old link in ferns. Science 0. Tryon, R. M. and A. F. Tryon. 1968. Edith Scamman (1882—1967). Amer. Fern J. 58:1—4. Tryon, A. F. 1968. Comparisons of sexual and apogamous races in the fern genus Pellnoe Rhodora 70:1—24. Tryon, A. F. 1970. A monograph of the fern genus Eriosorus. Contr. Gray Herb. 200:54—-174. Tryon, A. F. 1971. Structure and variation in spores of Thelypteris palustris. Rhodora 73:444—460. Tryon, A. F. 1972. Spores, chromosomes and relations of the fern Pellaea atropurpurea. Rhodora 74:220-241. Sia A. - - TRYON. 1973. ee in northeastern North America. Amer. Fern J. 63:65—76. N, JR., and A. F. 973. Geography, spores and evolutionary relations in the Sad po Bot. A. vas 67 supee 1:146-153. Tryon, R., B. VOELLER, A. F. Tryon and R. Ripa. 1973. Fern biology in Mexico (a class field program). BioScience 23:28-33. Tryon, A. F. and R. M. Tryon. 1974. ra sare patterns in temperate American ferns and some relationships in Thelypteris. Amer. Fern J. 64:99-104. Tryon, A. F. and L. J. FELDMAN. es Tree in indusia: studies of development and diversity. Can aa Bot. 53:2260-227 TRYON, “e (Ce 3 Bal pele: and A pA Sitva Araujo. 1975. Chromosome studies of Brazilian ferns. Acta Tryon, A. F. 19 iat gland sb redeicnats Rhodora 80:558—569. Tryon, A. F. — B. Luc structure and mineral content in Selaginella spores. Tryon, A. F. 1980. Foreword to the symposium “Rare and Endangered Plant Species in New England.” Rhodora 82:1-2. Tryon, A. F., R. Tryon and - Gee 1980. Classification, spores, and nomenclature of the marsh fern. Rhodora 82:461-4 Tryon, R. and A. Tryon. ee one and nomenclatural notes on n ferns. Rhodora 83:133-137. Tryon, R. and A. F. Tryon. 1982. Additional t notes on ferns. Rhodora 7125-130. Tryon, R. ae and A. F. Tryon. 1982. Ferns and Allied Plants with Special Reference to Tropical America. Springer-Verlag, New Yor TRYON, ra F. 1985. Spores of miyrmecophytic ferns. Proc. Roy. Soc. Edinburgh 86B:105-110. Tryon, A. F. 1986. Stasis, diversity and function in si based on an electron microscope surve form and garnet ‘Linguesn Society, London ER, E., C. ScHeete and A. F. Tryon. 1987. ntl and spores of Platyzoma aera ey an endemic fern of Australia. Amer. Fern 28-32. eee A. F. 1990. Fern spores: evolutionary levels and aaa differentiation. Pl. Syst. Evol. 5 upp pies Tryon, R. M., A. F. Tryon and K. U. Kramer. 1 se heresies Pp. 230—256, in K. Kubitzki, ed. The panies and Genera of Vascular Plants. Vol 1. Pteridophytes and Gymnosperms. Vol. eds. K. U. Kramer and P. S. Green. Springer-Verlag, how York. Tryon, A. F. and B. Lucarpon. 1991. Spores of the Pteridophyta. Springer-Verlag, New York. Tryon, A. F. and R. C. Moran. 1997. The Ferns ~~ sags Plants of New England. Massachusetts Audubon age South Lincoln, Massachus Tryon, R. and A. F. Tryon. 1999. Observations on oa phytogeography of eastern North American ferns. Pp. 2 250-273, in X-C. Zang and K-H. Shing, eds. Ching Memorial Volume. Institute of Botany, Chinese Acad. Sci., Beijing. American Fern Journal 99(4):236—237 (2009) Obituary: Prem Kumar Khare (1946-2009) RaMa SHANKAR and R. C. SRIVASTAVA Regional Research Institute (Ayurveda) and BSI Itanagar Born on 1* June, 1946 at Varanasi, Professor P. K. Khare, after completing his Master Degree from Gorakhpur University, Gorakhpur, started his research career from Allahabad University, Allahabad under esteemed guidance of late Professor Divya Darshan Pant, the then Head of Botany Department on various morphological and paleobotanical aspects of pteridophytes. During his research career he discovered Damudopteris polymorpha species from Raniganj hills. At the same time he has also discovered stomatal ontogeny of Psilotum, Tmesepteris species, Dipteris (wallichii), a rare fern. Simultaneously, he started his professional career as Lecturer in the same department in February, 1974 and continued as Reader as well as Professor of Botany. During 2007-08 he was honored with the prestigious chair of Head of the Botany Department of Allahabad University, Allahabad. During his professional career Professor Khare guided many research scholars. During his research career he made comprehensive studies on petiolar structure, phytochemical studies of economically and taxonomically important ferns as well as ecology of Pteridophytes of Western Himalaya and Central India. While studying petiolar structure he had given guidance for SHANKAR & SRIVASTAVA: PREM KUMAR KHARE 237 establishing petiolar characters as good tool for taxonomic identification of ferns, particularly Adiantum, Asplenium, Ophioglossum and Pteris species besides other fern species. For phytochemical studies he studied ferns from Western Himalayas and Central India. To his credit Professor Khare published over 50 research papers in Journal of Royal Society, Annals of Botany, Canadian Journal of Botany, American Fern Journal, International Journal of Pharmacognosy and other national journals like National Academy of Sciences, Indian Fern Journal, etc. During the year 2008 Professor Khare was graced with the deliberation of Dr. G. Panigrahi Memorial lecture at the occasion of the Indian Botanical Conference held at Allahabad. Professor Khare left this world on 3° May, 2009 with his sweet memory to science. He is survived by his wife, one married daughter and one son who recently joined Provincial Civil Service (PPS). American Fern Journal 99(4):238—243 (2009) Isoétes todaroana (Isoétaceae, Lycopodiophyta), a New Species from Sicily (Italy) ANGELO Trora* and FRaNcEsco M. RAIMONDO Dipartimento di Scienze Botaniche dell’Universita, via Archirafi 38, I-90123 Palermo, Italy BSTRACT.—Isoétes todaroana, a new species from western Sicily (Italy), is described. Morpholog- ical, anatomical and ecological characters are given. The main differential characters are the presence of only two leaf air chambers, rather than four as in all other known species of the genus, and the shape of the scales, which have two lateral rounded lobes and one central spine-like lobe, together with its peculiar calcophilic habitat. So far, the species is known from a single locality. Key Worps.—Lycopodiophyta, Isoétaceae, Isoétes, Mediterranean area, Italy, Sicily Four species of Isoétes have been previously reported from Sicily (Troia, 2005): Isoétes histrix Bory, I. sicula Tod. [= I. subinermis (Bory) Cesca & Peruzzi, =? I. gymnocarpa (Gennari) A. Braun], I. velata A. Braun, and I. duriei Bory. All of these grow on seasonally wet, siliceous soils, except for I. velata which colonizes temporary ponds. Over the last ten years, field, herbarium, and laboratory studies have been conducted by one of us on the genus Isoétes (e.g., Romeo et al., 2000; Troia and Bellini, 2001; Troia, 2005). As part of these studies, a population of Isoétes was located in a wetland near Mazara del Vallo, Western Sicily. Closer inspection of the specimens showed that they differed in several aspects from the other species occurring on the island. Analyses of living and dried plants confirmed that this population represents a unique and previously undescribed species of Isoétes, which is here named and described. For description and nomenclature of megaspores and microspores we followed Hickey (1986) and Musselman (2003), respectively. Isoétes todaroana Troia & Raimondo, sp. nov. TYPE.—ITALY. Sicily: contrada ‘“‘Critazzo”’ near Mazara del Vallo, 37°41'07”"N, 12°37’05”E, ca. 60 m a.s.l., 10 Apr. 2009, Angelo Troia (holotype: PAL; isotypes: PAL, FI). Figs. 1-10. Herba perennis amphibia, emergens aut submersa. Cormus trilobus, radicibus dichotomis. Folia 15-30 (—40), erecta vel patentia, 3-6 (—14) cm longa, inferne brevi tractu anguste alato-membranacea, basi usque 3-4 mm, in medio circa 1 mm lata. Duae magnae lacunae et 1-3 fasciculi fibrarum periphaerici (collenchymatosi) in trasversali sectione. Stomata elliptica, 37— 70 pm longa, 24-32 um lata, in facie abaxiali tantum obvia. Velum sporangium *Corresponding author: fax: +39 091 6238203, e-mail: angelo.troia@libero.it TROIA & RAIMONDO: ISOETES TODAROANA, A NEW SPECIES FROM SICILY 239 3b abaxial face adaxial face CO Fics. 1-3. Morphological and anatomical traits of I. todaroana. 1-2. Plants in their habitat (the coin is ca. 18 mm in diameter). 3 (a/b). Transverse section of the leaf: AC = air chambers (the one on the left partially covered by the translacunar diaphragm); CO = collenchymatous strands; C = vascular bundle (with at least one intrastelar canal). obtegens. Ligula membranacea, lanceolata, basi auriculata. Labium breve. quamae paucae, coriaceae, nigrescentes, lobis lateralibus rotundatis et dente medio aciculari. Macrosporae candidae, sphaeroideae, 420-460 um, annulo horizontali cinctae, hemispherio superiore tricostatae, undique tuberculatae. Microsporae ovatae, ca. 25 um longae, aculeatae. Habitat in humidis hyeme inundatis. Plants amphibious, emergent or submerged in temporary ponds, losing their leaves in the dry summer season. Stem (corm) trilobate, with dichotomous roots. Leaves 15—30(—40), patent to erect, narrowly lanceolate, 3-6 (—14) cm long, 3-4 mm wide at base, ca. 1 mm wide at mid-length. Alae proximally hyaline or transluscent, ca.1 mm wide at the sporangium, gradually narrowing distally. Subula semiterete, adaxially flat, abaxially convex. Leaf epidermis with cuticular ornamentation, “‘cuticular pegs” (sensu Prada and Rolleri, 2005, = “cufas cuticulares” sensu Rolleri and Prada, 2007) well developed, evident as continuous longitudinal ridges. Stomatal complexes in rows, elliptic, 37— 70 um long, 24-32 um wide, confined to the abaxial surface. Hypodermal collenchymatous bands one to three, the two marginal bands sometimes absent. Air chambers two, with translacunar diaphragms. Velum complete. Ligule ca. 1 mm long, membranaceous, broadly lanceolate, auriculate at base. 240 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Gs. 4-6. Morphological and anatomical traits of I. ee 4. Surface view of the leaf epidermal cells. Note the cuticular ornamentation, the cuticular pegs well developed, evident as continuous longitudinal ridges, and the epiphytic jae 5. Scales. 6 (a/b). Adaxial face of the leaf base: Li = ligula; La = labium; Sp = (mega)sporangium Labium shorter than ligule. Scales few, black, with two lateral rounded lobes and one (usually short) central spine-like lobe. Megaspores 420-460 um in diameter, white, subtriangular in polar view, tuberculate. Microspores ca. 25 um long, aculeate. TROIA & RAIMONDO: ISOETES TODAROANA, A NEW SPECIES FROM SICILY 241 a. pet sty 15 kK i nl Fics. 7-10. SEM images of I. todaroana megaspores and microspores. 7. Proximal view of megaspore. 8. Equatorial view of megaspore. 9. Megaspore (detail of a single tubercle). 10. Distal view of microspore. ErymoLocy.—This new species is dedicated to the Sicilian botanist Agostino Todaro (1818-1892), in recognition of his contribution to the pteridological flora of Sicily. EcoLocy.—The type locality is a temporary wetland that dries out during the summer. It is a remnant of a wider wetland that has been ‘“‘reclaimed”’ and converted to farming land that surrounds and encloses the type locality. The natural vegetation, although altered, is well represented with a mosaic of mmunities, with species such as Bolboschoenus maritimus (L.) Palla, Eleocharis palustris (L.) Roem. & Schult., Scirpus cernuus Vahl, Mentha pulegium L., Oenanthe sp., Lythrum sp., Tamarix sp., Romulea sp., etc. The wetland hosts the last remnants of a peculiar freshwater invertebrate fauna; a preliminary investigation has led, for example, to the discovery of a small population of the notostracan Lepidurus apus lubbocki (Crustacea), a “‘living fossil’ that was considered extinct in Sicily (F. Marrone, pers. comm The wetland exists on a peculiar geological substrate of calcareous sandstones with a thin layer of clays on top (hence the local name, ‘“‘Critazzo”’, which in Sicilian dialect refers to clays). The soil pH around the Isoétes (determined electrometrically with two replicates from 20 g of soil and measured in distilled water with a dilution ratio of 1: 2.5) was found to be 242 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) basic, 8.5-8.9. As far as we know, the other species occurring in Sicily consistently grow on acid soils, so that this site, although geologically complex, is unusual, a fact that deserves further investigations. DisTRIBUTION.—Isoétes todaroana is only known so far from the locus classicus, in an area of about 200 x 100 m. It is possible that other populations may be found in the future, in Sicily and elsewhere in the Mediterranean area. CONSERVATION STtatus.—The single known population is threatened by farming and other human activities (waste dumping, land reclamation, summer fires, etc.). On the basis of the current “IUCN Red List Categories and Criteria’ (IUCN, 2001), the species is rated ‘Critically Endangered” [CR B1ab(iii)]. rgent measures are needed to protect the site; considering that it is practically unexplored, it is possible that other rare or endangered species are present, in addition to Isoétes todaroana and Lepidurus apus lubbocki already mentioned. However, it is clear that it is a strategic area for migrating birds and it hosts communities that can be referred to the habitat “Mediterranean temporary pond’’, considered a “priority’’ habitat by the Council of the European Communities 92/43/EEC Directive. Our proposal is to include the site, currently not protected, inside the adjacent “Site of Community Interest” code ITA010014, established to protect habitat and species according to the mentioned Directive. Since this inclusion will not be made in a short time and will not automatically guarantee the protection of species and habitats, we also suggest considering other actions (e.g., agreements with the owners, territory planning restrictions, land purchase) as soon as possible. TAXONOMIC OBSERVATIONS.—The shape of the scales, and particularly the presence of only two leaf air chambers rather than four as in all other species of the genus (Rolleri and Prada, 2007), are the main differentiating characters of this species. Its systematic relationships are difficult to assess merely on the basis of anatomy and morphology, owing to parallel and convergent morphological evolution in the genus (Hickey, 1986; Hoot et al., 2006; Bolin et al., 2008). The presence of scales, sometimes transitioning into phyllopodia, and collenchymatous strands suggests a link between this species and the Mediterranean “‘terrestrial’’ taxa, and the megaspore ornamentation, in particular, vaguely suggests a connection with I. histrix and I. sicula. However, as shown by Bolin et al. (2008), the latter two species, although morpholog- ically similar, are not immediately related. On the other hand, the fibrillose megaspore surface background (Fig. 9) suggests a relationship with other species, e.g., the amphibious Isoétes velata. Further studies are in progress to add to the knowledge of the new species and shed light on its relationships. ACKNOWLEDGMENTS Thanks to Antonino Castelli from Mazara, who drew the pond of ‘‘Critazzo”’ to our attention, a M. Mannino for the assistance with scanning electron microscopy, Werner Greuter for his critical review of the manuscript and helpful suggestions, and the referees R. James Hickey and C. TROIA & RAIMONDO: ISOETES TODAROANA, A NEW SPECIES FROM SICILY 243 Cecilia Macluf for their valuabl s. Financial support from Regione Siciliana (L.R. 25/93) and Universita degli Studi di Saleen’ palate di Ateneo, ex 60%, titolare F.M. Raimondo) is gratefully acknowledged. LITERATURE CITED BOLIN, a F., R. D. Bray, M. Keskin and L. J. MusseLMan. 2008. The genus Isoetes L. (Isoetaceae, ype in South Wastern Asia. Turk. J. Bot. 32:447-457 nee R J. 1986. Isoétes megaspore surface morphology: nomenclature, variation and systematic Hoot, S. B., W. C. Taytor and N. S. Napier. 2006. Phylogeny i canara sed of Isoétes (Isoétaceae) based on nuclear and chloroplast DNA sequence dat he st. Bot. 31:449—-460. IUCN. 2001. IUCN Red List Categories and Criteria: Version 3. . IUCN Species Survival Commission. IUCN, Gland, Switzerland and ridge, MussELMAN, 03. Ornamentation of Isoetes (Isoetaceae, Lycouliyts) microspores. Bot. Rev. bLaticaster) 68:474—487 (2002). Prana, C. and C. H. Routeri. 2005. A new species of Isoetes (Isoetaceae) from Turkey, with a study of microptiyll intercellular pectic protuberances and their potential taxonomic value. Bot. J. Lin 47:213-22 Rotueri, C. H. and C. Prana. 2007. nee diagnosticos foliares en Isoetes (Pteridophyta, Isoetaceae). Ann. Missouri Bot. Gard. 94:202—235. Romeo, D., A. Trora, C. BurGARELLA and E. ee 2000. Casparian strips in the leaf “intrastelar of Isoetes duriei Bory, a Mediterranean terrestrial species. Ann. Bot. (London) 86:1051—-1054. Troia, A. and E. BeLuni. 2001. oe ee on Isoétes duriei Bory (Lycophyta, Isoetaceae) in Sicily. Bocconea 1 Troia, A. 2005. Note corologiche e alan sul genere Isoétes L. (Isoétaceae, Lycophyta) in Sicilia. Inform. Bot. Ital. 37:382-383. American Fern Journal 99(4):244—248 (2009) On Neolepisorus emeiensis and N. dengii (Polypodiaceae) from China XIAO-sI Guo College of Life Science, Northwest A & F University, Yangling, Shaanxi 712100, P. R. China BIN LI Xi’an Botanical Garden, Xi’an, Shaanxi 710062, P. R. China Asstract.—Neolepisorus dengii, N. dengii f. hastatus and N. emeiensis f. dissectus should be considered synonyms of N. emeiensis. This treatment is justified on the basis of complete intergradation of the frond forms that supposedly separate these taxa. The intergradation can be und on the same individual. Key Worps.—Neolepisorus, China, synonyms Neolepisorus occurs in tropical and subtropical Asia and Africa. It has one center of distribution in the Yangtze River area and south and southwest China. Its species are endemic to mainland China except for one endemic to Madagascar, one in Indo-Himalayas, upper Burma, northern Thailand, Indo-China and China, and a third in Japan, Philippines and China (including Taiwan). As originally proposed by Ching (1940), Neolepisorus consisted of three species: N. Jastii (Baker) Ching, N. ovatus (Bedd.) Ching, and N. ensatus (Thunb.) Ching. Subsequently Ching and sti (1983) published regional taxonomic revisions of Chinese Neolepi- orus and recognized 10 species, a conclusion with confirmed by Lin Folie (2000). Based on the study of specimens at KUN, PE, SZ and WUK, we came to doubt the establishment of a species and two forms that were named by Ching and Shing (1983). They claimed that Neolepisorus emeiensis and N. dengii differed in the shape of the lamina base. Furthermore, the published two forms, N. emeiensis f. dissectus and N. dengii f. hastatus, also based on differences in the shape of the lamina base. Our field observations and identification of Neolepisorus from southern Shaanxi Province, especially the Dabashan Mountain region, have confirmed these forms intergrade and therefore do not merit taxonomic recognition. We suggest that N. dengii, N. dengii f. hastatus and N. emeiensis f. dissectus be considered synonyms of N. emeiensis. Neolepisorus emeiensis Ching et K. H. Shing, Acta Phytotax. Sin. 21(3): 271, f. 2:1, 1983. TYPE.—China. Sichuan: Mount Emei, Hongchunping, 1150 m [erroneously as ‘1500’ m in the protologue], 27 August 1964, K. J. Kuan et al.1836 (Holotype, PE!) GUO & LI: NEOLEPISORUS FROM CHINA 245 Fic. 1. Herbarium specimens and epidermal structures of two species of Neolepisorus. A. The holotype of atih tice geod eatin (K. J. Guan & W. C. Wang 1836, PE). B. The holotype of N. dengii (S. W. Deng 90002, PE). C. The holotype of N. dengii f. hastatus (P. S. Wang 75473, PE). D. The holotype of N. emeiensis f. dissectus (G. X. Shing & K. Y. Lang 1143, PE). E. Upper epidermis of - Leoapepea (T. L. Dai 107331, WUK). F. Lower epidermis of N. emeiensis (T. L. Dai 107331, UK). G. Upper epidermis of N. dengii (T. P. Wang 8542, WUK). H. Lower epidermis of N. dengii He P. Wang 8542, WUK). Neolepisorus dengii Ching et P. S. Wang, Acta Phytotax. Sin. 21(3): 272, f. 2:3, 1983, syn. nov. TYPE.—China. Guizhou: Guiyang, 12 April 1936, S. W. Deng 90002 bis (Holotype, PE!) depend ete? dengii Ching et P. S. Wang f. hastatus Ching et P. S. Wang, Acta h . Sin. 21(3): 271, f. 2:4, 1983; TYPE.—China. Guizhou: Anshun, Pree m, ane December 1977, P. S. Wang 75473 (Holotype, PE!) 246 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) TABLE 1. Comparison of Neolepisorus emeiensis and N. dengii. Character N. emeiensis N. dengii Habitat wet forest floors _ Lee or on rocks Altitude 500-1800 m 500— Rhizomes long-creepin apes in Scales brown, ovate-lanceolate dark brown, oo Frond texture lightly coriaceus at dry thickly chartaceous at dry Frond length 24-28 cm 27 cm Frond shape broadly oblong-lanceolate, triangular-lanceolate, 5-8 cm wide, 6—7 cm wide, widest at base widest at base Frond base obliquely cuneate lightly hastate or obliquely cuneate Frond color yellow-green at dr brown or brown-green at dry Lateral veins mostly flat, Ligissis free lightly oblique, visible Sori round, larger, borne in 1-2 rows round, smaller, borne in 1-2 row between se lateral main veins between the lateral main veins Neolepisorus emeiensis Ching et K. H. Shing f. dissectus Ching et Shing, Acta Phytotax. Sin. 21(3): 271, f. 2:2, 1983. TYPE.—China. Sichuan: Mount Emei, 1000 m, 1 September 1963, K. H. Shing et K. Y. Lang 1143 (Holotype, PE!) DISTRIBUTION AND Hasirat.—China (Sichuan, Hubei, sa Guizhou, Southern Shaanxi); forests on hills or slopes or in valleys; 50 Om The protologue of Neolepisorus dengii (Fig. 1B) . that it differs from N. emeiensis (Fig. 1: A) by triangular-lanceolate fronds, slightly hastate lamina bases, thickly chartaceous, brown or brown-green laminae when dry, and lateral veins slightly oblique. Otherwise, the two species do not differ. After examining more material of N. emeiensis, we conclude that its frond shape falls within the range of variation of N. dengii (Table 1). To further test for differences, we studied the epidermis of Neolepisorus dengii and N. emeiensis with an optical microscope. We found that the two species have similarly shaped epidermal cells, including the distribution, structure, and type of stomata (Fig. 1: E, F, G, H). The arrangement of the upper epidermal cells in two species was also the same. There are copolocytic and coaxillocytic stomata in the lower epidermis. Thus we found no differences in epidermal characters between the two species. Neolepisorus dengii f. hastatus (Fig. 1: C) and N. emeiensis f. dissectus (Fig. 1: D) differ from N. emeiensis, respectively, only by the base of fronds hastate or with 1 or 2 pairs of long lanceolate segments. In observing many individuals in southern part of Shaanxi Province, we found that the fronds lobed or with 1—2 pair of segments (from lobate to triangular to lanceolate in shape) at base often occurs in N. emeiensis (Fig. 2). Through a wide range of herbarium and field investigations, two foliar variations in N. emeiensis have been determined: 1) from broadly oblong-lanceolate, triangular-lanceolate to halberd-shaped in shape, and 2) from obliquely cuneate to hastate at base. These two foliar variations are the basis of the two named forms. Thus we GUO & LI: NEOLEPISORUS FROM CHINA 247 Cc | d e f h 5cm in frond form i of Y. S. Chen et A pe WUK). A, B, Frond bony a base ake cuneat ea Frond lanceolate or triangular-lanceolate, base ete! hastate. G, H. Frond als ice. base with 1 or 2 pairs of long lanceolate segment m concluded that the shape of the lamina base is an unstable character, which could not be used to establish a new taxon. All supposedly seca characters of Neolepisorus dengii, N. dengii f. hastatus and N. emeiensis f. dissectus are variable and fall within the range of that found in N. emeiensis. Therefore, N. dengii, N. dengii f. hastatus and N. emeiensis f. dissectus should be considered synoymyms of N. emeiensis. ACKNOWLEDGMENTS The study was supported by funds from the National Natural Sciences Foundation of China (Grant No. 30370117, 39899400, 30499340) and the special support grants from the Chinese 248 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Academy of Sciences for Flora of China (KSXX-SW-122).We are most grateful to WUK, SZ, PE and KUN for providing specimens. We also express our deep gratitude to an anonymous reviewer for helpful comments on the manuscript. LITERATURE CITED Crinc, R. C. 1940. The studies of Chinese ferns. Bull. Fan Mem. Inst. Biol. Bot. 10:11. Cuinc, R. C. and Suinc, K. H. 1983. A monographic revision of the fern genus Neolepisorus China. Acta Phytotax. Sin. 21:266-276. Lin, Y. X. 2000. Neolepisorus. Pp. 36-42, in Y. X. Lin, X. C. ZHanc, L. Su, S. G. Lu, eds. Flora Reipublicae Popularis Sinicae 6(2), Science Press, Beijing. American Fern Journal 99(4):249-259 (2009) Botrychium ascendens W. H. Wagner (Ophioglossaceae) in Newfoundland and Notes on its Origin PETER F. ZIKA Herbarium, Box 355325, University of Washington, Seattle, WA 98195-5325 DONALD R. FARRAR Department of EEOB, 253 Bessey Hall, Iowa State University, Ames, Iowa 50011 Asstract.—Botrychium lens i d from Fogo Island in fe jland as an addition to the flora of the - province. Fogo Island oped are identical to pas in western North America, including those from the type locality, in pe size, and allozyme expression. Comparisons are made with related and siesst es taxa, B.c mpestre, B. crenulatum, the B. lineare/campestre complex. The current distribution of Botrychium ascendens and its putative parents suggest it probably originated in western North America and migrated across northern Canada to Newfoundland. Key Worps.—Botrychium ascendens, leaf morphology, allozymes, taxonomy Canada’s Maritime Provinces of Nova Scotia, New Brunswick, Newfound- land and Québec are reported to harbor 14 species of Botrychium (Cody and Britton, 1989; Wagner and Wagner, 1993; Kartesz, 1999), not including Botrychium ascendens W. H. Wagner, a species commonly found throughout western North America, east to central Alberta, with an outlier collection from northern Ontario (Wagner and Wagner, 1993). Wagner and Wagner (1990) tentatively reported a 1985 Britton and Anderson collection from Fogo Island in Newfoundland as Botrychium campestre W.H. Wagner & Farrar. The Wagners were uncertain because the pressed material was scanty. They wished to see a larger collection to study the morphological variation in the population. A traditional problem in the elucidation of species in Botrychium subgenus Botrychium is that much herbarium material consists of only one to a few plants, these often folded or shriveled, and thus difficult to classify. The most useful samples have leaves pressed with all pinnae flat and clearly visible, and have a minimum of 10-20 plants showing the variability within the population. (Underground parts are not diagnostic in moonwort species identification. Carefully harvesting the above-ground leaf by cutting at ground level allows the below-ground bud to produce new leaves in subsequent years.) Limited samples fail to show typical population variation, and if the individual specimens are small, they may resemble juvenile or small plants of other taxa. Diagnostic features observable in the field, such as stature, color, fleshiness and luster, are rarely noted by collectors on museum labels. 250 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) To overcome these difficulties, we visited the Fogo Island population to secure an adequate sample. Wagner and Wagner (1986) reported a chromosome number of n = 90 for B. ascendens, indicating it to be a tetraploid species. Using rbcL sequence comparisons Hauk (1995) found Botrychium ascendens clustered with the lineage including B. campestre and B. lineare W. H. Wagner, suggesting this lineage contributed the chloroplast genome to B. ascendens. Using allozymes at a smaller number of loci (6) than the current study, Hauk and Haufler (1999) found the non-chloroplast genome of B. ascendens clustered with B. crenulatum W. H. Wagner, thus suggesting an allopolyploid origin of B. ascendens through ancient hybridization between B. campestre/B. lineare and B. crenulatum. In this paper we examine the variation of the Newfoundland population, and compare it with the morphology and allozymes of B. ascendens and related species of known provenance from elsewhere in North America, to confirm the identity of the Newfoundland plants and provide further evidence of their origin. We also present morphological characters useful in separating B. ascendens from B. campestre and B. lineare. MATERIALS AND METHODS Plant collection.—Plants of Botrychium ascendens were obtained from Sandy Cove on Fogo Island in Newfoundland on June 26, 2001. Plants representing the observed variation were collected by cutting leaves at ground level and storing them in plastic containers in an ice chest until processing. The plants were divided into groups, one for immediate pressing and one maintained fresh for enzyme electrophoresis. After samples for electrophoresis were removed from the common stalk, the latter were also pressed for herbarium vouchers. Vouchers of all plants in the study are deposited in the Ada Hayden Herbarium of Iowa State University (ISC). Voucher specimens are labeled: CANADA: Newfoundland: Fogo Island, Sandy Cove, 7 July 1985, Britton 10671 & Anderson (MICH); Fogo Island, Sandy Cove, elev. ca. 3 m, 49° 42.5’ N, 54° 5.0’ W, 26 June 2001, Zika 16334 (CAN, ISC, MICH, MT, NFM, WTU). The population is quite local and restricted to 80 meters of low sand dunes southwest of Route 334, at Sandy Cove, by a sandy beach near Tilton. About 150 stems were seen on 26 June 2001. The fern’s associates included Carex nigra (L.) Reichard, Festuca rubra L., Achillea, Linnaea, Fragaria, Taraxacum, Aralia nudicaulis L., Ranunculus acris L., Equisetum arvense L., and Botrychium lunaria. Allozyme electrophoresis.—From an ongoing study of all moonwort Botrychium by Farrar, we constructed genetic profiles obtained through starch-gel enzyme electrophoresis for species relevant to this study. The source of genetically analyzed plants is listed in Table 1. The number of individuals sampled per site ranged from 1 to 92 depending on population size. The average number of plants per site was 10.6. Single leaves were cut at ground level and kept cool until processing. Approximately one-centimeter segments were removed from the common stalk ZIKA & FARRAR: BOTRYCHIUM ASCENDENS IN NEWFOUNDLAND 251 TaBLE 1. Source of collections used in genetic analysis, including the number of sites sampled and total number of plants analyzed. Details of site locations can be obtained from Farrar. Botrychium State or Province # Sites # Plants ascendens 6 84 California 1 12 Montana 5 19 a Zt ao Newfoundland 1 10 regon 2 34 Washington 3 24 campestre Alberta 1 3 Iowa 5 47 Michigan 1 16 Minnesota 7 74 ontana 4 17 South Dakota 3 38 Wyoming 1 a} crenulatum Alberta 1 6 California 9 41 Montana 5 19 crenulatum Nevada 3 15 Oregon 5 27 tah V's 25 lineare Alaska 4 2 Colorado 2 12 Montana 5 34 Oregon 2 15 South Dakota 3 11 Washington 1 1 Wyoming 3 11 Yukon 1 1 lunaria* Alaska (coastal) 18 476 2 51 — 3 25 Ont 1 15 Washingt 5 16 Total 113 1185 _ aiftt ] *The common American g from European B. Junaria suggesting it could be described as a new species (Stensvold 2008). J (petiole) and ground with mortar and pestle in a phosphate- ee done extraction buffer (Cronn et al., 1997). Grindates were stored at —70° until used, at which time they were spun at 12,000 rpm for two minutes e produce a clear enzyme-containing supernatant. The extracts were absorbed onto 2 X 8 mm wicks cut from Whatman 3u chromatography paper (Whatman International, Maidstone, UK). Allozyme variation was determined via horizontal starch-gel pe a enna Gel (11%) buffers and stain recipes followed Soltis et al. (1983). en enzyme systems stained for 22 putative loci found to be informative within the genus. System 7 (Soltis et al., 1983) was used to resolve 252 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) triosphosphate isomerase (Tpi-1, Tpi-2), aspartic acid transaminase (Aat-1, Aat-2, Aat-3, Aat-4), and phosphoglucoisomerase (Pgi-2). System 9 was used to resolve malate dehydrogenase (Mdh-1, Mdh-2, Mdh-3, Mdh-4), 6-phospho- gluconate dehydrogenase (6-Pgd-1), and phosphoglucomutase (Pgm-1, Pgm-2). System 11 was used to resolve aconitase (Aco-1, Aco-2), diaphorase (Dia-1, Dia-2, Dia-3, Dia-4), isocitrate dehydrogenase (Idh-1), and shikimic dehydro- genase (Skdh-1). Analysis of spores.—Spore sizes were measured as the longest diameter of a minimum of 20 spores from each of ten plants and compared with published values for Botrychium ascendens and related diploid species. Morphological comparisons.—Leaf morphology for Newfoundland plants was compared with Botrychium ascendens from western states and with B. campestre and B. lineare from all sites listed in Table 1. Illustrations were prepared to capture the range of morphology for each species. RESULTS Allozyme electrophoresis——Twenty enzyme loci were variable among the compared taxa (Table 2). The allelic composition of Botrychium ascendens from Newfoundland was identical to the combined expression of western B. ascendens populations from Oregon, Washington, Montana, Nevada, California and Alaska (Table 2). Plants of identical genotype were present within the Newfoundland populations and the type locality of B. ascendens in Oregon. Botrychium ascendens from all sites displayed fixed heterozy- gosity at seven of the 22 loci examined. Four additional loci varied among populations for fixed heterozygosity, with some plants expressing only one of the alleles of the heterozygous condition. At all loci, the alleles present in B. ascendens are also present in B. lineare, B. campestre and/or B. crenulatum. At five loci, Tpi-1, Tpi-2, Mdh-1, Mdh-2, and Skdh-1, the single allele present in the American genotype of B. Junaria (L.) Swartz is not present in B. ascendens. Analysis of spores.—Average spore size of Botrychium ascendens plants from Newfoundland was 41 um and ranged from 39 to 44 um. Wagner and Wagner (1986) reported spore sizes of 44-47 um for B. ascendens from western plants. Wagner and Wagner (1986) reported a range of 34-38 um for B. campestre. Wagner and Wagner (1994) did not include spore size in their description of B. lineare. Our measurements of spores of B. lineare averaged 36.2 um and ranged from 35 to 39 pm. Morphological comparisons.—Leaf morphology for Newfoundland plants was compared with Botrychium ascendens from western states and with B. campestre and B. lineare. Small Newfoundland plants closely resemble B. lineare and B. campestre (Figs. 1-3). Larger plants display features more typical of western plants of B. ascendens (Figs. 4-5). Typical plants of B. ascendens display a morphology intermediate between B. campestre/lineare and B. crenulatum (Fig. 6). ZIKA & FARRAR: BOTRYCHIUM ASCENDENS IN NEWFOUNDLAND 253 TasLe 2. Allozyme composition of Botrychium ascendens and putative parents. The number of individuals of each species analyzed is } indicated (in parentheses). Alleles for se locus are represented by numbers. N i n different individuals. Numbers joined by ‘‘+” are two alleles present (fixed) in all individuals of ie species or population. In Aco, Idh, and Pgi, B. ascendens displays fixed heterozygosity in most populations, but in some populations Pit ie only one of the alleles present in the heterozygous state. Allozyme B. campestre B. lineare B. ascendens — B. crenulatum B. lunaria (91) (186) (168) (583) Aat-1 2 2 +2 1,2 2 Aat-2 3 3 3 3 3 Aat-3 2 2,n 2 n n Aat-4 3 3 2+3 2,4 2,3 Aco-1 2 2 1,12 ae 1 Aco-2 t 3 3,143 ad 18 Dia-1 4 2,4 2+4 2 2 Dia-2 1 1 1 1 1 Dia 3 2,3 2,3 2+3 3 3 Dia-4 4 4 = n n Idh 2.3 1,2 wae a3, 204 3,4 2 Mdh-1 3 io Bs 2 - Mdh-2 3 3 Fo 2 5 Mdh-3 2° 5 ee Zz Ags 2 Mdh-4 2 2 2 n n 6Pgd 1 1 1+5** 5 5 Pgi-2 2 2 2,2+ 4,4 4,5 4 Pgm-1 id Based 1**F 12 * 4 1 Pgm-2 2,4 2 2 2 2 Skdh 1,2 1 1 1 2 Tpi-1 3 3 3 3 4 Tpi-2 3 3 3 3 4 *In these species an additional spot of unknown origin always trails the principal allelic spot at a uniform distance regardless of the allele. ** Activity of the homodimers varied among populations; in some, only the heterodimer expressed normal activity. ***In these species an additional spot, possibly from a locus duplication, always precedes the principal allelic. = null, i.e., no allele expressed. DISCUSSION Moonwort ferns of Botrychium subgenus Botrychium are notoriously difficult to identify with certainty by morphological characters alone. Th have been appropriately referred to as cryptic species (Hauk and Haufler, 1999). Because of their small size and simple morphology, differences between species are subtle and tend to be statistical rather than absolute. This problem is compounded in allotetraploids, in which the ranges of morphological characters overlap those of the parental diploids. In contrast to morphology, genetic markers clearly define species of moonworts and, for allotetraploids, provide evidence of the ancestral diploid 254 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) HpLigegye lcm lcm PSLeresy Fic. 1. Botrychium lineare trophophores, showing typical slender pinnae with margins essentially straight or narrowing to base, notched or forked at tip. A. Washington (Kirk 200 et al. WTU). B. Type locality, Oregon (Farrar 3918, E2235 ISG). C. Oregon (left to right: Zika 12906 OSC; Wagner 81128 & Wagner MICH; Zika 11353A OSC). D. Colorado (left to right: Farrar 3794, 3797, 3801 ISC). Fic. 2. Botrychium ascendens trophophores. Population variation showing narrow extremes from ZIKA & FARRAR: BOTRYCHIUM ASCENDENS IN NEWFOUNDLAND 255 species involved in their formation. In our comparison of genetic profiles determined for 22 loci in 10 enzyme systems, plants of B. ascendens from Newfoundland were consistent with those obtained for western plants from throughout the range of B. ascendens, including the type locality in northeastern Oregon. They differed from some (but not all) western populations only in not possessing fixed heterozygosity at Aco-2. This character state is also present in some populations in Oregon and Alaska. As with all plants of B. ascendens, in addition to possessing fixed heterozygosity, Newfoundland plants differed from both B. lineare and B. campestre in possessing many alleles not present in either of those species. Morphological comparison of the Newfoundland Botrychium ascendens with B. campestre and B. lineare is useful in field and herbarium identifications. Slender plants from Newfoundland with narrow pinnae resemble B. lineare (Fig. 1), but tend to have broadened distal segments with more dentate outer margins (Fig. 2). The larger Newfoundland plants (Fig. 4) compare favorably with western specimens of B. ascendens, including plants from the type locality. They show somewhat more highly divided segments than the type collection of B. ascendens from Wallowa Co., Oregon (W. H. Wagner 83363 et al. MICH). However, this variation in morphology is comparable to larger specimens collected subsequently from the type locality, as well as material collected in Washington and Alaska (Fig. 5). There also exist many individuals (not illustrated) with morphology intermediate and transitional between the plants depicted in Fig. 2 and Fig. 4 from Newfound- land, showing increasingly broad and fan-shaped proximal pinnae. Thus, as the plants increase in size, their morphology trends away from the characteristic narrowness of B. lineare n comparison to Botrychium campestre, B. ascendens has less fleshy central axes and has basal pinnae that are more uniformly and broadly fan-shaped. Pinnae of B. ascendens are also more evenly spaced along the rachis of the trophophore and are more regularly and more deeply cleft into spreading lobes (Figs. 2, 4, 5). Botrychium campestre (Fig. 3) sometimes shows partial fusion of adjacent pinnae, and a broadly decurrent basal margin to some of the basal pinnae, features absent in B. ascendens. The outer margins of pinnae and pinnae lobes are dentate in B. ascendens and usually entire to crenulate in B. campestre. The basal pinnae are usually the largest in B. ascendens and _ small plants in Newfoundland. Distal pinnae slender, as in B. lineare, but proximal pinnae WTU). Michigan (Wagner 85024 et al. MICH). E. Siinhancts (Farrar 3495 ISC). F. Nebraska (Farrar 86-6-2- 3D ISC). G. Minnesota (Farrar 3497 ISC). H. lowa (Farrar 952 ISC). I. Type locality, Iowa (Farrar 3484 ISC, on left; Farrar 86-5-31-1 ISC). 256 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) é 2a £ abt 'h py Se rote) Fic. 4. Botrychium ascendens SS SS variation showing large and deeply divided pinnae pi Newfoundland. A. Britton 10671 & Anderson (MICH); all others Zika 16328 (WTU). "IG. rychium ascendens rophophors Typic al arti from western North America. A. Type collection, oF tis (Wagner 83363 et al. CH). B. Type locality, Oregon (Zika 17090 OSC). C. Washington (Larson 254 WTU). D. Aas ‘Smith s.n. ALA). E. Washington (Buege s.n. WTU). “ry — 2m — — ZIKA & FARRAR: BOTRYCHIUM ASCENDENS IN NEWFOUNDLAND 257 B lcm & A Fic. 6. Proposed allopolyploid parentage of Botrychium ascendens. A. Botrychium crenulatum (Farrar 2031 ISC). B. Botrychium ascendens (Farrar 1818 ISC). C. Botrychium lineare (Farrar 3921 ISC frequently bear a few sporangia, whereas a non-basal pair is usually largest in B. campestre, and trophophore pinnae seldom bear supernumerary sporangia. Some B. campestre show basal pinnae erect in the axil between trophophore and sporophore, a feature not observed in B. ascendens. In large, well- developed plants, the non-basal pinnae of B. ascendens often tend to have a broader outer margin. An additional feature helpful in separating Botrychium ascendens from B. campestre and B. lineare is the length of the sporophore stalk, which, in B. ascendens, reaches 1/3 to 2/3 the length of the trophophore. In B. campestre and B. lineare the sporophore stalk is usually % or less the length of the trophophore. It must be noted, however, that this character is useful only in mature plants when (at spore release) the sporophore stalk has ceased elongation. The diploid species, B. campestre and B. lineare, also have smaller spores (34—39 pm) than does tetraploid B. ascendens, from Fogo Island and elsewhere (39-47 wm). All three can bear inconspicuous asexual reproductive gemmae on their subterranean stems (Johnson-Groh et al., 2002). Both B. campestre and B. lineare have been reported from northeastern North America (Kartesz, 1999; Hinds, 2000). Recombinational heterozygosity at a given locus segregates in meiosis and recombines in syngamy to produce a predictable proportion of individuals in a population that are homozygous. Occurrence of heterozygosity in all individuals of a population at a large proportion of loci is best explained as non-recombinational or fixed heterozygosity resulting from allopolyploidy. Plants of B. ascendens from Newfoundland and elsewhere display identical fixed heterozygosity at seven of 22 loci examined, and, in some populations, at four additional loci. Consistent with its chromosome number of n = 90 (Wagner and Wagner, 1986), this strongly indicates that B. ascendens is an allotetraploid derived through ancient hybridization between two diploid species. This is also supported by morphological evidence. The allelic composition of Botrychium ascendens matched an expected composition resulting from hybridization between either B. campestre or B. lineare and B. crenulatum (Table 2). Of the 11 loci displaying only a single 258 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) allele in B. ascendens, this allele is present in both of these putative parental lines (8) or null in B. crenulatum. In the loci displaying fixed heterozygosity, B. crenulatum possesses at all loci the alleles necessary to create, in combination with B. campestre/lineare, the allelic combinations present in B. ascendens. This includes an allele at Mdh-2 that, among diploid species of Botrychium, is found only in B. crenulatum. Close genetic similarity between B. lineare and B. campestre does not allow differentiation between these two species as to the most likely parent, but the B. campestre/lineare lineage is strongly implicated. Botrychium lunaria is the only diploid species other than B. crenulatum possessing the broad pinna shape predicted for the non-B. campestre/ lineare parent of B. ascendens. The American genotype of B. lunaria is homozygous at five loci (Tpi-1, Tpi-2, Mdh-1, Mdh-2, Skdh-1) for alleles not present in B. ascendens, and fails to provide the complimentary allele missing from B. campestre/lineare at an additional locus (Aat-1). The European genotype of B. lunaria is similar to that of B. crenulatum, but does not contain the Mdh-2 allele uniquely present in B. crenulatum and B. acendens (Stensvold 2008). The morphology of Botrychium ascendens is also consistent with parentage of B. crenulatum and B. lineare (Fig. 6). The ascending pinnae and their tendency to be bifurcate likely reflect tendencies inherited from B. lineare. The dentate outer margins of B. ascendens pinnae reflect the intermediacy between the crenulate margins of B. crenulatum and the entire margins of B. lineare. Botrychium lineare and B. campestre occur in both eastern and western North America. Botrychium crenulatum is known only in western North America, ranging in western mountains from southern California and Nevada to southern British Columbia and eastward to central Alberta. If these patterns represent distributions of the parent species at the time of the formation of B. ascendens, eastern Canadian populations of B. ascendens likely result from migration of the species from western to eastern North America. Botrychium ascendens is a fertile tetraploid. Gametophytes from a single spore are capable of producing sporophytes through self-fertilization in Botrychium, thus long distance migration via single spores is possible. The habitat of the plants in Newfoundland is remarkably similar to the back-beach sand dune habitat of B. ascendens and other Botrychium species in south coastal Alaska. A single collection of B. ascendens from the south shore of Hudson Bay (Moir 1444 CAN) suggests the possibility of a broader occurrence of B. ascendens in similar habitats across northern Canada. Both morphological and genetic evidence confirm that Botrychium ascen- dens is extant in the province of Newfoundland, in Fogo District, Fogo Island, off the northeast shore of the island of Newfoundland. We hope this discovery in Newfoundland will encourage botanists in eastern Canada to search for additional extant populations of Botrychium ascendens, a small and inconspicuous species that possibly has been overlooked in coastal and other habitats. ZIKA & FARRAR: BOTRYCHIUM ASCENDENS IN NEWFOUNDLAND 259 ACKNOWLEDGMENTS We would like to express our appreciation to Florence Wagner for her cheerful assistance, which included providing herbarium label details and maps to locate the Fogo Island population. We are grateful to Elizabeth and James Gould for their field assistance under difficult conditions. Funding to visit the type locality for Botrychium ascendens and B. lineare was provided by the Oregon Native Plant Society, Wallowa-Whitman National Forest, and Oregon Natural Heritage Program. Funding for genetic analyses was provided by the USDA Forest Service and by the USDI Fish and Wildlife Service. ous are indebted to the herbaria cited, as well as the curators at MICH, for access to loans and typ LITERATURE CITED Copy, W. J. and D. M. hes 1989. Ferns and Fern Allies of Canada. Research Branch Agriculture Canada, Ottaw Cronn, R., M. E poe K. Kure, J. F. Wenpet and P. K. Brettinc. 1997. Allozyme vowing in domesticated Helianthus annuus and wild relatives. Thee. Appl. Genet. 95:532-54 Hauk, W. D. 1995. A molecular assessment of relationships among cryptic species of pairs Subgenus Botrychium (Ophioglossaceae). Amer. Fern J. 85:375-394 Hauk, W. D. and C. H. Haurier. 1999. Isozyme ee among gil ai species of Botrychium subgenus Botrychium (Ophioglossaceae). Pane, J. Bot. 86:614-6 Hinps, H. R. 2000. Flora of New Brunswick, Ed. Univesity of New a. Fredericton. Jounson-Grou, : C. Rivet, L. ScHogss-er and K. Sxocen. 2002. Belowground distribution and abundance of Apa gametophytes and aes sporophytes. Amer. Fern J. 92:80—92. Kartesz, J. T. 1999. A Synonymized Checklist and Atlas with Biological Attributes for the Vascular Flora of the United States, Canada and Greenland, In: J. T. Karresz and C. A. MEacHaAM, eds ese of the North American Flora, version 1.0, CD-ROM. North Carolina Botanical arden, Chapel Hill. es D. E., C. H. Haurter, D. Darrow and G. Gastony. 1983. Starch gel electrophoresis of teridophytes: a compilation of slr es buffers, gels and electrode buffers, and staining cavede ne ae hive . 73:9-27 STENSVOLD, M. C. . A taxonomic and id aN ips of the Botrychium lunaria complex. Ph. a BEN Iowa State University, Wacner, W. H. and F. S. Wacner. 1986. Three new species er tmoonworts (Botrychium subg. Botrychium) wa deici in western North America. Amer. Fern J. 76:33—4 Wacner, W. H. and F. S. WacNer. 1990. Moonworts (Botrychium subs. kee of the upper Great Lakes region, U.S. A. i Canada, with descriptions of two new species. Contr. Univ. Mich. Herb. 17:313-325. Wacner, W. H. and F. S. Wacner. 1993. Ophioglossaceae. Pp. 85-106. In: Morin, N., ed. Flora of North America North of Mexico, Volume 2: Pteridophytes and Gymnosperms. Oxford University Press, New York Wacner, W. H. and F. S. WAGNER. 1 994. Another widely disjunct, rare and local North American moonwort Ophioglossaceae: Botrychium subgen. Botrychium. Amer. Fern J. 84:5—10. American Fern Journal 99(4):260—272 (2009) Spore Maturation and Release of Two Evergreen Macaronesian Ferns, Culcita macrocarpa and Woodwardia radicans, along an Altitudinal Gradient Maria L. AROSA Institute of Marine Research (IMAR/CMA), Department of Life Sciences, University of Coimbra, O. Box 3046, 3001-401 Coimbra, Portugal Luts G. QUINTANILLA Department of Biology and Geology, University Rey Juan Carlos, 28933 Méstoles, Spain JAIME A. Ramos Institute of Marine Research (IMAR/CMA), Department of Life Sciences, University of Coimbra, Box 3046, 3001-401 Coimbra, Portugal Ricarpo Ceia and Huco Sampaio Sociedade Portuguesa para o Estudo das Aves, Av. da Liberdade 105-2nd, 1250-140 Lisboa, Portugal .—The variables affecting spore phenology have been poorly studied in contrast with the abundant literature on leaf phenology. This paper deals with the influence of altitude and canopy cover on spore maturation and release of Culcita macrocarpa and Woodwardia radicans in the island of Sdéo Miguel, Azores. The study was conducted during one sporing season at three altitudes (400, 600, and 800 m). In both species spore maturation occurred in autumn and may be controlled by the previous accumulation of photosynthates. Spores were not released until late winter owing to a sg cesses ay weather conditions. Dispersal took place later at higher altitude, due to lower d higher humidity. This gradual liberation of spores along an altitudinal gradient is important for the endemic Azores bullfinch Pyrrhula murina (a bird that feeds on spores in winter), providing food over an extended period. Key Worps.—Azores, altitudinal gradient, spore phenology, laurel forest, Culcita, Woodwardia, Pyrrhula, Blechnaceae, Culcitaceae Diaspore (seed or spore) maturation and dispersal play a key role in plant population dynamics. Diaspore dispersal is a prerequisite for the establish- ment of new populations and a vehicle for gene flow between populations (Haufler, 2002). The selective forces that influence the timing of flowering, fruiting and seed dissemination have been widely investigated (Fenner, 1998). In contrast, few studies have dealt with fern spore maturation and dispersal (e.g., von Aderkas and Green, 1986; Durand and Goldstein, 2001; Sawamura et al., 2009). Spore production is affected by several environmental factors, such as temperature, humidity and canopy cover (Odland, 1998; Greer and McCarthy, 2000; Arens, 2001) Altitudinal gradients are powerful ‘natural experiments’ for testing ecolog- ical and evolutionary responses of organisms to abiotic factors. There are two categories of environmental changes with altitude: those physically tied to meters above sea level, such as temperature; and those that are not generally AROSA ET AL.: SPORE MATURATION AND RELEASE ALONG AN GRADIENT 261 altitude specific, such as hours of sunshine (Kérner, 2007). Altitudinal ecological gradients reflect differences in genetic (Herrera and Bazaga, 2008), vegetative (Scheidel and Bruelheide, 2004), reproductive (Dangasuk and Panetsos, 2004) and phenological traits (Schuster et al., 1989). In some fern species, spore production decreases toward higher and lower altitudinal distribution limits (Sato et al., 1989; Odland, 1998). We studied the influence of altitude on spore maturation and dispersal of Culcita macrocarpa C. Presl. (Culcitaceae) and Woodwardia radicans (L.) Sm. (Blechnaceae). These ferns occur in a warm-temperate range that extends discontinuously through Macaronesia (Azores, Madeira and Canary islands), the Atlantic coast of the Iberian Peninsula and, in the case of W. radicans, some locations in the Mediterranean region. Both species are considered relicts of the tropical flora that covered the Mediterranean area during the Tertiary period (Pichi-Sermolli, 1979). Culcita macrocarpa and W. radicans share the same life-form, with large shoots that grow above ground and evergreen leaves over two meters long, which makes them the largest ferns in Europe. Culcita macrocarpa and W. radicans abound in the Azores (Dias, 1996), the wettest and northernmost Macaronesian archipelago, where they occur along a large altitudinal gradient of 300-1000 m for C. macrocarpa and 50-950 m for W. radicans (Schafer, 2002). This makes the Azorean populations a suitable model to study the effects of altitude-correlated environmental factors on spore maturation and release. Additionally the sporangia of these two species are important winter food resources (Ramos, 1995, 1996a) for the critically endangered Azores bullfinch (Pyrrhula murina Godman). This bird is restricted to about 6000 ha in the east of the island of Séo Miguel, from which only 1675 ha correspond to native forest, largely invaded by exotic species (Ramos, 1996b; Ceia, 2008). We compared the spore phenology traits of C. macrocarpa and W. radicans at three altitudes in So Miguel island. Our specific questions were: (1) What are the effects of altitude on temperature, humidity and vegetation cover? (2) Is the timing and success of spore maturation influenced by temperature, humidity and vegetation cover? (3) Does the altitudinal gradient affect the dates of spore release? (4) What are the implications for the conservation of the Azores bullfinch? MATERIALS AND METHODS Study sites.—The study was conducted in Serra da Tronqueira, So Miguel Island, archipelago of the Azores (37°47'N, 25°13'’W). This area is a steep volcanic range with oceanic climate (Marques et al., 2008). Temperatures are mild throughout the year (mean annual temperature 17°C at sea level) and there is no frost. Yearly rainfall increases with altitude varying from ~1500 mm at sea level to >3000 mm at highest altitudes (~1100 m). The canopy of the natural laurel forest is dominated by evergreen trees and shrubs [Erica azorica Hochst. ex Seub., Frangula azorica V. Grubov, Ilex perado Aiton ssp. azorica (Loes.) Tutin, Juniperus brevifolia (Seub.) Antoine, Laurus azorica (Seub.) 262 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Franco, Myrsine africana L., Prunus lusitanica L. ssp. azorica (Mouillef.) ranco, Vaccinium cylindraceum Sm. and Viburnum tinus L. ssp. subcorda- tum (Trel.) P. Silva]. Most of the original forest has been converted to plantations of Cryptomeria japonica (L. fil.) D. Don (300-900 m) or invaded by alien species: Hedychium gardnerarum Sheppard ex Ker-Gwal. (0-950 m), Clethra arborea Aiton (500-900 m) and Pittosporum undulatum Vent. (50- 650 m) (Schafer, 2002). Ferns are rare to absent in patches of dense homogenous exotic vegetation. To study environmental and spore phenology variables (maturation and release), we selected three sites with both C. macrocarpa and W. radicans at 400, 600 and 800 m (hereafter referred to as low, mid and high altitude, respectively). The low altitude site was a laurel forest densely invaded with P. undulatum and Acacia melanoxylon R. Br., whereas, the mid altitude site was a laurel forest moderately invaded by C. arborea. The high altitude site was a laurel forest mixed with Pinus nigra Arn. and C. japonica plantations. At each altitude 12 mature individuals (i.e., with at least one fertile leaf) of each species were randomly selected and tagged, yielding a total of 72 marked individuals (12 individuals x 3 altitudes x 2 species). Environmental variables—Temperature and relative humidity measures were obtained with a thermohigrometer (HOBO Pro v2 logger, Onset Computer Corporation, USA) at each altitude (400, 600 and 800 m). These thermo- higrometers were placed 1.5 m above the ground under tree canopy. Data were hourly recorded for one year starting in April 2007. To determine canopy cover, hemispherical photographs at 1.3 m over each tagged individual fern were taken using a digital camera (Nikon CoolPix 995, Nikon, Japan) with a fish eye converter (FC-E8, Nikon, Japan). Photos were orientated to the magnetic north and horizontally located using a bubble level (Valladares, 2006). Images were processed with Gap Light Analizer 2.0 (Forest renewal BC, Canada). Canopy cover (%) was calculated as 100 — canopy openness (%), the later being percentage of open sky seen from beneath a forest canopy. Spore phenology variables.—From 30 October 2006 to 15 May 2007, the six study populations were visited every ca. 10 days to assess whether timing of spore maturation and release differed with altitude. At the base of a fertile pinna of each tagged individual, two opposite pinnules were marked, one to study spore maturation and the other to study spore release. Maturation was studied by collecting six sori per pinnule in each visit until the beginning of spore release (9 February 2007). Sori were stored in Eppendorf vials to keep sporangia hydrated and avoid spore release. In the laboratory, sporangia were opened with a lancet and their content was observed with a light microscope. A random sample of 400 spores per individual were sorted into three morphological groups: mature, immature and aborted. Mature spores have a two-layered wall, with both perispore and exospore, and their protoplast is fulfilled with lipid drops (Tryon and Lugardon, 1991). Immature spores lack a perispore and oil drops and aborted spores lack a protoplast and/or are collapsed. Spore maturation date was defined as the number of days since January 1 (i.e., Julian days) until an individual possessed 90% mature spores. AROSA ET AL.: SPOR JN AND RELEASE ALONG AN ALTITUDINAL GRADIENT 263 The percent of aborted spores was determined from the spore sample of the visit preceding the spore release date of each individual (see below). Indusia opening was used to estimate the timing of spore release. Both studied species have indusia that completely enclose the sori and as soon as indusia open, most spores are released out of the sori (pers. observation). During each visit we counted on the marked pinnules the number of sori with open indusia. Spore release date was defined as the number of Julian days until 50% of an individual’s indusia were open. Statistical analyses.—Generalized Linear Models (GLMs; McCullagh and Nelder, 1989) were built for the following variables: canopy cover percentage, spore maturation date, spore release date and spore abortion percentage using the GENMOD procedure of SAS 9.0 (SAS Institute, 2002). GLMs were used because these variables departed from the normal distribution. A binomial distribution with logit link function was used for canopy cover percentage and abortion percentage, and a Poisson distribution with log link for maturation date and release date because under these conditions the explained variation was maximal. The explanatory variables considered in the models were fern species (C. macrocarpa and W. radicans) and altitude (low, mid and high); canopy cover percentage was included as a covariate in the maturation date and release date models. All of these variables were considered as fixed effects. Subsequent pairwise comparisons were made using LSMEANS statement of SAS 9.0 (SAS Institute, 2002). The relationship between maturation date and release date was assessed by Spearman’s rank correlation coefficient within each species. This analysis was performed with SPSS 13.0 (SPSS, 2003). Data are shown as mean ~ SE unless otherwise specified. RESULTS Environmental variables.—Temperature decreased with increasing altitude. Yearly means were 15.5°C, 13.6°C, and 12.4°C in the low, mid and high altitudes, respectively. The average altitudinal gradient was —0.78°C/100 m [= (12.4°C-15.5°C)/(800 m—400 m)]. Temperatures were mild throughout the year (Fig. 1A), with only 7°C difference between the warmest (July, August) and the coldest (February, March) months in the three altitudes (F ig. 1A). The winter was frost free, with absolute minimums above 4°C. Relative humidity increased with increasing altitude (Fig. 1B). In the three altitudes monthly means were generally far above 85% although humidity decreased during spring and summer, with absolute minimums below 40%. Canopy cover percentage differed significantly among altitudes but not between fern species (Table 1, Fig. 2). Cover increased in the order: mid < low < high, with 63% + 2, 71% + 3 and 83% + 1, respectively (data for both fern species pooled). Spore maturation and abortion.—At the beginning of the study (October 30) C. macrocarpa had greater than 70% mature spores at the three altitudes (Fig. 3). Woodwardia radicans showed a similar percentage at low altitude, whereas percentages were lower at mid and, especially, low altitudes. This initial difference among altitudes disappeared with time and maturation date, 264 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) > 8 goLow (Mid o High w oa 8 i] oO = hy AYU BY, Q J Temperature (°C) No oO w —_ S | Sel a ae ey oO: °-o ESAS BPO aa a i Relative humidity (%) 8 6 3 10-4 oLow ©Mid o High geen DNs Weis Baca a, TORRE AE bias ay FA RIS Sere A. MJ. JAS ON DF EM 2007 2008 Fic. 1. Monthly temperature (A) and relative humidity (B) at the three study altitudes (mean + absolute maximum and minimum). Climatic data were recorded hourly with thermohigrometers placed 1.5 m above the ground under tree canopy. Bars are the highest and lowest value in each month .e., the number of days before reaching 90% mature spores, was not significantly affected by altitude (Table 1). Canopy cover also did not have a significant effect on maturation date. Differences between species were significant, with earlier maturation in C. macrocarpa [315 Julian days (= November 11) + 4 days, data from the three altitudes pooled] than in W. radicans [345 Julian days (= December 11) + 6 days]. Neither species reached 100% mature spores at the end of the study period (Fig. 3). This was mainly due to existence of some aborted spores in all study individuals. Abortion percentage did not differ significantly between species or among sites (Table 1). Both species had low abortion percentages at the three altitudes (means = 8%). AROSA ET AL.: SPORE MA El JN AND RELEASE ALONG AN ALTITUDINAI, CRADIENT 265 B Summary of GLMs for the effects of species (Culcita macrocarpa or Woodwardia radicans) and altitude (low, mid or high) on canopy cover percentage, spore maturation date, spore abortion percentage and spore rel date. C t idered a WallO in the models for maturation date and release date. S 10py cover a covariate CLUCIldSaoe ignificant values are in bold. d.f. = degrees of freedom. Variable Effect df ie P Canopy cover (%) species 5 01 0.9 altitude 2 54.02 <0.0001 species X altitude 2 1 0.559 Maturation date species 1 12.16 0. altitude 2 5.28 0.071 canopy cover Z 0.49 0.483 species X altitude 2 4.86 0.088 Abortion (%) species 1 0.01 0.922 altit 2 5,02 0.081 species X altitude 2 5.66 0.059 Release date species 1 0.66 0.416 altitude 2 6.11 0.047 anopy cover 1 0.38 0.536 species X altitude 2 0.06 0.968 100 BC. macrocarpa) []W radicans 90 e.¢ Canopy cover (%) oO oO Low Mid High Altitude Fic. 2. Percent canopy cover (mean + SE) for Culcita macrocarpa and Woodwardia radicans at three altitudes. Each mean represents twelve hemispherical photographs taken over each tagged individual (n = 12). Different letters indicate significantly different means (P < 0.05, LSMEANS; SAS Institute, 2002). 266 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Mature spores (%) 3 40 4 30 > 20°71 10 - : é -—O Low-O- Mid -O- High 0 Beer. DTS T ee T t T t i T T 1 BS > > = oO oO o | = oOo ee 1G a ee ee ; Oe 8 © fo eo 8 Se 2006 2007 B 100 - 4 4 = a= so peat ; OLY DO - KO T = 80 - + I de> “ Fe ( ) = wat f: os O ~~ % 8 604 4 O O S O 7) 50 a" @o 4 _— 2 40 = : 0 O e re 8 A : O au 4 10 + + Low-©- Mid -O- High 0 T T T tT T T T TT T T t S > > > oOo oO oO [eg Cc ins pe oS 2 2 8 & & §€ 3 § 2 GE 3 = & & oe & 8 i Ni io gS 2006 2007 Fic. 3. Seasonal changes in turati t SE) for Culcita macrocarpa (A) and Woodwardia radicans -(B) at throe altitudes. Each mean sate twelve permanently marked individuals (n = 12). Four hundred spores per individual plant were observed for each date. GRADIENT 267 AROSA ET AL.: SPORE MATURATION AND RELEASE ALONG AN -1t Low-©- Mid -O- High 100 - A (%) elsnpu! uedQ 2007 +O Low-O- Mid -O- High seW-SO qe4-2z 994-60 uep- Le uep-2z uep-Z1 90Q-0€ 980-02 980-01 AON-62 AON-0Z AON-OL 90-08 100 + (%) eIsnpul uedQ 2007 ‘006 N Fic. 4. Seasonal changes in opening of indusia (percent mean + SE) for Culcita macrocarpa (A) mI 1 41 Peliadhenuy ilatKhea } + + i = 32 zs (48 3h | PR ten Bry —s J. and W individuals (n = 12). One fertile pinnule per individual was observed. 268 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Release took place earlier at low altitude [54 Julian days (= February 23) + 8 days, data of both species pooled] than at high altitude [80 Julian days (= March 21) + 5 days], whereas release date at mid altitude [72 Julian days (= March 13) = 6 days]) was not significantly different from those at high and low altitudes (P > 0.05, LSMEANS). Release date showed a significant positive correlation with maturation date both in C. macrocarpa (r, = 0.530, P = 0.001, n = 35 individuals, Spearman rank correlation) and W. radicans (r, = 0.504, P = 0.007, n = 27 individuals). DISCUSSION Environmental variables.—As expected, we found a decrease in temperature with an increase in altitude. The average altitudinal temperature gradient (—0.78°C/100 m) at Serra da Tronqueira was higher than those of other similar latitude regions. For example, in the Iberian Peninsula the gradient is —0.59°C/ 100 m in the northwestern coast (Carballeira et al., 1983) and in the central mountain range (Wilson et al., 2005). We also found an altitudinal moisture gradient. As in other Azorean islands (Borges, 1999), humidity increased with increasing altitude. Canopy cover varied among the three study sites as a consequence of different forest managements. At low and mid altitudes, the cover of the laurel forests was 71% and 63%, respectively. These percentages are lower than laurel forests in the Canary islands, with >90% canopy cover (Delgado et al., 2007). At the low altitude site, the forest was highly disturbed, with a young native tree canopy invaded by P. undulatum and A. melanoxylon. At mid altitude, the management has opened gaps in the tree canopy where exotic species (mainly C. arborea) have been removed (SPEA, 2007). At high altitude, there was a dense mature C. japonica plantation creating a canopy cover of 83%, the highest of all the sites. Spore maturation and abortion.—We determined the timing of spore maturation on the basis of perispore formation and lipid-drops accumulation. The spores of C. macrocarpa and W. radicans matured by autumn (mean maturation date November 11 and December 11, respectively). In both species spores have a high lipid and caloric content (Arosa et al., 2009), as in ferns in general (Hew and Wong, 1974), but both spore mother cells and spores lack chloroplasts. Consequently, spore production may be largely controlled by the accumulation of enough photosynthates, as suggested for fruit production (French, 1992). In flowering plants, fruiting peaks generally occur during periods of low photosynthetic activity or after periods of high rates of reserve accumulation (Jordano, 1992). In temperate regions, fruiting has a unimodal peak in late summer or autumn (Jordano, 1992). The studied species mature spores in these seasons, as do many other temperate ferns (e.g., Page, 1997; Sawamura et al., 2009), suggesting that production of seeds and spores are governed by analogous selective pressures. Leaf expansion of C. macrocarpa and W. radicans ends in early summer (Quintanilla, 2002). Thus autumn spore maturation depends on photosynthate accumulation during summer and umn. AROSA ET AL.: SPORE MATURATION AND RELEASE ALONG AN ALTITUDINAL GRADIENT 269 Maturation date was neither affected by altitude nor by canopy cover for either species. This indicates that temperature, humidity and light variation along the altitudinal gradient did not constrain resource build-up for spore production. Kérner and Diemer (1987) found that the optimum geben e photosynthesis of lowland herbaceous flowering plants was ~23° temperate climate. In our study, temperatures were frequently close to ee optimum in the three altitudes, especially during the summer. Relative humidity was also high in the three altitudes during the summer and autumn months (means > 85%) and thus water availability must not be a limiting factor. The photosynthetic responses to light have been studied in very few fern species (Page, 2002). Hymenophyllum tunbrigense and H. wilsonii, filmy- ferns that grow on C. macrocarpa shoots in the study area, show photosyn- thetic saturation at low light levels (Proctor, 2003). A similar ecophysiological response in the study species would explain why maturation date was not affected by the differences in canopy cover among sites (maximum 83%, minimum 63%). Spore abortion can indicate environmental stress during sporogenesis. However, both species had few aborted spores at all three altitudes (means = 8%) suggesting that environmental conditions are optimal for their growth. Values were similar to those obtained from Dryopteris spp. (< 10%) in populations in northern Spain (Quintanilla and Escudero, 200 Spore release.—In both species there was a long period between spore maturation (autumn) and release (late winter). Since spore dissemination is the only function of the sporangia, we might expect its phenology to be influenced by selective pressures which would favor successful dispersal. Delayed release could be a strategy to avoid unfavorable winter conditions. However, mean temperatures during winter were 9°C to 12°C depending on altitude (Fig. 1A), which are suitable for spore germination of the studied species (Quintanilla et al., 2000). The long-term causes of the timing of spore maturation and release can also be biotic pressures such as seasonal presence of spore feeders. Some temperate ferns are attacked by a variety of spore-predator insects which occasionally cause severe spore reduction (Sawamura et al., 2009, and references therein). We have not observed spore-feeding insects in the study species but consumption by the Azores bullfinch is significant (Ramos, 1995; Arosa et al., in press). Given that Azores bullfinch consumes mature spores, we have studied its potential disperser role and the results will be reported elsewhere. In short, many droppings contained high amounts of viable (able to germinate) spores of C. macrocarpa and W. radicans and thus may provide a vehicle for dispersal. The pattern of autumn spore maturation and late winter release occurs throughout the range of both ferns (own observation), while the Azores bullfinch is present only in a small fraction (one island). Thus, the negative (predation) or positive (dispersal) interaction with the Azores bullfinch may not be important for determining the timing of spore dispersal. Spore dispersal may be largely influenced by evolutionary constraints. Related plants share similar inherent design constraints which would limit 270 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) their potential evolutionary response to selection (Fenner, 1998). In ferns, both indusia and sporangia openings are passively caused by evaporative forcing. This must be an adaptation to favor long-distance wind dispersal of spores in warm dry days. Culcita macrocarpa and W. radicans released spores a month earlier at low altitude than at high altitude. This is due to the humidity and temperature gradients (see above) that reduce the evaporative forcing at high altitude (Korner, 2007). Delayed spore release may be merely due to the absence of dry weather conditions during most autumn and winter days. Release date was positively correlated with maturation date, i.e., individuals with earlier spore maturation showed earlier spore release, indicating that there is some interdependence between these events. Implications for conservation of Azores bullfinch.—Culcita macrocarpa and W. radicans spores, together with seeds of the exotic C. arborea, are the main winter foods for the Azores bullfinch (Ramos, 1995; 1996a). The birds took the whole sorus, rejecting the indusium. After spore release, sori are not consumed since empty sporangia have negligible nutritional value. Food supply is at its lowest at the end of winter and the mortality of first-year birds appeared greater in this period (Ramos, 1995, 1996b). A gradual release of spores along the altitudinal gradient is important for the maintenance of a stock of spores throughout the winter. We can envisage that the Azores bullfinch distribution should be progressively pushed up to higher altitudes along the winter season following spore availability. For effective conservation of Azores bullfinches, the populations of C. marcrocarpa and W. radicans must be increased along a wide altitudinal range. This is significant in terms of habitat restoration for the Azores bullfinch because present management actions aim to control the expansion of C. arborea (Ceia, 2008). This invasive tree, although important in the winter diet, has a negative effect in spring because it outcompetes the native I. perado and P. lusitanica (the flower buds of both species are the main early spring foods for the Azores bullfinch; Ramos, 1996b). Conclusions.—Culcita macrocarpa and W. radicans have similar timing of spore maturation and release. Maturation is completed in autumn and is not affected by altitude nor by canopy cover. Spore production may be largely controlled by the previous accumulation of photosynthates. Spores of both species are not released until late winter due to a requirement for dry weather conditions. Dispersal occurs earlier at lower altitude, as a consequence of higher temperature and lower humidity. The present study is descriptive and thus cannot accurately establish cause-effect relationships. Transplant exper- iments moving individuals from one habitat to another or experiments manipulating the physical environment experienced by individual ferns will clarify the relative importance of environmental and genetic factors on spore phenology traits. ACKNOWLEDGMENTS We thank Fernando Valladares for useful information on hemispherical photography and four anonymous reviewers for useful comments on the manuscript. This work benefited from the AROSA ET AL.: SPORE MATURATION AND RELEASE ALONG AN GRADIENT 271 logistic support given by the project LIFE NAT/P/000013 “Recovery of Azores bullfinch’s habitat in the Special Protection Area of Pico da Vara/Ribeira do Guilherme’, taking place between October 2003 and October 2008. LITERATURE CITED Arens, N. C. 2001. Variation in performance of the tree fern Cyathea caracasana pice erian across a successional mosaic in an Andean cloud forest. Amer. J. Bot. 88:545— Arosa, M. L., J. A. Ramos, T. VALKENBURG, R. CEIA, H. Lasorpa, L. G. Bk and Go NO. 2009. Fern feeding ecology of the fs bullfinch (Pyrrhula murina): The selection of ra species and the influence of nutritional composition in fern choice. ous Borces, P. A. V. 1999. Plant and Arthropod species composition of sown and semi-natural pasture communities of three Azorean Islands (Santa Maria, Terceira and F Pico). Arquipélago. Life and Marine Sciences 17A:1—21. CarBALLEIRA, A., C. Devesa, R. RETUERTO, E. SANTILLAN soi F. Uctepa. 1983. Bioclimatologia de Galicia. Fundacién Pedro Bars de apa - A ste oru Cea, R. 2008 d da accao F6 do Projecto LIFE Priolo. Sociedade Portuguesa para Oo , Est udo oa Aves, Lisboa Dancasuk, O. G. and K. P. Panetsos. 2004. Altitudinal and longitudinal variations in Pinus brutia (Ten.) of Het Island, Greece: some needle, cone and seed traits under natural habitats. New Forests 27:269—284 Detcapo, J. D., N. L. Arroyo, J. R. AREVALO and J. M. FeRNANDEZ-PaLacios. 2007. Edge effects of roads on temperature, light, canopy cover, bots a. aay in laurel and pine forests (Tenerife, Canary Islands). Landscape Urban P 1:32 Dias, E. 1996. Vegetacgdo Natural dos Agores. Ecologi int ja das florest turais. Ph. D thesis, University of the Azores, Ponta Delgada. Duranp, L. Z. and G. Goipstemn. 2001. Growth, leaf pes and spore production in native and invasive tree ferns in anil Amer. Fern. J. 91:25—35. Fenner, M. 1998. The phenology of growth and dearest in plants. Perspect. Plant Ecol. 1:78-91. FrENcH, K. 1992. Phenology of fleshy fruits in a wet sclerophyll forest in southeastern Australia: Are birds an important influence? Rigid - eae 3. Greer, G. K. and B. C. McCartuy. 200! | juction in a natural population of the fern pee cota rere sens Amer. Fern. J. 9 Haur er, C. H. 2002. Homospory: an odyssey of progress in a ate genetics and evolutionary biology. BioScience 52:1081—1093. M. RRERA, C. M. and P, Bazaca. 2008. Adding a third dimension to the edge of a species’ range: altitude and genetic structuring structuring in mountainous landscapes. Heredity 100:275—285. Hew, C. S. and Y. S. Wonc. 1974. Photosynthesi 1 respirati f ferns in relation to their habitat. Amer. Fern. J. 64:40—48. JorpDANo, P. 1992. Fruits and pron gba dis 105-156, in M. FENNE eeds: the ecology of regeneration in natural plant comn Commonwealth polenta Bureau Internation- al, bilge os Korner, Cx. 2007. The use of ‘altitude’ in ecological research. Trends Ecol. Evol. 22:569-574. Korner, Cu. and M. Diemer. 1987. In situ photosynthetic responses to light, ee and carbon dioxide in herbaceous plants from low and high altitude. Funct. Ecol. 1:179-1 ass Maraues, R., J. Zézere, R. Trico, J. Gaspar and I. Trico. 2008. Rainfall osteres de cal values associated with landslides in Povoagéo Country (Sao Miguel Island, Azores): cag with the North Atlantic _ Hydrol. Process. 22:478—494 McCu.acu, P. and J. A. NELDER. 1989. Generalized nap ssiesnee cope ara and Hall, New York. Ob.anp, A. 1998. Size and ake of Thelypte Ae nee distentifolium along environmental gradients in Western Newey, Nord. J. Bot. 18:3 Pace, C. N. 1997. The ferns of Britain and Ireland, 2"? Ed. Cambridge sf i a Cambridge. 272, AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Pace, C. N. 2002. ioe came in fern evolution: a neopteridological overview. Rev. Palaeobot. Palynol. 1 PicHI-SERMOLLI, R. E. G. on. ms mavey of the pteridological flora of the Mediterranean region. Webbia 34:175-242. Proctor, M. C. F. 2003. Comparative ecophysiological measurements on the light responses, water relations and desiccation tolerance of the filmy ferns Hymenophyllum wilsonii Hook. and H. sagen ae sala Ann, Bot. 971-717-727: QUINTANILLA, L. . Germinaci6n, conservacién de las esporas, fenotipos sexuales y friabiliod es de los helechos amenazados Culcita macrocarpa C. Pres] y Woodwardia eas cans (L.) Sm. Ph. D. thesis, ates of Santiago de Compostela, Santiago de Composte and allotetraploid spenies of Dryoptens Dryopteriacoe Ann. Bot. 98:609-6 QuinraniLta, L. G., S. PajarOn, E. Pancua and J. Amico. 2000. Effect of temperature on germination in northernmost populations of Culcita riocnnie and Woodwardia radicans. Plant Bio 2:612-617. Ramos, J. A. 1995. The diet of the clei bullfinch Pyrrhula murina and floristic variation within its range. Biol. Conserv. 71: deen J. A. 1996a. The aes ae size, oo. and area content on the selection of winter oods by the Azores bullfinch. J. Zool. 238:415—4 Ee J. “i _ 1996 6b. Introduction of exotic tree eee as a threat to the Azores bullfinch population. J. Appt Ecol. 33:710-722. SAS na ete AS/STAT version 9.0. SAS Institute Inc., Car ary. Sato, T., G. GRABH plc K. WasHio. 1989. Quantitative comparison of fern-leaf development and wet te respect to ote in the Tirol, Central European Alps, Austria. J. Biogeogr. pea oy ee Kawakxita and M. Kato. 2009. Fern—spore-feeder interaction in — forests in Japan: Sporing phenology and spore-feeding insect community. Amer. J. Bot. 96:594-604. ScuArer, H. 2002. Flora of the fees A field guide. Margraf, Weikershei serail U. and H. BrugLHeme. 2004. The impact of altitude ee simulated h hisbivoly on the growth carbohydrate storage of Petasites albus. Plant Biol. 6:740—-74 Sia W. S., D. L. Autes and J. B. Mirron. 1989. Gene flow in ihe r pine: Evidence from pollination enemas and genetic differentiation along an elevational transect. Amer. J. Bot. 76:1395-1 SPEA. 2007. pase do habitat do Priolo na ZPE Pico da Vara/Ribeira do Guilherme. LIFE 03NAT/P/000013. Relatério de Progresso, 1 Novembro 2006 a 30 Setembro 2007. Sociedade Portuguesa para o Estudo das Aves, Lisboa SPSS. 2003. SPSS for — heges 13.0.1. SPSS Inc., Chicago Tryon, A. F. and B. Lucarpo 1. Spores of the Pteridophyta. Springer-Verlag, New Yor VALLapaRES, F. 2006. La ek de luz bajo el napa oe los bosques y matorrales théricos na gs mediante fotografia hemisférica. Ecologia 2 vON Aperkas, P. and P, REEN. 1986. Leaf development af the ostrich fern Matteuccia paar (L.) Vodain Bot. J. Linn. Soc. 93:307— Wison, R. J., D. Gutiérrez, J. Gutiérrez, D. MARTINEZ, R ia. nd V. J. MonserrRAT. 2005. Changes to the elevational limits and extent of species ranges SS with climate change. Ecol. Lett. 8:1138—1146. American Fern Journal 99(4):273—278 (2009) Nutrient Levels Do Not Affect Male Gametophyte Induction by Antheridiogen in Ceratopteris richardii Asya AyrapeTov® and MicHaeEL T. GANGER Department of Biology, Gannon University, 109 University Square, Erie, PA 16541-0001 Asstract—In the homosporous fern Ceratopteris richardii, sex is not determined chromosomally. Rather, hermaphroditic gametophytes produce a hormone called antheridiogen, which induces maleness in undifferentiated gametophytes. The percentage of males increases with increasing density of te phytes, presumably due to the cumulative effect of antheridiogen from multiple hermaphrodites Some have argued that antheridiogen lessens competition between gametophytes. Such competition is expected to be most intense between hermaphrodites given that they support zygote, embryo, and LS growth. Therefore, it is predicted that at lower nutrient levels, the Gametophytes were grown for four weeks at 28 degrees Celsius with a photoperiod of 14 L: 10 D. An ANCOVA found an overall positive relationship between gametophyte density and percentage of male gametophytes. However, the relationship between gametophyte density and percentage of male gametophytes did not differ among nutrient levels. Nutrient levels had no effect on the rate of male induction by antheridiogen. A post-hoc power analysis showed that the experimental power was 97%. Key Worps.—Ceratopteris, sex determination, gametophyte, antheridiogen The gender of sexually dimorphic plant species may be determined genetically, environmentally, or by a combination of the two (Lloyd and Bawa, 1984; Meagher, 1988). If sex in a species is determined by environmen- tal factors, males should be more common in low-resource environments (Schlessman, 1988). In flowering plants, this occurrence has been explained by a higher cost of producing ovules, seeds, and fruits versus producing pollen (Lovett Doust and Harper, 1980; Lovett Doust and Lovett Doust, 1983). A similar tendency is seen in seedless vascular plants, in which the female/ hermaphroditic gametophytes endure a higher reproductive cost because they are responsible for producing the egg, the zygote, and the embryo, as well as supporting the developing sporophyte (Sakamaki and Ino, 1999). Therefore, if sex determination in seedless vascular plants is analogous to flowering plants, then resource availability in the environment may be expected to have a direct effect on sex determination of gametophytes (Sakamaki and Ino, 1999). trent ycobing Department of Botany and Plant Pathology, Purdue University, West ane IN 479 274 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Specifically, males should be more common in environments with low resource levels. Sex determination in the homosporous fern Ceratopteris richardii Brongn. is plastic. Spores develop into gametophytes that are either hermaphroditic, containing both archegonia and antheridia, or exclusively male, containing only antheridia (Banks, 1997). Developing and mature hermaphrodites produce a gibberellin-like hormone (Warne and Hickok, 1989) called antheridiogen (Acg in C. richardii; Banks, 1997). Acg induces maleness in developing gametophytes (Banks, 1997), provided that exposure occurs between the third and the sixth day after spore inoculation (Banks et al., 1993; Eberle et al., 1995). In the absence of Ace, spores develop into hermaphrodites (Banks, 1997). This model of hormonally determined sex is not exclusive to C. richardii but is fairly common to many homosporous ferns (Haig and Westoby, 1988). Multiple experiments have demonstrated that spore density has an effect on sex determination in C. richardii (Warne and Lloyd, 1987; Hickok et al., 1995; Spiro and Knisely, 2008), as well as in another homosporous fern Osmunda cinnamomea L. (Huang et al., 2004). More specifically, the sex ratio is skewed toward males at high gametophyte densities. One explanation for this observation is that at higher densities, undifferentiated gametophytes are clustered closer together within the neighborhood of multiple hermaphrodites and are thus exposed to the cumulative effects of Acr- This hypothesis has indirect, empirical support in that experimentally elevating exogenous antheridiogen leads to male-skewed sex ratios in C. richardii (Warne an Hickok, 1991) and other species of homosporous ferns (Cousens and Horner, 1970; Stevens and Werth, 1999; Huang et al., 2004). However, there is a maximum concentration of antheridiogen above which increasing the amount of the pheromone has no additional effect on sex ratios (Cousens and Horner, 1970; Stevens and Werth, 1999; Huang et al., 2004). An alternative hypothesis for the skewed sex ratios with increasing density relates to the resource cost of being male versus hermaphroditic. If the sex of the gametophytes is based on the resource state of the environment, or if the resource state alters the effects of exogenous Acg, then the same pattern of gametophyte density and sex ratios would be expected: a higher ratio of males in low nutrient level culture and a lower ratio of males at higher nutrient levels for similar gametophyte densities. In high density cultures, resource levels per gametophyte are expected to be lower, while in low density cultures, resource levels per gametophyte are expected to be higher. The objective of this experiment is to determine whether the proportion of hermaphrodites in a culture of a given density may be altered by the level of nutrients. MATERIALS AND METHODS Ceratopteris richardii Petri dish cultures were established on nutrient agar following Hickok and Warne (2004) using wild type C. richardii spores and powdered media obtained from Carolina Biological Supply Company. AYRAPETOV & GANGER: NUTRIENTS AND MALE GAMETOPHYTE INDUCTION 275 Four experimental treatments were established using serial dilutions; 100% nutrient level, 50% nutrient level, 25% nutrient level, and 12.5% nutrient level. These nutrient levels are relative to the maximum level found in the powdered media. For a detailed list of nutrient components in media see Hickok and Warne (2004). Spores were sown on 35mm X 10mm Petri dishes containing the four nutrient treatments. Spore densities ranged from five to 50 spores per Petri dish and increased at increments of five he a This yielded an overall density of 0.52 spores/cm’—5.2 spores/cm*. Each of the four nutrient-level treatments contained 40 Petri dishes (four at each of the 10 spore densities) for a total of 160 dishes. Spores were incubated at 29 + 3°C under grow lights (24 W/m’) for 25 days following a 14 hours day/10 hours night cycle. At that time, determination of the sex of a high proportion of gametophytes was possible. The number of hermaphrodites, males, ungerminated spores, and spores of indeterminate sex were recorded. Hermaphrodites contain archegonia, antheridia, and a notch meristem, giving them a mitten-shaped appearance, whereas males contain antheridia, and are essentially oval (Banks, 1997). The shape of the gametophyte was the primary characteristic used for identification. The percentage of hermaphrodites in each Petri dish was determined by dividing the total number of hermaphrodites by the sum of the males and the hermaphrodites. To determine if nutrient levels affected the proportion of hermaphrodites, an Analysis of Covariance (ANCOVA) was performed using SYSTAT (Wilkins, 2002). This analysis determined 1) whether the density of gametophytes was related to the proportion of hermaphroditic gametophytes and 2) whether the different nutrient level treatments had an effect on this relationship. The ANCOVA model included nutrient level as a categorical variable and density of gametophytes as a covariate. After testing for the homogeneity of slopes (.e., the nutrient level* density of gametophytes interaction), the mean squares and degrees of freedom were excluded from the analysis (pooled into the error term) if p > 0.05. With the lack of significance for the ANCOVA, a Power Analysis using G*Power (Faul et al., 1996) was performed to assess the overall power of the experiment, as well as the probability of making a type II error (f). A type Il error is one in which no effect is detected when an effect really exists (Winer et al., 1991). The effect size necessary for power calculations followed Winer et al. (1991). RESULTS Severe agar desiccation in two Petri dishes made it impossible to determine the sex of the gametophytes. As a result, 158 Petri dishes were used in the statistical analysis. inety-nine percent of the gametophytes were identified as either males or hermaphrodites. The slopes of the covariate interactions (nutrient level*den- 276 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 0 10 20 30 40 50 Gametophytes / 10 cm? level; open square, dotted line = 50% nutrient level: open circle, dot-dashed line = 25% nutrient level; closed triangle, dashed line = 12.5% nutrient level. sity of gametophytes) were homogeneous (F359 = 1.932; p = 0.127). There- fore, the final analysis did not include this interaction term. There was an overall relationship between the density of gametophytes and the proportion of hermaphroditic gametophytes (Figure 1; p < 0.001, Adjusted R* = 0.353), in which the proportion of hermaphrodites declined with increasing gametophyte density. There was no effect of nutrient level on this relationship (F3 459 = 1.113, p = 0.346). The statistical power to assess whether nutrient levels affected the relationship between the density of gametophytes and the proportion of hermaphroditic gametophytes required the calculation of an effect size. Effect size was determined to be 0.357 following Winer (1991). The probability of making a type II error, 8, was determined to be less than 3%, making the power of this test greater than 97%. DISCUSSION Similar to previous studies of Ceratopteris richardii (Warne and Lloyd, 1987; Hickok et al., 1995; Spiro and Knisely, 2008), this experiment demonstrated a negative relationship between gametophyte density and the proportion of AYRAPETOV & GANGER: NUTRIENTS AND MALE GAMETOPHYTE INDUCTION 277 hermaphrodites that developed. Though this relationship was highly signif- icant (p < 0.001), the adjusted R* was 0.353, indicating that only 35.3% of the variation in the proportion of hermaphrodites is explained by the variation in gametophyte density. The large amount of unexplained variation (64.7%) indicates that factors other than the presence or absence of Aczg likely influence sex determination in this species. Indeed, hermaphrodites have been shown to develop from young C. richardii gametophytes even in the presence of substantial antheridiogen (Warne and Hickok, 1991) or hermaphrodites (Sayers and Hamilton, 1995). Other factors have been shown to override the effect of Acg in C. richardii. Light quality, biased toward red light, suppresses male development in favor of hermaphroditic development (Kamachi et al., 2007). Smaller spores tend to germinate later than larger spores, with the gametophytes of smaller spores tending to grow more slowly and to develop into males (Sayers and Hamilton, 1995). While nutrients have been shown to affect sex ratios in at least one other fern, Dryopteris filix-mas (L.) Schott. (Korpelainen, 1994), no such effect was shown here for C. richardii. The nutrient levels used in this experiment did not alter the effectiveness of Acg. Similar proportions of hermaphrodites were observed in cultures with similar gametophyte densities regardless of nutrient levels. The experimental power of this experiment was high (> 0.97) and therefore the probability of incorrectly concluding that nutrients have no effect on the proportion of hermaphrodites at similar densities is quite low (< 0.03). The possibility that nutrient levels used in this experiment were not limiting cannot be discounted, although the lowest nutrient level used was 12.5% of the maximum. It is also possible that even though nutrient levels are not important in determining gametophyte gender, they are important to gender allocation within gametophytes. The relative number of antheridia and archegonia in hermaphrodites, or the number of antheridia present in the male may change in response to nutrient levels in the environment. The period of susceptibility to Acg for undifferentiated gametophytes is between 3 and 6 days (Banks et al., 1993). It is possible that at this young age, the nutrient quality of the environment is not discernable by the gametophyte. Therefore, other indicators of environmental quality, such as light, may be more likely to factor into sex determination. AACKNOWLEDGMENTS The authors thank G. Andraso and two anonymous reviewers for their helpful comments. This research was supported in part by the Biology Department at Gannon University. LITERATURE CITED Banks, J. A. 1997. Sex determination in the fern Ceratopteris. Trends in Plant Science 2:175—180. Banks, J. A., L. Hickox and M. A. Wess. 1993. The programming of sexual phenotype in the homosporous fern Ceratopteris richardii. International Journal of Plant Sciences 154:522—534a. 278 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Cousens, M. I. and H. T. Horner Jr. 1970. yponeag ta ontogeny and sex expression in Dryopteris 3-—Z ri EBERLE, J., J. NEMACHECK, C.-K. WEN, M. HaSEBE ae AS ANKS. 1995. Ceratopteris: A model system for eed sex- determining mechanisms in plants. International Journal of Plant Sciences 156:359-36 FAUL,-E., 5: sia A.-G. Lane and A. Bucuner. 2007. G*Power 3: A flexible statistical power analysis program - the social, behavioral, and biomedical sciences. Behavior Research Methods 39:175-19 Haic, D. and M. Westosy. oer Sex erpreahan in homosporous ferns: An evolutionary perspective. Evolutionary Trends in Plants 2 Hicxox, L. G. and T. R. Warne. 2004. C- Foes Manual. Carolina Biological Supply Company. Hickok, L. G., T. R. Warne and R. S. Friourc. 1995, The biology of the — Ceratopteris and its use asa ree system. International Journal of Plant Sciences 15:332—345 Huan, Y.-M., H. M. Cuou and W.-L. Cuovu. 2004. Density affects Pecks growth and sexual Oo eR ee of Osmunda cinnamomea (Osmundaceae: Pteridophyta). Annals of Botany 1229-232. ce H., O. Iwasawa, L. G. Hickox, M. Nakayama, M. Nocucut and H. INouE. 2007. The effects of light on sex nearest in gametophytes of the fern Ceratopteris richardii. Journal of Plant Research 120:629-634. KorPELAINEN, H. 1994. Growth, sex determination and reproduction of Dryopteris filix-mas (L.) Schott gaan N under varying nutritional conditions. Botanical Journal of the Linnean Society 114:357—366. Lioyp, D. G. and K oe oan 1984. Modification of the gender of a plants in Heiss conditions. Pp. 255-338, in Hecut, M. K., B. Watace and G. T. PRrance (e Biology. Berlin: n : Lovett Doust, J. N. and J. L. Harper. 1980. The r der and maternal support in an andromonoecious umbellifer, Smyrnium olusatrum. New oe 85:251-264. Lovett Doust, J. and L. Lovetr st. 1983. Paternal strategy: gender and maternity in higher sont npr ga: 180-18 P Sayers, A. and R. Hamitron. 1995. The effect of neighbors on gametophyte development in — richardii. American chi Journal 85:47-53. ScHLESSMAN, M. A, 1988. Gender diphasy (‘‘sex choice”). Pp. 139-153, in Loverr Dousr, —e Doust (eds.). Plant fee Ecology: pitied and Strategies. Oxford Uaiverdty SPIRO, M. D. and K. I. KnisELy. 2008. Alternation of generations and experimental design: A guided- inquiry lab exploring the nature of the her1 developmental mutant of Ceratopteris richardii (C-Fern). eet Life Sciences Education 7:82-88. Stevens, R. D. and C. R. WertH. 1999. Int terpopulational comparison nd eee ais antheridiogen response in Onoclea sensibilis. American Fern Journal 89:2 is Warne, T. R. and L. G. Hickox. 1991. Control of sexual development in ene of Pinhopeiee: richardii: Antheridiogen and sehen ie I seseceiaiee — ied 148-15 53. Warne, T. and L, Hickox. 1989. Evidence for gibbere antheridiogen. fee falas 89:535-— 538 Warne, T. R. and R - 1987. Gametophyte density and sex expression in Ceratopteris. Canadian ae, oY pita 65:362-365. Witkinson, L. 2002. SYSTAT: The system aes statistics. Evanston, IL: SYSTAT, Inc Winer, B. J., D. R. Brown and K. M. MicueLs. 1991. Statistical Principles in Experimental Design, 3"¢ edition. McGraw-Hill, Inc. New sare American Fern Journal 99(4):279—291 (2009) Transplanting Tree Ferns to Promote Their Conservation in Mexico ANA ALICE ELEUTERIO* Postgrado en Ciencias Biolégicas, Universidad Nacional Autonoma de México, Aptdo. Post. 27-3, Xangari, CP 58089, Morelia, Michoacan, México Centro de Investigaciones en Ecosistemas, Universidad Nacional Auténoma de México, Aptdo. ost. 27-3, Xangari, CP 58089, Morelia, Michoacan, México DiEGO PEREZ-SALICRUP Postgrado en Ciencias Biolégicas, Universidad Nacional Auténoma de México, Aptdo. Post. 27-3, Xangari, CP 58089, Morelia, Michoacan, México Asstract.—Adult tree ferns of the genera Cyathea and Alsophila are frequently harvested from hope forest remnants near the city - Caen — cist is 1 artisans use the surround t In this region, tree ferns regenerate abundantly in disturbed areas such as roadsides, in which they suffer high mortality due to weeding and other road maintenance activities. Transplantation of young tree ferns from these areas to safe sites could contribute to the ex situ conservation of the species. The income. We identified and estimated the abundance of all tree fern species that occurred along roadsides in this region. We evaluated the viability of transplanting young tree ferns of Cyathea divergens and Alsophila firma to different conditions of light availability. While only 30% of the individuals naturally growing along roadsides survived for 1 year, C. divergens transplants experienced 73.3 and 86.7% survival and A. firma transplants experienced 93.3 and 40% survival when agaiveg in — cep Bader cen canopy and in 50% shade, respectively. Transplants of C. diverge faster in height than transplants of A. firma. individuals of she oo transplanted to 50% shade produced more fronds and grew faster than conspecifics transplanted to open canopy areas. Transplantation proved to be a low time- and cost-demanding strategy to promote conservation of native tree fern populations while providing local people with a potentially profitable alternative to replace handicraft production. Key Worps. ae ice Alsophila firma, management, Mexico, transplantation, tree fern, tropical montane fore, Disregarding law prohibitions, artisans in the region of Cuetzalan, Mexico harvest the stems of adult tree ferns of at least two species, Cyathea divergens var. tuerckheimii R.M. Tryon, and C. fulva M. Martens et Galeotti, to produce handicrafts (Eleutério and Pérez-Salicrup, 2006). Both species are listed in the exican law as threatened by land-use and land-cover changes (SEMARNAT, 2000). Adult tree ferns are mostly harvested from natural populations that occur in remnants of tropical montane forests. These forests are among the most * Corresponding author current address: Department of Botany, University of Florida, PO Box 118526, Gainesville, FL 32611, USA; e-mail: anaalice@ufl.edu 280 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) endangered ecosystems in Mexico (Diario Oficial de la Federacién, 2001; Luna et al., 2001). As a result, harvest contributes to increased mortality rates and jeopardizes the future regeneration of native tree ferns in forests that are already vulnerable (Eleutério and Pérez-Salicrup, 2006). Although tree fern species are often restricted to shaded and high moisture sites, at least three Cyathea and one Alsophila species commonly establish and grow in ruderal habitats, such as roadsides, near Cuetzalan (pers. obs.). Therefore, any management policy aiming to preserve tree fern species in this region must consider their occurrence in different habitats (Werth and Cousens, 1990), from forest remnants to disturbed areas. However, no study has yet documented the establishment requirements of tree fern species in this region. Moreover, few studies have looked at management requirements for conserving the 13 Cyathea spp. that are currently considered endangered by the Mexican law (Diario Oficial de la Federacién, 2001; Bernabe et al., 1999). Our study focused on providing basic information to allow the ex situ conservation of endangered species of the family Cyatheaceae. Tree ferns are usually propagated ex situ through spores, vegetative tissues (e.g., Finnie and Staden, 1987; Suzuki et al. 2005), and occasionally, by planting fronds (e.g., Cibotium splendens (Gaudich.) Krajina ex Skottsb.; Hensley, 1997) or the apical meristems of harvested adults (e.g., Dicksonia antarctica Labill.; Forestry Commission, 2001). Although not evaluated for the tree fern species that occur in the region of Cuetzalan, transplantation of seedlings and young plants is commonly performed to promote ex situ conservation and in situ enrichment planting of endangered species (Primack, 2002). Transplants are more likely to successfully establish and grow when environmental conditions of the sites where they naturally occur are similar to the ones of the areas they are relocated to (Jones and Hayes, 1999; Montalvo and Ellstrand, 2001). For the species of Cyatheaceae native to the region of Cuetzalan, transplantation from roadsides to safe sites could be an important strategy for ex situ conservation. Along these roadsides, young tree fern sporophytes (< 50 cm tall) normally experience high mortality rates due to annual cutting of herbaceous vegetation for road maintenance (pers. obs.). A bank of sporo- phytes could be created by transplanting them from exposed to protected areas under adequate environmental conditions. If tree fern transplantation proves successful, cost and time associated with spore or gametophyte germination could be avoided. The sale of young tree ferns for horticultural purposes could potentially substitute for income obtained by selling handicrafts fashioned from tree fern adventitious roots, and consequently contribute to their in situ conservation. To evaluate the feasibility of using transplants of Cyathea and Alsophila spp., extracted from roadsides for ex situ conservation, we first documented the tree fern species that naturally established along the margins of a major road used to access the city of Cuetzalan. We then transplanted young tree ferns to sites subjected to different light conditions to compare their survival and growth rates among species and treatments. Based on these data, we provide basic guidelines for tree fern conservation in the study region. ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 281 TaBLE 1. Maximum stem height (m) of adults and altitudinal ranges (meters above sea level) in which the studied species of tree ferns typically grow. Adult maximum Habitat Altitudinal Species stem height (m) Range (m.a.s.1.) Cyathea divergens var. tuerckheimii 12 450-2400 Cyathea fulva 12 800-2700 Alsophila firma 10.5 750-2000 Cyathea costaricensis 8 250-750 METHODS Study site—This study was conducted in the vicinities of the city of Cuetzalan (20° 01’ 33” N-97° 31’ 37” W), in the northern region of the state of Puebla, central-eastern Mexico. Study areas were located at elevations ranging from 500 to 1470 meters above sea level (m.a.s.l.). Annual precipitation averages 4141 mm, with all months receiving > 100 mm of rainfall. Mean annual temperature is 19.4°C, ranging from 14.3°C in January, to 22.9°C in June (Instituto Mexicano de Tecnologia del Agua, 2000). The landscape is dominated by shade coffee plantations with diversified overstory tree canopies, and tropical montane forest remnants. Study species.—Three species of the genus Cyathea and one species of the genus Alsophila were found along roadsides in a 670-1420 m altitudinal range: C. divergens var. tuerckheimii, C. fulva, C. costaricencis (Mett. ex Kuhn) Domin. (Mickel and Beitel, 1988), and Alsophila firma (Baker) D.S. Conant (Mickel and Smith, 2004). All species are protected by Mexican law (Diario Oficial de la Federacién, 2001). Cyathea spp. typically have trunks that range from approximately 10 cm diameter at breast height (DBH) to approximately 130 cm with the mantle of adventitious roots. Stems and stipes are scaly, and stipes may present spines (Mickel and Beitel, 1998). Adult stems are occasionally bent due to mechanical damage and to the posterior recovery of vertical growth (Seiler, 1981). Adults of both C. divergens var. tuerckheimii and C. fulva may present 12 m tall stems and grow in sites between 450-2400 and 800-2700 m.a.s.l., respectively. Cyathea costaricencis may grow to 8 m tall in relatively drier environments usually located between 250 and 750 m.a.s.l. (Table 1; Mickel and Beitel, 1998). Species of the genus Alsophila also present scaled stems and stipes, but petiole scales have characteristic apical setae (Korall et al. 2007). Most species grow between 1000 and 2000 m.a.s.1. of elevation, rarely occurring at altitudes below 250 m.a.s.l. Adults of A. firma grow up to 10.5 m tall, and are typically encountered between 750 and 2000 m.a.s.l. (Table 1). Stems may branch by adventitious buds (Mickel and Smith, 2004; Tryon and Tryon, 1982). Abundance of tree ferns along roadsides——We counted, identified and measured the height of all tree ferns taller than 0.5 m growing within distances = 2 m from the pavement along 16 km of the main highway to access the city of 282 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Cuetzalan. This highway connects the region to the state’s capital city, Puebla. We assigned each identified tree fern to the following five height categories: 0.5-1.0, 1.1—2.0, 2.1—-3.0, 3.1-4.0, and > 4.0 m. In addition, we divided the tree ferns encountered along the roadsides into three altitudinal ranges: 670-920 m.a.s.1., 921-1170 m.a.s.l., and 1171-1420 m.a.s.l. We only sampled tree ferns above 0.5 m in height because we were interested in quantifying the abundance of individuals that had successfully established along roadsides. Experimental transplants.—In October 2003, we collected tree ferns of the two species that presented a high number of individuals with a stem height between 10 and 50 cm, encountered within a 700-950 m.a.s.l. altitudinal range. Thirty C. divergens and 30 A. firma plants ranging 17-50 cm tall were excavated with spades from roadsides. Tree ferns were extracted with their entire root systems and approximately 1000 cm? of local soil. Plants within this altitudinal range were selected to minimize environmental heterogeneity between the site they were extracted from and the ones they were transplanted to, which were located at 700 m.a.s.]. Fifteen transplants of each species were planted into a 50% shade greenhouse, and fifteen were planted in an open (full sun exposure) garden. Plants were transplanted within two hours into holes 20-30 cm diameter and 30 cm depth. Holes were filled with local soil mixed with organic compost. We cut all fronds with fully expanded pinnae to minimize transpiration. We marked all fiddleheads (i.e., emerging leaves) at the beginning of the experiment and in subsequent censuses. To evaluate growth rates we measured stem height (to the nearest 0.5 mm), from the base of the newest crosier to the soil surface, monitored frond production every 2.5 months for 1 yr (from October 2003 to November 2004), and reported mean values + SE for the study period. Fronds with = 10% green tissue were considered alive (sensu Durand and Goldstein, 2001). To investigate the mortality of tree ferns along roadsides, in March 2003 we randomly selected 60 tree ferns < 30 cm tall of each C. divergens and C. fulva, the two species that presented a higher number of individuals in this size category, growing in altitudes ranging from 900 and 1200 m.a.s.l. This altitudinal range was selected to include the extension of roadsides that would be weeded during the period we performed our study. To verify the effect of the transplantation procedure, we used a spade to extract half of the plants of each species with their entire root systems. We subsequently replanted each plant into the same spot they had been extracted from (henceforth called transplanted control individuals). We d stem heights before transplanting, and cut all mature fronds with fully expanded pinnae to reduce water loss. We monitored plant survival for both transplanted control individuals and non-transplanted individuals, every 3 mo from March 2003—April 2004. We used failure time analyses to compare survival rates between species and between treatments in both experiments (Fox, 2001). We performed Cox proportional hazard model and log-rank tests (Pyke and Thompson, 1986) with SPlus 6.0 (Insightful Corp. Seattle, USA). We used linear and quadratic regressions between the initial and final stem heights to determine the model ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 283 40 Mmm 05m-1.0m Ma 1.14m-2.0m Mm 2.1m-3.0m a Stn 40m 30:4 Mmm >40m Number of tree ferns / size category 3 3 C. divergens C. fulva A. firma C. costaricensis Species Fic. 1. Number of tree ferns in several size categories found for the three species of Cyathea and one species of Alsophila encountered along the roadside near the city of Cuetzalan del Progreso. that best fit the patterns of stem growth (Sokal and Rohlf, 1995). Initial and final stem heights were linearly related, and therefore we performed a Pearson correlation analysis to evaluate the relationship between initial stems height and total number of fronds produced. We compared growth and frond production between species and treatments using a two way ANOVA without replication. RESULTS Abundance of tree ferns along roadsides.——Cyathea divergens and C. costaricensis were the most abundant tree fern species sampled along roadsides, with 96 and 34 individuals, respectively. Both species were represented in all height categories (Fig. 1). Only twelve plants of C. fulva and four of A. firma > 50 cm were encountered within the sampled area. Few plants taller than 3 m of both species were recorded (Fig. 1). Cyathea divergens was abundant along the whole altitudinal range sampled, while all the 34 plants of C. costaricensis were sampled within altitudes between 670 and 920 m (Fig. 2). Less than 10 individuals of C. fulva and A. firma were sampled in the whole study area. A. firma was restricted to altitudes between 921-— 1170 m, while C. fulva was only recorded in the other two altitudinal ranges. 284 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Gimme 670 - 920 mas. Gm 921-1170 m.as.l. Gm 1171-1420m.as.l. : . oO nl NO w oO oO n n Number of tree ferns / size category =) C. divergens C. fulva A. firma C. costaricensis Species Fic. 2. Number of tree ferns of the four studied species by altitudinal range growing along the roadside. Survival analyses.—While C. divergens experienced 73.3 and 86.7% survival, A. firma experienced 93.3 and 40% survival after 1 yr in open canopy and 50% shade, respectively. Survival curves did not differ between species (Fig. 3; log rank, x” = 1.2, df = 1, P> 0.25) or light treatments (Fig. 3; log rank, 77 = 3.2, df = 1, P > 0.05). Survival rates were not affected by the initial height of stems (z = —1.18, P > 0.2), species (z = 1.58, P > 0.10), or light treatment (z = —1.81, P> 0.05) (likelihood ratio test = 6.58, df = 3, P > 0.05). Tree ferns of C. divergens transplanted to safe sites, whether open or shaded areas, survived more than those left on roadsides (control and transplant control; see Figs. 3 and 4). Less than 50% of the transplants of both C. divergens and C. fulva survived for more than 6 mo when left along the roadsides. After 1 yr from the beginning of the experiment, approximately 37% of the control individuals of C. fulva survived, while less than 10% of C. divergens, or of the plants assigned to the other treatments, survived (Fig. 4). In general, plants kept as controls had greater survival than the controls for the transplantation method. Growth analyses.—Stem growth in height was between 4—5 mm/mo either in the sun or shade treatments and for both studied species in the first months after transplantation. Frond production was lower in the first three months than subsequent months (approximately 0.6 fronds/mo, in comparison to the 1.0—1.2 fronds/mo observed during the subsequent period). For these reasons, we decided to use data from the whole censused period to calculate total stem growth and frond production. Mean height growth rates for C. divergens were 8.0 + 1.0 mm/mo in open canopy, and 13 + 2.1 mm/mo in 50% shade. Mean ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 285 1007 @&——_ ¥ v ¥ e 90 - 80 + BS a = a @ 60- 20 7 —@— C. divergens shade 40 —O- C. divergens sun a —v— A. firma shade —L— A. firma sun 30 T T qT t T T 0 2 4 6 tee) ooh oO = N time (months) Fy Survival curves of transplanted Cyathea divergens and Alsophila firma individuals to a 50% shaded greenhouse and an open common garden near Cuetzalan del Progreso, Puebla. height growth rates for A. firma were 2.0 + 1.4 mm/mo in open canopy and 6.0 + 0.8 mm/mo in 50% shade. Height growth rates were higher for C. divergens than for A. firma (Fig. 5(A); ANOVA, F, 4, = 23.0, P< 0.001), and were higher under shade than in sunny conditions (Fig. 5(A); ANOVA, F,.4, = 9.0, P < 0.001). Frond production was not related to initial stem height (C. divergens: Pearson, r< 0.65, P > 0.5 in shade and r< 0.5, P> 0.5 in sunny conditions; A. rma: Pearson, r < 0.6, P > 0.25 in shade and r = 0.295, P > 0.95 in sunny conditions). Consequently, this variable was not considered as a covariate in the comparisons between species and treatments. Individuals of C. divergens produced 13.9 + 0.85 and 15.5 + 0.89 fronds/yr in open canopy and 50% shade, respectively, while A. firma individuals produced 6.8 + 0.97 and 10.4 + 0.53 fronds/yr in open canopy and 50% shade, respectively. Cyathea divergens produced more fronds per yr than A. firma (Fig. 5(B); ANOVA, F; 35 = 47.5, P< 0.001). Both species produced more fronds under 50% shade than in open canopy (Fig. 5(B); ANOVA, F,.3; = 7.8, P < 0.001). DIscussION Cyathea divergens was the most abundant tree fern species found along roadsides near Cuetzalan. This might result from a higher abundance of the 286 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 100 4 —@®— C. divergens - contro —O— C. divergens - parte (transplant) —v— C. fulva - control —A— C. fulva - control (transplant) 80 + S = 60 - = “a a ® 40; 20 - 0 T T T T T T T time (months) G.4. Survival of transplanted Cyathea spp. under two experimental treatments in Cuetzalan del Progreso, Sesmpi ——e Leper w: ) control ~ ‘individuals marked ” roadsides; (2) Ww they were extracted. species in the region, together with an adaptation to environmental conditions in disturbed areas. In contrast, we encountered only young individuals (< 1.0m in stem height) of A. firma, all located within an intermediate altitudinal range. Apparently, the conditions experienced in disturbed areas are not appropriate for the establishment and long term survival of this species. All studied species, except C. divergens, were unevenly distributed across altitudinal ranges. Air humidity and temperature, soil moisture content, and other environmental conditions that vary with elevation may restrict the habitat range of the less adaptable species. This distinct species’ abundance and the Lobe oe the number of tree ferns sampled in each altitudinal range have to be for selecting proper sites for extracting seedlings or transplanting them. In addition, species’ abundances in natural populations and in a broader range of disturbed areas should be assessed in order to provide reliable management and conservation strategies for tree ferns in the area. A higher number of transplants of C. divergens survived when planted in safe sites, whether they were located in sunny or shade conditions, compared to the controls left along roadsides (see Figs. 3 and 4). In such sites, tree ferns recurrently suffer damage due to weeding. Damage may have stressed young ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 287 1.6 Stem growth (cm/mo) Number of fronds produced C. divergens Species (A) Height growth (+ SE) for transplanted indiv soa of Cyathea divergens (N = 11 shaded, N = 13 in the open) and Alsophila firma (N = shaded, N = 6 in the open) near Cuetzalan del Progreso, Puebla. (B) Production of new oe i SE)i in aon arse individuals of Cyathea divergens (N = 11 shaded, N = 10 in the open) an nd A. firma (N = 12 shaded, N = 5 in the yee near Cuetzalan del Progreso, Puebla. All differences were ae sheers (P < 0.05). 288 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) tree ferns beyond their capability to recover, causing their death. Although not statistically significant, survival rates of C. fulva were higher in control than in control transplant treatments after 1 yr. Tree ferns of this species may not overcome the stress caused by transplantation. Further studies are necessary to assess the adequate conditions that would increase the survival of transplants of C. fulva. In general, survival rates were greater when tree ferns were transplanted to safe sites. However, because our experiments do not allow for comparisons among sites for species other than C. divergens, more solid information about the survival of transplants will depend on future experiments, in which each species is subjected to all treatments. Survival rates were statistically similar for tree ferns relocated to safe sites under sunny or shade conditions. Bernabe et al. (1999) observed the same pattern for two of the three tree fern species they studied in tropical montane forests of Mexico. As suggested by these authors, tree fern species vary in their tolerance to different light conditions. In our study, both C. divergens and A. firma seemed to tolerate a wide range of light availability. However, the survival rate for A. firma planted in the shade was higher, although not statistically significant, than in the sun treatment. The ability to branch by adventitious buds may confer this species a higher tolerance to the stress caused by transplantation. For A. firma, shade conditions, in which water stress is diminished, may be ideal for transplantation, at least during establishment, until the transplants produce their first expanded leaves. Further differences in survival could have been observed if younger and, therefore, more vulnerable, transplants were used, and if we had a bigger sample size or our observations were prolonged for more than 1 yr. Several studies on tree ferns have focused on understanding the plasticity of phenological responses of many species and the effects of such plasticity on population dynamics (e.g., Hunt et al., 2002; Mehltreter and Garcia-Franco, 2008). Adaptations to different light conditions have particularly been addressed for several species (Arens, 2001; Ash, 1987; Bernabe et al., 1999; Seiler, 1981, 1984; Walker and Aplet, 1994). In sunny conditions, for example, several Cyathea species often grow faster in height, produce more fronds and start to reproduce earlier than in shady conditions (Arens, 2001; Ash, 1987; Bittner and Breckle, 1995; Poulsen and Nielsen, 1995). On the other hand, Dicksonia antarctica Labill seems to grow slower in sunny sites, when compared to less exposed and more humid sites, where they are less likely to experience water stress (Hunt et al., 2002). Different light intensities can also cause changes in crown architecture of some Cyathea species (Arens and Sanchez-Baracaldo, 2000; Cox and Tomlinson, 1985; Tanner, 1983). In our study, transplants of both C. divergens and A. firma placed in the shade grew faster in height and produced more fronds than those planted in sunny sites. Our data is in contrast to what has been observed for other tree fern species, which grow faster when exposed to conditions of higher light availability (see Arens, 2001; Arens and Sanchez-Baracaldo, 1998; Ash, 1987; Bittner and Breckle, 1995; Bernabe et al., 1999; Seiler, 1981). These results may reflect a faster adaptation of transplants to shade conditions. When planted in ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 289 the shade, tree ferns would experience lower air temperature, higher air humidity and soil moisture availability. Under such conditions, water uptake would probably be adequate, and transpiration rates would not be elevated. Photosynthesis limitation by water availability would probably be lower when transplants are placed in the shade, in comparison to sunny sites, allowing tree ferns to grow faster and produce more fronds. Transplantation is a low cost- and time-demanding activity that would adequately enhance both in and ex situ conservation of tree fern species in the region of Cuetzalan. Potentially, young tree ferns transplanted from sites in which they experience elevated mortality could be used as ornamental plants, promoting the ex situ conservation of native Cyathea spp. in the region. Our study suggests that transplants of C. divergens could be successfully used in gardening. Under adequate conditions, this species showed high survival and growth rates. More concrete conclusions should rely on complementary studies with a larger number of transplants by species, divided into replicate sites. Additional support for the ex situ conservation of C. divergens comes from the fact that it is probably the most harvested species for handicraft production (Eleutério and Perez-Salicrup, 2006). Transplants could also be used for the conservation of rare species, such as A. firma, which is considered to be at risk of extinction (Diario Oficial de la Federacién 2001). Many local farmers, for example, have manifested interest in transplanting tree ferns to the understories of their shade coffee plantations. Given that our study shows that young tree ferns should be preferentially transplanted to sites where they are not exposed to direct sunlight, this use by local farmers may be successful. Moist and shaded conditions provided by the canopy of shade coffee plantations are probably adequate for a successful establishment and growth of transplants. Tree ferns could potentially survive and satisfactorily grow associated with this land use if they are protected from accidental damage during agricultural activities. If a few requirements are met, transplantation might also be the opportunity for local farmers to engage in the responsible management and trade of transplanted Cyathea spp. individuals. The trade of more abundant and less endangered tree fern species would additionally require more than current market assessments and future market predictions. Detailed studies about the state of native tree fern populations occurring in forest remnants in the region are essential to provide policies that benefit in situ conservation and limit tree fern exploitation. In addition to limiting tree fern exploitation, the extraction of tree ferns from disturbed areas and native populations in forest remnants should be exclusive to local landowners, who depend on the exploitation of forest natural resources for their livelihoods (see Pérez-Garcia and Rebollar-Dominguez, 2004). Finally, our study emphasizes the importance of disturbed areas for the conservation of endangered species. These areas may constitute not only important sources of young tree ferns, but also seedlings of other species. 290 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Therefore, the use of transplants for promoting conservation is worth testing for other species, sites, and environmental conditions. ACKNOWLEDGMENTS We are grateful to the Looprentive Tosepan Titataniske, and especially to Poncho and family and the workers at the “Mariposario” who assisted us many times during this study. We also thank LITERATURE CITED Arens, N. C. 2001. Variation in performance of the tree fern Cyathea caracasana (Cyatheaceae) across a successional mosaic in an pee cloud forest. Amer. J. Arens, N. C. and P. SANCHEZ-BARACALDO. 19 A eecrdcree of tree ferns cepecanpaet across a sucessional mosaic in an Andean cans feccat Narifio, Colombia. Amer. Fern J. 8 71, RENS, fa C. and P. SANcHEz-Baraca.po. 2000. Variation in . fern stipe length - sara ele tracking preferred habitat through morphological chan ange. Amer. Fern J. 90:1—15. AsH, at oT of Cyathea hornei (Cyatheaceae), a tropical tree-fern in Bee Aust. J. Bot. See He a ILLIAMS-LINERA and M. Patacios-Rios. 1999. Tree ferns in the interior and at the edge of a Mexican cloud forest remnant: spore germination and sporophyte survival and oe coma 31:83-88. ITTNER, J. and S. W. Bre oo The growth rate and age of tree fern trunks in relation to habitats. shane Pica 85: Pt Cox, P. A. and P. B. Tomunson. Ge Relationships between ea sian pattern po branching in the tree fern Lophosoria eprisipereia in Veracruz, Mexico. Amer. Fern J. 75:105—110. Diario OFiciaL DE LA FEDERACION. 2001. Norma oficial mexicana NOM. ECOL. pale ey Proteccién ambiental: Especies nativas de México de flora y fauna silvestres, categorfas de riesgo y ne Eee para su inclusién, exclusién o cambio, lista de especies en riesgo. México Duranb, L. Z. and G. Goupsrein. 2001. Growth, leaf Characteristics. and spore production in itive and { invasive tree ferns in Hawaii. Amer. Fern 5. Exeutério, A. A. and D. R. Pérez-SALicrup. 2006. Cig: see . tree eee (Cyathea spp.) for handicraft aiciarane in Cuetzalan, Mexico. Econ. Bot. 60: Finni, J. F. and J. V. STapen. 1987. shits of the tree ee: ie dregei. Hortscience 22:65. sacs Commission. 2001. Tree f lan for the h ting, transporting or trading of Dicksonia antarctica in Tasmania. Tasmania. 21 pp. Fox, G. A. 2001. cigar analysis: studying times to events and rates at which events occur. Pp. in S. M 235-266, - SCHEINER and J. Gurevitcn, eds. Design and analysis of ecological Perma Ae ed. Oxford University Press. Oxford, UK. Hens.ey, D., R. Stippe, N. Bezona and F. Raucu. 1997. Hapuu (Hawaiian Tree Fern). Ornamentals ge spears, OF-16. 1 Hunt, M. A., N. J. Davison, G. ‘ Unwin and D. C. Ciosg. 2002. Ecophysiology of the soft tree fern, Dickeonia antarctica labill. Aust. Ecol. 27:360—368. INstrruTO MEXICANO DE TECNOLOGIA DEL AGUA (IMTA). 2000. Programa Eric II. Mexico. Jones, A. T. and M. J. Hayes. 1999. Increasing floristic diversity in grassland: - — of management regime and provenance on species oreo Biol. Cons. 87:3 Korait, P., D. S. Conanr, J. S. Metzcar, H. ScHNEWeER and K. M. Prver. 2007. eae Sue of scaly tree ferns (Cyatheaceae). Amer. J. Bot. 94:873-886. ELEUTERIO & PEREZ-SALICRUP: MANAGEMENT OF TREE FERNS IN MEXICO 291 Luna, I., A. VELAquEz and E. VeLAsquez. 2001. México. Pp. 183-241, in M. Kappe.te and A. D. Brown, eds. Bosques nublados del neotrépico.1st ed. Inbio, San José, Costa Rica. MEHLTRETER, K. and J. G. GarciA-FRANcO. 2008. Leaf phenology and trunk growth of the deciduous tree fern Alsophila firma (Baker) D.S. Conant in a lower montane Mexican forest. Amer. Fern . 98:1-13. MickeL, J. T. and J. M. Berret. 1988. Pteridophyte flora of Oaxaca, Mexico. New York Botanical Garden. New York, USA. MicxeL, J. T. and A. R. Smiru. 2004. The pteridophytes of Mexico. New York Botanical Garden, New ork, US. Montatvo, A. M. and N. C. ELtstranp. 2001. Nonlocal transplantation and outbreeding depression n the subshrub Lotus scoparius (Fabaceae). Soo J. Bot. 88:258—2 Dee ads, M. and S. REBOLLAR-DOMINGUEZ. 2004. Reservas cctrativistan jAlternativa para la conservacidn de especies forestales? Madera : pean 10:55-69. Poutsen, A. D. and I. H. NieLsen. 1995. How many ferns are there in one hectare of tropical rain forest? Amer. Fern J. 85:29—35. Primack, R. B. 2002. Essentials of conservation biology. Sinauer Associates, Sunderland, EUA. Pyxe, D. A. and J. N. Lee 1986. Statistical analysis of survival and removal rate experi t Ecology 67:240— Sener, R. L. 1981. pi — rates and natural history of the Central American tree fern Also a seas Amer. Fern J. 71:75-81. Seer, R. L. . Trunk length and viieee size in a population of Nephelea tryoniana from El Sie “Ame. Fern J. 74:105-1 SEMARNAT. 2000. S . adel vulnerables al aprovechamiento forestal en bosques ae de Oaxaca. In: G. S. WARNHOLTZ. Pr ites o de conservacién y manejo sustentable de recursos forestales en es Oaxaca, Méxi Soka, R. R. and F. J. Rouir. 1995. Biometry: the can and seeigee * statistics in biological research, 3rd edition. W. H. Freeman and oe y, New York, U Suzuki, C. C. L. F., M. T. Pauuito and A. M. Ranoi. 2005. Substrate and ee affect the early growth of peer wtbke tropical tree fern Dicksonia sellowiana Hook. (Dicksoniaceae). Amer. Fern J. 95:155— TANNER, E. V. J. oe i pple! and growth of the tree-fern Cyathea pubescens Mett. ex Kuhn in Jamaica. Bot. J. Linnean Soc. 87:213—227,. Tryon, R. M. and A. F. Tryon. 1982. Fern and the allied reiu with special reference to Tropical America. Hecate saa York, New York, USA Wa ker, L. R. and G. H. ota 4. Growth and fartilizetion responses of Hawaiian tree ferns. Biotropica 26:378— Werth, C. R. and M. I. aan 1990. Summary: the contributions of population studies on ferns. Amer. Fern J. 80:183-190. American Fern Journal 99(4):292-306 (2009) Mycorrhizal Associations in Ferns from Southern Ecuador Marcus LEHNERT* Albrecht-von-Haller-Institut fiir Pflanzenwissenschaften, Abt. Systematische Botanik, Georg-August-Universitaét Gottingen, Untere Karspiile 2, D-37073 Géttingen, Germany INGRID Korrke and SasriNA SETARO Botanisches Institut, Spezielle Botanik, Mykologie und Botanischer Garten, Eberhard-Karls-Universitat, Auf der Morgenstelle 1, D-72076 Tubingen, Germany Linpa F. PazmiNno and JuAN PaBLo SUAREZ Escuela de Ciencias Ambientales, Universidad Técnica Particular de Loja, Ecuador MicHacL Kessier? Albrecht-von-Haller-Institut fiir Pflanzenwissenschaften, Abt. Systematische Botanik, Georg-August-Universitat Gottingen, Untere Karspiile 2, D-37073 Gottingen, Germany mycorrhizal fungi (AMF) and 36 were infected by dark septate endophytes (DSE), which are identified as ascomycetes and here considered as a kind of mycorrhiza similar to the ericoid type. Th ts of 30 species (including all grammitid Polypodiaceae and half of the Elaphoglossum species) were free of evident fungal infection. AMF were frequent in terrestrial species (29.10% of species, or 48.49% of infected terrestrial samples). DSE prevailed in epiphytic species (58.62% of species, or 96.15% of infected epiphytic samples) and were also common in terrestrial samples of predominantly epiphytic species. Key Worps.—Andes, arbuscular mycorrhizal fungi (AMF), ascomycetes, dark septate endophytes (DSE), grammitid ferns, Hymenophyllaceae, vesicular arbuscular mycorrhizae (VAM) Mycorrhiza, the symbiosis between fungi and plant root, is known to enable plants to survive in the harshest environments by mediating nutrient and water fluxes (Allen et al., 2003; Cairney and Meharg, 2003; Cooke and Lefor, 1998). Despite the evident advantage, there are conditions under which plants may dispense of a fungal partner and thrive, especially if they are growing on substrates with easy nutrient availability. Since most plant groups have a preference for one type of substrate, it does not surprise that mycorrhizae are “Corresponding author new address: Staatliches Museum fiir Naturkunde Stuttgart, Am Léwentor, Rosenstein 1, D-70191 Stuttgart, Germany; email: lehnert.smns@naturkundemuseum- bw.de ‘New address: Systematic Botany, University of Ziirich, Zollikerstrasse 107, CH-8008 Ziirich, Switzerland. LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR 293 unevenly distributed among the plant families (Newman and Reddell, 1987; Wang and Qui, 2006). Each new screening for fungal infections helps to understand the relationship between substrate type and mycorrhizae, especially if they include exceptions from the rule (e.g., Gemma et al., 1992; Moteetee et al., 1996). Mycorrhization is common and diverse among landplants (Brundrett, 2002, 2004; Allen et al., 2003) but only two types have been confirmed for ferns and lycophytes. The arbuscular mycorrhizal fungi (AMF) belong exclusively to the Glomeromycota (SchiiBler et al., 2001; Brundrett, 2004) and are the oldest form of the symbiosis (Pirozynski and Malloch, 1975; Blackwell, 2000; Brundrett, 2002). They are prevailing among ferns, lycophytes, and most other groups of vascular land plants (Brundrett, 2004). AMF are unable to grow without the association to a green plant (Brundrett, 2002), and are not easily dispersed from the soil to other habitats (Janos, 1993). The other group is the dark septate endophytes (DSE), which is a polyphyletic compound of several more derived fungal lineages. Contrary to the AMF, their spores get airborne more easily and are thus more readily available in the epiphytic habitat. The symbiotic character of DSE associations is still discussed controversially because the taxa involved are closely related to non-symbiotic endophytes, pathogens, and litter decomposers (Jumpponen and Trappe, 1998). However, most DSE found in ferns are apparently related to the ascomycetes (Schmid et al., 1995) that form the well-studied Ericoid mycorrrhiza (Cairney and Meharg, 2003). Basidiomycetes (i.e., the known showy mushrooms) are commonly associated with northern temperate tree species and most orchids, including the epiphytic species (Brundrett 2004). Although they can also be found in liverworts (Kottke and Nebel, 2005), they are not confirmed as fungal partners of ferns and lycophytes (Kottke et al., 2008). Compared to the overwhelming diversity of green plants in the tropics, the studies on tropical mycorrhizae are relatively few (Wang and Qiu, 2006). One area worthy of such investigations is the Reserva Biolégica San Francisco in southern Ecuador (Prov. Zamora-Chinchipe), where we conducted ecological studies on ferns and lycophytes (Gradstein et al., 2007). The 1000 ha large reserve contains mature montane rain forest at 1800-3150 m and harbors 247 species of ferns (incl. horsetails; Smith et al., 2006) and lycophytes (Lehnert et al., 2007). The rugged topography of the area creates a mosaic of different substrate properties, with nutrient deficient soils on the ridges (Gradstein et al., 2008) and slopes that receive a downhill flow of nutrients (Wilcke et al., 2001). The divergent soil properties should also influence the mycorrhization of the plant species, given the fact that mycorrhizae enable plants to prosper in harsh nutrient deficient environments (Cairney and Meharg, 2003). Surpris- ingly, many usually epiphytic species in the area also colonize the ground on the ridges (Kessler and Lehnert, 2009), although epiphytic ferns are considered to be less dependent on mycorrhizae than terrestrial ones. Highly abundant groups with numerous epiphytic species in the area are the filmy ferns (Hymenophyllaceae), grammitid ferns (Polypodiaceae), and the genus Elapho- glossum (Dryopteridaceae). 294 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Looking for a reference on the mycorrhizal status for these fern groups, we found that most available reports are for smaller regions outside of South America (e.g., Berch and Kendrick 1982; Cooper 1976; Gemma et al., 1992: Iqbal et al., 1981; Moteetee et al., 1996; Nadarajah and Nawawi, 1993), and the few surveys cover only a fraction of the ferns and lycophytes worldwide (Boullard, 1958, 1979; Hepden, 1960; Newman and Reddell, 1987). No treatment for tropical Andean ferns was found; the few studies in South and Central America had either no overlap in the investigated species (Andrade et al., 2000; Ferndndez 2005), or they had contradicting results for the same species (Lesica and Antibus, 1990; Schmid et al., 1995). Compared to the general diversity, the number of investigated species from our three focal groups (filmy ferns, grammitid ferns, and the genus Elaphoglossum) is rather low. The present account aims to increase the investigated species number of these groups in order to have a more representative basis for future comparative studies. Boullard (1958) included several Neotropical species in his survey but these were sampled either from herbarium specimens or from cultivated material. Drying reduces the ability of the hyphae to take up the dye, so that the mycorrhization of the plant may be rated too low or may go undetected. In cultivation, the kind or degree of mycorrhization may depend on the fertilization of the substrates (Entry et al., 2002). Species that otherwise are mycorrhizal may completely dispense of the symbiosis in cultivation. Therefore, root samples are best taken directly from nature and preserved specifically for later dyeing. As far as we know, this is the first survey on mycorrhizae in tropical Andean ferns sampled in situ. MATERIALS AND METHODS Root samples were collected at different sites in SE Ecuador: A) along the Gualaceo-Limon road (3100-3300 m, Prov. Azuay), B) the mountain pass El Tiro between the towns of Loja and Zamora (2600-2800 m, Prov. Loja/Zamora- Chinchipe), C) the area of Cerro Toledo, situated E of the town of Yantzatza (2900-3100 m, Prov. Loja), D) Reserva Bioldégica San Francisco (1800-2600 m, Prov. Zamora-Chinchipe), E) Reserva Cajanuma (2750 m, Prov. Loja), F) Reserva Tapichalaca (2450-2650 m, Prov. Zamora-Chinchipe), and G) the Campamento Indigena Shaimi on the shores of Rio Nangaritza (900-1200 m, Prov. Zamora-Chinchipe). The study sites span an elevational gradient of 2400 m and range from lower montane forest to paramo vegetation. All sample areas face east and receive heavy precipitation all year round (Richter, 2003). Sampling was focused on previously rarely investigated taxa. The substrates of the ferns were categorized as terrestrial, epiphytic, and saxicolous = epilithic, rupicolous). Voucher specimens were deposited at Pontificia Universidad Catdélica del Ecuador, Quito (QCA). Duplicate collections of M. Lehnert were further distributed to Géttingen (GOET) and Berkeley (UC), and a set of specimens collected by L. Pazmifio is deposited at the herbarium of Universidad Técnica Particular de Loja (UTPL), Ecuador. LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR 295 Sample plants were carefully removed and cleaned mechanically from the substrate, then rinsed with water to remove smaller litter parts and mineral compounds. At least 10 cm of roots from each specimen were preserved in 70% ethanol; of plants which we suspected to harbor DSE, additional 5-10 cm of the roots were preserved in 10% aqueous glutardialdehyde for transmission electron microscopy (TEM) preparation and stored at 8-10°C. Preparation of the ethanolic samples for light microscopy followed Grace and Stribley (1991) and Haug et al. (2004). The samples were cleared in 10% KOH for ca. 24 h at 60°C; if the roots were still dark, the KOH was changed and the sample was kept at 60°C for another 12-24 h. Then the roots were rinsed twice with water and acidified with 1 N HCl. Staining was done with 0.05% methyl blue in lactic acid for at least 3 h. The stained roots were examined with a dissecting microscope at 30-60 x; promising young roots were cut into portions, mounted on slides in lactic acid and examined at 100-400 x. If mounted roots turned out to be insufficiently cleared, they were bleached with 3 % H,O2 for 2-5 min, rinsed with water and acidified with 1 N HCl. Then they were covered with same staining solution as before and heated over a small flame for 1-3 min. Excess staining solution was washed off with 90% lactic acid. Preparation of the TEM samples followed Schmid et al. (1995). We opted for the fixation with 1% osmiumtetroxid for 1 h at 20°C, then 1% uranylacetate for 1 h at 20°C. Samples and slides are stored at the Georg-August-Universitat Gottingen, Germany. F were screened in the light microscope for presence. AMF are recognizable as relatively strong, aseptate hyphae with irregular diameter, forming terminal and lateral vesicles (Boullard, 1958). These infections were counted as real mycorrhizae if arbuscules were visible in the cortex (Gemma et al., 2002). Dark septate endopyhtes (DSE) were assigned to ascomycetes (Schmid et al., 1995) if the characteristic Woronin bodies at the porate septa in the hyphae were visible in the TEM (Fig. 1D; Haug et al., 2004). Fungal infection was considered as mycorrhiza if hyphal coils were developed in host cells that were still intact and showed some response to the infection, i.e., thickening of the cell walls where the hyphae penetrated the cell and thickening of host cell cytoplasma as indicator of increased cytological activity (Fig. 1C). The frequency of infections in the roots was quantified under the light microscope, preferably on a single root with a minimum length of 10 cm measured from the root tips. In cases where the plants developed only considerably shorter roots, we combined several complete roots to reach the minimum length of 10 cm. The frequency of stained hyphae was categorized in three classes (Gemma et al., 1992) to give an impression of the extent of the infection: Present in 1) <25%, 2) 25-75%, and 3) >75% of investigated root length. Presence of single hyphae or vesicles in the outer cortex as well as infection rates below 5% were considered as erroneous infections and not counted as mycorrhizal association. We did not distinguish between “obligately” and ‘‘facultatively mycorrhizal” because we usually sampled 296 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 10 ym Fic. 1. Fungal infections in fern roots. A) AMF infection in young roots of Loxsomopsis pearcei; arbuscules fill out most of the cortical cells. Photograph M. Lehnert; B) DSE infection of root cortex in Lellingeria major, visible as external net and dense, internal coils of hyphae. Photograph M. Lehnert; C) Infection by ascomycete in Melpomene firma; the hyphae enter the root through the characteristic Woronin-bodies, visible as darker dot on each side of the septum. TEM-photograph I. Kottke. only one specimen per species and habitat. Since degree and frequency of mycorrhization is dependent on external factors, such a categorization would be misguiding. RESULTS Among the 101 Ecuadorian fern samples, 85 species from 10 families were represented (Table 1). A total of 63 samples were infected by mycorrhizal ngi. AMF occurred in 19 species (22.35%) represented by 19 samples, and 36 species (42.35%) represented by 44 samples were infected by dark DSE (Table 2). Identified DSE always turned out to be ascomycetes that probably form a mycorrhizal association similar to the ericoid mycorrhiza (Sc‘:mid et al., 1995; Kottke, 2002). Since it was not possible to process all specimens in question adequately, we retain the more general term DSE in the following LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR 297 passages. The roots of 35 samples were free of evident fungal infection. Three specimens (Arachniodes denticulata, Elaphoglossum lloense, Micropolypo- dium sp.) had only a weak peripheral infection by DSE. They were regarded as dubious and are included in the non-mycorrhizal species (35.30% of the species). Mixed infections cannot be confidently reported. AMF were found in 29.10% and 28.57% of the terrestrial and saxicolous species, respectively, but only in 3.45% of the epiphytes (Table 2). DSE showed a similar presence in terrestrial and saxicolous species (30.91% and 28.57%, respectively), but they dominated over AMF in the epiphytic species with 58.62%. Hymenophyllaceae were represented with 18 species in our sample and showed a high presence of mycorrhization (78%). The mainly epiphytic species of Hymenophyllum were colonized by DSE (80%), whereas the predominantly terrestrial or saxicolous species of Trichomanes s.1. (Tricho- manes, Abrodyctium) had more cases of AMF infection (50%). One unidentified Trichomanes grew epiphytically and had DSE like the epiphytic Hymenophyllum species. The only terrestrial Trichomanes s.1. with DSE was Trichomanes dactylites Sodiro. Grammitid ferns (Polypodiaceae; Schneider et al., 2004, Smith et al., 2006), represented by 24 species, had an infection rate of 75%. Only ascomycetes (i.e., DSE) were found as fungal partner, even in terrestrial and saxicolous species (L. Pazmifio, unpubl. data). Non-grammitid Polypodiaceae were completely free of evident fungal infections. Among the 23 species of Elaphoglossum, we found only 12 (52.20%) with fungal infection. DSE accounted for 75% of the infections. The remainder of the investigated species showed mycorrhizal associations as was more or less expected from previous accounts. All three species of Asplenium (Aspleniaceae) were terrestrial and free of fungal infection. Of the two terrestrial species of Blechnum (Blechnaceae), only one had a low AMF infection. The investigated Pteridaceae showed a medium to strong infection by AMF (2 species, 100% infection). Although they have been cited as examples for high infection rates (Boullard, 1958, 1979), only 50% of the species in the Cyatheaceae and 40% of the species in the Gleicheniaceae had mycorrhizal associations (Table 1). However, the exclusive colonization by AMF could be confirmed in both families. Our sample size was not sufficient for a statistical analysis of changing mycorrhization along an elevational gradient. The localities of the samples are included in Table 1 for future studies focusing on this topic, which may want to include the data presented here. DISCUSSION The overall infection by confirmed and putatively mycorrhizal fungi among our samples was 62.38% (64.70% at the species level). These percentages are lower than those reported for angiosperms or land plants in general. Trappe AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) ce) jo) N 3 Z9PL WW Heuye'T JWV GZ-S 1 aloo “| (YOszjO[y) WnydiowoJajay wunssojsoydviq ad €6PL ‘W Weuya'y asa GL—-GZ a ‘IMD ‘5D (O11pog) wnuprupurpns umnssojsoydnjyq ad cSSt ‘W Heuye'y 4Sd S7-S a ‘uorolp umnyyAydossojs urnssojsoydnjyq d TSST ‘W Meuye'y] - a ‘uololy] umnyjAydossojs urnssojsoydnjq d Z8EL ‘W Meuya'y asa GZ-S a aloo “|, (894) WNaapuLa uInssojsoydnvyjy =) ZSPL ‘W Meuyea'y asd GL-SZ } YD “H (3s1ey “H) Ujesua urnssojsoydvyjyq 2 8SPL ‘W Heuye'y = } ISWYD *H (leyeg) Djoolpuap umnssojsoydvlyq a, €9FL ‘W Heuyey] - - } JSWYD “H (O11pos) urnaprojyjap urnssojsoydvyjyq a O9FL ‘W Neuye'y . . } SWI *H (O1pos) urnapiojap uinssojsoydnyjq qd O6FL "W Weuye'y : > a ‘paul “quioo ‘uURIO|y ‘2 ‘Y (O1rpos) wmnyjjAydo.sAs10 urnssojsoydnyjy ad S8hL ‘W Neuyey - e) JSWYD *H (O11pos) apupsyuD urnssojsoydnjyq d 966 ‘WMeuye'y é4Sd ¢> } surly) (‘MS) DIDjNIYWUap saporuyooIy avaoep Hoyo ad ¢Sg ‘W NeuysT AWV GZ—-S } urwmlog (‘UOoJet}{) sisuappjad payyDAD d OZFL ‘W Nauye'y - - } Weuya’] Drxougo payyDAD ad PEPL ‘WW HeuyeT : } pliqdy payyDAy d OSST ‘W Neuya'y SAV SZ-S } uOALL, ‘W"Y IAaypnp payywAD d SEPL ‘W NoeuyeT - } urulog (1ayDg) Dprf{tjouurdiq vayywAy d PLPEL ‘W Neuye'y JINV G@-S } Weuye'] DupyuDuOS vjIYydosfy avaovaTeAD d OFFL ‘W NouyeT - - } L ‘ds wmuyoarg qd P8PL (W Neuye'y] AWV Gé-S } ‘IMD “D (YoszjoO[y) (pysanquroyos urnuyoayg aeaoeuypal gq a PEEL "W Houya'y - } ‘YOST B ‘pssue'] dias wnruaydsy a S6EL ‘W HeuyeT] - } *‘yooH mjjoy umiuaydsy ral EZPl (W Weuye'Ty . - } ‘MS urInjlinp wintualdsy ‘00'] uonoa][0D uoroeyut jo addy, (%) Woroeyut jesung aye.s-qnsg satveds ‘(WI 00ZT—-006) MuTeYs eUssIpuy O}UeUTeduIeD aq} (5 ‘(We OS9z—0S¢tZ) eoeyeyorde | PAIOSOY (J “(Ul OGZZ) eurnuele) PAIasay (WJ ‘(Ul 009Z—O008T) OOsI9URIY URS Rolso[olg PAIosOy (C ‘(ploy ‘AOIg ‘Ul OOTE —006Z) Opejo], O11eF (FD ‘(Ut 0O8Z—009Z) CALL, [9 ssed urejunow (gq ‘(Ul OOSE—-OOTE) peor UOUIT']-oaoR[eNy (Y :set}I[eoO'] “piooed snoiqnp = j ‘seyAydopua ayejdas yrep = ASC ‘!sunj [eztys09AW Je[NOsnqie = JPY ‘snojoorxes = s ‘ayAydida = @ ‘[RINSaIa} = } :suotetAiqqy ‘seydures poyeSyseauy "| ATAV 299 LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR d Z9EL “W Weuye'y asa GL-GZ a ‘ABID) BF ‘YOOH Muerumnyd urnjyAydouaurApy € ZbbL (W WouyeT asd GL-SZ a uoWoW ‘A ‘D unyoyoyynur wnyyAydouautAyY d P6PL W Heuyey] asa ¢z-S } Aseq uindip20s9nw ~umnjyAydouaurAzy a perl W weuyeyT asa GZ-S a Mg (‘MS) saproonf urnjjAydouaurAyy ad ZPSL ‘W Meuya'yT asa GZ-S a AaID ¥ “YOOH wmnjojst9 urnjyAydouawuApy a EPL ‘W MeuyeyT asa GZ-S ) yosog uoAjoIpojpo urnjjAydoueurAyy 5 CISL ‘W Mouye'T AINV GL-SZ } uossinqng ¥% ereyiqg (“Mg) UINpIsIa uNAjIIpoIqy d ZZb1 (W MeuyeyT - - } ‘Wg “Y V (‘MS Xa ‘AeD) snsojuauIO) sniayoyg A 6ZPL ‘W Weuyey AY SZ-S } L ‘ds snaayons A 8ZPL (W WeuyeT SAV Sc-S } EXPN (HEN) snsousiqns snsayoyg i 89cL ‘W Weuye'y : . } TEYPN (“HA)) snsousiqni snsayons d 9ZPL ‘W weuye'y] = * } SII ‘A B paeesieys—y SnjsD/qOuvjaul snsayoyg ad 6PSL ‘IW MeuyeT z - } ‘BIO “A ¥ preesiaysg snysppqouvjaur snseyons A O8FL ‘(W Heuye'y = - } preesi9\sQ ¥ ‘3][Q “q snsojuaurojraasq snsayons aeadRIUayIIa]s) d ZLPL WW Wouye'y AAV GL-SZ 1 [slg °D (PITIM) wnypAydAjoyd urnyoysAjog A 086 ‘W Meus] AIANV GZ—SZ } ayepuly (uoxey)) Wddiyry stsdoaysvT ea LOFL ‘WW Weuye'y asd GZ-SZ } ISNYD 'H (O1tpog) fsajDA unssojsoydvjq d SZPL ‘(W Weuya'T > a ISNYD *"H wUNIupINA urnssoysoydviyq d 88FL ‘WW Meuya'y SINV ¢z-S ? e100 ‘1 (Y9SZJO[Y) wNsowoNbs unssopsoydnyy ad cOSt ‘W Houya'y . } ¢ ‘ds umnssojsoydnjq da 98FL W Heuya'T . . a z ‘ds uwnssojsoydp/q d Z8PL ‘W Weuye'y asd GZ-S e) t ‘ds urnssojsoydplq J 6SPL ‘W euya'T asd GL-GZ } ‘IYO ‘D (4eyxeg) asuazinb unssojsoydniq ai €Sst ‘W HeuyeT : - a ‘ysussoy UNjonpold urnssojsoydniq a LPol [pueyy asa G7z-S a e100 ‘J, (‘ASe(]) UNsojoyad urnssojsoydniq N 8 ‘W Weuyey A eZbl (W Mouya'Ty AINV GZ-S } SHY “H (texeg) wnsopidod unssojsoydniy d T6F7L ‘W HeuyeT éasa ¢> c) a100W ‘J, (OOH) asuaoyy] umnssojsoydnjq ad Z6PL ‘W Meuya'y ANY GL-SZ e) ‘wg ‘{ (“Mg) uNnjof{14yD] Unssojsoydnyq ‘00'T uonoayjop uonseyut jo addy, (%) WoNoeyur jesung aye.ys-qng satsedg ‘panurjuo) ‘L ATV, AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 300 d ‘u's “] OuTUIZeg asd GZ-S 1 UBIO “DY 8 “WS “Y “V (48xeq) syrssasqns DuesuT][a] a ‘u's "] OUTUIZEg asa GL-SZ a UBIO] "DY RUS “YY (sexe_) sI[Issasqns puasuTjaT dq 6671 ‘We Weuya'T is - c) uBIOW “) ‘YR “WS “YY (1eyxeg) SIIssasqns DLAasUTJaT a 86PL ‘IW Houye'y asa GL-SZ a ueIOW “OY B “Ws “Y “V (‘jedoD) sofour vesuT[aT 4, 99PL ‘WW Weuye'y asa GL-SZ 1 uBio] “YY 8 ‘WS “YY (‘[edoy) sofour DiuasuTyaT ‘al ‘u's “"] ouTUIzeg asa GL-GZ } doysig “q “| pjoorurpand sryrururmmary fal ‘u's “"T OUTUIZed ASA GL-SZ a doystg “4 “| pjoorurpind syrururmpsy d Z6¢1 ‘W Moeuye'y " - cs) doystg "4" (Y98z}0[y) DUaLInd p1osolajuy qd ‘u's "7 OUTUIZEG 4qSa Gz-S a doysig “4 “T (‘MS) UINjDyN4as UINIpITy90) “6 ZL9PL ‘W Weuya'y asda G7-S } doysig “4 “J (Msg) uNjpjn4ias uINIpITy90y ad S6PL ‘WW Weuya'y asd G7-S c) Ja[ssay “WI 8 YUIS “YY Diqnjs DIuapp.ay ad ‘u's "J OuTUIzeg aSd GZ-G } yney (Yssi0.J) DsouLmp{ pruappsay a ‘u's “"] OUTUIZed asa GL-GZ a ‘J[ney (“Yssi04) psouLmp{ pruappsay (al ‘U's “"] OUTUIZeg - - } "AOID) B “YOO aan mice: ‘al ‘u's "J OUTUIZeg - - } Jesul[e'] unurtssjpjaundoqp urnrprydiny ‘al ‘u's “"] ouTWIZzeg - - 1 99,J uouajsoydurp urnsnauojAdurpy A 9SOL ‘W WeuyeT AAV Gl< } Jayeg cao; 1ao1pad poe ara ABAIR}LULOXO'T ad Z8hL ‘WW Weouyey 2 - } ¢ ‘ds saupuroyoity a, BOTSL ‘WW WeuyeT 2 - 8 z ‘ds saupuroyorly d E8PL ‘W Moeuya'y asa GZ—-S a 1 ‘ds saupwoyoiy, i) PISL ‘W HeuyeT AWV G7-S } azuny suaonjyjad saupuroyorsy 3] OTST ‘W Woeuys'T ANV GZ-S s yory supsaja saupuloyaity, d LOST ‘WHeuye'y asd G7-S } OIIpOg sajljAjapp saupuloyoisy, ad L8PL "W Mouye'y AWY Gz-S } YOSZJO]Y UINsoyNjjaod sauDUIOYOIL], a 9PPL ‘W Moule] asd SZ-S c) yosog saproupuoyoi4) urnjyAydouautAyy Vv 99ST ‘W WeuyeT ‘ - s z ‘ds urnyjAydouautAyy 2 SSPL ‘W Weuye’] . - e) t ‘ds urnpAydouaurAy a SPPL ‘WW Heuye'y] asa GZ-S e) ‘MS (‘Ms) soyjuDdjod umnyyAydouautA}y 00'T uoT9aT[0D uonseyut jo adAy, (%) worjezyut jesung a}e]s-qng seeds ‘penuruo7 ‘L ITV], 301 LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR a ZEEL ‘W Woeuya'y - - s uo}_OW “A *D DjNynurur srszajdAjay J, avaoepiia}dAyoy [, ‘al CEPL ‘W Weuye'T JNV GL-SZ s Jesur]a'y ("UIs “5 “y) sudd{tAalg UINIUOZOLAa}g q LZSL ‘W WouyeT ANY ¢z-S } ‘YOOH DIDILINUT Sia} aRadePLAld q LISL ‘W Houye'T asa G7-S 1 ‘WS “YM “V (Y9SzZ}0]y) Djnsuryruras aroyotsd.a J q 60ST ‘W Wouys'y asa G7-S } ‘Wg “A 'V (‘Wg “f) DJINs0aNa aroyatsdia a 96FL "W Wouya'y - - a ‘WS “YW 'V (‘ASeq]) D1asruD] aroyotsdia a ‘U's “T OUTUIZeg asa SZ-S f) L ‘ds urnspodAjodosapy a ‘u's "J OUTUIZeg asa co 1 t ‘ds urmrpoddjodoso1ypy ad ‘W's "T OUTUIZzed 4sd Gz-S } URIOW "DY 8 “US “Y “V (‘UOIeTH) Hf/jom auaurodjay ad ‘u's "J ouTUIzeg asa GL-SZ a uRIOW “DY 8 “Wg “Y “VY (‘UOIeT}) 1/jom auaurodjapy 5 S9OFL ‘WW Mouye'y asa ¢Z-S } Wouye’] [.ipualys auaurodyjapy a ‘u's "] OUTUIZeg asa GL—-GZ a ueIOW ‘OY 8 “WIS ‘UV (}sUasoy ¥ Jst1yD) suDjnuopnasd auautodjay o vOFL ‘W Weuye'y asd Sc-S } uBIOW ‘D'U 8 ‘WS ‘UV (jsuasoy ¥ yS1IyD) suDjnuopnasd auaurodjapy V 8ST ‘WW Neue] 4sd Se-sg 8 UBIO ‘OY 8 “WIS UV (Jsuesoy ¥ Is) suDjnuopnasd auauodjay d 80ST ‘W }euye'T . S } Weuye'y syjpjuapi990 auaurodjapy a ZOST ‘(W Weuye'T : : } Weuye’] sijojuapl990 auaurodjap a OTST "W WeuyaT 4sa Se-s } uBIOW ‘O'U 8 WS “Y'V (‘Ms Xo eoseSe']) sruO/1ruoW auauodjapy V 6SST ‘W weuyeT asd GZS 8 uel0oW “DY 8 WS “Y'V (‘Mg xo BOsese']) sruof/7ruoUT auautodjapy V 69ST 'W Neuye'T asa GZ-S s uBIOW ‘DY 8 US “Y “VY (‘YOOH) syropi8 auaurodjay d 8ZEL ‘W NeuyeT qsa GL-SZ BS) ueIOW ‘DY 8 ‘WS “Y “V (‘UIs ‘[) DUL/ auaWOdjay Vv OZSL ‘W Meuyey asa SZ-S 1 ueloW ‘OA B WS “YA V (UO_OW ‘A °D) DJ9a10 auautodjay a L7PL ‘W Wouye'] aASad GL-GZ a URIOW “DY ‘Wg “Y “Vy (UOXe|) suasinssv auauodjay ‘00'T uonoeTjop uonoeyut jo adj, (%) Woroeyut pesung ayeis-qng ‘panunuoy *L VIaVvy, 302 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) TasLE 2. Distribution of the 85 investigated species onto the registered categories: total numbers are followed by percentages per life forms ee and infection types (rows) in brackets. Abbreviations are the same as in Table 1; NM = non-mycorrhizal; ** six species occurred on more an one substrate, adding 7% to the total count ae 20% to N Species All t e s total 85 (100/100**) 55 (100/64.71**) 29 (100/34.12**) 7 (100/8.24**) AMF 19 (22.35/100) 16 (29.10/84.21) 1 (3.45/5.26) 2 (28.57/10.53) DSE 36 (42.35/100) 17 (30.91/47.22) 17 (58.62/47.22) 2 (28.57/5.55) NM 30 (35.30/100**) 22: (35.29/73.337* *) 11 (37.93/36.67**) 3 (42.86/10.00**) (1987) estimated that 82% of angiosperms host mycorrhizae; Wang and Qiu (2006) concluded that 80% of all land plants are mycorrhizal. Studies focusing on ferns and lycophytes found similar results to ours. Values gathered from literature (Boullard, 1958; Cooper, 1976; Berch and Kendrick, 1982, Iqbal et al., 1981, Gemma et al., 1992, Lesica and Antibus, 1990, Moteetee et al., 1996; Ragupathy and Mahadevan, 1993, Schmid et al., 1995, Muthukumar and Udaiyan, 2000; Zhao, 2000; Zhang et al., 2003) sum up to 68% of general fungal colonization and to 53% of AMF in ferns and lycophytes (M. Lehnert, unpubl. data). Wang and Qiu (2006), considering only AMF, found a comparable 52% of the species of ferns and lycophytes to be mycorrhizal. Despite the congruence in general mycorrhizal infection, our survey found AMF in only 22.35% of the species, including 29.10% of the terrestrial, 28.57% of the saxicolous species, and only 3.45% of the epiphytes. In contrast, DSE showed a similar presence in terrestrial and saxicolous species (30.91% and 28.57%), but they dominated over AMF in the epiphytic species with 58.62%. The discrepancy in AMF percentages between our study and the average is likely due to our selective sampling. We laid the focus on predominantly epiphytic taxa, and although we still examined more terrestrial than epiphytic samples, we evidently included a higher percentage than previous studies. The epiphytic habitat is rarely colonized by AMF because their spores are not easily dispersed from the soil. Furthermore, most AMF are dependent on their host, requiring the presence of a facultatively mycorrhizal plant for successfully establishing the symbiosis on a chorophyte (Janos, 1993). Thus the low presence of AMF in epiphytes (3.45%) is not surprising. DSE, however, have spores that get airborne and are thus more likely to contact the roots of epiphytic plants. Epiphytic plant species are well known to suffer from nutrient shortages and should greatly benefit from a fungal symbiont (Lesica and Antibus, 1990). If DSE are excluded in surveys as potential mycorrhizal partners in ferns and lycophytes (Lesica and Antibus, 1990; Michelsen, 1993), the recorded mycorrhization is low or absent, in our case only 22.4%. If they are regarded as mycorrhizae, the mycorrhization level will increase (Schmidt et al., 1995; Kottke, 2002), in our case to 42.35%. Overall, the degree of LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR 303 infection by DSE was higher in our study than in any other previous study on ferns and lycophytes. Beyond these general patterns, it is worthwhile to focus on individual study groups. The Hymenophyllaceae nicely mirror the general distribution pattern of the fungal infections. Terrestrial and saxicolous species have predominantly AMF, whereas DSE prevail in epiphytes. Gammitid ferns (Polypodiaceae), however, have almost exclusively DSE, no matter if they grew as epiphytes or as terrestrials. This apparent conflict with the general trend is due to the microhabitats inhabited by the species. The investigated terrestrial grammitid ferns usually grew in thick moss cushions like their epiphytic kin and by this means under very similar ecological conditions, which may lead to maintaining the type of mycorrhiza. Furthermore, most of the species sampled as terrestrials are either potentially epiphytic or closely related to epiphytic species. Only the samples of Melpomene occidentalis Lehnert rooted directly in mineral soil and showed no fungal infection. Opposed to this, the samples of eleven terrestrial and epiphytic species of non-grammitid Polypodiaceae from the investigated area are free of fungal infections, which is congruent with previous reports (Lesica and Antibus, 1990; Schmid et al., 1995). Since grammitid ferns represent a clade nested deeply within the Polypodiaceae, it is likely that the original condition in the family is a lack of mycorrhization and that mycorrhization has been secondarily regained in grammitid ferns. Apparently, this symbiosis was developed with DSE rather than with glomeromycetes. A similar situation of loss of AMF mycorrhization and secondary gain of DSE mycorrhization, also related with shifts between the terrestrial and epiphytic habitat, has been reported in liverworts (Kottke and Nebel, 2005). The genus Elaphoglossum showed no clear correlation between the types of substrate and fungal infections. The genus Asplenium is not very diverse or abundant in the study sites and occurred only on the lower slopes where nutrients are accumulated (Gradstein et al., 2008). The absence of mycorrhizae in our samples may be related to the improved availability of nutrients at their microhabitats. Previous studies found generally low infection rates in the Aspleniaceae (e.g., Boullard, 1958) and often varying results within a species, indicating that most species may be only facultatively mycorrhizal. Gleicheniaceae are usually axiomatic for strong presence of mycorrhizae (100%; Boullard, 1958, 1979). It is assumed that this affects both their ability to row on nutrient deficient soils and their inability to be transplanted and cultivated. Surprisingly, we found only 40% of our samples infected by AMF. Their root samples, however, were difficult to prepare because of a tough texture and dark, persistent cortical colorants. The necessary clearing with hydrogen peroxide may have affected the colourability of fungal hyphae with dye. Possibly a higher percentage of fungal infections was present but not detectable in our samples of Gleicheniaceae. Our results for the Cyatheaceae are much lower (50% of specimens infected) than the results of previous surveys (100% of specimens infected; Boullard, 1958; Hepden, 1960). The tree ferns (families Cyatheaceae and Dicksoniaceae) 304 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) bear the difficulty of acquiring fine roots from the compact subterranean root system that many species develop. Aerial roots from the trunks are easier to harvest but are expected to lack mycorrhizae because they are less likely to get in contact with inoculum of soil fungi. In order to bypass this sampling artefact, the plants included in this study were either small species or young plants of easily assignable larger species, which can be uprooted with most of their roots. One explanation for the low infection rate could be that these juvenile plants of Cyathea are less dependent on mycorrhizae than mature plants. The trunk-less tree ferns dwell in the shade where these often sun- loving species are under lesser drought stress but presumably achieve only a part of their potential photosynthetic rate. The profits of better supply with water and micronutrients may not compensate the cost of sharing assimilates with symbiotic fungi. e are aware that negative results in any species here included do not exclude the potential occurrence of mycorrhiza in other individuals of the same species. We aim to widen our sample size and want to include conspecific samples from sites with different substrate chemistry. This should allow us not only to distinguish between facultative and obligatory mycorrhizae but also about the conditioning factors. ACKNOWLEDGMENTS We thank our colleagues of the Research Unit of the DFG 402 “Functionality in a Tropical Mountain Rainforest: Diversity, Dynamic Processes and Utilization Potentials under Ecosystem Perspectives” for various help and fruitful discussion, especially Nicki Mandl and Rob Gradstein: we are indebted to our Ecuadorian counterparts in Loja (Fundacién Cultura y Naturaleza; Herbario LITERATURE CITED ALLEN, M. F., W. Swenson, J. I. QuereyeTa, L. M. EGERTON-WarBuRTON and K. K. TRESEDER. 2003. Ecology of mycorrhizae: A conceptual framework for complex interactions among plants and fungi. Annu. Rev. Phytopathol. 47:271-303. ANpRADE, A. C. S., M. H. Queiroz, R. A. L. Hermes and V. L. Ouiveira. 2000. Mycorrhizal status of some plants of the Araucaria forest and the Atlantic rainforest in Santa Catarina, Brazil. Mycorrhiza 10:131-136. Bercu, S. H. and B. Kenrick. 1982. Vesicular-arbuscular mycorrhi f southern Ontario f 1 fern allies. Mycologia 74:769-776. BiackweLL, M. 2000. Terrestrial life - Fungal from the start? Science 289:1884—1885. BoutLarp, B. 1958. La mycotrophie chez les ptéridophytes. Sa fréquence, ses caractéres, sa signification. Doctor thesis, Université de Caen. (Imprimerie E. Droulliard, Bordeaux BouLarD, B. 1979. Consideration sur la symbiose fongique chez les Pteridophytes. Syllogeous 19:1-59. BrunpbreTT, K. 2002. Coevolution of roots and my hi f land plants. New Phytol. 154:275-304. Brunprett, M. C. 2004. Diversity and classification of mycorrhizal associations. Bot. Rev. 79:473—495. LEHNERT ET AL.: MYCORRHIZAL FERNS FROM ECUADOR 305 Carney, J. W. G. and A. A. MEHarRG. 2003. Ericoid ieee a partnership that exploits harsh edaphic ie Eur. J. Soil Sci. 54:735-7 Cooke, J. C. and M. W. Leror. 1998. The ees, status of pee Drees species from Conneticut wetlands and transition zones. aesepeneee! Ecology 6:214— Cooper, K. M. ne A field survey of mycorrhizas in New ati bee 2: : Bot. 14:169-181. ENTRY, . Ryciewicz, L. S. Warrup and P. K. le 2002. Influence of adverse soil iene alee on the formation and function of arbuscular mycorrhizas. Adv. Environ. Res. 7:123-138. marie N., S. Fonrenta and M. I. Messuti. 2005. Micorrizas en pteridofitas de los bosques emplado- Ataviosos del an de Patagonia. IJ Convencién Ambiental Universitaria i Hee Gemma, J. N., RE . Koske and T. FLynn. 1992. Mycorrhizae in Hawaiian pteridophytes: occurrence and evolutionary eeeen ney Riles J. Bot. 79:843-852 Grace, C. and D. P. Srripiey. 1991. nee Dota aetae for routine staining of vesicular-arbuscular mycorrhizal ae “Myool. ng 95:1160-116 peenae S. R., M. Kessier, M. LEHNERT, ML ABIY oY Manp., F. MaKescHIN and M. RIcuTer. 2008. Vegetation, ilinaate and soil of the unique fae forest of southern Ecuador. Ecotropica 5—26. Haus, L, M. Weis, J. Hometer, F. OBERWINKLER and I. Korrke. 2004. Russulaceae and Thelephoraceae form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical mountain rain forest of southern Ecuador. New Phytol. 165:923—-936. Heppen, P. M. 1960. Studies in vesicular-arbuscular endophytes. II. Endophytes in ne fea with special reference to leptosporangiate ferns. Trans. Br. Mycol. 43:5 IQBAL, — H Pal "Yous and M. Younus. 1981. A field survey of mycorrhizal associations in ferns of New Phytol 87:69-89. — D. " er Vesicular-arbuscular Ss gous of epiphytes. Mycorrhiza 4:1-4. Jumeponen, A. and J. M. Trappe. 1998. Dark se endophytes: a review of facultative biotrophic root-colonizing fungi. N epi 140:295-310 Kess.er, M. and M. LEHNERT. fete Do ridge ree pon tefbaits to pteridophyte sitet in tropical m doe Kore, I. 2002. Mycorrhizae - Rhizosphere determinants of plant communities. Pp. oe in: WaiseL, Y., EsHEL, A. and U. Karxari, (eds.), Plant Roots: The Hidden Half. 3rd ed. Marcel Korrxe, I. and M. NEBEL. 2005. hee evolution of mycorrhiza-like associations in liverworts: An update. New Phytol. 167:330-334. Korrke, I., A. Beck, I. Hauc, S. ae V. JESKE, J. P. SUAREz, L. PazmiNo, M. Preusinc, M. NeseEL and F. BERWINKLER. 2008. Mycorrhizal state and new and special features of mycorrhizae of trees, ericads, aha ferns and liverworts. Pp. 137-148, in: Beck, E., J. Benpix, I. Korrxe, F. MakescuIn and R. Mosanp1, (eds.), Gradients in a Tropical Mountain Ecosystem of Ecuador. Series peu Studies 198, Springer Verlag, Berlin, Heidelberg. LEHNERT, Kesser, L. I. SALAZAR, H. NAVARRETE, F. A. WERNER ap S. R. Grapstein. 2007. Pterido ‘ a. Pp. oe | in: LrepE-SCHUMANN, S. and S.-W. Breckie, (eds.), Provisional ire ir of fauna and flora of the San Francisco valley and its sounding s (Reserva San Francisco/Prov. a southern Ecuador). Ecotrop. Mono. Lesica, P. and R. K. Antipus. 1990. occurrence of mycorrhizae in vasculas ne of two osta Rican rain foreste. Sissies 33:250—258. MIcHELsEN, A. 1993. The were status of vascular epiphytes in Bale Mountains National Park, thiopia. coe 4:11- MotezteE, A., J. G. Ducketr and A a RussgLL. 1996. Mycorrhizas in the ferns of Lesotho. Pp. 621 631, Camus, J. M., M. Gipsy and R. J. Jonns, (eds.), Pteridology in perspective. Royal Rotaric Gardens, Kew MurtHukumar, T. aad K. Uparyan. 2000. Arbuscular ibaa of plants growing in the Western Ghats region, Southern India. Mycorrhiza 9:297-3 306 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) NaparajaH, P. and A. Nawawi. 1993. Mycorrhizal status of epiphytes in Malaysian oil palm plantations. Mycorrhiza 4:21—-24. Newman, E. I. and P. REDDELL. 1987. - distribution of mycorrhizas among families of vascular : Zz Prrozynski, K. A. and D. W. Mattocn. ite The origin of land plants: A matter of mycotrophism. Racupatny, S. an . Manan DEVAN. 1993. Distribution of vesicular-arbuscular mycorrhizae in the plants and hzospho soils of the tropical plains, Tamil Nadu, India. Mycorrhiza 3:123-136. RicuTer, M. 2003. Using epiphytes and soil temperatures for eco-climatic interpretations in southern Bousdor. aan 57:161-181. Scum, E., F. OBERWINKLER and L D Comm. 1995. Light and electron epragee ed ofa Srna perecsieie: in the roots ote some spine. — from Costa Rica. Can. 73:991— SCHNEIDER, H., . SMITH, R. Cranritt, T. J. Hi-pepranp, C. H. HAuFLER ao 4 be Unraveling the phylogeny of aty ygram ced ferns (Polypodiaceae and Grammitidaceae): exploring — of diversification of epiphytic plants. Mol. Phylo. Evol. 31:1041-1063. ScHisier, A., D, Scuwarzcorr and C. Waker. 2001. A new fungal phylum, the Glomeromycota: pene eo pie Mycol. Res. 105:1413-1421. SMITH, » E. Scuuerrretz, P. Koratt, H. ScHNewer and P. G. Wo r. 2006. A as he eee ferns. Taxon 55:705-731. Trappe, J. M. 1987. Phylogenetic and ecologic ae ‘ mycotrophy in the angiosperms from an evolutionary standpoint. Pp. 5-25, in: Sarr, G. R., (ed.), Ecophysiology of va mycorrhizal lants. Boca Raton, FL, USA.CRC Press. Wane, B. and Y.-L. Qiu. 2006. ocleuaetig distribution and evolution of mycorrhizas in land plants. i opatag 16:299— Wiucke, W., S. Yasin, C. ania and W. ZecH. 2001. yioc oo of three microcatchments under ‘ropical ssecesteine forest in Ecuador. Die Erde 132 ZHANG, Y., L.-D. Guo and R.-J. Liu. 2004. Arbuscular eer ae associated with common pte ridophytes i in Dujiangyan, southwest China. Mycorrhiza 14:25-30 HAO, Z. W. 0. The arbuscular mycorrhizas of ipa in Yunnan, southwest China: evo sb aia interpretations. Mycorrhiza 10:145— American Fern Journal 99(4):307—322 (2009) Differences In Post-Emergence Growth Of Three Fern Species Could Help Explain Their Varying Local Abundance Kar RUNK* and MarTIN ZOBEL Institute of Ecology and Earth Sciences, Department of Botany, University of Tartu, 40 Lai St., 51005 Tartu, Estonia Asstract.—Despite the large number of comparative studies on species with different distribution and abundance, no clear general pattern of attributes explaining species’ rarity has yet been found. The relationship between different life-history traits of a species and abundance tend to be conditional and context dependent. We were interested in whether the local relative population density of three fern species in Estonia is related to post-emergence growth of their young sporophytes, i.e., that the locally abundant species, D. carthusiana, has the highest vegetative growth in its first growth periods and the two less abundant species, D. dilatata and D. expansa, have lower. We were also interested in differences between generative traits of young sporophytes of three species, specifically in the number of spores. We grew the species in a garden experiment for two vegetation periods, 2004-2005, until the first sporulation. The relative population density of the three Dryopteris species was related to the relative post-emergence growth of the species. The most abundant species D. carthusiana, exhibited the highest values of vegetative growth parameters in the first growth period. The less abundant D. dilatata and D. expansa both had shorter fronds, shorter intensive growth periods and lower leaf elongation rates. Dryopteris dilatata had a different vegetative growth strategy compared to the other two species; it differed in timing of intensive growth of frond length and increase of frond number and had the lowest values of generative parameters among the three species. Key Worps.—Dryopteris, Post-emergence growth, Rarity Ecology is aimed at detecting factors and processes that control the relative abundance and distribution of species (Kunin and Gaston, 1997; Crawley, 1997). Understanding why some species are more common than others provides us with basic information about the distribution and _ regional dynamics of different species. Such understanding is essential for the practical conservation and management of rare species, i.e., species with a low relative abundance/distribution at continental, and particularly at regional and local levels. One possible approach for investigating the mechanisms behind rarity is through the comparison of taxa with contrastingly different distribution and abundance patterns (e.g., Baskauf and Eicmeier, 1994; Sultan, 2001; Simon and Hay, 2003; Pohlman et al., 2005). The study of pairs or even larger numbers of closely related taxa with common genetic heritage may more easily reveal factors limiting rare species (Baskin and Baskin, 1986; Silvertown and Dodd, * Corresponding Author: (E-mail: kai.runk@ut.ee; phone: +3727376381; fax: +3727376222) 308 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Bradfield, 2000; Brown et al., 2003; Rymer et al., 2005), no clear pattern of general attributes or one specific feature explaining species’ rarity has yet been found. Relationships between different life-history traits of a species and abundance tend to be conditional and context dependent (Murray et al., 2002). Several recent studies have focused on the relative importance of dispersal and environmental determinants of fern distribution. Evidence has been found that habitat availability, at a local scale (Richard et al., 2000; Wild and Gagnon, 2005) and a regional scale (Guo et al., 2003), and not dispersal capability is responsible for fern distribution. Karst et al.’s study (2005) at two contrasting local spatial scales (local mesoscale and local fine) showed that fern distribution at the local mesoscale (135-3515 m) was linked to environmental factors, but at the local fine scale (4-134 m); both dispersal and abiotic environment were jointly responsible for fern distribution. 1988; Riink et al., 2006). The current study is a part of a larger project investigating the possible reasons of different regional frequency and local abundance of three closely related co-occurring fern species: Dryopteris carthusiana, Dryopteris expansa and Dryopteris dilatata. Dryopteris carthusiana is common in Estonia; D. expansa is distributed in scattered localities throughout Estonia, while D. dilatata is rare, being close to its north-eastern distribution limit. According to our earlier study (Riink et al., 2004); the different competitive abilities of D. carthusiana and D. expansa might help explain their different relative regional frequency, but not in the case of D. dilatata, which is near its distribution border as tolerant to competition as the most frequent D. carthusiana. Climatic factors are a likely limitation to distribution of D. dilatata in Estonia. The northern distribution limit of this species is approximately 300 km from Estonia, in southern Finland (Hultén and Fries, 1986), and shadows the RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE = 309 isothermal line along which the coldest month is between 5 and 8°C (Boucher, 1987). Still, the particular mechanism behind climatic restrictions remains open to debate. The results of field survey of the three species on permanent plots showed the higher local relative population density of D. carthusiana compared to D. dilatata and D. expansa (Riink et al., 2006). The order of the species’ rankings could be explained by the competitive ability of the three fern species. Therefore we hypothesized that the local relative population density of the three fern species is related to the success of post-emergence growth of their young sporophytes, i.e., that comparatively more abundant species have the highest vegetative growth in their first growth periods. We were also interested in whether there were differences in the generative traits of the three species’ young sporophytes, and erected the hypothesis that D. dilatata had the lowest number of spores than D. carthusiana and D. expansa. MATERIAL AND METHODS Study species——The three species studied are closely related from an evolutionary point of view (Gibby and Walker, 1977) and are morphologically similar (Fraser-Jenkins and Reichstein, 1984; Page, 1997). All three species are medium-sized, rhizomatous, herbaceous plants with 3-pinnate fronds and orbicular sori covered with reniform indusia (Fraser-Jenkins, 1993). Tetraploid (2n = 164) Dryopteris carthusiana (Vill.) H.P. Fuchs is the most common of the three species, and can be found throughout Europe, North America, West and Southeast Asia (Hultén and Fries, 1986; Fraser-Jenkins, 1993). Dryopteris expansa (C. Presl) Fraser-Jenkins and Jermy can also be found in North America and Asia. Tetraploid (2n = 164) Dryopteris dilatata (Hoffm.) A. Gray is distributed mostly in Western and Central Europe (Hultén and Fries, 1986; Fraser-Jenkins, 1993). Diploid (2n = 82) D. expansa is mainly restricted to mountainous regions of Europe, and has a more northerly and easterly distribution than D. dilatata (Fraser-Jenkins and Reichstein, 1984; Hultén and Fries, 1986). Piekos-Mirkova (1991) found D. expansa at 2098 meters above sea level, above the timberline in the Poland’s Tatra Mountains. In Scandinavia, the distribution limit of D. expansa is the northernmost of the three species (Jonsell, 2000). In Western and Central Europe, D. dilatata is a more common species than D. expansa (Fraser-Jenkins and Reichstein, 1984; Page, 1997). In Estonia the opposite is true; D. expansa is distributed in scattered localities throughout Estonia (Kukk and Kull, 2005), while D. dilatata, close to its north- eastern distribution limit (Page, 1997; Jonsell, 2000), is rare. Dryopteris carthusiana possesses the highest regional frequency of the three species, and is evenly distributed across the country. Similarly, the local abundance (population density) of D. carthusiana is the highest among the three species (Riink et al., 2006). According to the Atlas of the Estonian Flora (Kukk and Kull, 2005), in which Estonia is divided into a grid of 513 (6 x 10 minute squares), D. carthusiana was recorded in 441, D. expansa in 145 and D. dilatata in 20 of the squares. While D. expansa, like D. carthusiana, is distributed 310 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) evenly, most of D. dilatata populations are situated in the northern and western part of the country. In Estonia all the species can be found growing in mesic woodlands (Riink, 2002), mostly in mixed populations. All three fern species (D. carthusiana, D. dilatata and D. expansa) are sexually reproducing species (Manton, 1950) with sporangia that contain 64 spores (Widén et al., 1967; Schneller, 1975: Fraser-Jenkins and Reichstein, 1984) with a similar size per sporangium (Piekos-Mirkova, 1979; Seifert, 1992). Species nomenclature follows Fraser-Jenkins (1993). Experimental design.—Vegetative growth, reproduction, morphology and biomass were assessed in a common garden experiment conducted in 2004 and 2005. Spores of all fern species were collected in the wild in July 2003 and stored in a refrigerator (at 2 + 1°C) until the beginning of the experiment. The substrate used for spore germination was sterilized and consisted of 3 parts horticultural peat and 1 part fine-grade sand. Spores were sown on October 20, 2003 and sporophytes emerged in March 2004. Young sporophytes were planted, nine evenly spaced per plastic box (12 X 8 X 8cm deep), on May 16, 2004. The specimens were replanted individually in plastic pots (10 cm diameter, 8 cm deep) on August 2. Initially all three species were represented by 60 individuals, but for the final harvest and analysis, 15 individuals per species were randomly selected. The soil mixture used for receiving sporophyte plants consisted of 4 parts horticultural peat and 1 part fine-grade sand. The boxes were placed in a greenhouse at 22 + 2°C with a photoperiod of 12:12 h (fluorescent light: daylight tubes, photon flux density 40 umol s~'m~2) and watered as needed to keep the soil moist. On August 10 the pots were relocated to the experimental garden and grown in shaded light for another 14 months, In order to minimize possible differences in illumination, the positions of all pots were changed weekly. To imitate the species’ natural Estonian environment a screen with a shade value of 65% was used, as all three species can be found growing mainly in mesic woodlands. Shade treatment was provided using a screen made of aluminum-coated shade cloth (spectrum neutral; Ludvig Svensson, Kinna, Sweden). During the winter of 2004/2005, plants were covered with horticultural peat imitating fallen leaves and their decayed remnants. The experimental garden was located in Tartu (58°21'25"N, 26°42'5’E, 68 meters a.s.l.), in south-eastern Estonia, where the average annual temperature is 5.0°C and the average amount of annual precipitation is 550 mm (Jaagus, 1999). Data collection.—During the two growing seasons, a total of nine measurements were conducted every 28-34 days. Five measurements were made in 2004 (on June 9, July 9, August 9, September 10, October 8) and four in 2005 (on June 22, July 27, August 25, September 30), the first measurement of each year occurring when the fronds had rolled out and the last just before the first autumn frost. For each individual, the number of fronds was counted and the length of the longest frond was measured. In generative individuals, the number of fertile (spore-bearing) fronds was also counted. In the case of the length of the longest frond the frond was measured to the nearest millimeter on RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 311 each individual fern between the base of the stipe (stalk of the frond) and the tip. Those measurements allowed us to calculate leaf elongation rate (LER) and frond number increase rate (NIR). Leaf elongation rate (LER, mm/day) were calculated using the following basic equation: (Mn+1 — Mn) LER = ——————— D (1) where M,,; is the current measurement in millimeters; M, is the previous measurement in millimeters and D is the number of days between measurements. Frond number increase rate (NIR, number of fronds/day) was calculated using the following basic equation: (Fyi1 ies Fy) NIR = +—__* . D (2) where F,,; is current measurement (number of fronds); F, is the previous measurement (number of fronds); D is the number of days between measurements. LER and NIR were calculated for all seven time intervals between the measurements; four in 2004 (June, July, August and September) and three in 2005 (July, August and September). In generative individuals, the number of fertile (spore-bearing) fronds was also counted. After the final harvest in October 2005, fronds, rhizomes and roots were separated and dried at 75°C for 48 hours. Biomass fractions were determined by weighing the parts separately. The length of all fronds and frond laminae (the leafy part of the frond) were measured to the nearest millimeter before the final harvest. The length of the stipe was obtained by subtracting lamina length from frond length. Lamina area and lamina area (pinnae) covered with sori were measured using a scanner (ScanJet5p), DeskScan II 2.9, and Pindala 1.0 software (designed by I. Kalamees, Eesti Loodusfoto, Tartu, Estonia). Specific leaf area (SLA) was calculated as lamina area (cm”) per unit of lamina dry mass (g). Statistical analysis.—Differences in and the timing of vegetative growth (length of the longest frond and the number of fronds) during the both growth periods were tested separately for each year with repeated measures of ANOVA (using the Statistica software version 6.0; StatSoft Inc., 1998) with the species (three levels) as fixed factors and measurement time (five levels in 2004 and four levels in 2005) as a repeated factor. Differences in vegetative growth rate, LER and NIR, between D. carthusiana, D. dilatata and D. expansa during the growth periods in the years 2004 and 2005 were tested separately for each year with repeated measures of ANOVA with the species (three levels) as fixed factors and period of time between measurements (four levels in 2004 and three levels in 2005) as a repeated measurement factor. 312 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Taste 1. Results of repeated measures ANOVA: effects of species, measurement time and their interaction on the length of the longest frond and on the number of fronds of Dryopteris carthusiana, D. expansa and D. dilatata in 2004 and in 2005. Species Time Species*time Source of variation Df F e Df F-ratio P rt. F P Length of the longest frond in 2004 2 5.315 0.009 4 414.98 <0.000 8 12.63 <0.000 Length of the longest frond in 2005 2 9.448 <0.000 3 532.35 <0.000 6 5.937 <0.000 Number of fronds in 2004 2 21.88 <0.000 4 418.52 <0.000 8 4.865 <0.000 Number of fronds in 2005 2 11.88 <0.000 3 251.58 <0.000 6 1.508 0.181 Differences in the length of the longest frond and the number of fronds at the end of both growth periods and other morphological, biomass and reproduc- tive parameters between D. carthusiana, D. dilatata and D. expansa at the end of the experiment were tested by one-way ANOVA with the species (three levels) as fixed factors. In the case of LER and NIR the equation X’ = /X+ /X+ 1 was used for transformation (Zar, 1999). All other variables were log transformed, except in the case of relative biomass allocation, for which the data (as proportions) was arcsine square root transformed. Differences between mean number and length of fertile and sterile fronds among species were tested by Students’ t-test. The significance of the differences among all other parameters means was estimated with a Tukey HSD multiple-comparison test with a 0.05 significance level (Sokal and Rohlf, 1995). RESULTS Vegetative Growth Traits Vegetative growth and timing of the vegetative growth.—During both growth periods in 2004 and 2005, there were differences in length of the longest frond and in number of fronds between the three species (Table 1). Dryopteris carthusiana and D. dilatata were characterized by longer fronds and by a higher number of fronds than D. expansa; all differences were significant except in the case of the length of fronds between D. dilatata and D. expansa in 2004. There were also differences in the timing of vegetative growth between the species in 2004 and 2005 (Table 1), except in the case of the number of fronds in 2005. In 2004, D. carthusiana had the longest period of intensive growth when the increase in number of fronds and length of the longest frond between measurements were significant. The production of new fronds and the growth of the longest frond continued until September. Dryopteris expansa had the shortest period of intensive growth of the three species; the number of leaves increased until August and the length of the longest frond increased only until July. Dryopteris dilatata produced new fronds even in September, however the growth period of the longest frond matched that of D. expansa; it took place only in June. RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 313 TasBLeE 2. Results of repeated measures ANOVA: effects of species, period of time between measurements and their interaction on the LER and NIR of Dryopteris carthusiana, D. expansa and D. dilatata in 2004 and 2005 tin of Species Time period Species* time period variation Df F r Df F P Df F F LER 2004 2 22.66 <0.000 3 42.73 <0.000 6 4.704 <0.000 LER 2005 2 8.181 0.001 2 70.03 <0.000 4 0.848 0.499 NIR 2004 2 7.000 0.003 3 10.15 <0.000 6 1.638 0.144 NIR 2005 2 11.81 <0.000 2 17.53 <0.000 4 1.264 0.291 LER (leaf elongation rate) and NIR (fronds number increase rate).— Differences in LER (Table 2, Fig. 1) were more distinct than in growth of the longest frond or number of fronds; D. carthusiana had significantly the highest LER in 2004 and D. dilatata in 2005; the differences between the other two species were non-significant in both years. The timing of LER was different only in 2004; D. carthusiana had significantly higher LER in August 2004, compared to the two other species, and in July 2004, compared to D. dilatata. There were also differences in LER between 2004 and 2005. At the beginning of the experiment in 2004, LER of D. expansa and D. dilatata dropped during July, while in the case of D. carthusiana, high LER continued up to September. h 25+ = D. expansa --§-- D. dilatata pee es D. carthusiana = 20} 2 1.5} 3 2 C3 Fy 1.0F ir) Ge o 3 ost Oo} June 04 July04 Aug04 = Sept 04 July 05 Aug05 Sept 05 Period of time Fic. 1. Mean + SE of the LER /day) jlatata and D. carthusiana in June ayant fe ot aues August ae ae (10/ 09-08/10) 2004 and in July (22/06—27/07), August (27/07—25/08), September (25/08-30/09) 2005. Whiskers with the same letter are not significantly different (P < 0.05, Tukey test; separately for 2004 and 2005). X-axis breaks between the results of different analysis. 314 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) TaBLE 3. Results of one-way ANOVA: effects of species on the morphological, biomass and reproductive traits of Dryopteris carthusiana, D. expansa and D. dilatata at the end of the first growth period (October 2004) and at the final harvest (September 2005). Species (Df = 2) Source of variation F P October 2004 Length of the longest frond 13.60 <0.000 No of fronds 13.52 <0.000 September 2005 Length of the longest frond 172 <0.000 Mean frond length 6.653 0.003 Mean lamina length 4.508 0.017 Mean stipe length 23.93 <0.000 No of fronds 24.54 <0.000 No of fertile fronds 3.754 0.033 No of sterile fronds 12.79 <0.000 Total mass 13.85 <0.000 Rhizome mass 5.968 0.005 Root mass 13,35 <0.000 Frond mass 17.67 <0.000 Relative biomass allocation to lamina 14.20 <0.000 Relative biomass allocation to rhizome 13.97 <0.000 i 22.91 <0.000 3.413 0.042 Pinnae area covered with sori 5.472 0.009 In 2005, LER of all three species was the highest at the beginning of the vegetation period and fell significantly in September, at the end of the growth period. The differences in NIR (Table 2) were similar in both vegetation periods. The increase in number of fronds of D. carthusiana and D. dilatata was higher than that of D. expansa. — 15.42, Df = 12, P = <0.000; D. dilatata: t = —7.010, Df = 11, P = <0.000 and D. expansa: t = —16.82, Df = 13, P = <0.000). Dryopteris carthusiana and D, dilatata both had significantly higher biomass in regard to all fractions studied RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 315 d snl 4 [oO] D. expansa => A D. dilatata E RN D. carthusiana 4 200} 5 ° 2 = C=) = : 150 + Es = 100} el ° 3 oe b j 5 50 | aa N i F : N BR N N ‘ SS ZS FL 04 FL 05 SLi Parameter Fic. 2. Mean + SE of length of fronds of Dryopteris expansa, D. dilatata and D. carthusiana: length of the longest frond in October 2004 (FL 04) and length of the longest frond (FL 05) at the final harvest; length of the fronds (FLi), length of the lamina (LLi) and length of the stipe (SLi) per fern individual (mm) at the final harvest. Bars with the same letter are not — different (P < 0.05, Tukey test). X-axis breaks between the results of different analysis (total, frond, rhizome and root) and also larger lamina area compared to D. expansa. In the case of rhizome mass, the difference between D. expansa and D. dilatata was marginally non-significant (P = 0.09). There were no differences in SLA between species. The relative biomass allocation pattern was different between species; D. expansa allocated significantly more biomass into the rhizome and less into the laminae than D. dilatata and D. carthusiana (Fig. 4). Reproductive Traits Dryopteris dilatata had the lowest proportion of fertile individuals in the final harvest (80.0%), whereas D. expansa and D. carthusiana had more (93.3% and 86.7% respectively). Dryopteris dilatata had significantly fewer fertile fronds compared to the number of its own sterile fronds (t-test: t = 3.178, Df = 11, P = 0.01) and fewer fertile fronds per fertile individual than D. carthusiana at the end of the experiment (Fig. 3). Dryopteris dilatata also had a smaller pinnae area covered with sori per fertile individual at the final harvest compared to D. carthusiana and D. expansa (Fig. 5). There was no significant difference between the number of fertile and sterile fronds between the other two species. In the case of D. carthusiana and D. dilatata, vegetative reproduction was also observed; D. 316 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 14 d [a] D. nsa 12 D. dilatata D. carthusiana 10 h Cc Zs BS b aro} : b f ° Zi a o gs 4 e 2 #F04 #F04 #FFOS #SFO5 Parameter significantly different (p < 0.05, Tukey test). X-axis breaks | tk Its of different analy carthusiana had an average of 1.07 vegetative offspring per plant individual and D. dilatata 0.07. There was no difference among the species for the time when the first fertile frond appeared; all appeared in August 2005. Discussion During the first growth period all three species showed differences in vegetative growth. Intensive growth of D. carthusiana for a longer period of time than the other two species resulted in the tallest plants (the longest fronds) by the end of the first growth period and the longest fronds per fern individual by the second growth period. All morphological and. biomass parameters, recorded at the end of the experiment, showed that individuals of D. carthusiana were larger than those of D. expansa. The most successful post- emergence growth may be the crucial precondition for D. carthusiana’s high frequency in natural ecosystems. The first vegetation period of young D. carthusiana sporophytes was characterized by the longest period of intensive vegetative growth (from June until September), the highest LER, and as a result probably the largest biomass. Achieving higher fertility or utilizing more resources for reproducing could support the finding that the LER of D. carthusiana in 2005 was lower than that of D. dilatata. RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 317 100 Percentage of biomass D.expansa D2.dilatata D. carthusiana Species . Mean relative biomass allocation pattern in Dryopteris carthusiana, D. expansa and D Lae Proportions with the same letter are not significantly different (P < 0.05, iy test). hey 5 S Pinnae area covered with sori (cm?) 7 8 8 : 20 D. expansa D. dilatata D. carthusiana Fic. 5. Mean + SE of pinnae area covered with sori (cm”) per generative individual of Dryopteris expansa, D. dilatata and D. carthusiana at the final harvest. Bars with the same letter are not significantly different (P < 0.05, Tukey test). 318 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Although none of the reproductive traits of D. carthusiana were significantly higher than those of D. expansa in the present experiment, the ability of D. carthusiana to self-fertilize (in experimental conditions 55% of singly isolated gametophytes grown on soil and even 79% on decomposed wood formed sporophytes; Seifert, 1992) provides the species with a high potential for establishment (Flinn, 2006) and may be an important factor behind its broad distribution. In addition, comparatively high values of vegetative parameters in different light conditions (Riink and Zobel, 2007), and therefore the high competitive ability (Riink et al., 2004) may help to explain the highest local (Riink et al., 2006) and regional frequency (Kukk and Kull, 2005) of D. carthusiana among the three species in Estonia. In the first growth period D. expansa, compared to other two species, had the lowest values of frond number parameters (number of fronds in October 2004, increase in number of fronds and NIR in 2004). Dryopteris expansa, compared to D. carthusiana, had a shorter period of intensive growth, lower LER and lower values of frond growth parameters (length of the longest frond in October 2004 and increase in number of fronds in 2004). The biomass results of the present, two-year experiment related to D. expansa were analogous to results of our earlier one-year experiment (Riink et al., 2004); D. expansa had the smallest biomass parameters (total mass, roots mass and frond mass), except in the case of rhizome biomass. We also found a significant difference between D. expansa and the other two species in relative biomass allocation, where D. expansa invested more biomass in its storage organ, the rhizome, and less in the laminae. The different allocation strategy may be connected with the habitat preferences of this species such as better tolerance to severe climatic factors in mountains or in extreme northern regions of Europe. The relatively short period of intensive vegetative growth (only in June and July) may also have the same explanation. Although the reproductive success of D. expansa in terms of fertile fronds, both in natural (Riink et al., 2006) and experimental conditions, as well in number of spores, were not lower than of D. carthusiana, a low mean intragametophytic selfing rate of 0.34 (Soltis and Soltis, 1987) and thus low establishment ability may have an effect on the distribution frequency of the species. The lower vegetative growth of diploid D. expansa and hence lower competitive ability (Riink et al., 2004) and lower post-emergence growth compared to tetraploid D. carthusiana could be connected to the diploid origin and mating system (comparatively low intragametophytic selfing rate) of the species. The differences between diploid and tetraploid species may partly be based on higher levels of inbreeding depression in the case of diploid species (Masuyama and Watano, 1990). Tetraploid fern species are generally larger (Page, 2002), due to heterosis, and have higher rates of spore germination and faster growth rates (Kott and Peterson, 1974). Considering that D. dilatata is a tetraploid, its potential growth ability should be as high as D. carthusiana. Still, according to the results of the present experiment, D. dilatata had slower leaf elongation rates of young sporophytes during the first growth period, specifically in July and August, RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 319 which resulted in shorter plants by the end of September. Dryopteris dilatata, compared to D. expansa, had taller and a faster increasing number of fronds. Dryopteris dilatata had a different growth strategy compared to the other two species. Growth of the longest frond of D. dilatata was intensive for only a very short time, in June during the first growth period, similar to D. expansa. By contrast D. dilatata had an intensive increase in the number of fronds during almost the whole growth period until October, an even longer duration than D. carthusiana. The ability of D. dilatata to maintain intensive vegetative growth of the longest frond for a longer time may be restricted by some climatic factor. The notable difference in the timing of these two parameters may be connected with the different type of parameters under discussion. Since the frond size is more plastic than the number of fronds, an increase in the number of the fronds was preferred by the trade-off between the two parameters. Conse- quently, that ability of D. dilatata to establish in local vegetation very probably depends on some climatic factor. In better weather conditions D. dilatata may grow larger than D. expansa in the first growth period (Riink et al., 2004) and have better post-emergence growth ability. The growth of the species may be slower in less ideal conditions, as in the first growth period and continued in the second of the present experiment. In the second growth period D. dilatata achieved the highest LER, had a larger biomass, more and longer fronds than D. expansa; however this may occur too late for the successful establishment of the specific cohort and as well for the species. With regards to the reproductive parameters, D. dilatata had the lowest number of spores, the lowest number of fertile individuals and a lower relative number of fertile fronds, compared to the other species. The number of fertile fronds per fertile individual of D. dilatata was also the lowest among the three species, although the difference with D. expansa was not significant. Taken all together, those differences indicate that in given conditions, the reproductive success of D. dilatata might be the lowest. Not only may the unstable establishment abilities limit the distribution of D. dilatata, but also its comparatively low self-fertilization rate (only 19.2% gametophytes on soil and 35.2% on decomposed wood produced sporophytes; Seifert, 1992). Therefore a low establishment potential may contribute to the low frequency of this species in Estonia In conclusion, the relative population density of the three Dryopteris ee is related to the relative establishment abilities of the species. Dryopteri carthusiana had the highest values of the length parameters of cues growth and growth rate in the first growth period and has the highest local population density, while D. dilatata and D. expansa, both with shorter fronds, shorter intensive growth periods and lower leaf elongation rates, have lower population densities. Although the short time period of our observatory studies did not allow for any assessment of the dynamics of the distribution of D. dilatata in the region, the dynamic population structure (Riink et al., 2006) and high plasticity (Riink and Zobel, 2007) of the species might indicate that those species have a good perspective to expand their distribution in the future. 320 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Data made available in 2003 (Blamey et al.) has already shown expansion of the distribution of D. dilatata in Great Britain and Ireland during the last 40 years. Explanations for the distribution expansion may be the relatively young age of D. dilatata (allotetraploid, originated from D. expansa and D. intermedia), or expansion due to climate warming as already predicted (Bakkenes et al., 2002). ACKNOWLEDGMENTS We thank E. Toomiste for .* care of the plants in the experiment and Marcus Denton for editing the language of the manuscript. We would like to express gratitude to two anonymous referees and Editor in Dogs jennifer Geiger, for their constructive comments on the preliminary version of this manu This study was Randel by the Estonian Science Foundation (grant 5535) and Tartu University (grants 1896 and 2540). LITERATURE CITED BakkeENEs, M., J. R. M. ALKEMADE, F. THLE, R. LEEMANs and J. B. Latour. 2002. Assessing effects of forecasted climate change on the eae and distribution of European higher plants for 2050. Glob. Change Biol. 8:390-407 Baskaur, C. J. and W. G. ErckMerer. 1994. ea pe a of a rare and a widespread species of Echinacea (Asteraceae). Amer. J. cue 61: Baskin, J. M. and C. C. Baskin. 1986 ti 1 monitoring populations of rare plants in successional environments. Nat. Areas J. 6: 26-30. Bevitt, R. L. and S. M. Loupa. 1999. pe moenien of related rare and common species in the study of ea rarity. ~~. Biol. 13:493-498. Binney, E. P. an RADFIELD. 2000. initial comparison of growth rates in the rare grass Achnatherum — and its common associate Poa secunda. Ecol. Research 15: 181-185. Biamey, M., R. Firrer and A. Firrer. 2003. Wild flowers of Britain & Ireland. A & C Black Publishers Ltd., London. Boucuer, K. R. 1987. Climate of Europe. Pp. 428-445, in J. E. Oliver and R. W. Fairbridge, eds. The epee aba of Climatology. Van Nostrand Reinhold, New Les Brown, J. H., N. J. EnricHt and B. P. Miter. 2003. Seed production ination in ty d ee common emgertenini Acacia species from south-east Australia. Austral Ecol. 271-280. Cousens, M. I. . Blechnum spicant: habitat and vigor of optimal, so and disjunct postions, ond field observations of gametophytes. Bot. Gaz. 142:2 Craw ey, M. J. 1997. The structure ns plant communities. Pp. 475-531, in M. I. les ed. Plant ecology. Blackvel Oxford, U Finn, K. M. 2006. Reproductive ates of three fern species ge aoe to differential colonization success in Seah danobe: forests. Amer. J. Bot 289-1294. FRASER-JENKINS, C. R. and T. REICHSTEIN. 1984. opteris. Pp. dagen ce J. Conert, U. Hamann, W. Schultze-Motel and G. 3. Wagent eds. Illustrierte Flora von ead Verlag Paul Parey, Berlin oe pags FRASER-JENKINS, C. R. 1 gua Adanson. Pp. 27-30, in T. G. Tutin, V. H. Heywood, N. A. B D. H. — S. M. Walters and D. A. Webb, eds. Flora Europea. Cambridge University Press, Canbaidg. Gipsy, M. and S. WaLker. 1977. Further tudi isal of the diploi? ancestry in the Dryopteris carthusiang aii Fern Gaz. 11:315-324. GiTzeNDANNER, M. A. and P. S. Soxtis. 2000. Patterns of genetic variation in rare and widespread congeners. Amer. J. Bot. 87:783-—792. RUNK & ZOBEL: POST-EMERGENCE GROWTH AND VARIATION IN LOCAL ABUNDANCE 321 Grime, J. P. 1985. Aasipaec orn the contribution of pteridophytes to a local flora. Proc. Roy. Soc. ee 86B Griv_, J. P., J. fcr a R. Hunt. 1988. Comparative plant ecology. A functional approach to common British species. Unwin Hyman, London. Guo, Q. F., M. Kato and R. E. Rick.ers. 2003. Life history, diversity and distribution: a study of Ja epanese teridophytes. Ecography 26:129-138. Hu, R. H.1 mpara bitat requirements for spore cones and prothallial growth of three oes in Sprctrs ae Michigan. Amer. Fern J. 61:171-182. Huttén, E. and M. Fries. 1986. Atlas of North European Ns Plants. Koeltz Scientific Books, K6nigstein Jaacus, J. 1999. usi andmeid Eesti kliimast. (New data about the climate of Estonia). Publicationes nstituti oe Universitatis Tartuensis 85:28-38. JonseLL, B. (ed.) 2000. Flora Nordica 1. Bergius Foundation, The Royal Swedish Academy of Sciences, — Karst, J., B. Giipert and M. J. Lechowicz. 2005. Fern community assembly: the roles of chance and e sa acme at — ae! intermediate scales. Ecology 86:2473—2486. Kort, L. S. and R. S. Peterson. 1974. A c sear study of ie oo of the diploid and tetraploid races of Polypodium virginian anad. Kuxk, 7: and 7 L. (eds.) 2005. Atlas of ike Estonian Flora. secant " gical and a maneiory oy of the Estonian University of Life Sciences, Tartu Kunin, W. E. and K. J. Gaston. 1997. Rare-common differences: an overview. Pu. 12-29, in W. E. unin and K. J. Gaston, eds. The biology of rarity: causes and consequences of rare-common differences. — & Hall, London, UK. LeIsHMaN, M. R. 1999. How well do plant traits correlate with establishment ability? Evidence from a study of a guraniali grassland species. New P as Manton, I. 1950. Problems of cytology and evolution in iia phieidiohyte. University Press, Cambridge. Masuyama, S. and Y. Watano. 1990. Trends for inbreeding in polyploid pteridophytes. Pl. Spec. Biol. 5: par Murray, - R., P. H. THRaAxt and B. J. Lepscui. eee aap species rarity to life history in plants of tern opine Evol. Ecol. Research 4:937—9 PacE, e N. 19 he ferns of Britain and led a University Press, Cambridge. Pace, C. N. ude e _ Bolg strategies in fern evolution: a neopteridological overview. Rev. Palaeobot. Palynol. 1 Prexos-Mirxova, H. 1979. a z grupy D; dilatata complex in Poland). Monae f Bot. 50:1- Piexos-Mirkova, H. 1991. The distribution of the ne dilatata complex in Poland and in Slovakia. Ver6ff. A ge veiaies Inst. Riibel. 106: so PoHLMaAN, C. Nicorra and B. R. Murray. 2005. Geographic range size, wana ecdphysiclony ae phenotypic plasticity in pone oe Acacia species. J. Bio 1-351 Prapa, C., E. Pancua, S. PajaROn, A. ee A. Escupero and A. Rusio. 1995. A comparative study of gametophyte morphology, gametangial ontogeny and sex expression in the Asplenium adiantum-nigrum complex incolenionae Pteridophyta). Ann. Bot. Fenn. 32:107—115. RicHarp, M., T. Bernuaror and G. Beit. 2000. Environmental heterogeneity and the spatial structure of fern species diversity in one hectare of old-growth forest. Ecography 23:231-245 Rink, K. 2002. Initial survey of the sida geoisy carthusiana complex in Estonia. Fern Geax 16:450. Ronk, K., M. Moora and M. ZoseL. 4. Do different rapa abilities of three fern species explain psa different regional perenne J. Veg. Sci. 15:351-356. Rtnk, K., M. ra and M. Zope. 2006. Population stage structure of three congeneric Dryopteris species in agotant Proceedings of the Estonian Academy of Sciences. Biology. Ecology Be aarti w Polsce (The ferns of the Dryopteris 30 Ronx, K. and K. Zoset. 2007. Phenotypic plasticity and biomass allocation patt n three Dryopteris i species on an experimental light-availability essary Plant Ecol. 193:8 322, AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Rymer, P. D., R. J. WHELAN, D. J. Ayre, P. H. Weston and K. G. Russet. 2005. Reproductive success and sillier ps ata differ i in common and rare Persoonia species (Proteaceae). Biol. Conservation 123:521-532. Sato, Y. and A. Saxar. 1981. Cold ion of gametophytes of some cool temperature ferns native to Hokkaido. Canad. J. Bot. 5 —608 —. J. J 1975. Untersuchungen ae ciaheivais chen Farnen, insbesondere der Dryopteris filix- ruppe 3. Teil. Okologische Untersuchungen. Ber. Schweiz. Bot. Ges. 85:110-159. Gane M. 1992. Populationsbiologie und Aspekte der ee zweier Wurmfarne, Dryopteris carthusiana und Dryopteris dilatata. Univetsitat Ziirich, Z setae tue J. and M. Dopp. 1996. Comparing plants and cagnecnt traits, Phil. Trans. Roy. Soc. London, = 351:1233—1239. Siwon, M. F. J.D. Hay. 2003. m of a common and rare species of Mimosa PANE ae in Central Brazil. Pees a. 28:315-326. Soka, R. R. and F. J. Rouir. 1995. Biometry. Freeman & Co, San Fransisco. apie D. E. and P, S. remett 1987. oe system of the fern Dryopteris expansa: evidence for ed mating. Amer. J. Bot. 74: Ske Inc. 1998. STATISTICA for gouiinva (Computer program manual). StatSoft Inc., Tulsa, K. Suttan, S. E. 2001. Phenotypic plasticity for fitness components in Polygonum species of cntasting ecological breadth. Ecology 82:328-343. Won, C.-J., J. SaRvera and T. Anti. 1967. The Dryopteris spinulosa complex in Finland. Acta Bot. Fenn. 77:3-24. . GaGNoN. 2005. Does lack of available sano — explain the patchy ‘disttbutinns of rare calcicole fern species? Ecogra 2 Zar, J. H. 1999. Biostatistical Analne 4th edition. Prentice a aa New Jersey. American Fern Journal 99(4):323—332 (2009) The Function of Trichomes of an Amphibious Fern, Marsilea quadrifolia Tal-Cuunc Wu Inst. of Ecology and Evolutionary Biology, National Taiwan University Wen-YuaNn Kao* Inst. of Ecology and Evolutionary Biology, National Taiwan University, epartment of Life Science, National Taiwan University Asstract.—Marsilea ONG an atau asin fern, has the ability to develop heterophyllous, aerial and submerged leaves submerged leaves, aerial shied have Sirharnes ¢ on ot surfaces. To examine if the presence of tricho reflect excess light a i of being dam — ee excess light, we compared the _ optical caine cgi crenata ge a significantly increased in transpiration rates and decreased em in WUE were found in dc. enies nesaneed in pea rae to intact ones. These results imply that the presence of trichomes n reducing water loss than in reflecting light and protecting M. quadrifolia seis the potentially oo effect of photoinhibition in aerial environments. RDs.—amphibious fern, gas exchange, Marsilea quadrifolia, optical property, photoinhibi- tion, trichome How amphibious species cope with contrasting environmental conditions between aquatic and terrestrial habitats is of interests to researchers. Marsilea, an amphibious fern genus, has the ability to oe heterophyll. Marsilea quadrifolia L. experiencing extreme variation in environment develops submerged, floating, emergent and terrestrial wee (Liu, 1984; Lin et al., 2007). These different forms of leaves have different morphological and physiological characteristics. For example, in contrast to the glabrous surface of submerged and floating leaves, the adaxial and abaxial surfaces of emergent leaves are covered with dense trichomes. In addition, leaves of terrestrial grown M. quadrifolia (terrestrial leaves) have more trichomes than emergent leaves of aquatic grown (Lin et al., 2007). The ecological importance of these trichomes has not been studied. The two commonly cited functions of trichomes are to increase reflectance and to increase boundary layer resistance (Lambers et al., 1998). Increasing * Corresponding author: Wen- = Kao; Address: Department of Life Science, National — University, Taipei, Taiwan; Fax: 886-2367-3374, Phone: 886-3366-2511; e-mail: 1.tw 324 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) reflectance would reduce incident light on leaf surfaces and might reduce the risk of overheating and the potentially harmful effects of excessive light on leaves (Ehleringer, 1984). Increasing boundary layer resistance would reduce transpirational water loss. A combination of drought, temperature and light stress would greatly increase in terrestrial environments in comparison to that in aquatic habitats. Hence, the production of trichomes may represent one of the acclimation responses conferring M. quadrifolia the ability to grow in terrestrial conditions (Lin et al., 2007). The phenomenon of photoinhibition occurs when leaves are exposed to light levels in excess of what can be utilized in photosynthesis (Powles, 1984). Photoinhibition leads to decreases in photon yield and photosystem II is considered the primary site of photoinhibition (Barber and Andersson, 1992). The yield and kinetics of chlorophyll a fluorescence emitted from leaves upon illumination with actinic light have been used as a probe for the primary photochemistry of photosynthesis (Krause and Weis, 1991). In particular, the linear relationship between quantum yield and the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) (Kao and Forseth, 1992), suggests that Fv/Fm can be used as a probe to monitor the activity of photosynthetic carbon assimilation. A decrease in the values indicates reduction in photosynthetic activity. The objective of this study is to investigate the ecological significance of trichomes in leaves of M. quadrifolia. To examine if the presence of trichomes can reflect excess light and hence reduce the risk of being damaged by excess light, we compared the optical properties and chlorophyll a fluorescence of M. quadrifolia intact leaves (with trichomes) with leaves having trichomes removed (by de-trichomed treatment). Photosynthetic gas exchange measure- ments were also conducted to quantify transpirational water loss and instantaneous water use efficiency (WUE) of M. quadrifolia intact and de- trichomed leaves. We test the hypothesis that to reduce the risk of drought stress and being damaged by excessive light, M. quadrifolia develops trichomes, reducing transpirational water loss and/or reflecting excess light. MATERIAL AND METHODS Rhizomes of M. quadrifolia were planted in 2 L plastic pots filled with a mixture of vermiculite: soil of 1:1 by volume. Plants were grown in a glasshouse receiving natural daylight, watered to soil saturation every other day, and fertilized using inorganic fertilizer (N:P:K of 20:20:20) once every two weeks. The plant produces leaves with four leaflets expanded on a plane perpendicular to the petiole, resembling a four-leaf clover, which is connected to the rhizome. The following measurements were conducted on leaflets. The morphology of trichomes was observed and the length measured under a scanning electron microscope (TM 1000, Hitachi). The optical properties were measured on the same leaflet before and after partial trichomes being removed. To remove trichomes, we gently brushed both surfaces of leaflets. Trichome density was estimated on both surfaces with a dissecting microscope WU & KAO: TRICHOMES OF MARSILEA QUADRIFOLIA 325 before and after the de-trichomed treatment. Leaf optical measurements on adaxial surfaces were made using a custom-built dual integrating sphere system following the method described by Runcie and Durako (2004). Briefly, leaf spectral transmittance (T(A)) and reflectance (R(A)) were measured from 400 nm to 700 nm at 0.5 nm resolution using a fiber-optic spectrometer (HR2000, Ocean Optics) interfaced with a FOIS-1 (for T(A) measurement; Ocean Optics) or ISP-REF (for R(A) measurement; Ocean Optics) integrating spheres. Light source provided by a collimated beam from a tungsten-halogen light (LS-1, Ocean Optics) was directed into the entrance port and to an exit port of the opposite side of the sphere through an optical fiber. For measuring reflectance, measurements were calibrated against a 99% reflectance standard (WS-1-SS, Ocean Optics). After measurements of T(A) and R(A), we then calculated leaf absorptance (A(X) = 1 — T(A) — R(A)). To evaluate if the presence of trichomes can reduce photoinhibition, we measured the characteristics of fluorescence induction on intact and treated leaflets in situ using a portable, pulse amplitude modulated fluorometer (Mini- PAM, Walz, Effeltrich, Germany). Fluorescence was measured on the adaxial side of leaflets with or without trichomes removed (n = 6) at 10, 12 and 14h on a clear day. Leaves were adapted to darkness for 30 minutes before the measurement was taken. Photosynthetic photon flux (PPF) on a horizontal surface at the same height of the leaves and air temperature were also monitored. Photosaturated photosynthetic rates (A,,ax) and transpiration rates (E) were measured with an LI-6400 infrared gas exchange system (LI-Cor, Lincoln, Nebraska, USA) on the most recently expanded, intact and de-trichomed leaflets. The intercellular CO, concentration (Ci) was calculated according to Farquhar and Sharkey (1982). Measurement conditions within the cuvette were: photosynthetic photon flux density (PPF) of 1200 umol m~2 s~?, cuvette temperature 30°C, leaf-to-air water vapor pressure difference (VPD) 1.5— 1.6 kPa, and ambient CO, concentration 360 + 5 cm® m~®. The de-trichomed leaflets remained green and looked healthy a few days after the experiment indicating that the brushing treatment did not damage the leaflets. To further make sure that the de-trichomed treatment did not damage the epidermis, we also made paraffin sections of leaflets and found that the epidermis cells remained intact after the detachment of trichomes (data not shown). All statistical tests were performed with the computer software SYSTAT (Statistical Solution, Cork, Ireland). Significant differences are reported as P < RESULTS Characteristics of trichomes.—The morphology of trichomes is shown in the SEM picture (Fig. 1). Grown in terrestrial condition, M. quadrifolia produced multicellular, 2-3 cells, trichomes (Fig. 1). Before making the SEM scan, we used liquid nitrogen to fix the samples. Some of trichomes were detached from the surface by the treatment. Hence, we estimated trichome density with a 326 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Fic. 1. Adaxial surface of M. quadrifolia leaflet with trichomes. dissecting microscope instead of calculating the number of trichomes by SEM scanning. The result showed that there was no significant ane in trichome density between the adaxial and abaxial surfaces (Table 1). Leaf optical properties.—In general, intact and de-trichomed Tecdlats had the highest reflectance and transmittance and the lowest absorptance at ca. 550 nm in the wavelengths ranging from 400 to 700 nm (Fig. 2). In a comparison of intact and de-trichomed leaves, no significant difference was found either in T(A) or in R(A). Consequently, both types of leaves had similar A(A). As a result, ABLE 1. Trichome density (cm *) on adaxial and abaxial surfaces of intact and de-trichomed leaflets and the optical properties of M. quadrifolia (mean + S.E., n = 5). biecaep within the same row followed by different superscripts represent significant difference at P = Parameters Intact leaflets De-trichomed Trichome density Adaxial surface 1433 + 69° 149 + 47” Abaxial surface 1594 + 99° 159 + 39° Total 3027 + 164° 308 + 77° Optical properties Reflectance (%) 5.5 + 0.4° 5.3 = 1.0. Transmittance (%) 6.5 + 0.9% 78+ 1.1" Absorptance (%) 88.0 + 1.0° e704 17° WU & KAO: TRICHOMES OF MARSILEA QUADRIFOLIA 327 100 BOTH O he we 6 \ fs) 8 os > — ss Peal —®— Intact-R Fs) 50 - --4%--- Intact-T = —@— _ Intact-A o (=) OQ —O— De-trichome-R +h - De-trichome-T —— De-trichome-A Wavelength (nm) = c. 2. Average (n = 5) spectral reflectance (R), transmittance (T) and absorptance (A) of M. quadrifolia leaftlets before (Intact) and after the removal of tichomes (De-trichomed). reducing trichome density did not affect the average reflectance, transmittance and absorptance of visible light in M. quadrifolia leaflets (Table 1). Chlorophyll fluorescence measurement.—The diurnal courses of PPF at plant height and air temperature (Ta) were recorded on the same day as leaf fluorescence was measured (Fig. 3a). Initial and continuous dark adapted measurements of maximum PSII photochemical efficiency (Fv/Fm) indicated that the leaves were healthy and not experiencing stress due to the removal of trichomes (Table 2). Midday depression of Fv/Fm values was found in the leaflets when exposed to solar irradiation (Fig. 3b). In comparison to continuously dark-adapted leaves, illuminated leaflets showed significant reduction in Fv/Fm at 1000h, 1200h and 1400h when PPF and air temperature were highest during the day (Fig. 3). No significant difference was found in Fv/ Fm values between intact and de-trichomed leaflets. Gas exchange measurement.—Transpiration rate (E) increases with Amax in both intact and detrichomed leaflets; as a result, a significant, positive, linear relationship was found between Amax and E (Fig. 4a). No significant difference was found between the slopes of these positive relationships for intact and de- 328 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) 45 a 2000 - } 42 1500 4 a i] 2 e S L39 = = a = 3 1000 a oar O 2 = ot ee d 2 it. = & by Tag i. 500} [hy rt Gi 3s g ir a ~@- prr a -} Temp 04 - - 33 8 10 12 14 0.7 - Intact -CO- De-trichome E & 06 - > _ 0.5 8 10 12 14 16 Time (h) Fic. 3. Diurnal changes in air shoe ase and PPF incidence on a horizontal surface (a) and the ratio of Fv to Fm (mean + s. e., n = 6) measured at 1000, 1200 and 1400 h on meen —— leaflets of M. quadrifolia ak (De- sichowad and without (Intact) removal of trichom WU & KAO: TRICHOMES OF MARSILEA QUADRIFOLIA 329 TasLe 2. Maximum PSII photochemical efficiency (Fv/Fm) of initial (before leaflet being exposed to solar irradiation) and continuous dark adapted intact and de-trichomed leaflets of M. quadrifolia used in the measurement of chlorophyll fluorescence (mean = s.e., n = 6 Treatments Initial Continuous dark Intact 0.76 + 0.01 0:77: O41 De-trichomed : O77 20.01 0.77 + 0.01 trichomed leaflets. However, de-trichomed leaflets had significantly higher E relative to intact leaflets for a given Amax- The calculated Ci values of de-trichomed leaflets were higher than those of intact leaflets (Fig. 4b). DISCUSSION Knowledge of how fern species cope with excess light or drought stress in terrestrial environments is of ecological and evolutionary importance. Biochemical mechanisms, such as xanthophyll-mediated energy dissipation, and/or morphological mechanisms, such as leaf curing and laminar scales, have been demonstrated in pteridophytes (Eichmeier et al., 1993; Tausz et al., 2001; Watkins et al., 2006). To our knowledge, the influence of pubescence on the incident radiation and water budget has not been studied in any fern species. Among other functions (Johnson, 1975; Zvereva et al., 1998), pubescence had been shown to increase reflectance (Ehleringer, 1984; Holmes and Keiller, 2002) and afford protection against excess radiation (Ripley et al., 1999; Morales et al., 2002; Manetas, 2003) in seed plants. Our results, however, showed that M. quadrifolia leaflets have a very high absorptance and de- trichomed treatments did not affect the visible part (400—700 nm) of optical properties of the leaflets (Fig. 2). Additionally, the Fv/Fm values of leaflets of M. quadrifolia at the midday were not affected by the removal of trichomes (Fig. 3b). It is therefore possible that trichomes on leaflets of M. quadrifolia are of less importance in reflecting light and in protecting the plant against the potentially damaging effect of photoinhibition. However, leaf temperatures in de-trichomed leaflets may be reduced due to their increased transpiration rates (Fig. 4a), which may ameliorate the damaging effect of high light and high air temperature on de-trichomed leaflets. For example, the result that the de- trichomed/orienting leaflets had less reduction in Fv/Fm, though not significant, than intact/orienting leaflets (Fig. 3b) might result from the increased transpiration rate in the former. Accordingly, we cannot completely exclude the role of trichomes in providing photoprotection for M. quadrifolia leaflets. Few studies have also shown that the presence of trichomes can reduce leaf transpiration rates (Ripley et al., 1999). The significantly increase in transpiration rates, about 30%, measured in de-trichomed M. quadrifolia leaflets compared to intact ones (Fig. 4a) suggests that the presence of E (mmol m? s") we AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) et Pa Intact De-trichome O 1 - - —_—_—_——, 8 12 16 20 280 - O 260 - On a 240 4 ie = = C) 5 O O 7 ame} e ad E) O ee , 2 200 e 180 T T T Tea eee Se 8 12 16 20 Fic The relationship between photosaturated photosynthetic rate (A... bates (E) (a) and intercellular CO, concentration ( and with removal of trichomes (De-trichome d) 2 Amax (Hmol m™ s’) ) and transpiration Ci) (b) of M. quadrifolia leaflets without (Intact) WU & KAO: TRICHOMES OF MARSILEA QUADRIFOLIA 331 trichomes is of more importance in reducing water loss than in providing photoprotection. At constant leaf-air vapor pressure deficit, Ci values may be used as a measure of the instantaneous water use efficiency (WUE) of the leaf (Farquhar and Sharkey, 1982), with a lower Ci indicating a higher instantaneous WUE. De-trichomed treatments resulted in leaflets with higher Ci (Fig. 4b) implying a lower WUE. These results reveal that the reduction in water loss from M. quadrifolia leaflets with trichomes also resulted in increased WUE. he hair layer on leaves may lead to higher leaf temperatures caused by reducing the transpirational water loss (Ripley et al., 1999). We have observed that leaflets of M. quadrifolia have the ability of performing tropic movements (pers. obs.). It is possible that M. quadrifolia adjust leaflet angle and azimuth to intercept a smaller quantity of radiant energy, which would allow the plant to moderate leaflet temperature without excessive transpi- ration. The function of tropic leaf movements in protecting soybean leaves from photoinhibition has been documented (Kao and Forseth, 1992). Thus, the presence of trichomes on both surfaces combined with leaflet movements may provide M. quadrifolia mechanisms against drought stress. Accordingly, we hypothesize that the combination of the avoidance mechanisms, leaf movements and the production of trichomes are very important adaptations, conferring the amphibious M. quadrifolia ability to grow in terrestrial conditions. The effect of the interaction between water availability and light intensity on trichome density and leaflet movements in M. quadrifolia are currently under studied. AACKNOWLEDGMENTS ank Dr. Bai-Ling Lin, for the inspiration of this study and providing rhizomes of M. quadrifolia, Dr. Shiang-Jiuun Chen for providing technique support in taking the SEM picture, and Yih-Chi Chang, for help in taking chlorophyll fluorescence and gas exchange measurements. LITERATURE CITED Barser, J. and B. ANDERSSON. 1992. Too much of a good thing: light can be bad for photosynthesis. Trends in oe Sci. 17:61-66. EHLERINGER, J. R. 1984. Ecology and esi elisa of leaf pubescence in North American desert plants. In E. hui P. L. Healey and I. Mehta, eds. Biology and Chemistry of Plant Trichomes. Plenum Press, New cae EIcHMEIER, W. G., C. CASPER and C B. a oats pnts des fluorescence in the resurrection plant Selaginella lepid g high-light a seusuaian stress, and evidence for ee, grind beet Planta 189:30— Farqunar, G. D. and T. D. SHarKEy. 1982. Stomatal conductance and ie Ann. Rev. Plant Pigeiol: 33:317-345. Jounson, H. B. 1975. Plant pubescence- ecological perspective. Bot. Rev. 41:233—-258. Ho.mes, M. G. and D. R. Kemer. 2002. Effects of pubescence and waxes on the reflectance of leaves in the ultraviolet and photosynthetic wavebands: a comparison of a range of species. Plant Cell Environ. 25:85—93. 332 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) Kao, W.-Y. and I. N. ForserH. 1992. Diurnal leaf movement, chlorophyll fluorescence and carbon assimilation in (oc grown under different nitrogen and water availability. Plant Cell Environ. 15:703—71 Krause, G. H. and E es 1991. ae nae and photosynthesis: the basics. Ann. Rev. Plant Piysiol. Plant Mol. Biol. 42:313-349 Lampers, H., F. S. Cuapin III and T. L. Pons. on Plant Physiological Ecology. Springer, New York. Lin, C.-H., B.-L. Lin and W.-Y. Kao. 2007. Leaf characteristics and photosynthetic performance of floating, emergent and terrestrial leaves of Marsilea quadrifolia, Taiwania 52:195-200. Lv, B. L. eee 1984. Abscisic acid induces land form characteristics in Marsilea quadrifolia L. Am. J. B 7638-644. Maneras, & 2003. The importance of being hairy: the adverse effects of hair removal on stem photosynthesis of Verbascum speciosum are due to solar UV-B radiation. New Phytol. 158:503-—508 igcrsieoe ?> AS ApRapia, J. ApBapia, G aad ae and E. Gri-PELecrin. 2002. Trichomes f Quercus ilex subsp. billota ‘Das ) Sam amp. an and Quercus coccifera L. to Mediterrane ean stress conditions. Trees 16:504—51 Powtss, S. B. 1984. Photoinhibition of photosynthesis induced by visible light. Ann. Plant Physiol. 35:15-44 Riptey, B. S., N. W. Pameonee and V. R. Smrrx. 1999. Function of leaf hairs revisited: the hair layer on leaves of Arctotbeca popula reduces photoinhibition, but leads to higher leaf Speer caused by lower transpiration rates. J. Plant Physiol. 155:78-85. Runciz, J. W. and M. J. Durakxo. at 004. Among-shoot variability and leaf-specific absorptance characteristics affect diel estimates of in situ electron transport of Posidonia australis. Aquat. Bot. 80:200-209. Tausz, M., P. Herz and O. Briones. 2001. The significance of carotenoids and tocopherols in photoprotection of Sie epiphytic fern species of a Mexican cloud forest. Aust. J. Plant Physiol. ou 75-78 Watkins, J. E., A. Y. Ea S. T. Leicut, J. R. Autp, A. J. BicksLer and K. Kaiser. 2006. Fern aminar scales na leanne photoinhbition a excess light. Am. Fern J. 96:83—92. ZveREVA, E. L., M. V. Koztov and P, NiEMELA. 8. Effects of leaf pubescence in Salix borealis on host- plant choice sey feeding leh oe de leaf ioke Melasoma lapponica. Ento. Exp. Appl. 89:297-303 American Fern Journal 99(4):333—334 (2009) SHORTER NOTES Isoetes duriei New to Lebanon.—In a recent paper, we (Bolin et al., Turkish Journal of Botany 32:447-457. 2008) discussed the taxonomy and distribution of the quillworts (species of the genus Isoetes, Lycophyta) in Western Asia. In this supplementary note, we record the presence of three quillworts new to Lebanon —one a widespread Mediterranean species, one known from only a single site in Turkey and two in Syria, and an undescribed new species. With this report, the number of documented species in Lebanon has increased from one to three. Voucher specimens will be deposited at BEI, E, and ODU. In his flora, Mouterde (Nouvelle Flora du Liban et de la Syria. Beirut: Editions de L’Imprimerie Catholique. 1966) included two species of Isoetes from Syria and Lebanon- Isoetes olympica A. Braun known from only a few sites on Jebel Al Arab (historically known as Jebel Druze) in extreme southeastern Syria, and what Mouterde called I. histrix Bory forma subinermis Durieu from the Akkar region of northern Lebanon. He separated the two species chiefly on the basis of velum coverage—I. olympica with an incomplete velum and I. histrix forma subinermis has complete velum coverage. Musselman (Fern Gaz. 16(6, 7 & 8):324—3 29. 2002) noted the impending demise of the Jebel Al Arab populations due to habitat destruction. However, in 2007 populations of I. olympica were found at several sites in the vicinity of Homs, Syria (Bolin et al., 2008). In April 2009, we located thousands of quillworts at several different sites in the Akkar region of extreme northern Lebanon, a region of basalt derived soils. Examination of the megaspores showed clearly that they are J. duriei Bory, a Mediterranean species previously unknown in the eastern Mediterranean with the closest populations in Turkey (Bolin et al., 2008). Unlike most species of quillworts, I. duriei is terrestrial and grows in typical garrigue (degenerated Mediterranean forest) vegetation. Plants were small and initially difficult to locate among the grasses and forbs. Based on the large number of plants we saw, it is hard to understand how this plant could have been overlooked after more than a century and a half of botanical studies in Lebanon. This may be due to their maturation as early as mid-April, plants were beginning to senesce and had mature spores at this stage. The large megaspores of I. duriei with distinctive alveolate ornamentation are easy to recognize, being among the most distinctive in the genus. Near the village of Kfar Noun, especially robust plants of I. olympica, readily discerned by the incomplete velum and much smaller tuberculate megaspores, were abundant in a vineyard among numerous I. duriei. Isoetes olympica has never been reported from Lebanon. For almost a century, I. olympica was known only from the type locality near modern day Bursa, Turkey. In the 1930’s several populations were found at Jebel Druze (Musselman, 2002). The discovery of large populations near Homs and the recently discovered Akkar plants strongly suggests that I. olympica has a much wider distribution and is 334 AMERICAN FERN JOURNAL: VOLUME 99 NUMBER 4 (2009) more abundant than previously thought. It should be sought at additional sites in Syria, as well as eastern Turkey and Iraq. Images of I. olympica and IJ. duriei from Akkar are at: http://www.odu.edu/ Imusselm/plant/index.php In addition to I. olympica and I. duriei we found a third species which is apparently new to science. Hybrids are known from most places in the world where two or more species grow together and we have recently noted the first hybrids involving I. duriei (with I. histrix)(Bolin et al., 2008). The addition of these species to the flora of Lebanon is significant for two reasons. It is the first report of I. duriei in the eastern Mediterranean. We have also documented new populations of I. olympica formerly thought to be of great conservation concern. The only quillwort we have not yet found in Lebanon is J. histrix forma subinermis (sometimes known as I. subinermis (Bory) Cesca & Peruzzi, see Bolin et al., 2008). Because this taxon, sensu Mouterde, has a complete velum, it must include I. duriei and I. olympicaa. It is likely that additional quillwort species could be found in the eastern Mediterranean and we hope that this note will help botanists be aware of these easily overlooked plants.—Lyrron J. MusseLMAN and Monammap S. AL- ZeIN, Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529-0266, USA. American Fern Journal 99(4):335 (2009) ERRATA AFJ volume 98, issue 4, pp. 214-250 (October-December 2008) The following errors have been kindly pointed out by Alan R. Smith, UC voD te Mud . sD SU UU Berkeley, in Lehnert, M. (2008). Eleven new species in the grammitid fern genus Melpomene (Polypodiaceae). American Fern Journal 98(4): 214— 250. . 219 — M. albicans: Lehnert 512, 599, 601, ere not at UC. 219 — M. albicans: Lehnert 707 should be 7 . 221 — M. caput-gorgonis: the type Brae Oe Lehnert 367 (p. 219) is cited also as an additional specimen examine . 219 — M. caput-gorgonis: isotype Lehnert 367 not at UC, but LPB. . 221 — M. caput-gorgonis: Lehnert 386 not at UC but MO, Lehnert 392 not at UC . 222 — M. caput-gorgonis: Lehnert 496a, 586 not at UC. 224 — M. flagellata: Jiménez I. & Gallegos 527 must read 725. . 229 — M. jimenezii: In the additional specimens examined, the collector Perea is misspelled Perera. . 230 — M. michaelis: In order to conserve the writing of the epithet, the dedication of this species is restricted to Michael Sundue. A singular of the name as pars pro toto is apparently not tenable. 231 — M. michaelis: The type collection Lehnert 519 (p. 229) is also cited as an additional specimen examined. 234 — M. occidentalis: Lehnert 1464a, 1558a not at UC. 236 — M. paradoxa: the type collection Kessler et al. 11717 (p. 235) is cited also as an additional specimen examine 236 — M. paradoxa: Lehnert 536, 542 not at UC. 241 — M. personata: Kessler et al. 6862 must be 6862B, not at UC. 241 — M. personata: Lehnert 404 not at UC. 244 — M. sklenarii: Sklenar & Sklenarova 2803 not at UC. 249 — M. vulcanica: Lehnert 168, 174 not at UC. Marcus Lehnert, Staatliches Museum fiir Naturkunde Stuttgart, Am Lowentor, Rosenstein 1, D-70191 Stuttgart, Germany. American Fern Journal 99(4):336 (2009) Referees for 2009 All papers submitted to the journal are peer reviewed. Members of the editorial board and the Journal and to its continued success. The American Fern Society and I extend our thanks to the following reviewers for the assistance, sre ae and patience in the year 2009 (I apologize if I inadvertently omitted anyone from this ALDRI VictorR AMOROSO DANIEL BALLESTEROS MIKE BARKER Davip BARRINGTON DaMIEN BONAL GaBRIELA GIUDICE MicHAEL MESSLER JOHN Micke Maria MOLINE RICHARD Moore Rossin Moran JEFFERSON PRADO Tom RANKER SHANE SHAW HITT GEORGE YATSKIEVYCH X. C. ZHANG Z RADHANATH MUKHOPADHYAY Jose Maria GasrieL y GALAN LyTToON JOHN MussELMAN GERALD GaASTONY JENNIFER Ramp NEALE WANG ZHONGREN STATEMENT OF OWNERSHIP, MANAGEMENT, AND CIRCULATION Publication title and number: American Fern Journal (0002- 8444), Date of filing: 63166-0299. Editor: Jennifer Geiger. The American Fern Journal is wholly owned by the American Fern Society, Inc., with no bond holders. Physical address of the Society: c/o Missouri Botanical Garden, 4344 Shaw Blvd., St. Louis, MO 63110. The purpose, function, and nonprofit status of the Society and its tax exempt status for Federal income tax purposes remains the same as in past years. The average press run for Volume 99 is 912, and was 900 for the issue appearing immediately prior to the filing date, for which 760 copies were mailed as paid circulation and 0 copies were mailed as free distribution, leaving 134 copies for office use and back-issues sales. I certify that these statements are correct and complete. GzorcE YATsKiEVycH, Membership Secretary of AFS Table of Contents for Volume 99 (A list of articles arranged alphabetically by author) A.-Hampanl, S. H. and J. J. GHazat. Selected Physiological Responses of Salvinia minima to Various Temperatures and Light Intensities ................. Al-Zen, M.S. (eee L. |, MUSSRIMAN) 0.6 a ee Anperson, O. R. Eukaryotic Microbial Communities Associated with the Rhizo- sphere of the Temperate Fern Thelypteris noveboracensis (L.) Nieuwl. Arosa, M. L., L. G. Quintanita, J. A. Ramos, R. Ceta and H. Sampaio. Spore Maturation and Release of Two Evergreen Macaronesian Ferns, Culcita macrocarpa and Woodwardia radicans, along an Altitudinal Gradient ... AyrapeTov, A. and M. T. Gancer. Nutrient Levels Do Not Affect Male Gametophyte Induction by Antheridiogen in Ceratopteris richardii .................. Barker, M. S. and G. YarskievycH. Summary of the 2008 AFS Symposium: From Gels to Genomics: The Evolving Landscape of Pteridology. A Celebration of Gerald Gastony’s Contributions to Fern Evolutionary Biology ........ Barker, M. S. and G. Yarsktevycu. A Brief History of Gerald J. Gastony’s Botanical COMOOP ie Barker, M. S. Evolutionary Genomic Analyses of Ferns Reveal that High Chromosome Numbers are a Product of High Retention and Fewer Rounds of Polyploidy Relative to Angiosperms ............0..cecceeeeceeccencs BARWNGCTON. . S, teon G, f..Gastony). 32. 6 Bax, | tee Ml 0 WAM) a ee ke Ca.urr, M. G. A New Species of Adiantum from Cuba ................00+-000: (CaAnpeius, C. (aoe J.B. Warts te) 660... es Coun, Bipot LC AROSAD 2 i Cuanc, H.-M., W.-L. Cxrou and J.-C. Wanc. Molecular Evidence for Genetic Heterogeneity and the Hybrid Origin of Acrorumohra subreflexipinna mu TRIWE CuristeNHusz, M. J. M. Type Specimens of Dracoglossum sinuatum Uncovered in the Rio Ge lane Heras cc Conant, i. S (eee) Gasman). gw Lmomuen 1k Oe A PRAGA) a ery, A eee FS We) 8. a EpiwarA, A., J. H. Nitra, D. Lorence and J.-Y. Dusuisson. New Records of Polyphlebium borbonicum, an African Filmy Fern, in the New World MN PIRYROMA ga se i a 200 ELeurério, A. A. and D. Pérez-Sauicrup. Transplanting Tree Ferns to Promote Their CAO AUN it MAONIGD i i ic Viet ies ce 279 PAveas, 2 BR ieee PF PA) eee 249 Fiori, C. C. L., M. Santos and A. M. Rano. Aspects of Gametophyte Development of Dicksonia sellowiana Hook (Dicksoniaceae): an Endangered Tree Fern Indigenous to South and Central America ..............ccecesesececees 207 ReaD OO I PPRAORTON i oko ie inscsee ce 273 GancuLy, G., K. Sarkar, and R. Muxuopapuyay. In vitro Study on Gametophyte Development of an Epiphytic Fern Arthromeris himalayensis (Hook.) ine OF OOntn Mike Windia) 5666 ii isla 217 Gastony, G. J., D. S. BarriNcTON, and D. S. Conant. Obituary: Alice Faber Tryon Re i a. 231 CHAZAL, 7.7: fone S. TL, AL-PEAMOANY) oe sa ee, 154 Comuez, Ac Li; (eek MOD WINDHAM) oo cs oa ea 128 Guo, X.-s. and B. Li. On Neolepisorus emeiensis and N. dengii (Polypodiaceae) ORE CRI es ee es a a 244 Haur er, C. H. Gels and Genetics: The Historical Impact of Isozymes on Paradigm Shifts in Hypotheses about Fern Evolutionary Biology ................. 125 Hickey, R. J., C. C. Mac.ur and M. Link-Pérez. Isoetes maxima, a New Species from Brat oo a a ia 194 Hosuizaxi, B. J. Illustrated Flora of Ferns and Fern Allies of South Pacific Islands 59 Hau, B teee Cy BR. MARTIN (ee a 145 Hua, W., C. Pinc-Tinc, Y. Li-Pinc and C. Lonc-Qinc. An Efficient Method for Surface Sterilization and Sowing Fern Spores in vitro ...............+- 226 Hur, i. (eg MD War) «2 a 128 Kao, W.-Y. (eee TC Wo) 323 Ressumn, M. (sea: Mi. Peineet) 3 ee 292 KOrrke, & gee Mo LeANERT) (2. ee 292 LABIAK, F. He (sep Fo BuMATOS) og a 101 LABIAK. Po He isee RU, MORAN) 2. Se 1 LEHNERT, M., I. Kottke, S. Seraro, L. F. Pazmino, J. P. SuArez, and M. KeEssLer. Mycorrhizal Associations in Ferns from Southern Ecuador ............. 292 ba, B. (eee Sie, Gn os is a ee a a 244 Dh TAG. (ape G. BE Magi) 3. sos oo ioe eS ee ee 145 bm Pome, Vo leee Bei) a ce 194 Lonc-Ome. Go teoe W. Mua) ee 226 Losocs, 2. ene A. Remagal ae ee 200 Macy, G. Gea KR. a 194 Martin, C. E., R. Hsu, and T.-C. Lin. Comparative Photosynthetic Capacity of Abaxial and Adaxial Leaf Sides as Related to Exposure in Two Epiphytic Ferns in a Subtropical Rainforest in Northeastern Taiwan .............. Matos, F. B., P. H. Lasiak, and L. S. Sytvestre. A New Brazilian Species of the Conus Aaplonium L. (Asploniacene) 2.200.650 6.446505 06 101 Mee 1 t ieee LD. eee) 109 Mouina, M., V. Reyes-Garcia and M. Parpo-pe-SantayaNna. Local Knowledge and Management of the Royal Fern (Osmunda regalis L.) in Northern Spain: Implications for Biodiversity Conservation ...............e.ceeeeceeees 45 Mora-O.ivo, A. and G. YatskiEvycH. Salvinia molesta in Mexico ............... 56 Moran, R. C., J. Prapo and P. H. Lasiak. Megalastrum (Dryopteridaceae) in Brazil, Paraguay, and Uruguay. 206 ce es ea BAUR PANVAY, (0004s, GANGULY) 62250... 6 237 MussELMAN, L. J. and M. S. AL-ZENN. Isoetes duriei New to Lebanon ............ 333 Nakazato, T. Fern Genome Structure and Evolution ..............0.eceeceeeee 134 INITIAL J, 01, (OOO A BAARAD oo a 200 PAzinG, LF (606 ME Tee) 292 PARDO-De-SANTAYANA, M. (s66 M. MoOUNA) ... 2... 0 es 45 Phae-SaAucme. D. (see A. A. Rimvis) 279 PinG- Tig, (ieee W, HUA) 6 a 226 PADD, } i900 Bt MORAN) (0 1 reves, &. M. (see M.D. WitowaM) «24. es 128 Qi, X., ¥. Yano, Y. Su, and T. Wane. Molecular Cloning and Sequence Analysis of Cyanovirin-N Homology Gene in Ceratopteris thalictroides ............ Compania, LG. (966 MUL. Anoaal 2 260 RErEetsAmA, ¥, ee Me Mola) ©. 45 Raber. Fo Ms eee A. Ta) 238 Kaos, 1 A ieee A Abe) ee s. 260 Sean A A foe ©. GL. Pie) ge iS Borns, {; M. leee F. G. Wore)... porupaa, 1, (966 M.D: Weuwian) | ose i i a ee Runx, K. and M. Zoset. Differences In Post-Emergence Growth Of Three Fern Species Could Help Explain Their Varying Local Abundance .......... Saino, A. New Combinations in Pleopeltis (Polypodiaceae) from Southeastern RN i PRADA Po Oe TL A ro ir ee A Bintoe. NE gee US eee ie a SAREAR, ©. (oon G GAN a ig as i ererrees, OOO L.A) et RTARO Oo LGO0 (Vi LEHNERT) (000 Ge a eae SHANKAR, R. and R. C. Srivastava. Obituary: Prem Kumar Khare (1946-2009) MiNi, Pu, BR MUO 1. 1D, SE ee oo i is oi a Situ, A. R. Flora de Nicaragua. Tomo 4. Helechos ................2.-0eeeeee: Srivastava, A. and S. CuHanpra. Structure and Organization of the Rhizome Vascular System of Four Polypodium Species ..............+s+seeeeeee MRIVARTAVA. OO COOGAN) 6 es i ee ea a OO a a a mianee, 7. P. eee A. Le mviyeen i ee Be a TeyERO-Dfez, J. D., J. T. Micke, and A. R. Smirx. A Hybrid Phlebodium (Polypodiaceae, Polypodiophyta) and Its Influence on the Circumscription ioc i eee, Troia, A. and F. M. Raimonno. Isoétes todaroana (Isoétaceae, Lycopodiophyta), a New Species fram Sicily (tely) ..0 6655 sk cae ei i es Wana, 14. eee FM, Cea) aes oe a Wane, T. (eee OO oie oa ke a i a Watkins, J. E. Jr. and C. Carpe.ts. Habitat Differentiation of Ferns in a Lowland Tropical Bain Fore i is ese es oe WinpuaM, M. D., L. Huet, E. Scnuetrperz, A. L. Grusz, C. Rorure.s, J. Beck, G. YaTskiEvycu, and K. M. Pryer. Using Plastid and Nuclear DNA Sequences to Redraw Generic Boundaries and Demystify Species Complexes in Chellentinid FOO 656s or a ee Wor, P. G., A. M. Durry, and J, M. Roper. Phylogenetic Use of Inversions in Fern CRIONOIIBNE GONE in cies cece ceca cee cc east Wu, T.-C. and W.-Y. Kao. The Function of Trichomes of an Amphibious Fern, PRIETO TION si cee eel 323 Tae, 1 A Oe eo 78 Varoaevecs, G gee A. Masai). 6... 56 wAToEVIGH, G, (non MS. BAgees) oi... oo ee ee 417 WAvericH, (tee MD WINDEAM) .....5.. 02.02... a 128 Zika, P. F. and D. R. Farrar. Botrychium ascendens W. H. Wagner (Ophioglossa- ceae) in Newfoundland and Notes on its Origin ................00e000s 249 cre ee eee Rie) ee 307 Volume 99, number 1, January—March, pages 1-60, issued 1 June 2009 Volume 99, number 2, April-June, pages 61-144, issued 1 September 2009 Volume 99, number 3, July-September, pages 145-230, issued 1 February 2010 Volume 99, number 4, October—December, pages 231-342, issued 31 March 2010 Hea eek a INFORMATION FOR AUTHORS Authors are encouraged to submit manuscripts pertinent to pteridology for publica- tion in the American Fern Journal. Manuscripts should be sent to the managing editor at amerfernj@hotmail.com. Acceptance of papers for publication depends on merit as judged by two or more referees. Authors are encouraged to contribute toward publishing costs; however, the payment or non-payment of page charges will affect neither the accept- ability of manuscripts nor the date of publication. Authors should adhere to the eat guidelines; manuscripts not so prepared sends be returned for revision prior to revie ever if it is necessary to submit hard copy, please submit one copy of the ee, phi include a review copy of illustrations and cae eases of illustrations. After apse please submit final versions of manuscripts via FTP (contact tl email, or on diskette or CD ROM (see below ie figure formatting). If eranrre hard copies, use standard 8. 5 by 11 inch paper of good quality, not “erasable” paper. Double- t, including title, author’s names and full addresses, a short, informative abstract, key words, text (including heads and keys), literature cited, tables (separate from text), and figure captions (grouped as consecutive paragraphs separate from figures). Arrange parts of manuscript in order just given. Include author’s name and page number in the upper right corner of every sheet, and provide an abbreviated running title. Provide margins of at least one inch (25 mm) all around on typed pages. Do not submit right-justified text, avoid footnotes, and do not break words at end of lines. Make table headings and figure captions self-explanatory. Use S.I. (metric) units for all measures (e.g., distance, elevation, weight) unless quoted or cited from another source (e.g., specimen citations). For nomenclatural matters (i.e., synonymy and typification), use one paragraph per basionym (see Regnum Veg. 58:39—40. 1968). Abbreviate titles according to Botanico- Periodicum-Huntianum (Lawrence et al., 1968, Hunt Botanical Library, Pittsburgh) and its supplement (1991). References cited only as part of nomenclatural matter are not included in literature cited. For shorter notes and reviews, omit the abstract and put all references parenthetically in text. Use Index Herbariorum (Regnum Veg. 120:1-693. 1990; or http:// websun.nybg.org/bsci/ih/) for designations of herbaria. For more detailed instructions on manuscript preparation, see http://amerfernsoc.org/. Illustrations should be proportioned to fit page width (5 inches or 12.5 cm) with caption ultimately to be included on the same page. Halftone and color images should be scanned at a minimum of 300 pixels per inch (ppi). Line art should be scanned at 1200 ppi when- ever possible. Please note that nearly all images that are downloaded from the Internet or that are in JPEG or GIF format will be 72 dpi and not acceptable for the printing process. Indicate the file format of the graphics. Please submit image files in TIFF (preferable) or EPS format. Provide margins of at least 25 mm on all illustrations. For continuous-tone illustrations, design originals for reproduction without reduction or by uniform amount. In composite blocks, abut edges of adjacent photographs. Avoid combining continuous- tone and line-copy in single illustrations or blocks. Coordinate sequence and numbering of figures (and tables), with order of citation in text. Explain scales and symbols in figures themselves, not in captions. Include a scale and reference to latitude and longitude in each map. ford loadi t f int order fi t to authors by the printer. Authors should ‘send proof corrections of corrected Proofs to the editor and reprint orders to the printer. Authors will after type has been set. For other matters of form or style, consult recent issues of the American Fern Jour- nal and The Chicago Adana of Style, 14" ed. (1993, Univ. of Chicago Press, Chicago). Occasionally, departure from these guidelines may be justified. Authors are eubtaiectts to consult the editor for assistance with any aspect of manuscript Preparation. — Papers of longer than 32 printed pages may be sent to th Editor, see journal cover page 2). PTERIDOLOGIA ISSUES IN PRINT The following issues of Pteridologia, the memoir series of the American Fern Society, are available for purchase: 1. Wagner, David H. 1979. Systematics of Polystichum in Western North America North of Mexico. 64 pp. $10.00 plus postage and handling. 2A. Lellinger, David B. 1989. The Ferns and Fern-allies of Costa Rica, Panama, and the Chocé6 (Part 1: Psilotaceae through Dicksoniaceae). 364 pp. $32.00 plus postage and handling. 3. Lellinger, David B. 2002. A Modern Multilingual Glossary for Taxonomic Pteri- dology. 263 pp. $28.00 plus postage and handling. For orders and more information, please contact our authorized agent for sales at: Missouri Botanical Garden Press, P.O. Box 299, St. Louis, MO 63166-0299, tel. 314-577- 9534 or 877-271-1930 (toll free). For online orders, visit: http://www.mbgp org AMERICAN FERN JOURNAL ON MICROFICHE Volumes 1-61 of the American Fern Journal are available as archival quality, silver positive microfiches. Single volumes or the entire run may be purchased. The fiches are easily read with 10x or greater magnification (using a dissecting microscope and transmit- ted illumination or a fiche reader). Silver negative microfiches of vols. 1-50 are also avail- able. The price is $4.00 per volume or $244.00 per set of 61 volumes, postpaid. Send your inquiry or order with a check or money order to: American Fern Society, Inc., % Ecology II, 804 Salem Blvd., Berwick, PA, 18603-9801. FIDDLEHEAD FORUM The editor of the Bulletin of the American Fern Society welcomes contributions from members and non-members, including miscellaneous notes, offers to exchange or purchase materials, personalia, horticultural notes, and reviews of non-technical books on ferns. SPORE EXCHANGE Ms. Denia Mandt, 12616 Ibbetson Ave., Downey, CA 90242-5050, is Director. Spores exchanged and lists of available spores sent on request. http://amerfernsoc.org/sporexy. html GIFTS AND BEQUESTS gq ts to the So iety enable it t Pp dit i t others interested in ferns. Back issues of the Journal and cash or other gifts are always welcomed and are tax-deductible. Inquiries should be addressed to the Membership Secretary. VISIT THE AMERICAN FERN SOCIETY’S WORLD WIDE WEB HOMEPAGE: http://amerfernsoc.org/ Ci3fte Ak A+ py ie