AICS AMERICAN sy FERN miner J O UJ R N A I January-March 2004 QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY — Interactions Among Cordate Gametophytes _ om Sap Fern, Athyrium filix- femi K. Greer and Dorothy Curry 1 Spore Morphology of the Polypodiaceae from Northwestern Argentina Gabriela E. Giudice, Marta A. Morbelli, Maria R. Pineiro, Manuel Copello and Goergina Erra 9 Spore spend under Different Storage Conditions in Four Rupicolous Asplenium L. Tax Cristina F. Aragon and Emilia Pangua 28 A Comparison of Useful Pteridophytes between Two Amerindian piste 28 from Amazonian Bolivia and Ecuador nuel J. Macia 39 Influence of Copper on Selected Physiological Responses in Salvinia minima and Its otential Use in Copper Remediation Safaa H. Al-Hamdani and Stacy L. Blair 47 The American Fern Society Council for 2004 TOM RANKER, University Museum, Campus Box 265, University of Colorado, Boulder, CO 80309-0265. President DAVID S. CONTANT, Dept. of Natural Sciences, Lyndon State College, Lyndonville, VT 05851. President-Elect W. CARL TAYLOR, 800 W. Wells St., Milwaukee Public Museum, Milwaukee, WI 53233-1478. Secretary JAMES D. CAPONETTI, Dept. of Botany, University of Tennessee, Knoxville, TN 37916-1110. Treasurer GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299 Mem uae Secretary JAMES D. MONTGOMERY, Ecology III, 804 Salem Blvd., Berwick, PA 18603-9801. Curator of Publications R. JAMES HICKEY, Botany Dept., Miami University , Oxford, OH 45056. : Journal Editor R. JAMES HICKEY, Botany Dept., Miami University , Oxford, OH 45056. Memoir Editor CINDY JOHNSON-GROH, Dept. of Biology, Gustavus Adolphus College, 800 W. College Ave., St. Peter, MN 56082-1498. Bulletin Editor American Fern Journal EDITOR R. JAMES HICKEY Botany Department,Miami University, Oxford, OH 45056, ph. (513) 529-6000, e-mail: hickeyrj @ muohio.edu ASSOCIATE EDITORS GERALD J. GASTONY. .......:....: Dept. of Biology, Indiana University, Bloomington, IN aihpag eat GARY E. GREER ors geste ys Dept., Grand Valley State University, Allendale, MI 4940 CHRISTOPHER H. HAUFLER .... Dept. of Ecology and Becknone) stow pauses es of Kansas, e, KS 66045-2106 ROBBIN C. MORAN New York Botanical Garden, Bran ‘NY 10458-5126 z H. PECK Dept. es ang ta Peseta of Arkansas—Little Rock, versity Ave., Little Rock, AR 72204 Daag “American Fern Journal” (ISSN 0002-8444) is an illustrated quarterly devoted to the general tudy of ferns. It is owned by the American Fern Society, and published at The American Fern Society, sates Botanical ear P. O. Box 299, St. Louis, MO 63166-0299. Periodicals postage paid at MO, and gras? on missing issues, a 6 months (domestic) to 12 months (foreign) after the date of issue, and orders for back issues pt be addressed to Dr. James D. Montgomery, Ecology II, 804 Salem Bivd., ee. PA 1860 address, and applications for membership should be sent to the Membership eakral inquiries concerning ferns should be addressed to the Secretary. Back volumes are avalible for most years as printed issues or on microfiche. Please contact the Back Issues er for prices and availability. Sub: Society Membership - USA, ¢ Canada, \ i (includes Journal 1 Fiddlet . \ $25 Society Membershi Snip (includ Tanrnal and Biddlch Aan \ $32 Society Life Membership $300 (add $140 mailing surcharge for outside USA, Canada, Mexico Regular Membership — USA, ¢ Canada VMeecicn Grille Piidichest $12 lar Membership ther $19 Institutional ‘Membership "335 to USA, —— Mexico; $45 elsewhere —— agency fee) changes t n, P.O. Po L. Missouri Box 299, St. Louis, MO 63166-0299. American Fern Journal 94(1):1-8 (2004) Pheromonal Interactions Among Cordate Gametophytes of the Lady Fern, Athyrium filix-femina Gary K. Greer’ and DorotHy CuRRY Department of Biology, West Virginia State College, Institute, WV 25112-1000 ApstTRACt.—Pheromonal interactions between cordate gametophytes of the lady fern, A. filix- femina, were assayed using a protocol typically used for detecting water-soluble pheromones such as antheridiogen. Three week-old, cordat tophyt e transferred from ti It oO | a WL t rown on nutrient agar to agar containing extracts from a previous generation of gametophytes re gametophytes that were six weeks old at time of transfer to treatment and control plates. Treatment gametophytes in the second experiment did not differ significantly in size (area) or length from control gametophytes; however treatment gametophytes were more circular and possessed greater widths and length : width ratios, deeper notches, and fewer archegonia. We present a model in which one or more phytochemicals released by cordate gametophytes increase rates of anticlinal division in the apical meristem. The possibilities that the substances involved are phytohormones involved in the development of a notch meristem and cordate morphology in the source gametophyte, and that antheridiogen may be involved, are explored. Ferns are not as phytochemically diverse as seed plants (Cooper-Driver, 1985). Nevertheless, the ability of many ferns to interact phytochemically with neighboring plants has been well established. The sporophytes of some species, notably Dennstaedtia punctilobula and Thelypteris normalis, produce allelo- paths that suppress germination and growth of gametophytes of the same or other species of ferns (Munther and Fairbrothers, 1980; Raghavan, 1989; Wagner and Long, 1991) or the germination and growth in neighboring seed plants (Horsley, 1977, 1986; Davidonis and Ruddat, 1974; Lyon and Sharpe, 1996). e most extensively documented phytochemical interactions among gametophytes involve antheridiogen. Antheridiogen is a water-soluble phero- mone produced by cordate gametophytes that induces dark germination, precocious maleness, and subsequently retards growth and morphological development in less-developed, acordate neighbors. First observed by Dépp (1950), antheridiogen has been subsequently documented in numerous families of filicalean ferns (Raghavan, 1989; Chiou and Farrar 1997). Along with genetic load, antheridiogen has been suggested as a mechanism promoting relatively 1 Author for correspondence: Biology Department, Grand Valley State University, Allendale, MI U. S. A. 49401; ph. (616) 331-2470; fax (304) 766-3446. MISSOURI BOTANICAL JUL 0 6 2004 GARDEN LIBRARY 2 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) high rates of cross-fertilization in a group of plants that, given their potentially bisexual gametophytes, appear predisposed to selfing (Soltis and Soltis, 1987: Schneller et al. 1990). Allelopathic interactions among fern gametophytes have also been reported in which gametophyte development was retarded in crowded conditions relative to less crowded conditions (Smith and Rogan, 1970) or cordate gametophytes in particular retarded spore germination, growth, and survival in acordate neighbors (Bell, 1958; Bell and Klikoff, 1979: Peterson and Fairbrothers, 1980). In these studies, and those regarding antheridiogen, the possibility of phytochemical interactions between cordate gametophytes has remained unexplored. Cordate gametophytes that can perceive the presence of other cordate gametophytes could enhance their own fitness by accelerating their own growth rate, morphological development, or production of archego- nia. Such interactions may be particularly relevant to understanding gameto- phyte ecology and the evolution of phytochemical interactions in ferns. We report the results of a bioassay for water-soluble phytochemical interactions between cordate gametophytes of Athyrium filix-femina var. asplenoides, a species with a previously documented antheridiogen system (Schneller, 1979). In isolation, gametophytes of A. filix-femina remain asexual until they develop a cordate morphology, when they produce archegonia (Schneller 1979). In multispore populations, acordate gametophytes often become male in response to native antheridiogen and both male and female gametophytes are capable of becoming hermaphrodites following prolonged growth (Schneller, 1979). Thus, A. filix-femina possesses a Category B pattern of gender expression (Klekowski and Lloyd, 1968). MATERIALS AND METHODS To collect water-soluble pheromones, spores of A. filix-femina were collected in the summer of 1996 from Kanawha State Forest in Charleston, WV, and stored in glass vials at room temperature. The spores were then surface sterilized (Dyer 1979) and sown on nutrient agar containing Parker’s macronutrients and Thompson’s micronutrients (Klekowski, 1969). The resulting gametophtyes were grown for fifteen weeks at a mean temperature of 19.6°C (+ 0.64) and under a bank of grow lights with a mean light level of 27.0/m?/sec. (+ 2.8) and a sixteen hours light : eight hours dark regimen. At the end of fifteen weeks, the gametophytes were discarded and an extract from the agar was obtained by a freeze-thaw process. Suspended matter was removed from the extract by centrifugation and the supernatant was diluted by fifty-percent using Parker- Thompson’s nutrients. Fresh agar was then added and the resulting solution was used to make a treatment agar. In the first experiment, over one hundred three-week old cordate game- tophytes (i.e., exhibiting conspicuous apical notches) were transferred to petri- plates containing either treatment agar or a control containing basal nutrient agar (Figure 1). No antheridia or archegonia were observed in cordate gametophytes at the time of transfer. Gametophytes were evenly spaced at GREER & CURRY: PHEROMONAL INTERACTIONS 3 ae SF = 0.35 ‘Siem mm (c) SF = 0.46 extension of the length line from the deepest point in the notch to the line connecting the most distal points of the apical lobes (dashed line). a density of approximately one gametophyte per square centimeter. Petri-plate lids remained unsealed to facilitate gaseous exchange and reduce ethylene buildup. Twenty-one days after transfer, fifty gametophytes per control and treatment were harvested and mounted on microscope slides using permount. In a second experiment, the entire procedure was repeated using six-week old cordate gametophytes (Figure 1a) at time of transfer and harvesting fifty-five gametophytes per control and treatment. Archegonia in various states of development were observed on all individuals at time of transfer in this experiment. No antheridia were observed. Each gametophyte was photographed at 8.5 using a digital camera attached to a dissection microscope. Gametophyte photographs were magnified 2X on a computer and size (area in pixels) and shape (circularity) of each gametophyte was measured using Sigmascan Pro 4.0 (Fox and Urich, 1993). Circularity was analyzed using the shape factor function and was calculated using the formula (4x X Area) / Perimeter”. Shape factor values range from zero to one, indicat- ing linearity and circularity, respectively. In addition to size and shape, gametophyte length, width and notch depth were also measured in the second experiment using the linear distance function in Sigmascan Pro 4.0. Gameto- phyte length was measured from the base of the gametophyte to its notch (solid white arrow) and width was measured as the longest line (dashed white arrow) perpendicular to the length line (Figure 1). Notch depth (solid black arrow) was measured as an extension of the length line from the deepest point in the notch 4 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) TABLE 1. Equal variance t-tests and Mann-Whitney U rank sum tests of size and shape, and chi- square tests of frequency of antheridia-bearing individuals and archegonia per gametophyte, of three-week old and six-week old A. felix-femina gametophytes transferred to basal nutrient agar (control) versus a nutrient agar that supported a previous generation of gametophytes (treatment). Shape factor values approaching zero indicate a linear morphology, whereas those approaching one indicate a circular morphology. Trait Control Treatment Statistic, P-value Three-week old gametophytes (at transfer) # of Gametophytes 50 50 — Size (cm*)* 1.10+0.46cm* 0.71+0.36cm? T=5.01, P < 0.001 Shape factor 0.28 + 0.18 0.26 + 0.18 U 116. F = 0.023 Antheridial gametophytes/ population 5 19 X? = 10.44; P < 0.005 Archegonia/population (per gametophyte) 759 (14.3 + 9.3) 405 (7.6 + 7.2) X? = 109.38; P < 0.001 Six-week old gametophytes (at transfer) # of Gametophytes 55 55 — Size (cm*)* 1.09+0.32cm* 1.02+0.23cm* T=1.35, P = 0.090 Shape factor 0.40 + 0.24 0.49 + 0.22 U = —2.44, P = 0.007 Length 46,8 = 12.7 434 4 12:11 iL =O51 P= 0350 Width 152.0. 19:2 165.8 > 31.3 U = 1980, P = 0.006 Length : width ratio 0.51 + 0.09 0.47 + 0.02 T= 11.98,.P =:0:03 Notch depth 3122: 7.6 37:6: 10.2 i= 13.35, P= 0,001 Antheridial gametophytes/ opulation 0 a — Archegonia/population (per gametophyte) 1688 (33.1 + 13.2) 1461 (28.7+ 11.5) X? = 16.38, P < 0.001 * Converted from image area in pixels to reflect true size. to the line connecting the most distal points of the apical lobes (dashed line, Figure 1). Numbers of archegonia and antheridia on each gametophyte were counted directly using a compound microscope in both experiments. Size and shape data were analyzed using equal variance t-tests, except when data failed to meet assumptions of normality and homoscedasticty, in which case Mann-Whitney U rank sum tests were performed. Data sets were tested for normality using Pearson-D’Agostino omnibus tests and for homoscedasticity using modified-Levene tests. A critical value of P = 0.05 was used for all statistical procedures. All statistical analyses were conducted using NCSS 97 (Hintze, 1997). RESULTS In the first experiment, individuals from the treatment population were not significantly different in shape, but were significantly smaller, possessed fewer archegonia, and contained more antheridial individuals (both as males and bisexuals), than individuals from the control population (Table 1). Thus, at three-weeks from germination, 38% of cordate gametophytes were still able to respond to antheridiogen by producing antheridia. GREER & CURRY: PHEROMONAL INTERACTIONS 5 In the second experiment, in which six-week old gametophytes were transferred, the treatment population was more circular, possessed greater widths, smaller length : width ratios and deeper notches than the control population (Table 1). Collectively, these observations indicate that lateral growth generated by the apical meristem was greater in the treatment population than in the control population. The treatment population also possessed significantly fewer archegonia (13.3%) than the control population (Table 1). Differences in length and the proportion of males in each population were not significant (Table 1); however, the marginal P-value (0.09) associated with the t-test for difference in size (Table 1) may indicate a weak negative treatment effect. No antheridia were found in the control population and only one gametophyte (1.8%) possessed antheridia in the treatment population. DISCUSSION Pheromonal interactions between cordate and acordate gametophtyes, mediated through antheridiogen secretion by the former and uptake by the latter, are known for many filicalean ferns (Naf et al., 1975; Raghavan, 1989). Cordate gametophytes undoubtedly release numerous other substances into their surroundings that may include water-soluble regulators of their own growth and production of gametangia. These substances may influence neighboring cordate gametophytes. Such interactions may provide insight into the phytochemical regulation of gametophyte development and reproductive ecology. Results from our second experiment demonstrate that cordate gametophytes of A. filix-femina produce one or more water-soluble substances that accelerate lateral growth, and subsequent development of a circular profile and deeply recessed notch meristem, and retard production of archegonia in cordate gametophytes of the same species. The ecological relevance of these cordate- cordate interactions remains unclear, however, it is not difficult to envision a fitness advantage to reducing the risk of polyembrony in dense populations with a high likelihood of fertilization success. Concentrations of the phytochemicals used in this study are unlikely to occur in nature. Although the treatment agar was diluted by fifty percent, it represented the accumulation of fifteen weeks of water-soluble metabolites. In the wild, drainage and biotic and abiotic interactions within soil probably reduced concentrations of these phytochemicals well below those used here. Nevertheless, Greer and McCarthy (1997) observed a peak in cordate males in populations growing on soil at the periphery of the antheridiogen neighborhood (the horizontal range of effect from a source gametophyte) of Polystichum acrostichoides (Michx.) Schott. Thus, the substance(s) responsible for the effects observed in this study may reach sufficient levels in nature to induce the responses we observed. The seemingly contradictory effects of increased circularity without in- creased size may be the result of one or more water-soluble hormones involved in gametophyte morphogenesis. Morphological development in most filicalean 6 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) gametophytes is a function of the planes of division occurring in the meristem (Raghavan 1989). Transition from a one-dimensional filamentous morphology to a two-dimensional spathulate morphology involves the initiation of oblique and anticlinal divisions, as opposed to periclinal divisions, in the single-celled meristem (von Aderkas and Cutter, 1983; Raghavan, 1989). Likewise, transition from a spathulate to a cordate morphology results from an increase in anticlinal divisions in the meristem and its derivatives (von Aderkas and Cutter, 1983; Raghavan, 1989). In cordate gametophytes, anticlinal divisions in the meristem tend to produce small, columnar cells, whereas periclinal and oblique divisions tend to produce larger cells that have a greater impact on overall size (von Aderkas and Cutter, 1983). Thus, the presence of one or more phytochemicals in the treatment may have accelerated anticlinal divisions at the expense of oblique or periclinal divisions, resulting in a deeper notch and wider, more circular profile without a corresponding increase in size. This may also explain the decreased production of archegonia in the treatment in the experiment using six-week old gametophytes, because archegonia are ultimately derived from oblique and periclinal divisions of the meristem. Although the identity of the substance or substances inducing the effects observed in this study remain unknown, antheridiogen is a candidate. Antheridiogen is a stable, water-soluble compound produced by cordate gametophytes that overrides the light requirement for spore germination, induces precocious maleness in filamentous and spathulate gametophytes, and subsequently retards growth and morphological development in acordate neighbors (Naf et al., 1975; Raghavan, 1989). Structurally, antheridiogen is similar to gibberellin (Naf et al., 1975; Yamauchi et al., 1996; Nester-Hudson et al., 1998), which has similar effects on many seed plants; i.e., it stimulates seed germination and induces maleness in flowers. Recognizing the structural and functional similarity between many known antheridiogens and gibberellin, and the ability of gibberellins to substitute for the antheridiogen of some species of ferns (Naf et al., 1975; Raghavan, 1989), a few authors have speculated about a hormonal role for antheridiogen (Willson, 1981; Voeller and Weinberg, 1969; Schraudolf, 1985; Greer and McCarthy, 1997). Greer and McCarthy (1997) advanced two hypotheses that may be relevant here, the hormone-pheromone and multiple-signal hypotheses. The hormone- pheromone hypothesis suggests that antheridiogen is a hormone involved in the development and persistence of a cordate morphology in a source gametophyte. In addition to the similarities between antheridiogen and gibberellin listed above, this hypothesis emphasizes the correspondence between the develop- ment of a cordate morphology and the production of antheridiogen, and the ability of gibberellin to stimulate cell division in the shoot apex of seed plants. According to the hormone-pheromone hypothesis, response to antheridiogen changes following attainment of a cordate morphology from production of antheridia, reduced growth, and delayed attainment of circularity, to acceler- ated attainment of circularity and reduced production of archegonia. This model may explain the inhibitory effect of GA, on archegonial development in Lygodium japonicum (Takeno et al., 1979) and prolonged production of GREER & CURRY: PHEROMONAL INTERACTIONS i antheridia and delayed production of archegonia by cordate gametophytes of Onoclea sensibilis exposed to antheridiogen of Pteridium aquilinum beginning during acordate stages (Naf et al., 1975). It is noteworthy here that genes (TRA and MAN) that control gender expression and are indirectly responsive to antheridiogen in gametophytes of Ceratopteris richardii appear to also regulate sporophyte development in this species (Banks 1997 Alternatively, the multiple- signal hypothesis suggests that cordate gameto- phytes release two or more water-soluble p! Ifa compound other than antheridiogen is responsible for accelerated attainment of a circular profile, it has no apparent effect on acordate gametophytes. Under this scenario, receptivity to pheromonal influences on gametophyte development gradually shifts from antheridiogen during acordate phases to one or more other pheromones that affect cordate gametophytes In conclusion, cordate gametophytes of “Athyziam filix-femina produce a eee compound that accelerates the development of a circular profile and retards production of archegonia in well-developed cordate gametophytes. The identity of the substance eliciting these effects remains unknown, however antheridiogen or another gibberellin like substance is a possibility. To answer the many questions raised by this study, similar experiments need to be conducted using isolated antheridiogen and other water-soluble substances released by fern gametophytes. ACKNOWLEDGMENTS We thank Christopher Haufler and an anonymous reviewer for helpful comments on a previous version of this manuscript. REFERENCES Vs Banks, J. A. 1997. The transformer genes of the and archegonia development and ioe antheridia ericiannis in the pierre —1897. ameto BELL, .. R. oy Variations in the germination-rate and development of fern spores in culture. Ann. Bot. 22:503-511. Bett, S. and L. G. Kucxorr. 1979. Allelopathic and autopathic relationships among the Polystichum acrostichoides, Polypodium vulgare and Onoclea sensibilis. Amer. Midl. N 10 ‘ge Cuiou, - sat D. R. Farrar. 1997. Antheridiogen production and response in polypodiaceous species. Amer. J. Bot. 84:633-640. Cooper-Driver, G. 1985. Anti-predation in pteridophytes—a biochemical approach. Proc. Royal Acad. Edin. 86:397-402. DavImponIs, G. H H. and M. Ruppart. 1974. Growth inhibition in gametophytes and oat a aa by thelypertin A and B released from roots of the fern Thelypteris normalis. Amer. J. Bot. 61:925— Dopp, bi 1950. Eine die antheridienbildung bei farnen férdernde oe in den prothallien von eridium aquilinum (L.) Kuhn. Ber. Deutsch Bot. Ges. 63:1 DYER, - F. 1979. The culture of fern gametophytes for ac og investigation Pp. 253-305, in A. F. Dyer. The experimental biology of ferns. Acad. Press, ork. Fox, E. and C. G. Uricu. 1991. Sigma-scan User’s manual. Jandel cia San Rafel, California. 8 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Greer, G. K. and B. C. McCartuy. 1997. The santa ig neighborhood . oe t. J. ee teed pie ig ania on a native substrate Plant Sci. 158:7 sag . H. and T. A. Ran NKER. 1985. Differential wart eee response and Jaliiie: 72:659-665. anisms in tah opteris. Amer. J. Bot. 7 — ‘i a 1997. NCSS 97 Statistical system for windows. NCSS systems, Kaysville, Utah. Hors ey, S. 1977. Alelopathic inhibition of black cherry by fern, grass, goldenrod, and aster. Can. J. For. Res. 7:205— Horsey, S. 1986. Sectiwicn of hayscented fern influence with black cherry. Amer. J. Bot. 73: 668-6 KLEDOwskI, E. I, JR. 1969. Reproductive biology in the pteridophyta III. A study of the Blechnaceae. KLEKOwskI, E. J. and R. M. Luoyp. 1968. Reproductive biology of the oo I. General conclusions bys a study of Onoclea sensibilis L. Bot. J. Linn. Soc. 60:315-324. Lyon, J. and W. E. SHarre. 1996. Hay-scented fern (Dennstaedtia eatin (Michx.) Moore) len Sill with growth of northern red oak (Quercus rubra L.) seedlings. Tree Physiol. 16:923—932. Muntuer, W. E. and D. E. FAIRBROTHERS. igh aes ame and autotoxicity in three Eastern North American ferns. Amer. Fern J. 70:124— Nar, U., K. NAKANISHI and M. ENpo. 1975. On = physiology and chemistry of fern antheridiogens. Bot. Rev. 41:315-359. NesTER-Hupson, J. E., L. J. CREAcy and J. L. PALMER. 1998. Gibberellins A45 and A61, were essen or the first time from irae ag culture media extracts of Anemia mexic 49-14 PETERSON, R. L. and D. E. usa 1980. Reciprocal prencueser between the gametophytes of Osmunda cinnamomea and Dryopteris intermedia. Am —78. RacHavan, V. 1989. Developmental biology of fern goatee cae Guiibenien University Press, New SCHRAUDOLF, a 1985. Action and phylogeny of antheridiogens. Proc. Roy. Soc. Edinburgh 86: sais . J. 1979. i a investigations on the lady fern (Athyrium filix-femina). Plant Syst. Evol. ny ei hi, Gow. a and T. A. RANKER. 1990. Antheridiogen and natural gametophyte populations. Amer. Fern J. 80:138-147. aca D. L. and P. G. Rocan. 1970. The effects of population ened on gametophyte morphogenesis in Polypodium vulgare L. New P Phytol. 69:1039-10 Soitis, D. E. and P. M. Sottis. 1987. Polyploidy and breeding exten in homosporous a se reevaluation. Amer. Nat. 130:219— TAKENO, K., M. uya, H. YAMANE and N. TAKAHASHI. 1979. Evidence for naturally occurring inhibitors - pina differentiation in Lygodium japonicum. Physiol. Plantarum 45: 305-310. VoELLER, B. R. and E. S. WEINBERG. 1969. Evolutionary and physiological aspects of antheridium induction in ferns. Pp. 77-93 in J. Gunkel. Current topics in plant science. Acad. Press, New York. VON AbDERKAS, P. and E. G. Currer. 1983. The role of the meristem in gametophyte development of osmunaceous fern Todea seen un ) ae + Bat, Gaz. 144:519-524. Wacner, H. B. and K. E. Lonc. 1991 f O da ci three species of Dryopteris. Amer. Fern J. 81: 134-138. WILLSON, M. F. 1981. Sex expression in fern gametophyt lutionary possibilities. J Theor iol 93:403—409 oe. TN. Ovnama, H. YAMANE, N. Murorusu, H. ScHRAUDOLF, M. Pour M. Furser, and L. N. MANDER. 1996. fsttentitiction. we Sg iata in Lygodium circinnatum and Lygodium flexuosum. Plant Physiology 111:741-745 American Fern Journal 94(1):9—27 (2004) Spore Morphology of the Polypodiaceae from Northwestern Argentina GABRIELA E. GIUDICE Catedra De Morfologia Vegetal, Facultad de Ciencias Naturales y Museo de La Plata, Paseo del Bosque s/n, 1900, La Plata, Argentina Marta A. Morseii, Maria R. PINEIRO, MANUEL CoPELLO and GOERGINA ERRA Catedra de Palinologia, Facultad de Ciencias Naturales y Museo de La Plata Bosque s/n, 1900, La Plata, Argentina AssTRAcT.—The spores of the following genera of Polypodiaceae growing in northwest Argentina were analyzed: Campyloneurum, Microgramma, Pecluma, Phlebodium, Pleopeltis and Polypo- dium. The study involved analyses of herbarium material using light microscopy and scanning electron microscopy. The spores are monolete, 40-90 um in major equatorial diameter, eliptic to oblong in polar view and plane to concave-convex in equatorial view. The exospore ranges from 2— thick, is apparently double-layered, with a verrucate or tuberculate surface that is usually analyzed have globules on the surface. These are single or associated in masses and irregularly distributed. Characteristics such as size, shape and exospore and perispore sculpture allow us to differentiate among some of the genera as well as recognize species groups. Microgramma, ampyloneurum, Pecluma, Pleopeltis and Polypodium have verrucate spores whereas those of Phlebodium are tuberculate. This study forms part of a project dealing with the palynological flora of Northwest Argentina. According to de la Sota (1973), this region comprises the provinces of Jujuy, Salta , Tucuman, Catamarca, the eastern part of La Rioja, and southwestern Santiago del Estero (Fig. 1). The following members of Polypodiaceae grow in this region: Polypodium argentinum Maxon, P. bryopodum Maxon, P. chrysolepis Hook., P. lasiopus Klotzsch, P. loriceum L., P. pleopeltidis Fée, P. squalidum Vell., P. tweedianum Hook., Campyloneurum aglaolepis (Alston) de la Sota, C. lorentzii (Hieron.) Ching, C. major (Hieron. ex Hicken) Lellinger, C. tucumanense (Hieron.) Ching, Microgramma squamulosa (Kaulf.) de la Sota, Pecluma filicula (Kaulf.) M. G. Price, P. oranense (de la Sota) de la Sota, P. venturi (de la Sota) M.G. Price, Phlebodium pseudoaureum (Cav.) Lellinger and Pleopeltis macrocarpa (Bory ex Willd.) Kaulf. (de la Sota, 1960, 1977; Ponce,1996). Polypodium loriceum L. recently collected by Martinez and de la Sota, (Sota, et al. 1999) in Salta province, was also included in the study. According to Ponce (1996), Polypodium hirsutissimun probably grows in the region, however, no material documenting its occurrence was found in the herbaria. According to Tryon and Tryon (1982) and Tryon and Lugardon (1991), American Polypodiaceae are mostly diploid. The spores of the Polypodiaceae have been described and illustrated with LM by Nayar and Devi (1964), Lloyd (1981) and Pal and Pal (1970), with SEM 10 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Fic. 1. Study area (Northwest Argentina). and TEM by Tryon and Lugardon (1991), Tryon and Tryon (1982), van Uffelen (1992, 1993, 2000), van Uffelen and Hennipman (1985) and Hennipman (1990). However among these contributions there are not many references to the spores of species that grow in Argentina. Tryon and Tryon (1982) differentiated 6 informal groups of Polypodium species from tropical America. Those groups were based on a combination of characters such as stem scales, lamina indument, venation, soral arrangement and spores. The aim of this study is to analyze the spores of the Polypodiaceae that grow in northwestern Argentina in order to add to the existing information about these taxa and to assess the systemic value of palynological data. MATERIALS AND METHODS Spores were obtained from herbarium (BA, LP, LIL and SI) specimens and were studied using light (LM) and scanning electron microscopy (SEM). For LM the spores were treated with hot 3% sodium carbonate for 2 minutes and acetolyzed according to the method of Erdtman (1960). For SEM, the material was treated with hot 3% sodium carbonate, washed, dehydrated, suspended in 96% ethanol and then transferred to acetate plates. After dryng they were coated with gold. Wall fractures obtained using ultrasound for 10 minutes were also used in order to study the sporoderm structure. All the observations were made with Olympus BH2 and BHB light microscopes and a JEOL JSMT- 100 scanning electron microscope at the Museo de Ciencias Naturales de La Plata. GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 141 The terms proposed by Hennipman (1990), Tryon & Lugardon (1991), van Uffelen & Hennipman (1985) and van Uffelen (1993) were used for spore descriptions. Spores were characterized for: color of acetolyzed material, shape, diameters, laesurae, sporoderm thickness, and ornamentation, structure and stratification of the wall layers. In some species of Campyloneurum, the number of spores produced per sporangium was considered in order to understand spore irregularities related to size and morphology. The letters MP, associated with the list of specimens investigated (Table 1) indicate the reference number of each palynological sample as filed in the Laboratorio de Palinologia, Facultad de Ciencias Naturales y Museo de La Plata. RESULTS Campyloneurum (Table 2; Fig. 2, A-L) This genus is represented by four species in the study area: C. aglaolepis (Alston) de la Sota, C. Jorentzii (Hieron.) Ching, C. major (Hieron. ex Hicken) Lellinger and C. tucumanense (Hieron.) Ching. All are epiphytic and characterized by an entire lamina, anastomosing veins and round sori borne in a marginal or submarginal position. Campyloneurum tucumanense is the largest species of the genus in the northwestern of Argentina. The lamina is soft, membranaceous and has the most complex and evident venation of the Campyloneurum studied. Ponce (1996) considered this species to be endemic to the region of study although it has also been reported for Bolivia by Lellinger (1988). The spores are ellipsoidal or oblong in polar view (Fig. 2 A, D and J) and plane to concave-convex in equatorial view (Fig. 2 B, F, G and K) . In Campyloneurum aglaolepis, C. lorentzii, and C. major, equatorial diameters range between 70 and 80 um and polar diameters between 40 and 60 um. In Campyloneurum tucumanense the equatorial diameter ranges between 85 and 94 1m and 46-60 um in polar diameter. The exospore is the thickest wall layer, ranging between 1.5 and 3 um in all species except in Campyloneurum tucumanense in which it reaches 4 um. Spores are verrucate with a dense compact wall (Fig. 2 I). With LM it is apparently double-layered in section with a compact structure. The inner layer (ie) is thiner than the outer (ie: oe ratio 1:2—1:3). The outer layer (oe) forms the elements of the sculpturing (verrucae). The verrucae have a circular or polyhedral outline and diminish in size toward the proximal face. In C. aglolepis (Fig. 2 A—C) the verrucae are obscure. The verrucae of C. aglolepis (Fig. 2 A-C) and C. lorentzzi (Fig. 2 D-F) are larger than in C. major (Fig. 2 G—I) and C. tucumanense (Fig. 2 J-L). The perispore is 0.4—-1 pm thick, smooth and perforated. With LM it is aparently single-layered in section and follows the verruca contours (Fig. 2 C, E and H). Most of the species analyzed have irregularly distributed globules on AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) TABLE 1. Specimens studied. Taxon Voucher specimens Polypodium L. P. argentinum Maxon P. bryopodum Maxon P. chrysolepis Hook P. lasiopus Klotzsch P. loriceum L. P. pleopeltidis Fée P. squalidum Vell. P. tweedianum Hook. Campyloneurum C. Pres/ C. aglaolepis (Alston) de la Sota C. lorentzii (Hieron.) Ching C. major (Hiero: ex Hicken) Lalieais Jujuy: Dpto. Yala, Eskuche 119 (LP), MP 3853 Salta: Dpto. ne Victoria, Hurrel 51 (LP), MP 3852 Tucuman: Dpto. Tafi, Maruriak, Olivia & Puezo 345 (LP), MP 3847 Jujuy: Dpto. — Volcan, Cabrera, Torres, Tur & Kiesling 18353 (LP), M Tucuman: phi ical Estancia Santa Rosa, Venturi 4802 (LP), MP Salta: Dpto. ae cerro La cueva, (SI, 1321), MP 3913. Tucuman: Dpto Tafi, La Ventanita, Castillo 35 (SI), MP 3914 Jujuy: at, Capital, Laguna de Yala, Palacr et al. 893 (SI), MP Salta: ae Guachipas, Estancia Pampa Grande, Hawkes et al. 3976 (LP), MP 3887 Salta: Dpto Santa Victoria, Los Toldos, Martinez 641 et al. (LP), 3886 Salta: Dpto Santa Victoria, Los Toldos, Martinez et al. 595 (LP), P 3957 M Jujuy: Dpto. Ledesma, Cabrera, Kiesling & Zardini 24008 (LP), MP 3849 Jujuy: Dpto. Ledesma, Abra de las Cafias, de la Sota 4428 (LP), a ae Santa Barbara, Zuloaga & Deginami 327 (LP), MP ie -_ Ledesma, Calilegua, Arroyo del medio, Cabrera et al 30363 (LP), MP 3956 Salta: Dpto ai Parque Nac. El Rey, Arroyo La Sala, Brown 90 (LP), MP 384 Jujuy: er Capital, Lozano, Krapovickas & Schinini 35825 (LP), MP Salta: Dpto. roe Aguas Blancas, Palacr 104 (LP), MP 3885 Tucuman: Dpto. Monteros, Quebrada Pueblo Viejo, de la Sota 4066 (LP), MP 3848 Salta: Dpto. Capital, San Lorenzo, Cabrera 3061 (LP), MP 3888 Salta: Dpto. Capital, Quebrada San Lorenzo, Cabrera 9118 (LP), MP 3916 i lal Capital, Quebrada San Lorenzo, Palacr 160 (LP), MP Pa Dpto Capital, Parque Nacional El Rey, Brown 983-2 (LP), Cedi libaaates Los Pinos, Borsini s/n (LP), MP 3952 Jujuy: pen a Quebrada Yala, Cabrera y Kiesling 25227 (LP), Jujuy: hg ae Mesada de las Colmenas, de Ja Sota 4483 (LP), MP 3941 GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 13 TABLE 1. Continued. Taxon Voucher specimens C. tucumanense _ Dpto. Ledesma, 10 a 20 km de Libertador Gra (Hieron.) Ching San Martin, Krapovickas, Schinini & C. Quarin 26641 (LP), 3900 Salta: Dpto. sy i Baritu, Marmol, Legname & Cuezzo 8762 (LP) M Tucuman: ad ce Quebrada de Tafi, Venturi 871 (LP), MP 3901 Pecluma M.G. Price P. filicula (Kaulf.) Jujuy: gay sen Grande, Mesada de las Colmenas, Fabris 3425 M.G. Price (LP), MP Salta: a Orin Aguas Blancas, Quebrada El Nogal, Palacr 92 (LP), M P. oranense (de la Sota) si Dp. rare Cerro Labrado, de la Sota 4310 (LP), MP la Sota sees a Santa he tea Marmol, Cuezzzo (h) & Cuezzo 9209c (LP), MP 3 P. venturi (de la Sota) Salta: Dpto. ae eoae San Lorenzo, Palacr 166 (LP), MP M.G. Price 3890 Salta: Dpto. Santa Victoria, Los Toldos, Quebrada E] Astillero, Palacr 499 (LP), MP 3907 Tucuman: Dpto. Monteros, Quebrada Pueblo, de la Sota 4059 (LP), MP 3908 Microgramma C. Presl M. squamulosa (Kaulf.) si begs Capital, La Cuesta, Cabrera et al. 18856 (LP), MP Sota ei oe Nac. 9, Pampa aan — s/n (LP), MP 3903 Tucuman: Dpto. Monteros, Quebrada de los Sosa, Casa de Piedras, Sais & see 20456 (LP), MP 3905 Phlebodium (R. Brown) J. Smith P. pseudoaureum Jujuy: Dpto. Capital, Cabrera 8178 (LP), MP 3906 (Cav.) Lellinger Salta: Dpto. Rosario de Lerma, Venturi 8227 (LP), MP 3850 Salta: Dpto. Oran, Aguas Blancas, Palacr 96 (LP), MP 3918 Pleopeltis Humb. & Bonpl. ex Willd. P. macrocarpa (Bory Salta: Dpto. Santa Victoria, Los Toldos, Martinez et al. 653 (LP), ex Willd.) Kaulf. MP 3891 Salta: Dpto. Santa Victoria, camino a Los Toldos, Martinez et al. 644 (LP), MP 3915 the spore surface (Fig. 2 C, D, G, J and K). In some samples (de Ja Sota 4483, LP; Fig. 2 H) small perforations were observed across the perispore surface. The number of spores produced per sporangium was estimated in several specimens in order to check for the possiblity of apogamy in Campyloneurum tucumanense. In Krapovickas et al. 26641 (LP) and in Venturi 871 (LP), 64 spores per sporangium were estimated and, apart from mature spores, hyaline TABLE 2. Spore morphological data of the Polypodiaceae from Northwestern Argentina (sizes in ym, mean value in parentheses). Major inor equatorial equatorial Polar Laesura xospore Taxon diameter diameter diameter length Exospore Perispore ornamentation Polypodium tin 76.6 (86.2) 95.6 54 (61.3) 68.5 46 (54.8) 64 46 (52.7) 58.6 2.5 (3.3)4.3 0.4 (0.8) 1 Verrucate, with globules P. lasiopus P. loriceum P. pleopeltidis P. squalidum P. tweedianum Campyloneurum aglolepis C. lorentzii C. major . tucumanense Pecluma Digan Po P. venturi Microgramma macrocarpa 61.9 (75.6) 87.1 54.7 (61.9) 72 34.9 (45.2) 61.5 44.8 (55.4) 55.2 3.1(3.9)4.9 0.4 (0.7) 0.8 Verrucate, with globules 71.4 (78.4) 82.1 48.1 (54.6) 61.8 38.9 (48.1) 56.2 34.4 (40.8) 46.3 1.8 (3.0) 3.4 0.4 (0.6) 0.6 Verrucate, with globules 74 (83) 94.8 43.3 (57.2) 64.1 39.9 (52.1)59.8 30(38) 41.5 2.1 (3.3) 4.1 0.4 (0.7) 0.9 gaicinane with large errucae and ridges 51.6 (63.5) 74.5 37.2 (40.5) 45 30.4 (36.7) 47.8 41.5 (45.2) 49.8 2.9 (3.4) 4.2 0.4 (0.6) 0.8 Hunn with large verrucae and ridges 58.5 (66.1) 75.7 37.2 (42.1) 49.5 32.5 (42.6) 46 31.6 (34.5) 38.6 2.1 (2.9) 3.9 0.8 (1.1) 1.2 Verrucate, with.large verrucae 47.8 (57.2) 64.7 35.1 (40.4) 46.7 33.3 (39.6) 45.2 22.5 (26.9) 31.2 1.8 (2.15) 2.5 0.57 (0.7) 1 Verrucate, with globules 59.5 (69.6) 80.7 41.8 (47.4) 51 30.5 (37.4) 44.4 44.8 (50.1) 58 1.7 (2.1) 2.5 0.3 (0.4) 0.6 Verrucate, with. low verrucae, and globules 67.6 (73.8) 83 47.7 (54.9) 64.7 47.3 (51.1) 55.6 29.5 (34.6) 39.4 2.1 (2.4) 2.9 0.7 (0.8) 0.9 Verrucate, with low verrucae, and globules 64.9 (71.5) 79.1 54.8 (59.2) 63.9 41.8 (48.9) 55.9 26.0 (30.9) 39.1 2.2 (2.9) 3.3 0.5 (0.7) 0.8 Verrucate, with globules 71.4 (71.8) 72.2 39.0 (45.2) 51.5 37.3 (42.5) 45.6 44.8 (45.2) 45.6 1.5 (1.6) 1.7 0.4 (0.5) 0.8 Verrucate, with few globules 85.1 (91.0) 93.8 57.3 (63.6) 71.4 46.5 (54.3) 59.3 45.6 (53.9) 61.4 3.3 (3.7) 4.1 0.8 (0.9) 1.3 Verrucate, with few globules 45.1 (51.9) 58 31.7 (37.7) 40.2 26.9 (32.5) 39.1 21.2 (27.1) 30.7 1.4 (1.7) 2.1 0.6 (0.8) 0.95 Verrucate, with few globules 44 (49.6) 54 27.5 (28.5) 33 26 (27.9) 30.5 20.5 (25.65) 30 2(2.6)3.4 0.5 (0.8) 1 Verrucate, with large verrucae in two levels and ridges, with globules 43.6 (48.7) 54 28.1 (33.1) 36.4 26.5 (30.3) 35.9 21.6 (26.31) 28 1.8 (2.14) 2.3 0.45 (0.6) 0.8 Verna with large verrucae n two levels and ridges, with ke les 70.5 (75.9) 80.9 52.9 (55.8) 60.5 42.1 (46.6) 52.1 35.6 (38.1) 43.6 3.1 (4.1) 4.9 0.4 (0.5) 0.7 Verrucate, with few globules 35.6 (38.2) 41.3 21.8 (23.8) 26.7 21.1 (23.5) 25.9 19.4 (20.8) 25.1 3.2 (3.6) 4.1 0.9(1.0) 1.2 Tuberculate, with few globules 74.7 (81.6) 86.3 45.6 (52.1) 64.3 77.6 (80.1) 81.7 41.5 (45.4) 50.6 1.7 (2.0) 2.3 0.9(1.2) 1.5 Verrucate, with few globules (#002) | YAHWON 6 ANNTIOA “TVNYNO!L NAdd NVORINV GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 15 and small immature spores were also observed. In Legname & Cuezzo 8762 (LP) 32 spores per sporangium were estimated. Pecluma (Table 2; Fig. 3 A-K) This genus is represented by three species in the northwest of Argentina: P. filicula (Kaulf.) M. G. Price, P. oranense (de la Sota) de la Sota and P. venturii (de la Sota) M. G. Prince. Pecluma oranense is endemic to Salta and Jujuy, growing as an epiphyte in the basal forest and in Podocarpus parlatorei dominated forest. This genus is characterized by pinnatifid, pubescent lamina, sori borne at the tip of a vein and nonclathrate basifixied rhizome scales. Pecluma filicula is the smallest of the species studied. It reaches ca. 20 cm in length and has a pubescent and scaly rachis, whearas P. oranenese and P. venturi have longer lamina and have a pubescent, but never scaly rachis. The spores are monolete and yellowish in Pecluma oranense and P. venturi and light-brown in P. filicula. They are ellipsoidal to oblong in polar view (Fig. 3 A, E, G and H), and concave and convex distally in equatorial view (Fig. 3 B, D and I). They are 44-58 ym in major equatorial diameter and 26—40 um in polar diameter. The exospore is 1—2 ym thick in Pecluma filicula and 2-3.4 «um thick in P. oranense and P. venturi . With LM it is apparently double-layered in section and with a verrucate surface. The inner layer (ie) is bright yellow and the outer one (oe) light yellow. In P. filicula the ie: oe ratio is 1:1 to 1:2, while in P. oranense and P. venturi the ie: io is 1:4—1:5. The exospore of Pecluma filicula has heterometric, low verrucae arranged on one level (Fig. 3 A-C). When observed with LM in equatorial view, exospore thickness increases toward the proximal face. The perispore is 0.6-0.9 um thick, verrucate and perforate. The perforations are located between verrucae (Fig. 3 C). The perispore is uniformly adhered to the exospore. In Pecluma oranense (Fig. 3 D-F) and P. venturi (Fig. 3 G-K) the verrucae are arranged in two levels. The upper level has spheres and large verrucae, which are sometimes laterally fused. The lower level has heterometric verrucae, which are sometimes fused but are smaller than the upper ones. In both species verruca size diminishes toward the proximal pole. When observed with LM in equatorial view, exospore thickness diminishes toward the proximal pole. As seen with LM the perispore is 0.4 to 1 ym thick, apparently single- stratified and adhered to the exospore. It has a micro-verrucate and baculate surface (more evident in Pecluma oranense and P. venturi;Fig. 3 F, J and K). The globules in Pecluma venturi (Fig. 3 J) showed a micro-verrucate surface like that of the exospore. Microgramma (Table 2; Fig. 4 A—D) Only one species, M. squamulosa (Kaulf.) de la Sota, is reported for Northwest Argentina. A hybrid, Microgramma x mortoniana, was reported for 16 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) ores. Fic. 2. SEM micrographs of Campyloneurum s A-C, Campyloneurum aglolepis (Cabrera 3061). A. Proximal view. B. Equatorial view. C. Detail of he surface, showing single or grouped globules on the abraded perispore. D-F, Campyloneurum lorentzii (Kiesling 25227). D. Proximal view, isolated globules are present on the surface. E. Dista surface in detail, the sculpture is slightly verrucate. F. Equatorial view, the verrucae are polyedric aa GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 17 Salta by de la Sota, et al. posite The hybrid was not included in this study because the specimen was sterile. The spores of Microgramma squamulosa are monolete, yellowish to light brown, ellipsoidal or oblong in polar view (Figs. 4 A, B) and plane/ concave-convex in equatorial view, 70—80 sm in major equatorial diameter and 42-52 um in polar diameter. The exospore is 3-5 ym thick and apparently double-layered in section when observed with LM. The inner layer (ie) is bright yellow and the outer (oe) light brown. The ie: oe ratio varies from 1:4 to 1:6. The exospore sculpture is verrucate. The verrucae have a micro-verrucate surface (Fig. 4 C—D). The verrucae are single, or fused to form ridges (Fig. 4 A-D) which diminish in size toward the proximal face (Fig. 4 A—B). The perispore is 0.4—0.7 \tm thick, apparently single-layered in section with the LM closely adhering to the exospore and smooth or micro-verrucate with perforations (Fig. 4 C—D). Occasionally, globules are observed on its surface In spores of Krapovickas & C.L. Cristobal 20456 (LP) granular material was observed on the surface (Fig. 4 C). Phlebodium (Table 2; Fig. 4 E-1) One species of this genus, P. pseudoaureum (Cav.) Lellinger, is present in the northwest of Argentina. It grows as a deciduous epiphyte in the basal forest. The laminae are large, pinnatifid and glabrous with anastomosing venation. The spores are monolete, yellowish, oblong to ellipsoidal in polar view (Fig. 4 E and G) and plane to concave and convex distally in equatorial view (Fig. 4 F). Their diemnsions are 35—42 um in major equatorial diameter and 21-26 um in polar diameter. The exospore is 3—4 pm thick, at LM apparently double-layered in section, the inner layer (ie) being lighter than the outer layer (oe). The ratio ie: io is 1:3 to 1:5. It is compact as seen in fractures observed with SEM (Fig. 4 I). The exospore is tuberculate (Fig. 4 E-H) and the tubercles are single or fused, with a blunt or truncate apex. They seem to be formed by coalescent roads (Fig. 4 F— H). — in shape and heterometric, the size of the verrucae diminish toward the proximal pole at both sides of the lesura. G-I, Campyloneurum major (de la Sota 4483). G. Equatorial view, the verrucae are round, small, and densely packed. H. Distal surface in detail. Irregularly distributed perforations (arrow head) are present and some verrucae are fused. I. Fracture through the sporoderm. J-L, Campyloneurum tucumanense. (J and L: Schinini & C. Quarin 26641; K: Legname & Cuezzo 8762). J. Distal view, globules are occasionally present. K. Equatorial view. Verruca size diminishes toward the proximal pole which is at the bottom. L Detail of the distal surface, the verrucae are heteromorphic and heterometric and some of them fused. Their surfaces are slightly verrucate. Scale bars: A, B, D, F, G, J and K 10 pm; C, E, H and L: 5 pm; I: 2 ym.; e: exospore, p: perispore. 18 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Fic. 3. SEM micrographs of Pecluma spor A-C, Pecluma filicula (Palacr 92), : iach view. Verruca size diminishes toward the asvsinngie pole. The laesura ridge is thick. B. Equatorial view. C. Detail of the proximal surface; verruc surface is smooth and perfo ee are present between verrucae at the sides of the aan (arrowhead). D-F, Pecluma oranense (de la Sota 4310) D. Equatorial view, showing verruca GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 19 The perispore is up to 1 pm thick, with LM it is apparently single-layered in section, smooth with few perforations (Fig. 4 H) and adherant to the exospore. Pleopeltis (Table 2; Fig. 4 J—M) One species, P. macrocarpa (Bory ex Willd.) Kaulf., is reported (Ponce, 1996) for the northwest of Argentina. It grows as an epiphyte or is epipetric in the basal forest between 1000 and 2000 a. s. 1. It is characterized by a scarcely pubescent lamina, ellipsoidal sori, and peltate scaly paraphyses. The spores are monolete, light brown, ellipsoidal to sub-spheroidal in polar view (Fig. 4 J) and plane/concave-convex in equatorial view (Fig. 4 K), 75-86 uum in equatorial diameter and 46—64 um in polar diameter. The exospore is 1.7—2.3 ym thick and verrucate. The verrucae are very low, isolated, and have a micro-verrucate surface (Fig. 4 J—-L). With LM it is apparently double-layered in section, the inner layer (ie) being brighter than the outer layer (oe). The ie: oe ratio is 1:3-1:4. The exospore is apparently compact, as seen in fractures with SEM (Fig. 4 M). The perispore is 1 um thick, and apparently single-stratified with LM and adhered to the exospore. Its surface is smooth or micro-verrucate and perforated (Fig. 4 L). Globules were observed on the surface (Fig. 4 K). In some specimens (Martinez et al. 644, LP), there were abundant perforations distributed on the whole surface of the perispore (Fig. 4 Our material of Pleopeltis macrocarpa has spores with characteristics similar to those described by Tryon & Tryon (1982) based on material from Peru. These authors illustrated the spores of P. macrocarpa with SEM and described the surface as verrucate, with globules and ca. 70 um Polypodium (Table 2; Figs. 5, 6) Eight species of Polypodium grow in the northwest of Argentina. They are P. argentinum Maxon, P. bryopodum Maxon, P. chrysolepis Hook., P. lasiopus Klotzsch, P. loriceum L., P. pleopeltidis Fée, P. squalidum Vell, and P. tweedianum Hook. These species are epiphytic or rupestral, rarely terrestrial, and are characterized by a pinnatifid lamina, with scales or glandular hairs and with sori borne at the tip of a vein. ee ' + diameter di the proximal pole. E. Distal view, showing verrucae at different levels; some of the verrucae are Gateritiatiie and fused. F. Magnification of the distal surface of the spore in picture 4; verruca surfaces are micro-verrucate and the verrucae laterally fused. G-K, Pecluma venturii (de la Sota 4059) G. Proximal view, the verru r here than in the rest of the spore surface and the laesura is membranaceous and high, H. Distal view showing verrucae at different levels with some of them fused. I. Equatorial view, the verrucae are so densely packed that most of them have a polygonal outline, and some are fused. Verrucae diameter diminishes toward the proximal pole. J and K. Magnifications of the surface, showing micro-verrucation of surface. In J, isolated and grouped globules are present on their surfaces; note the similarity between globule and verruca surface. Scale bars: A, B, D, E, G, H, I: 10 um; C, F, K and J: 5 uum, 0 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Fic. 4. SEM micrographs of sac iricee Apion and siruongrs spores. A-D, Microgramma eG (Krapovickas & al 20456) A. Proximal view, the verrucae are smaller on both sides of the laesura. B. ‘Distal view, ke verrucae are both irregularly-shaped and irregularly-sized. Some ae them are fused, forming ridges. C. Detail of the proximal surface of the pore in A with polygonal verrucae and micro-verrucate surface. ‘Small globules and granular GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 21 The spores are monolete, light brown to yellowish, ellipsoidal to oblong in polar view (Fig. 5 A, D, G, and L; Fig. 6 B, C and F) and plane-convex to plane- hemispherical in equatorial view (Fig. 5 B, E, H, K and N; Fig. 6 A, E, H and J), 52-98 um in equatorial diameter and 29-65 um in polar diameter. The exospore is generally 2-4 ym thick, but range to 8 wm thick in Polypodium Jasiopus, and verrucate. As seen with LM it is apparently double- layered in section and in equatorial view it increases in thickness toward the proximal face. Variation in size and degree of verruca fusion were observed in different specimens In all the species the perispore is up to 1 pm thick. With LM it is apparently single-layered in section and smooth, rugulate or micro-verrucate according to the species (Fig. 5 C, F, I, J, M and O; Fig. 6 D, G and J). There are perforations on the exospore and perispore surfaces in all the analized species, located on and between verrucae (Fig. 5 C and F; Fig. 6 D and J) Sporopollenin globules either single or associated in masses are adherant to the perispore in most of the species analized. They differ in size, number and distribution. These globules show a structure similar to that of the sporoderm (Fig. 5 B-E, G, H, J—K; Fig. 6 A-C, E-J). In Polypodium argentinum the verrucae are uniform in shape and size, and isoleted or grouped globules are adherant to the perispore ( Fig. 5 A-C). The spores of Polypodium chrysolepis (Fig. 5 G—J) have low verrucae. The verrucae in Polypodium lasiopus (Fig. 5 K—-M) and P. loriceum (Fig. 5 N—O) are circular to polyhedral and fused, to form radial ridges across the proximal face. The spores of P. squalidum are the smallest within the Polypodiacea studied here, the verrucae are uniform in size and shape, and globules are adherant to the perforated perispore (Fig. 6 E-G). The spores of Polypodium tweedianum (Fig. 6 H-J) have verrucae that are perforated, polyhedral and variable in size. According to observations with LM, the globules of Polypodium bryopodum (Fig. 5 D-F) and P. pleopeltidis ( Fig. 6 A-D) show a central zone much dense — material are abundant on the whole surface. D. Detail of the surface of the spore in B showing micro-verrucate verruca surface and scattered globules (arrow head) of different sizes are present on and between the larger verrucae. E-I, Phlebodium pseudoareum (Cabrera 8178). E. Proximal Detail of the equatorial surface with several large, grouped tubercles. Several ridges form the bases of the grouped elements and the spaces between tubercles are deep; perforations indicated by arrow head. I. Fracture across the sporoderm that exposing the juncture between exospore and perispore. J—M, Pleopeltis macrocarpa (Martinez et al. 644). J. Proximal view. The laesura is a short and the sculpture is verrucate; the verrucae are smaller on the sides of the laesura. K. Equatorial view. The verrucae are low and mainly rounded; their surfaces are micro-sculptured. Globules of oe — are — to the perispore ones — are more evident on the left. L. Detail of =~ ic £ f oo £ to tn + th is ‘ices sckrecabe and perforated (arrow head). M d A dlcen zone in the exospore a its inner surface can be Paneer Scale bars: A, B, E, F, G, J, K: 10 pm; C, D, H, L and M: 3 pm 22 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Fic. 5. SEM micrographs of Polypodium spores. A-C, Polypodium argentinum (Hurrel 51) A. Proximal view. the laesura is a straight, thick ridge. The size ae shape - ae elements are uniform. B. Equatorial view, showi ing numerous globules attached to perispore surface. C. The surface in detail with abraded perispore evident on the left and Dae globules of vais sizes fused to the perisy Perforations are also GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 23 and so could be classified as “‘captive type’’ (sensu Lugardon, 1981). When observed with SEM, these globules seem to be sculptural elements. (Fig. 5 F; Fig. 6 D). In both species exospore ornamentation is verrucate. The verrucae are heterometric and located on a lower level than the globules. (Fig. 5 F; Fig. 6 D) DISCUSSION AND CONCLUSIONS The large spore size in Campyloneurum tucumanense, together with 32 or 64 spores per sporangium, and the greater plant size are characteristics probably related to polyploidy (Tryon & Lugardon, 1991). Walker (1985) detected polyploids in Jamaica and diploids among the South American species of this genus. Cytological studies are needed in order to determine if C. tucumanense, apparently endemic to the northwest of Argentina, is a polyploid. Variations from 49 um up to 95 um in spore size were ggebiie within the genus Polypodium. According to Tryon & Tryon (1982), the Polypodium species from America are diploid or tetraploid with breed stable chromosome numbers. Further cytological studies of the material from Argentina would explain if the size differences are associated with differences in ploidy levels. Phlebodium pseudoaureum has the smallest spores among the Polypodia- ceae analyzed in this work and its tuberculate exospore is the thickest. These features, together with its greater plant size and pinnatifid glabrous lamina with anastomosing venation, differentiate it from other species in the study area. The general features of the exospore in the species analyzed are in agreement with those of the “Polypodium vulgare” type as described by Hennipman (1990). —s present on the perispore (arrow head). The exospore is exposed on the right. The verrucae are different in size and shape, laterally fused and relatively smooth surface. Perforations are evident mostly at the junctions between the verrucae. D-F, Polypodium bryopodum (D: Venturi 4802, E-F: Tur & Kiesling 18353) D. Distal view with verrucate ornamentation. The verrucae are heterometric, mainly poligonal in outline, densely packed and laterally fused. E. sc anatee’ view with globules on the perispore (arrow head). F. Detail of a spot of the spore in figure E showing a micro-verrucate surface of the verrucae a perforations. G-J, Polypodium oe (Castillo 35) G. Distal view with single or grouped globules off different sizes. H. Equatorial view, the verrucae are low and verrucae are more evident than in J. J. masses of globules on the perispore surface, some of them are fused to the perispore (arrow head). The perispore surface is fairly smooth. K-M, Polypodium lasiopus (Hawkes et al. 3976) K. Equatorial view, the verrucae are tetagentially elongated at the equatorial zone, forming true ridges. Small granules are present mainly at the junction places. L. Distal view, granules are present between sculpture elements. M. Detail of the surface showing irregular sizes and polygonal verrucae, mostly fused forming ridges. The perispore surface seems to be fairly smooth to scabrate. N—-O, Poly, podium loriceum (Martinez 641 et al.). N. Equatorial view, the verrucae are numerous per area unit, pene? in shape and low, but there is a general tendency to verruca fusion in the form of ridges. O. Detail of the es waaises: tueed verrucae in the form of ridges. The perispore surface is rugulate. Small-sized g d to the ger Re surface in some places (arrow). Scale bars: A, B, D, E,G, H, K, L, N: 10um; C, F, I, J, M an 24 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Fic. 6. SEM micrographs of Polypodium spores. A-D, Polypodium pleopeltidis (Cabrera, Kiesling & Zardini 24008). A. Equatorial view, the verrucae are smaller toward the proximal pole. Scattered globules can be appreciated at different laces B. Distal view, the surface is uneven and it has verrucae of different sizes and shapes in different levels. The large verrucae are laterally fused but each of them keeps the original outline. GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 25 Variations in sporoderm thickness, size of sculptural elements, number of perforations, and presence of granular material on spore surfaces were not considered to be as diagnostic characteristics because, they may be related to different stages of development (van Uffelen, 1992). Perforations observed in the perispore and exospore of the analyzed spores could be associate with microchannels traversins the exospore; such structures were described by Lugardon (1974), van Uffelen (1992) and Hennipman (1990). The latter author classified the channels according to their size, shape and location within the exospore. In all of the analyzed taxa the spore surface is covered with irregularly nya globular bodies with a smooth or micro-verrucate surface, according axa. These globules were acetolysis resistent and showed variable phan among spores of the same taxa. With LM they showed a density similar to that of the sporoderm. In some samples, Polypodium bryopodum and P. pleopeltidis, these globules were mistaken for elements of the exospore ornamentation. According to Lugardon (1981), these sporopollenin globules are usually present in the Filicopsids, and they were described as having a smooth surface and the same structure as the sporoderm. The globules were also analyzed using TEM in other taxa of Polypodiaceae (van Uffelen & Hennipman, 1985; Hennipman, 1990; van Uffelen; 1993 and Tryon & Lugardon, 1991). e would noted in our analysis that the spores of some Polypodium species from the study area ( P. argentinum, P. squalidum and P. chrysolepis) have similarities to those of Pleopeltis, some others ( P. bryopodum and P. pleopeltidis) are similar to those of Pecluma, and others, such as P. lasiopus and P. loriceum, to those of Microgramma. These observations are in agreement with de la Sota (1977), who suggested that Polypodium is not a well delimited genus, and that certain taxa are closer to Microgramma whearas some others are closer to Pleopeltis. Later systematic studies grouped several species of Polypodium within Pecluma (Price, 1983), while others species were transferred to Pleopeltis (de la Sota, in press) and the species Polypodium — Small, single globules attached to the surface can be seen in several places (arrow head). C. Proximal view. The laesura is —_ = a straight — bial sesiant ged are small on cee nutes of the laesura. D: Detail of the surface. Th masked possibly Py the perispore. Peniandiinns of different sizes are located areas on and between the verrucae (arrow heads). The perispore is micro-verrucate. E~G, Polypodium squalidum (Zuloaga & parine 327). E. heae sai “ee, hg — are “sig ROP as oe area unit, uniform in shape and size. Scattered urface. F. Proximal view. The laesura appears as a stick ridge. There are also aie at the igen pole. G. Detail of se surface. The limits between the verrucae of the exospore below are obscure. The perispore surfac is rugate and perforations are evident (arrow heads). Single and grouped globules of different ‘eee with a smooth surface are fused to the perispore. H-J, Polypodium tweedianum (Palacr 104). H and I. Equatorial views. The presence of globules is variable. Some verrucae are fused, forming short ridges. J. Detail of surface. Verrucae are clearly defined although laterally fused. Globules of different sizes are fused to the surface. The perispore surface is relatively smooth, although some perforations are present (arrow heads). Scale bars: A, B, C, E, F, H, I: 10 um; G, D and 26 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) chrysolepis was considered as Microgramma by Tryon & Stolze 1993 based on some venation characteristics. The spores of Polypodium loriceum and P. lasiopus differ from other studied Polypodium species in exospore ornamentation. In the former species the verrucae are generally fused and form radial ridges proximally. These species belong to the “Polypodium-loriceum group” that is represented by three species in Argentina (de la Sota, Giudice & Gaute, pers.comm.). Our ob- servations suggest that spores are systematic of value at infrageneric level. Within Pecluma, P. fillicula is differentiated from other species on the basis the small size and a pubescent-scaly rachis. The thin exospore, low verrucae and smooth perispore allow it to be differentiated from others species within the genus. CONCLUDING REMARKS Five of the genera of Polypodiaceae from Northwestern Argentina, Campyloneurum, Microgramma, Pecluma, Pleopeltis and Polypodium, have verrucate spores, whereas the spores produced by Phlebodium are tuberculate. Within the verrucate spores taxa, variations were observed in verrucae size, shape, surface pattern and degree of fusion. The large size, together with other characteristics of Campyloneurum tucumanenese spores, may be related to polyploidy. In all the taxa studied the exospore was the thickest wall layer. As revealed by LM it is apparently double-layered and shows wide variation in ornamentation and structure. The inner exospore layer is thin, generally 1/3 to 1/6 the thickness of the outer layer. In acetolyzed material, as observed with LM, the outer exospore appears lighter than the inner one and, apparently, the verrucae or tubercles are restricted to the outer exospore layer. In all cases the perispore is difficult to distinguish and measure using LM because it is thin and adhered to the exospore. However, it was possible to distinguish it in fractured spores with SEM. It is micro-ornamented, generally perforated and covered without any modification to exospore ornamentation. The presence of sporopollenin globules is frequent on most of the spores analyzed. They are single or grouped and are either attached or fused to the perispore surface. We noticed that there is little systematic agreement between systematists and palynologists when considering the significance of spore characteristics in the Polypodiaceae. Nevertheless, in the taxa analyzed here, palynological characteristics, together with morphological data, allow us to identify some genera such as Pecluma and Phlebodium, as well as recognize species groups within the genus Polypodium, e.g. the Polypodium-loriceum group. We are continuing this study on the spore wall of the Polypodiaceae with TEM analyses to determine the structure and stratification of the spore wall, the relationship between perforations and channels within the exospore, and to characterize the globules. GIUDICE ET AL.: SPORE MORPHOLOGY OF POLYPODIACEAE 27 ACKNOWLEDGMENTS The authors wish to thank the institutions which provided herbarium material and the seeder Rafael Unrejola from the Servicio de Microscopia Electrénica del Museo de Ciencias Naturales de La Plata. This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET, PIP5044 ) and the Universidad Nacional de La Plata (UNLP, Project N 363). LITERATURE CITED RDTMAN, G. 1960. The acetolysis method. A revised description. Svensk. Bot. Tidskr. 54:561—564. LuGarpon, B. 1974. La structure fine de lexosporio et = la périspore des Filicinéesisosporées. II- Filicales. ee Pollen et Spon 16: >i 226. LUGARDON, B. 1981. I Filicinées g p dUbisch des Sr t Pollen et Sea 23(1): 93-124. LELLINGER, D. B. 1988. Some new species of Campyloneurum and a provisional key to the genus. Am. Fern J. 78(1):14— Lioyp, R. M. 1981. The as in Polypodium and related genera (Polypodiaceae). Can. J. Bot. 59:175—-189. Saar ms K. and S. Devi. 1964. Spore morphology of indian ferns. III. Polypodiaceae. Grana 5:342- a 7 Pat, : er N. Pat. 1970. Spore morphology and Taxonomy of Polypodiaceae. Grana 10:141-148. Ponce, M.M. 1996. Pteridophyta, en F.O. Zuloaga & O. Morrone, eds. Catdlogo de las Plantas vasculares de la Flora Argentina. Monogr. Syst. Bot. Missouri Bot. Gard. 60:1-79. Price, M. G. 1983. Pecluma, a new tropical American fern genus. Amer. Fern. J. 73:109-116. Sora, E. R., DELA. 1960. Polypodiaceae y Grammitidaceae Argentinas. Opera Lilloana 5. Tucumaén, Argentina. Sora, E. R., DELA. 1973. La weer — de las Pteridofitas en el cono sur de América meridional. Bol. Soc. Arg. Bot. 15(1):2 Sora, E. R., DELA. 1977. Pisidaulets en A, as (ed.), Flora de la Provincia de Jujuy, Colecc. Ci. Inst. Tecnol. Agropecu. 13:1-275. Sora, E. R., DELA and O. MarTINEz, 1998. F — ag Valle de Lerma, Salta. Polypodiaceae Bercht. Et J. Pres]. Ap. Bot. Salta, ser. Flora 5(8 € Sora, E. R., DELA O. G. MarTINEz, and M re ANEM, 1999. Diversidad pteridofitica en pircas de Los Toldos y Eee i canainenas Santa Victoria, Salta, Argentina). Ap. Bot. Salta, ser. oe 1(8 Sora, E. R., s Nuova a. en Pleopeltis (Polypodiaceae).In ee »Hickenia. TRYON, R. wad B UGARDON ores of Pteridophyta. suena Verlag, New York. Tryon, R. and R. ro ang “Pteridophyta of Peru, Part V, 1 es os 21. Polypodiaceae. Fieldiana Botany, new series Tryon, R. and A. TRYON pai Ferns son Allied Plants, with special reference to Tropical America. Springer-Verlag. New York, Heidelberg, Berlin UFFELEN, VAN. 1992 sly ate in Polypodiaceae (Filicales). II. The genera Microgramma Pres] ond sapien Mirbel. Blumea 36:515— UFFELEN, G. A.,VAN. 1993. au in aaee — Ill. Species of ae genera. Spore esate and their value in Phylogenetic analysis. Blumea 37: 529-56 UFFELEN, G. A.,vAN. 2000. Studying spores of the Polypdticen a comparison of — with other micrsocope techniques. In M.M. Harley, C.M. Morton & a a4 Pollen and Spores: Morphology and Biology: wine rg Botanic Garden UFFELEN, G. A.,VAN. and E. HENNIPMAN. 5. The spores of =p a (Polypodiaceae), a SEM study. Pollen et Spores 27(2): satin Water, T. G. 1985. Cytotaxonomic studies of the ferns of Trinidad: The cytology and taxonomic implications. Bull. Br. Mus. Nat. Hist. 13:149-249. American Fern Journal 94(1):28—38 (2004) Spore Viability Under Different Storage Conditions in Four Rupicolous Asplenium L. Taxa CristTINA F. AraGon! and Emitia PaNncua? Departamento de Biologia Vegetal I, Facultad de Biologia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid A. ruta-muraria. subsp. ruta-muraria) was determined after 1, 6, and 12 months of storage in Eppendorf tubes (dry storage) or on agar plates (wet storage) at —20, 5 and 20°C. In general, technique and temperature factors and the moisture-temperature interaction, had a significant account when designing spore conservation programs. Spores of A. ruta-muraria yielded better results in wet storage. In dry storage its response was different from that of the other three taxa. Wet storage at —20°C killed all or most spores of all taxa. Interest in the conservation of pteridophyte spores has become evident in recent decades, because they are easy to obtain, can be stored in large quantities, and can germinate rapidly in simple media (Dyer, 1979). Spores are of interest not only in ex situ conservation programs, but also, as Page et al. (1992) show, in taxonomic studies in the broadest sense, and as a commercial source in horticulture. However, in contrast to seed conservation (Baskin and Baskin, 2001 and references therein), little is known about the factors that affect spore viability during storage. Lloyd & Klekowski (1970) calculated the variation in viability of chloro- phyllous (green) and non-chlorphyllous spores over storage periods of 2 months to 3 years, noting the marked contrast between Equisetum (12 to 24 days viability) and Asplenium (up to 48 years). The conditions under which spores are stored have a notable impact on their viability. Generally, to avoid deterioration, they are stored in dry, ambient or low temperatures, although in some cases this has resulted in loss of viability (Beri and Bir, 1993; Camloh, 1999). Another option that has been tried is storage of spores in a hydrated state (Lindsay et al., 1992), analogous to conditions prevailing in natural spore banks in which spores of some species can remain viable for long periods (Lindsay and Dyer, 1990). It has been ‘Current address: Area de Biodiversidad y Conservaci6n. Escuela Superior de Ciencias Experimentales y Tecnologia. Universidad Rey Juan Carlos. E-28933 Mostoles, Madrid. ? Corresponding Author. ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM 29 observed that this type of storage may be more effective than dry storage for certain species. Pteridophyte spores may remain viable, in a metabolically inactive state, when conditions are not adequate for germination (Page, 1979). The length of time over which spores can maintain viability varies enormously from species to species (Miller, 1968) and it has been shown that other characters, such as spore age (Raghavan, 1989 and references therein), ploidy level (Kott & Peterson, 1974; Kott & Britton, 1982), and the presence of chlorophyll are influential. Even though they can survive desiccation (Lloyd and Klekowsky, 1970; Lebkuecher, 1997), chlorophyllous spores have generally limited viability, compared with pteridophytes with much longer-lived non-chloro- phyllous spores. Page et al. (1992) point out the need to investigate storage conditions that guarantee the maintenance of spore viability for the longest possible time, their genetic integrity, and their developmental capacity. Having available collec- tions of adequately stored spores is of interest in order to avoid the loss of species in nature, in the case of threatened species, while at the same time offering the possibility of having subsequent developmental phases with which to investigate other aspects of the biology of the species. In the present study, various storage conditions were tested on the spores of four Asplenium taxa: A. septentrionale (L.) Hoffm. subsp. septentrionale, A ruta-muraria L. subsp. ruta-muraria, A. adiantum-nigrum L. var. adiantum- nigrum, and A. adiantum-nigrum var. silesiacum (Milde) Viane & Riechstein. These taxa constitute a homogeneous biogeographical and ecological group. They are circumboreal species (Pichi Sermolli et al., 1998) orophyllous, rupicolous, and all are tetraploids. Asplenium septentrionale subsp. septen- trionale is an autotetraploid derived from Sepa caucasicum Fraser- Jenkins & Lovis (Lovis, 1964) and is widely distribu t Eurasia as well as disjunctly in North Africa and on the Pacific coast of North America, where it preferentially inhabits acid substrates. Asplenium ruta-muraria subsp. ruta-muraria is also an autotetraploid but derived from subspecies dolomiticum Lovis & Reichst. (Lovis, 1964) and is distributed across a broad belt in the northern hemisphere, in Europe, Asia and America, having its southern limit in North Africa. It prefers basic substrates. Finally, A. adiantum-nigrum is an allotetraploid arising from crossing and subsequent chromosomal duplication of A. cuneifolium Viv. and A. onopteris L. (Shivas, 1969). It has a wide range throughout Europe, Macaronesia, Asia, Africa, North America and Australia, where it colonizes cracks and fissures preferentially in siliceous rocks. Two varieties are recognized: the typical variety described above, and the variety silesiacum (serpentinicolous ecotype), of ultrabasic substrates in northern and western Europe (Salvo, 1990). Different degrees of hydration (wet and dry) have been combined with different temperature regimes in order to analyse the percentage germination after varying periods of storage. This same methodology has been used in a previous study (Quintanilla et al., 2002) of a group of relict Macaronesian species that inhabit forest floors. These authors aimed to optimise the method 30 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) TABLE 1. Location of populations studied, collector and date of collection. Coordinates Collection Taxa Location Altitude UTM data Collector(s) A. septentrionale Madrid. Manzanares 1300 30TVL2310 July C. F. Aragon & El Real. La Pedriza 2000 R. G. Camacho A. adiantum- A Corufia. A Bafia. 225 29TNH2153 October L. G. Quintanilla nigrum var. 2000 adiantum- nigrum A. adiantum- A Corufia. Sierra 280 29TNJ8139 October J. Amigo & nigrum var. de la Capelada. 2000 L. G. Quintanilla silesiacum Chao do Monte A. ruta-muraria Guadalajara. 1000 30TVL9263 July E. Pangua & Somolinos. 2000 S. Pajaron of storage of viable spores as part of a conservation strategy. Although the taxa included in this study present no problem from a conservation point of view, our objective was to establish whether the optimal spore storage method varies among the taxa. MATERIAL AND METHODS Spores were obtained from populations of each taxon (Table 1). Fertile fronds of 15 sporophytes were collected per population and transported to the laboratory where they were washed under tap water and pressed for two weeks until the spores were released. Each sample was obtained from a mixture of spores from all sporophytes gathered in each population. The spores of these four taxa were subjected to different storage conditions in which different degrees of hydration (wet and dry techniques) and temperatures were combined. With the wet technique, spores were sown directly on a mineral agar medium (Dyer, 1979) that had been sterilized in an autoclave at 20 atm and 125°C for 20 minutes, on sterile plastic Petri dishes (5.5 cm diameter), which were sealed with Parafilm (American National Can, Chicago) to avoid desiccation. To prevent contamination, the antifungal agent Nystatin (100 U ml’) was added to the culture medium after autoclaving and also, in all sowings the spore samples were passed through two layers of lens cleaning tissue (Whatman International Ltd. Maidstone, n° 2105841) to eliminate impurities, remains of sporangial walls, etc. With the dry technique, spores were kept in Eppendorf tubes until germination tests were carried out at which time they were plated out as above. The dishes and tubes were stored at temperatures of 20, 5 and —20°C. They were kept in the dark by wrapping them in aluminium foil to avoid germination during the storage period. Germination tests were carried out after 1, 6 and 12 months of storage. All dishes were incubated for 30 days in a culture chamber at 21°C and 30 pmol m~* s * intensity of photonic flow, with a 16 h light: 8 h dark photoperiod. Four replicates were incubated for each combination of technique and temperature ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM 31 and the percentage germination was assessed after 30 days. This same germination test was carried out before storage to establish a control group. Germination was considered to have occurred if the spore wall was broken and the first rhizoid had emerged. Germination rate (%) was calculated on the basis of a count of 100 randomly chosen spores from each dish. To determine the effects of technique (wet and dry) and temperature (20, 5 and —20°C) on germination rate, the percentages were arcsine-transformed and their means compared by a two-way analysis of variance (Zar, 1999). The analyses were repeated for 1, 6 and 12 months of storage. The multiple com- parisons among means for the identification of homogeneous groups, wherein the effect of a factor was significant (p < 0.05), were made using the Tukey test (p < 0.05). All analyses were done with the SPSS statistical program (1999). RESULTS As in the majority of pteridophyte species, spores of the taxa studied required the presence of light to germinate. The effect of technique and temperature factors, and their interaction, upon spore germination was statistically significant in all cases, except for the hydration factor after one month of storage in the case of A. ruta-muraria (F = 0.059; Table 2). The existence of a significant interaction between factors implies that the effect of each is different for each level. Thus, multiple comparisons between media were made for each possible combination of hydration and temperature. The response of A. adiantum-nigrum var. adiantum-nigrum, A. adiantum- nigrum var. silesiacum and A. septentrionale to the different storage conditions was similar (Fig. 1A, B and C), whereas those of A. ruta-muraria were more variable (Fig. 1D). In general, high percentages of germination were found in the first three taxa, irrespective of the storage conditions, except for wet storage at —20°C, in which case only a small percentage of spores of these three taxa germinated after the first month. In these three cases, dry storage was fairly effective, although there was a slight decrease in the percentage of viable spores after 12 months of storage when kept at —20°C (Fig. 1A, B and C). Dry storage at 5°C (Table 3) was significantly higher (p < 0.05) than for dry-storage at 20 and —20°C, in the case of A. adiantum-nigrum var. silesiacum, and at —20°C for A. septentrionale, after 12 months storage (see Table 3). With respect to A. ruta-muraria, the best results were obtained with wet storage at 20 and 5°C; no spores germinated at —20°C. Percentages achieved with the dry technique were generally lower than under humid conditions and lower than in the other taxa, except for the results after 6 months’ storage at 5 and —20°C (Fig. 1D), where similar percentages were obtained to those with wet storage (Table 3). DISCUSSION Our results indicate that for the taxa studied, except A. ruta-muraria, storage under any of the tested conditions allowed relatively high percentages of viable spores, except with wet storage at —20°C. Under those conditions there : TABLE 2. Levels of significance of the effects of tech p t percentage spore germination after 1, 6 and 12 months of storage. MS, mean squared; df, degrees of freedom; *, p < 0. 05: ** p< 0.001; ***, p < 0.001 Storage time 1 month 6 months 12 months Taxa Source of variation d.f. MS F MS F MS F A. septentrionale ment 1 2,303,118 91,188*** 1,583,659 77,204" ** 849,024 23,600*** Temperatur 2 2,167,125 85,603°*= 3,168,146 154,609*** 3,727,413 115,463" ** et dain x Temperature 2 1,945,107 77,013"** 1,829,107 89,262*** 1,503,217 46,565*** Error 18 25,2 bes 20, Bee 32,282 ate A. adiantum-nigrum pee 1 =: 1,983,658 88,865*** 2,361,546 214,497*** 1,960,820 207,121*** var, adiantum nigrum Temperatur 2 2,513,572 112,603*"* 3,809,133 o40,077 °°" " 3,316,035 350,274" ** echnique x < tale 2 1,824,572 845 737°"" 2,859,518 250,720" 2,600,584 274,695 *** ror 18 22,3 wa 11,010 ee 9,467 oh A. adiantum-nigrum ence a 2,963,416 75,1067" = 3,358,753 126,386* ** 2,335,769 207,865*** var. silesiacum Temperatur Pe 2,506,259 64,104* ** 2,917,486 109,782*** 2,674,307 237:922°"** Daciene § x Temperature 2 1,605,239 41,058"** 1,899,082 71,460°** 1,775,800 156,092" ** Error 18 39,09 oe 26, ae 11,237 nee A. ruta-muraria Technique A 15,918 0.059 ns 455,188 8,684** 2,263,204 22,964"*" Temperature yi 1,977,344 tar 1,941,044 37,032*** 3,208,584 32,557*** Technique X Temperature 2 1,305,054 4,865* 2,477,879 42,274*** 1,491,976 15;430°%% Error 18 68,264 52,4 98,553 (f00Z) | MHAWAN $6 AWNTIOA “TVNUNOL NYA NVOMINV ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM 33 A B woo 10 80 4 60 ang M6 40 O12 20 0 0 control W20 WS W-20 D20 DS D-20 control W20 ws W-20 D20 DS D-20 8 D 100 100 80 80 60 + = 60 Bo 40 O12 40 20 20 0 0 control W20 W5 W-20 D20 D5 D-20 control W20 ws W-20 D20 DS D-20 Fic. 1. Germination percentage after 1, 6, and 12 months’ storage with different techniques (W, wet; D, dry) at temperatures of 20°, 5°, and —20°C. (A) Asplenium adiantum-nigrum var. adiantum- nigrum, (B) A. adiantum-nigrum var. silesiacum; (C) A. septentrionale; (D) A. ruta-muraria was practically no germination after one month of storage. This combination of hydration and temperature was generally inefficient at maintaining spore viability, confirming observations under identical conditions by Quintanilla et al. (2002) on relict forest species. This implies that this combination is not efficient irrespective of species ecology. Pangua et al. (1999) also noted a decrease in germination for spores of Cryptogramma crispa (L.) R. Br. kept in the wet at —18°C. Germination percentage in that species varied among populations. In this species wet spores subjected to a temperature of 70°C yielded higher germination percentages than did dry spores when subjected to the same temperatures after 24 h of treatment (Simpson and Dyer, 1999). Given that a dry —20°C treatment did not result in such drastic reduction in ermination, it is obvious that previous hydration renders the spores more sensitive to freezing. Hill (1971) showed that spores of Adiantum pedatum L. and Thelypteris palustris Schott, after a month of freezing in liquid medium, had higher percentages of germination for the characteristic periods of time than when spores were kept at ambient temperature. Although it has not been shown that longer preservation times yielded the same results, it nevertheless appears that these spores may require chilling in order to germinate. Hill (1971) did not specify whether the spores were frozen immediately after their inclusion in the medium or if there was a time of imbibition. Wet storage at 20 and 5°C maintained the viability of a large number of spores of all taxa studied and hence represents an effective method of storage. TABLE 3. Percentage germination (mean + standard error) of spores without previous storage (control) and after 1, 6, and 12 months storage, with wet (W) and dry (D) techniques at rare of 20°, 5° and —20°C. The vertical lines indicate those groups with homogenous means, between which there are no significant differences Storage time 1 month 6 months 12 months Taxa Control Germination Treatment Germination Treatment Germination Treatment A. septentrionale 72a Gl 0:5: = 0:5 W —20 0.0 + 0.0 W —20 | 0.0 + 0.0 W —-20 | 70:2 21:5 W 20 60.5 + 6.8 D —20 47.0 £8.7 D —20 7O.7 5.8 D —20 FAD = 2.6 D 20 | 66,7 = 5.7 D 20 | 71.0. 5:5 W5 74.0 + 1.4 W 20 73,0 1.6 W 5 72,0 2-46 D 20 7o0 S34 D5 73.5 +44 Tass 77.0 2 3.1 | Bs Via = 26 W5 79:2 247 W 20 A. adiantum-nigrum 92.0 = 1.1 4.5 + 1.9 W —20 0.0 + 0.0 W —20 | 0.0 + 0,0 W —20 | var. adiantum 7935 = 44) D —20 70.7 2 a1 D —20 74:5 = 1.6 D—- nigrum 84.0 + 0.7 W 20 4.7 = 1.8 D 20 80.0 + 2.0 D 20 84.7 + 2.4 D 20 67.7 = 29 D5 60.5 = 3.0 D5 65.7 = 2.6 W 5 68:2 + 1.0 W5 63:2 = 2:3 W5 66.7 = 14 D5 G9,2-= 15 W 20 84.5 + 2.1 W 20 A. adiantum-nigrum 79,0 = 0:6 202 1.7 W —20 0.0 + 0.0 W —20 0.0 + 0.0 W —20 | var. silesiacum 74,24 3.5 W 20 69.7 + 6.4 W5 64:2 $12 D —20 CO STs D —20 P20 2 Bel D —20 66.7 2 2.1 D 20 76:2 2 2:8 W5 76:7 = 3:9 D 20 67.0 = 2:8 W5 80.2 = 2.3 D 20 40.7 = a, W 20 fan 2 2.5 W 20 | 86.2, 2 2:5 D5 OF 2 27 81.0 = 4.1 D5 A, ruta-muraria 73.0 = 2.6 0.0 + 0.0 W -20 0.0 + 0.0 W -—20 | 0.0 + 0.0 W -20 25 17 By D —20 SL 1S D 20 | 7.22 4.7 D —20 | 26.7 2-64 D 20 54.7 + 6.8 D —20 10.5 = 7.4 Ds | S45 = 17.1 DS bo 2 33 D5 26.0 = 10,7 D 20 55.0 + 10.4 W5 60.5: = 1.0 W 20 69.5 = 32 W5 Bhs 2-74.27 W 20 67.2 22 Sei W5 70:5 = 135 W 20 | (F002) L MASINON #6 ANNTIOA “TVNUNOl NYAAA NVORMANV ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM ao Lindsay et al. (1992) studied the response to spore hydration of four species with non-chlorophyllous spores and one with chlorophyllous spores, all of which were hygrophilous. Fully hydrated spores were capable of germinating at ambient temperature after two years of storage at 20°C at much higher percentages than those preserved dry but under otherwise identical con- ditions. Other hygrophilous species, such as Woodwardia radicans (L.) Smith and Culcita macrocarpa C. Presl., show a marked sensitivity to desiccation, such that only those spores that had been maintained in a wet medium germinated after 12 months’ storage, 60% and 84% respectively, compared with those kept in the dry, 1% and 0% respectively, at the same temperature (Quintanilla et al., 2002). These results are interesting because spores of natural spore banks would be in a wet state (Page et al., 1992), especially those from species that inhabit places where the soil is very wet throughout the entire year. Dyer and Lindsay (1992) have shown the persistent presence of A. adiantum-nigrum, A. ruta-muraria and A. septentrionale (L.) Hoffm. in British spore banks. However, in the latter two strictly rupicolous species, the presence of spores in these banks is of low importance, because, although the gametophyte is already established, new sporophytes cannot be established, possibly due to a problem of competition with other species. Furthermore, these two species require a minimum temperature for germination (Young, 1985; Pangua et al., 1994; Dyer and Lindsay, 1996), which they may encounter within cracks or other protected places. Nevertheless, they can tolerate temperatures of up to 70°C for at least 24 h (Simpson and Dyer, 1999). These results may represent an adaptive efficiency in these species for the media they inhabit — exposed rocks with large temperature differences throughout the day and the year. In our study, for A. septentrionale, A. adiantum-nigrum var. adiantum- nigrum and A. adiantum-nigrum var. silesiacum dry storage gave results similar to those with wet storage, although perhaps longer-term storage would have revealed greater differences. Nevertheless, in light of the results, it appears that these rupicolous taxa, have a relatively high capacity to withstand desiccation. Therefore it appears that ecological requirements of species can indeed result in taxa specific adaptations in terms of spore viability, although it must be born in mind that few species have been studied and considerable variability may exist in this respect. In A. ruta-muraria wet storage, with the general exception of —20°C for all the taxa studied, was significantly more effective than dry in maintaining spore viability. Results obtained at 5 and 20°C are essentially the same, showing a germination capacity that increased slightly with time of storage. However, the response obtained with dry storage at different sampling times is difficult to explain. Only after 6 months of storage at 5°C was the germination comparable to that of wet storage. These results might be explainable by a need to go through a cold period before germination. For A. ruta-muraria, despite not being a hygrophilic species, the most suitable preservation method is wet at 5 or 20°C. 36 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) In Polystichum setiferum (Forsskal) Woynar and Athyrium filix-femina (L.) Roth, typical of wet woodlands, it has been observed that dry storage at 4°C results in increased spore viability after 12 and 24 months, respectively, whereas at 20°C, spore viability is practically lost (Lindsay and Dyer, in Simpson and Dyer, 1999). Spores of Cyathea delgadii Sternb. (Simabukuro et al., 1998) and Pteridium aquilinum (L.) Kuhn remain viable for years after dry storage at 4°C (Ashcroft and Sheffield, 2000), for which reason the authors have proposed the routine use of this storage technique and temperature. Some species with chlorophyllous spores, such as those of Osmunda, also retain their viability after years of dry storage at temperatures of 2° and 6°C (Stokey, 1951). In our case, therefore, it appears that the dry technique would be a good option for the conservation of the spores since, although the two techniques have yielded favourable results, the dry technique has some advantages, such as saving space, time and materials. The ideal storage temperature, in this case, would be 5°C, bearing in mind the results of our experiments, where the spores kept dry at this temperature had somewhat higher germination percentages in A. adiantum-nigrum var. adiantum-nigrum, and significantly higher percent- ages in A. adiantum-nigrum var. silesiacum and A. septentrionale. Recent studies (Agrawall et al., 1993; Pence, 2000) have demonstrated the effectiveness of preservation of dried chlorophyllous and non-chlorophyllous spores at —196°C in liquid nitrogen. Pence (2000) observed germination rates of spores of A. ruta-muraria stored under these conditions that were similar to that of the control population. These results may imply that imbibed spores are affected by very low temperatures, but that keeping them dry is a good conservation technique. The time of storage and the processes of sterilization bring about alterations in germination and subsequent development of the gametophytes. Smith an Robinson (1975) studied germination of Polypodium vulgare L. using spores dry-stored at 4°C for 7 years. They observed a decrease in germination and an increase in the proportion of abnormal gametophytes. Similar results were obtained by Beri and Bir (1993) for Pteris vittata L., stored at room temperature for 100 days; spores lost germination capacity in association with total loss of sugars, amino acids and proteins. Camloh (1999) observed in Platycerium bifurcatum (Cav.) C. Chr. that sterilized spores lost viability and that with age there were fewer and shorter rhizoids. It would be interesting, in addition to germination studies for conservation, to study the impact on the development of the gametophyte. In our work, although we have not carried out a thorough post-germination study, the plates used in the various experiments remained in culture chambers at 20°C for 6 months and the gametophytes appeared to develop normally. This suggests that storage time may not affect the subsequent development of the prothalli. ACKNOWLEDGMENTS We thank Drs. Santiago Pajarén, Stuart Lindsay and Luis G. Quintanilla for suggestions on an early draft of this paper. We also thank two anonymous reviewers and Dr. R. James Hickey for ARAGON & PANGUA: SPORE VIABILITY IN ASPLENIUM 37 comments and suggestions, and the latter also for his help with the manuscript. This study has been partially funded by the Spanish Ministry of Science and Technology Project PB97-0307 and the Universidad Complutense de Madrid Project PR78/02-11020. LITERATURE CITED AcrawaL, D. C., S. S. Pawar and A. F. MASCARENHAS. 1993. Cryopreservation of ra bias spinulosa Wall. Ex. Hook. F. An endangered tree fern. J. Plant Physiol. 142:124— AsucrorT, C. J. and E. SHEFFIELD. 2000. The effect of spore density on germination and development in Pteridium, monitored using a novel culture technique. Amer. Fern J. 90:91 BaskKIN, C. C. and J. M. BasKIN. 2001. Seeds. Ecology, Biogeography, and Evoluti vie y and Germination. Academic Press. San Diego. Beri, A. and S. S. Bir. 1993. Germination of stored spores of Pteris vittata L. Amer. Fern J. 83:73—-78. Camiou, M. 1999. Soar age and sterilization affects ge peouigees and early gametophyte development of Platycerium bifurcatum. Amer. Fern J. 89:124—132. Dyer, A. F. 1979. The culture of fern ee for ke a? 7 In Dyer, A. F., ed. The experimental biology of ferns, pp. 253-305. Academic Pre ondon Dyer, A. F. and S. Linpsay. 1992. Soil spore banks of temperate ferns. ne pe I. 82:89-122. Dyer, - F. and S. Linpsay. 1996. Soil spore banks, a new resource for conservation. In J. M. Camus, M. Gibby and R. J. Johns, eds. Pteridology in Perspective pp: 153-160. Royal Botanic Gardens, ew. Hit, : H. 1971. Comparative habitat ts fi tion and prothallial growth of ree pines in south eastern cra Amer. Fern J. 62 1:171-182. Kort, i S. R. L. PETERSON. 1974. A comparative study of gametophyte ape cia of the diploid a and tetraploid races of Polypodium virginiantm. Can. J. Bot. 52:91-9 sane d 82. A tion of som tes sp of n eee North America. Can. J. Bot. 60:1 1679-1687. prrnrc J. 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American Fern Journal 94(1):39—46 (2004) A Comparison of Useful Pteridophytes between Two Amerindian Groups from Amazonian Bolivia and Ecuador MANUEL J. Macia Real Jardin Botanico de Madrid (CSIC), Plaza de Murillo 2, E-28014 Madrid, Spain AsstTract.—An ethnobotanical study of the pteridophytes used by the Tacana and Huaorani indigenous groups from Amazonian forests of Bolivia and Ecuador is presented. Twenty-four useful species, eleven for Bolivia and fourteen for Ecuador, are reported. The only species used by both groups is Cyathea pungens. Most of the recorded uses (76%) are medicinal. Whereas the Tacana use most medicinal pteridophytes by external administration, to heal wounds, swelling, boils, and as eyewash, the Huaorani use them by internal administration, mainly to cure diarrhea, stomachache, body-pain, toothache, and colds. Three species are recorded for veterinary use (12%), to heal wounds and to expel intestinal parasites of domestic animals. Tree-ferns were widely used by all the interviewed informants. Ferns and lycophytes have been employed for a wide variety of uses all over the tropics: cosmetics, dyes, fibers, folklore, flavorings and foods, medicines, and other minor products (e.g., Sodiro, 1893; Copeland, 1942; May, 1979; Murillo, 1983; Schultes and Raffauf, 1990; Ortega and Diaz, 1993; Nwosu, 2002). Although in Amazonia, rural, mestizo, and indigenous people have also used pteridophytes for those purposes, medicinal uses were the most important category (e.g., Davis and Yost, 1983; Murillo, 1983; Boom, 1985; Bourdy et al., 2000). There is little published about useful pteridophytes in the Bolivian Amazon. Fifteen medicinal species are reported for the Chacobo indigenous community (Boom, 1985) and four medicinal species for the Tacana ethnic group (Bourdy et al., 2000). Information from Amazonian Ecuador is more complete. There are five indigenous groups known to use pteridophytes, mainly for medicinal purposes. The Quichua use 12 species (Alarcén, 1988; Marles et al., 1988; Baez, 1998); the Cofan 10 species (Cerdn et al., 1994; Cerén, 1995); and the Huaorani (Davis and Yost, 1983; Cerén and Montalvo, 1998), the Shuar (Baez and Backevall, 1998; Bennett et al., 2002), and the Siona-Secoya (Vickers and Plowman, 1984) use four species each. In this study, I record and compare the uses of pteridophytes by two indigenous people from western Amazonia: the Tacana from Bolivia and the Huaorani from Ecuador to determine whether the uses, applications, and administration of ferns and lycophytes follow the same general pattern of utilization for the two indigenous ethnic groups. METHODS In Ecuador, fieldwork was carried out in Orellana province, from April 1997 to May 1998, near the Huaorani communities of Tiputini (0°36’S; 76°27’W) and 40 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Dicaro (0°56’'S; 76°12’W). The first community is located within the limits of the Yasuni National Park; the second is located within the Huaorani Ethnic Reserve. The study area is tropical evergreen rainforest at 200-300 m elevation. According to drainage and flooding, three broad forest types (tierra firme, floodpain, and swamp) can be recognized (for a specific description of the area, see Romero-Saltos et al., 2001). The indigenous Huaoranis are hunters and fruit-gatherers, and were first contacted less than 50 years ago (Yost 1991; Cabodevilla 1994). They have a deep knowledge of the biology of the forests and their useful plants (Macfa et al., 2001) Fieldwork in Bolivia was conducted in Abel Iturralde province, Departamento La Paz, from.April 2001 to April 2002, in various areas of the Madidi National Park and in the Area Natural de Manejo Integrado Madidi. The study site is a transitional area between Amazonian forest and low montane forest from 260 to 1070 m elevation. Ethnobotanical in- formation was obtained from five pilot-study remote areas (13°53’S— 68°09’W; 14°10’S—67°54’W). The indigenous Tacanas were contacted in the 17" century by Franciscan missionaries (Wentzel, 1989; Hissink and Hahn, 2000) and today they are mainly farmers, although hunting and fishing are occasional activities. Five male informants (>40 years old) were separately interviewed about useful pteridophytes in each of the two study sites; the participation of women was not possible. The informants were chosen by villagers as the most plant- knowledgable people within their own communities. In Bolivia, the infor- mants came from three Tacana communities (Carmenpecha, Macahua, and Tumupasa), and in Ecuador from two Huaorani communities (Dicaro and Tiputini). A semi-structured interview was followed for ethnobotanical queries (Alexiades, 1996). All interviews and fern collections were conducted in the field with the informants. In this paper, I follow the taxonomic system of Tryon and Tryon (1982) for ferns and lycophytes. Vouchers from Bolivia have been deposited in LPB, MA, and MO;’and vouchers from Ecuador in AAU, MA, QCA, and TUR. RESULTS The generic vernacular name for pteridophytes is ‘atarisi’ in the Tacana language whereas in the Huaorani language it is ‘toyuba’. Twenty-four pteridophytes were used by both groups: 11 species for the Tacana and 14 species for the Huaorani. The tree-fern Cyathea pungens was used by both. Most uses (76%) were for medicinal purposes. In Bolivia, four species were used to heal wounds and as an antiseptic (including two Campyloneurum species), and two more species as an anti-inflammatory for boils and swelling. In Ecuador, four species were used to alleviate diarrhea and stomachache (including two Adiantum species), three species to cure general bodypain (including two Polybotrya species), and two species to alleviate toothache. Three species were used for veterinary medicine (12%). MACIA: USEFUL PTERIDOPHYTES OF BOLIVIA AND ECUADOR 41 Adiantum humile Kunze [Pteridaceae] Huaorani vernacular name: Toyuba. Vouchers: Macza et al. 874, 2841, 3388. Tierra firme, floodplain, and swamp (Ecuador). Uses: A decoction of crushed fronds is drunk to cure diarrhea and stomachache. One informant reports that this plant was only used by shamans as a medicinal remedy. Adiantum obliquum Willd. aaa Tacana vernacular name: Atari Voucher: Macia et al. 5945. Tierra firme in high Amazonian forest (Bolivia). Uses: Crushed fronds are directly applied to stop hemorrhaging and heal wounds. Adiantum platyphyllum Sw. [Pteridaceae] Tacana vernacular name: Cucubio ina. Voucher: Macia et al. 4455. Tierra firme in high Amazonian forest (Bolivia). Uses: Some drops from crushed fronds are used as eyewash when vision is not clear. Adiantum pulverulentum L. [Pteridaceae] No vernacular name given. Voucher: Macia et al. 2899. Tierra firme (Ecuador). Uses: A decoction of the fronds is drunk to cure diarrhea. Alsophila cuspidata (Kunze) D. S. Conant [Cyatheaceae] Tacana vernacular name: Atarisi. Vouchers: Macia et al. 4017, 4424, 6492. High Amazonian and low montane tierra firme forests (Bolivia). Uses: Mucilage from the apical part of the cut stem applied to boils. A poultice made from this sap is externally applied to reduce swelling in any part of the body. Bolbitis nicotianifolia (Sw.) Alston oe Huaorani vernacular name: Acaguem Vouchers: Macia et al. 2861, 3218, 3668. Floodplain and swamp UsEs: Boiled crushed rhizome with one pinna is drunk to cure stomachache; a decoction of the rhizome is drunk to alleviate body pain and fever (‘calentura’). Campyloneurum fuscosquamatum Lellinger [Polypodiaceae] Huaorani vernacular name: Toyuba. Vouchers: Macra et al. 1592, 2982. Tierra firme, floodplain, and swamp (Ecuador). 42 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Usss: A decoction of the crushed fronds is drunk to cure colds and coughs. Campyloneurum repens (AubI. : be Pres] [Polypodiaceae]} Tacana vernacular name: Atar: Voucher: Macia et al. ae Tierra firme in high Amazonian forest (Bolivia). Uses: Crushed fronds are applied directly to heal wounds, fronds may also be placed under bandages for several hours. Campyloneurum sphenodes (Kunze ex Klotzsch) Fée [Polypodiaceae] Tacana vernacular name: Chati ina Voucher: Macia et al. 5174. Low Sueitiainn tierra firme forests (Bolivia). Usss: Crushed fronds are directly applied to stop hemorrhaging and heal wounds. When dogs have been bitten by wild animals, people chew the fronds and apply them to the dogs wounds. Cyathea amazonica R. C. ye acces Tacana vernacular name: Ata Voucher: Macia et al. fe Tierra firme in high Amazonian forest (Bolivia). Uses: Crushed apical part of the stem is macerated in cold water or urine and applied directly to scabby dogs. According to our informant, it cannot be used on people because it is too “strong”’. Cyathea delgadii Sternb. [Cyatheaceae] Tacana vernacular name: Atarisi. Vouchers: Macia et al. 5357, 6183. Low montane tierra firme forests (Bolivia). Uses: Mucilaginous sap from central apical part of the cut stem is directly applied to boils. Cyathea lasiosora (Mett. ex Kuhn) Domin [Cyatheaceae] Huaorani vernacular name: Toyuba, toyuto. Vouchers: Macia et al. 400, 655. Tierra firme and swamp (Ecuador). Uses: Drops of mucilaginous sap, from the basal part of a cut pinna or apical part of the cut stem, are used to alleviate toothache, placing them directly on the gum. Five informants from two Huaorani communities reported this use. Well-dried stems are occasionally used for firewood. Cyathea pungens (Willd.) Domin [Cyatheaceae] Huaorani vernacular name: Toyuba, to : Vouchers: Macia et al. 309, 2441, 2721. Swamp forest (Ecuador). Uses: Drops of mucilaginous sap from the basal part of a cut pinna are used to alleviate toothache by placing them directly on the gum. Tacana vernacular name: Atarisi. Voucher: Macra et al. 4127. Tierra firme in high Amazonian forest (Bolivia). MACIA: USEFUL PTERIDOPHYTES OF BOLIVIA AND ECUADOR 43 Uses: Mucilaginous sap from central apical part of the cut stem is applied directly on skin in cases of swelling. Equisetum giganteum L. [Equisetaceae] Spanish vernacular name: Bigote de tigre. Voucher: none. Floodplain in Amazonian forest (Bolivia). Uses: A decoction of crushed aerial stems and whorls of branches is drunk to alleviate kidney and bladder pain. Lomariopsis japurensis (Mart.) J. Sm. [Dryopteridaceae] Tacana vernacular name: Chati ina Voucher: Macia et al. 3880. Tierra firme in high Amazonian forest (Bolivia). Usss: Dried fronds are pulverized and put directly on wounds to heal them. Melpomene melanosticta (Kunze) A. R. Sm. and R. C. Moran [Grammitidaceae] Tacana vernacular name: Afarisi. Voucher: Macia et al. 6224. Low montane tierra firme forests (Bolivia). Users: Whole plant is used for womens’ necklaces because their fresh rhizomes are fragant for a long time. Microgramma fuscopunctata eon Vareschi [Polypodiaceae] Huaorani vernacular name: Guimipume. Vouchers: Macia et al. 1535, = Swamp forest (Ecuador). Uses: Boiled fronds are rubbed on joints (knee, elbow, shoulder) to alleviate aching. Polybotrya crassirhizoma Lellinger [Dryopteridaceae] Huaorani vernacular name: Toyuba, toyuba bengana. Vouchers: Maczia et al. 623, 684. Tierra firme and floodplain (Ecuador). Uses: A decoction of croziers is drunk to alleviate body pain. Polybotrya osmundacea Humb. & Bonpl. ex Willd. [Dryopteridaceae] Huaorani vernacular name: Toyuba. Vouchers: Macia et al. 605, 3377. Tierra firme forest (Ecuador). Uses: A decoction of croziers is drunk to alleviate body pain. Saccoloma inaequale (Kunze) ina [Dennstaedtiaceae] Huaorani vernacular name: To Vouchers: Macia et al. 1521, a Tierra firme and swamp (Ecuador). Uses: Crushed rhizome is fragrant and used as deodorant. Selaginella exaltata (Kunze) _— ‘caiman Huaorani vernacular name: To Vouchers: Macia et al. 311, py ogee forest (Ecuador). 44 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Uses: Crushed rhizome is macerated in cold water, mixed with chicha (traditional beverage made from cassava), and drunk to cure stomachache and diarrhea. Selaginella geniculata (C. Presl) Spring [Selaginellaceae] Huaorani vernacular name: Toyuba. Voucher: Macia et al. 2555. Floodplain (Ecuador). Uses: Fronds are used in ceremonial forehead bands for adornment at traditional Huaorani feasts. Selaginella parkeri (Hook. & Grev.) Spring [Selaginellaceae] Huaorani vernacular name: Toyotome. Voucher: Yanez, Macia et al. 2231. Tierra firme (Ecuador). Uses: A liquid decoction of crushed rhizomes is given to dogs to expel intestinal parasites. Thelypteris macrophylla (Kunze) C. V. Morton [Thelypteridaceae] Huaorani vernacular name: Toyuba. Voucher: Macia et al. 2980. Tierra firme, swamp (Ecuador). Uses: A decoction of crushed rhizome is drunk to cure stomachache. DISCUSSION There are clear differences in the use of pteridophytes in Bolivia and Ecuador. The medicinal and veterinary species used by the Tacanas were administered externally (except for Equisetum giganteum), whereas those used by the Huaoranis were administered internally (except for Microgramma fuscopunctata). This differentiated medicinal pattern seems to be exclusive to pteridophytes, because medicinal administration of other vascular plants is not as specific for these two Amerindian people (Davis and Yost, 1983; Cerdén and Montalvo, 1998; Bourdy 1999; Bourdy et al., 2000). Other indigenous groups also show preferences for medicinal administration of ferns and lycophytes: the Chdcobo from Bolivia and the Quichua from Ecuador, mostly administered their preparations internally (Boom, 1985; Alarcén, 1988; Marles MACIA: USEFUL PTERIDOPHYTES OF BOLIVIA AND ECUADOR 45 et al., 1988), whereas the Cofan from Ecuador administer their preparations externally (Cerén et al., 1994; Cerdn, 1995). The fragant rhizomes of two species are used as a perfume or a deodorant. Furthermore, the rhizomes of Melpomene melanosticta have been reported to maintain a sweet spicy fragance for tens of years (Smith and Moran, 1992); chemical analysis of this fragance should be of interest. ACKNOWLEDGMENTS I am deeply grateful to the Tacana communities of Carmenpecha, Macahua and Tumupasa in Bolivia, and to the Huaorani communities of Dicaro and Tiputini in Ecuador, for sharing their knowledge and for their hospitality; to the staff of Herbario Nacional de Bolivia (LPB), especially to S.G. Beck; to the staff of Herbarium QCA at Pontificia Universidad Catélica del Ecuador in Quito, especially to R. Valencia. To H. Tuomisto (TUR), I. Jiménez (LPB), and M. Lehnert (GOET), who project by the Consejeria de Educacién, Comunidad Auténoma de Madrid (Spain), and to the Ecuadorean project by the European Comission (INCO-DC, IC18-CT960038) is acknowledged. LITERATURE CITED ALARCON, R. 1988. Etnobotdnica de = Quichuas de la Amazonia ecuatoriana. Misc. Antropol. Ecuatoriana, song ogr. ser. 7:1— ALEXIADES, M. N. 1996. state ee data: an introduction to basic concepts techniques. : . 53-94 in M. N. Alexiades, ed. Selected Guidelines for tahadhebaiitea! Research: a Field Manual. The New York Botanical Garden, New Yor BAgz, S. 1998. Dictionary of plants used by the Canelos-Quichua. Pp. 64—70 in 1H. Borgtoft, F. Skov, J. Fjeldsa, I. Schjellerup and B. Ollgaard, eds. People and Biodiversity - Two Case Studies from the Andean Foothills of Ecuador. Centre for Research on Cultural and Biological Diversity of Andean Rainforests (DIVA), Tech. Report 3. Arhus, Denmark. BAgz, S. and A. BACKEVALL. 1998. Dictionary of plants used by the Shuar of Makuma-Mutints. Pp. 125-133 in H. Borgtoft, F. Skov, J. Fjeldsa, I. Schjellerup and B. Oligaard, eds. People and Biodiversity - Two Case Studies from the Andean Foothills of Ecuador. Centre for Research on Cultural and Biological Diversity of Andean Rainforests (DIVA), Tech. Report 3. Arhus, Denmark. BENNETT, B. C., M. = fig and P. GOMEz-ANDRADE. 2002. Ethnobotany of the Shuar of Eastern Ecuador. Adv. 299 Boom, B. M. 1985. Panter iss of the Chacobo indians in Amazonian Bolivia. Amer. Fern J. 75:19-21 Bourpy, G. 1999. Tacana—Conozca tros drboles, nuestras hierbas. Universidad Mayor de San Andrés (UMSA), Consejo indigen de los Pueblos Tacana (CIPTA), and Institut de Recherche pour le pp ute (IRD). La Paz, cag Bourpy, G., S. J. Dewaut, L. R. CHAvEZ DE Mic: L, A. Roc A, E. DEHARO, V. MuNoz, L. BALDERRAMA, C. UENEVO iy A. GIMENEz. 2000. a eee uses of the Tacana, an Amazonian Bolivian ethnic group. J. Ethnopharm. 70:87—109 CaBopevitLa, M. A. 1994. Los Huaorani en Ia Historia de los Pueblos del Oriente. Editorial Cicame, Coca, Ecuador Cron, C. E. 1995. Etnobiologra de los Cofanes de Dureno. Publicaciones del Museo Ecuatoriano de Ciencias Naturales, crane International, and Ediciones Abya-Yala, Quito, Ecuador. Crron, C. E. and C. G. Monravo, 1998. ugha tg de los Huaorani de Quehueiri-Ono, Napo- Ec sete Ediciones eae Yala, Quito, Ecuado Ceron, C. E., C. G. MonTALvoO, J. UMENDA and E. CHICA- een 1994. Etnobotdnica y Notas sobre la Diversidad Vegetal de la en Cofdn Sinangtié, Sucumbios, Ecuador. Ecociencia, Quito, Ecuador. 46 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) CopELAND, E. B. 1942. sei —_ Amer. Fern J. 32:121-126. Davis, E. W. and J. A. 3. The ethnomedicine of the Waorani of Amazonian Ecuador. a. yy Hissink, K. and A. HaHN. 2000. Los Tacana- Datos sobre la Historia de su Civilizacién. APCOB- Plural Editores, La Paz. Macia, M. J., H. ROMERO-SaTos and R. VALENCIA. 2001. Patrones de uso en un bosque primario de la mazonia ecuatoriana: comparacién entre dos comunidades Huaorani. Pp. 225-249 in J. F. Purarara H. Balslev, - —_ 5 Se a dez, H. a and R Valencia, eds. uacion Veg la Am nstitute for RE ter and Ecosystem eng miki Universiteit van ie oe. The Netherlands. Marts, R. J., D. A. New and N. R. FARNsworTH. 1988. A contribution to the e = age of the ediaad Quichua people of Amazonian Ecuador. Revista Acad. Colomb. Ci. Exact. 16:111-120. May, L. W. 1979. The economic uses and associated folklore of ferns and fern allies. Bot. Rev. 44:491-528. MvriL_o, M. T. 1983. Usos de los Helechos en Suramérica con Especial Referencia a Colombia. Universidad Nacional de Colombia, Bogota. Nwosu, M. QO. 2002. Ethnobotanical studies on some Pteridophytes of southern Nigeria. Econ. Bot. 56:255—259. Orteca, F, and W. Diaz. 1993. Ethnopharmacological notes on two Venezuelan Asplenium. Amer. Fern J. 83:71. Romero-Satros, H., R. VALENCIA and M. J. Macia. 2001. Patrones de diversidad , distribucion y rareza de plantas lefiosas en el Parque Nacional Yasuni y la Reserva Etnica ‘Huaoran ani, Amazonia ecuatoriana. Pp. 131-162 in J. F. Dui eestor ig H. Balslev, J. Cavelier, C. Grandez, H. Tuomisto and R. Valencia, eds. Evaluacidn de Recursos Vegetales No Maderables en la Amazonia iashiatidietcs Institute for Biodiversity and Ecosystem Dynamics (IBED), eaieh heey van Amsterdam, The Netherlands. ScHULTEs, R. E. and R. F. RaFFaur. 1990. The Healing Forest-Medicinal and Toxic Plants of the Northwest Amazonia. Dioscorides Press, Portland, Oregon Smitu, A. R. and R. C. Moran. 1992. Melpomene, a new genus of Grammitidaceae. Novon 2:426— 432 Sopio, A. 1893. ee Vasculares Quitensis. Typis Universitatis, Quito, Ecuador Tryon, R. M. and A. F. Tryon. 1982. Ferns and Allied Plants with Special Reference to eccteat America. ction Velie, New York. Vickers W. T. and T. PLowman. 1984. Useful plants of the Siona and Secoya indians of eastern Ecuador. Ficldiane, Bot. 15:1-63. WENTzEL, S. 1989. Tacana and highland migrant land use, living conditions and local organizations in the Bolivian Amazon. Ph.D. dissertation, University of Florida. Yost, J. A. 1991. Los Waorani: un pueblo de la selva a. Pp. 95-115 in Anonymous, ed. Ecuador a la sombra de los volcanes. Ediciones Libri Mundi, Quito, Ecuador. American Fern Journal 94(1):47—56 (2004) Influence of Copper on Selected Physiological Responses in Salvinia minima and Its Potential Use in Copper Remediation SAFAA H. AL-HAmpDaNnNI and Stacy L. BLAIR Jacksonville State University, Biology Department, 700 Pelham Rd. N, Jacksonville, AL 36265 (USA) ABSTRACT.—This study was designed to evaluate selected hs papers responses of Salvinia minima to copper (Cu**) concentrations of 0.06 (control), 1.0, 2.0, 2.5, and 3.0 mg |’. The aa were grown under laboratory conditions of 25 + rC. a light oo. of 120 pmol a 4 ,anda h photoperiod. After seven days of exposure to the Cu, Salvinia growth ae | gradually geet an increase in Cu concentration resulting in a significant decline at 3.0 mg I ‘Cu. Similar results increase of Cu concentration in the growth media. This study demonstrated the potential of Salvinia to remediate Cu in concentrations 100 times what is currently found in freshwater environments. Toxic heavy metal contamination is common in aquatic ecosystems due to both anthropogenic and natural sources. Runoff, industrial waste discharge and sewage effluent are the most frequent anthropogenic sources of aquatic contamination (Lee et al., 1998). pper (Cu), an essential metal for plant growth is required in trace amounts (Guilizoni, 1991). Copper is a constituent of the chloroplast protein plastocy- anin, which forms part of the electron transport chain linking the two photochemical systems of photosynthesis (Bowyer and Leegood, 1997). In addition, Cu functions as an activator or component of certain enzymes that are involved in a variety of biochemical processes, such as cytochrome c oxidase, and Cu-Zn superoxide dismutase (Linder, 1991). Copper uptake appears to be a metabolically mediated process and there is evidence that Cu strongly inhibits the uptake of zinc (Zn) and vice versa (Hawf and Schmid, 1967). Generally, Cu toxicity causes chlorosis (Lewis, 1993; Vavilin et al., 1995) and iron (Fe) deficiency by inhibiting translocation of Fe through the plant (Chaney, 1970; Lingle et al., 1963; Wallace and DeKock, 1966). In addition, toxic levels of Cu inhibit root growth by damaging plasma membrane integrity (Marschner, 1995). Copper is one of 13 metals listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA) (Salomons et al., 1995) and is among one of the most frequently discharged elements into the environment. It has been estimated that the global discharge of copper in aquatic systems is near 112 x 10° metric tons per year (Moore, 1991). Conventional remediation 48 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) methods such as precipitation, chemical oxidation or reduction, ion exchange, filtration, or evaporation processes are generally inefficient for removing metals in aquatic systems (Bervoets et al., 1994). In contrast, the use of aquatic plants is currently under investigation as a viable alternative for remediation of a wide range of contaminants including heavy metals (Lee et al., 1998). This cost- effective, plant-based approach to remediation takes advantage of the remark- able ability of plants to concentrate elements and compounds from the environment and to metabolize various molecules in their tissues (Salt et al., 1998). Selecting plants as suitable candidates in phytoremediation must satisfy certain criteria such as reasonable tolerance to the contaminant in question, relatively high growth rate, and the ability to uptake and preferably metabolize the contaminant (Salt et al., 1998). The genus Salvinia (Salviniaceae) is comprised of one genus and 10 known species (Nauman, 1993). Salvinia minima Baker is a small, free-floating freshwater fern found in tropical and temperate regions of the world (DeBusk and Reddy, 1987) in areas such as North, South, and Central America, the West Indies, and Central America (Nauman, 1993). This plant can be found floating near the edges of slow moving streams and in nutrient enriched ponds. It is commonly referred to as water spangles and floating fern (Nauman, 1993). Salvinia minima demonstrated the ability to withstand aluminum (Al) concentrations of 20 mg |’ through the manipulation of the media pH from 3.9 to near 7 within 24 hours (Gardner and Al-Hamdani, 1997). In addition, Salvinia showed considerable ability to accumulate cadmium (Cd II), 10,930 mg kg’; therefore it was suggested as a Cd II hyperaccumulater (Olguin et al., 2002). Salvinia has the potential to double its population in approximately 3.5 days (Nichols et a/., 2000) making it a suitable candidate for phytoremediation. This study was designed to evaluate the impact of Cu 2* concentrations of 0.06 (control), 1.0, 2.0, 2.5, and 3.0 mg |? on various physiological responses of Salvinia including plant growth, photosynthetic pigments, and CO, assimila- tion. The 0.06 mg 1“ concentration designated as the control was selected based on the average concentration of Cu in uncontaminated freshwater (Boyd, 1990). Copper uptake by Salvinia, grown at the different treatments, was determined. MATERIALS AND METHODS The Salvinia minima utilized in this study was taken from stock material grown under greenhouse conditions for four years. Dr. David Whetstone, at the Jacksonville State University Herbarium, identified the plants, which were originally collected at a drainage ditch near Sanford, Florida (USA). Plants with a total of 15 fronds were placed into 60 (250 ml) Erlenmeyer flasks containing 125 ml of various Cu concentrations dissolved in 10% Hoagland solution with a pH of 6.5 (Hoagland and Arnon, 1938). Twelve flasks, samples, were used for each of the selected Cu concentrations, control (0.06); 1.0; 2.0; 2.5; and 3.0 mg I. The initial fresh weight of the plants was recorded for each flask. The samples were placed randomly in the growth chamber and allowed to grow under conditions of 25 + 2°C, a light intensity of 120 pmol m~ s~! and a 14-h AL-HAMDANI & BLAIR: INFLUENCE OF COPPER IN SALVINIA MINIMA 49 photoperiod. On day seven of the experiment, plant fresh weight and total frond number of six randomly selected flasks were assayed. In addition, 0.1 g fresh weight of tissue from each flask was used for chlorophyll aand b, and carotenoid determination. The remaining samples from these flasks were oven dried at 80°C for 48 hrs to be used later for Cu uptake determination. The media of the remaining six samples of each treatment were replaced with fresh solutions on day seven and the plants were allowed to grow for an additional seven days. On day 14 of the experiment, the same physiological parameters were determined as was CO, assimilation. arat t, as described above, was conducted with the exception that the media were not replaced at day seven. The existing media was filtered twice to reduce algal contamination. These plants were harvested on day 14 and oven dried at 80°C for 48 hrs to be used later for total Cu uptake. In addition, the medium of each sample was collected and the filters were analyzed for total Cu concentrations to insure a total accounting for Cu partitioning. The data obtained from this experiment was used to determine bioconcentration factors (BCF) and percent of Cu uptake. Salvinia growth was expressed as doubling time (DT) in days. The doubling time was determined using the following equation: DT= t log 2 [log (w,w, ‘)]"' (Moretti and Gigliano, 1988), where DT is the doubling time (days), t is the experiment duration (days), w; is the final weight (or number of fronds), and w, is the initial weight (or number of fronds). Approximately 0.1 g fresh weight of each sample was used for measuring chlorophyll a, b, and carotenoid concentration. The plant was 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 spectrophoto- metrically at wavelengths of 647 and 664.5 nm (Inskeep and Bloom, 1985). Carotenoid concentrations of the DMF extract were determined spectrophoto- metrically at a wavelength of 470 nm and the concentration was calculated using the formula of Doong et al. (1993). Carbon dioxide assimilation and internal CO, concentrations of six randomly selected samples from each treatment were measured four hours after the onset of the light period on days seven and 14 of the treatments application. The selected plants of each sample were 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 aprcta conditions were 120 pmol m~ s ' photon flux density, 45 to 50% RH, and 2 Oven ie plant samples, ranging from 0.01-0.07 g, were digested according to procedures for Cu sampling outlined in the Buck Model 210 VGP Atomic Absorption Spectrophotometer Operating Manual (Buck Scientific, 1996). The samples were refluxed in 10 ml of 6N nitric acid for 15 min, just below the boiling point, and then 5 ml concentrated nitric acid (15.8N) was added. The reflux process was continued until the sample volume was reduced to approximately 5 ml. The samples were allowed to cool after which 2 ml H,O and 5 ml 30% H.O, were added to each sample. The samples were warmed 50 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) fasts tC trati Salvinia growth. Plant growth is expressed as doubling time (DT) based on fresh weight and frond number for both seven and 14 days of exposure. LSD (P = 0.05) = Least Significant Difference value for difference between means within a column. Same letter denotes no statistical difference. Lower case letters denote differences between treatments within a day and upper case letters denote differences between days within treatments. N = 24 DT based on mean frond number (days) DT based on mean fresh weight (days) Length of exposure (days) Cu (mg I~’) ? 14 7 14 0.06 (control) 7.61 aA 8.23 aA 5.70 aA 7.68 aB 1.0 8.37 abA 8.44 aA 6.03 aA 8.39 abB 2.0 8.10 abA 8.65 abA 6.83 aA 8.79 abB 2.5 8.34 abA 9.17 abA 7.87 abA 9.22 bB 3.0 9.84 bA 9.82 bA 8.92 bA 10.06 bB slowly adding 1 ml of 30% H,O, as needed until effervescence subsided. After cooling again, reflux of the samples was continued for 15 min. using HCl in aratio of 1 ml for each 2 ml of sample. After cooling, the samples were brought to 25 ml with distilled H,O. Standards were established using Cu concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 mg |? and absorbance was measured for each sample using the atomic absorption spectrophotometer. Copper concentrations in the growth media were determined following the digesting procedure outlined in the Buck Model 210 VGP atomic absorption spectrophotometer-operating manual (Buck Scientific, 1996). To the sample media, 2 ml nitric acid (15.8N) and 5 ml HCl was added. The samples were refluxed until approximately one quarter of the media remained and the volume brought back to 125 ml with distilled H.O. The Cu concentration of each sample was determined as described above. The BCF was determined as the ratio of Cu concentration in the plant tissue to the concentration in the external media (Spacie et al., 1995). The experiments were 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. The block in this study represent the replicate series of each experiment 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). RESULTS After seven days of exposure to the various Cu concentrations, the only significant reduction in Salvinia growth was obtained on the medium containing 3.0 mg l"* Cu (Table 1). Similar results were obtained at the end of day 14 of treatments exposure. Additionally, using fresh weight to calculate DT, the data showed that Cu concentrations of 2.5 mg I? significantly reduced AL-HAMDANI & BLAIR: INFLUENCE OF COPPER IN SALVINIA MINIMA 51 TaBLeE 2. Concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and a ceil in a day and upper case letters denote differences between days within treatments. N = 24 Chl a Chl b Carotenoid (mg g ’ fr wt.) (mg g’ fr wt.) (mg g' fr wt Length of exposure (days) Cu conc (mg 17’) 7 14 7 14 7 14 0.06 (control) 11.67 aA 12.82 aB 6.93aA 7.01 aA 3.14aA 3.61 aA 1.0 6.02 bA 5.98 bA 3.82 bA 3.09 bB 0.47 bA 0.37 bA 2.0 5.10 bA 3.67cB 3.25 Ch. 1.86cB 0.30 bA 0.07 bA 2.5 5.60 bA 4.36 cB 3.66bcA 2.40 dB 0.42 bA 0.16bA 3.0 5.29 bA 4.57 cB 3.35 bcA 2.43 dB 0.33bA 0.17bA Salvinia growth at the end of day14 of treatment (Table 1). Salvinia growth, measured as frond number DT, was not significantly different in the presence of 1.0, 2.0, 2.5 mg 1‘ Cu from that of 3.0 mg I‘ Cu. Comparing the same treatments, day 14 data analysis was shown that significant reduction in growth at 3.0 mg 1 Cu in contrast to that of 1.0 mg l~* Cu. Slight deviation from these findings was obtained from data analysis using plant fresh weight DT that reveled growth was significantly less in 3.0 mg |-* Cu than at 1.0 and 2.0 mg I? Cu. Using frond number DT, no differences were noted in Salvinia growth between day seven and 14 at each of the various Cu concentrations. However, there was a significant increase in fresh weight DT values, which corresponds toa significant reduction in growth, at the end of 14 days for all treatments in comparison to those obtained at day seven (Table 1). After seven days exposure, the increase in Cu concentrations from 1.0 to 3.0 mg |' had similarly influenced chlorophyll a and b and carotenoid concen- trations and tly reduced these photosynthetic pigments, relative to the control. The only exception to this finding was Chlorophyll b, which was significantly higher at 1.0 than at 2.0 mg!~' Cu (Table 2). In general, a reduction in Salvinia photosynthetic pigments obtained at day 14 reflected that seen at seven days (Table 2). However, at 1.0mg1™' Cu, chlorophyll a concentration was significantly higher than that of the other treatments except for the control, 0.06 mg |’. The gradual decrease in chlorophyll b was interrupted by an increase in Cu from 2.5 to 3.0 mg |“ in comparison to 2.0 mg |. Chlorophyll a accumulation in Salvinia significantly increased at day 14 in comparison to day seven within the control (Table 2). However, chlorophyll b and carotenoid concentrations were not significantly affected. In comparison the photosyn- thetic pigments between days seven and 14 for each treatment, carotenoid concentrations showed no significant difference whereas Salvinia grown at Cu concentrations of 1.0 mg |’ and higher revealed a significant reduction in chlorophyll a and b, with the exception of chlorophyll a at 1.0 mg I’ After fourteen days of Cu exposure there were significant decreases in CO, assimilation for all Cu concentrations from 1.0 to 3.0 mg | * as compared to the 52 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) TasLe 3. Carbon dioxide assimilation and internal CO, in Salvinia after 14 days of varying Cu exposure. LSD (P = 0.05) = Least Significant Difference value for difference between means within column. Same letter denotes no statistical difference. Lower case letters denote differences between treatments within a day and upper case letters denote differences between days within treatments. N = 12. Cu conc CO, assimilation Internal CO, (mg 1~*) (umol m~?s?) (ul 1”) 0.06 (control) 232 a 351.37 a LO 1.51b $51.77 a 2.0 Lie 350.92 a 25 110'¢ 350.92 a 3.0 LOT ¢ 350.06 a control (Table 3). However, treatments receiving 2.0, 2.5, and 3.0 mg I did not differ. Furthermore, there was a 25.8, 27.1, and 33.1% increase in CO, assimilation in plants grown at 1.0 mg 1’ Cu when compared to that obtained in plants receiving higher Cu concentrations. Internal CO, concentrations were not different among plants grown in all treatments (Table 3). After seven days of growth, Cu accumulation was significantly higher in plants receiving 1.0 to 3.0 mg 1’ Cu in comparison to the control (Table 4). Furthermore, Cu accumulation in Salvinia grown at 1.0 mg 1! was significantly lower than those plants receiving higher concentrations. At the end of 14 days, Salvinia still showed an increase of Cu uptake correlated with increasing Cu concentrations in the growth media. However, examining the individual treatments showed that at 1.0 mg 1™' Cu, Salvinia accumulation was similar to that of the 0.06 and 2.0 mg |’ concentration. Copper uptake of the media containing 3.0 mg 1‘ was the highest in comparison to the other treatments with a 43.9% increase in comparison to the nearest treatment, 2.5 mg 1‘. Whereas, Cu accumulation in Salvinia was similar in those plants grown at 2.0 and 2.5 mgl™. With the exception of those plants grown at 0.06 and 3.0 mg I! Cu, Salvinia uptake of Cu was significantly higher during the first seven days in comparison to 14 days of the experiment (Table 4). These results coincide with the BCF calculation of each treatment after 14 days of Cu exposure that was nearly 20- fold higher in plants at 3.0 mg 1“! Cu in comparison to the control and twice as high in plants grown in 2.5 mg 1! Cu (Table 4). DISCUSSION The association between reduced growth and increased Cu concentration (Table 1) has also been observed in several other aquatic plants such as Lemna minor L. (Teisseire et al., 1998), Potamogeton pectinatus L.,Vallisneria spiralis L., Hydrilla verticillata (L.f.) Royle (Guilizzoni 1991), and Elodea nuttallii (Planch.) St. John (Van der Werff and Pruyt, 1982). Sarkar and Jana (1986) reported that Azolla pinnata R. Br. growth was significantly reduced following exposure to 2.0 mg |~' Cu, whereas plants at 1.0 mg 1”! exhibited growth similar AL-HAMDANI & BLAIR: INFLUENCE OF COPPER IN SALVINIA MINIMA 53 Tape 4. Cu uptake (ug Cu g™' d. wt) in Salvinia after seven and 14 days of exposure to varying concentrations. LSD (P = 0.05) = Least Significant Difference value for difference between means within a column. Same letter denotes no statistical difference. Lower case letters denote differences between treatments within a day and upper case letters denote differences between days within treatments. N = 12. Bioconcenration factor (BCF) was determined as the ratio of Cu concentration in the plant tissue relative to the concentration in the external media after 14 days of exposure. N = 6. Length of exposure (days) Cu (mg 17’) 7 14 BCF 0.06 (control) 1.50.35 aA 167.33 aA 308.91 a 1.0 1833.54 bA 735.62 abA 1390.08 ab 2.0 2934.90 cA 1545.49 bcB 2132.87 be 2.5 3111.67 cA 2413.75 cB 3111.63 c 3.0 3519.20 cA 4304.44 dB 4304.44 d to the control after 28 days of incubation. A possible explanation of decreasing Salvinia growth with increasing Cu concentrations might be attributed to Cu- induced ethylene production. Matoo et al., (1986) reported that intercellular membrane and organelle disintegration in giant duckweed (Spirodela oligor- rhiza (Kurz) Hegelm) resulted from induced ethylene production by copper. In our study, reduction in Salvinia growth was directly related to a decline in CO, assimilation as a function of Cu increase (Table 3). The negative impact of increasing Cu concentration on CO, assimilation was reported to be due to the inhibitory effect on the electron transport of photosystem II (PS II) (Sarkar and Jana, 1986; Renganathan and Bose, 1989). The decline in PS II was attributed to degradation and leakage of the chloroplast membrane induced by Cu (Ouzounidou et al., 1993). Furthermore, the decline in CO, assimilation might be influenced by the reduction in photosynthetic pigments, which was associated with increasing Cu concentrations (Table 2). A reduction in photosynthetic pigment concentration in Salvinia has been associated with an increase in metal contamination (Gardner and Al-Hamdani, 1997; Nichols et al., 2000). Sarkar and Jana (1986) attributed the reduction in chlorophyll concentration to the influence of Cu on declining chlorophyllase activity. Furthermore, the decline in CO, assimilation and photosynthetic pigment might be related to membrane destruction by lipid peroxidation, which was found to be associated with an increase in Cu accumulation (Halliwell and Gutteridge 1984). Mattoo et al. (1986) reported that free radical formation was induced by an increase in Cu concentration that later reformed into H2O>. As a defense mechanism against increasing free radicals, plants usually respond by increasing catalase activity (Foyer et al., 1994). Catalase activity was found to decline gradually in duckweed as Cu concentrations increased above 0.2 mg 1“? in the nutrient media (Teisseire et al., 1998). Copper uptake by Salvinia was significantly higher with an increase in Cu concentration in the growth media (Table 4). With the exception of those plants grown in 3.0 mg |‘ Cu, the concentration of Cu in Salvinia was significantly higher at the end of seven days of plant growth in comparison to that at day 14. 54 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) In calculating Cu uptake as pg g’‘ plant dry weight, growth should be considered a factor in interpreting the values at each individual treatment. This conclusion equally reflects Cu uptake in plants grown in 3.0 mg I* Cu, where growth was severely affected at the end of seven days with a chlorotic and necrotic appearance that advanced with time. However, the BCF was highest for the plants grown in 3.0 mg |‘ Cu followed by a decreasing order as the Cu concentration decreased in the media. In comparison with other species, Salvinia uptake of Cu was comparable to that obtained with monkey flower (Mimulus guttatus DC.; Tilstone and MacNair, 1997) and iceplant (Mesembry- anthemum crystallinum L.; Thomas et al., 1998) using equivalent Cu concentrations and incubation periods. 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 and duckweed (Reddy and DeBusk, 1985). This can be used as an additional indication that Salvinia can be considered an essential agent in phytoremediation. In conclusion, this study demonstrated that increases in Cu concentration from 1.0 to 3.0 mg | * negatively impacted plant growth, photosynthetic pigments, and CO; assimilation. However, the reduction in plant growth was not severe enough to totally inhibit plant growth even at Cu concentrations of 3.0 mg 1’. Salvinia demonstrated the ability to accumulate significant concentrations of Cu in its tissues. This, in addition to its high growth rate and ease in harvesting, make it a possible candidate for phytoremediation. However, further research should be implemented to investigate the perfor- mance of Salvinia under field conditions. ACKNOWLEDGMENTS The authors wish to acknowledge Dr. James Rayburn for his technical assist d Jacksonville State University for supporting this project. LITERATURE CITED Bervorts, L., L. Panis and R. VERHEYEN. 1994. Trace metal levels in water, sediments, and Chironomus GR. Thurni from different water courses on flanders (Belgium). Chemosphere 29:1591— Bowyer, J. R. and R. C. Leecoop. 1997. In, P. M. Dey and J. B. Harborne (eds.). Plant Biochemistry. Academic Press, Inc., San Diego, Boyp, C. E. 1990. Water quality in ponds for aquaculture. Birmingham Publishing Company, irmingham, Buck SCIENTIFIC. 1996. Puck wince 210 vgp atomic absorption spectrophotometer operating | Buck Scientific, Inc. East Norwalk, CT Cuaney, R. L. 1970. Effect of nickel on iron inetabolian: by soybean. Ph. D. Dissertation, Purdue University, —— Ind. Diss. Abstr. Int. 31:1692-93. Desusk, W. F. and K. R. Reppy. 1987. Growth and nutrient uptake potential of Azolla — Willd. and Salvinia rotundifolia Willd. as a function of temperature. Environ. Exp. B 27:215-221. Doone, R. L., G. E. Macponatp and D. G. SuiLuinc. 1993. Effect of fluoridone on chlorophyll, carotenoid, and anthocyanin content of Hydrilla. J. Aquat. Plant Manag. 31:55-59 Foyer, C. H., M. Letanpais and K. Kunert. 1994. Photooxidative stress in plants. Physiol: Plant. 92:696—717. AL-HAMDANI & BLAIR: INFLUENCE OF COPPER IN SALVINIA MINIMA 55 Garpner, J. L. and S. H. AL-Hampanl. 1997. Interactive effects of aluminum and humic substances on Salvinia. “i Aquat. Plant Manag. 35:30-34. GulizzonI, P. 1991. The role of heavy metals 4 toxic materials in the physiological ecology of submerged macrophytes , nee joni 9. Pe cote B. and J. oe gent ity, oxyg dicals, transition metals, and diseases. ene he Hawr, L. R. and W. E. esha rool Uptake and translocation of zinc by intact plants. Plant Soil Hoac.anp, D. R. and D. I. ARNON. 1938. The isonge culture method for growing plants without soil. Univ. _ Agri. Exp. Stn. Cir. No. 347:1-32. INsKEEP, W. P. and P. R. BLoom. 1985. “nates aprarcan . rn a and b in N, N- inet fomamid and 80 % ree Plant Physiol. 7 Lee, C. L., T. C. Wane, C. H. Hsu and A. A. Cuiou. peg “Heavy pales cE by aquatic plants in aha Hts ane Contam. Tostea!. 61:49 Lewis, M. A. 1993. Freshwater primary produc ash — in, P. Calow (ed.). Handbook of pres vol. 1. Blackwell Scientific, rarted England. Linper, M. C. 1991. Biochemistry of copper. Plenum Press, New A LINGLE, J. C., on TIFFIN and J. C. Brown. mee Iron uptake- pheiall init of soybean as influenced by other pom Plant Physiol. 38:71-76. Marscuner, H. 1995. Mineral Nutrition of Higher Plants. 2"¢ed. Academic Press, New Yor k, NY. Mattoo, A. K., e pee and H. E. Mouine. 1986. Induction by copper ions of ethylene e production in Spirodela oligorhiza: aden, ve a a independent of 1-aminocyclopropane-1- carboxylic acid. J. Plant Physiol. 123:193-2 Mopernrrt, D. K., J. M. Norman, J. T. Davis, J. aaa . . ARKEBAUER, J. M. WELLEs and S. R. Ror 1989. CO, resp a ee neg ia with a field-portable closed-loop ae dl system. Ann. Sci. For. 46:4 Moorg, J. W. 1991. = contaminant in surface water. Springer-Verlag, New York, NY Morettl, A. and G. S. GIGLIANO. 8. Influence of light . ide growth and nitrogenase activity on aE Nc azo ia git Fertil. Soils 6:131- Nauman, C. E. 1993. Salviniaceae. Pp. 336-337, in Flora ae America Editorial Commi ttee. Flora of Nort Nico s, P. B., J. D. CoucH and S. H. AL-Hampanl. 2000. serge cs a responses of Salvinia minima to different chromium concentration. Aquat. B Oucutn, E. J., E. HERNANDEZ and I. Ramos. 2002. The effect of nk nae light condition and the pH value on the capacity of pedi minima Baker for removing cadmium, lead and chromium. as Biotechnol. 22:121-131. Ouzountbou, G., R. LANNoyYE and S. spanner 1993. Photoacoustic measurements of photosyn- thetic activities in intact leaves under copper stress. Plant Sci. 89:221-226. RENGANATHAN, M. and S. Bose. 9, Inhibition of primary photochemistry of photosystem II by Copper IN IsoLATED PEA pans PLASTS. BIOCHIMICA ET BiopHysica Acta. 9 SaLomons, W., U. ForstNer and P. Maper. 1995. Heavy metals: Problems and eee Spring Sarkar, A. and S, JANA. ogg Heavy metal pollutant tolerance of Azolla pinnata. Water, Air, and Soil Pollut. 27:15— SPACIE, A., L. S. MccarTy aa G. M. Ranp. 1995. Bioaccumulation and bioavailability in multiphase systems. P. 493 in Fundamentals of Aquatic Toxicology. G. M. Rand (ed.). Taylor and Francis, Pare ps STEEL, R. G. D. and J. D. Torr. 1980. Principles and procedures of statistics: a biometrical its To: McGraw-Hill, New York, NY. TEISSEIRE, H., M. CouDERCHET and G. VERNET. 1998. Toxic responses and catalase activity of Lemna minor L. exposed to folpet, copper, and their combination. Ecotox. Env. Saf. 40:194-200. 56 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 1 (2004) Tuomas, J. C., F. K. Matick, C. ENpReszL, E. C. Davies and K. S. Murray. 1998. Distinct responses to copper stress in the halophyte MeceutinaitNenans crystallinum. Physiol. Plant. 102:360- ie G H. and M. R. Macnar. 1997. Th of selection for su tolerance on the uptake and accumulation of copper in Mimulus ee uttatus. Ann. Bot. 80:747-751. VaN Der Werrr, M. and M. J. Pruyt. 1982. Long-term effects of heavy eens on aquatic plants. Chemosphere 11:727-—739. VaviLin, D, V., V. A. Potynov, D. N. Matorin and P. S. VENEDIKTOV. 1995. Sublethal concentrations of copper stimulate photosystem II photoinhibition in Chlorella pyrenoidosa. J. Plant Physiol. 146:609-614 Wa ttace, A., and P. C. DeKock. 1966. Translocation of iron in tobacco, sunflower, and bush bean plants. Pp. 3-9, in A. Wallace (ed.). Current Topics in Plant Nutrition. Edward Bros., Ann Arbor. Li NT INFORMATION FOR AUTHOR Authors are encouraged to submit manuscripts pertinent to pteridology for pub- lication in the American Fern Journal. Manuscripts should be sent to the Editor. 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 acceptability of manuscripts nor the date of publication Authors should adhere to the following ‘euidelines: manuscripts not so prepared may be returned for revision prior to review. 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Van den heede, Santiago Pajarén, Emilia Pangua, and Ronald L. L. Viane Shorter Notes Nomenclatural Corrections in Adiantum Jefferson Prado 81 112 The American Fern Society Council for 2004 TOM RANKER, University Museum, Campus Box 265, University of Colorado, Boulder, CO 80309-0265. President DAVID S. CONTANT, Dept. of Natural Sciences, Lyndon State College, Lyndonville, VT 05851. ioe W. CARL TAYLOR, 800 W. Wells St., Milwaukee Public Museum, Milwaukee, WI 53233-14 ecretary JAMES D. CAPONETTI, Dept. of Botany, University of Tennessee, Knoxville, TN 37916-1110. GEORGE YATSKIEVYCH, Missouri Botanical Garden, P. O. Box 299, St. Louis, MO 63166-0299. embership Secretary JAMES D. MONTGOMERY, Ecology III, 804 Salem Blvd., Berwick, PA 18603-9801. Curator of Publications R. JAMES HICKEY, Botany Dept., Miami University, Oxford, OH 45056. Journal Editor R. JAMES HICKEY, Botany Dept., Miami University, Oxford, OH 45056. 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Please contact the Back Issues Hated for prices and availability. ription Society Maaeiikin USA, Canada, Mexico (includes Journal and Fiddlehead Forum) $25 Society Membership — “All other er countries (includes Journal and Fiddlehead Forum) $32 Society | embershi 2 for outside USA, Canada, Mexico) Regular Membership — USA, Canada, Mexico (includes Fiddleh $12 — Membership — All other Fiddlehead Forum) $19 embership — $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. sido uate sy 166-0299. American Fern Journal 94(2):57—69 (2004) Bark Spore Bank of Ferns in a Gallery Forest of the Ecological Station of Panga, Uberlandia-MGdtpazid TaniCAL Mart A. RANAL 4 Instituto de Biologia, Universidade Federal de Uberlandia, AUG ) 5 200 Caixa Postal 593, 38400-902, Uberlandia, MG, Brasil GARDEN LIBRARY AsstTract.—Information about fern spore banks is restricted to the soil systems. As the dispersion of spores occurs by means of air, it is possible to have viable spores on tree bark. Thus, it is desiccation than the soil, the spores can survive for any length of time, forming transient or persistent spore banks. Samples of bark were collected from ten angiosperm trees in March 1997 and from fifteen trees in February and September 1998. The samples collected in March 1997 contained from 0.05 to 7.19 gametophytes cm “ of cultured bark, those of February 1998 from 0.11 to 4.22 gametophytes cm *, and in September 1998 from 0.32 to 5.0 gametophytes per square cm. Although the cerrado region is characterized by climatic seasonality, this seasonality was not observed in relation to number of viable spores on barks. As a consequence of the casual spore epiphytic (Phlebodium areolatum (Humb. & Bonpl. ex Willd.) J. Sm.) and the others terrestrial species. Thelypteris was the most frequent genus in the analyzed samples. The results obtained show the potential for these substrates to retain viable spores that can participate in the regeneration process and population d i f the pteridophyte fl Moreover, th on £ viable spores of terrestrial species on tree bark does not answer an important question—why do terrestrial species not establish themselves on trees? Information about fern spore banks is restricted to the soil (Carroll and Ashton, 1965; Wee, 1974; Strickler and Edgerton, 1976; Pérez-Garcia et al., 1982; During and ter Horst, 1983; During et al., 1987; Leck and Simpson, 1987; Hamilton, 1988; Lindsay and Dyer, 1990; Milberg, 1991; Dyer and Lindsay, 1992; Milberg and Anderson, 1994; Penrod and McCormick, 1996; Raffaele, 1996; Schneller and Holderegger, 1996; Simabukuro et al., 1998, 1999; Ranal, 2003). As the dispersion of spores occurs by means of the air, it is possible that viable spores present on the bark of trees could germinate under appropriate conditions. Thus, it is important to know if on this kind of substrate, which is thinner and apparently more susceptible to desiccation than soil, the spores can survive long enough to form a transient or persistent bank. These spores can participate in the population dynamics, particularly of epiphyte species. The role of these fern spores could be amplified after the fall of trees, if spores of terrestrial species could survive in this kind of substrate. In vertical position the spore reception is maximized on the trunks. On the other hand, in horizontal positions (dead trees), wind action and self-defense of trees decreases. As a consequence, water retention and decomposition activities increase, making the germination process on this new substrate easier. In this sense, it will be possible to consider that this bark spore bank has the same role recognized for soil spore bank, that is, this bank could take an important role in 58 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) propagation and in preservation of fern species as pointed out for soil spore bank by Lindsay et al. (1992), Dyer (1994), and Dyer and Lindsay (1996); in sexual process and in genetic variability (Milberg, 1991); and in regeneration process of forest gaps. In this context, the aim of this study was to investigate the existence of a bark spore bank on trunks of angiosperm trees in the gallery forest of the Ecological Station of Panga, Uberlandia—MG, Brazil. This being the case, the purpose was to characterize this bank in relation to quantity of viable spores and fern species composition. MATERIALS AND METHODS Bark was collected from ten angiosperm trees in March 1997, and from fifteen in February and September 1998. These trees are growing in the gallery forest of the Ecological Station of Panga, Uberlandia, Minas Gerais, Brazil, situated at 19°09'20’—19°11'10” S, 48°23'20’—48°24'35” W, at an elevation of approximately 800 m. This Station has 409.5 ha occupied by cerrado sensu Jato (Schiavini and Aratijo, 1989; Ratter, 1992). The region is characterized by an Aw climatic pattern (K6ppen, 1948) with a wet and hot season from October to March and a dry and cold season from April to September (Ranal, 2003). Random sampling was used to mark the trees for the bark collection. All trees were adult individuals, with more than 15 cm in circumference at 1.30 m height and with sufficient bark to extract by scratching with a knife, without reaching live tissues. It means that dead bark was extracted (outer bark or rhytidome according to Esau, 1977), a perennial rhytidome with slow detachment. Bark samples were collected at about 1.30 m height around the trunks. As in 1998 the two collections were done on the same trees, the bark extraction was done at about 1.30 m height in February and at about 1.50 m in September. After collection, each bark sample was placed in a plastic bag which was labelled and immediately closed to prevent contamination. In the laboratory bark samples were manually mixed and converted into small pieces and powder, inside the bags. Each bark sample was divided into sub-samples and spread over sterile sand in quadrangular, transparent, covered plastic boxes of 50 cm® (experimental units), moistened with nystatin suspension (10,000 units nystatin per mL in DPBS—Dulbecco’s Phosphate Buffered Saline; 1 mL per 100 mL of distilled water) and later, if necessary, with distilled water. Near the end of the experiments, when young sporophytes presented the first signals of chlorosis, the cultures were moistened with nutrient solution (Meyer et al., 1963). Each experimental unit received 15 g of sterile sand and 1 g of bark. This quantity of bark formed a layer of approximately 5 mm thickness. Samples were maintained at 20.9-23.2°C, 35.77 + 6.71 nmol m~ s? PAR (mean + standard deviation) for the March 1997 collection, at 21.4—24.4°C, 35.77 + 6.71 umol m~’* s PAR for the February 1998 collection, and at 21.8—22.9°C, 30.84 + 6.09 pmol m™ s* PAR for the September 1998 collection. All samples were subjected to continuous white fluorescent light. Radiation measurements were made using a LI-COR LI-250 light meter and a LI-190SA quantum sensor. RANAL: BARK SPORE BANK IN BRAZIL 59 Sterilized soil samples (10 replicates) were maintained under the same manipulation conditions to assess the level of contamination by foreign spores. The superficial area of cultured barks was used to calculate the number of ga- metophytes and sporophytes formed per square centimeter. As bryophytes were the first colonizer of the barks, fern gametophytes were counted 2-3 months after each collection, when they reached adult form, becoming easily visible. The number of gametophytes formed was the criterion used to evaluate viable spores on the bark samples. Sporophytes were counted when cultures were two (February collection) or three months old (March and September collections). The criterion to count sporophytes was the presence of a perceptible crozier when viewed under stereomicroscope. At the end of the experiments young sporophytes were transplanted to bags containing soil and were maintained under greenhouse conditions until the production of fertile leaves when they were collected. The sporophytes collected were prepared and deposited at HUFU and SP. The experimental units were randomly distributed in laboratory conditions, with two sub-samples per tree for March 1997 collection (20 cells) and four subsamples per tree for February and September 1998 collection (60 cells for each collection date). The number of gametophytes and sporophytes formed per square centimeter of cultured bark, as well as the percentage of gametophytes forming sporophytes were submitted to the Shapiro-Wilk (normality of populations) and Bartlett or Cochran tests (homogeneity between variances). If the original data exhibited normality and homogeneity, they were submitted to analysis of variance and Tukey test. If the original and transformed data showed non-normality and/or heteroscedasticity, non-para- metric statistical tests were used (Kruskal-Wallis and Wilcoxon-Mann- Whitney tests). Comparisons between the bark spore bank of the wet and dry seasons were carried out using the Mann-Whitney test. Pearson correlations were made to detect associations between number of gametophytes formed on cultured barks and tree characteristics (height, circumference, N, P, K, Ca, Mg, S, Fe, B, Cu, Mn, and Zn content). Bark samples collected were chemically analyzed in the Laboratory of Leaf Analysis of the Federal University of Uberlandia, according to Miyazawa et al. (1999). RESULTS All analyzed trees presented viable spores in their outer bark (Tables 1-3). The size of the bark spore bank, evaluated in relation to gametophytes formed, varied from 0.05 to 7.19 gametophytes cm ~ of cultured bark. There is no seasonality in the bark spore bank (Table 4). Half of the analyzed trees showed no differences between February (wet season) and September (dry season) collections; three showed increase in number of viable spores and four showed decrease in number of viable spores at the end of the dry season. There is no association between angiosperm species, tree size or chemical composition of the bark and the number of viable spores on barks (Tables 1-3, 5, 6). The same 60 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) TaBLE 1. Gametophytes and sporophytes formed per square centimeter of cultured barks of angiosperms occurring in a gallery forest of Ecological Station of Panga, Uberlandia—MG, Brazil and their reproductive success measured by percentage of gametophytes forming sporophytes (mean + standard deviation). March 1997 collection. Angiosperm Gametophytes Sporophytes % Species Family cm * cm * sporophytes Psidium rufum Matt. ex Dc. Myrtaceae 7.19+ 0.49 a 2.68 + 0.38 a 37.24 + 2.76 ab tree in decomposition a 215 + 0.30 b 0.82 + 0.43 b 39.82 + 25.56 ab Luehea divaricata Mart. Tiliaceae 1.93 + 0.41 be 0.37 +0.23b 18.16 + 8.00 ab tree in decomposition — 1.56 + 0.47 bed 0.33 + 0.20b 20.19 + 6.80 ab Unidentified sp." as 0.89 + 0.87 bed 0.00+0.00b 0.00 + 0.00b Cupania vernalis Cambess. Sapindaceae 0.59 + 0.22 bed 0.00+0.00b 0.00 + 0.00b Chrysophyllum marginatum Radlk. Sapotaceae 0.48 + 0.005cd 0.05 +0.07b 10.00 + 14.14b undentified sp.' _— 0.49+0.39cd 0.29+0.26b 56.25 + 8.84a Matayba guianensis Aubl. Sapindaceae 0.09 +0.0d 0.00 + 0.00b 0.00 + 0,00 b Terminalia brasiliensis Eichl. 1d) + I+ Combretaceae 0.05 + 0.07 d 0.00 + 0.00b 0.00 + 0.00 b Ww 0.9813 0.9433 0.9301 Cochran 0.4473 0.3718 0.6227 9; 10 53.29** 27.55** 7.78** W: Shapiro-Wilk test (« = 0.05); boldfaced values indicate normality of populations (P > 0.05). Boldface values for Cochran test indicate homogeneity between variances. F: value of Snedecor’s distribution, including the degrees of freedom; ** P < 0.01. Means followed by the same letter in each column are not significantly different based on the Tukey test (a = 0.05). ' Tree with Microgramma persicariifolia. angiosperm species presented high or low number of viable spores, trees with different dimensions presented similar number of viable spores without any significant correlation between tree size and bark bank size, and weak tendency related to chemical composition could be observed. Moderate to substantial negative correlations (Table 6), according to the criterion adopted by Miller (1994), were detected for nitrogen (February collection), magnesium, and copper content (September collection). Sporophyte production ranged from zero to 2.68 sporophytes cm™~ of cultured bark and the reproductive success (percentage of gametophytes forming sporophytes) from zero to 62.10 % (Tables 1-3). Host trees of viable fern spores are presented on table 7. Ten fern species were recognized in barks collected in February 1998 and 15 species in barks collected in September 1998. Each analyzed tree presented from two to ten fern species in their barks. Pityrogramma calomelanos (L.) Link var. calomelanos and Thelypteris opposita (Vahl) Ching were broadly distributed, occurring in 13 trees, from the 25 analyzed; Phlebodium areolatum (Humb. & Bonpl. ex Willd.) J. Sm., Pteris vittata L., Thelypteris burkartii Abbiatti, and T. mosenii (C. Chr.) C.F. Reed appeared only in one tree. Phlebodium areolatum is epiphyte and the others are terrestrial species. Microgramma persicariifolia (Schrad.) Presl was found growing as an epiphyte in the forest, but no RANAL: BARK SPORE BANK IN BRAZIL 61 TABLE 2. Gametophytes and sporophytes formed per square centimeter of cultured barks of angiosperms occurring in a gallery forest of Ecological Station of Panga, by percentage of gametophytes forming sporophytes and their reproductive success measured (mean + standard deviation). February 1998 collection. Uberlandia—MG, Brazil Angiosperm Gametophytes §Sporophytes % pecies Family cm” on" sporophytes Copaifera langsdorffii Desf.” Caesalpiniaceae 4.22+0.42a 2.29+0.30a 54.22+2.51a Eugenia ligustrina Miq. yrtaceae 2.80 + 0.42ab 1.22+0.23b 43.78 + 6.54 ab Endlicheria paniculata (Spreng.) Macbride® — Lauraceae 2.68 +0.99ab 1.59+0.70ab 58.67 + 10.36a Eugenia ligustrina 2.26 + 0.54 abc 0.91 + 0.32 bc 40.86 + 12.94 ab Aspidosperma a rt iat uell. Arg. Apocynaceae 2.06 + 0.36 abc 0.514 0.14cd 24.70 + 4.41 ab Pest guianensis Aubl. Anacardiaceae 1.94 + 0.54 abc 0.91 +0.20bc 49.07 + 15.61 ab Coussarea hydrangeae- folia Benth. & Hook Rubiaceae 1.92 + 0.59 abc 0.32 + 0.14 def 16.76 + 6.59 ab Eugenia florid rtaceae 1.55 + 0.57 abc 0.28 + 0.11 def 19.64 + 8.27 ab Inga affinis Mimosaceae 1.49 + 0.30 abc 0.45 + 0.16 cde 30.27 + 6.32 ab Luehea divaricata Mart. _ Tiliaceae 1.32 + 0.22 abe 0.56+0.09cd 43.14 + 7.94 ab dead tr —_ 0.83 + 0.39 bc 0.02 + 0.04 f 1.56 = 3:12 6 Tapirira guianensis Anacardiaceae 0.75 + 0.29bce 0.32 + 0.22 def 44.17 + 24.10 ab Aspidosperma cylindrocarpum Apocynaceae 0.50 + 0.08 bc 0.15 + 0.11 def 29.64 + 21.61 ab rira guianensis Anacardiaceae 0.17 +0.10c 0.02 + 0.04f 25.00 + 50.0 ab Luehea divaricata Tiliaceae 0.11+0.11c 006+ 0.08ef 41.67 + 50.0 ab WwW 0.9435 0.9728 0.8774 Bartlett 24.1624 Kie _ 31.42** _ H 52.11** _ 30.32** W: Shapiro-Wilk test (a = Boldface value for Bartlett test indicates homogeneity between variance om P< Wilcoxon-Mann-Whitney or Tukey test (# = 0.05). * transformation for the adjustment to normality an $s; numbers and with Microgramma persicariifolia. are original number: paae ‘Wallis per not sgnttianll different based on the 0.05); boldfaced value indicates normality of pace (P > 0.05). value of Snedecor’s ** P< * Bark collected of the horizontal part of the stem gametophyte of this species was found in the analyzed barks. Thelypteris was ) the most frequent genus among the analyzed samples (Table 8). DISCUSSION The bark spore bank of the analyzed trees is smaller than the soil spore bank of the middle and edge of the same gallery forest, at the first centimeters of the soil column (2—7 cm depth), in the wet season, and similar to that soil spore bank in deeper soil column (15-22 cm depth). Soil samples of the gallery forest of the Ecological Station of Panga could reach 29.52 gametophytes cm of 62 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) TaBLE 3. Gametophytes and sporophytes formed per square centimeter of cultured barks of angiosperms occurring in a gallery forest of Ecological Station of Panga, Uberlandia—MG, Brazil and their reproductive success measured by percentage of gametophytes forming sporophytes (mean + standard deviation). September 1998 collection. Gametophytes Sporophytes % Angiosperm Species Family cm * em~* sporophytes Copaifera langsdorffii Desf.” Caesalpiniaceae 46.81 + 8.80a 2.526 O32:ab > 146 0.22 b Eugenia ligustrina Miq. Myrtaceae 0.83 £0.36be 0.23+0.10d 29:38 +:15.60a Endlicheria paniculata (Spreng.) Macbride) Lauraceae a%.12-= 7:82) 8 5.00 + 0.73 a 1:86 = 0.53 a Eugenia ama Myrtaceae 0.73 + 0.39 be 0.26 +0.19d 35.02 + 13.54 a Aspidospe Seliiiesetaptha Muell. Arg. Apocynaceae 30.15 + 17.84a 1.54 + 0.35 abe 0.45 + 0.26 cd Tapirira guianensis Aubl. Anacardiaceae 1.75 + 0.50 abc 1.02 + 0.23 bc 62.10 + 25.65 a Coussarea Anse th. & Hook. f. Ben Rubiaceae 14.45 + 14.08 a 0.93 + 0.28be 0.11 + 0.09d Eugenia apn DC. Myrtaceae 1.30 + 0.05 abc 0.70 + 0.28 bcd 54.06 + 21.82 a Inga affinis DC. imosaceae 1.95 + 0.43 abc 0.24 + 0.15 d 14.09 + 11.86 a Luehea varia Mart. Tiliaceae 2.47+049ab 1.10+0.56bc 46.54 + 25.60a dead tr - 2.28 + 1.45 abc 0.70 + 0.24 bcd 34.78 + 13.26 a hapless guianensis Anacardiaceae 0.88 + 0.12 bce 0.52 + 0.24 bcd 58.61 + 20.19 a Aspidosperma cylindrocarpum Apocynaceae 56.75 + 15.61 a 1.84 + 0.36 abc 1.00 + 0.13 be Tapirira guianensis oa apse 0.432 0.290 O13 =O011d 30.42 + 2750-4 Luehea divaricata Tiliac 0:32 > 019% 0.17 + 0.12d 47.5 + 41.13 a WwW 0.9078 0.9708 0.9837 Bartlett — 23.9762 14.8281 Fra; 45 14.1975** 2.1431* H 51.6850** —_ a W: Shapiro-Wilk test (« = 0.05); boldfaced values indicate normality of populations (P > 0.05). cultured soil, in the wet season, when collected at 2-4 cm depth, and ranged from 0.13 to 6.84 gametophytes cm ~ from 15 to 22 cm depth (Ranal, 2003). Dyer and Lindsay (1992) registered more than 30 gametophytes cm * from surface to 2.5 cm depth of soil collected in North Carolina and 0.46 gametophytes cm” at 20.0—22.5 cm Similar results in relation to reproductive success were obtained for soil spore bank studies (0.76 to 63.33 % gametophytes producing sporophytes) of the same gallery forest (Ranal, 2003) Periodic observations indicate that for some species of the Ecological Station of Panga, production of new leaves occurs in October-November, at the beginning of the rainy season, and the production of fertile leaves occurs in RANAL: BARK SPORE BANK IN BRAZIL 63 LE 4. Simple comparisons for gametophytes formed in bark samples collected in February and maar 1998, in the gallery forest, Ecological Station of Panga, Uberlandia-MG. The mean values and the dispersion measurements are included on tables 2 and 3 Angiosperm Species U value P value Copaifera Pee Desf. 16.0 0.0286 Eugenia ligus 16.0 0.0286 Endlicheria paniculata (Spreng.) Macbride 16.0 0.0286 Eugenia ligustrin 16.0 0.0286 Aspidosperma uitiocmad Muell. Arg. 14.0 0.1140 Tapirira guianensis Aubl. 8.5 0.8860 Coussarea i at Benth. & Hook. f. 16.0 0.0286 Eugenia florida DC 8.0 1.0000 Inga affinis DC. 13.0 0.2000 Luehea divaricata Mart. 16.0 0.0286 dead tree 15.0 0.0571 Tapirira guianen 11.0 0.4860 Aspidosperma eu 16.0 0.0286 Tapirira guianensis 14.0 0.1140 Luehea divaricata 12.0 0.3430 U: statistic of ss Sener’ “Whitney eats PR: prokability’ to accept or reject the null hypothesis. P > 0.05 ignificantly different. P < 0.05 means that the two medians are significantly different. December—January. Spore dispersal occurs from December for precocious leaves to March—April for late leaves, depending on the annual rainfall distribution. This seasonality in spore production was not observed in the bark spore bank, but was detected for soil spore bank in the first centimeters of soil TasLe 5. Bark chemical composition of angiosperm species occurring in the gallery forest of Ecological Station of Panga, Uberlandia—MG, Brazil. g kg" mg Kg" Angiosperm Species NP "K) 6Ga> Me US Fe B Cu Mn Zn Aspidospe inocu Muell: Are. 17.2 12 1.5 6:6.1:2 1.9 17508:0 15.0 °18.0 252.0 23:0 pidosperma sae aged 19.0 1.5 1.5 10.3 1.3 2.0 15030.0 26.0 17.0 203.0 21.0 rat "angsdorffii D esf. 11.4 0.9 15 45 1.0 1.3 11503.0 23.0 13.0 350.0 20.0 Coussarea hydran oti Benth. & Hook. f. 13:9 09 0.5 47 O83 Tt. 6015.0 17:0 100 703.0 4150 Endlicheria aenbits (Spreng.) Macbride 1541.5 20° 68 1 2:1 270000 17:0 25.0 1051.0 61.0 Eugenia florida DC. 12:1. 0.8 1.5 26.8 1:2 2.4 16005.0 21.0' 11.0 293:0 42.0 Eugenia ligustrina Miq. 13:5 0.7 10 249° 1.6 1:3. 7012.0 18.0 12.0 125.0 22.0 Eugenia ligustrina 12.1 0.7 1.0 20.8 1.2 1.0 6511.0 13.0 10.0 133.0 22.0 Inga affinis DC. 16.3 1.7 7.0 10.6 1.7 2.1 ‘2000.0 21.0 25.0 675.0 -40.0 Luehea s oieane Mart. 13.9 0.8 1.0 48.3 3.6 1.4 10000.0 11.0 17.0 1105.0 240.0 Luehea divaricata 15.7 0.9 1.0 43.3 2.3 6.9 16550.0 18:0 25.0 551.0 115.0 are peace Aubl. 13.2 0.8 1.0 43.9 1.9 1.0 5750.0 28.0 15.0 425.0 20.0 Tapirira guianensis 15.4 1.2 1.5 25.2: 1.9 2.0 2250.0 23.0 22.0 675.0 55.0 Tapirira guianensis 13.9 0.9 1.0 26.8 2.6 1.0 5020.0 18.0 15.0 148.0 23.0 dead tree 11.7 0.8 3.0 19.3 1.1 0.9 7750.0 22.0 8.0 177.0 47.0 64 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) TaBLE 6. Coefficients of the linear correlation (r) among tree characteristics and gametophytes per ec i + £ quare r of cultured bark collected in February and September 1998 in the gallery forest of the Ecological Station of Panga, Uberlandia—MG, Brazil. Tree characteristics r values’ P values r values” P values Height (m) —0.1014 0.3596 0.0932 0.3706 Circumference (cm) 0.1580 0.2869 0.0728 0.3983 N —0.5003 0.0288 —0.3538 0.0979 P —0.1228 0.3314 —0.1615 0.2827 K 0.3403 0.1073 0.0634 0.4112 Ca —0.2815 0.1548 —0.0691 0.4033 Mg —0.3348 OATS —0.4716 0.0380 S —0.0176 0.4752 —0.0736 0.3971 Fe O.1775 0.2634 0.0889 0.3763 B —0.0678 0.4051 0.2245 0.2106 Cu —0.3978 0.0710 —0.4591 0.0426 Mn 0.0298 0.4581 —0.2133 0.2227 Zn 0.0817 0.3861 —0.2651 0.1698 * linear correlation for gametophytes per square centimeter of cultured bark collected in February 1998. * linear correlation for gametophytes per square centimeter of cultured bark collected in September 1998. P > 0.05 means that null hypothesis was accepted and r was not considered as significantly different from zero by ‘‘Student’s” t test. P < 0.05 means that null hypothesis was rejected and r was considered as significantly different from zero by “Student’s” t test. column, with high quantity of viable spores occurring at the end of the wet season and low quantity at the end of the dry season (Ranal, 2003). Although Copaifera langsdorffii Desf. contains alkaloids and terpenoids in its bark (Souza and Silva, 2001), these substances apparently did not influenced the spore germination and gametophyte development of Pity- rogramima calomelanos var. calomelanos, Pteridium aquilinum (L.) Kuhn var. arachnoideum (Kaulf.) Brade, Thelypteris burkartii, T. conspersa (Schrad.) A. Sm., T. dentata (Forssk.) E. St. John, T. hispidula (Decne) C. F. Reed, T. opposita (Vahl) Ching, and T. patens (Sw.) Small because viable spores of these species were maintained on its bark, with normal development until sporophyte production. Alkaloids and terpenoids are related to allelopathic mechanisms that inhibit the germination process (Inderjit and Dakshini, 1995). Five of the species of the bark spore bank did not occur in soil samples of the allery forest of the Panga Stream (Phlebodium areolatum, Pityrogramma trifoliata (L.) R.M. Tryon, Pteridium aquilinum var. arachnoideum, Pteris vittata, and Thelypteris burkartii). On the other hand, three of the 13 species registered by Ranal (2003) in the soil of the gallery forest of Panga did not occur in bark cultures (Lygodium venustum Sw., Blechnum brasiliense Desv., and Blechnum occidentale L.). Thus, there were 10 common species in the soil and bark spore bank. The interpretation of these data is limited because these are the first results about bark spore banks and few angiosperm species were analyzed. Moreover, there is no assurance that all fern spores present on the barks could germinate and form sporophytes under the experimental conditions used in this study. Several environmental factors such as wind currents, rainfall, gravity, and RANAL: BARK SPORE BANK IN BRAZIL 65 :& j | f, kK 1 1 5 Oe | i W ra = fal. n ] . 1 . that t t t ig Station 7. Sp of Panga, Uberlandia, MG. Fern species Host tree Macrothelypteris torresiana Coussarea hydrangeaefolia Benth. & Hook. f. (Gaud.) Ching dead tree Eugenia florida DC. Inga affinis Luehea are Mart. Tapirira guianensis Aubl. Phlebodium areolat Tapirira guianensis (Humb. & a hoadl.G ex aan )J. Sm. Pityrogramma calomelanos (L.) Aspidosperma cylindrocarpum Muell. Arg. ink var. calomelanos ie langsdorffii Desf. Coussarea hydrangeaefolia rae tree Endlicheria oe (Spreng.) Macbride Eugenia flori Eugenia ligustrina Mig. Inga affinis Luehea divaricata Tapirira guianensis dead tree Endlicheria ee Eugenia aie Inga a Luehea ae ricata Tapirira guianensis Pityrogramma trifoliata (L.) Aspidosperma cylindrocarpum R. M. Tryon Pteris vittata L. Tapirira aa Pteridium aquilinum (L.) Copaifera langsdorffii Kuhn var. arachnoideum Endlicheria nice on (Kaulf.) Brade Eugenia er Inga a Tapirira joy nes Thelypteris burkartii Abbiatti Copaifera langsdorffii Thelypteris conspersa (Schrad.) Aspidosperma cylindrocarpum A. R. Sm. Copaifera langsdorffii Endlicheria oT Eugenia = id Inga a Luehea an Tapirira guianensis Thelypteris dentata (Forssk.) Pentti langsdorffii E. St. John creche paniculata Eugenia flori Eugenia ligustrina Tapirira guianensis Thelypteris hispidula Aspidosperma cylindrocarpum Decne) C.F. Reed sit a langsdorffii dea 66 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) TABLE 7. Continued. Fern species Host tree Endlicheria paniculata Eugenia ligustrina Inga affini Luehea divaricata Tapirira guianensis Thelypteris interrupta (Willd.) Iwats. Aspidosperma cylindrocarpum Eugenia florida Eugenia ligustrina Tapirira guianensis Thelypteris mosenii (C. Chr.) C.F. Reed dead tree Thelypteris opposita (Vahl) Ching Aspidosperma cylindrocarpum Copaifera langsdorffii Coussarea hydrangeaefolia dead tree Endlicheria paniculata Eugenia florida Eugenia ligustrina Inga affinis Luehea divaricata Tapirira guianensis Thelypteris patens (Sw.) Small Aspidosperma cylindrocarpum Copaifera langsdorffii ueh ivaricata Thelypteris sp. Coussarea hydrangeaefolia Eugenia florida Eugenia ligustrina Luehea divaricata Tapirira guianensis temperature can participate in the spore dispersion (Page, 1979) and several factors act in the trees, modifying their barks and preparing them to shelter epiphytes (Barkman, 1958). Among them are light affecting the temperature and evaporation, rainfall, atmospheric humidity, and characteristics of the trees such as water and vapour capacity of bark, colour influencing the warmth capacity, hardness, presence of fissures, acidity and chemical composition of the bark. As no association between the size of this spore bank and angiosperm species, tree dimensions or chemical composition of the bark was observed, perhaps the wealth or poverty of these trees in relation to the number of viable spores depends on the dispersal spore processes as an important factor. As a consequence of the casual spore dispersion pattern, the bark spore bank has a random distribution among the trunks. The influence of the physical characteristics of the outer bark needs to be studied for a complete un- derstanding of this bank. The data obtained in this study show that barks retain viable spores that can participate in the regeneration process and population dynamics of the environment. Probably the spores with greater longevity could participate in TasLe 8. Sporophyte frequency (mean percentage) registered in barks extracted from angiosperms occurring in the gallery forest of the Ecological Station of Panga. Values were calculated in relation to the total number of sporophytes formed in the replications. Data obtained two (February collection) or three months (March and September collections) after the collection of barks Fern species Thelypteris sp. Pteridium aquilinum Pityrogramma calomelanos Pityrogramma trifoliata Pteris vittata Angiosperm Species Feb98 Sep98 Feb98 Sep98 Feb98 Sep98 Feb98 Sep98 Feb98 Sep98 Copaifera arene oe Desf. 43.14 88.93 2.56 5.42 2.56 5.95 0.78 0.00 0.00 0.00 Eugenia ligustrina M 59.79 66.67 0.00 25.00 14.58 8.33 3.33 0.00 0.00 0.00 Endlicheria pepe oi (Spreng.) Macbride 73.28 86.60 2.50 1.47 4.50 9.31 0.00 2.60 0.00 0.00 Eugenia ligustrina 84.25 91.67 Le 0.00 4.70 8.33 0.00 0.00 0.00 0.00 Aspidosperma cylindrocarpum Muell. Arg. 52.50 94.44 0.00 0.00 10:71 5.56 5.00 0.00 0.00 0.00 Tapirira guianensis Aubl 77,38 80.32 0.00 1.92 2.78 14.98 0.00 0.00 0.00 0.00 Coussarea a enth. & H of 29.16 75.00 0.00 0.00 25.00 0.00 4.17 0.00 0.00 0.00 Eugenia frida DC. 35.00 93.06 0.00 0.00 6.33 6.94 0.00 0.00 0.00 0.00 Inga a 81.2 45.00 0.00 0.00 0.00 17.50 6.25 0.00 0.00 0.00 Luehea ae Mart. 100.00 = 98.75 0.00 0.00 0.00 1.25 0.00 0.00 0.00 0.00 dead tree 0.00 94,44 0.00 0.00 0.00 5.56 0.00 0.00 0.00 0.00 Tapirira guianensis 50.00 88.75 0.00 0.00 8.34 11.25 0.00 0.00 0.00 0.00 Aspidosperma cylindrocarpum 66.67 —- 95.58 0.00 0.00 0.00 4.42 0.00 0.00 0.00 0.00 Tapirira guianensis 0.0 66.67 0,00 0.00 0.00 0.00 0.00 0.00 0.00 8.33 Luehea divaricata 25.00 75,00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 UZVad NI ANVA AWOdS WAVE “TVNVU 68 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) the recomposition processes mentioned above when the tree falls on the soil. In this sense, the participation of the spores included in the soil in these processes can be higher than that included in the barks. The greater quantity of spores on the soil surface and the infrequent fall of trees in the studied forest make the establishment of gametophytes and sporophytes arising from the soil spore bank faster than those originating from bark spore bank. The existence of viable spores of terrestrial species on tree bark does not answer an important question—Why do terrestrial species not establish themselves on trees? ACKNOWLEDGMENTS Statistical information and suggestions were provided by Dr. Denise G. Santana and Paulo angearo Peres. The identifications of the species were done by Dr. Jefferson Prado and the confirmations of some Thelypteris species by Dr. Alan R. Smith and Dr. M. Monica Ponce. The field work was done with the help of Mr. Hélio Pereira. Review of the English text was done by Mr. John David Bagnall. Important constructive criticism was done by the anonymous referees and by Dr. James Hickey. The author registers her sincere thanks. LITERATURE CITED BARKMAN, J. J. 1958. Phytosociology and ecology of cryptogamic Ps te including a taxonomic survey and Pein ih of ee vegetation units in Europe. V. orcum & Comp. N.V., Assen. CarroLt, E. J. and D. H. AsHTon. 1965. Seed storage in soils of ape Victorian plant communities. Victorian ctl 82:102—110. Durinc, H. J., M. Brucugs, R. M. Cros and F. Lioret. 1987. The diaspore bank of bryophytes and ferns in ie soil in some contrasting habitats around Barcelona, Spain. Lindbergia 13: Durinc, H. J. and B. TER Horst. 1983. The diaspore bank of bryophytes and ferns in chalk grassland. Dyer, A. F. 1994. Natural nae spore banks: can they be used to retrieve lost ferns? Biodiversity and Conservation 3:16 Dyer, A. F. and S. fin 1 992. Soil spore banks of temperate ferns. Amer. Fern J. 82:89-123. Dyer, A. fe and S. Linpsay. 1996. Soil spore banks—a new resource for conservation. Pp. 153-160 in . Camus, M. Gibby, and R. J. Johns, eds. Pteridology in perspective. Royal Botanic Ga i s, Ke Esau, K. eens Anatomy of seed plants, 2”*. ed. John Wiley & Sons, Inc., New AMILTON, R. G, 1988. The significance of spore banks in natural scene of Athyrium pycnocarpon ata . thelypterioides. Amer. Fern J. 1 INpeRyIT and K. M. M. Daksuini. 1995. On laboratory nrititionag in aliglopathy. Bot. Rev. 61:28—44. Koppen, W. 1948. Climatologia: con un estudio de los climas de la Tierra. Trad. P.R. Hendrichs Pérez. Fondo de Cultura Economica, Mexico. Leck, M. A. and R. L. Simpson. 1987. Spore bank of a Delaware River freshwater tidal wetland. Bull. Torrey Bot. Club 114:1-7. Linpsay, S. and A. F. Dyer. 1990. Fern spore banks: implications for gametophyte establishment. axonomia, Biogeografia y Conservacién de Pterid6fitos. Soc. d’Hist. Nat. de les Illes Balears - IME. Palma de Malliceve: 243-253. — S., N. Wittiams and A. F. Dyer. 1992. Wet storage of fern spores: unconventional but far re effective! Fern Hort.:285—294. Mave, B. in D. B. ANDERSON and C. A. opens 1963. Laboratory plant physiology. 3". ed. D. Van and Company, Inc., Prince Se ss 1991. Fern spores in a eae soil. Can. J. Bot. 69:831—-834. RANAL: BARK SPORE BANK IN BRAZIL 69 MiLserG, P. and L. ANDERSON. 1994. Viable fern spores in an arable soil. Fern Gaz. 14:299-300. Miter, L. E. 1994. Correlations: description or inference? J. Agric. Educ. 35:5-7. Miyazawa, M., M. A. Pavan, T. Muraoxa, C. A. F. S. Carmo and W. J. MELLo. 1999. Andalises quimicas de tecido vegetal. Pp. 173-223 in F. C. Silva, ed. Manual de andlises quimicas de solo, — e fertilizantes. EMBRAPA, Brasilia Pac, C. N. . Experimental aspects of fou ecology. Pp. 552-589 in A. F. Dyer, ed. The ain biology of ferns. Acacias shin ss, London Penrop, K. A. and L. H. McCormick. 1996 f viable hay-scented fern apenes germinated from hardwood forest soils at various distances from a source. Amer. Fern J. 9-79. PeREZ-GarciA, B., A. OROZCO-SEGOVIA and R. Ripa. 1982. El banco de esporas de helechos en el suelo e los Tuxtlas, Bol. Soc. Bot. México 43:89-92. RaFFAELE, E, 1996. Relationship between seed and spore banks and vegetation of a mountain flood meadow (Mallin) in Patagonia, unig Wetlands 16:1-— RANAL, M. A. 2003. Soil spore bank of ferns in a eas forest of the Ecological Station of Panga, Uberlandia—MG, Brazil. Amer. Fern J. 93:1— Rartter, J. A. 1992. Transitions between cerrado ye: a vegetation in Brazil. Pp. 417-429 in P. A. Furley, J. cero and ae . Ratter, eds. Nature and dynamics of forest-savanna boundaries. Chapman & Hall, Lon SCHIAVINI, I. an Ae M. sari ae Consideragoes sobre a vegetacao da Reserva Ecoldégica do Panga (Uberlandia). Sociedade & eng naa ony se m 61 66. SCHNELLER, J. J. and R. HOLDEREGGER. of Athyrium filix-femina. Pp. 663-665 a M. Camus, M. ony and R. I. see ‘eae cnenians in perspective. Royal Botanic Gardens, Kew. SIMABUKURO, E. A., A. Becovacz, L. M. Esteves and G. M. FELIPPE. sno Fern spore bank at Pedregulho Gttirailins, Sao Paulo, Brazil). Rev. Brasil. Biol. 59:131- aor hery E. A., L. M. ESTEVE “aia M. FE.iPpPe. 1998. Analysis of a can spore bank in Southeast ea Hoehnea 25:45-5 ee ec . M. F. and R. 7 G. Sitva. 2001. Determinagao do perfil fitoquimico de Copaifera Saat os Desf. (Caesalpiniaceee. III Simpésio Brasileiro de Farmacognosia. Sociedade Brasileira ee — armacognosia. Setembro, Curitiba, Parana, Brasil: QN-26 (Abstract). a= G. S. and P. J. EDGERTON. Hes Emergent seedlings from coniferous litter and soil in astern Oregon Ecology 57:80 WEE, "5 C. 1974. Viable seeds al sme of weed species in peat soil under p ppl ltivati Weed “denies 14:193— American Fern Journal 94(2):70-76 (2004) The Occurrence of Trichomanes godmanii (Hymenophyllaceae) on Welfia georgii (Arecaceae) at the La Selva Biological Station, Costa Rica RoBBIN C. MorAN The New York Botanical Garden, Bronx, New York 10458-5126, USA ROMINA VIDAL RUSSELL Department of Plant Biology, Southern Illinois University at Carbondale, Carbondale, Illinois 62901-6509, USA AssTract.—Field observations suggested that the epiphytic fern Trichomanes godmanii occurred more frequently and abundantly on the trunks of the palm Welfia georgii than on the trunks of dicotyledonous trees. We tested this observation statistically by randomly selecting 25 individuals of W. georgii and the nearest dicotyledonous tree of similar dbh, for a total of 50 trunks. For each trunk up to a height of three meters, we recorded the presence or absence of T. godmanii and, if present, we visually estimated percent cover using a ranked scale. We found that the fern occurred more frequently and abundantly on the palm than on dicotylendonous trees. No relationship was found between the diameter of the trunks and vegetati . This is one of the few host-specific preferences recorded among epiphytic ferns. We cannot fully explain why the fern occurs more frequently and abundantly on the trunks of Welfia georgii instead of dicot trees, but the fern’s adhesive hairs on its rhizomes and petioles probably help attachment to the smooth trunk of the palm. epiphytes such as Tmesipteris in Australasia (Brownsey and Smith- Dodsworth, 1989), and Blechnum fragile (Liebm.) C. V. Morton & Lellinger, Costaricia werckleana H. Christ, Terpsichore leamanniana (Hieron.) A. R. Sm., T. semihirsuta (Klotzsch) A. R. Sm., and Trichomanes capillaceum L. in Mesoamerica (Moran and Riba, 1995, p. 399). The only statistical demonstra- tion of species-specific host relationships in ferns was by Moran et al. (2003). They studied low-trunk epiphytic ferns on tree fern root mantles versus angiosperms at four sites in Costa Rica. They found that of the 31 species that occurred frequently enough in their samples to be tested statistically, 11 (35%) occurred more frequently on tree fern root mantles. MORAN & RUSSELL: TRICHOMANES GODMANII ON WELFIA GEORGII 71 The present study was prompted by an observation made by Grayum and Churchill (1989) at the La Salva Biological Station in Costa Rica about the frequency of occurrence of Trichomanes godmanii Hook. on the trunks of the palm Welfia georgii H. A. Wendl. ex Burret. They observed that T. godmanii was frequently found as a low-trunk epiphyte on the palm—a palm ubiquitous at the La Selva—and that it is one of the few epiphytes seen on the palm (Fig. 1). This observation was subsequently confirmed by the senior author at the La Selva Biological Station and at other nearby lowland forests in Costa Rica. We decided to test these observations by sampling and analyzing the results statistically. We tested the following two null hypotheses: first, that there is no difference in the frequency of occurrence of T. godmanii on Welfia georgii vs. dicotyledonous trees; second, that there is no difference in the abundance, expressed as percent vegetative cover, of T. godmanii on Welfia georgii versus dicotyledonous trees. Besides these two hypotheses about host preference, we examined the possible influence of the palm’s dbh on the percent cover of Trichomanes godmanii. We expected no influence because the age of the palm is not correlated with the diameter of its trunk (Rich, 1986). Therefore, larger diameter trunks would not necessarily be available for a longer time for the fern to colonize and form a greater percent cover. The null hypothesis we tested was that of no correlation between percent vegetative cover of the fern and the trunk diameter of the palm. METHODS The La Selva Biological Station is located in Heredia Province, at the confluence of the Puerto Viejo and Sarapiqui rivers, near Puerto Viejo de Sarapiqui, on the Caribbean side of Costa Rica, 10°26'N, 83°59'W. The elevation is about 50 m, and the vegetation is relatively aseasonal, tropical wet forest, with an average annual rainfall of 4,000 mm (McDade and Hartshorn, 1994). We sampled along an established trail called the Camino Experimental Sur beginning at a point where it meets the Sendero Tres Rios in front of the clearing that harbors the laboratory buildings. We sampled 25 pairs of trees, each pair consisting of one Welfia georgii and one angiosperm. We first sampled a palm and then selected the nearest dicotyledonous tree of similar (+ 10 cm) dbh. We sampled pairs to control for microclimatic effects, and we sampled trunks of similar dbh to control for differences in presence or percent cover that might be associated with trunk width. Only the lower three meters of the trunks were sampled. When the fern was present, percent vegetative cover on each trunk was visually estimated and then scored using a ranked scale where 0 = absent, 1 = 1-10% cover, 2 = 11-25%, 3 = 26-50%, 4 = 51-75%, 5 = 76-100%. To test the first hypothesis (no difference in the frequency of occurrence of Trichomanes godmanii on Welfia georgii versus dicot trunks), we used Fisher’s Exact Test (Langley, 1971). This test is a 2 X 2 contingency table that should be used instead of a chi-square contingency table when N is between 8 and 50 (we had N = 50; i.e., the 25 pairs). The probabilities were calculated for a 72 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) A. Welfia georgii without Trichomanes godmanii, showing smooth surface of trunk. B. Trunk of Welfia georgii covered with T. godmanii. C. Close up of T. godmanii colony. D. Fertile frond of T. godmanii (about 1 cm long). All photographs taken at the L Costa Rica. a Selva Biological Station, MORAN & RUSSELL: TRICHOMANES GODMANII ON WELFIA GEORGII 73 Contingency table showing the occurrence of Trichomanes godmanii on the trunks of Welfia georgii and dicotyledonous trees at the La Selva Biological Field Station, Costa Rica. According to a Fisher’s exact test, T. godmanii occurred more frequently on the trunks of W. georgii (P = 0.0014). Dicot W. georgii Total Absent 20 8 28 Present 5 17 22 Total 25 25 50 two-tailed test because we were interested in whether T. godmanii occurred more frequently on either Welfia georgii or angiosperms trunks. To test the second hypothesis (no difference in the percent vegetative cover of T. godmanii on Welfia georgii versus dicot trunks), we used a Wilcoxon/ Kruskal-Wallis rank sum test. Included in this test were only those trunks where the fern was present (17 palms, 5 dicots). For the third hypothesis (no correlation between percent vegetative cover of T. godmanii with the dbh of the palm trunks), we included only those 17 trunks where the fern was present. We then tested for a correlation between dbh of the palm and percent cover of the fern using Spearman’s rank coefficient. The significance level for all three hypotheses was set at P < 0.05. The statistical tests described in this paragraph were performed using the JMP statistical package, version 3.3.2 (Sall and Lehman, 1996). Several species of Trichomanes with leaves less than 3 cm long occur at the La Selva Biological Station, and these can be easily confused with T. godmanii (Grayum, 1989). During sampling we examined suspected individuals of T. godmanii for the presence of cross-connections between the false veins. This characteristic distinguishes T. godmanii from all other similar small species of Trichomanes (Wessels Boer, 1962). Other characteristics helpful in identifying the species were glabrous lamina margins (i.e., without black paired or stellate hairs) and green-margined involucres (not black margined; Fig. 1D). At the La Selva Biological Station, none of the other small species of Trichomanes form large extensive mat-like colonies that T. godmanii does on the trunks of Welfia georgii. RESULTS In the 25 paired samples, Trichomanes godmanii was present on 17 palms and 5 dicot trees. The mean dbh of the palms sampled in this study was 17 cm (s.d. 2.2), with a range of 13-25 cm. The mean for the nearest dicot trees of similar dbh was 17.7 cm (s.d. 5.5), with range of 9-26 cm dbh. Hypothesis 1.—no difference in the frequency of occurrence of Trichomanes godmanii on Welfia georgii versus dicot trunks. The null hypothesis was rejected (P = 0.0014; Table 1). The fern occurs more frequently on the palm. Hypothesis 2.—no difference in the percent vegetative cover of T. godmanii on Welfia georgii versus dicot trunks. The null hypothesis was rejected 74 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) BB Welfia georgii [ ] Dicot trees No. of Trunks on re) 1-10 11-25 26-50 51-75 76-100 Percent Cover Fic. 2. Comparison of percent vegetative cover of Trichomanes godmanii on 25 trunks of Welfia georgii and 25 trunks of dicotyledonous trees at the La Selva Biological Station, Costa Rica. According to a Wilcoxon/Kruskal-Wallis rank sum test, T. godmanii had greater percent cover on Welfia georgii than on dicotyledonous trees (P = 0.0186 (P = 0.0186; Fig. 2). The fern is more abundant (higher percent cover) on the palm. Hypothesis 3.—no correlation between percent vegetative cover of T. godmanii with the dbh of the palm trunks. The null hypothesis was retained (r, = 0.37; P=0.149). There was no correlation between the abundance of fern and the dbh of the palm. DISCUSSION The results support the observations of Grayum and Churchill (1989) that Trichomanes godmanii occurs more frequently on the trunks of Welfia georgii than on dicot trees (hypothesis 1). This is one of the few host-specific relationships that have been documented statistically in ferns (for others see Moran et al. 2003). Trichomanes godmanii seems to be one of the few ferns capable of colonizing the trunks of Welfia georgil. Grayum and Churchill (1989) noted that an unnamed species of Elaphoglossum also occurred rarely on the trunks, but we did not find this species in or outside our samples. The only other fern we found on the palm was Trichomanes (sect. Didymoglossum) angustifrons (Fée) W. Boer. It occurred as isolated individuals near the base of three trunks. MORAN & RUSSELL: TRICHOMANES GODMANII ON WELFIA GEORGII 75 The results also showed that Trichomanes godmanii was not only more frequent on the palm, but also more abundant (hypothesis 2). The colonies could be so dense that the surface of the trunk was obscured. When present, they always occurred around the base of the trunk (Fig. 1B) and diminished upward, but in some cases dense colonies extended five meters above the ground (at La Selva the palm can attain a height up to 23 meters; Rich, 1986). The large extensive colonies of the fern (Fig. 1B) are formed by the plants’ long- creeping rhizomes that occasionally branch and run horizontally or upward around the trunk. There was no correlation, however, between percent cover of the fern and dbh of the palm (hypothesis 3). Like many palms, Welfia georgii begins vertical growth with a stem girth that is sufficient to support its maximum height, and it maintains the same or nearly same width as it grows taller (Rich, 1986). Thus wider trunks do not necessarily represent older individuals that have been available for colonization for a longer time by epiphytes. Given this, one would expect an equal amount of the fern in terms of percent cover on both narrow and wide diameter trunks, and this is what we found. Why is Trichomanes godmanii most frequent and abundant on Welfia georgil? Perhaps allelopathy plays a role, with the palm trunk presenting a chemical that inhibits epiphytes other than the fern. This idea, however, cannot be assessed because allelopathy has never been investigated in the trunks of W. georgii nor any other palm (Andrew Henderson, pers. com.). More likely in promoting growth of the fern is the smooth texture of the palm trunk. This might favor the fern two ways. First, it could hinder other epiphytes from establishing and attaching the trunk. This might be because of the smooth surface itself or because such surfaces retain less water or nutrients than rougher surfaces. In either case, fewer epiphytes would free the fern from competition, allowing it more space, light, and nutrients. Second, unlike other epiphytes, the smooth surface of the palm might be an easy substrate for T. godmanii to grasp. Like all members of Trichomanes sect. Didymoglossum, T. godmanii is rootless, but its rhizomes, petioles, and sometimes basal portions of the midrib, bear abundant specialized hairs called ‘‘adhesive hairs” (Schneider, 2000). These usually form a dense mat surrounding the rhizome and are dark, stiff, and several-celled. They have a cuticle (as do the rhizomes) and apparently do not absorb water or mineral nutrients, but they might hold water by capillary action and gradually release this water to the lamina as it dries. Many species of Trichomanes absorb water and mineral nutrients directly through their leaves, which are only one cell layer thick between the veins and lack a cuticle or nearly so (Haertel, 1940). The main function of the hairs, however, appears to be for attachment. The presence of these numerous hairs greatly increases the surface area for clinging to the substrate. In some species of Trichomanes sect. Didymoglossum, the adhesive hairs branch or enlarge at the tip when they touch the substrate (Duckett et al., 1996), increasing adhesive ability. Trichomanes godmanii has such branched or swollen hairs (pers. obs.) that would facilitate its colonizing smooth surfaces. Trichomanes angustifrons, the only other fern we found on the trunks, is also 76 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) a member of sect. Didymoglossum and has the adhesive hairs, suggesting the importance of this type of indument for growing on smooth surfaces. Nevertheless, adhesive hairs cannot be the only reason why T. godmanii flourishes on palm. Four other species of Trichomanes sect. Didymoglossum occur at the La Selva Biological Station (Grayum and Churchill, 1989), and although they have adhesive hairs, only one of them (T. angustifrons) was found on the palm. All of the other four grow primarily on dicots. Thus, although adhesive hairs probably play an important role, they are not the entire reason why T. godmanii prefers the trunks of Welfia georgii. ACKNOWLEDGMENTS This study was done while the junior author was a student in Sistemdtica de Plantas Tropicales, a course sponsored by the Organization for Tropical Studies (OTS). This course was coordinated by the senior author, and funding for the course came primarily from the Andrew G. Mellon Alvaro Idarraga for their help with sampling. We also thank Andrew Henderson for generously sharing his extensive knowledge of palms, and Harald Schneider for his comments on adhesive hairs in Trichomanes. LITERATURE CITED eicitcstcas P. J., and J. C. Smirx-Dopswortn. 1989. New Zealand Ferns and Allied Plants. David Bateman Ltd., Auckland. DUCKETT, 7. G, A. J. RussELL and R. LicRone. 1996. Trichomes in the Hymenophyllaceae. Pp 511-514. In: J. M. Camus, M. Gibby and R. J. Johns, eds. Pteridology in Perspective. Royal Botanic Garden, Kew, UK. Kress, W. J. 1986. The systematic distribution of vascular a hig an update. Selbyana 9:2-22. Kress, W. J. 1990. The systematic distribution of vascular epiphytes. Pp. 234-261. In: U. Liittge, ed. Vascular Plants as = = Evolution and Ecophysiology. Becloicd Studies 76. Springer- Verlag, New York, New Y: GrayuM, M. H. and H. W. ee 1987. An introduction to the pteridophyte flora of Finca La Selva. Amer. Fern J. 77:73-89. Grayum, M. H. and H. W. CuurcHm. 1989. The vascular flora of the La Selva Biological Station, Costa Rica. Polypodiophyta. Selbyana 11:66-118 HaerTEL, O. 1940. Physiologische studien an Hymenophyllacese. Il. Wasserhaushalt und resistenz. Protoplasma 4:489-514. LANGLEY, R. 1971. Practical Statistics Ard Explained. Dover Publications, New York. McDape, L. A. and G. S. HARTSHORN. ge Biological Station. Pp. 6-18. In: L. A. McDade, . S. Bawa, H. A. Hespen eran pies G. Hartshorn, eds. La Selva a Ecology and Namal History of a Niclieeicad Rain Forest. The ena of Chicago, Illinois, USA Moran, R. C., S. Kuimas and M. Cartsen. 2003. Low-trunk epiphytic ferns on true ferns versus angiosperms in Costa Rica. Biotropica 35:48-56. Ricu, P. M. 1986. Mechanical architecture of arborescent rain forest palms. Principes 30:117-131. SaLt, J. and A. LEHMAN. 1996. JMP Start — A Guide to ge and Data Analysis using MP and JMP IN Software. SAS Inst e, Cary, North Carolina. USA. SCHNEIDER, H. 2000. Morphology and a she of roots in the film ¥ form tribe Trichomaneae H. ooo fitymenophyllansae, F aa and the evolution of as taxa. Bot. J. Linn. Soc. Wires sith om , . The New World a of Trichomanes sect. Didymoglossum and Microgonium. Pi ne Neerl. 11:277-3 American Fern Journal 94(2):77—80 (2004) A New Species of Adiantum (Pteridaceae) from Thailand PIYAKASET SUKSATHAN Herbarium, Queen Sirikit Botanic Garden, P.O. Box 7, Mae Rim, Chiang Mai 50180, Thailand ABSTRACT.—A new species, Adiantum thongthamii (Pteridaceae), known only from a small island in southeastern Thailand is described and illustrated. Adiantum is a genus of ca 150 species widely distributed pantropically but extending as far as southern South America, New Zealand, Newfoundland, Alaska, and northeastern Asia, (Tryon & Tryon, 1982; Mabberley, 1997). Tagawa & Iwatsuki (1989) record ten species of Adiantum from Thailand. During a field trip to southeastern Thailand in March 2002, an eleventh species, Adiantum thongthamii, was found in Koh Chang, a small island close to Cambodia. Adiantum thongthamii Suksathan sp. nov. TYPE:—Thailand: Trat Province: Koh Chang, 600 m alt., 24 March 2002, P. Suksathan 3303 (holotype: QBG; isotypes: AAU, L, US). Figs. 1, 2. Species nova, Adianto erylliae C.Chr. & Tard. affinis a qua differt squamibus rhizomatis concoloribus rufis, foliis dense pubescentibus, rachidi non prolifera, petiolulis ca 1 mm, pinnis late flabellatis usque rotundatis, coriaceis, soris minoribus ca 1 mm latis, 8-21 in quaque pinna, pseudoindusiis minoribus obovatis 0.5—1.0 mm latis. Plants terrestrial. Rhizomes short-creeping, erect to sub-erect, ca 5 mm diam; scales copious, narrowly lanceolate to linear, 4-5 X 0.1-0.9 mm, lustrous, concolorous, reddish brown, margins entire to sparsely minutely toothed in the upper half. Fronds monomorphic, simply pinnate, with or without a (smaller) conform terminal pinna, 7-20 cm long; stipes 3-10 cm long, lustrous, reddish brown to nearly black, sparsely to densely covered with grayish to reddish brown multicellular hairs; the hairs, spreading, 1.5-2 mm long, decreasing to ca 1 mm long toward the apex. Lamina oblong, to 2.5 cm wide; rachis with indument similar to the upper part of the stipe, neither prolonged nor proliferous; pinna pairs 3-6 (—9), opposite to alternate, basal pinnae often slightly reduced in size, upper pinnae rarely gradually reduced in size. Pinnae; stalks ca 1 mm long, with short hairs, flabellate-cuneate to flabellate-truncate or suborbicular, 7-15 x 9-20 mm, green when young becoming dark bluish green with age; distally lobed to 1/3 the length of the pinna, the sinuses typically narrow, lobes rounded to truncated, entire in sterile pinnae, coriaceous, idioblast absent, both surfaces densely hairy; the hairs ca 1 mm long, adaxial hairs gray, abaxial hairs AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) <= = Se — me ss 7 com Oe eae Fic. 1. Adiantum thongthamii Suksathan. A) Habit; B) rhizome scale; C) stipe hairs; D) frond and frond apex; E) pinna, abaxial surface with hairs removed; F) sorus with hairs removed. Drawn from the holotype (P. Suksathan 3303, QBG). SUKSATHAN: A NEW SPECIES OF ADIANTUM 79 80 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) brown; veins free, forked, prominulous on lower surfaces, in dry specimens. Sori round, 8-21 per pinna; pseudoindusia small, obovate, 0.5-1 mm long, margin entire, hairy. Spores trilete, dark yellow to brown, the rugose, ca 50 mm. DIsTRIBUTION.—Known only from the type locality at Koh Chang (12° 0’ N, 102° 22’ E), an island in SE Thailand. Hasirat.—Occurs in full sun-xeric habitat on exposed sandstone outcrops along the Khao Laem mountain ridge between 500-640 m. Adiantum thongthamii was found growing with Melastoma spp. (Melastomataceae), Nepenthes spp. (Nepenthaceae), Doritis spp. (Orchidaceae), Adiantum capillus-veneris L. (Pteridaceae), Selaginella siamensis Hieron. (Selaginella- ceae), and others. Adiantum thongthamii is very distinct from other known species of Adiantum and is easily recognized by its once pinnate fronds with 3-6 (9) pairs of broadly fan-shaped pinnae and by its dense wooly pubescence. Adiantum thongthamii differs from A. erylliae C. Chr. & Tard., and A. capillus- junonis Ruprecht by having hairy fronds and many smaller sori per pinna (8— 21 versus 2—7) and from A. caudatum L. by its concolorous scales and pinna shape (broadly fan-shape versus parallelogram-shaped in A. caudatum). Tryon and Tryon (1982) divided the genus Adiantum into eight groups based on morphology. Adiantum thongthamii appears to be belong to the A. philippense Group, in having simply pinnate fronds, flabellate segments, and free veins. The subdivision by Tryon and Tryon has, however, strongly emphasizes the American species and does not include all taxa in the genus. Some species are also placed with uncertainty. Nevertheless, ongoing work at the DNA level should hopefully reveal more insight into natural groups and species relationships in the genus (A.R. Smith, pers. com.). The species is named in honor of Associate Prof. ML Charuphant Thongtham of Thailand, an expert pterido-horticulturist who inspired my interest in ferns and plants in general. ACKNOWLEDGMENTS I am grateful to Dr. Benjamin Qllgaard for his kind suggestions and for supplying the Latin diagnosis. I thank Dr. Alan R. Smith for comments. Thanks also to Ratchada Pongsattayapipat of the Queen Sirikit Botanic Garden, Chutchai Wiboonronnarong for sending me specimens from Thailand, and Apichai Engkhavut for photographs. LITERATURE CITED MapserLey, D. J. 1997. The plant-book, a portable dictionary of the vascular plants, 2nd ed. Cambridge University, Cambridge. Tacawa, M. and K. Iwatsuxki. 1989. Parkeriaceae. Pp. 183-216 in T. Smitinand, ed. Flora of Thailand vol. III: part 2. Chutima Press, Bangkok. Tryon, R. M. and A. F. Tryon. 1982. Ferns and allied plants, with special reference to tropical America. Springer-Verlag, Berlin. American Fern Journal 94(2):81—111 (2004) Asplenium ceterach and A. octoploideum on the Canary Islands (Aspleniaceae, Pteridophyta) CAROLINE J. VAN DEN HEEDE ca Section, Department of Biology, Ghent University, L. Ledeganckstraat 35, B-9000 Ghent, Belgium aia PajJARON and EMILIA PANGUA Departamento de Biologia Vegetal I, Facultad de Biologia, Universidad Complutense, E-28040 Madrid, Spain RONALD L.L. VIANE! ee Section, Department of Biology, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium ABSTRACT.—Isozyme ee plastid DNA analysis iso that true A. ceterach occurs on the Canary Islands, in addition to A. aureum and an octoploid taxon. Combining morphological and cytological jie leads to correct Nie ate but the exospore length alone also allows reliable identification of these Canarian species. Our allozyme data suggest that the Canarian A. ceterach population is not completely genetically isolated from the European ones. The holotype of Ceterach aureum var. parvifolium, formerly regarded as an octoploid taxon, proved to be A ceterach, leaving the octoploid without a correct name. The recently described A. octoploideum hows monomorphic, presumably fixed heterozygosity for a combination of the patterns seen in A. ceterach and A. aureum at four loci (Aat, Sk sh Me, and Pgi-2) confirming its allo-octoploid nature. It most probably originated by ch ling in a tetraploid hybrid between A. aureum and A. ceterach or via the union of their unreduced gametes. Pgi-2 indicates multiple origins of the allo- octoploid, implicating recurrent gene flow from tetraploids to octoploids. Asplenium subgenus Ceterach (Willd.) Bir et al. is a small group of about nine fern taxa within the large (720 species), subcosmopolitan genus Asplenium L. (Kramer and Viane, 1990). This subgenus contains xerophytic rock ferns with the dorsal side of the lamina densely covered with reddish- brown scales (= paleae). Van den heede et al. (2003) have shown that the group must be restricted to the Eurasian and Macaronesian species. Ever since the description of Asplenium aureum Cav. from Tenerife by Cavanilles (1801), it has been unclear how many “‘Ceterach”’ species are extant in the Canarian Archipelago, and whether the ‘“‘European”’ A. ceterach L. (Syn.: Ceterach officinarum Willd.) occurs in Macaronesia (Table 1). This confusion was caused by the lack of distinctive characters to distinguish both species. Cavanilles (1801) and Bory de St. Vincent (1802) mentioned only the much larger size of A. aureum compared to that of A. ceterach. Willdenow (1810) introduced the concept of ‘‘toothed scales” as a diagnostic feature, whereas Milde (1865) claimed that ‘‘Cuticularstreifen” (cuticular lines or ridges on the periclinal cell walls of the scales) could be used to distinguish A. aureum from * Author for correspondence. 82 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) .aur.=A. aureum, A. lol.=A, lolegnamense, A. par.= HA, cakes sensu Vado a Reichst” ~ = A. po Be . the filled symbol indates sa the taxon was included in this author’s concept of the species mentioned in column 2; mbol riod that this taxon was included in the author’s concept of the species mentioned in paris 2 prior to its formal description. Taxa included in this name Literature reference Name used or published Avces -Acaur -A.lol. “A. par” Linnaeus pie A. ceterach a Cavanilles 1801 A. aureum a Bory de St. ae 1802 =A. cetera a A. latifolium Bg Desvaux 182 C. aurea a von magi 128 (1825 1° C. aureum a Moor C. aureum a C. officinarum Lowe, E. J. 1858 C. officinarum a Oo Hooker 1860, 1861 A. ceterach A. ceterach var. aureum | | ‘a Bolle 1864 G.au | C. officinarum a O fa] Milde 1866b, 1867a,b C. aureum a Oo C. officinarum a O O Kuhn 1868 C. aureum I a Sauer 1880 C. aureum fl C. officinarum a Luerssen 1889 C. officinarum i] a D Oo Schneider 1892 A. ceterac gs A. ceterach var. aureum | 0 Christ 1897 G. a a C. officinarum & O Burchard 1929 C. aureum a C. officinarum g Oo Chevalier 1935 C. officinarum a C. officinarum var. aureum a Oo Tardieu-Blot 1946 C. officinarum a a oO Copeland 1947 C. aureum a a C. officinarum a Manton 1950 C. aureum a C. officinarum ] Romariz 1953 C. aureum |] O Lems 1958, 1960 C. aureum a O C. officinarum a Ol Dansereau 1961 C. aureum s | | O Fabbri 1965 C. aureum 1 | C. officianarum a Kunkel 1965 C. aureum a O O Benl and Kunkel 1967 C. aureum var. aureum a C. aureum var. parvifolium a a Lid 1967 C. aqureum a a Hansen 1969 C. aureum | Benl and Sventenius 1970 C. aureum var. aureu a C. aureum var. parvifolium a a VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 83 TABLE 1. Continued. Taxa included in this name Literature reference Name used or published A.cet. A.aur. A. lol. “A. par.” Kunkel 1971 C. aureum var. aureum a C. aureum var. parvifolium a a Hansen and Sunding 1979 _C. aureum var. aureum a = C. aureum var. parvifolium a a Reichstein 1984 C. aureum var. aureum a C. aureum var. parvifolium a C. offinarum a Bir et al. 1985 A. aureum a O a A. ceterach ssp. ceterach a Manton et al. 1986 C. aureum var. aureum a C. aureum var. parvifolium g Gibby and Lovis 1989 C. lolegnamense a Ormonde 1990 C. aureum var. aureu a C. aureum var. madeirense a C. aureum var. parvifolium a | Viane and Reichstein 1992 _A. parvifolium a A. lolegnamense a Griffiths 1997 A. aureum a A. ceterach a Hoshizaki and Moran 2001 C. a a a C. officinarum a * According to Stafleu and Cowan (1976) this book was may published after 28 May 1828, it is not clear whether Link or von ‘ioe ade the combination “C. aureum’”, which is, in any case, antedated by Desvaux (1827 its continental counterpart, though he soon (Milde 1866a, 1866b, 1867a, 1867b) cast doubt on the utility of this character. As early as May 1866, Milde admitted that “die Cuticularstreifen, welche die Spreuschuppen von C. aureum stets zeigen, fand ich nun auch an exemplaren, die sich von C. officinarum nicht unterscheiden aa Finally, he came to the conclusion that the character was useless to discriminate A. ceterach from A. aureum (Milde, 1867b). Nevertheless, Bornmiiller (Plantae exsicc. Canarienses—1901), and Benl and Kunkel (1967) heavily relied on this character to recognize taxa. Since 1970, chromosome numbers were used to distinguish species in this group (T. Reichstein, pers. comm.), and morphological characters became less important (e.g., Bir et al., 1985; Manton et al., 1986; Gibby and Lovis, 1989). In 1967, Benl and Kunkel considered all Canarian plants that looked like A. ceterach to be a dwarfed variety of A. aureum. Unfortunately, their variety Ceterach aureum (Cav.) Desv. var. parvifolium Benl and G.Kunkel was published without cytological information. In March 1967, T. Reichstein collected living “A. ceterach’” on Gran Canaria, and sent material for chromosome counts to G. Vida. In 1970, these plants were found to be octoploid, but because good cytological photographs were lacking the results were not published (T. Reichstein, pers. comm.). From then onwards, but without studying the type of A. parvifolium (Benl and G.Kunkel) Vida and 84 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) Reichst., octoploid status was attributed to it. To clarify the origin of A. parvifolium, a hybridization program was started by G. Vida in Budapest; results were partly published in Manton et al. (1986). Meanwhile, T. Reichstein had informed many pteridologists about the putative allo-octoploid nature of A. parvifolium and briefly mentioned it in Hegi (1984). To date two cytologically different endemic species are generally accepted to occur on the Canary Islands: A. aureum and A. parvifolium. Asplenium aureum was found to be tetraploid by Manton (1950). Vida and Reichstein (Vida, 1972; Viane and Reichstein, 1992) suggested A. aureum to be allotetraploid, which was confirmed by ITS analysis (Van den heede et al., 2003). The name A. parvifolium was used for the allo-octoploid that prob- ably formed by chromosome doubling of the tetraploid hybrid between A. aureum and A. ceterach (Vida, 1972; Viane and Reichstein, 1992). After 1970, all small Canarian plants that looked like A. ceterach were considered to be a) A. parvifolium and b) octoploid. According to Manton et al. (1986) “C. officinarum is not positively recorded from Macaronesia, but its former presence, at least in the Canaries, is suggested by the morphology of some representatives of C. aureum sens. lat. from these islands.”’ Within A. ceterach sensu lato three cytotypes are known, and according to the Biological Species Concept (Mayr, 1942, 2000; see review in King, 1993), autopolyploids should be considered separate species because they produce sterile hybrids with their parents from which they are reproductively isolated. Diploid A. javorkeanum Vida [Syn.: A. ceterach ssp. bivalens (D.E. Mey.) Greuter and Burdet; C. officinarum Willd. ssp. bivalens D.E. Mey.] is known from Albania, Bulgaria, Croatia, Greece, Hungary, Italy, Romania, and Slovenia (Vida, 1963; Reichstein, 1984), and should be looked for in northern Algeria and Turkey, because the triploid hybrid A. Xmantoniae Varoczy and Vida was found there (Greuter, 1980; Viane et al., 1996). Tetraploid (Manton, 1950; Vida, 1963) A. ceterach [Syn.: C. officinarum Willd. ssp. officinarum] is supposed to have originated via chromosome doubling in A. javorkeanum: its autopoly- ploid status was confirmed cytologically by Rasbach et al. (1987). The autotetraploid is the more common species, occurring throughout Europe see maps in Jalas and Suominen, 1972; Pichi Sermolli, 1979; Reichstein, 1984), southwestern Asia and the western Himalayas. Asplenium ceterach is more rare in northern Africa (Jahandiez and Maire, 1931; Maire, 1952; Quezel and Santa, 1962; Siddiqi, 1989), but extends into Eritrea and Somalia (Viane et al., 1996), the Arabian Peninsula (Collenette, 1985), and Yemen (Christ, 1900; Wood, 1997). The autohexaploid A. cyprium Viane and Van den heede (Syn.: A. ceterach ssp. cyprium Viane) was described from Cyprus (Van den heede and Viane, 2002; Viane and Van den heede, 2002), and is also known from Greece and Sicily (Viane et al., 1996; Van den heede et al., 2002). For the biosystematic revision of the Ceterach group (Van den heede, 2003), field trips were organized to study the Macaronesian representatives. Plants that we could not distinguish from the European A. ceterach were tentatively called A. parvifolium, and assumed to be octoploid. To our great surprise many of them turned out to be tetraploid. VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 85 In order to clear up the A. ceterach—A. parvifolium muddle on the Canarian Archipelago, we studied type material, and cytologically checked 145 samples from Gran Canaria, La Palma, and Tenerife. Because the type of A. parvifolium turned out to be A. ceterach, the octoploid taxon needed a new name and was described as A. octoploideum Viane and Van den heede (Van den heede and Viane, 2002). Because electrophoretic analysis of isozymes has been successfully used in studies of reticulate complexes of Pteridophyta (Werth et al., 1985a, 1985b; Werth, 1991; Haufler et al., 1995) and has been applied at population and species levels (see Haufler, 1985b, 1997), we tried this method together with DNA sequencing, to determine whether true A. ceterach grows on the Canary Islands. A combination of morphological, cytological, and biogeographical data and isozyme markers can determine whether taxa are auto- or allo- polyploid (Crawford, 1985; Haufler, 1985b; Bryan and Soltis, 1987; Weeden and Wendel, 1989; Crawford, 1990; Pryer and Haufler, 1993). An overview of the literature about DNA sequencing in Pteridophyta is given in Van den heede et al. (2003). MATERIAL AND METHODS Between April 1995 and May 1999, field trips were organized to three Canary Islands from which A. parvifolium was known in the literature: Gran Canaria, La Palma and Tenerife. From 145 specimens, fronds with ripe spores were collected by C.V. and R.V, and ecological notes were made. Voucher information (Appendix 1, 2, and 3) is given only for specimens from which we were able to raise progeny and obtain aia data. The following localities are shown in Fig. 1 1) Gran Canaria, S of Moya, ‘‘Los Tilos’” Reserve, W exposed slopes of Barranco del Laurel, degraded laurel forest, in fissures of volcanic rocks; 28°05'03”"N, 15°35'28”W, 600 m alt. 2) Gran Canaria, 4 km from junction Tejeda—San Mateo—Las Mesas, E exposed slopes of ‘El Nieblo’’ Nature Reserve, in fissures of volcanic rocks; 28°01'03”N, 15°36'07”W, 1550 m alt. 3) Gran Canaria, lava field near Cueva Corcho, along road GC110 from Artenara to Valleseco, 4 km NW of junction Artenara—Valleseco—Tejeda, in fissures of volcanic rocks; 1350 m alt. 4) Gran Canaria, 900 m S of Valsendero, W exposed cliff sides of narrow gully with laurel forest remnants; 28°02'48"N, 15°34'27”W, 900 m alt. 5) La Palma, c. 3 km E of Tijarafe, Pinar Lomo del Horno; 28°42’N, 17°55’W, 1140 m alt. 6) La Palma, S of Gallegos, Barranco Lomo de los Machines, Laurel forest W of tunnel El Envetadero, E exposed slope; 28°48’N, 17°50’W, 390 m alt. 7) La Palma, volcanic rocks above roadside to Fuencaliente, S of Monte de Luna; 28°31'N, 17°49’W, 710 m alt. 8) La Palma, footpath to Monte de Luna in Pinar S of Flores, in fissures of volcanic rocks; 28°31'N, 17°49’W, 810 m alt. 86 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) 28°.N 25 km Gran Canaria 17° W_ G. 1. Map of the western Canary Islands with localities of voucher specimens (see also Appendix 1, 2, and 3). 9) La Palma, along track to Pinar de la Virgen at junction with track to Caldera Los Arreboles, in fissures of volcanic rocks; 28°30’N, 17°50’W, 920 m alt. 10) La Palma, along track to Refugio de Tigalate from Zona Recreativa Fuente de los Roques, above ‘‘Malpais’’ W of Monte de Luna, in fissures of volcanic rocks; 28°31'N, 17°49’W, 1070 m alt. 11) La Palma, Caldera de Taburiente, track from La Cumbrecita to Hoyo de los Pinos, Pinar in Barranco de la Faya, in fissures of volcanic rocks; 28°43’N, 17°50’W, 1200 m alt. 12) La Palma, lava field 2.5 km E of El Paso church, in fissures of volcanic rocks; 28°38'N, 17°51’W, 800 m alt. 13) La Palma, lava field E of El Paso, NE of Montana Las Moraditas, in fissures of volcanic rocks; 28°39'N, 17°50’W, 800 m alt. 14) Tenerife, along road from Vilaflor to Pico del Teide, ca. 8.1 km from junction Vilaflor-Santiago del Teide-La Orotava, under disc-like, SW exposed volcanic rocks; 28°10'55”"N, 16°39'17”W, 1850 m alt. 15) Tenerife, Barranco de las Gambuesas above Arafo, N exposed slopes; 28°20'29"N, 16°26’02”W, 710 m alt. 16) Tenerife, Barranco del Espigon de Tea, NE exposed slopes; 28°20'44’N, 16°26'33"W, 825 m alt. 17) Tenerife, Montana de la Hoya, ridge S of Las Manchas, above Ermita de la Santa Angel del Guardo, in fissures of volcanic rocks; 28°16'34’N, 16°48’05”W, 1120 m alt. 18) Tenerife, Teno, Barranco head between Tierra del Trigo and Ruigomez, along track above Tierra del Trigo, 1.8 km NW of Ruigomez, NW exposed basaltic slopes; 28°20'48”N, 16°48’28”W, 800 m alt. VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 87 19) Tenerife, Pinar above Vilaflor, Bandes de Chasna, c. 2.5 km NNW of Vilaflor, E exposed, in fissures of volcanic (phonolite) rocks; 28°10'48’N, 16°38'36”"W, 1880 m alt. 20) Tenerife, Pinar above Vilaflor, W exposed slopes of small Barranco, in fissures of volcanic rocks; 28°10'48’N, 16°38'39”W, 1900 m alt. 21) Tenerife, Barranco de la Piedra Cumplida above (NW) Arafo; 28°21'17’N, 16°26'05”W, 900 m alt. 22) Tenerife, volcanic outcrop along footpath between Santiago del Teide and Arguayo, SE of El Retamar, between Montana de la Hoya and La Hoya; 28°16"N, 16°48”W, 920 m allt. 23) Tenerife, lava field NW of Montana de las Flores, ‘“‘Vuelta Grande”, along track from El Portillo del Rastrojo to Llanos del Hospital; 28°18’N, 16°45”"W, 1410 m alt. 24) Tenerife, Chio Street, direction Canadas, Restaurant ‘‘De Evora’’. Vouchers listed in Appendix 1, 2 and 3, are deposited in the personal herbarium of Viane and Van den heede (including the T. Reichstein herbarium), with duplicates in GENT. Between 1992 and 2001, R.V., C.V., and W. Bennert gathered additional material in Europe, Madeira, and Turkey (Appendix 4). Voucher information about 108 Cypriot samples is published in Van den heede et al. (2002). Our living European and Macaronesian Asplenium subg. Ceterach collection contained up to 550 specimens. All material for this study has been cultivated in Ghent University Botanical Garden (Belgium). Spores were sown on agar-solidified medium containing a nutrient solution recommended by Dyer (1979). The cultures were stored in continuous light at room temperature. After formation of mature gametophytes, distilled water was added to achieve fertilization. If necessary, prothallia were transplanted onto fresh agar. Young sporophytes were planted individually in pots kept in a temperate greenhouse (minimum temperature 12°C). An air- and water-permeable soil mixture was required for these xerophytic rock ferns. Full-grown maturity was reached after approximately two years. For chromosome counts, immature spore mother cells were fixed in the field, or in the greenhouse, using freshly prepared 3:1 absolute ethanol:glacial acetic acid, and stored at freezing-temperature until required. Acetocarmine squash preparations were made as described by Heitz (1925, 1950) and Manton (1950). Photographs were taken with an Olympus BH2 phase contrast microscope. Preparations were made permanent by dehydrating cover slip and slide in graded mixtures of acetic acid and absolute ethanol, followed by mounting in Euparal (T. Walker and H. Rasbach, pers. comm.). All permanent preparations are kept in the Pteridological Section of the Department of Biology at Ghent University. Sixteen cytologically checked plants (five tetraploids identified as A. aureum, seven tetraploids identified as ‘A. parvifolium sensu Benl,”’ and four octoploids, (Appendix 1, 2, 3) were used as standards to compare the nuclear DNA content of the remaining specimens by a flow cytometer (Partec PA-1), using the manufacturer’s protocol (Partec GmbH, Miinster, 88 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) Nordrein-Westfalen, Germany). Both nuclei extraction solution and DAPI staining dilution were provided by Partec (Germany). Methods for making permanent epidermis preparations and for measuring stomatal guard cells and spores, are described by Viane (1990, 1992). For exospore measurements untreated, fresh spores mounted in DePeX were used. Spore size is unaffected by DePeX, whereas in some other mounting media, e.g., glycerin-gelatin (Ormonde, 1990), spores expand by 5-15 %. The values of microcharacters are extracted from our regularly updated database, presently containing 110 different specimens of the European “A. ceterach” group, and 56 specimens of the Macaronesian “A. aureum’’ group (raw data available upon request). Only vigorously growing plants were included in the allozyme study. Fresh leaves from 105 Canarian (Appendix 1, 2, 3) and 220 European and Turkish specimens (Appendix 4) were collected in the greenhouse, where the ferns were growing under the same conditions. Sporulating fronds of similar age were wrapped in wet tissue, stored in plastic bags, and kept refrigerated at 4°C for maximum 0.5—2 days (until extraction). Polyacrylamide gel electrophoresis (PAGE) was performed by C.V. at the laboratory of ‘General Botany and Nature Management” of the Free University Brussels (Belgium). Starch gel electro- phoresis (SGE) was done by S.P. and E.P. in the “Departamento de Biologia Vegetal I’ of the Universidad Complutense in Madrid (Spain). All specimens from the Canarian Archipelago were analysed by starch gel electrophoresis. Equal amounts of tissue and extraction buffer were used to obtain uniform concentrations of extracts. Cooling (4°C) was applied during both homogeni- zation and electrophoresis. Acquaah (1992) was consulted for the Enzyme Commission (E.C.) numbers. PAGE procedures mentioned in Triest (1989) and Van den heede et al. (2002) were used, whereas SGE protocols followed Soltis et al. (1983) and Haufler (1985a). In a preliminary survey 19 enzyme systems (G-3PDH, G-6PD, GDH, IDH, MDH, ME, 6-PGD, SkDH, SOD, XDH, ACO, AAT, HK, PGM, B-EST, LAP, ALD, PGI, and TPI) were checked for polymorphism. Because the primary goal of this isozyme research was to test the hypothesis that tetraploid A. ceterach occurs on the Canary Islands in addition to A. aureum and related taxa, it was necessary to identify unique ‘‘marker” alleles characterizing each species or its progenitors. Finally, only five enzyme systems were suitable: aspartate aminotransferase (AAT = GOT, E.C. 2.6.1.1), shikimate dehydrogenase (SkDH, E.C. 1.1.1.25), malic enzyme (ME, E.C. 1.1.1.40), phosphoglucose isomerase (PGI, E.C. 5.1.3.9), and triosephosphate isomerase (TPI, E.C. 5.3.1.1). All pictures and dried gels are kept in the Pteridological Section of the Department of Biology at Ghent University. Band homologies were determined by running samples side-by-side on the same gel (see Haufler et al., 1995). Allelic variants within loci were distinguished from the products of different loci by assuming that Asplenium enzymes conformed to established models of organellar compartmentalization (Gottlieb, 1982; Gastony and Darrow, 1983; Soltis, 1986: Weeden and Wendel, 1989). Presumed loci were numbered sequentially, with the most anodally VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 89 (i.e., the fastest band) migrating one designated ‘‘1.”’ Similarly, different alleles of the same gene locus (i.e., allozymes, Crawford, 1990) were denoted alphabetically with the most anodal being ‘“‘a Sequencing work was done by C.V. at the Jodrell Laboratory in Kew (United Kingdom). Nineteen cytologically and isozymically interesting, vigorously growing plants, including three A. aureum specimens (CV164, CV670, CV712) from Gran Canaria, Tenerife, and La Palma, one putative A. ceterach from Tenerife (CV187), and one octoploid from La Palma (CV709), were selected to generate DNA sequences from the plastid trnL-trnF intergenic spacer. European material for comparison included two A. javorkeanum specimens from Italy and Slovenia (CV14 and CV85b), two A. ceterach samples from Italy and Cyprus (CV494 and CV225), and a hexaploid A. cyprium plant (CV249) from Cyprus (see Appendix 4). Sequences of the closely related A. Jolegnamense (Gibby and Lovis) Viane from Madeira (CV985 and CV993), of the less related A. dalhousiae Hook. from Ethiopia and Pakistan (CV318 and TR7634), and of the more distantly related A. nidus L. (AF425118), and A. scolopendrium L. and A. unilaterale Lam. (R. Cranfill, University of California, Berkeley, California, USA, unpublished data) were included as outgroups. The trnL-F sequence of a species of Dennstaedtia (R. Cranfill, unpublished data) was used to represent a group basal to the Aspleniaceae (e.g., Bower, 1928; Christensen, 1938; Copeland, 1947; Pichi Sermolli, 1977; Kramer and Green, 1990; Hasebe et al., 1995; Pryer et al., 1995). Methods are explained in Van den heede et al. (2003). RESULTS AND PRELIMINARY DISCUSSION To avoid prolixity, we have combined both the results and the interpretation of the isozyme phenotypes. Chromosomes were counted for 16 specimens collected on Gran Canaria, La Palma, and Tenerife. In addition to five tetraploid A. aureum (n = 72") plants, eleven small specimens that we could not distinguish from European A. ceterach, were examined. Seven of them turned out to be tetraploid (n = 72") and four were octoploid, having a meiotic chromosome number of n = 144" (Fig. 2). Meiosis in all cells examined was regular, showing only bivalents, and giving no indication about the polyploid status of the species. This agrees with Lovis’ (1977) statement that most autopolyploid ferns possess diploidized meiosis (only bivalent formation), and ‘“‘that the absence of multivalents is no valid evidence of allopolyploidy.”’ The counted samples were used as standards to determine the ploidy level of the remaining 146 specimens using a flow cytometer. Results are given in Appendix 1, 2 and 3; localities of cytologically checked material are indicated on the map of the Canarian Archipelago (Fig. 1). In May 1995 (RV6135) and 1997 (CV165-170; CV183-187), we discovered tetraploid A. ceterach specimens on both Gran Canaria and Tenerife (see Appendix 1 and 3). The three species (A. aureum, the “small tetraploid”, and the octoploid) cannot always be distinguished macromorphologically, but can be identified 90 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) r) eae ie ee» 4 a % . os fF & Ty hm @ wae a. & e >> A? ry ty tod 2 ag Pq ed a > r , Hd B’ Fic. 2. Cytology showing spore mother cells in first meiotic division. A, B= photographs; A’, B’ = explanatory diagrams with bivalents in black A, A’: A. ceterach (CV 170b), metaphase I showing n = 72". B, B’: A. octoploideum (CV 188, holotype}, cell showing n = 144”. Scale bar = 10 pm. (preparations, photos and diagrams: C.V.). by measuring exospore length (Table 2). Stomatal guard cell length can only be used to distinguish the ‘‘small tetraploid” (45 + 3.8 um) from the octoploid (52 + 4.8 um). We found no differences in perispore morphology, stomatal type, or epidermal cell pattern. Perispores have costato-cristate folds with few perforations, stomates are mesopolocytic, and epidermal cells mostly sinuous. Polyploidy factors (Viane, 1986, 1990) in A. ceterach are Peet, exo = 1.25 (for the exospore) and Peet, sto = 1.16 (for the stomates), and Paur, exo = 1.18 and Paur, sto= 1.07 in A. aureum. Using these P-values, the theoretical spore and stomate sizes calculated for the octoploid are less than 1 s.d. different from their actual means (Table 2), thus supporting the proposed ancestry (Viane, 1990). Westress the presence of a small indusium in all taxa; it can best be observed in epidermis preparations (Viane, 1990). Our observations show that the (toothed) margin and the “cuticular lines’’ of the scales, are unreliable characters to VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 91 ABLE 2. Microcharacters differentiating taxa within the A. aureum-—ceterach group on the Canary Islands. All measurements are based on cytologically checked material. Additional information about material and the number of measurements is available from the authors. Mean exospore Mean guard cell Taxon Ploidy length + s.d. length + A. 4x 32 + 1.9 pm 46 + 4.4 um A. aun 4x 39 + 2.6 pm 45 + 3.8 pm A: ais 8x 44 + 3.1 um 52 + 4.8 um discriminate A. aureum and relatives from A. ceterach. All taxa have scales with more or less dentate margins, and periclinal cell walls with or without ‘cuticular lines’. These “‘cuticular stripes”’ are folds in the periclinal cell wall (Fig. 3), and the bigger the cell the more folds seem to be present. However, A. aureum scales usually show numerous folds, whereas A. ceterach (from its entire range of distribution) paleae possess only few. In the octoploid the number of folds is usually intermediate between that in A. aureum and A. ceterach. Isozyme analysis can be used to determine whether taxa are auto- or allopolyploid. The electrophoretic phenotype of an autopolyploid should show a subset of the isozymes present in its progenitor, assuming no mutation subsequent to the origin of the polyploid (Weeden and Wendel, 1989; Crawford, 1990; Pryer and Haufler, 1993). An allopolyploid should display fixed heterozygous (i.e., nonsegregating) banding patterns for many loci, resulting from the combination of different parental genomes (Gottlieb, 1982; Werth, et al. 1985b; Pryer and Haufler, 1993; Soltis and Soltis, 2000). Fixed heterozygous banding patterns differ from normal heterozygous zymograms, because the bands do not segregate among progeny and remain fixed in all ee Nineteen enzyme systems were tested in a preliminary survey. The low tee of ALD, and the smeared patterns of XDH made both unusable. IDH, Fic. 3. Folds (“cuticular lines”) in periclinal cell walls of A. aureum paleae. Phase contrast micrographs of goon scales (CV 157). A: scale margin with several cells. B: ingle cell with folds. ar: A= 50 um, B= 92 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) I ll HI IV Vv + a a od ees ae b es ae eee ane as c em ciaiemenel genotypes: bbbc bbbb aaaabbbb aacc aaaa taxa “small tetraploid” “smail tetraploid” “octoploid” A, aureum A, aureum A. celerac. distribution: Canary Islands Canary Islands Canary Islands Canary Islands — Canary Islands Europe Europe Turkey Fic. 4. Diagrams of electrophoretic AAT phenotypes, showing 3 alleles and the corresponding genotypes, as observed in the European—Canarian Asplenium ceterach group. Zymotype II was also found in all samples of A. javorkeanum (bb) and A. cyprium (bbbbbb) examined MDH, ACO, and SOD yielded unclear patterns with limited variation. G-6PD and G-3PDH gave inconsistent zymograms. Consequently, these enzyme systems were not retained. To test our hypothesis that in addition to A. aureum and the octoploid, true A. ceterach occurs on the Canary Islands, the following four enzyme systems were suitable: AAT, SkDH, ME, and PGI. These enzyme systems yielded reproducible, well resolved banding patterns discriminating specimens representing A. aureum, A. ceterach and the allo-octoploid hybrid. These enzymes, encoded by four putative loci, were also used to get an idea of the variation of the species. AAT or GOT: this dimeric enzyme was studied using both PAGE and SGE (system 8 of Haufler, 1985a). We observed only one activity zone, which agrees with Gastony and Darrow (1983), who proved that this single enzyme activity is chloroplastic. Most of the specimens studied are homozygous, showing a single well- resolved AAT band (Fig. 4, zymotype II). This applies to all 50 A. javorkeanum specimens (Italy and Slovenia), most (102) A. ceterach plants (Belgium, Croatia, Cyprus, France, Italy, Slovenia, Spain, Turkey, and the United Kingdom), 23 ‘small tetraploids” from the Canary Islands, and all (44) A. cyprium samples tested (Van den heede et al. 2002). Ten A. ceterach individuals from Croatia, France, Italy, and Slovenia, and two ‘“‘small tetraploids” from the Canary Islands, showed the rarer three-banded zymotype I with skewed staining intensities, interpretable as heterozygous for a dimer. VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 93 Corresponding genotypes are bb, bbbb, or bbbbbb for zymotype II of the homozygous (diploid to hexaploid) plants, versus bbbc for zymotype I of the heterozygous tetraploid specimens. Identical banding patterns in the Canarian ‘small tetraploid” and in European A. ceterach plants indicate that true A. ceterach is growing on the Canary Islands. Because neither European nor Canarian samples reveal unique electrophoretic phenotypes, the Canarian populations do not seem to be ge- netically isolated. Though more sampling of A. javorkeanum is needed, these preliminary results (Fig. 4, zymotype II) seem to confirm the autotetra- ploidy of A. ceterach. The a allele (Fig. 4, zymotype V) can be used as a ‘“‘marker”’ allele characterizing A. aureum. Some plants, showing a single band corresponding to genotype aaaa, are homozygous, whereas others, with a balanced three- banded zymogram corresponding to genotype aacc, are heterozygous. To explain zymotypes I and IV, an extra genotype cc is postulated and expected in A. javorkeanum, which was studied only on the basis of Italian and Slovenian material. All 54 octoploid specimens (from Gran Canaria, La Palma, and Tenerife) show a monomorphic, presumably fixed heterozygous banding pattern of genotype aaaabbbb (Fig. 4, zymotype III), which we postulate to be derived from a combination of zymotypes II and V (Fig. 4). This would agree with the suggestions of Reichstein (1984) and Viane and Reichstein (1992), that the Canarian octoploid is an allo-octoploid, which originated either by chromo- some doubling in an unknown tetraploid hybrid between A. aureum (with zymotype V) and A. ceterach (with zymotype ID), or via unreduced gametes of each species (Fig. 9). The fact that, in all the octoploids (from 15 different localities) only one AAT zymotype was detected can be explained by the preponderance of the ‘‘small tetraploid” with zymotype II. It may also reflect incomplete sampling of the variation present in the octoploid. SkDH: resolution for this monomeric enzyme was superior on SGE (system 2 of Weeden and Wendel, 1989). In our study, the enzyme was represented by a single locus, which agrees with Gastony and Darrow (1983). As expected for a monomeric enzyme, homozygotes had a typical one-banded pattern whereas heterozygotes showed two or more bands. We detected four alleles in the European—Macaronesian Asplenium ceterach—aureum group. Two of these, a and b, were observed in A. aureum, whereas c and d characterized the “A. ceterach’’ group. Although SKDH was polymorphic in A. ceterach (Fig. 5), only zymotype V (cccd) was found on the Canary Islands. This two-banded pattern with unequal staining intensities forms also part of zymotype VI found for all 54 octoploid specimens. Thus the octoploid is monomorphic and presumably heterozygous for this locus, showing a four-banded zymogram corresponding to genotype aabbcccd. The a and b alleles are unique ‘marker’ alleles for A. aureum, one of the progenitors of the octoploid. This monomorphic pattern showing presumed fixed heterozygosity seems to confirm the putative allo-octoploid origin of this species (Reichstein, 1984; Viane and Reichstein, 1992). The cccd SkDH 94 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) I iT il IV v Vi Vil rg a al so b eae ral c a ees ee aa aT - @ ——s oe eee — cece ccdd cddd cccd aabbcccd aabb taxa (abbreviated) “small 4x7 —~octoploid™ A. aur. A. jay. A. cet. A. cel. A. cet. A. cet. distribution: ! Cyprus Italy Croatia Canarian Archipelago Slovenia France Spain Slovenia Croatia Italy Turkey Cyprus Slovenia Italy Fic. 5. Diagrams of electrophoretic SkDH phenotypes, with corresponding genotypes, as observed in the Soave ara i Asplenium ceterach-aureum group. Zymotype I ici also found in some A. javorkeanum (genotype: cc); zymotype V was present in all A. cyprium (genotype: ccecdd) samples checked. The Canarian small tetraploid is abbreviated as: “small 4x.’ genotype (zymotype V) of the ‘‘small tetraploids”’ is not limited to the Canaries, but was also found in A. ceterach from Croatia, Cyprus, and Italy. These results again both prove the occurrence of A. ceterach on the Canary Islands, and the fact that the populations in this Archipelago are not genetically isolated. Three additional zymotypes were detected in continental A. ceterach: a single- banded (cccc), a balanced two-banded (ccdd), and an unbalanced two-banded pattern (cddd). The presence of the unbalanced patterns (cccd, cddd) in tetraploid A. ceterach at Skdh can be explained by tetrasomic inheritance (see discussion). Diploid A. javorkeanum from Italy and Slovenia showed a single- banded pattern of either genotype cc (zymotype II) or dd (zymotype J). ME: this tetrameric enzyme was studied only by PAGE. The single enzyme activity visible was shown to be cytosolic by Gottlieb (1982), Gastony and Darrow (1983), and Soltis (1986). was monomorphic in each of the three species, and thus can be used to distinguish them from each other (Fig. 6). All A. ceterach specimens (Europe) and ‘‘small tetraploids’’ (Canary Islands) were heterozygous showing an identical five-banded zymogram, typical for a tetrameric enzyme controlled by one locus with two alleles, a and d. Heterozygous A. aureum was characterized by a five-banded pattern controlled by the same locus, but with two different alleles, b and c, and corresponding to genotype bbcc. All octoploid plants VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 95 I x Ul —> =i aaaa af 2 ea oe oe scoaneeetinn (nn bbbb b bbbb ee ee ‘anaes —- — becc ane — aaad = — becc mee aadd — Wee pac cams DDEd ecm ee —— beec a c — addd — ccce — ams a = am accc + bbdd ae | —_ dddd ome — becc genotypes bbcc abbccddd —_— addd + eeee =——— | taxa — coon “small 4x” A. aqureum “octoploid” — (coud A. ceterach ae distribution: — dddd anarian Archipelago E Turkey Fic. 6. Diagrams me ees the ME zymotypes showing 4 alleles, with corresponding cee ti as observed in the European—Canarian Asplenium ceterach-aureum group. For each band fou letters — association of an eerie by alleles) joined to form this bia enzyme. The homotetramers in the brid’’ pattern are in boldface. Zymotype III confirms the allopolyploid status of the anions Dotted iaee indicate very faint bands. The Canarian small tetraploid is abbreviated as “small 4 showed a complex zymogram, and conform to the expected hybrid phenotype resulting from the cross between A. ceterach and A. aureum. The “hybrid” had the four parental alleles, and since ME is a tetramer, each of the six pairs of alleles (a X b, aX c, aX d, bX c, b X d, c X d) formed three heterotetramers of intermediate mobility. Theoretically this results in a 22-banded pattern (4 homotetramers plus 6 X 3 = 18 heterotetramers, makes 22 bands), but because twice two bands have the same mobility, a maximum of 20 bands was visible (Fig. 6). The monomorphic and presumably fixed banding pattern of the “hybrid” zymogram is in agreement with the putative allopolyploid origin of the octoploid (Reichstein, 1984; Viane and Reichstein, 1992). PGI: this dimeric enzyme was studied by both PAGE and SGE. Because the resolution was much better with SGE, all results shown were obtained using starch gel electrophoresis (system 6 of Soltis et al., 1983 Two loci were present: Pgi-1, most probably chloroplastic, and Pgi-2, cyto- 96 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) Pgi-2 Il TI IV Vv VI I (I x TI) (x ID ? + a imme: 68 — aa — aa mae = AC mee §=6aC b — ad == ad Cc me = CC mm =(CC me CC — cc mmm = CC ce ein ed ed ean ed a ced d =_— dd ame CO+dd a dd ame «dd —_— dd seamen de _ Sc seman ee genotypes: aacc ccc ccdd cdde eceeccdd aaccecdd aaaadddd taxa: all 4x” A, aureum A. aureum “octoploid” “octoploid” — “octoploid” A. ceterach A. ceterach distribution: CC .4°2) 2 ff 2 a. i Aer Ge a i ap Cede 8 age 26) Italy Europe IG. Diagrams of electrophoretic Pgi-2 phenotypes, — 5 alleles, with gettin genotypes and their distribution, as observed in the European—Canarian Asplenium ceterach— aureum group. Zymotype II was also ‘soa in most A. kaa (cc) i Each band is indicated by two letters representing the association of subunits, pees to form this dimeric enzyme. The Canarian small tetraploid is abbreviated as ‘‘small 4x solic (Gastony and Darrow, 1983; Soltis, 1986). Consistent with observations on other ferns (Gastony and Gottlieb, 1985; Werth, 1991; Haufler et al., 1995; Hauk and Haufler, 1999), resolution of the more anodal locus Pgi-1 was inferior to that of Pgi-2. Because Pgi-1 appears invariant across all taxa, it will not be discussed. Among the European and Macaronesian samples studied, five allozymes were observed at Pgi-2 (Fig. 7). Although the continental A. ceterach, with its six different zymotypes, was highly polymorphic for this locus (Van den heede et al., 2002), only a single banding pattern was detected for the 22 ‘small tetraploids’’ from the Canary Islands, corresponding to genotype cccc. This widely distributed zymotype was also found in A. ceterach specimens from Belgium, Croatia, France, Italy, Slovenia, Spain, and the United Kingdom. We obtained two electrophoretic phenotypes for the 28 A. aureum plants, with corresponding genotypes ccdd (25 specimens) and cdde (3 specimens). The octoploid was the most variable taxon in the Canarian Archipelago, showing three different zymotypes translated into genotypes (Fig. 7) ccccccdd (zymotype V), aaccccdd (zymotype VI), and aaaadddd (zymotype VII). Zymo- type V (found only on La Palma) most probably resulted from hybridization VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 97 TABLE 3. Bc ee accession numbers for trnL-trnF nucleotide sequences of newly sequenced Asplenium specimens. CV and TR are abbreviations es gia Van den heede and Tadeus schol maaide Localities are given in Appen Voucher GenBank accession Species number Data of collection number A. aureum CV164 25 May 1997 AY160993 CV670 12 Jan. 1999 AY160994 CV712 3 Apr. 1999 AY160995 A. ceterach CV187 27 May 1997 AY162333 CV225 11 June 1997 AY162334 CV494 17 Aug. 1998 AY162335 A. cyprium CV249 12 June 1997 AY162337 A. dalhousiae CV318 13 Jan. 1998 AY161000 TR7634 27 Aug. 1990 AY161001 A. javorkeanum CV14 24 July 1996 AY162330 CV85 30 Aug. 1996 AY162331 A. lolegnamense CV985 29 May 2000 AY160998 CV993 1 June 2000 AY160999 A. octoploideum CV709 2 Apr. 1999 AY161003 between a “small tetraploid’? with genotype cccc and an A. aureum with genotype ccdd, which are both abundantly present on the Canaries, followed by chromosome doubling, or via unreduced gametes of each species. Zymotype I, though presently known only from Italy, can be used to explain zymotype VI, which was found only on La Palma. Octoploids with this genotype (aaccccdd), expressed three homodimeric bands (Fig. 7, aa, cc, dd) plus three hetero- dimeric bands (ac, ad, cd). More sampling is desirable and ue detect other genotypes such as aacc in the “small tetraploid,”’ as well as dddd needed to explain zymotype VII from Gran Canaria and Tenerife. Pgi-2 suggests that the formation of the allo-octoploid happened at least three times. Because plastid DNA is uniparentally inherited, it discloses only the maternal lineage (Stein and Barrington, 1990; Gastony and Yatskievych, 1992). GenBank accession numbers for trnL-trnF nucleotide sequences of newly sequenced specimens are listed in Table 3. Analysis of the plastid trnL-trnF intergenic spacer sequences resulted in the clustering of the “small tetraploid” Tenerife (CV187), A. ceterach from Italy and Cyprus, and A. cyprium, with their diploid ancestor A. javorkeanum (Fig. 8). We found no chloroplast variation (with the exception of CV494) between specimens sampled from the Mediterranean (Cyprus, Italy, Slovenia) and Tenerife. Asplenium javorkeanum, A. ceterach, and A. cyprium form a cluster of their own, different from the ‘‘A. aureum clade,” which includes all the A. aureum specimens, A. lolegnamense, and the octoploid (CV709) from the Canaries. Identical groups are obtained by analysing rbcL gene sequences. The position of A. lolegnamense and the octoploid, in the plastid trees, suggests that A. aureum acted as the maternal parent in the formation of the specimens used. These molecular data independently prove that in addition to an octoploid species, true A. ceterach 98 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) A. aureum CV 164 Gran Canari A, aureum CV670 Tenerife A. M octoploid CV709 La Palma 9 1A. dalhousiae CV318 Ethiopia 100 | A. dalhousiae TR7634 Pakistan 7 A. javorkeanum CV 14 ie 2_| tetraploid ec 6 100 7 ceterach CV225 Cyprus 70 A. ceterach CV494 Italy a A. cyprium CV249 Cyprus | te | — A. scolopendrium 37 A. nidus Bar| A. unilaterale Dennstaedtia — 5 changes Fic. 8. Tree randomly selected from the 73 shortest trees of European—Canarian Asplenium ceterach—aureum taxa and A. dalhousiae, resulting from parsimony analysis of our 14 trnL-trnF intergenic spacer pik nea (Table 3) and 4 trnL-trnF sequences of other species available in GenBank; length = 227 steps, CI= 0.92, and RI = 0.91. Based on rbcL evidence (Hasebe et al., 1995; Pryer et al., 1995), = sequence of the more distantly related Dennstaedtia was specified as outgroup. Fitch branch lengths (ACCTRAN optimized) are shown above and bootstrap percentages (1000 replicates) below the branches. Other sequencing results are described extensively in Van den heede et al. (2003). (= the ‘‘small tetraploid’’) is growing on the Canary Islands. Other sequencing results are described extensively in Van den heede et al. (2003) Because Gran Canaria, La Palma, and Tenerife are of volcanic origin, these epilithic ferns mainly grow on rocks of basaltic types, like phonolites, rhyolites, trachytes and olivine basalts (Page, 1979). Asplenium aureum prefers moister, shady habitats at lower altitudes, whereas the “small tetraploid” and octoploid plants share more exposed, drier habitats. However, on both Gran Canaria and Tenerife, only single localities were found where ‘‘small tetraploids” and octoploids grew together (loc. 2 and 19; see Appendix 1 and 3). Though forty-five specimens from nine different localities on La Palma were cytologically checked (see Appendix 2 and Van den heede and Viane, unpublished data), we could not detect any “small tetraploid” specimen. We intensively looked for it in the field, especially in the higher regions of La Palma. Whereas we discovered “‘small tetraploids” between 1500 and 1900 m altitude on Gran Canaria and Tenerife, the highest altitude we found ferns of the Ceterach group on La Palma was near 1200 m We found A. aureum between 300 and 1000 m altitude in valleys and sheltered ravines (‘‘barrancos’’) with remnants of (degraded) evergreen laurel VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 99 forest dominated by broad-leaved trees: Laurus azorica (Seub.) Franco, Persea indica (L.) Spreng., Ocotea foetens (Aiton) Berthel., Apollonias barbujana (Cav.) Bornm., Ilex canariensis Poir., Ilex platyphylla Webb and Berthel., and Arbutus canariensis Vieill. (Bramwell and Bramwell, 1974). Asplenium aureum usually grows in humus-rich soils, often together with Adiantum capillus-veneris L., A. reniforme L., Anogramma leptophylla (L.) Link, Asplenium aethiopicum (Burm.f) Bech., A. hemionitis L., Cheilanthes pulchella Bory ex Willd., Davallia canariensis (L.) Sm., Polypodium cambricum L. ssp. macaronesicum (A.E. Bobrov) Fraser-Jenk., and Selaginella denticulata (L.) Spring. Asplenium ceterach and A. octoploideum were found in the natural pine forests (‘‘Pinar’’) at 900-2000 m on Tenerife, and at 1200-1600 m on Gran Canaria. The octoploid was observed on La Palma at 700-1200 m. The open savannah-like vegetation is dominated by Pinus canariensis C. Sm. and a few shrubs, such as Adenocarpus foliolosus (Aiton) DC., Cistus symphytifolius Lam., Daphne gnidium L., Micromeria species, and Rumex lunaria L. (Bramwell and Bramwell, 1974). Asplenium ceterach and the octoploid grow in rock fissures, often together with Monanthes laxiflora (DC.) Bolle, Aeonium species, Asplenium aethiopicum, A. trichomanes L., Anogramma leptophylla, Cheilanthes guanchica Bolle, C. pulchella, Cosentinia vellea (Aiton) Tod., Notholaena marantae (L.) Desv. subsp. subcordata (Cav.) G. Kunkel, and Polypodium cambricum ssp. macaronesicum. Where A. ceterach and A. octoploideum grow together abundantly, we discovered their sterile hexaploid hybrid, A. Xchasmophilum Van den heede and Viane (Van den heede and Viane, 2002) DISCUSSION In combination with morphological, cytological, and biogeographical data, isozyme markers can determine whether taxa are auto- or allopolyploid (Crawford, 1985; Haufler, 1985b; Bryan and Soltis, 1987; Weeden and Wendel, 1989; Crawford, 1990; Gastony, 1990; Pryer and Haufler, 1993). Electrophoretic analysis of isozymes is an ideal way to investigate the origin of allopolyploid taxa because parental loci are expressed as stable marker bands in the progeny (Haufler, 1985b; Werth et al., 1985b; Gastony, 1986). The potential of isozyme data to clarify relationships in fern complexes is dependent upon the degree of differentiation among the ancestral genomes. In the present study, four loci (Aat, Skdh, Me, and Pgi-2) showing a unique set of bands characterizing A. aureum and different from the banding patterns found in European A. ceterach, proved adequate to disentangle the “Ceterach” complex in the Canarian Archipelago. All zymograms present in the Canarian “small tetraploid”, were also observed in A. ceterach, and confirm that true A. ceterach is growing on the Canary Islands. Moreover, this suggests an occasional spore flow from Europe towards the Canaries. A flow in the opposite direction is less likely because the western islands are dominated by the northeast trade wind system. Conse- 100 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) quently, the Canarian A. ceterach population cannot be considered genetically isolated. No local zymotypes seem to have originated in this taxon in the Canary Islands, contrary to the situation on Cyprus, which is much older than the Canary Islands (Van den heede et al., 2002). For example, the unique Tpi-2 zymogram, present in all A. ceterach and A. cyprium specimens from Cyprus, suggests the local origin of the Cypriot taxa (Van den heede et al., 2002). All four loci (Aat, Skdh, Me, and Pgi-2) of the Canarian octoploid show monomorphic heterozygosity for a combination of the patterns seen in A. ceterach and A. aureum. Our allozyme data confirm the allo-octoploid nature of this species, which most probably originated by chromosome doubling in a tetraploid hybrid between A. aureum and A. ceterach (Viane and Reichstein, 1992). Theoretically, though less parsimoniously, the formation of this taxon could also happen directly via the union of unreduced (4x) gametes (on gametophytes resulting from unreduced spores) of both species. All allozymes observed in the allo-octoploid were electrophoretically identical to those found in the parental tetraploids. However, in some octoploid samples from Gran Canaria and Tenerife, Pgi-2 expressed a zymotype (corresponding to genotype aaaadddd) resulting from the combination of two undetected genotypes (aaaa) in A. ceterach and (dddd) in A. aureum. The occurrence of this putative ‘‘orphan” genotype may reflect incomplete sampling of the variation present in the tetraploids, or alternatively these genotypes may no longer be present in extant A. ceterach and A. aureum specimens. The variation in the allo-octoploid seems to be related to the mono- or ’ polymorphism (and its abundance) in the parental tetraploids. Thus, at the two loci (Skdh and Me) showing a single octoploid genotype, only one genotype was observed in each of the Canarian parents. At Aat two different genotypes were found for the Canarian A. ceterach, though only a single zymotype was detected for the octoploid. However, the A. ceterach genotype not detected in any octoploid, was found in only ca. 10% of the population. On the other hand, this may also be the result of limited sampling of the variation present in the octoploid. The allo-octoploid species showed three different isozyme profiles at Pgi-2, a locus that is polymorphic in its tetraploid progenitors, indicating that the octoploid probably originated at least three times. According to Werth et al. (1985a) such patterns of variation in allopolyploids are almost certainly the result of repeated allopolyploidizations involving pairs of different genotypes. Thus, each of the octoploid zymotypes may have arisen from a separate hybridization event. The present observations, demonstrating multiple origins of allopolyploids, are similar to those of Werth et al. (1985a, b) for Asplenium, Soltis et al. (1987) for Polystichum, and Ranker et al. (1989) for Hemionitis. Recurrent origins of the allo-octoploid species implicate a repeated gene flow from tetraploids to octoploids, and mean a continued gain of genetic diversity by the allopolyploid. Our electrophoretic data also provide evidence for the operation of tetrasomic inheritance in natural populations of autotetraploid (Rasbach et VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 101 al., 1987) A. ceterach. At Skdh, for which only two allozymes were observed, three types of heterozygotes were present: balanced heterozygotes (ccdd) and two types of unbalanced heterozygotes (cccd, cddd). The presence of these three types in tetraploid A. ceterach at Skdh is suggestive of the three possible classes of heterozygotes expected in an autotetraploid at a locus having two alleles (Weeden and Wendel 1989). Unbalanced staining activities indicate multiple doses of individual alleles. Tetrasomic inheritance with chromatidal segregation explains the arrays of homozygous, balanced heterozygous, and unbalanced heterozygous banding patterns observed in A. ceterach (Weeden and Wendel 1989). Tetrasomic inheritance implies that a chromosome can pair with any of its three homologous chromosomes (e.g., Soltis and Rieseberg, 1986; Weeden and Wendel, 1989; Crawford, 1990), and that there is apparently no strict preferential chromosome pairing. Consequently, the present isozyme analysis confirms the autotetraploid status of A. ceterach, which was cytologically proven by Rasbach et al. (1987). The fact that isozyme studies point to tetrasomic inheritance and that we found only bivalents during meiosis in autotetraploid A. ceterach, suggests that both processes are controlled by different (sets of) genes. Similar unbalanced patterns found in allotetraploid ferns have been explained also by segregating intralocus heterozygosity and fixed interlocus heterozygosity (Gastony, 1990). We were able to prove, by isozyme and plastid DNA analysis, that in addition to A. aureum and the octoploid, true A. ceterach occurs on Gran Canaria and Tenerife. A combination of morphological and cytological analysis leads to correct determination, but even the exospore length alone allows reliable identification of the three Canarian species: A. aureum (32 + 1.9 um), A. ceterach (39 + 2.6 wm), and the octoploid (44 + 3.1 pm). As mentioned in the introduction, Ben] and Kunkel (1967) published C. aureum var. parvifolium without cytological investigation. Plants collected in 1967 by T. Reichstein and G. Kunkel were found to be octoploid, leading T. Reichstein and other European pteridologists to attribute octoploid status to A. parvifolium (including all small Canarian “Ceterach” specimens), but without having checked the holotype. We repeatedly visited the type locality of A. parvifolium, the Pinar above Vilaflor (Tenerife), and found several taxa (see Appendix 3) growing together. As soon as we were convinced that two kinds of ‘‘small Ceterach”’ species were growing at the locus classicus (and on Gran Canaria), we decided to study the holotype (Ben! s.n., 26/12/1966, M) of C. aureum var. parvifolium. This holotype consists of one single plant. We studied its microcharacters (see also Table 4) and found a mean exospore length (38 + 2.8 um) and very few folds (‘‘cuticular lines’) in the scales characteristic for true A. ceterach. The values for the exospore and stomate length (38 + 3.7 um) prove that the holotype was not an octoploid, but a tetraploid plant! Consequently, C. aureum var. parvifolium Benl and G.Kunkel and A. parvifolium are synonyms of A. ceterach, and the octoploid had no correct name and was described as A. octoploideum (Van den heede and Viane, 2002). Asplenium octoploideum is morphologically intermediate between A. aureum and A. ceterach, from which 102 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) . Comparison of mean exospore length (LEXO) of various types to that of cytologically checked vouchers (see Table 2). LEXO = s.d. LEXO = s.d. Taxon Voucher, status, and herbarium (types) (cytol. checked) A. aureum Broussonet s. n., iso-:P 3t > 1.6 am 32. 1.9 pm A. ceterach Hort. Cliff. Aspl. 4, lecto-: BM 39 + 2.8 um 39 + 2.6 pm A. parvifolium Benl s. n., holo-: 38 + 2.8 um A. octoploideum CV 188, holo-: GENT 42 + 3.4 um 44 + 3.1 um it can be distinguished by its different mean exospore length (44 um) and mean stomate length (52 pm), and its octoploid chromosome number n = 144" (Fig. 2B + B’). It is endemic to the Canarian Archipelago but presently confirmed only (cytology) for Gran Canaria, La Palma, and Tenerife, and to be expected on La Gomera and El Hierro. In addition to the holotype from Gran Canaria [lava field near Cueva Corcho, in fissures of volcanic rocks, 1350 m alt, 28th May 1997, leg. Van den heede and Viane CV 188 (Holo-: GENT, iso-: personal herbarium of Viane and Van den heede)], the following collections (paratypes) were also made (for localities see Appendix 1, 2, and 3): CV 171, CV 172, CV 173, CV 175, CV 176, CV 177, CV 179, CV 672A+B, CV 674, CV 686, CV 687, CV 695, CV 708, CV 709, CV 715, CV 716, CV 717, CV 718, CV 719A+B, CV 720, CV 721, CV 723, CV 724, CV 725, CV 726, CV 727, CV 729, CV 730A+B, CV 731, CV 732, CV 733, CV 734, CV 740, CV 741, CV 742, CV 743, CV 744, CV 745, CV 746, CV 747, CV 748, CV 749, CV 750, CV 751A+B, CV 752, CV 754A+B+C, WB 22/93. Many herbarium specimens still need to be inspected before the ranges of the “small Canarian Ceterach’’ taxa can be established. Literature references and information on herbarium labels are often unreliable, e.g., Bornmtuller 3094 (P), labeled as C. officinarum f. typica (cellulis palearum non striatis!) collected on Gran Canaria, has a mean exospore length of 44 + 3.0 um and numerous folds in the scale cells: it is without any doubt an octoploid. As far as is known, the octoploid is endemic to the Canary Islands, but because Madeira could also harbor this species (climatologically and topographically), we studied 30 specimens from five Madeiran localities. However, all of them turned out to be hexaploid and were identified as A. lolegnamense. Both in the Madeiran and the Canarian Archipelago the northeast trade wind prevails during the year. This phenomenon may help to explain the restricted range of several Macaronesian taxa, because most propagules fall into the Atlantic Ocean. Even when spores occasionally reach the African continent, the Western Sahara and Mauritania offer no appropriate habitats for these ferns, because of their ultra dry climate. The derivation we hypothesize for the allo-octoploid species is presented in Fig. 9. It is generally accepted (e.g., Burchard, 1929; Lems, 1960; Page, 1973; Bramwell and Bramwell, 1974; Page, 1977, 1979) that many of the Canarian ferns are endemic relicts of the Tertiary fern flora that existed in southern Europe during the Miocene and Pliocene. These ferns form an important part of the original vegetation of the Canary Islands, especially of the evergreen VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 103 bridization followed by chromosome doubling OR via unreduced gametes 4 ee A. ceterach A. aureum A. octoploideum JSIJ JIXX Fic. 9. Scheme of relationships explaining the origin of the Canarian Asplenium octoploideum (Van den heede et al., 2003) suggest that A. aureum is an allotetraploid, involving A. javorkeanum (JJ) and ‘‘A. semi-aureum” (XX, unknown) as ancestors, its genome formula is given as JJ forests (Page, 1977). Unfortunately, these habitats, if not totally destroyed today, are greatly endangered by modern tourism (building, water supply). Several mountain areas need further research, and new species and hybrids await description. Undoubtedly, this Tertiary (fern) flora forms an irreplace- able genetic resource that should be conserved. ACKNOWLEDGMENTS We are very grateful to L. Triest (Free University, Brussels), M. Chase (Jodrell Laboratory, Royal Botanic Gardens Kew, UK), and R. Johns (K) for providing laboratory facilities, and to the curators of various herbaria (BM, M, P) for permission to study type material. We tha providing trnL-F sequences of some outgroups; G. Van der Kinderen for his technical assistance GE) =] ~_ zA oF hy ies) | eh S 5 cultivating sporophytes. We thank W. Bennert for extra material from Turkey, and P.S. and D.E. Soltis for a and encouragements, T. Ranker and an anonymous reviewer for constructive comments on the manuscript. The management of Ghent Botanical Garden (Belgium) kindly provided slanditciie facilities. LITERATURE CITED Acquaan, G. 1992. Practical protein electrophoresis for genetic research. Dioscorides Press, Portland, Oregon BENL, G. and G. Kuna. 1967. Zur ee der Gattung Ceterach auf den Kanarischen Inseln. Ber. Schweiz. Bot. Ges. 77:257—265. BENL, G. and E. R. SVENTENIUS. 1970. ett zur Kenntnis der Pteridophyten-Vegetation und -Flora 104 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) n der aaa Westprovinz (Tenerife, La Palma, Gomera, Hierro). Nova Hedwigia moe 413 Sanam Bir, S. S., C. R. FRraser-Jenxins, and J. D. Lovis. 1985. 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Voucher Meiotic chromosome number Taxon Locality _—_ Date of collection (n), or ploidy CV 157 A. aureum 1 25 May 1997 72 CV 158 A, aureum 1 25 May 1997 4x CV 159 A, aqureum 1 25 May 1997 CV 160 A. aureum 1 25 May 1997 Py CV 161 A. aureu 1 25 May 1997 72 CV 162 A. aureum 1 25 May 1997 4x CV 163 A. aureum 1 25 May 1997 CV 164 A. aureum 1 25 May 1997 7a" CV 165 A. ceterach 2 25 May 1997 72" CV 166 A. ceterach 2 25 May 1997 4x CV 167 A. ceterach 2 25 May 1997 4x CV 168 A. ceterach Z 25 May 1997 CV 169 A. ceterach 2 25 May 1997 4x CV 170a A. ceterach 2 25 May 1997 x CV 170b A. ceterach 2 25 May 1997 7 CV 171 A. octoploideum 2 25 May 1997 8x CV 172 A. octoploideum 2 25 May 1997 8x CV 173 A. octoploideum 3 26 May 1997 8x CV 175 A. octoploideum 3 26 May 1997 8x CV 176 A. octoploideum 3 26 May 1997 8x CV 177 A. octoploideum 3 26 May 1997 8x CV 179 A. octoploideum a 26 May 1997 CV 188 A. octoploideum 3 28 May 1997 144” CV 180 A. aureum 4 26 May 1997 4x CV 181 A.a 4 26 May 1997 4x CV 182 A. aureum 4 26 May 1997 VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 109 AprenpDix 2. Vouchers from La Palma, with corresponding taxa, locality numbers, and chromosome numbers. CV is the Simatic for Caroline Van den heede. The description of the localities and er number 4 is “aa in Material and Methods. For oe — chromosomes counted, the number ven. Counted sonia level (indicated by 4x and to by flow cytometry. All specimens were used for isozyme analysis Voucher Meiotic chromosome number number Taxon Locality —_ Date of collection (n), or ploidy CV 708 A. octoploideum 5 2 Apr. 1999 8x CV 709 A. octoploideum 5 2 Apr. 1999 144" CV 711 A. aureum 6 3 Apr. 1999 4x CV 712 A. aureum 6 3 Apr. 1999 4x CV 713 A. aureu 6 3 Apr. 1999 CV 715 A. octoploideu 7 4 Apr. 1999 8x CV 716 A. octoploideum 7 4 Apr. 1999 8x CV 7I7 A. octoploideum 8 4 Apr. 1999 8x CV, 718 A. octoploideum 8 4 Apr. 1999 8x CV 719A _ A. octoploideum 8 4 Apr. 1999 8x CV 719B A. octoploideum 8 4 Apr. 1999 8x CV 720 A. octoploideum 8 4 Apr. 1999 8x CV 721 A. octoploideum 8 4 Apr. 1999 8x CV 723 A. octoploideum 9 4 Apr. 1999 8x CV 724 A. octoploideum 8 4 Apr. 1999 8x CV 725 A. octoploideum 10 4 Apr. 1999 8x CV 726 A. octoploideum 10 4 Apr. 1999 8x CV 727 A. octoploideum 10 4 Apr. 1999 8x CV 729 A. octoploideum 11 5 Apr. 1999 8x CV 730A A. octoploideum 11 5 Apr. 1999 8x CV 730B _ A. octoploideum 11 5 Apr. 1999 CV 731 A. octoploideum 12 5 Apr. 1999 144" CV 732 A. octoploideum 12 5 Apr. 1999 8x CV 733 A. octoploideum 12 5 Apr. 1999 8x CV 734 A. octoploideum 12 5 Apr. 1999 8x CV 740 A. octoploideum 13 9 Apr. 1999 8x CV 741 A. octoploideum i 9 Apr. 1999 8x CV 742 A. octoploideum 13 9 Apr. 1999 8x CV 743 A. octoploideum 13 9 Apr. 1999 8x CV 744 A. octoploideum 13 9 Apr. 1999 8x CV 745 A. octoploideum 13 9 Apr. 1999 8x CV 746 A. octoploideum 13 9 Apr. 1999 8x CV 747 A. octoploideum 13 9 Apr. 1999 8x CV 748 A. octoploideu 13 9 Apr. 1999 8x CV 749 A. octoploideum 13 9 Apr. 1999 8x CV 750 A. octoploideum 13 9 Apr. 1999 8x 110 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 2 (2004) APPENDIX 3. Vouchers from Tenerife, with corresponding taxa, locality numbers, and chromosome specimens served as standards to determine the ploidy level (indicated by 4x and 8x) by flow cytometry. All specimens were used for isozyme analysis. Voucher number Taxon Locality Date of collection n or ploidy RV 6135 A. ceterach 14 7 May 1995 CV 183 A. ceterach 14 27 May 1997 ry CV 184 A. ceterach 14 27 May 1997 2 CV 185 A. ceterach 14 27 May 1997 CV 186 A. ceterach 14 27 May 1997 = CV 187a A. ceterach 14 27 May 1997 12° CV 187b A. ceterach 14 27 May 1997 4x CV 187c A. ceterach 14 27 May 1997 4x CV 663B A. ceterach 14 11 Jan. 199 4x 665 A.a m 15 12 Jan. 1999 4x CV 666 A. aureum 15 12 Jan. 1999 4x CV 667 A. aureum 16 12 Jan. 1999 CV 668 A. aureum 16 12 Jan. 1999 CV 669 A. aureum 16 12 Jan. 1999 CV 670 A. aureum 16 12 Jan. 1999 72" 671 A. aureum 16 12 Jan. 1999 4x CV 672A A. octoploideum 17 13 Jan, 1999 8x CV 672B A. octoploideum 17 13 Jan. 1999 8x CV 674 A. octoploideum 47 13 Jan. 1999 8x CV 675 A. aureum 18 14 Jan. 1999 4x CV 676 A. aureum 18 14 Jan. 1999 4x CV 677 A. aqureum 18 14 Jan. 1999 4x CV 678 A.a m 18 14 Jan. 1999 4x CV 683 A. ceterach 19 15 Jan. 1999 4x CV 684 Av€ 19 15 Jan. 1999 4x CV 686 A. octoploideum 19 15 Jan. 1999 8x CV 687 A. octoploideum 19 15 Jan. 1999 8x CV 695 A. octoploideum 19 15 Jan. 1999 8x CV 696 A.C 19 15 Jan. 1999 4x 701 A. ceterach 20 15 Jan. 1999 4x CV 702A A. ceterach 20 15 Jan. 1999 4x CV 702B A. ceterach 20 15 Jan. 1999 4x CV 704 A. cet A 20 15 Jan. 1999 4x CV 705 A.a 21 16 Jan. 1999 4x CV 706 A. aureum Zz 16 Jan. 1999 4x CV 707 A.a 21 16 Jan. 1999 4x CV 751A A. octoploideum Pie 10 Apr. 1999 8x CV 751B A. octoploideum 22 10 Apr. 1999 CV 752 A. octoploideum 22 10 Apr. 1999 8x CV 754A A. octoploideum 23 10 Apr. 1999 8x CV 754B A. octoploideum 23 10 Apr. 1999 8x CV 754C A. octoploideum 23 10 Apr. 1999 8x WB 22/93 A. octoploideum 24 15 Apr. 1993 144" VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP 111 1x 4. Alphabetical list of mat lf this study. CV, RV, TR, and WB are Sie of C. Van den heede, R. Viane, T. Reichstein, and W. Bennert. Vouchers are deposited in GENT and in our personal herbarium at Ghent University. Additional information about vena ties is available from the first and the last author (lienvdheede@hotmail.com; ronnie.vian UGent.be). Voucher information about 108 Cypriot samples is given in Van den heede et al. a Asplenium ceterach: CV25b+ CV25b, cvs0a+b CV31 CV36a+ CV. CV41,CV42b, CV44,CV48, CV49b CV64, CV65, CV66, CV67, CV494 CV225 oie CV276; GV277 CV278, sain CV282, — CV285, CV286 V659 CV767, CV768, CV769, CV770, CV771 CV774, CV775, CV776 WB5c/97 WB12c+d/97 Asplenium cyprium: CV249 Asplenium dalhousiae CV318 TR7634 Asplenium javorkeanum: , CV4, CV7a+b+c, CV8a+b+c CV10, CV11, CV12, CV14, CV412, CV414 CV20a+b+c+d, CV21a+b+c+d+e CV81, CV82a+b, CV83, CV84, CV85a+b CV86, CV87, CV88 CV89, CV90, CV91, CV92, CV93 CV94a+b, CV95 CV404, CV405 CV410 CV480 CV483, CV484 CV504, CV506 Asplenium lolegnamense: CV985 CV993 Slovenia, Kal Croatia, Roé Croatia, Bassania Slovenia, Korte Italy, Valle della Marossa Italy, Termine di Roverano Cyprus, Troodos Mts., Chandria Belgium, Marcourt Italy, Cannero Riviera United Kingdom, Wales, Snowdonia Crotia, Losin} Italy, Berceto Italy, Boio Spain, Torrelodones Vv ul France, NNE of Montpellier, La Pene Turkey, Karaoba Turkey, Manisa Turkey, Okgular Turkey, Mugla Cyprus, Troodos Mts., Tsakistra-Vroiska road Cyprus, Kyrenia Mts., Kyrenia-Kythrea road Ethiopia, Harerge Province, Asbe Teferi Pakistan, Swat Province, Ambela Italy, Stupizza Slovenia, 1 km E of Bata towards Podbrdo Slovenia, Bata-valley, Kneza-Klavze road eee ‘inde taly, a ria Freddone, 730m Slovenia, Ljubinj Italy, N slope of Pania Secca Italy, Fosso di Antona Italy, E slope of Monte Corchia Madeira, SW slope of Pico Ruivo Madeira, N of Serra de Agua American Fern Journal 94(2):112 SHORTER NOTES Nomenclatural Corrections in Adiantum.—Two Brazilian species of Adian- tum recently published by Prado in two different papers and journals need corrections. It has been brought to my attention that Adiantum pulcherrimum Prado is a later homonym of A. pulcherrimum Copel. (Philipp. J. Sci. C. 6:138, tab. 22. 1911), a fern that occurs in Borneo. Consequently, a new name is required for the Brazilian species: Adiantum mynssenae Prado, nom. nov. Replaced synonym: Adiantum pulcherrimum Prado, Amer. Fern J. 93:76, fig. 1. 2003, non Copel. (1911). The species is named for Claudine Mynssen, currently doing research on pteridophytes at the Jardim Botanico do Rio de Janeiro (RB Herbarium) and collector of the holotype. During my last visit to RB she called my attention to this beautiful species from the Atlantic forest. It is in recognition of her friendship and invaluable assistance that I name it for her. Adiantum giganteum was published by Prado (Fern Gazette 16:209, figs. 1, 2. 2001), based on a holotype collected by Ynes Mexia from the state of Para, Brazil, and cited as Mexia 6031 (UC). However, the correct number of the holotype is Mexia 6013 (UC). I thank Alan R. Smith and Layne Huiet, both UC, for directing my attention to these problems and for Smith’s suggestions on an initial draft of this note.—JEFFERSON Prapo, Instituto de Botanica, C. P. 4005, 01061-970, Sao Paulo — SP, Brazil. vit 753 INFORMATION FOR AUTHORS Authors are encouraged to submit manuscripts pertinent to pteridology for pub- lication in the American Fern Journal. Manuscripts should be sent to the Editor. 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 acceptability 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. 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Inquiries should be addressed to the Secretary. ‘VISIT THE AMERICAN FERN SOCIETY’S WORLD WIDE WEB HOMEPAGE: -“http:/ Td. wr me : : org/ gk RI9G AM ERICAN Volume 94 FERN Number 3 JOURNAL See QUARTERLY JOURNAL OF THE AMERICAN FERN SOCIETY Distribution, Ecology and Cytology of gas pings azoricum Lovis, Rasbach & Reichstein (Aspleniaceae, Sepoes hyta) and Its H Fred Rumsey, Stephen russell Hanno Schdfer and Helga Rasbach 113 Phylogenetic Relationships of the Subfamily Taenitidoideae, Pteridace Patricia cine Baracaldo 126 A Contribution to the Gametophyte Morphology and Development in Several Species of Thelypteris, Thelypteridaceae Blanca Pérez-Garcia and Aniceto Mendoza-Ruiz 143 Shorter Notes Botrychium pallidum Newly Discovered in Maine Arthur V. Gilman 155 Asplenium ruta-muraria L. in lowa Thomas F. Cady and Diana Horton 157 Vitexin 7-O-rhamnoside, a New Flavonoid from Pteris vittata Filippo Imperato 159 The American Fern Society Council for 2004 TOM RANKER, University Museum, Campus Box 265, University of Colorado, Boulder, CO gress 0265. esident DAVID S. CONTANT, Dept. of Natural Sciences, Lyndon State College, Lyndonville, VT ney 1. President-Elect W. CARL TAYLOR, 800 W. Wells St., Milwaukee Public Museum, Milwaukee, WI 53233-1478 e JAMES D. CAPONETTI, Dept. of Botany, University of Tennessee, Knoxville, TN 37916-1110. 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 R. JAMES HICKEY, Botany Dept., Miami University, Oxford, OH 45056. Journal Editor R. JAMES HICKEY, Botany Dept., Miami University, Oxford, OH 45056. Memoir Editor CINDY JOHNSON-GROH, Dept. of Biology, so pated Adolphus College, 800 W. 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Box 299, St Li Louis, MO 63166-0299. American Fern Journal 94(3):113-125 (2004) Distribution, Ecology and Cytology of Asplenium azoricum Lovis, Rasbach & Reichstein (Aspleniaceae, Pteridophyta) and Its Hybrids FRED RUMSEY and STEPHEN RUSSELL Department of rp The Natural History Museum, Cromwell Road, -London SW7 5BD, United Kingdom HANNO SCHAFER Institut fiir Botanik, AG ae & his aap eau Universitat Regensburg, 93040 Regensburg, Germ HELGA RASBACH Datscherstr. 23, D-79286 Glottertal, Germany AssTRACT.—New data on distribution, ecology and cytology of the Azorean endemic Asplenium also new to science and is described here as A. diasii. It is confirmed cytologically as triploid with two genomes from A. azoricum and one from A. onopteris. Asplenium diasii and an experimental hybrid show that A. azoricum is an allotetraploid species. The parentage and directionality of hybridization for both hybrid taxa have been established using uniparentally inherited plastid genome markers. The Azores is an isolated archipelago of nine inhabited, volcanic islands in the Northern Atlantic Ocean. The shortest distance to the European coast, Cabo da Roca, Portugal, is almost 1,300 km. The American coast, Newfoundland, is about 1,700 km distant (Fig. 1). Four morphologically similar, simply-pinnate species of the genus Asple- nium have been reported in the Azores: the almost cosmopolitan tetraploid A. trichomanes L. ssp. quadrivalens (D. E. Mey.) Lovis; the widely scattered Neotropical triploid apomict A. monanthes L.; the locally rare Macaronesian endemic diploid A. anceps Lowe ex Hook. & Grev. (Rasbach et al., 1981), two plants of which were reported from Pico island in 1973 (Lovis et al., 1977) and the endemic tetraploid A. azoricum Lovis, Rasbach & Reichstein. Although A. azoricum was collected by K. Hochstetter in 1838 on Faial Island (Seubert, 1844), its specific distinction was not recognized until the work of Lovis et al. (1977). Key distinguishing features had already been commented upon by Wilmanns and Rasbach (1973), and earlier authors, e.g., Milde (1867), and Trelease (1897), obviously recognized that Azorean material differed from typical A. anceps, referring their gatherings to a forma (f. azorica Milde) of that species. While previously confused with both A. anceps and A. trichomanes, its separation in the field generally is quite easy. Asplenium azoricum differs from both in the more elongated, broadly-triangular, often MISSOURI BOTANICAL 1 1 2004 114 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) biauriculate, more conspicuously dentate pinnae, and from the latter in its glossier, bright green frond colour. Typical luxuriant material from shaded, sheltered environments is considerably larger than any of the taxa listed above, with fronds sometimes exceeding 35 cm long. Confusion is possible, particularly with A. trichomanes ssp. quadrivalens, when comparing small examples from dry, exposed environments. Asplenium anceps, which is implicated in the parentage of A. azoricum but currently is not sympatric with it, differs in having a very pronounced additional third rachis wing on the abaxial side. A greater potential confusion is with another recently described taxon, A. trichomanes ssp. coriaceifolium H. & K. Rasbach, Reichstein & Bennert (syn.: A. azomanes Rossello, Cubas & Rebassa). This highly restricted tetraploid was described from walls terracing olive groves in the Balearic Islands, where it is also found in sheltered rocky gulleys of seasonal watercourses close to sea-level. It, however, also occurs on montane karstic limestones in Southern Spain and the Rif mountains of Morocco (Rumsey and Vogel, unpubl.). Its genomic constitution and relationship to A. azoricum are currently being explored. Natural hybrids of A. azoricum have not previously been reported (Reich- stein, 1981) although they have been actively searched for (Lovis et al., 1977). Plastids have been shown to be maternally inherited in Asplenium (Vogel et al., 1998a), and fragment length polymorphism and sequence data for this moderately variable plastidic region allows for unequivocal identification to the species level (Vogel et a/., unpubl.). Thus, molecular studies of this sort facilitate both the determination of hybrid parentage and the establishment of hybrid directionality, i.e., which parental taxon was maternal. MATERIAL AND METHODS The distribution and ecology of A. azoricum was studied between 1998 and 2001 on the nine islands of the archipelago, during field work by H.S. for a project mapping all vascular plant species. Distribution maps based on the UTM 1 X 1 km grid have been created for islands representative of the western, central and eastern groups of the archipelago: Flores, Faial and Santa Maria (Fig. 2). During the course of our fieldwork, a number of plants with abortive spores were detected whose morphology suggested possible hybridity. Fronds with premature sporangia were fixed in a mixture of acetic acid and ethanol (1:3) in the field. Preparations of meiosis were made following Manton (1950). A few mature fronds of each potential hybrid were collected as voucher specimens and will be deposited in the herbaria of Universidade dos Agores, Terceira (AZU) and Natural History Museum, London (BM). Live material was not collected as all hybrids are rare and should be protected in their natural habitat on the islands. Total DNA extractions were made from small (c. 20 mg) portions of each of the herbarium specimens using the method of Rogers & Bendich (1994) and amplified using the universal plastid primers C and F of Taberlet et al. (1991). RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS 115 Bermuda +*. Bohamas GQ 500 1000 km i) Fic. 1. Location of the Azores in the Atlantic Ocean. RESULTS ASPLENIUM AZORICUM LOvIS, RASBACH & REICHSTEIN.—Asplenium azoricum (Fig. 5a) was found on all nine islands of the archipelago, the only member of the A. trichomanes group for which this is true. Unlike A. trichomanes ssp. quadri- valens, which in the Azores is largely restricted to human-made structures and most abundant on the most populous islands, A. azoricum is widely distributed in a range of natural and semi-natural environments. Although it has been able, like many rock ferns, to exploit built structures it is not restricted to them. Usually a species of somewhat humid environments, paradoxically it is most common and shows its widest ecological amplitude on the driest, easternmost island of Santa Maria. Here it effectively replaces A. trichomanes ssp. quadrivalens (a species with very few individual plants on the island), existing in large numbers from coastal areas up to c 575 ma.s.l. on Pico Alto (Fig. 2). Throughout the archipelago, the species achieves its greatest abundance at lower altitudes, declining markedly above the lowland forest zone. Elsewhere in the eastern group, it is locally frequent and often luxuriant in the central low-lying valley between the major volcanic peaks of So Miguel Island. In the central group of the Azores it is more restricted to west and north-west exposed 116 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) 1 4041 4243.44 45 46 47 48 49 5051 5253 54 555657585960 61 62.63 64 65 66 67 6869 7071 7273747576 76 76 7al | 78 99 [ l LI |99 75 75 «77/ oe 7 98 98 74 74 «76 ° a 7 97 ° 97 73 7s 7st | |75 96 e 96 72 72 mer ° 74 95 ee 95 1 1 73 a i 73 o4{(] || lob plelegie 94 70 70 72[_™ 5 72 93 N00 93 69 es 711 1 eletel S 4 AT {i719 elele ele 92 68 es vol | [| a i 70 91 a e elele 91 67 67 68 LEIA RET A et 69 90 0 OOO 90 66 66 eal | Si Vie, Ses il \ jes 89 89 65 65 67 (BRL Whoae ie 67 sal] | 88 84 64 66] | aa 4888C8 66 =. 61 6263 64. 65 68.67 68.69 7071 7273747576 63 63 65| ine: ob | 65 62 62 64{ | | | CE Bee | | je a1 61 40.41 42 43.44 45 46.47 48 49 5051 52 53 54 55 5657 5859 60 60 60 58 1 Fic. 2. Distribution of A. azoricum on sb islands of Flores (left), Faial (center), and Santa Maria (right) in UTM 1 X 1 km grid, WGS 8 lines along 200 m pensangs Symbol size indicates abundance within that 1 x 1 km square: r rare *, occasional ® or commo slopes between 100-500 m a.s.]., although it ascends to at least 700 m on Pico (Rumsey et al. 96-10-3-3, BM!). In the western group, Flores and Corvo Islands, A. azoricum exists in the absence of A. trichomanes ssp. quadrivalens. On Flores, it is widespread (see Fig. 2) but uncommon; on Corvo, it should be considered very rare. On these islands, the species is usually found in small populations (<20 individuals), or as single plants and only at altitudes below 00 m. Restriction to particular site aspects is not so marked as in the central group, presumably because the more generally humid climate reduces the need for shelter. Asplenium azoricum is most common on steep, humid slopes of shady ravines at low altitudes. In these places it forms large populations on bare soil, often mixed with other ferns like A. scolopendrium or A. onopteris. However, judging by earlier literature reports (e.g. Lovis et al., 1977) and herbarium gatherings, it is most likely to be found on or at the base of walls by roadsides and field-margins. It is one of several shade tolerant fern species that predominantly occurs in lowland areas dramatically influenced by the almost complete destruction of the natural vegetation following human arrival on the islands in the 15th and 16th centuries. Asplenium azoricum has faired better than some, and is now one of the very few native species that can be found in lowland forests and plantations dominated by the Australian neophyte Pittosporum undulatum. In this environment, it occurs in deep shade in small soil pockets on volcanic rocks on the forest floor, or rarely on the ground. In the coastal zone, it is absent in sea-wind exposed sites but grows in stands of Erica azorica or Arundo donax together with Asplenium marinum. Inland, while often found on mossy rocks at the base of walls, it can also be found higher on the wall proper, although typically only in soil filled crevices. A requirement for humidity throughout the year means it usually is absent from dry, south-exposed or mortared walls where it is replaced by the more xerophytic and calcicolous A. trichomanes ssp. quadrivalens. Soil backed retaining walls, or old walls which have accumulated substrate in the RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS Fic. 3. Silhouettes of fronds. A. Asplenium ai anceps (Madeira, TR-25 anceps X A. azoricum (TR-5188 and 5188/7, in B 200125235/37, azoricum - Azores, TR-3335/2). A and C bitte Lovis et al., 1977; B Reichstein. 59). Cc ) sha B. ai yerimental hybrid 4 A. § K. Rasbach). ( noto HH Ki: TR stands for T. AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) oat e4, ° i =e ~~ o ® e*s@ ¢ ot i ue z: es % “ T. \ > a} 2,90 z, @e og : *® Ss oo ss) , " ¢ ft ef J.- 975 . . ig Td 3 i og ee B » co — “, " ba om ", "2 Pr) . i, F »* 3 Pm te | °,* » » 4 a Re *s ae 2, _» Cge ¢ y# oN : ~ 00 ° al? ie ae ie Ci Vitale Pirie TTF Ya) el es | ‘ OM 9° -. a, , & - ° = i ¢ ZX . by Hap oo oa : oa o%; SoM oo Ae of qj ® P °8 ’ yg ™ one &® \ ooes ve i \ A Sy 7 etaRe Boe Pa . a ee ~ 8 7 O° So 8 nar \. o ae ¢ : nese : fay 9°? Oo Fic. 4. A. Cytology of Asplenium. a. Photomicrograph of A. anceps x A. azoricum; spore mother cell in meiosis with n = 36II and 36I. b. Explanatory diagram of a; scale bar 10 ym, pairs black, univalents outlined (TR-5188, in B, collection H. & K. Rasbach: 200125235/37). c. Photomicro- graph of A. Xdiasii; spore mother cell in meiosis with n = 108]; d. Explanatory diagram of c; scale bar 10 um; prep. and photographs (HR Az-Ma-927 Schafer). interstices and are partially shaded by other vegetation, are thus most likely to support the endemic taxon. In summary, the ecological range of A. azoricum seems to be rather broad and ees the few existing relevés it is not possible to describe vegetation associatio Cytological ais noe ty ren et al. (1977) showed that A. azoricum is tetraploid with 2n = 144. To determine the nature of its polyploidy and re- lationship to other fae taxa, a program of experimental hybridization RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS 119 SS Maria, 2001 (e). 120 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) and cytological investigation was begun. In 1978, the late Prof. T. Reichstein hybridized A. anceps Lowe ex Hooker & Grev. from Madeira and A. azoricum from the Azores (for method see Rasbach et al., 1994). This hybridization program produced 35 plants of A. anceps, five plants of A. azoricum, and 22 hybrids (fig. 3). Cytological study of one of these hybrids by Prof. J. J. Schneller (Ziirich) revealed that it was triploid with n = ca. 36 II and 36 I (Schneller, in litt. 23.01.1995). As part of this study H. Rasbach analyzed meiosis in another hybrid plant. The chromosome number of ten spore mother cells was n =ca. 36 II and 36 I (fig. 4A). The most parsimonious explanation of this pairing behavior is that tetraploid A. azoricum shares a genome with A. anceps and that these homologous n chromosome sets undergo synapsis in the hybrid to form the 36 pairs. The 36 unpaired chromosomes would thus represent a non-homologous genome contributed by the second diploid parent of A. azoricum. Under this hypothesis, Asplenium azoricum is an allotetraploid and chromosome pairing observed in the hybrid A. anceps x A. azoricum is allosyndetic. The alternative hypothesis, that A. azoricum is an autopolyploid and that pairing in the hybrid is autosyndetic (occurring between the two genomes contributed by A. azoricum) can be rejected based on cytological study of the natural hybrid A. Xdiasii reported below. We therefore conclude that A. anceps is one of the parental species of A. azoricum and suggest that the genomic constitution of this allotetraploid be represented by the formula AnAnUnUn, where An = anceps and Un is unknown. On morphological grounds we hypothesize that the unknown parent of A. azoricum is a member of the A. trichomanes group sensu lato. Allozyme and DNA studies (Vogel et al., unpublished) preclude the possibility that the missing parent (UnUn) is an extant European taxon. Lovis et al. (1977) commented on the morphological similarities with the Neotropical A. heterochroum Kunze. However, the material of this or similar species they examined was tetraploid and hexaploid (unpublished records), not diploid as would be required of the missing parent. Although the missing parent remains unknown, the likelihood of a Neotropical origin is perhaps strengthened by the growing list of Macaronesian cryptogamic species disjunct to the Neotropics, or with their closest relatives there. These include the pteridophytes Ceradenia jungermannioides (Klotzsch) R. C. Ching, Grammitis marginella (Sw.) Sw. (Schafer, 2001), Isoétes azorica Dur. ex Milde (Britton and Brunton, 1996), and Huperzia dentata (Herter) J. Holub, and the bryophytes Jamesoniella rubricaulis, Radula nudicaulis (Sjégren, 2000), Plagiochila retrorsa Gottsche, P. virginica A. Evans, P. stricta Lindenb., P. papillifolia Steph. and P. longispina Lindenb. & Gottsche (Rycroft, 2002). Asplenium xdiasii Schaefer, Rumsey & Rasbach, hybr. nov. (Asplenium azoricum Lovis, Rasbach & Reichstein x A. onopteris L.) TYPE:—Acgores (Portugal), Ilha de Santa Maria, Sao Lourengo, 150 m a.s.l., in Pittosporum undulatum forest, n = 1081, triploid. 01.08.2001, H. Schdfer, Az-Ma-749 (holotype, BM). Fig. 4c-d, 5c-e. RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS 121 Planta hybrida Asplenio azorico simillima; sed rhizoma caespitosa; petiolus rachisque basi ferrugineo-fuscis apice viridibus; folia 6-18 cm longa, 3-3.5 cm lata, lanceolato-acuminata, basi bipinnata; pinnae oblongo-ovatae, brevissime petiolatae, margine dentatae; sporae omnes abortivae; chromosomatum numerus 108, meiosi chromosomatibus 108 univalentibus; differt. Rhizome caespitose. Fronds 6-18 cm, stipe to 5 cm, stipe and rachis black or dark reddish brown, the uppermost 2 cm green. Pinnae numerous (ca. 15-37), up to 16 X 12 mm, light green, short petiolate, entire or the lower bipartite, triangular to oblong-ovate, margin sharply dentate. Sori ca 1.0-3.3 mm. Triploid hybrid of the tetraploid A. azoricum and the diploid A. onopteris (maternal parent). The hybrid contains one genome of A. onopteris and two of A. azoricum that do not form pairs, i.e. are not homologous. Named after the Azorean botanist Prof. Dr. Eduardo Dias. PaRATYPES.—Acores (Portugal), Ilha de Santa Maria: Feteiras de Cima, 220 m a.s.l., roadside slope in pasture, 13.06.2001, H. Schdfer Az-Ma-915 (AZU, BM) Fig 5d; Loural, 350 m a.s.1., Erica shrub on roadside, 15.06.2001, H. Schdfer Az- Ma-926 (AZU, BM); Loural, 350 m a.s.l., W exposed slope in pasture, n = 108I, 15.06.2001, H. Schdfer Az-Ma-927 (AZU, BM), (Fig. 4c); Acgores (Portugal), Ilha de Santa Maria, Loural, 350 m a.s.l., Erica shrub on roadside, n = 1081,15.06.2001, H. Schdfer Az-Ma-928 (AZU, BM); Loural, 360 m a.s.l., Erica shrub on roadside, 15.06.2001, H. Schdfer Az-Ma-929 (AZU, BM); Loural, 360 m a.s.l., Erica shrub on roadside, 15.06.2001, H. Schdfer Az-Ma-1200 (AZU, BM); NE Cruz dos Picos, roadside slope in pasture, 22.06.2001, H. Schdfer Az- Ma-983 (AZU, BM); Santa Barbara, 200 m a.s.1., Pittosporum undulatum forest on roadside slope, 17.07.2001, H. Schdfer Az-Ma-1036 (AZU, BM) Fig. 5e; Feteirinha, 310 m a.s.]., E exposed slope in pasture, 24.07.2001, H. Schafer Az- Ma-1072 (AZU, BM); Cardal, 350 m a.s.l., SW exposed slope in pasture, 05.08.2001, H. Schdfer Az-Ma-1108 (AZU, BM). As the only simply pinnate member of the A. trichomanes group present in the vicinity of these hybrids, A. azoricum is almost certainly one of the parents. The other parent was less easily determined. Because Asplenium hybrids generally show clear morphological intermediacy (Wagner, 1954), the second parent was presumed to have more dissected, bi- to tripinnate fronds. In the field it was assumed that at least some of the hybrid plants could have A. obovatum Viv. ssp. lanceolatum (Fiori) P. Silva as the second parent, it being present in the majority of hybrid sites. Subsequent cytological examination revealed that the three plants investigated were triploid, not tetraploid, as would be expected from a hybrid between tetraploid A. azoricum and tetraploid A. obovatum ssp. lanceolatum. Further investigation was therefore needed to establish the identity of the second parent(s). DNA extraction and PCR amplification of a portion of the plastid genome was carried out on five of the individuals identified as hybrids: HS Az-Ma 749, 927, 928, 1014 and 1036. Each generated identical fragments of c 1000 bp which differed markedly in their base sequence from both A. obovatum and A. azoricum but which matched that of A. onopteris, another 122 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) 61 62 63 64 65 66 67 68 69 70 71 7273 7475 76 9 99 98 age 98 97 A Bim wn 97 96 tah 17, 96 95 |} dca} 95 94 S aks al 94 93; \ ~ B New| gle i 93 92 vy le. ~ Ped | 92 91/ |} |e oe ca % | 91 90 ee al A *>\| 90 89 2 a Keo ithe Lals¥] 89 88 ‘ 88 61 62 63 64 65 66 67 68 69 70 71 7273 7475 76 Fic. 6. Distribution of Asplenium Xdiasii on Santa Maria island in UTM 1 X 1 km grid, WGS 84 (contour lines along 200 m isohypses). species present at the majority of hybrid sites. The second (and consistently maternal) parent of A. Xdiasii is therefore concluded to be A. onopteris. This fits with the cytological data because A. onopteris is diploid. The considerable variability of A. onopteris may help explain the range of morphologies shown by the hybrid, A. azoricum being rather invariable. Strong unidirectionality in hybrid formation has previously been reported for A. Xalternifolium (Vogel et al., 1998b) and is true for a range of hybrids in which A. onopteris and its polyploid derivatives take part (Rumsey and Russell, unpubl.). Altogether, 13 individual plants of this hybrid were found in seven locations of the eastern part of Santa Maria Island (Fig. 6). It has yet to be detected on any other island of the archipelago. The likelihood of the formation of this hybrid on the other islands is undoubtedly lessened by the reduced abundance of A. azoricum elsewhere. We might speculate that as the paternal species, hybrid formation is dependent on the presence of extensive growths of A. azoricum gametophytes and limited numbers of those of the maternal parent such that the ratio of A. azoricum to other species’ antherozoids favors interspecific matings. The hybrids were usually found in large mixed populations of the parents. Hybrid plants were restricted to more humid situations than the parents, often growing below them and near the foot of slopes, in communities dominated by Pittosporum undulatum, or Erica azorica. Asplenium Xsantamariae Schaefer, Rumsey & Rasbach, hybr.nov. (Asplenium azoricum Lovis, Rasbach & Reichstein < A. scolopendrium L.). TYPE:— Acores (Portugal), Ilha de Santa Maria, Santo Espirito, 280 m a.s.l., W. exposed slope in pasture. 21.07.2001, H. Schifer, Az-Ma-1064, (holotype, BM; isotype, AZU). Fig. 5b RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS 123 61 62 63 64 65 66 67 68 69 70 71 7273 7475 76 99 : 99 98 es 98 96 ap : ane 96 95 Y | deel ¥ 95 94 a . AON 94 93 | at er | | 93 92/ |{ wre, N, | 92 91 Lie tT ef | 91 << 90 ach, a X Dd) 90 89 A Ry 89 88 Po 88 61 62 63 64 65 66 67 68 69 70 71 7273 7475 76 Fic. 7. Distribution of Asplenium Xsantamariae on Santa Maria island in UTM 1 X 1 km grid, WGS 84 (contour lines along 200 m isohypses). Planta hybrida ex A. azoricum et A. scolopendrium exorta; rhizoma caespitosa; folia 10-15 cm longa, 2 cm lata; petiolus 3-5 cm longus, fuscus; rachis basi ferrugineo-fusca; pinnae cordatae-ovatae, 10 X 9 mm, crispatae; superiores approximatae; sporae omnes abortivae. Fronds 10-15 cm; stipe ca 5 cm, rachis ca 8 cm, black except uppermost 2 cm; pinnae up to 33, slightly crispate, confluent and crowded towards the tip of the frond; margin entire to shallowly dentate up to 10 X 9 mm, ovate- elliptical to cordate, light green; sori ca 1 mm. Hybrid of A. azoricum and A. scolopendrium (the latter the maternal parent) with abortive spores. Given the ploidy levels of the two parents this taxon is assumed to be triploid. Fig. 5b. In appearance A. Xsantamariae very closely resembles A. Xconfluens T. Moore, the extremely rare hybrid between A. trichomanes ssp. quadrivalens and A. scolopendrium. It differs from that hybrid in its less distinctly stalked pinnules with more crenulate-dentate margins and a somewhat thicker overall texture. As with A. Xdiasii, it is clear that the A. trichomanes group parent present in this hybrid is A. azoricum, it being the only simply pinnate species present. The confluent tip and broader, more decurrent pinnule attachments clearly indicate that the second parent of this hybrid would have fronds less divided than that of A. azoricum. The only Macaronesian species with less divided fronds are A. scolopendrium and A. hemionitis L. The latter is very rare on Santa Maria and has never been reported to hybridize with other species. It has a very distinctive, acutely lobed, palmate leaf, no sign of which is present in A. Xsantamariae. The participation of A. scolopendrium, in the origin of A. santamariae is confirmed by examination of the hybrid’s plastid genome. Only one small individual of this hybrid was found in the archipelago. It grows in the south-eastern part of Santa Maria island (Fig. 7). The hybrid was 124 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) found in a large mixed population of the parents growing on a steep, west exposed slope with some Pittosporum undulatum. The slope and the Pittosporum stands are part of a large pasture that is, in some places, heavily grazed by cattle. Due to the animals and strong rainfall, erosion is a common phenomenon on the steep slopes. The resulting bare soil is soon colonized by a number of A , including A. adiantum-nigrum, A. azoricum, A. marinum, A. obovatum ssp. lanceolatum, A. onopteris, and A. scolopendrium. Similar conditions, with periodic disturbance of soil banks, previously has been shown to promote hybrid formation between Asplenium species (Jee, 1994). DISCUSSION AND CONCLUSIONS e endemic Asplenium azoricum is widely distributed throughout the Azorean archipelago and is the most common representative of the A. trichomanes group on these islands. As it is able to survive even in dense plantations of invasive species, it cannot be considered endangered, although on those islands where it is very rare, e.g., Corvo, its few localities should be afforded protection. It is one of several fern species endemic to the acaronesian region that almost certainly have evolved in the region but are not now sympatric with both their putative parents. It is hoped that ongoing phylogenetic studies will elucidate the relationship of A. azoricum and A. trichomanes ea coriaceifolium to the, as yet unknown, non-European parent they ma The merit of phen to protect sterile fern hybrids is contentious, especially when resources are limited and many other endemic species are under threat, as in the Azores. Often the formation of the hybrids has been dependant on disturbance to sites through human activities. Arguably, if conditions and healthy populations of the parental taxa are maintained then it is likely that hybrids will continue to be sporadically produced. However, a strong case can be made for their conservation as unique biological entities with considerable evolutionary potential. Hybridization followed by poly- ploidy is the most rapid route to the generation of novel species and would seem to be the prevalent mode of speciation within the pteridophyta. These natural hybrids give us a rare opportunity to observe the process of alloploid speciation and the mechanisms and controlling factors behind it. ACKNOWLEDGMENTS We are grateful to Prof. Dr. J. J. Schneller (Ziirich) for permission to use his cytological result in this publication, to Dr. Norman Robson (NHM) for his assistance with the latin descriptions and to the director of the Botanical Museum, Berlin-Dahlem (B) for the loan of specimens. HS thanks the German National Merit Foundation for a PhD grant LITERATURE CITED Britton, D. M. and D. F. Brunton. 1996. Spore morphology and cytology of Isoétes azorica phere Isoétaceae) and its affinity with North America. Fern Gaz. 15:113-118. RUMSEY ET AL.: ASPLENIUM AZORICUM AND ITS HYBRIDS 125 Jez, N. 1994. The Guernsey Fern—xAsplenophyllitis microdon. La Soc. Guernesiaise Rep. & Trans. 23:724-749, Lovis, J. D., H. RasBacu, K. RasBacH and T. REICHSTEIN. 1977. Asplenium azoricum and other ferns of the A. trichomanes group from the Azores. Am. Fern J. 67:81—93. Manton, I. fone. Problems of cytology and evolution in the pteridophyta. Cambridge Univ. Press. &- RASBACH, H., K. RasBacu and J. J. SCHNELLER. 1981. A ch t for Asplenium anceps from the Canary Islands. Fern Gaz. 12:157—159. Raspacu, H., K. RaspacH and H. W. BENNERT. 1990. New records and new cytological results for the sag ae Madeira. Fern Gaz. 13:391-395. RasBAl REICHSTEIN and R. ViANE. 1994. Asplenium chihuahuense (Aspleniaceae, Meidphyta an allohexaploid species and the description of a simplified hybridisation technique. Am. Fern J. 84:11—40. unelare iE ia Hybrids i in European Aspleniaceae (Pteridophyta). Bot. Helv. 91:89—-139. ocers, S. O. and A. J. BeNpicH. 1994. Extraction of total cellular DNA from plants, algae and fungi. Pp 1-8, in B. S. Gelvin and R. A pars a, eds. Plant molecular biology manual. D1, 2. ed. Kluwer ee Publishers, Dordrec Rycrort, D. S. 2002. Plagiochila in Europe ner beyond). Bull. Brit. Bryol. Soc. 78:21— ScuArer, H. 2001. The Grammitidaceae, Pteridophyta, of Macaronesia. Feddes sail 112: seicoener, M. 1844 . Flora Azorica quam ex collectionibus schedisque Hochstetteri patris et filii el iecigeye ‘Bonna ae. SJOGREN, E. A. 0. Aspects on the biogeography of Macaronesia from a botanical point of view. Arqui feorhey Life & Marine Sci. Suppl. 2(A):1-9. TABERLET, P., L. Getty, G. Paurou and J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Mol. Biol. 17:1105—1109. TRELEASE, W. 1897. Botanical eau on the Azores. Ann. Rep. Missouri Bot. Gard. 8:77—220. VocEL, J. C., S. J. RUSSELL, F. J. Rumsey, J. A. BARRETT and M. Gipsy. Ht ol On hybrid formation in the rock fern Asplenium obrcsmnersit ea oe Pteridophyta). Bot. Acta 111:241—246. VocEL, J. C., S. J. Russet, F. J. Rumsey, J. A. BARRETT an . Gipsy. 1998b. Evidence for maternal transmission of chloroplast DNA in the estes Asplenium (Aspleniaceae, Pteridophyta). Bot. Acta 111:247-249. Witmanns, O. and H. Rassacu. 1973. Ob ti the pteridophytes of Sao Miguel, Acores. Fern Gaz. 10:315-329. American Fern Journal 94(3):126-142 (2004) Phylogenetic Relationships of the Subfamily Taenitidoideae, Pteridaceae PATRICIA SANCHEZ-BARACALDO! Department of Integrative Biology and University Herbarium, University of California, Berkeley, CA 94720, USA Asstract.—Thirteen genera are traditionally recognized in the subfamily Taenitidoideae, Pteridaceae. A phylogenetic study of this subfamily, based on both morphological and molecular included in the analyses: Jamesonia, Eriosorus, Pterozonium, Syngramma, Taenitis, Austro- intergenic spacer rps4-trnS. The results reject the hypothesis of monophyly of the subfamily as presented by Tryon et al. (1990). However, the results support the monophyly of a well-supported clade consisting of Jamesonia, Eriosorus, Pterozonium, Austrogramme, Syngramma, Taenitis, Pityrogramma, and Anogramma. The New World genera Jamesonia and Eriosorus form a mono- phyletic group, and Pterozonium is more closely related to the Old World genera, Austrogramme, gramma, and Taenitis. Although, ferns are the second most species-rich group of land plants, they have been relatively understudied compared to the largest group, the flowering plants. Several comprehensive studies on pteridophytes have looked at morphological and/or molecular characters from a phylogenetic perspective (Hasebe et al., 1993, 1994, 1995; Pryer et al., 1995; Schneider, 1996; Wolf et al., 1998; Pryer et al., 2001). While the majority of studies have focused on establishing higher-level relationships (Wolf et al., 1998: Pryer et al., 2001), an increasing number of studies have looked closely at lower-level relationships (Conant et al., 1995; Haufler et al., 1995; Gastony and Rollo, 1995; Pryer, 1999; Gastony and Johnson, 2001; Smith and Cranfill, 2002; Ranker et al. 2003). Such phylogenetic studies at lower taxonomic levels are sorely needed to facilitate understanding of evolutionary processes of diversification and biogeographic patterns among pteridophytes. The Pteridaceae is a large and diverse family of homosporous ferns with a nearly worldwide distribution (Tryon et al., 1990). The family comprises 34 genera that are mostly restricted to the New World (Tryon et al., 1990). A considerable number of species are found in exposed and rocky environments, although some members of the family are found in a diverse array of mesic habitats (Tryon et al., 1990). Phylogenetic relationships within the Pteridaceae * Current Address: School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK. SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 127 are poorly understood. Of the six subfamilies recognized in the family (Tryon et al., 1990), only the Cheilanthoideae has been extensively studied from a phylogenetic perspective, using plastid rbcL and nuclear ITS nucleotide sequences (Gastony and Rollo, 1995, 1998). Some members of subfamily Taenitidoideae were included in a larger phylogenetic analysis based on rbcL nucleotide sequences (Gastony and Johnson, 2001; Nakazato and Gastony 2003), and more studies are needed to understand the phylogenetic relation- ships amongst its members. Phylogenetic relationships within and among the other five subfamilies are yet to be resolved. Subfamily Taenitidoideae is difficult to circumscribe morphologically. Some of the most distinctive and diagnostic morphological characters are not consistent across all members of the group. In general, the sporangia are borne along the veins in exindusiate soral lines, or on an inframarginal commissural vein, but in some genera the sporangia are borne at the leaf margin and are protected by a false indusium. Most genera in the Taenitidoideae have paraphyses associated with the sporangia, but some genera completely lack paraphyses. According to the most recent taxonomic review (Tryon et al., 1990) thirteen genera belong to subfamily Taenitidoideae, which has a worldwide distribu- tion. Jamesonia, Eriosorus, Pterozonium, and Nephopteris are primarily neotropical (Tryon et al., 1990). Pityrogramma and Anogramma are mostly restricted to the Neotropics, but the former extends to Madagascar, and the latter is subcosmopolitan. Actiniopteris is primarily African, and Syngramma, Taenitis, and Austrogramme are centered in southeastern Asia. Afropteris is distributed in tropical West Africa and the Seychelles. Onychium is found from northeastern Africa, from Iran eastward to China, as well as in New Guinea. Cerosora is a native of Borneo, Sumatra, and the Himalayas. The traditionally recognized members of subfamily Taenitidoideae exhibit a di- versity of habitats, ranging from moist and sheltered to dry and open, and from terrestrial to rupestral. Some species live in forests, in the understory growing along streams, and a few species are rheophytes Historically the Old World genera Syngramma, Taenitis, and Austrogramme have been considered to make up a natural group based on a number of shared morphological characters such as disposition of sporangia and paraphyses (Copeland, 1947; Holttum, 1959, 1960, 1968, 1975; Walker, 1968; Hennipman, 1975). The New World genera Jamesonia, Eriosorus, and Pterozonium have also been considered closely related to each other based on venation, blade indument, and spores (A. Tryon, 1962). A. Tryon (1970) postulated that Eriosorus represents the least advanced group among the mainly American genera, and that its relationship to Pterozonium is not as close and is without a clear lineal derivation. She also suggested that Jamesonia and Eriosorus are closely related, and that Jamesonia is derived from more than one element in Eriosorus. A. Tryon (1962) pointed out morphological similarities shared between the Old and New World genera of the Taenitidoideae, characters that suggested a close phylogenetic relationship. Recent studies have suggested that the Old World genera of the Taenitidoideae and Pterozonium originated in 128 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) southern Gondwana region before South America and Antarctica-Australia separated during the Lower Cretaceous (Schneider, 2001). Furthermore, Pity- 2 1 Anog | been hypothesized to be closely related to each other based on lamina architecture, sorus type, and spores (A. Tryon, 1962). The general aim of this study was to elucidate phylogenetic relationships within the subfamily Taenitidoideae based on both morphological and molecular data. As a working hypothesis, monophyly of the Taenitidoideae as presented by Tryon et al. (1990) was assumed. A second aim was to test various hypotheses of relationships within and among the Old and New World genera of the subfamily. A third aim was to establish the closest relatives of Jamesonia and Eriosorus. MATERIAL AND METHODS SPECIMENS EXAMINED.—Using the exemplar approach, 20 species were chosen to represent eleven of the thirteen genera currently recognized in the subfamily. Vouchers and DNA samples were not available for Cerosora and the monotypic genus Nephopteris, so they were excluded. A complete list of the taxa used in this study is presented in Table 1, which refers to vouchers that were used to generate sequence data for rps4 as well as morphological characters. Morphological characters were also corroborated with other specimens housed at the University Herbarium, University of California, Berkeley (UC). Most of the DNA vouchers included in this study are housed at UC. Two specimens are at the Nationaal Herbarium Nederland, Leiden (L), and one specimen is at Institut fiir Systematische Botanik der Universitit Ziirich (Z). A multiple outgroup approach was used to resolve plesiomorphic characters within the ingroup (Maddison et al., 1984). Pteris multifida and P. quadriaurita from the subfamily Pteridoideae were included based on broader-scale previous phylogenetic studies (Hasebe et al., 1995: Pryer et al., 1995). Coniogramme fraxinea was also included as a more distantly related outgroup (Hasebe et al., 1995; Pryer et al., 1995; Gastony and Rollo, 1995: Gastony and Johnson, 2001). Coniogramme was initially placed in the cheilanthoids by Tryon et al. (1990) but subsequently shown to be the sister to other traditional Pteridaceae plus Vittariaceae (Hasebe et al., 1995: Gastony and Rollo, 1998; Nakazato and Gastony 2003). All three outgroups are restricted to the Old World. MORPHOLOGICAL CHARACTER ANALYSIS.—The following criteria were considered when selecting morphological characters for this study: 1) characters should exhibit greater degree of variability among OTUs than within, thus providing discrete character states; 2) characters should lack variability due to ecophenotypic factors; 3) characters should be independent of each other (Wiley, 1981); and 4) there should be a good basis for hypothesizing homology across the study group. As a first approach, literature on traditional classifications of genera in the Taenitidoideae, and previously published morphological descriptions were SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE TABLE 1. 129 Species used as a source for DNA and rps4 sequence data for this study. Most material was preserved in silica gel, two were herbarium specimens, and a few were fresh material. Collector/Source/ Geographic Accession Genus Species Herbarium origin number te det australis Sanchez-Baracaldo oo native AF321693 ink 360 (UC) of A Penenen barklyae Kramer 11086 (Z) dct: Islands AF544984 (Baker) Anogramma chaerophylla Sanchez-Baracaldo Unknown AY357705 (Desv.) Link Anogramma eon Smith 2586 (UC) Costa Rica AF321699 (Domin) C Pi ccenn tat —— van der Werff 16114 New Caledonia AF321702 (Mett.) Hennipman Austrogramme marginata D. Hodel 1454 (UC) New Caledonia AY357704 . Fo Coniogramme fraxinea UC Bot. Gard 58.0375 Java AF321696 (D. Don) Fée ex Diels C) Eriosorus flexuosus Copel. Sanchez-Baracaldo Colombia, AF321710 z Eriosorus insignis (Kuhn) A. Salino 3010 (UC) Brazil, Minas Gerais AF321708 A, EF. Tryon Eriosorus a Ngai (Fée) Sanchez-Baracaldo Colombia, Antioquia AF321719 AcE. iy 268 (UC) Jamesonia vlopete Sanchez-Baracaldo Colombia, Cocuy AF321747 . Tryon 246 (UC Jamesonia imbricata Sanchez-Baracaldo Colombia, Guantiva AF321756 (Sw.) Hook. & Grev. 252. Onychium ga B. Ornduff 10278 (UC) China, Yunnan AF321697 (Thunb.) K Pityrogramma petleomner aa UC Bot. Gard. 98.0063 Unknown, native of AF321698 omin Neotropics ee multifida Poir. UC Bot. Gard. 80059 Unknown, native of AF321695 (UC) Old World Pteris quadriaurita Retz. UC Bot. Gard. 67.1645 Unknown, pantropical AF321694 Pterozonium cyclosorum Brewer et al. 1006 (UC) Venezuela, Bolivar AF321703 A. G, Sm, Pterozonium reniforme Brewer et al. 1005 (UC) Venezuela, Amazonas AF321704 Syngramma quinata M. Kessler 2273 (L) Borneo, West AF321701 (Hook.) Carr Kalimantan Taenitis intenepte H. Schneider 1031 (L) Borneo, Sarawak AF321700 . et Grev. reviewed (Ching, 1934; Pichi Sermolli, 1962; A. Tryon, 1962; R. Tryon, 1962; Lellinger, 1967; Holttum, 1968; Atkinson, 1970; Holttum, 1970, 1975, Tryon and Lugardon, 1991). From this list, those characters that met the above criteria were selected and modified. In addition, some new characters not previously considered were examined and included. In the present study, non-applicable characters were coded as missing data, an approach previously discussed in 130 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) LE 2. Morphological data matrix for the cladistic oo of subfamily Taenitidoideae, bonne Saf See Appendix 1 for characters and characters state Character number i 2 Taxa a He’ aes Gee irs Pi oar, ta a eel Vapor Gr 2 Br a@ 12 4.506 Jamesonia imbricata LT Ole 2 eo tt 2 Pit 2 Oot Yr oA Jamesonia alstonii Ha 9 is Ws Dies Nis a WR Hs Wa #0 et Ves Ooh 12-0 Ob ee O41 Eriosorus insignis LO OAV OCE hatte OO See 2 Oo Pale os Eriosorus rufescens LOO 1B ka 2 Or Ot Or0ed 6) s0h4 2 OO 1 25250) 1 Eriosorus flexuosus LOO OR Att 1 O..0 50.8 -O oOFt 10 tO OC 20. 4 Pterozonium reniforme Pe Ae) Lee 2 er et et i eo Pterozonium cyclosorum COC ZOE ti 2 2a re eae 2 Oe ee Ot Austrogramme decipiens LY Pa2 OC LAL Orie Oo ft LOO Oe T PF 2°0'0 Austrogramme marginata Dao 2,200 bat Oi 20-10 FV oO 8 0 7 4010 a quinat Le a2 OO bbe AO) 0 £206 0.0 181 Taenitis interrupta OO 22 O02 bot et oO Uh ts OOO O48. 2 7-04 nogramma guatemalensis £02070) 136) ae Oo <6 tL 20r0:0 2 O370-1 Ff Anogramma chaerophy: 22022 O00-E Ohi (0 0-0.) 2 10070 4 O30, 1? ityrogramma austroamericana10200101011700 0 £ £000 1020.1 7 Onychium japonic be T02 3 OC: eO 4 200.0 w 1030 22 8 20a ¢ Actiniopteris australis POU te oie tse 8 t too 1101 8 te Afropteris bar. OOe 22 70 0101 F000 0 OO Lt Ooo 7 7 Coniogramme fraxinea Pat kOe a Ea eo O10 a 10-60 6.0.2.1, 1 7 Pteris multifida C00 OOF CO1LTOOTOO O 1 00:0 0-0-0501 7 Pteris quadriaurita 0-0:0:0' 0 7:0':0 1:00 70:0) 0.1 4.000000:0 1 7 Maddison (1993). Non-applicable characters occur when taxa lack the structure in question, for instance, in the present morphological data set, color of scales was scored only for Anogramma, Pityrogramma, Onychium, Actiniopteris, Afropteris, Pteris, and Coniogramme because the other genera lack scales. Some morphological characters were sought from cleared leaves mounted on slides (Arnott, 1959) from each exemplar. Two to three slides were mounted per exemplar. A total of 26 characters were included in the analyses. The data matrix with the characters and character states is shown in Table 2, and a detailed description of the characters used is presented in Appendix 1. MOLECULAR CHARACTERS.—A list of the taxa sr and their respective vouchers is presented in Table 1. Total genomic DNA was extracted using Chies et al., 1997). PCR reaction mixtures each contained 0.5 ern “of AmpliTaq Gold my eG (PE Applied Biosystems), 5 wL of the supplied 10x Buffer II (2.5 mM MgCl,), 0.1 mM of each dNTP, 2.5 mM of each primer, ~50 ng of total genomic DNA and purified water to volume. PCR cycles (Perkin Elmer GeneAmp PCR System 9600 thermocycler) were programmed as follows: an initial hot start of 95°C for 10 min to activate the AmpliTaq Gold polymerase, 40 cycles (94°C for 30 s, 60°C for 45 s, and 72°C for SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 131 2 min), and a 7 min final extension step at 72°C. PCR products were visualized with ethidium bromide on 1% agarose gels which were run in 1 X Tris-borate/ EDTA electrophoresis buffer (pH 7.8). Amplicons were purified with the QIAquick PCR purification kit (Qiagen, Chatsworth, CA) following the manufacturer’s protocol, and then processed by cycle sequencing and BigDye-terminator chemistry (PE Applied Biosystems) on an ABI model 377 automated fluorescent sequencer in the Molecular Phylogenetics Laboratory at the University of California, Berkeley. Sequence files were edited by visual inspection of electropherograms, and mutations or changes were verified using the program Sequence Navigator (PE Applied Biosystems). Alignments were performed by eye in a nexus file. The final aligned data matrix consisted of 993 characters; 578 from the rps4 coding region, and 415 from the intergenic spacer rps4-trnS. For Eriosorus and Jamesonia, 413 bp from the intergenic spacer rps4-trnS were included; twelve distinct shared insertion/deletion regions were recognized in the final alignment and each region was coded as a single binary character for the maximum parsimony analyses. For Pterozonium, Austrogramme, Syngramma and Taenitis, 252 bp were included from the intergenic spacer rps4—trnS; nine distinct shared insertion/deletion regions were recognized in the final alignment and each region was coded as a single binary character for the maximum parsimony analyses. The whole intergenic spacer rps4-trnS region was excluded due to ambiguity in the alignment for the following taxa: Anogramma chaerophylla, A. guatemalensis, Pityrogramma austroamericana, Onychium japonicum, Actiniopteris australis, Afropteris barklyae, and the three outgroups. PHYLOGENETIC ANALYSIS.—The morphological data set was compiled using MacClade 4.0 (Maddison and Maddison, 2000). All maximum parsimony and bootstrap analyses were run in PAUP* 4.0b10 (PPC; Swofford, 1999). Multistate characters were unordered, and uninformative characters were excluded in all analyses. For each analysis, maximum parsimony analyses were performed, and stepwise addition searches were conducted with the following specifications: 1000 random additions, tree-bisection- reconnection (TBR) branch- “swapping, and MULPARS. Equally most f strict consensus tree. Bootstrap values were calculated (Felsenstein, 1985; Sanderacti, 1989; Hillis and Bull, 1993) to provide a measurement of support. Bootstrapping of all data sets used 1000 replicates, with 100 random addition starting trees implemented for each replicate, TBR branch swapping, and MULPARS. The three analyses that were carried out in this study are as follows: 1) morphological data; 2) molecular data; and 3) both morphological and molecular data. RESULTS The morphological data set included 26 characters; of these, 25 were parsimony-informative and one was autapomorphic. The molecular data set 132 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) included 993 characters; of these, 309 sites were variable of which 186 were parsimony-informative and 123 were parsimony-uninformative. MorPHOLOoGICAL ANALYsIS.—A single most parsimonious tree (Fig. 1) was found at 50 steps (CI = 0.70; RI = 0.86). Only bootstrap values higher than 50% are reported. Results of this analysis weakly supported the monophyly of the Neotropical genera, Jamesonia, Eriosorus, and Pterozonium, plus the Old World genera Austrogramme, Syngramma, and Taenitis. Within this clade, Pterozonium was basal to Eriosorus and Jamesonia. Eriosorus and Jamesonia together formed a monophyletic group. Eriosorus appeared to be paraphyletic containing a monophyletic Jamesonia. Onychium japonicum, and Actiniopteris australis formed a monophyletic group within a weakly supported clade including also Afropteris barklyae, Pteris multifida, and P. quadriaurita. MOLECULAR AND ComBINED Data ANALYSES.—The parsimony analysis of molec- ular characters generated a total of two equally most parsimonious trees found at 481 steps (CI = 0.76; RI = 0.83); the strict consensus is shown in Fig. 2. The parsimony analysis of morphological and molecular characters combined resulted in a total of four equally most parsimonious trees found at 539 steps (CI = 0.75; RI = 0.82); the strict consensus is shown in Fig. 3. Only bootstrap values higher than 50% are reported. Results for the molecular data and the combined data sets are described together because the strict consensus topologies of both analyses agreed in almost every aspect (Figs. 2, 3), except for an unresolved node of the clade containing Afropteris barklyae, Pteris multifida, and P. quadriaurita (Fig. 3). Both analyses support the monophyly of clades containing Austrogramme, Syngramma, Taenitis, and Pterozonium, as well as the Neotropical clade of Jamesonia and Eriosorus. Eriosorus itself appears to be paraphyletic and includes a monophyletic Jamesonia. In both analyses, Anogramma and Pityrogramma form a monophyletic group, which is sister to the monophyletic group that includes Jamesonia, Eriosorus, Pterozonium, Austrogramme, Syngramma, and Taenitis, with high bootstrap support. Pteris multifida, P. quadriaurita, and Afropteris barklyae formed a highly supported monophyletic group in both analyses (Figs. 2, 3). In the molecular analysis, Afropteris barklyae is nested within Pteris (Fig. 2), while in the combined analysis the relationship of Afropteris barklyae, Pteris multifida and P. quadriaurita is unresolved (Fig. 3). Onychium and Actiniopteris form a well- supported monophyletic group, that appears sister to the Afropteris-Pteris clade (Figs. 2, 3). Outgroups.—Even if all analyses were rooted with both species of Pteris and Coniogramme fraxinea, Pteris multifida and P. quadriaurita were consistently nested with Afropteris (Figs. 1-3). In the morphological analysis, P. multifida and P. quadriaurita form a monophyletic group that is sister to A. barklyae, although with very low bootstrap support (Fig. 1). The relationship of the Afropteris-Pteris clade to Onychium japonicum and Actiniopteris australis is weakly supported (Figs. 1-3). Both molecular and combined data sets strongly SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 133 3 Jamesonia imbricata 95 Jamesonia alstonii 81 3 ; oF Eriosorus insignis 66 2 Eriosorus rufescens Eriosorus flexuosus Pterozonium reniforme Pterozonium cyclosorum 60 3 a Austrogramme decipiens 1 i Austrogramme marginata 1 Syngramma grande abe Taenitis interrupta 4 51 Anogramma guatemalensis Anogramma chaerophylla Pityrogramma austroamericana 3 m os Onychium japonicum : = Actiniopteris australis 3 Afropteris barklyae Le Pteris multifida Pteris quadriaurita Coniogramme fraxinea Fic. 1. Parsimony analysis of morphological data set. Single most parsimonious tree of 50 steps (CI = 0.70; RI= 0.86). Numbers —. branches Rutioate aiiebsinkc, percentage values based on 1000 replicates of 100 random additio of character state changes per branch are indicated below the aa The tree was rooted using the outgroups Pteris multifida and P. quadriaurita from subfamily Pteridoideae, and a more distantly related member, Coniogramme fraxinea, as explained in the text. 134 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) Jamesonia imbricata 99 10 11 7 Jamesonia alstonii 94 57 r Eriosorus insignis 12 100 26 3 Eriosorus flexuosus 65 Eriosorus rufescens 96 Pterozonium reniforme 37 77 [8 9 oe Pterozonium cyclosorum 98 Austrogram me decipiens 37 90 Austrogram me marginata 52 Syngramma grande Taenitis interrupta yi) Anogramma guatemalensis 9 | 22 7 Anogramma chaerophylla 11 16 Pityrogramma austroamericana 99 a Onychium japonicum 13 | Actiniopteris australis 20 54 61 65 10 Afropteris barklyae Zz 100 6 Pteris multifida 6 10 Pteris quadriaurita Con Parsimony analysis of molecular data set. Strict consensus of tw oniogramme seme most i aaails Fic. parsimonious trees at 418 steps (CI = 0.76; RI=0.83). Numbers above branches ssi bootstrap percentage values based on 1000 replicates of 100 random addition ange replicates each. Numbers of character state changes per branch are indicated below the lines. Trees we ere rooted using the outgroups Pteris multifida and P. quadriaurita from subfamily Pridden and a more distantly related member, Coniogramme fraxinea, as explained in the te SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 135 support the relationship between Afropteris barklyae, Pteris multifida, and P. quadriaurita (Figs. 2, 3). DISCUSSION PHYLOGENETIC RELATIONSHIPS.—Bootstrap values robustly support the topologies generated by the analyses of the molecular data alone and the combined data sets, but are weak in the analysis of the morphological data set. Clade support values for the combined data sets are slightly higher than for the molecular data set alone. The outgroup rooting employed rejects the hypothesis of monophyly of subfamily Taenitidoideae as defined by Tryon et al. (1990). In this study, Pteris appears to be closely related to a member of the ingroup (e.g. Afropteris) suggesting that it would be more appropriately classified with the pteridois as initially proposed by Tryon and Tryon (1982). However, all analyses agree on the monophyly of a highly supported clade including: Jamesonia, Eriosorus, Pterozonium, Austrogramme, Syngramma, Taenitis, Anogramma, and Pityrogramma (Figs. 1-3). The most robust analyses, the molecular and combined data sets, recover a well supported monophyletic group including: Jamesonia, Eriosorus, Pterozonium, Austrogramme, Syn- gramma, Taenitis, Anogramma, and Pityrogramma (Figs. 2, 3). In addition, all analyses performed in this study indicate that Jamesonia and Eriosorus form a monophyletic group (Figs. 1-3). Based on the analysis of morphological characters alone, Pterozonium is sister to the clade consisting of Jamesonia and Eriosorus (Fig. 1); this clade is defined here by acropetal (outward) sporangial maturation (character 7, Appendix 1) shared by these three genera. In contrast, the most robust analyses based on DNA sequences alone and the combined data sets suggest that the New World genus Pterozonium is more closely related to three Old World genera, Austrogramme, Syngramma, and Taenitis (Figs. 2, 3); a number of morphological characters states are shared by this clade, e.g., spore ornamentation, sporangial disposition, and paraphyses disposition (characters 4,7 and 12 respectively, Appendix 1). Previous HyPoTHEsEs OF RELATIONSHIPS.—The topologies presented in this study prompt discussion of several previously proposed phylogenetic hypotheses. The Old World genus Austrogramme is closely related to Syngramma and Taenitis, as postulated by Walker (1968). In all analyses, Syngramma and Taenitis are closely related, as hypothesized by Copeland (1947) and Holttum (1960, 1975), with Taenitis being basal to Syngramma and Austrogramme (Figs. 2, 3). Although, A. Tryon (1962) postulated that the neotropical genera Pterozo- nium, Eriosorus, and Jamesonia constitute a natural group, she later (1970) stated about Eriosorus that, ‘‘the relationship to Pterozonium is not as close and is without clear linear derivation.” The results in this study (Figs. 2, 3) suggest that Pterozonium is actually more closely related to the Old World genera Austrogramme, Syngramma, and Taenitis (Figs. 2, 3) as proposed by Schneider AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) Jamesonia imbricata 100 | G)4 VI (1)2 IX Jamesonia alstonii (3)41V 86 (6)3] XXVI Gri Eriosorus insignis 100 ” (4) 19 VII Qi Eriosorus flexuosus 38 XXVII Eriosorus rufescens 3) ~~ 88 [oor Pterozonium reniforme (3) 3 VI Pterozonium cyclosorum ba tad (1) 61 Austrogramme decipiens 94 Austrogram me marginata (6) 48 IV Syngramma grande Taenitis interrupta Anogramma guatemalensis ——— (3) 22 - 77 Anogramma chaerophylla 11 Pi : : ityrogram mericana (2) 16 tyrogramma austroameric: Onychium japonicum 100_| (1) /2 — (1) /3 Actiniopteris australis 7 (2) 20 9) 61 (9) 10 Afropteris barklyae 100 |? fee Ran G)6 7 teris multifi (3) 2 70 Pteris quadriaurita Coniogramme fraxinea Fic. 3. Parsimony is of hol nd molecular combined data sets. Strict consensus of four equally most parsimonious trees at 539 pen (CI= 0.75; RI= 0.82). Numbers above branches ar cei percentage values based on 1000 replicates of 100 random addition sequence ers of morphological steps per branch are indicated in parentheses. Numbers wal supporting molecular characters per branch, derived from the coding region of rps4, are written SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 137 (2001). Moreover, A. Tryon (1970) stated that ‘‘Eriosorus represents the least advanced element among the five, mainly American genera, Pityrogramma, Anogramma, Eriosorus, Jamesonia and Pterozonium.” Evidence presented in this study suggests that Pityrogramma and Anogramma are sister to the clade containing both New World genera Jamesonia, Eriosorus, and Pterozonium, and certain Old World genera, Austrogramme, Syngramma, and Taenitis (Figs.1—3). Jamesonia and Eriosorus form a monophyletic group, supporting A. Tryon’s (1962) hypothesis that both genera might belong to a single genus, in which Jamesonia represented the more specialized elements of the larger unit. These hypotheses have been tested and subsequently supported by a more detailed phylogenetic study including 16 species of Jamesonia and 14 species of Eriosorus, based on a total of 1152 bp from the nuclear External Transcribed Spacer (ETS) of 18S—26S rDNA, and the plastid gene rps4 and the intergenic spacer rps4-trnS (Sanchez-Baracaldo, 2004). Furthermore, it was concluded in that study that neither genus is a natural group: Jamesonia is polyphyletic and Eriosorus is paraphyletic. Jamesonia’s polyphyly had been implicitly hypoth- esized by A. Tryon (1970): “‘Jamesonia is derived from more than one element in Eriosorus.” In all analyses, the species of Anogramma and Pityrogramma examined here form a monophyletic group as originally postulated by R. M. Tryon (1962). The close relationship between Anogramma and Pityrogramma species is strongly supported by phylogenetic analyses based on rbcL sequence data (Nakazato and Gastony, 2003). They examined more species of Anogramma and Pityrogramma than here, however, finding that Anogramma sensu R. Tryon (1962) is polyphyletic, with A. osteniana more closely related to Eriosorus and Jamesonia then to other species of traditional Anogramma. The results of the present study suggest a very strong phylogenetic relationship between the genera Onychium and Actiniopteris, with a weakly supported pee grat to other traditionally recognized taenitidoids, as previously found by Gastony and Johnson (2001), and Nakazato and Gastony (2003). Afropteris was treated with the pteridoids (Tryon and Tryon, 1982), before it was reclassified with the subfamily Taenitidoideae (Tryon et al., 1990). Evidence presented here suggests that Afropteri barklyae is indeed more closely related to Pteris multifida, and P. quadriaurita than to the taenitidoids, and suggests that A. barklyae would be more accurately classified within the pteridoids as in Tryon and Tryon (1982). Further phylogenetic studies, including broader taxonomic sampling are needed to clarify how this species relates to other — in italic script. Numbers of supporting molecular characters, derived from the intergenic spacer rps4-trnS, are indicated in roman script. Trees were rooted using the outgroups Pteris multifida and Pteris quadriaurita from subfamily =e and a more distantly related member, Pein ree fraxinea, as explained in the te 138 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) members of the Pteridaceae. Gastony and Johnson (2001), and Nakazato and Gastony’s (2003) work pointed out a close relationship between a clade of P. fauriei and P. cretica and the taenitidoids, as well as a distant relationship to the outgroup Coniogramme japonica. Hypotheses presented in this study are open to further testing with additional taxa. More morphological characters and data from other genes could also help to resolve the history of this group. ACKNOWLEDGMENTS is a portion of a Ph.D. thesis completed at the University of California, Berkeley under the supervision of Brent D. Mishler, to whom I am most grateful for his unconditional support and advice on phylogenetic methods. I thank Alan Smith for his advice, taxonomic expertise, and continued s support. I also thank Pedro Sénchez-Baracaldo, Augusto Repizo, Alvaro Cogollo, and the field, and Martin Grantham, Holly Forbes (UC Botanical Garden), Mark W. Moffett, Robert Ornduff, Alexandre Salino, and Harald Schneider for providing DNA specimens, as well as Kath vans and two anonymous reviewers for helpful comments on this manuscript. I thank John Wheeler for advice on molecular techniques and for introducing the use of rps4 in Mishler’s lab. This work was supported by an NSF Doctoral Dissertation Improvement Grant (9801245), a Vice Chancellor for Research Fund Award, and several grants from the Department of Integrative Biology, University of California, Berkeley. I received financial support from Colciencias, Colombia, and a Rimo Bacigalupi Fellowship from the Jepson Herbarium. LITERATURE CITED Arnott, H. 1959. Leaf clearings. Turtox News 37:192-194. ATKINSON, L. R. 1970. Gametophyte of Taenitis pinnata and development of a hig gana plate in Taenitis a epesieeag and Syngramma alismifolia. Phytomorphology Cuinc, R. C. 1934. On the genus Onychium Kaulf. from the far Orient. “Sines Sci. J. 13:493-501. Conant, D. S., a Rauseson, D. K. 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Molecular ela i and evolution of oi endemic Hawaiian genus Adenophorus sens rae Molec. Phylogen. Evol. 26:337-347. SANCHEZ-BARACALDO, P. 2004. Phylogenetics - “boweornphy of the neotropical fern genera ‘a nia and Ketone: (Pteridaceae). Am . Bot. 4-28 SANDERSON, M. J. 1989. Confidence limits in aE hl the avai steel. Cladistics 5:113- 129. ScHNENIDER, H. 1996. Vergleichende Wurzelanatomie der Farne. Ph. D. dissertation, Universitat Zurich, Ztirich. SCHNENIDER, H. 2001. Biogeography, ecology and phylogeny of Pteridaceae (Filicatae) in a - Taenitidoideae and Cheilanthoideae. Pp. 211-224, in Saw ua, eds. Taxonomy: The Cornerstone of cae hing ae ‘a the Fourth Pieiecent Flora Malesia ngs m 1998. Ampan, s, Lum SmitH A. R. and R. CRANFILL. 2002. tnfaia iataeahias of the thelypteroid ferns ania Amer. Fern J. 92: Souza-Curs, T. T., G. Brrrar, S. oe “4 en R, E. Besin and B. LEjEUNE. 1997. Phylogenetic analysis of — with parsimony and eae methods using the plastid gene rps4. PI. Syst. Evol. 204:109-123. priaaine Db. poi PAUP*: Phylogenetic oe Using Parsimony, Version 4.0 (* and other age Version 4. Sinauer, Sunderland, M as. y F, 1962. A monograph of the fern genus tail Contr. Gray Herb. 1991:109—203. 140 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) Tryon, A. F. 1970. A monograph of the fern genus Eriosorus. Contr. Gray Herb. 200:54—174. Tryon, A. F. and B. LuGARDON. 1991. Spores of the Pteridophyta. Springer-Verlag, New York. Tryon, R. M. 1962. Taxonomic fern notes. II. Pityrogramma (including Trismeria) and Anogramma. Contr. Gray Herb. 189:52—76. Tryon, R. M. and A. F. Tryon. 1982. sch and Allied Plants with Special Reference to Tropical America. Springer-Verlag, New Tryon, R. M., A. F. Tryon and K. U. esti 1990. Pteridaceae. Pp. 230—256 in K. U. Kramer & P. S. Green, eds. The Families and Genera of Vascular Plants. Vol. I. Pteridophytes and Gymnosperms. Springer-Verlag, Berlin. Watker, T. G. 1968. The anatomy of some ferns of the Taenitis alliance. Proc. Linn. Soc. London 179:279—286. Wiey, E. O. 1981. iii aa the theory and practice of phylogenetic systematics. J. Wiley & Sons, New Wor, P. G., K. M. om AiR SMITH gy M. Hasse. 1998. Phylogenetic studies of extant pteridophytes. Pp. 541-556 in D. Soltis, P. S. Soltis. & J. J. Doyle, eds. Molecular Systematics of Plants, II: DNA oar Kluwer Academic Publishers, Norwell. APPENDIX 1 Morphological character list. Characters states follow criteria discussed in text. 1. SPORE SHAPE: electron micrographs are well documented for most genera and many species of ferns. Most taxa included in this study are documented in Tryon and Lugardon (1991). [tetrahedral-deltoid = 0; go ee eae ae 2. EQUATORIAL RINGE: an equatorial flange is defined here as a prominent structure (ring) surrounding a ae at the equatorial plane (Tryon and Lugardon, 1991). [present = 0; abse 3. NUMBER OF EQUATORIAL RIDGES IN SPORES: Spores in Taenitidoi- deae exhibit variation in the number of equatorial ridges in different taxa; some taxa completely lack ridges (Tryon and Lugardon, 1991). [one equatorial ridge = 0; two equatorial ridges = 1; three equatorial ridges = ] 4. SPORE SURFACE: There is great variation in spore ornamentation among members of the Taenitidoideae (Tryon and Lugardon 1991). [extremely verrucose with spines = 0; moderately verrucose = 1; slightly verrucose = 2; smooth = 3] 5. SPORE COLOR: Spore color is a discrete character among members of the Taenitidoideae. This character has been previously used as diagnostic for some genera of the Dheagriragn (A. Tryon, 1962; 1970). [dark brown = 0; light pees = 1; white SPORANGIAL meek Exindusiate ferns can exhibit scattered or es sporangia along veins. For instance, Pityrogramma and Anogramma have evenly scattered sporangia in contrast with genera that have clustered sporangia such as Austrogramme, Syngramma, Taenitis, and Pterozonium. Afropteris, Onychium, and Pteris were not scored because it was hard to discern the distribution of their sporangia due to their false indusium. [scattered sporangia = 1; clustered sporangia = 2] SANCHEZ-BARACALDO: RELATIONSHIPS OF SUBFAMILY TAENITIDOIDEAE 141 7. SPORANGIAL MATURATION: This character refers to sporangial maturation on a fertile leaf. In some fern genera with linear sori, sporangia develop in an outward (acropetal) sequence, along the vein towards the margin. Other genera exhibit mixed sporangial maturation (A. Tryon, 1970). [mixed maturation = 0; acropetal maturation = 1] 8. SPORANGIAL STALK LENGTH: Sporangial stalks vary in length. This character exhibits discrete character states. Taxa with sporangial stalks that were equal to or greater than the capsule length were scored as long. Taxa with sporangial stalks that were extremely short (sessile capsule) or less than half the length of the capsule were scored as sessile to short. Only fully mature sporangia were measured. [long = 0; sessile to short = 1] 9. FARINA: Farina is a waxy-appearing exudate of glands believed to protect young sporangia (Lellinger, 1985). This character can be present in exindusiate and indusiate ferns. [present = 0; absent = 1] 10. INDUSIUM: An indusium is a scale-like structure partially or fully covering and protecting the young sporangia (Lellinger, 1985). In some members of the Pteridaceae, the inrolled lamina edge is modified and called a false indusium. [false indusium = 0; exindusiate = 1] 11. PARAPHYSES: Hairlike structures borne on the soral receptacles or on sporangial stalks or capsules (Lellinger, 1985). Paraphyses are believed to provide protection for young sporangia. [present = 0; absent = 1 12. PARAPHYSIS ARRANGEMENT: Paraphyses can be densely packed around the sporangia. In contrast, some genera have loose and more relaxed paraphyses associated with their sporangia. [loose=0; densely int i=1] 13. FROND DISSECTION: [bipinnate or more = 0; pinnate = 1; simple = 2; pedate = = 3] 14. DETERMINATE GROWTH: This character refers to mature fronds bearing sporangia, either maintaining a fiddlehead-like morphology at the tip as adults or not. [determinate = 0; indeterminate = 1] 15. LEAF MARGIN: In some genera, pinna margins are fully extended when mature; in other genera, the pinna margins are more or less incurved, thus protecting the sporangia. In the latter case there is no scale like structure developmentally derived from the leaf margin protecting the sporangia (e.g., false indusium). This character exhibits discrete states. [fully extended = 0; mildly incurved = 1; 1/4 strongly curved = 2] 16. LEAF HAIRS ON ABAXIAL LEAF SURFACE: Hairs are defined as epidermal outgrowths composed of a single elongated cell or a single file of cells. Some species exhibit uniseriate hairs on veins or on abaxial sides of blades. [present = 0 ; absent = 1 17. STELLATE ARRANGEMENT OF CELLS ON THE LEAF: Cellular configuration of cells associated with only some epidermal hairs on the adaxial side. a character can be observed only with cleared leaves. [present = 0; absent = 18. VEIN et WITH RESPECT TO LEAF MARGIN: Strands of vascular tissue can reach or stop before the leaf margin. [veins ending before margin = 0; veins reaching margin = 1] 142 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) 19. VEIN ENDS: Vascular strands may keep their width or become reduced or enlarged at vein ends. This character is easily observed with cleared leaves. [reduced = 0; same width = 1; enlarged = 2 20. CELL LENGTH ON ADAXIAL SURFACE OF LEAF: Cells vary in length. In this study, short cells are defined as three to four times longer than wide, and long cells are defined as six to eight times longer as they are wide. This character can be observed only in leaf clearings. [short = 0; long = 1 21. SHAPE OF CELL WALL ON ADAXIAL SURFACE OF LEAF: Cell wall borders vary among genera; some cell walls are straight while others are sinuous. Among species of Jamesonia the degree of sinuosity varies with respect to its position on the leaf (A. Tryon, 1962). However, the cell wall shape, sinuous vs. straight, is a discrete character among genera. The adaxial cells observed for this character were equidistant between veins and margins. This character can be observed only in leaf clearings. [sinuous = 0; straight = 1] 22. SCALES: Scales are defined here as multicellular, bi- to multiseriate epidermal outgrowths (Kubitzki, 1990). In some cases they can also be found on the rhizome, at the base of petioles, and on leaf blades. [present =0; absent=1] 23. COLOR OF SCALES: Color of scales is a discrete character among members of this group. [very dark=0; bicolorous = 1; brown = 2; very pale=3] 24. SHAPE OF SCALES: Scales exhibit a variety of shapes that seem to be consistent within species but variable across species lines. [elongate = 0; lanceolate = 1] 25. HAIRS ON RHIZOME: Hairs are defined here as uni- to multicellular, uniseriate, epidermal growths (Kubitzki, 1990). [present = 0; absent = 1] 26. HAIR CELLS (ON RHIZOME): Hairs vary in the number of cells at their base. [two cells wide at base = 0; one cell wide at base = 1] American Fern Journal 94(3):143—154 (2004) A Contribution to the Gametophyte Morphology and Development in Several Species of Thelypteris, Thelypteridaceae BLANCA PEREZ-GaRCIA and ANICETO MENDOZA-RUIZ Departamento de Biologia-Botanica Estructural y Sistematica Vegetal Universidad Auténoma Metropolitana — Iztapalapa Apdo. Postal 55-535, 09340 México, D. F. Asstract.—A contribution to the study of the gametophyte’s morphology and development of some species of Thelypteris Schmidel (Thelypteridaceae). The development and morphology of the sexual phase of five species of the Thelypteridaceae family is described and compared. Spores are of the common type of the leptosporangiate ferns. Sporophytes in Thelypteris reptans var. reptans and T. tetragona appear 90-285 days after sowing; T. dissimulans, T. piedrensis and T. oviedoae did not develop any sporophytes. The sexual phase of these species has many morphological characteristics in common with species of the Old and New Worlds. The genus Thelypteris has ca 1000 species distributed in tropical and subtropical regions; the American species have been taxonomically studied by Smith (1971, 1973, 1974, 1980). Approximately 300 species are found in Neotropical areas, ca 60 of which are known from Mexico (Smith, 1973, 1974, 1995). Some pteridologists have subdivided Thelypteris sensu Jato into natural groups as genera, subgenera or sections. Many of the New World taxa can be circumscribed using different combinations of characters and thus be treated as genera. In this paper we treat Thelypteris in the broad sense, but recognize several subgenera. As construed, Thelypteris is distinguished by the presence of two vascular bundles in the petiole (Dryopteris and other closely related groups have many vascular bundles), acicular hairs on many parts of the blade, spores usually bilateral with a prominent perispore, and a chromosomal base number of 27 to 36 (x = 40, 41 in Dryopteris, Athyrium, and close relatives). Although the determination of the Thelypteridaceae is based on the sporophyte’s morphological characters such as, mainly, the type of indument, for example: bifurcated or stellate hairs (subg. Goniopteris); acicular, unicellular or pluricellular hairs (subg. Goniopteris), stellate or no furcate hairs (subg. Macrothelypteris; subg. Meniscium and subg. Amauropelta); setose sporangia (subg. Stegnogramma); sporangia without setae (subg. Cyclosorus, and subg. Steiropteris), when doing a follow up of the morphogenetic development, we would expect to find that the gametophyes have the same type of indument, a diagnostic character that would help us to support the segregation at the subgeneric level. Nevertheless, in the studied species we could only observe in the laminar phase, unicellular, capitate hairs, with 144 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) a waxy secretion. For this reason we suggest a more detailed study with a larger number of species in order to learn if the indument is a useful character, helpful in separating the species into well defined subgenera, or to join them in infraspecific taxa. Details of the plants’ indument provide important taxonomic characters. The indument includes: sessile or pedicellate stellate hair, bifurcate hairs, anchor shaped, fasciculate or hooked hairs; sessile or stipitate glands, setose sporangia or sporangia with stellate hairs or glands. We would expect the plant’s gametophytes to share these characteristics. Although the Thelypteridaceae is very large, its prothallial morphology is poorly known. Its gametophytes can be described as follows: epigeous, chlorophyllous, at maturity cordiform or elongated-cordiform, symmetrical, often with wide wings, fast growing, without a thickened cushion or lacking one, with colorless or light brown rhizoids abundant on the cushion’s ventral surface, frequently with unicellular hairs or rarely pluricellular and/or glandular hairs on the margin and on both surfaces. Gametangia are of the advanced leptosporangiate type: the antheridia are characteristically three- celled and dehiscence occurs when the opercular cell detaches; the archegonia are ventral surface, especially on the lower region, the slim neck points towards the meristematic notch (Tryon and Tryon, 1982). Previous work on Thelypteris gametophytes, mainly Asian species include: Schmelzeisen (1933), Momose (1938, 1941), Kachroo (1963), Nayar and Chandra (1963, 1965, 1966), Chandra and Nayar (1968), Devi (1966), Nayar and Devi (1963, 1964), Nayar and Kaur (1969), Mittra and Sen (1981) and Tigerschidld (1989a, 1989b, 1989c,1990). Details on the prothallia of New World Thelypteris are mentioned by Stokey (1960), Atkinson (1971, 1973, 1975a, 1975b), Atkinson and Stokey (1964, 1973). Huckaby and Raghavan (1981b) worked with 16 Jamaican species; Reyes-Jaramillo and Pérez-Garcia (1991) worked with T. patens and T. puberula var. puberula; Pérez-Garcia et al. (1994) worked with T. rhachiflexuosa and Nester-Hudson et al. (1997) worked with T. ovata var. lindheimerii. This paper is a contribution to the study of the morphology and development of the gametophytes of Thelypteris (Amauropelta) piedrensis (C. Chr.) Morton, T. (Cyclosorus) oviedoae C. Sanchez & Zavaro, T. (Goniopteris) dissimulans (Maxon & C. Chr. ex C. Chr.) C. F. Reed, T. (Goniopteris) reptans (J.G. Gmelin) Morton var. reptans and T. (Goniopteris) tetragona (Sw.) Small. MATERIALS AND METHODS Material for research was collected from the “Jardin Botdnico de los Helechos”’, in Santiago of Cuba and from several different Cuban localities (Table 1). Vouchers are kept at this Herbarium (BSC). Spores were obtained from the fertile leaves of different individuals and leaves with spores were kept in paper envelopes to be released at room temperature. The samples were put through a metallic sieve, with pores 0.074 mm in diameter to remove excess non-spore material. Spores of each species were sown, without sterilization, in 5 Petri dishes, 5 cm in diameter (3 replicates for each species), onto Thompson GARCIA AND MENDOZA-RUIZ: THELYPTERIS GAMETOPHYTES 145 BLE 1. Sites of origin of the taxa under research. Caluff = Manuel Garcia Caluff; Shelton = pce Shelton Serrano, all vouchers have been deposited alive at the BSC (Fern Garden), Santiago de Cuba, Cuba. Taxa Vouchers Locality Habitat/Altitude Thelypteris Margins of the ‘“‘Barao del Gallery forests on (Goniopteris) Shelton 4204 Banao” river, Santispiritus, limestone rocks, dissimulans Prov. de Santiepiitns, Cuba 250 m asl Thelypteris No date “Jardin de los Helechos No date (Amauropelta) (Fern Garden) piedrensis Thelypteris Mogotes of boigne, La Tabla, Perennial vegetation, (Goniopteris) Shelton 2963 3er frente, Prov. de Santiago on limestone rocks reptans de Cuba, Cuba and slopes, 600 m asl reptans Thelypteris Caluff 47 A-B Altos de Villalén, “La Gran Secondary growth, coffee (Goniopteris) Piedra”’ mountain range, plantations, 500 m asl tetragona Prov. Santiago de Cuba, Cuba Thelypteris Caluff s/n “Lomas del Solén”, las Secondary growth on road (Cyclosorus) Terrazas, Prov. Pinar margins, 600-650 m asl oviedoae del Rio, Cuba sowing medium (Klekowski, 1969), under aseptic conditions into 5 cm Petri dishes; spores were spread across the medium surface with a brush of scarce bristles. The density of the sown spores varied from 100—150/cm’. The cultures were kept under lab conditions inside transparent polyethylene bags to avoid contamination and dehydration, under a 12 hr light/12 hr dark photoperiod, with artificial light (Solar 75 Watts, day light) and a 25—28°C temperature. One dish of each taxon was kept in the dark to check for photoblastism. Spores of the five species were also sown separately in soil in 3 small pots, 5 cm in diameter (2 replicates for each species). Photomicrography of all gametophytes was performed on specimens grown on agar in Petri cultures and sporophytes grown in soil samples. RESULTS The spores of all species are monolete, with a single leasura and brown to dark-brown. Spores of T. piedrensis measure 45 um X 29 pm (51 pm X 30 um) 55 um X 32 um, of T. reptans var. reptans, 50 um X 30 um (52 pm X 34 pm) 58 um X 38 um, of T. tetragona 42 um X 28 um (44 um X 29 um) 47 pm X 32 pm, of T. dissimulans 48 um X 30 pm (49 um X 33 pm) 50 pm X 35 pm, the perine can be wide or narrow and it has various ornamentations. In T. dissimulans (Fig. 1) the perine is quite thick, in T. piedrensis (Fig. 2) and T. oviedoae it is very thin, and in T. reptans var. reptans (Fig. 3) and T. tetragona it is intermediate in thickness. In all species germination began 8—11 days after sowing. Germination is first evidenced by the development of the very long, hyaline rhizoidal cell 146 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) Fics. 1-9. Monolet , germination and filamentous phases of Thelypteris. Fig. 1-3. Spores. eed dssimlans. 2.7 inca 3. T. reptans var. reptans. 4. Germination in T. lige (11 ys). 5-9. Filamentous phases. 5. T. dissimulans (26 days). 6. T. tetragona (11 days). 7. T. pidrnsie (a (14 da sie 8: T. om (5 days). 9. T. piedrensis (14 days). p = perine, pc = peeballial cell, r=r GARCIA AND MENDOZA-RUIZ: THELYPTERIS GAMETOPHYTES 147 containing limited cytoplasm and the prothallial cell which develops inside the spore coat (Fig. 4). This type of germination is the Vittaria-type, characterized by the first division giving rise to an initial rhizoid oriented perpendicular to the long axis of the spore. The second division produces an initial prothallial filament at the base of the rhizoid but at right angles to it (Fig. 5). The filament phase begins between days 11 and 26. During this phase a sequential series of cell divisions occur in essentially the same plane to produce a slim, pluricellular germinal filament of three to seven barrel shaped cells (Figs. 5—9). All of these prothallial cells have numerous chloroplasts. This uniseriate condition is only a temporary phase. Thelypteris oviedoae, T. piedrensis, T. reptans var. reptans and T. tetragona have the same de- velopment period of 11-15 days; the development of this phase takes longer in T. dissimulans (11-26 days). The development time of the plate phase varies among species: in T. oviedoae and T. piedrensis it is 26—50 days long, in T. reptans var. reptans and T. tetragona 26-60 days, and for T. dissimulans it is 70 days. All species have an Aspidium-type prothallial development (Fig. 10-13), in which the uniseriate filament and plate phase development begins before the appearance of hairs, the meristematic cell is located centrally (Fig. 11) along the distal margin, the prothallus grows due to this cell’s activity and later acquires a notch and becomes apical, a pluricellular meristem becomes apparent and, later, a cushion develops; young prothallia begin to grow unicellular (Fig. 12), capitate hairs. The Aspidium-type is variable with regard to sequence of cell divisions. The sequence of cell divisions and development of the young thallus are conditioned here by early hair formation in the young prothalli. Plate phase formation is initiated by cells behind the terminal cell (sometimes the penultimate cell of the germ filament in also sluggish) dividing longitudinally. Marginal hairs are produced continuously. The prothallus grows by the activity of the meristematic cell and soon the meristematic region becomes notched and apical by unilateral growth of the thallus. A pluricellular meristem is established in the usual way and soon afterwards a midrib is formed. The mature prothallus is cordate and profusely hairy. The adult phase corresponds to days 60 to 150 in T. reptans var. reptans and T. tetragona. It has a cordiform-spatulate shape with an indistinct notch, an elongated, thickened cushion over which the gametangia and rhizoids are found, and short, more or less isodiametric, undulating wings with marginal and superficial capitate hairs (Figs. 14-15). There is variation in the shape of the gametophyte in T. oviedoae and T.piedrensis (50-75 days), T. reptans var. reptans (80-150 days), and T. dissimulans and T. tetragona (60—200 days) where the gametophytes have a cordiform-reniform shape, with a dense cushion, a marked notch, wide and isodiametric wings, with undulate margins and the same type of hairs (Figs. 16-17). Rhizoids of all gametophytes are ventral, unicellular, hyaline to light brown, and abundant, intermixing with the reproductive structures. 148 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) Fics. 10-17. Developmental — of the eT of Thelypteris. 10-13. Young plate phases. 10. T. piedrensis (15 d . 11. T. tetragona (15 days). 12. T. tetragona (26 days). 13. T. piedrensis (32 days). 14-17 Auta gametophytes, spatulate to cordiform. 14. T. tetragona (89 days). 15. T. tetragona (70 ia 16. T. reptans (89 days). 17. T. piedrensis (50 days). an = antheridia, h = secretory hairs, mc = meristematic cell, mz = eodetian zone. GARCIA AND MENDOZA-RUIZ: THELYPTERIS GAMETOPHYTES 149 According to Atkinson, thelypteridoid ferns are generally characterized as having pubescent gametophytes; glabrous gametophytes have never been observed. Hairs are commonly simple and non-chlorophyllous, with a yellow or transparent secretion. Hairs of the studied species were found on the margin and on both surfaces of the gametophyte; they are hyaline unicellular, and capitate, with an extracellular waxy coat on the apex (Figs. 18-19). These hairs develop during the young plate phases in T. oviedoae and T. piedrensis, in T. reptans var. reptans at 26 days and in T. dissimulans and T. tetragona at 35 days. They are abundant on the adult phase, and are distributed along the margin and on both surfaces. The gametangia are characteristic of typical leptosporangiate ferns. Arche- gonia are located distally on the cushion intermingled with the rhizoids. The long neck, which is oriented toward the meristematic zone, is composed of four rows of neck cells and a mouth of four cells (Figs. 20-21). Archegonia develop in T. reptans var. reptans at 67—75 days, in T. piedrensis at 89 days and in T. tetragona at 70-108 days. The antheridia are small, globose, with a basal cell, an annular cell and an opercular cell (Figs. 22-23). They develop in T. tetragona at 70-108 days, in T. piedrensis at 89 days and in T. reptans var. reptans at 67-108 days. Gametangia did not develop in either T. disimulans or T. oviodeae during the 300 days of cultivation. Sporophyte initiation was observed in T. tetragona at 89-166 days, and in T. reptans var. reptans at 137-285 days. Sporophytes did not develop in cultures of T. dissimulans, T. piedrensis or T. oviedoae, even after 300 days of cultivation. First leaves of young sporophytes had a petiole and a 2-, 3-, or 4- lobed blade. The venation was open and dichotomous (Figs. 24-26), All leaves had abundant hairs, marginally and on both surfaces. The hairs were unicellular, capitate, hyaline, achlorophyllous and secretory, similar to those on young and adult gametophytes. Stomata are of the policitic- type, with one or two subsidiary cells (Kondo, 1962; Thurston, 1969; Smith, 1990). DISCUSSION All spores are monolete, with ornamentation ranging from reticulate-pitted in Thelypteris subgenus Amauropelta, to winged in T. subgenus Goniopteris, to low ridged or ridged-papillate in T. subgenus Cyclosorus (Wood, 1973; Tryon and Tryon, 1982; Tryon and Lugardon, 1991). The gametophyte phase of the Thelypteridaceae, especially subgenera Amauropelta, Cyclosorus, Goniopteris and Thelypteris, is notoriously uniform, both in relation to spore germination, the Vittaria-type (Nayar and Kaur, 1971; Huckaby and Raghavan, 1981a) and prothallial plate development, the Aspidium-type. In the Vittaria-type germination, the spore’s first division produces two unequal cells, a smaller one that immediately elongates and differentiates into the first rhizoidal cell, and a second larger cell which eventually gives rise to cells of the prothallial plate. 150 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) eI Fics. 18-26. Secretory hairs, gametangia and first leaves in Thelypteris. 18-19. Unicellular secretory hairs. 18. T. piedrensis (89 days). 19. T. reptans (75 days). 20-21. i nia. 20. Nec archegonia in T. reptans (67 days). 21. Mouth of archegonia in T. tetragona (70 days). 22-23 Antheridia. a T. reptans (67 days). 23. T. tetragona (108 days). 24—26. First jaan aioe leaves. 24. T. reptans (137 days). 25. T. tetragona (166 days). 26. T. reptans. (166 days). GARCIA AND MENDOZA-RUIZ: THELYPTERIS GAMETOPHYTES 151 Seven different types of prothallial development in the homosporous ferns are described by Nayar and Kaur (1969). The critical elements which define these types are differences in the sequence of cell divisions during de- velopment, the location at which a meristematic cell or pluricellular meristem is established, and the ultimate shape of the thallus. Although the taxa studied by us show a delay in prothallial hair development according to Nayar and Kaur (1969), we consider that they have an Aspidium-type development because the meristematic cell has a very active role in the growth and expansion of the young prothallus and this Aspidium- type of development is seen in more advanced genera such as Thelypteris. During the each developmental stage, the plate of the gametophyte originates from three divisions on the same plane - two lateral and one horizontal - of the inferior face of an initial meristematic cell. That cell is subsequently replaced by a group of meristematic cells which contribute to the development of the cushion and to the expansion of the gametophyte wings. The substitution of the initial cell by a pluricellular meristem is common in other subgenera of Thelypteris (Kachroo, 1963; Nayar and Chandra, 1963; Atkinson, 1971; Reyes- Jaramillo and Pérez-Garcia, 1991). A distinctive characteristic of the gametophytes of the different subgenera of the Thelypteridaceae is the differentiation of unicellular, short, capitate hairs, with a wax secretion at the apex, during the blade development. This was clearly seen in all species. Various authors have studied the gametophyte morphology of various species of Thelypteridaceae. They are all pubescent, with a relatively long- lived thallus that become cordate at maturity, delicate for their size, with a cushion and wide wings, abundant colorless or light brown rhizoids and advanced-type sexual organs. Although the differences regarding spore ornamentation, length of simple hairs and antheridia shape are insignificant, these characteristics vary in different species; the taxa we studied share the characteristics previously mentioned. We summarize that the morphology of the gametophytes in Thelypteris have a Vittaria type germination pattern, an Aspidium type prothallial development, that the gametophytes are generally uniform in form and development, and show enough diversity to suggest that they are useful in understanding taxonomic boundaries. In the species studied here we only observed the most common type of hair, which is unicellular, capitate, secretory and with a waxy secretion; we did not observe other types of hairs mentioned in the literature, for example, secretory hairs, long, glandular-septate hairs, and acicular hairs (Kachroo, 1963; Nayar and Kaur, 1971; Tigerschidld, 1989b, 1990). Prothallial hairs have developed only in some subgenera, possibly the presence of similar types of hairs in groups apparently unrelated gave origin to the idea that epidermal derivates are of little value in taxonomic and phyletic studies. Nevertheless, the restricted distribution of pubescent gametophytes among several phyletic groups, and the presence of certain characteristic types of hairs in other groups (for example: acicular hairs in the Thelypteridaceae) apparently indicate that these are important characteristics in comparative 152 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) studies. As is common with other aspects of gametophyte morphology, the tendency to have hairs seems to have developed independently in the different phyletic groups of ferns. Regarding the gametangia, our results indicate that the antheridia have the characteristic type of structure and ontogeny characteristic of advanced ferns such as the Thelypteridaceae (Davie 1951) and Stone (1960). Tigerschiéld’s (1989a) contributions in which she mentioned four different dehiscence types in antheridia: 1) irregular split on the wall of the opercular cell, 2) a circular split similar to a pore, 3) the opercular cell is expulsed completely to the exterior and 4) the slit opens laterally (like a lid), the operculum remains joined to the annular cell. Although it has been stated that dehiscence is specific to each species, Tigerschiéld (1989a) observed that this is not always true, the same species can have two or three of these dehiscence mechanisms. All of the gametophyes in this study dehisced by means of the separation of the whole opercular cell, which in this case corresponds to Tigerschidld’s type three. In other studies, Tigerschiéld (1989b, 1990) showed that his thelypteroid ceylandes gametophytes are similar in most respects. Nevertheless, we did find some aietinelanitie characteristics among t species. Variation was present in thallus margin shape, hair length, Adi bice of secretory hairs, presence or absence of acicular hairs, number of archegonial neck cells, and opercular cell form. Just as antheridial anthesis and antherozoid liberation varies in each of the species studied, this study demonstrates that it is possible to identify species based on characteristics of the gametophyte alone. Stokey (1951), Atkinson and Stokey (1964) and Smith (1971) state that fern gametophytes have only recently begun to be considered as a possible source of taxonomic characters, or as a comparative morphological tool. Gametophytes seem to be useful in systematics at the family level, and in certain cases, at the generic level, but they are most frequently used, on a smaller scale, to distinguish species within the same genus. The gametophyte’s uniform development in species of different subgenera of the Thelypteridaceae has been mentioned by several authors (Nayar and Chandra, 1963; Huckaby and Raghavan, 1981a; Tigerschidld, 1989a, 1989b, 1990; Reyes-Jaramillo and Pérez-Garcia, 1991). Nevertheless, the comparative study of the gametophyte development of American species helps define, based on hair type and position, margin, antheridial structure and shapes of the anteridial slit, the combination of characters that will delimitate subgenera, species, or groups of species within the Thelypteridaceae (Atkinson 1973; Pryer et al., 1995). This study of the gametophytes of five species of thelypteroid ferns from the New World indicates that they can be very diverse and that they are distinctive. Nevertheless, because the sampling was scarce, this conclusion could be premature or it could only represent a record of the gametophyte’s characteristics and an effort to accommodate or adjust them within today’s classification. This research could be useful as a comparative basis in which many thelypteroid gametophytes could begin to be known. GARCIA AND MENDOZA-RUIZ: THELYPTERIS GAMETOPHYTES 153 ACKNOWLEDGEMENTS e wish to thank the anonymous reviewers for their suggestions and criticisms of the Helechos” (Fern Botanical Garden) at Santiago de Cuba, Cuba. We thank Jorge Lodigiani for photographic assistance. LITERATURE CITED ATKINSON, L. R. 1971. The gametophyte of Thelypteris erubescens. Amer. Fern J. 61:183—186. ATKINSON, L. - 1973. The Gametophyte and Family Relationships. J. Linn. Soc. Bot., Suppl. No. ‘L,G7: ATKINSON, L. . 1975a. The gametophyte of five Old World Thelypterioid ferns. Phytomorphology 25:38-54 ATKINSON, he R 1975b. The gametophyte of five Old World thelypteroid ferns. Phytomorphology 25:3 ATKINSON, ee - and A. G. STOKEY. 1964. es morphology of the gametophyte of osporous ferns. Phytomorphology 14:5 ares, L. R. and A. G. Stoxey. 1973. The shnistesini of some Jamaican thelypteroid ferns. J. n. Soc., Bot. 66:23—36. i "P. and B. K. Nayar. 1968. Morphology of the edible fern Ampelopteris Kunze. Proc. Indian Acad. Sci., B. 68:25-36. vig, J. H. 1951. Development of antheridium in the Sy | Aaa Amer. iad hie 38:621-628. bie S. 1966. Spore morphology of Indian Ferns. Ph. D. Thesis. Agra Univ Huckasy, C. S. and V. RAGHAVAN. 1981a. The spore- seantntted: te of ie ferns. Amer. . Bot. 68:517-523. Hucxasy, C. S. and V. RAGHAVAN. 1981b. Photocontrol of spore germination in the fern Thelypteris kunthii. Physiol P]. (Copenhagen) 51:19—22. Kacuroo, P. 1963. Observations on certain aspects of the sane ape of the gametophyte of Pe ctor (Wall.) Ching. J. Indian Bot. Soc. 42:19 KLekowskl, E. 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Pollen et Spores 5:354-372 ite B “a and S. Devi. 1964. Spore morphology of Indian ferns-I: Aspidiaceae. Grana Palynol. Give i ps and S. Kaur. 1969. Types of prothallial development in homosporous ferns. Phytomorphology 19:179-188. 154 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) aoe: se K. and S. Kaur. 1971. Gametophytes of homosporous ferns. Bot. Rev. (London) 37:295- eck eee J. E., C. Lapas and A. McC.urp. 1997. pigrarestir a development and antheridiogen activity in Thelypteris ovata var. lindheimeri. Amer. Fern J. 87:131—142. PEREZ-GarciA, B., R. RisA and A. MENDOozA. 1994 a del gametofito de Thelypteris srry pasa Riba sea eerie hike Bot. Mex. 2 siden K. M., A. R. Smiru and J. K. Skoc. 1995. ae relationships of extant based on ks nce from morphology and rg poe pense Amer. Fern J. 85:205— REYES- alas I. and B. Pérez-Garcia. 1991. Desarrollo de los gametofitos ie Thelypteris patens (Sw.) Small y de T. wailenuta es Morton var. puberula. Acta Bot. Mex. 16:7—1 SCHMELZEISEN, W. 1933. Beitrage zur Enticklunggiscicht der prothallien einiger na 27:23 Smitu, A. R. 19 pa gg of the neotropical species of Thelypteris section Cyclosorus. Univ. Calif. Pub Bot. —143. Smit, A. R. ip mexican species of Thelypteris subgenera Amauropelta and Goniopteris. Amer. Pattee 63:116-127. Soir, A. R. 1974. A revised classification of Thelypteris subgenus Amauropelta. Amer. Fern J. or 83- Agi 0. Taxonomy of Thelypteris oe Steiropteris, including Glaphyropteris “eteridpiyt Univ. Calif. Publ. Bot. 76:1 Hest 0. Thelypteridaceae. Pp. 263-272, in K U. Kamer and P. 8S. rset, eds. The oa. ine Genera of Vascular Plants. I. P: j Verlag, k. New Yor Situ, A. R. 1995. Thelypteris Schmidel. Pp. 164-165, in R. C. Moran and R. Riba, eds. hag he to hoorapeains Flora Mesoamericana Vol. 1. Universidad Nacional Auténoma de Méxic STONE, L G. 1960. Observations on the gametophytes of Grammitis billardieri Willd. and Ctenopteris heterophylla (Labill.) Tindale (Grammitidiaceae). Austral. ee A. G. 1951. The anpesgein n by the gametophyte to classification af the homosporous rns. Phytomorphology 5 rane A. G. 1960. Multicellular and —— hairs on the fern gametophyte. Amer. Fern J. 50: 78-87. THuRsTON, E. L. 1969. Taxonomic significance of stomatal patterns in the ferns. Amer. Fern J. 59: 68-79. TIGERSCHIOLD, E. 1989a. Micromorphology of gametophytes and antheridial dehiscence in The oe Tesis Doctoral, University of Stockholm. TIGERSCHIOLD, E. . Scanning electron microscopy of gametophyte characters and antheridial opening in some Ceylonese species of Thelypteridaceae. Nordic. J. Bot. 8:639-648. TIGERSCHIOLD, E. 1989c. Dehiscence of spalnaaiicgers in esemnan ferns. Nordic. J. Bot. 9:407—412. TIGERSCHIOLD, E. 1990. G y f some Cey f Thelypteridaceae. Nordic. J. Bot. 9:657-664. TRYON, A. F. and B. LucarpoN. 1991. Spores of the Pteridophyta: vig Wall Structure, and Diversity Based on Electron Microscope Studies. Springer-Verlag, N linia R. M. and A. F. Tryon. 1982. Ferns and Allied Plants with paltry jieferenee to Tropical erica. Springer-Verlag. New York. we C. C. 1973. Spore variation in the Thelypteridaceae. J. Linn. Soc., Bot. 67, Suppl. 1:191-202. American Fern Journal 94(3):155—-162 (2004) SHORTER NOTES Botrychium pallidum Newly Discovered in Maine.—A recent report of Botrychium pallidum W. H. Wagner from Maine (Wagner, Jr., W. H. and F. S. Wagner, 1993, Ophioglossaceae. Pp. 85-105 in FNA Editorial Commit- tee, Flora of North America north of Mexico, vol. 2. Pteridophytes and Gymnosperms. Oxford University Press, New York) was apparently erroneous (W. H. Wagner, pers. comm.) and was subsequently removed in the second printing of early 1994. It is not known on what basis the report was made, and no specimens have been seen in regional herbaria to support it. Now, however, the species has been discovered at a site in Washington County, Maine, the first in the northeastern United States. The site is located approximately 440 km south of the nearest known station at Bic, Saguenay County, Quebec. The Maine population was observed first on 3 July 2000 and again on 17 June 2001. On both dates, only two plants were observed. Given the difficulties inherent in identifying moonworts (Williston, P. 2001. The Botrychiaceae of Alberta. Alberta Natural Heritage Information Centre, Edmonton), the identification awaited confirmation until another visit was made on 14 June 2003, when 60 well-formed, unequivocally identifiable plants were observed. They occurred singly and in small clumps (2-4 stems per clump), scattered over an area approximately 12 m X 60 m. On a return visit on 24 June 2003, fewer plants were observed, perhaps due to herbivory; plants on that date were observed to be sporulating and most were already senescent. One plant (above ground parts only) was collected for isozyme analysis and two for vouchers. Isozyme analysis also confirmed the identity (Don Farrar, Iowa State University, pers. comm.). VOUCHER MATERIAL: USA: Maine: Washington County: Steuben, Petit Manan National Wildlife Refuge, 60 plants in old field, with Botrychium simplex and Botrychium matricariifolium, among low grasses, forbs, mosses, and lichens, 14 June 2003, Gilman 03008, Norm Famous & Marcia Spencer-Famous (IA); 24 June 2003, Gilman 03009, Arthur Haines, Sally Rooney, Linda Welch & Michael Langlois (MAINE, Herb. Petit Manan National Wildlife Refuge). Botrychium pallidum was originally described from Quebec, where it was collected near Baie St. Catherine (Wagner, Jr., W. H. and F. S. Wagner. 1990. Notes on the fan-leaved group of moonworts in North America with descriptions of two new members. Amer. Fern J. 80:73—81). In Quebec, it has also been collected at Bic (Wagner 90010 & Wagner, MICH, image seen; Coursol et al., DAO, image seen), and Forillon Federal Park, Gaspé County (J. Labrecque, Ministére de l’Environement du Quebec, pers. comm.). It is also known from the Great Lakes region in Michigan (Upper and Lower Peninsulas), Ontario, and Minnesota, and occurs in Saskatchewan, Alberta, Montana, and Colorado (Wagner and Wagner, 1990; Wagner and Wagner, 1993; Williston, 2001). Nevertheless, it is a rare species with widely scattered populations normally 156 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) consisting of few individuals. It currently has a Global Heritage Status Rank of G3 (globally vulnerable) taxon (NatureServe, 2003, NatureServe Explorer: an on- line encyclopedia of life [web application]. Version 1.8. NatureServe, Arlington, VA. http: www.natureserve.org/explorer accessed 6 April 2004). Williston (2001), however, suggested it may be even rarer, i.e., G2 (globally imperiled). The Maine population occurs on the Petit Manan National Wildlife Refuge (PMNWR), a federal holding dedicated primarily to preserving migratory bird habitat and enhancing the success of seabird nesting (http: petitmananfws.gov). The Botrychium pallidum colony is approximately 400 m from the ocean, in a dry field with a sparse to thin stand of grasses, notably Festuca filiformis Pourret and F. rubra L., and a sparse cover of mostly non-native forbs, especially Hieracium pilosella L., H. praealtum Gochnat, Taraxacum laeviga- tum (Wild.) DC., Rhinanthus crista-galli L., Rumex acetosella L., and an undetermined annual species of Trifolium. Co-occurring in the habitat are Botrychium matricariifolium A. Br. and B. simplex E. Hitchc. Other fields on the Refuge show a significant or dominant cover of ericaceous shrubs, e.g. Vaccinium angustifolium Aiton, V. vitis-idaea L., V. myrtilloides Michx., Kalmia angustifolia L., and Rhododendron canadense (L.) Torr. However, only a few scattered plants of Vaccinium angustifolium and V. vitis-idaea occur in the area of the Botrychium colony, evidence perhaps of a less acidic soil reaction or different history of disturbance in the immediate area. Areas within the colony not covered with vascular plants support patches of mosses, including Aulacomnium palustre (Hedw.) Schaegr. and Poly- trichum piliferum Hedw., and lichens, Peltigera sp. and Cladonia spp. The discovery presents challenges. One is to discover other regional populations. Botrychium matricariifolium, B. simplex, and B. lunaria (L.) Sw. all occur along the immediate coast and on islands in eastern Maine and the Maritime Provinces, and additional populations of B. pallidum are to be expected. Accurately characterizing the micro-habitat of this population with respect to soil type, soil chemistry, nutrient input (perhaps including ions from storm spray), disturbance regime, and other factors may prove helpful in searching for other regional populations. Another challenge devolves upon the US Fish and Wildlife Service: to manage the habitat in a manner that will maintain this population. Some type of long-rotation disturbance regime may be necessary to retard habitat succession and provide colonizable sites (Gilman, A. V. 2002. Botrychium lunaria (L.) Sw. (Moonwort) Conservation and Research Plan for New England. New England Wild Flower Society, Framingham, MA). Thanks are extended to Linda Welch, Refuge Manager, for permission to enter protected areas and to collect specimens, to Michael Langlois (PMNWR) for aid and information, to Norm Famous and Marcia Spencer-famous for assistance in fieldwork, and to Don Farrar, Iowa State University, for confir- mation of a specimen through isozyme analysis. Janis Lesbines, Arthur Haines, and Sally Rooney also accompanied me in the field at various times.—ARTHUR V. Giman, William D. Countryman Environmental Assessment & Planning, 868 Winch Hill Road, Northfield, VT 095663. SHORTER NOTES 157 Asplenium ruta-muraria L. in lowa.—A small population of Asplenium ruta- muraria L., a species previously unrecorded from Iowa (Eilers, L. J. & D. M. Roosa. 1994. The Vascular Plants of Iowa. University of Iowa Press, Iowa City; Peck, J. H. 1982. Ferns and fern allies of the Driftless Area of Illinois, lowa, Minnesota and Wisconsin. Milwaukee Public Mus. Contr. Biol. Geol. 53:1-140; Peck, J. H. 1989. Additions to the Iowa Pteridophyte Flora, III. J. lowa Acad. Sci. 96:54—56), was recently discovered by the first author in Clinton County in the east-central part of the state. This locality is some 300 miles north of its nearest known location in the Missouri Ozarks (Wagner, Jr., W. H., R. C. Moran and C. R. Werth. 1993. Aspleniaceae Newman. Pp 228-245 in FNA Editorial Committee. Flora of North America North of Mexico. Vol. 2 Pteridophytes and Gymno- sperms. Oxford Univ. Press, New York; Yatskievych, G. 1999. Steyermark’s Flora of Missouri, Vol 1. Revised edition. Missouri Botanical Garden Press, St. Louis.). Since the Iowa population is small (> 50 plants), only portions of a few plants were collected and these were deposited in the University of Iowa Herbarium (Clinton Co., Iowa: T. F. Cady s.n., May 18, 2001, IA). Primarily an Appalachian species in North America (Fig. 1), the main range of A. ruta-muraria is from southeastern Ontario, south-central Quebec, New York, Vermont, New Hampshire and Massachusetts southwest into Kentucky, Tennessee, Georgia and Alabama (Wagner, et al., 1993; Monro, D. 1988. A disjunct station of Asplenium ruta-muraria, with Pellaea atropurpurea and P. glabella, in Eastern Ontario. Amer. Fern J. 78:136—138). Additionally, there are a number of disjunct populations. The most extensive of these are in the Ozarks in southeastern Missouri (Yatskievych, 1999) and along the Niagara Escarpment in Lake Huron. In the nineteenth century, the Ozark population was reported to extend into adjacent Arkansas, but no specimens or extant populations are known (Taylor, W. C. 1984. Arkansas Ferns and Fern Allies. Milwaukee Public Museum, Milwaukee). The Ozark population also may have reached into Illinois. Mohlenbrock (Mohlenbrock, R. H. 1999. The Illustrated Flora of Illinois. Ferns. Second Edition. Southern Illinois University Press, Carbondale) reported that A. ruta-muraria was collected from an unspecified location in southern Illinois in the mid 1800’s, but it is now presumed extirpated from the state. The Niagara Escarpment populations in southwest- ern Ontario extend through the Bruce Peninsula and Manitoulin Island to Drummond Island in Michigan’s Upper Peninsula (Monro, 1988; Drife, D. C. & J. E. Drife. 1990. Oliver A. Farwell’s early Pteridophyte records from the Keweenaw Peninsula. Michigan. Bot. 29:90—91; Gart Bishop pers. com.). An additional Michigan population previously reported from Keweenaw County (Monro, 1988; Wagner et al., 1993) is discounted (Drife & Drife, 1990). Another remarkable disjunction was recorded recently by Gart Bishop and Bruce Bagnell from New Brunswick at Kennebecasis Bay, Kings County (Sheppard, M. 2001. In awe of minister’s face, trust buys rare plant habitat. Refuge 11(2):1; Gart Bishop, pers. com.). The Iowa population augments the picture of a species characterized by widespread disjunctions well outside the core Appalachian range. 158 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) rt NN Iowa population New Brunswick population = A ave : SI ARG \ Ce aV; * @ WN Us \ Fic. 1. North American distribution of Asplenium ruta-muraria, including the newly discovered Iowa population. Map based on sources cited in text. The Iowa population of A. ruta-muraria occurs along the edge of a dolomite prairie characterized by Schizachyrium scoparium (Michx.) Nash (little bluestem), Bouteloua curtipendula (Michx.) Torrey (sideoats grama), Spor- obolus heterolepis (Gray) Gray (prairie dropseed), Carex richardsonii R. Br., C. umbellata Schkuhr ex Willd., Liatris cylindrica Michx. (blazing star), Arabis lyrata L. (rockcress), Dalea purpurea Vent. (prairie clover), Aster sericeus Vent and A. azureus Lindley, to name but a few. The southern and western perimeters of this prairie are defined by dolomitic outcrops; A. ruta-muraria occurs near the southeastern edge on small cliffs approximately 2.5-3.5 meters high. Although there has been some minor woody encroachment in recent history, these bluffs were historically surrounded by prairie, based on descriptions in the General Land Office Survey of 1837 (General Land Office Survey Field Notes, Iowa City, IA, State Historical Society of Iowa). The main population occurs on a section line, so the surveyor, John Wharry, would have walked right through this site and his notes recorded no trees or shrubs, only SHORTER NOTES 159 ‘Broken along the line 2"? rate Land generally, Lime stone rock along the ravines”. The largest concentration of A. ruta-muraria at this site occurs in a small, exposed area at the eastern terminus of a south-facing outcrop. On the south and east facing bluffs, A. ruta-muraria grows with the dominant fern encountered on these outcrops, Pellaea glabella Mett. ex Kuhn (smooth cliff- brake), whereas on the small, sheltered northern face it occurs with Cystopteris bulbifera (L.) Bernh. (bulblet fern). Other plant species co-inhabiting the shelves and crevices of these bluffs include Arabis lyrata (rock cress), Euphorbia maculata L. (spurge), Minuartia michauxii (Fern.) Farw. (=Arenaria stricta Michx.; rock sandwort), Nepeta cataria L. (catnip), Parietaria pensyl- vanica Muhl. ex Willd. (pellitory), Aquilegia canadensis L. (columbine), Sporobolus neglectus Nash (small rush grass) and Schizachyrium scoparium (little bluestem). We thank Gart Bishop, Doug Larson, Jim Peck, Tony Reznicek, Carl Taylor, and George Yatskievych for information or advice.—TuHomas F. Capy, Iowa City, IA and Diana Horton, Biological Sciences Department, 143 BB, University of Iowa, Iowa City, IA 52242. Vitexin 7-O-rhamnoside, a New Flavonoid from Pteris vittata.—Previous work on the flavonoids of Pteris vittata L. has led to the identification of luteolinidin 5—O-glucoside by Harborne (Phytochemistry 5:589-600, 1966); in addition acid hydrolysis of extracts of this fern has led to the identification of kaempferol, quercetin, leucocyanidin and leucodelphinidin by Voirin (Ph. D. thesis, University of Lyon, p. 151, 1970). More recently 3-C-(6’"-acetyl-B— cellobiosyl)-apigenin (Amer. Fern J. 89:217—220, 1999) and 6-C-B-cellobiosy]- isoscutellarein-8-methyl ether together with quercetin 3-O-glucuronide and rutin (Amer. Fern J. 90:42—45, 2000) have been identified by Imperato and Telesca. In addition three kaempferol glycosides (3-O-glucoside, 3-O-glucuro- nide and 3-O-(X",X"-di-protocatechuoy])-g] ide), two di-C-glycosylfla- vones (3,8-di-C-arabinosylluteolin and 6-C-arabinosyl-8-C-glucosylluteolin) and three flavonol glucosides acylated with hydroxycinnamic acids (kaemp- ferol and quercetin 3-O-(2", 3’-di-O-p-coumaroyl)-glucosides together with kaempferol 3-O-(X"-O-p-coumaroyl-X”-O-feruloyl)-glucoside) have been found by Imperato (Amer. Fern J. 90:141-144, 2000; Amer. Fern J. 92:244—246, 2002; Amer. Fern J. 93:157—160, 2004). In the present paper two flavonoids (I and II) have been isolated from aerial parts of Pteris vittata L. collected in the Botanic Garden of the University of Naples. The fern was identified by Dr. R. Nazzaro (University of Naples); a voucher specimen (149.001.001.01) has been deposited in the Herbarium Neapolitanum (NAP) of the University of Naples. Flavonoids (I and II) were isolated from an ethanolic extract of aerial parts of Pteris vittata by preparative paper chromatography in BAW (n-butanol-acetic acid-water, 4:1:5, upper phase), 15% HOAc (acetic acid) and BEW (n-butanol- 160 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) TaBLE 1. '%C- and ‘H-NMR spectral data (DMSO-dg) of flavonoid (I). Carbon 5. ppm dy ppm (J in Hz) Apigenin 164.6 0.97 (3H, d, J = 6, rhamnosy! methyl group) 3 102.7 2.95—-4.11 (10 H, m, glucosyl 6 ie + rhamnosyl 4 protons) 4 182.4 4.52 (1 H, d, J = 8, glucosyl H 5 161.6 5.51 (1 H, d, J = 2, rhamnosyl] i 6 99.1 6.33 (1 H, s, H-6) 7 163.2 6.77 (1H, s 8 104.4 6.91 (2 H, d, J = 8..6,(??) H-3’ and H-5’) 9 156.8 7.97 (2 H, d, J = 8.6, H-2’ and H-6’) 10 103.2 sy 121.6 28 128.9 Es a 215.7 4' 160.5 O-Rhamnosyl 5 99.2 a 70.4° a 70.5" 4” 71.8° 5” 70.8° Ge 18.2 C-Glucosy] i a 73.6 2 As 3 79.6 4” 69.9 a” 82.1 Gi 62.2 «> Assignments with the same superscripts may be interchanged. ethanol-water, 4:1:2.2). Further purification was carried out by Sephadex LH-20 column chromatography eluting with methanol. Color reactions (brown to yellow in UV+NH3.), chromatographic behaviour (R; values on Whatman No 1 paper: O.37 in BAW; 0.54 in 15% HOAc) and ultraviolet spectral analysis in the presence of usual shift reagents (A,;,ax(nm) (MeOH) 273, 332; +AICl, 280, 302, 344, 383; +AlCl3/HCl 281, 300, 341, 381; +NaOAc 273, 391; +NaOMe 274, 391) suggested that flavonoid (I) may be a flavonoid glycoside with free hydroxyl groups at positions 5 and 4’. Acid hydrolysis (2N HCl; 1 hr at 100°C) gave L-rhamnose, vitexin (8-C-glucosylapigenin) and isovitexin (6-C-gluco- sylapigenin). These results show that flavonoid (I) may be a C-glucosylapige- nin 7-O-rhamnoside and this was confirmed by the FAB mass spectrum which showed a quasimolecular ion at m/z 579 [M+H]* and an ion at m/z 601 [(M+H}+Na]*. "H- and '°C- NMR spectral data (Table 1) showed that D-glucose is attached at position 8 o f the flavone since H-6 appeared as a singlet at 5 6.33; in addition C-8 showed a downfield shift of 10.4 p.p.m., in comparison with the corresponding carbon atom of apigenin; this shift of glycosylated carbon SHORTER NOTES 161 OH Fic. 1. Vitexin 7-O-rhamnoside. atom is an effect of C-glycosylation on the aglycone spectrum as shown in the review by Markham and Chari (pp. 19-134 in J. B. Harborne and T. J. Mabry, eds., The Flavonoids: Advances in Research, Chapman and Hall, London, 1982). Hence the presence of isovitexin among the products of acid hydrolysis of flavonoid (I) is due to a Wessely-Moser acid isomerization. The combined data show that flavonoid (I) is vitexin 7-O-rhamnoside (Fig 1), a new natural product; 'H- and '*C-NMR spectral data (Table 1) support this structure. A large number of C-glycosylflavonoids have been found in ferns as shown in the review by Markham (pp. 427-468 in J. B. Harborne, ed., The Flavonoids, Advances in Research Since 1980, Chapman and Hall, London, 1988) and in a review by Imperato (pp. 39-75 in Current Topics in Phytochemistry, Research Trends, Trivandrum, 2000). However C-glycosylflavonoid O-glycosides are rare in ferns. A C-glycosylflavonoid O-glycoside in which the hydrolyzable sugar is attached to a phenolic hydroxy group was found for the first time in ferns by Hiraoka (Bioch. Syst. and Ecol. 6:171-175, 1978) who identified vitexin 7-O-glucoside in the genus Dryopteris; subsequently 8-C-rhamnosylluteolin 7-O-rhamnoside was found in Pteris cretica (Phytochemistry 37:589-590, 1994) by Imperato. C-Glycosylflavonoid-O-glycosides in which the hydro- lysable sugar is attached to a hydroxy group of a C-glycosyl moiety were found for the first time in ferns by Markham and Wallace (Phytochemistry 19:415— 420, 1980) who found apigenin and luteolin 8-C-glucoside 2”-O-xylosides in Trichomanes venosum; two further C-glycosylflavone O-glycosides of this type have subsequently been found in Pteris vittata by Imperato and Telesca who identified these flavonoids as 3-C-(6’”-acetyl-cellobiosyl)-apigenin (Amer. Fern J. 89:217-220, 1999) and 6-C-B-cellobiosyl-isoscutellarein 8-methyl ether (Amer. Fern J. 90:42—45, 2000). Flavonoid (II) has been identified as kaempferol 3-O-rutinoside by UV spectral analysis with the customary shift reagents, acid hydrolysis, "H-NMR 162 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 3 (2004) spectrum, '°C-NMR spectrum and co-chromatography with an authentic sample. As shown in the review by Markham (1988), kaempferol 3-O- rutinoside has previously been identified in Adiantum capillus-veneris (Adiantaceae), another of the 53 species of Adiantum, Loxsoma cunninghamii, L. costaricensis (Loxsomaceae), all four species of Bommeria (Sinopterida- ceae), four species of Gymnopteris (Sinopteridaceae), two species of Hemi- onitis (Sinopteridaceae) and the genus Trachypteris (Sinopteridaceae); more recently kaempferol 3-O-rutinoside has been identified in Diplazium nippo- nicum (Athyriaceae) and Thelypteris palustris (Sinopteridaceae) as shown in the review by Imperato (2000). The author thanks Murst (Rome) for financial support. Mass spectral data were provided by SESMA (CNR, Naples).—Fiuipro Imperato Dipartimento di Chimica, Universita della Basilicata, 85100 Potenza, Italy. 7 : i : , + Ru ; A ° D 7 a = - i ns Ve i Raat an ae 2 y 7 i. , i Fe ena . ore _ : 7 - a a 5 Reyes _ 7 a " rae = A 7 : 3 ae | aa°9 sce 7 oe? ae A =) 0 el) a 7 s eat 7 Pas a a : f aioe ’ in oe CF an eee a a. ae a a r : - a aan “ 7 , hae 3 a oo. i Mea } hd : : 7 P ( ; : ; ie ee : rs a ° 7 Las ee noe 7 Ss : m 7 : ewe! a LE iy. -_ 7 say i ; yy as > a if : . Serres os _ eee 7 7 ies 7 oo : 7 7 - if een Pere ae a ke a ae a 77 ; 1 oe is 7 7 _ _ - ': Say ro 7 on be We | - 7 w a - 7 Bat = ies 4 0 De en ; 7 Soe Ui 3 jor 7 ee. sy at : ; - - Sean fon fo epic. = 7 a ie _ : = 1. er aC “ 7 7 _ : Wee : 7 7 7 ban — _ ee ae ce - i 7 a = ‘ 5 - - : : ’ 7 a =. =e : ; ; : : il _ : } et a aa 7: = 7 i" ; 7 _ — : 7 a 7 , — ide pee - 7 : 7 - eee ; - i a. a : x 7 7 : : - 7 i 7 as - 7 a) : 7 > . 1? 7 4 > : = : - ’ i¢ i = ‘ 7 c vf : ° oe Gane TT. 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MISSOURI BOTANICAL American Fern Journal 94(4):163-182 (2004) GMANL, $2005 A Taxonomic Study of the Fern Genus Arachniodes Blume (Dryopteridaceae) from China Hat HE Ecological Restoration Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China AssTract.—The taxonomy of the fern genus Arachniodes Blume in China is rather complicated with the creation of many new names since the 1960’s. The purpose of this article is to make a clarification of the genus as a whole from China and provide an enumeration of what is known at present. Through herbarium studies and field observations, the distribution, morphological criteria and subdivision of the genus Arachniodes from China are discussed. The total number of species prove their a A subdivision of four sections is . ane further completed, i.e. sect. alphabetical order with information about their synonyms, their distribution and the sections and groups in which they are categorized. The definition of the fern genus Arachniodes Blume is rather confused in that its species share some key characteristics with both Dryopteris Adans. and Polystichum Roth, the two largest genera of the family Dryopteridaceae. Arachniodes was established by Blume in 1828; however, the genus was not recognized by other pteridologists for nearly one and a half-centuries. Some of its species have experienced a lot of changes in nomenclature before Tindale (1961, 1965) resurrected Arachniodes as the acceptable generic name (Ching, 1934, 1938, 1962; Holltum, 1954; Morton, 1960; Ohwi, 1962). The subsequent circumscription and delimitation of the genus by Serizawa (1976), Proctor (1985, 1989), Wu and Ching (1991), and Hsieh (2000) is still incomplete; Sledge (1973) has called into question the naturalness of the genus and Tryon and Tryon (1982) put it in an expanded Dryopteris. However, the genus has general acceptance among world pteridologists (Pichi-Sermolli, 1977; Fraser-Jenkins, 1984, 1986; Jarrett, 1985; Gibby et al., 1992; Iwatsuki, 1992; Nakaike, 1992, 2001; Ammal and Bhavananda, 1993; Shieh et al., 1994; Kumar et al., 1998; Moran and Ollgaard, 1998; Antony et al., 2000; Hsieh, 2000) though some discrepancy about the scope of the genus exists. In the present paper the author adopts the generic concept of Arachniodes sensu Ching (1978), leaving Leptorumohra (H. It6) H. It6, Acrorumohra (H. It6) H. It6 and Phanerophlebiopsis Ching, three small genera closely related to Arachniodes, as separate genera. Due to the different criteria used to define species, it is very difficult to provide an exact number of species in the genus worldwide. There is little doubt that most species occur in southern China. The first checklist made by Ching (1962, 1964) recorded 22 species names from China; but since then, many new taxa have been described in the Chinese literature (Anonymous, 164 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) 1974; Anonymous, 1977; Ching, 1964, 1982, 1986; Ching and Wang, 1964; Ching and Wu, 1983; Ching and Zhang, 1983; Hsieh, 1983a, 1984a, 1984b, 1986, 1991a, 1991b; Ching and Liu, 1984; Wu, 1995). To date the number of names under Arachniodes from China has increased to nearly 130, of which 103 species names, 2 variety names, and 4 questionable species names were documented in the Chinese version of the Flora of China (Hsieh, 2000). This has made the classification and identification of the genus very difficult in China and worldwide. It is for these reasons that the current paper has been written. It is hoped that outlining what is known about the genus will aid in the further study and enumeration of the genus. DISTRIBUTION OF ARACHNIODES In general Arachniodes is a pantropical genus (Proctor, 1985; Wu, 1997) and is distributed in the subtropical to tropical forest regions of the world, mostly in China and southern to southeastern Asia. Only a small number of species are found in Central America. About 11 species are listed by Ching (1962), Proctor (1985), and Moran and Ollgaard (1998); but only 4 species are accepted by Nakaike (2001) who excluded three African and one Australian species (Ching, 1962; Gibby et al., 1992; Nakaike, 2001) in Polystichopsis (J. Sm.) Holttum. A comparison of Japanese ferns (Kurata, 1962; Nakaike 1975; 1992; Iwatsuki, 1992) revealed that China and Japan have the greatest species diversity as well as the most species in common. The present-day distribution of Arachniodes is centered in the Sino-Japan region, not the Sino-Himalayan region (Ching, 1962; Wu and Ching, 1991). In China this genus mainly occurs along the drainage area of the Yangtze and southern provinces. Its northern boundary does not exceed that of the subtropical area, to about 34°N, except for Arachniodes exilis, which extends northward beyond 36°N in Shandong province (Li, 1990); its western boundary is in southeastern Tibet (95°E). Most species are concentrated in southwestern and southeastern China and grow at altitudes lower than 2000 m; a few species can reach an altitude higher than 2700 m. TAXONOMIC CRITERIA The taxonomy of Arachniodes is complicated by its decompound fronds and multiple, minor morphological changes in almost all species. For a fern student who studies herbarium specimens only, it is difficult to identify species correctly. Most herbarium sheets consist merely of fronds without an attached rhizome, and without habit descriptions. The latter is important in this genus as will be discussed below. The majority of new names in the genus have been described on the basis of subtle differences in shape and other minor variations of the frond. This has led to a misleading comparison of species and has contributed to the creation of many synonyms. The most dangerous of all is the new taxa being published that are based only on single collections. For example, most of the 63 names described by Ching (1986) were only HE: ARACHNIODES FROM CHINA 165 accompanied by one cited collection and one or several duplicate sheets deposited in PE and other herbaria in China; the same is true for most of Hsieh’s (1983a, 1984a, 1984b, 1986, 1991a) descriptions. By examining more than 1250 collections of specimens in herbaria (CDBI, CTC, HITBC, KUN, PE, PYU, SZ, WNU, WUK, YAF, and some Japanese plants borrowed from TNS) and through field observations of habit in Yunnan, Sichuan, Guizhou, western Hunan and Hubei, southern Shaanxi, southern Gansu, southeastern Tibet as well as Chongqing Municipality, the author has found that the most stable and useful characters in this genus are rhizome habit and scale type. The rhizome habit can be categorized as either ascending or creeping (either short or long). The rhizome scales of most species are more or less lanceolate in shape, entire or sometimes with teeth on the margin. However, the scales found in Arachniodes globisora and A. amoena are quite specialized as will be noted below. Other useful characters include frond scales or indument, the degree of division of the lamina and each level of segmentation, shape of the lamina apex or that of the basal pinnae, shape and dissection of the ultimate pinnules, texture and luster of lamina, position of sori on the ultimate segments, and various aspects regarding the indusium. Some of the most unreliable features are the size of the frond, lamina and pinnae (especially in young fronds); the angle between rachis and the pinnae rachides; and the distribution of sori on the lamina. These characters should not be used as the sole basis for defining species. Moreover, slight to obvious morphological differences between the sterile and the fertile fronds do occur in most species, of which an extreme example is Arachniodes dimorphophylla. Based on these findings, species from Yunnan and Sichuan provinces have been clarified and more than 60 names have been reduced to synonymy (He and Wu, 1996; He, 1997). But for the genus Arachniodes as a whole in China, it is still in need of a general revision. SUBDIVISION OF THE GENUS IN CHINA A system proposed by Hsieh (1983b) divided the genus into two sections, i.e. Sect. Cavaleria Ching et Y. T. Hsieh, and Sect. Arachniodes. The latter was further subdivided into two subsections and 11 series. This system attaches importance solely to the position of the sorus on veinlets of the ultimate pinnules when recognizing sections. As for the recognition of subsections and series, characters such as shape of apical pinnae, degree of frond complexity, shape and size of basal pinna pairs, that of the basiscopic pinnule of basal pinnae and that of ultimate pinnules or segments, and so on are used. In the system proposed by Hsieh (2000), some closely related species or even mor- phological variations within one species are placed into different subsec- tions or series; whereas species with more fundamental differences such as habit and scale character s are put together in one section. Therefore, it is neces- sary to make some revision and clarification of this system. Mainly based on the habit of rhizome, characters of rhizome and stipe base scales, and the position of sori on the veinlets of the ultimate pinnules, the 166 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) revised subdivision of Arachniodes categorizes the genus into four sections (He an u, 1996). Three of the sections have ascending rhizomes, and especially some species of sect. Globisorae S. K. Wu et H. He bear nearly erect ones; whereas the majority of species in the sect Arachniodes have creeping rhizomes. The four sections are well distinguished on the basis of rhizome and stipe base scales as described in Table 1. Sect. Cavaleria is the only group in which the sori are positioned dorsally on the veinlets of the ultimate pinnules. Though some species such as Arachniodes globisora and A. henryi were de- scribed as having have dorsal posited sori, observations of specimens re- vealed that the sori are only occasionally dorsal and are most often terminal on the veinlets. The erection of sect. Globisorae and sect. Amoenae (Ching et Y. T. Hsieh) S. K. Wu et H. He has taken into consideration their entire geo- graphical distribution (Table 1) as well as characters of rhizomes and scales. Moreover, plants of sect. Amoenae are much more glabrous above the base of stipes. Table 1 provides a comparison of the four sections of Arachniodes based mainly on plants from China and adjacent regions. Relatively few species are in the first three sections, i.e. only one species in sect. Cavaleria, five species in sect. Globisorae, and two species in sect. Amoena. Analyses of specimens in PE indicate that the African species A. foliosa (C. Chr.) Schelpe is quite similar to A. spectablis and could be placed in sect Globisorae and the Central American A. denticulata (Sw.) Ching could be safely treated in sect. Amoena. There is no doubt that most species worldwide should be placed in sect. Arachniodes. For the Chinese plants as a whole, 50 acceptable species names enumerated in this article belong to the section Arachniodes though some of them are still not satisfactory. To leave the problem open and for the purpose of convenience and further comparison, ten species groups are proposed for sect. Arachniodes based mainly on the rhizome habit, color of stipe scales, shape and division of the lamina, shape of pinnae and texture of the frond. Table 2 provides a comparison of these ten species groups in sect. Arachniodes from China. ENUMERATION OF ARACHNIODES FROM CHINA The following is an enumeration of names belonging to the genus Arachniodes Blume known from China. They are arranged in alphabetical order with original reference of publication. Accepted named are accompanied by synonyms, distribution, as well as sectional and group classificati Accepted names are in bold type; synonyms are in italics. Some of the presently accepted names, those marked with an asterisk have very few specimens available and more collections are required to prove their acceptance. For the distribution in China, the provinces listed are based on specimens checked in herbaria, unless relevant literature is cited. Arachniodes abrupta Ching, Bull. Bot. Res., Harbin 6(3):35. 1986. = Arachniodes chinensis HE: ARA FROM CHINA 167 Arachniodes acuminata Ching et C. H. Wang, Acta Phytotax. Sin. 9:367. 1964. — Arachniodes cavalerii Arachniodes ailaoshanensis Ching, Bull. Bot. Res., Harbin 6(3):60. 1986. Sect. IV. Arachniodes, Group Arachniodes nipponica. SYNONYMY: Arachniodes jingdungensis Ching 1986. DISTRIBUTION: Central Yunnan. Arachniodes amoena (Ching) Secs Acta Bot. Sin. 10: 256. 1962. Rumohra amoena Ching, Sinensia 5: 40, pl. 1. 1934. Sect. III. Amoenae._DISTRIBU- TION: Yunnan (Lu and Zhang, 1994), Guizhou, Hunan, Guangxi, Guang- dong, Jiangxi, Fujian, Zhejiang, and Anhui (Chen, 1985). *Arachniodes anshunensis Ching et Y. T. Hsieh, Bull. Bot. Res., Harbin 6(3):67, pl. 8, f. 3. 1986. Sect. IV. Arachniodes, Group Arachniodes henryi. DISTRIBUTION: Central Guizhou. Arachniodes aristatissima Ching, Bull. Bot. Res., Harbin 6(3):1, ma. 1.8 h 1986. Sect. IV. Arachniodes, Group Arachniodes simplicior. DISTRIBU- TION: Zhejiang (Hsieh, 2000; Ching, 1986). Arachniodes assamica (Kuhn) Ohwi, J. Jap. Bot. 37:76. 1962. Aspidium assamicum Kuhn, Linnaea 36:108. 1869. Sect. IV. Arachniodes, Group Arachniodes assamica. SYNONYMY: Arachniodes leuconeura Ching 1986, Arachniodes suijiangensis Ching et Y. T. Hsieh 1986, Arachniodes xinpingensis Ching 1986, Arachniodes yaomashanensis Ching 1986, Arachniodes basipinnata (Ching) Ching et Y. T. Hsieh 1991. DISTRIBU- TION: Sichuan, Chongqing, Yunnan, Guizhou, and Guangxi; Northern Thailand, Burma, Northeastern India and Sikkim. *Arachniodes attenuata Ching, Bull. Bot. Res., Harbin 6(3):2, fe Ay | 1986. Sect. IV. Arachniodes, Group Arachniodes simplicior. DISTRIBU- TION: Southern Yunnan (Ching, 1986; Hsieh, 2000) and Zhejiang (Zhang, 1993). Arachniodes australis Y. T. Hsieh, Bull. Bot. Res., Harbin 11(3):27. 1991b. = Arachniodes caudata Arachniodes austro-yunnanensis Ching, Bull. Bot. Res., Harbin 6(3):3, pl. 1, f. 86. = Arachniodes sporadosora Arachniodes baiseensis Ching, Bull. Bot. Res., Harbin 6(3): 25. 1986. = Arachniodes cavalerii Arachniodes basipinnata (Ching) Ching ex Y. T. Hsieh, Bull. Bot. Res., Harbin 11(3):27. 1991b. = Arachniodes assamica Arachniodes calcarata Ching, Bull. Bot. Res., Harbin 6(3):30. 1986. = Arachniodes simplicior Arachniodes caudata Ching, Acta Phytotax. Sin. 9:384. 1964. Polystichum simplicius (Makino) Tagawa var. majus Tagawa, Acta Phytotax. Geobot. 1:90. 1932. Sect. IV. Arachniodes, Group Arachniodes simplicior. SYNON- YMY: Arachniodes caudata Ching var. kansuensis Ching 1974, Arachniodes kansuensis (Ching) Y. T. Hsieh 1984b, Arachniodes australis Y. T. Hsieh TABLE 1. Comparison of the four sections of Arachniodes mainly based on plants from China and adjacent regions Sect. I. Cavaleria Ching et Y. T. Hsieh Sect. II. Globisorae S e Sect. Il. Amoenae i et Hsieh) H. He Sect. IV. Arachnoides Section type Rhizome habit Scales on rhizome and on the base of stipe Sori position Species Distribution st cere ne (Christ) O fcaacei Long-lanceolate (up to stipe and turn dark- brown upward Dorsal on veinlets and se to the costa of the ultimate pinnule Only
28 Fics. 21-28. Microsporogenesis and Megasporogenesis in Isoetes sinensis. 21. Late Diplotene. (arrow). 26. Cytomixis at tetrad stage (arrows). 27. Diakinesis in megaspore mother cells. 28. Diakinesis in microspore mother cells. Scale bars in Figs. 27 =5 ym; Figs. 21, 25 & 28=10 um; Figs. 22-24 & 26 = multivalents were not seen. In microspore mother cells, secondary synapsis was observed. The configurations formed by these synapses were dependent on the number and position of the chiasmata present in the bivalents. For example, Fig. 28 shows a rhombic bivalent configuration created by two 190 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) chiasmata located at the end of the chromosome arms and two at the center of the arms. Nucleoli gradually disappeared during diakinesis. During metaphase I (Figs. 29-31), all chromosomes arrayed along the metaphase plate. As megaspore mother cells continued to develop and enlarge, the cell wall became thicker and more rigid. In addition, the cytoplasm became filled with metabolic products that obscured the chromosomes and the thicker wall made it difficult to flatten and spread preparations in order to see chro- mosomes. Therefore, following diakinesis, it was necessary to focus on micro- spore mother cells to describe cytological changes during the succeeding stages of meiosis. Pairing of bivalents was frequently observed in microspore mother cells (Figs. 29-30). Sometimes two or more bivalents associated to form a short or long chain (Figs. 30-31). At anaphase I (Figs. 32-33), chromosome configurations in the microspore mother cells were clearly seen. Of the 118 cells evaluated in anaphase I, 24.4% showed abnormalities such as the presence of lagging chromosomes (Figs. 34, 36), chromosome fragments, or chromosome bridges (Figs. 34-35). It was un- certain if there were such abnormalities in megaspore mother cells since the densely stained cytoplasm obscured the chromosomes (Fig. 37). During telophase I, chromosomes uncoiled into chromatin, nucleoli and nuclear membranes reappeared, and dyads formed. The Second Meiotic Division.—In prophase II, chromatin in the daughter nucleus of each dyad condensed and coiled into chromosomes again. At metaphase II, the centromere of each chromosome split and the chromatids separated. During anaphase II, the four daughter nuclei became arranged as if they were at the ends of a cross. Lagging chromosomes and micronuclei were found in 26.9 % of 191 microspore mother cells observed (Fig. 38). In a few of these cells, unbalanced divisions occurred yielding two larger and two smaller daughter nuclei (Fig. 39). In such case, the resulting tetrad of microspore mother cell daughter nuclei formed a one-dimensional tetragonal configuration. In contrast, the tetrad of the megaspore mother cell daughter nuclei formed a three- dimensional tetrahedral configuration (Figs. 42-43), and the walls became further thickened. Approx. 8.4 % of 152 randomly counted megaspores appeared to be irregular in size and form (Fig. 44). Approx. 14.8% of 266 microspores were observed to be irregular in size and form (Figs. 40-41). This percentage of irregular microspores was considerably lower than the 24.4% incidence of abnormal chromosomal behavior noted in microspore mother cells during anaphase I (Figs. 34—36). DISCUSSION Seven species of Isoetes are currently recognized in East Asia (Liu et al., 2002, Takamiya et al., 1997). These include four basic diploid species (2n = 22) I. asiatica (Makino) Makino, I. hypsophila, I. taiwanensis and I. yunguiensis and three polyploid species I. sinensis (2n= 44), L. japonica A. Braun (2n=66), and I. pseudojaponica M. Takamiya, Mitsu Watan. & K. Ono (2n = 88). Chung and Choi (1986), described I. coreana Y. H. Chung & H. K. Choi (2n = 66), but HE ET AL.: CYTOGENETICS OF ISOETES SINENSIS * ". & € Z * ae ~. es” 29 31 4 » Yat, 4 M3, ; es 4 L es 34 35 37 Fics. 29-37. Microsporogenesis and Megasporogenesis in Isoetes sinensis. 29-31. Metaphase I in microspore mother cells. 29. 22 bivalents. 30-31. Secondary synapsis (arrows). 32-37. Anaphase in microspore mother cells. 32. Early anaphase I, showing sister chromatids together only at their centromeres. 33-37. Anaphase I, 33. Chromosomes at poles. 34. Mother cells, showing a chromosome bridge (hollow pant and lagging chromosomes (arrows). 35. Chromosome heidge (hollow arrow) and chromosome fragment (arrow). 36. Lagging chromosomes (arrows). 37. se, -aeage lin poral mother cells. Scale bars in Fig. 32 = 5 ym; Figs. 29-31, 33 & 35-37 = 10 m; Fig. 34 = 20 192 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) - @ ‘ Fics. 38-45. ea sages and Megasporogenesis in Isoetes sinensis. 38-39. Anaphase II in microspore mother cells. 38. Lagging chromosomes (arrows). 39. Unbalanced divisions. 40-41. If in megaspore mother cells. 43. Tetrad of m egaspores. 44. Megaspores, irregular megaspores (arrows). 45. Megaspore and tetrads of microspores (arrow) in a — citi Scale bar in Figs. 39 = 5 um; Figs. 38 = 10 pm; Figs. 40-43 = 20 um; Figs. 44-45 = 40 um. HE ET AL,: CYTOGENETICS OF ISOETES SINENSIS 193 Takamiya et al. (1997) reduced this hexaploid taxon to I. sinensis var. coreana. Takamiya et. al. (1996) reported on chromosome behavior during meiosis for Japanese species of Isoetes. In this paper, we provided a more detailed analysis of chromosome behavior in J. sinensis with an emphasis on prophase I chromosome behavior in both megaspore and microspore mother cells. We observed the mixed sporangia in Isoetes sinensis, and this result was consistent with the reports in I. pantii Goswami & Arya (Goswami, 1975) and I. yunguiensis (Wang et al., 2002). Isoetes sinensis showed chromosome pairing and bivalent formation similar to that observed in basic diploid species except that twenty-two bivalents formed in this tetraploid instead of the eleven bivalents observed in basic diploid species. These observations of bivalent formation in J. sinensis were consistent with those previously reported for allopolyploids by Takamiya et al. (1996). In Isoetes sinensis, megasporogensis appears to be a more reliable process than microsporogensis in the production of uniform meiotic products. During the early stages of megasporogenesis where chromosomes could be clearly observed, chromosome behavior appeared normal. Only about 8% of the megaspores produced appeared to be irregular in size and form. In contrast, during microsporogenesis, microspore mother cells showed secondary pairing of bivalents and abnormal chromosome behavior in nearly 25% of the cells examined during anaphase I. About 15% of the microspores produced appeared to be irregular in size and form. Cytomixis, the transfer of cytoplasmic and nuclear contents from one cell to another, has been observed in pollen mother cells of angiosperms (Cheng et al., 2001; Malallah and Attia, 2003), but cytomixis has not been reported in Isoetes. Cytomixis can lead to the production of abortive pollen grains (Sapare, 1978; Samushia et al., 1979). Drugs or hanical f J cytomixis (Bobak and Herich, 1978; Morisset, 1978). However, cytomixis can occur naturally to produce variations in cl ber (Cheng et al., 1980; Cheng et al., 1982). Whatever its cause, cytomixis in I. sinensis | terized by: (1) an occurren during all stages of meiosis; (2) a frequency of incidence which varied among individuals, sporangia, and locations in the same sporangium, and (3) the presence of intercellular spaces along the perine of microspore mother cells undergoing cytomixis. Abnormalities observed as a consequence of cytomixis included unbalanced chromosome numbers, micronuclei, and enucleate cells. We cautiously assert that cytomixis is a natural phenomenon in J. sinensis. We do not believe that our experimental methods caused cytomixis since we have used the same methods to study meiosis in members of the Taxodiaceae, Magnoliaceae, Styracaceae, and Actinidiaceae where cytomixis was not always seen. Isoetes sinensis is an element of the Sino-Japan forest subkingdom (Wu and Wu, 1998). Generally, in Isoetes, each megasporangium contained 40-60 megaspore mother cells and each microsporangium contained ca 75, 000 microspore mother cells (Smith, 1900). We do not believe that the incidence of abnormal chromosome behavior we observed in our study could greatly impede the reproduction and dispersal of I. sinensis. We have observed that in 194 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) native populations and in botanical garden cultures, J. sinensis reproduces effectively. Isoetes sinensis is known from more than thirteen populations in ve Chinese Provinces, but field studies have revealed that eleven of these populations, one in Nanjing, Jiangsu (the type locality), four in Zhejiang, four in Anhui, one in Guangxi, and another in Jiangxi, may no longer exist. Of the remaining four sites, the Songyang and Jiande, (Zhejiang) populations cover less than 1000 m? (Ye and Li, 2003), the Xiuning, Anhui population covers less than 900 m’, and the Guilin (Guangxi) population has only a few individuals remaining. Although cytomixis, lagging chromosomes, chromosome bridges, chromosome fragments, and micronuclei observed during meiosis in J. sinensis may have some effect on sexual reproduction, they probably play only a minor factor in population loss. The main cause of population decline of I. sinensis appears to be from human disturbance. With increasing urbanization, de- struction of wetlands, and water pollution, habitats for I. sinensis are becoming degraded and fragmented and, as a result, populations are dwindling. Intensive searches to locate additional native populations as well as strict conservation measures to safeguard the remaining known populations are urgently needed. AACCKNOWLEDGMENTS The study was supported by grants from the Chinese Academy of Sciences (KSCX2-SW-104), the State Key Basic Research and Development Plan of P. R. China (G2000046806), and the Wuhan Botanical Garden, CAS (01035123).\ LITERATURE CITED Buu, I. and H. K. Goswami. 1990. A new line of chromosomal evolution in Isoetes Bionature 10: 5 5— Bosak, M. ae R. HericH. 1978. Cytomixis as a manifestation of pathological changes after the application of trifluraline. Nucleus India 21:22-26. CHEN, J. K., H. Y. Wanc and G. Q. HE. 1998. A survey on the rpm . Oryza rufipogon and Isoetes sinensis in Jiangxi Province. 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J. 1986. Isoetes Tapani aie morphology: nomenclature, variation, and systematic mportance. Amer. Fern J. 76:1— HE ET AL.: CYTOGENETICS OF ISOETES SINENSIS 195 Hickey, R. J., C. Mac.ur and W. C. Tay.or. pie hos re-evaluation of Isoetes savatieri Franchet in ees os Chile. Amer. Shige 93:1 Hickey, R. J., W. C. TAyLor and N. T. LUEBKE. 188, tthe species concept in Pteridophyta with special reference to mone ba mer. Fern | pers HOLsINcgER, K. E i aiomgaaia systems aa evolution in vascular plants. Proc. Natl. Acad. US, AL GF: 7037-70 Jermy, A. C. 19 es. Pp. 26-31, in K.U. Kramer and P.S. Green, eds. The Families and Genera of ene Plants. pons ; Peridophyte and Gymnosperms. Springer-Verlag, Berlin. Kort, L. S. and D. M. Brirron. 1980. Chromosome numbers of Isoetes in northeastern North Liu X., Y. WaNG, Q. F. Wane and Y. H. eri 2002. Chromosome numbers of the Chinese Isoetes and their taxonomical significance. Acta Phytotax. Sin. 40:351-356 Love, A., D. Love and R. E. G. PicHI SERMOLLI. 1977. 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P., "4 Timotny and W. J. Younc. 1981. pes 2" ed. Prentice-Hall, Englewood Cliffs. TaKAMIYA, M., M. WaTANABE and K. Ono. 1994. Biosystematic studies on the genus Isoetes (Isoetaceae) in Japan. I. Variations of the somatic chromosome numbers. J. Plant Res. 107: Takamiya, M., M. WaTANABE and K. Ono. 1996. Biosystematic studies on the genus Isoetes sgn in Japan. II. Meiotic ‘behavior and reproductive mode of each cytotype. Amer. J. ot. 83:1309-1322. ae miva, M., M. WaTANABE and K. Ono. 1997. Biosystematic studies on the genus siti Isoetaceue) in Japan. IV. Mor eS $Y on of sporophytes, phytogeography an taxono ta Phytotax. Geobot. 48:8 Tanaka, R. 19 71, oa of resting nuclei in ee Bot. Mag. (Tokyo) 84:118-121. Tanaka, R. 1977. Recent karyotype studies. In K. Ogawa et al., eds. Plant Cytology, Asakura Shoten, Tokyo ) THOMAS, 8. and P. J. KA.Tsikes. 1976. A bouquet-like attachment plate for telomeres in leptotene of rye revealed by heterochromatin staining. Heredity 36:155-162. Tayor, W. C. and R. J. Hickey. 1992. Habitat, evolution and speciation in Isoetes. Ann. Missouri ot. Gard. 79:613-622 Wane, Q. F., X. Liu, W. C. Taytor and Z. R. HE. 2002. Isoetes yunguiensis (Isoetaceae), a new basic diploid quillwort from China. Novon 12:587-591. Wu, G. Y. and S. G. Wu. 1998. A proposal for a 5 ne Kingdom (Realim) — the E. Asiatic Kingdom its delineation and characteristics. In A.L. Zhang and S.G. Wu, eds. age Characteristic and saa of East Asian Plants. CHEP, Beijing and Springer-Verlag, B Ye, Q. G. and . Li. 2003. Distribution status and causation of endangerment “of iii sinensis Palmer in | Zhejiang Province. J. Wuhan Bot. Res. 21:216—220. American Fern Journal 94(4):196—205 (2004) Phylogenetic Relationships of Isoétes (Isoétaceae) in China as Revealed by Nucleotide Sequences of the Nuclear Ribosomal ITS Region and the Second Intron of a LEAFY Homolog W. Cari TaAyLor and ANGEL R. LEKSCHAS Department of Botany, Milwaukee Public Museum, Milwaukee, WI 53233 Qinc FENG Wanc and XING LIu a of Plant Systematics and Evolutionary Biology, Wuhan University, Wuhan 430072, People’s Republic of China Nancy S. Napier and Sara B. Hoor Department of Biological Sciences, University of Wisconsin, Milwaukee, WI 53201 Asstract.—Isoétes is an ancient lycopod lineage with a highly conserved morphology that provides few morphological characters to resolve the phylogeny of its species. Species appear to indicates that the Chinese Isoétes species are part of an Australasian clade including I. brevicula from Western Australia and I. kirkii from New Zealand. Two distinct cloned sequences of the second intron of a LEAFY homolog were recovered from I. sinensis supporting the ene that I. sinensis is an allotetraploid. One of the I. sinensis cloned sequences was similar t sequences recovered from J. sinensis were recombined parts of the two distinct sequences. Morphological evidence supporting an allotetraploid origin of J. sinensis is found in its larger microspore size and intermediate megaspore texture compared to J. taiwanensis, and _ I. yunguiensis Isoétes L. is a cosmopolitan genus of heterosporous lycopods containing hundreds of species. Plants usually appear as tufts of linear leaves arising from an underground, corm-like rootstock. Ellipsoidal sporangia occur in expanded leaf bases. Species range from evergreen aquatics to ephemeral terrestrials. Although Isoétes is an ancient lineage with its distinctive morphology rec- ognizable in the Triassic (Retallack, 1997), few characters have been found in its highly conserved morphology to resolve the phylogenetic relationships of its species. Distinguished by their habitat preference, megaspore morphology, and chromosome numbers, species appear to have evolved by ecological isolation and genetic divergence as separated populations adapted to ter- restrial or aquatic habitats and by interspecific hybridization and chromosome doubling (allopolyploidy) when divergent species were dispersed into the same sites (Taylor et al., 1993). Interspecific hybridization and allopolyploidy are well documented for Isoétes. Many interspecific hybrids have been recognized by their production of irregular spores and confirmed by their chromosome numbers (Brunton and TAYLOR ET AL.: PHYLOGENY OF CHINESE ISOETES 197 Britton, 1999). In several cases, interspecific hybrids and their suspected allopolyploid derivatives have been verified by chromosome counts, isozyme profiles, and DNA sequences (Taylor and Hickey, 1992; Hoot and Taylor, 2001; Hoot et al., 2004). A polyploid series ranging from 3x = 33 to 12x = 132 is known for Isoétes. Over 60% of Isoétes taxa, for which chromosome counts have been published, are polyploid (Troia, 2001). Therefore, not only is there documentation that interspecific hybridization and allopolyploidy occur in Isoétes, but there is also evidence that allopolyploidy is an equally important mechanism of speciation in this genus. Recently, herbarium, field, and laboratory studies have been conducted to learn more about the Isoétes of China. These studies have provided an opportu- nity to determine the status of historical populations, discover new popula- tions and new taxa, and obtain live specimens from which root tips could be harvested for chromosome counts and fresh leaves could be collected for DNA isolations. Four species of Isoétes have been described for China. All are believed to be rare and endangered. Isoétes hypsophila Handel-Mazzetti is known from the Hengduan Mountains in northwestern Yunnan Province and southwestern Sichuan Province. In this region, I. hypsophila occurs in the shallow water of lakes and ponds about 3600 m above sea level. Isoétes sinensis T. C. Palmer has been found at about ten sites in and along rivers and lakes of the middle and lower Yangtze River system. At present, only three populations are known to remain in China. Isoétes sinensis has also been reported from the Kyushu and Chubu Districts Japan (Takamiya et al., 1997) and Cheju Island, South Korea (Takamiya, 2001). Isoétes taiwanensis DeVol is known only from Menghuan Lake near the foot of Zhixing Mountain in the Yangming Mountains National Park, north of Taipei in northern Taiwan. Isoétes yunguiensis Q. F. Wang and W. C. Taylor is known from the Yunnan—Guizhou Plateau in southwest China. In this region, plants have been recorded at four sites, but only two small populations, totaling about 400 individuals, are known to remain. Liu et al. (2002) reported that J. hypsophila, I. taiwanensis, and I. yunguiensis are basic diploids (2n = 2x = 22) and Isoétes sinensis is a tetraploid (2n = 4x = 44). Nucleotide sequences from the nuclear ribosomal ITS region, the chloroplast atpB-rbcL spacer region, and the second intron of a LEAFY homolog have been used to determine phylogenic relationships of Isoétes, delimit species, and reveal an interspecific hybrid and its allotetraploid derivative (Hoot and Taylor, 2001). Hoot et al. (2004) used cloning to separate homoeologous sequences of the second intron of a LEAFY homolog for several Isoétes allotetraploids. By comparing these cloned sequences to those of putative parents, some of the parent species could be identified. The goals of this paper were to use nucleotide sequences from the nuclear ribosomal ITS region and the second intron of a LEAFY homolog to: (1) determine the relationships of the Chinese Isoétes species, (2) test the hypothesis that the tetraploid J. sinensis is an allotetraploid and, if this hypothesis is correct, (3) identify the basic diploid parent species of I. sinensis. 198 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) LE 1. Specimens sampled. Columns indicate species, oe collector, collection number- nc isolation number, date of collection, and herbarium acronym for location of voucher. Collections are from Mainland China unless otherwise noted. Isolation Species Voucher Collection Number Isoétes brevicula Rock pool, summit of Lily McCarthy Rock, Western 189 Australia, 25 Sep 2002, W. C. Taylor & N. T. Luebke 6383 (MIL) Isoétes hypsophila Tu-er-sjan, Dao-cheng County, Sichuan Province, 01 Aug 127 2001, Wang Aing-Feng, Liu Xing, Liu Hong & Yang Xiao-Lin 2 (WH) Isoétes kirkii Lake Brunner, South Island, New Zealand, 27 Mar 2004, kiNZ D. W. Woodland & Felicity Cutten s.n. (MIL) Isoétes sinensis Xing-an-jiang, Jiande City, Zhejiang Province, 19 Oct 129, 131 001, Liu Xing & Pang Xin-An 3, 4 (WH Isoétes taiwanensis Menghuan Lake, Yangming Mountains, Taiwan, May 78 1998, Chiou Wen-Liang s.n. (MIL) Isoétes yunguiensis Sha-shi-chong, Ping-ba County, Guizhou Province, 15 130 Aug 2001, Liu Xing & Yang Xiao-Lin 5 (WH MATERIALS AND METHODS Species sampling.—Table 1 contains locality, collector, collection number, and location of voucher specimens for plants used in this study. Specimens were identified to species using the original descriptions of the species (Handel- Mazzetti, 1923; Palmer, 1927; DeVol 1972a; Wang Q. F. et al., 2002) and by comparison with authentic and type specimens. Diagnostic morphology for megaspore textures was evaluated using an Olympus SZX12 stereomicroscope. DNA isolation and amplification —DNA was isolated from 20 mg of silica dried leaves from each sample by grinding the leaf tissue, frozen in liquid nitrogen, to a powder with a disposable 1.5 pellet pestle (Kimble-Kontes) in a 1.5 ml snap-cap microcentrifuge tube (Eppendorf) and using the DNeasy® Plant Mini Kit (Qiagen) following the manufacturer’s protocol. The ITS region for all samples was amplified with the primers ITS-I (5’- GTCCACTGAACCTTATCATTTAG-3’; Urbatsch, et al. 2000) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’; White et al. 1990). The LEAFY intron for all samples was amplified with the primers 30F (5’- GATCTTTATGAA CAATGTGG-3’) and 1190R (5'- GAAATACCTGATTTGTAACC-3’); Nancy s. Napier designed both LEAFY primers. PCR reaction mixtures followed manufacturer protocols using Ready-To-Go™ PCR Beads (Amersham Bio- sciences). PCR amplification for the ITS region began with denaturation for 60 s at 97°C followed by 40 cycles of denaturation for 10 s at 97°C, annealing for 30 s at 48°C, and extension for 20 s at 72°C with 4 s added to extension time each cycle and ending with a final extension of 7 min at 72°C. P amplification for the LEAFY intron began with denaturation for 5 min at 94°C followed by 40 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 50°C, and extension for 1 min at 72°C, and ending with a final extension of 5 TAYLOR ET AL.: PHYLOGENY OF CHINESE ISOETES 199 min at 72°C. PCR products were concentrated via electrophoresis in a 2% agarose gel containing ethidium bromide and visualized with transilluminated UV. Bands were cut from the gel and purified using the QlAquick® Gel Extraction Kit (Qiagen). Cloning and sequencing.—Sequencing of the ITS PCR products was performed on both 5’ and 3’ DNA strands using the amplification primers ITS-I and ITS4 as cited above. Ligation of the purified LEAFY PCR product and subsequent transformation, cloning, and visualization of transformed clones followed manufacturer protocols using the pGEM®-T Easy Vector Systems (Promega) with LB Amp 100 X-gal plates (Teknova). For basic diploid species, I. taiwanensis and I. yunguiensis, eight clones (colonies) for each species were sequenced. For the tetraploid species, I. sinensis, 16 clones (eight from each of two plants) were sequenced to increase the odds of capturing all parental cloned sequences. For the outgroup species, I. brevicula E. R. L. Johnson, I. hypsophila, and I. kirkii A. Braun, four clones from each species were sequenced. Cleaning and concentration of the vector DNA followed manufacturer protocols using the QIAPrep® Spin Miniprep Kit (Qiagen). Sequencing was performed on both 5’ and 3’ DNA strands using sequencing primers M13F and M13R in conjunction with the ABI Big Dye® Terminator Cycle v 3.1 Sequencing Kit (Applied Biosystems) following the manufacturer’s protocol. Sequencing products were resolved on an ABI (model 377) DNA sequencer at the Iowa State University DNA Sequencing and Synthesis Facility, Ames, Iowa. Sequence alignment and phylogenetic analysis.—Nucleotide sequences were aligned and edited using Sequencher 4.1 (Gene Codes Corp.). Gaps were treated as additional presence/absence characters, with one or multiple base gaps scored as a single character (Baldwin et al. 1995). Maximum parsimony analysis of the data was conducted with PAUP* version 4.0b10 (Swofford, 2002) using the heuristic search option for the ITS data set and the LEAFY data set, maximum trees = 4000 for the ITS data set and aximum trees = 100 for the LEAFY data set. PAUP* was used to run 500 bootstrap replicates for each data set to estimate reliability of the clades (Felsenstein, 1995). RESULTS ITS sequences of Isoétes hypsophila, I. taiwanensis and I. yunguiensis from China, I. brevicula from southwestern Australia, and I. kirkii from New Zealand form an Australasian clade with other species and clades previously reported by Hoot and Taylor (2001). The data set analyzed consisted 16 ingroup species and two outgroup species with a total of 826 characters; 313 characters were variable and 239 were parsimony informative. The ITS tree illustrated is a bootstrap 50% majority-rule consensus tree of the 39 most parsimonious trees retained (Fig. 1). Tree topology shows a Close relationship 200 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) lsoétes taiwanensis yunguiensis — Australasian — hypsophila 81 echinospora Ta 100 2 8 flaccida engelmannii melanopoda See 4 2 bolanderi nuttallii fe) © N ——— American olympica abyssinica — Mediterranean — histnx 14 capensis Fa stellenbossiensis Fic. 1. naincrs ITS region tree. Bootstrap 50% vg -rule consensus tree of 39 _ resulting from maximu analysis using | earch of ITS region data for eighteen basic diploid species of Isoétes. Tree length is orn steps of equally aan nucleotide substitutions and gaps, CI (excluding uninformative characters) = 0.76, RI = 0.86. Numbers above the branches are the number of nucleotide substitutions. Numbers below the branches are bootstrap values. Major clades are labeled on the right. The tree is rooted with I. capensis and I. stellenbossiensis from South Africa. exists between I. taiwanensis, and I. yunguiensis. All species in the tree are basic diploids (2n = 22). Defined by the 30F and 1190R Napier primers, the LEAFY region for aligned sequence clones of I. sinensis, I. taiwanensis, and I. yunguiensis was 1105 bases long and included all of the second intron and parts of the flanking exons. All of the eight sequence clones of I. taiwanensis were 1075 bases long and all of the eight sequence clones of J. yunguiensis were 1072 bases long. Two of the 16 I. sinensis clones did not amplify for the LEAFY intron. Therefore, 14 aligned, cloned sequences were compared for informative sites. At 15 informative sites that included 12 substitutions, two one base gaps, and one three base gap, six cloned sequences of I. sinensis, each 1076 bases long, matched or nearly matched the cloned sequence type of I. taiwanensis and five cloned sequences of I. sinensis, each 1079 bases long, matched or nearly matched the consensus cloned sequence type of I. yunguiensis (Table 2). Seven I. sinensis cloned sequences showed evidence of recombination i.e., part of the PCR amplified, cloned sequence first matched either the I. taiwanensis or the I. yunguiensis sequence type, but further on matched the other sequence type. TAYLOR ET AL.: PHYLOGENY OF CHINESE ISOETES 201 TABLE 2. Comparison of fourteen cloned sequences of I. sinensis with cloned sequences of I. taiwanensis and I. yunguiensis at fifteen informative sites. Sites are numbered sequentially beginning with the 30F Napier primer. Isoétes sinensis cloned sequences are identified by collection number-DNA isolation number-clone number. Nucleotides i in italic compare to those of from the data set before the final analysis. Clones 4-131-6 and 4-131-7 did not amplify for the LEAFY region and are not included in the table. site/clone 205 289 290 357 525 549 660 741 776 827 840 865 965-967 987 1059 famonensis G Gf TT €¢ G Tr T A. A A A --- - A 3-129- SG a WG oe I ae Ae Ae A --- - A 3-129-7 G06 1 ER MEA FBO A) Sa) A --- - A 4-131-1 Ct Se I oii Til a a, aA) A --- - A 4-131-3 Se | NG SE IE ANS “CGE SE adi, ea oil, 0 ee --- - A 4-131-4 CG OS AO Ae i ar a a --- - A 3-129-5 Go Sed UP MG baie a a ak --- - G 3-129-2* CG 3G a. a a ae ae RS --- - A 3-129-1* GSE Ere Se age Be ee AEG ocd A 4-131-8* i» “ee, OC cea ce Oey Se Me een Gee | A --- - A 4-131-2 GC GAR Uy A EE NE Be eB a> a AG. © 3 G 3-129-4 5 area "Saami, ae Oca! Vee! eee: Oma Poe, Caf G ATG... '- G 3-129-8 A PR GR Oe NE Ge Rr Gee oN Sons rier» OP Th © G 3-129-6 A A Ge a a a is et er a ee G 4-131-5 fame : gee, “mw, ee Lem Some: Osage Cee ete © eee Mies em a igh ee G yout: T A ££ G 7 - 7 Bo. &. oo. 8 ATO F G Three sequences showing evidence of extensive recombination were removed from the data set evaluated by PAUP*. Based on the relationships indicated in the ITS tree (Fig. 1), I. brevicula, I. kirkii, and I. hypsophila were chosen as outgroup species to root the LEAFY second intron tree. The sequenced LEAFY region for I. brevicula was 1035 bases, for I. kirkii it was 1090 bases, and for I. hypsophila it was 1095 bases. The LEAFY second intron data set analyzed contained 16 sequences with a total of 1125 characters; 171 characters were variable and 54 were parsimony informative. The data set included consensus sequences from clones of I. brevicula, I. hypsoj I. kirkii, I. taiwanensis, and I. yunguiensis and 11 cloned sequences of I. sinensis. The LEAFY second intron tree illustrated is a bootstrap 50% majority-rule consensus tree of the two most parsimonious trees retained (Fig. 2). Six ofthe I. si is cloned sequences formed a clade with I. taiwanensis and five of the I. sinensis cloned sequences formed a clade with I. yunguiensis. DISCUSSION Although Isoétes is Paleozoic in origin (Pigg, 1992), worldwide in distribution, and over time, undoubtedly adapted to changing climates and aquatic to terrestrial habitats on every continent many times, the morphology of Isoétes has been remarkably conserved. Thus, morphology provides few characters that can be used to reliably reconstruct phylogenetic relationships. Nevertheless, pteridologists have speculated about the relationships of Isoétes 202 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) yunguiensis 5-130 sinensis 3-129-4 sinensis 4-129-8 sinensis 4-129-6 sinensis 3-131-5 sinensis 3-131-2 2 taiwanensis s.n.-78 4 4 sinensis 3-129-3 100 9 sinensis 3-129-7 al 4 1 sinensis 4-129-5 0 sinensis 4-131-3 0 sinensis 4-131-1 Oo _sinensis 4131-4 42 hii s.n.-kiNZ 95 138 _previcula 6383-189 38 hypsophila 2-127 Fic. 2. Isoétes LEAFY second intron homolog tree. Bootstrap 50% majority-rule consensus tree of four trees resulting from maximum parsimony analysis using heuristic search of LEAFY second represent the tetraploid genome of J. sinensis. Tree length is 183 steps of equally weighted nucleotide substitutions and gaps, CI (excluding uninformative characters) = 0.89, RI = 0.96. Numbers above the branches are the number of nucleotide substitutions. Numbers below the branches are bootstrap values. Figures to the right of the specific epithets are the collection and clone identification labels. The tree is rooted with I. hypsophila from China, I. brevicula from Western Australia, and J. kirkii from New Zealand. species based on ecology, morphology, and biogeography. Britton and Brunton (1991) reevaluated the spore morphology of I. taiwanensis, concluding that it was not related to taxa from southwestern Australia as proposed by Marsden (1979), but instead appeared to have its closest affinity to I. kirkii from New Zealand. The ITS tree (Fig. 1) shows that both I. brevicula from southwestern Australia and I. kirkii form a sister clade to the Chinese species and all are members of an Australasian clade. Based on spore morphology, habit, and habitat, Huang et al. (1992) concluded that I. taiwanensis is probably closer to I. asiatica than it is to I. sinensis, but I. asiatica (I. echinospora subsp. asiatica (Makino) A. Love is a member of the I. echinospora species complex, a group of circumpolar taxa with echinate megaspores (Love, 1962, Takamiya, 1997). The ITS tree (Fig. 2) shows that I. echinospora, a member of an American clade, is only distantly related to members of the Australasian clade, which includes I. taiwanensis. DeVol (1972b) mentioned that I. taiwanensis seemed nearer to I sinensis than any other species. Takamiya (2001) saw similarities in the spore morphology of I. taiwanensis and I. sinensis and concluded that phylogenetic comparisons of these two taxa were needed. TAYLOR ET AL.: PHYLOGENY OF CHINESE ISOETES 203 |. taiwanensis I. yunguiensis ¥¥ 2n= 22 § 2n= 22 Tr Chromosome Doubling |. sinensis 2n = 44 Fic. 3. Hypothetical phylogeny of Isoétes sinensis involving interspecific hybridization and chromosome doubling of the basic diploids I. taiwanensis and I. yunguiensis. Data presented in this study supports the hypothesis of an allotetraploid origin for I. sinensis. The recovery of two, distinct, LEAFY second intron sequence types from the tetraploid I. sinensis supports the hypothesis that I. sinensis is an allotetraploid (Table 2). To clarify the results, it was assumed that some of the sequenced clones recovered from [. sinensis were recombinations of the two distinct sequence types and three of the recombinant sequences were removed from the data set evaluated by PAUP*. Since recombination occurs from crossing over between chromosomes during meiosis, it is possible that the observed re- combined sequences were products of natural events. If the recombined sequences detected were the result of crossing-over during meiosis, we would predict that identical crossover sequences would be recovered as clones. All seven of the recombinant cloned sequences from JI. sinensis were different, indicating that these recombined sequences more likely occurred during the R amplification reaction. Whatever their source, including recombinant sequences in a cladistic analysis will affect results and therefore, they need to be recognized and removed from the data set before the final analysis. Comparison of the two distinct cloned sequences from the allotetraploid I. sinensis with the cloned sequences of J. taiwanensis and I. yunguiensis indi- cates that either these two basic diploid species, or closely related taxa, likely participated in the formation of I. sinensis (Table 2). Although the I. yunguiensis clade, including five I. sinensis cloned sequences, and the I. taiwanensis clade, including six I. sinensis cloned sequences, are both well supported with high bootstrap percentages, the I. yunguiensis sequence is distinguished from its sister I. sinensis clones by sixteen autapomorphies and the I. taiwanensis sequence is distinguished from its sister I. sinensis clones by two autapomorphies (Fig. 2). These unique nucleotide substitutions could be due to (1) sequencing nucleotides of taxa different from those of the parent taxa, (2) continued evolution of the progenitor parent species and the allotetraploid species following allopolyploidy, or (3) copy errors during PCR. Causes for the autapomorphies might be determined by additional sampling and repeated PCR of the same clones. In addition to the molecular characters, morphological characters indicate that I. sinensis, I. taiwanensis, and I. yunguiensis are distinct, but closely 204 AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) related species and provide some evidence ing the allopolyploid origin of I. sinensis. All three species are amphibious fe with tri-lobed rootstocks. They all have a rudimentary velum that covers only the upper edge of the sporangium. The microspores of I. taiwanensis and I. yunguiensis range from 20-26 ym in length whereas, those of J. sinensis range from 26-30 um in length (Britton and Brunton, 1991; Wang et al., 2002; Palmer, 1927). The larger size of I, sinensis microspores is attributed to its increased chromosome number. Increases in chromosome number are usually accompanied by larger spore size (Kott and Britton, 1983). In contrast, megaspores of the tetraploid I. sinensis and the basic diploid I. yunguiensis average about 400 um in diameter, pea megaspores of the basic diploid I. taiwanensis average about 300 pm diameter. Megaspore texture appears to be the most distinctive character ia separates these three species. However, in view of the results presented here, the cristate to verrucate megaspores of I. sinensis can also be interpreted as subtly combining the textures of the rugulate to reticulate megaspores of I. taiwanensis and the cristate to reticulate megaspores of I. yunguiensis. ACKNOWLEDGMENTS The authors thank Chiou Wen-Liang, Felicity Cutten, Gerald J. Gastony, Kuo Chen Meng, Neil T. Luebke, Lytton J. Musselman, and Dennis W. Woodland for providing plant specimens analyzed in this study. NSF grants DEB-9981460 to Sara B. Hoot and DEB-9981501 to W. Carl Taylor and the State Key Basic Research and Development Plan of China (G2000046805) supported parts of this study. LITERATURE CITED BALDwIN, B. G., M. J. SANDERSON, J. M. Porter, M. F. hi Sealine an - ns CAMPBELL and M. J. DonoGHuE. 1995. The ITS region of nuclear ribosomal DN of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82 ais Britton, D. M. and D. F. BRuNTON. 1991. The spores and affinities of Isoétes tai is (I Pteridophyta). Fern Gaz. 14:73-83. uNTON, D. F. and D. M. Britron. 1999. Isoetes xechtuckerii, ean nov., a new triploid quillwort m northeastern North America. Canad. J. Bot. 77:1662—16 DEVOL, C E. 1972a. sas found on Taiwan. Tucweta 17:1-7. Devo, C. E. 1972b. A correction for Isoetes taiwanensis DeVol. Taiwania 17:304—305. FELSENSTEIN, J. 1995. Confidence limits on phylogenies: An approach using the bootstrap. Evolution Praag net H. 1923. eign hie Hand.-Mazt. Akad. Wiss. Wien 13:95. Hoot, S. B. and W. C. Taytor. . The arate . uied HS, a mnie as ae — ge me pig tae spacer regio da Isoétes. Pe geress m J. 91:1 Hoor,’S. B:, No S. ie and w ie eine 2004. Revealing unknown or extinct lineages within Isoétes (Isoétaceae) using DNA sequences from hybrids. Amer. J. Bot. 91:899-904. Huane, T. C., H. J. CHEN and L. C. Li. 1992. A palynological study of Isoetes taiwanensis DeVol. mer. Fern J. 82:142—150. Kort, L. S. and D. M. Brirron. 1983. Spore oe and taxonomy of Isoetes in northeastern North America. Canad. J. Bot. 61:3140-316 Love, A. 1962. epee ee of the Isoétes echinospor complex. Amer. eore }. 52: 113-123. Liu, X., Y. WANG, Q. F. Wanc and Y. H. Guo Isoetes and their taxonomical significance. Acta seis Sin. 40:351-356. TAYLOR ET AL.: PHYLOGENY OF CHINESE ISOETES 205 Marspen, C. R. 1979. Morphology and Taxonomy of Jsoetes in Australasia, India, north-east and south-west Asia, China and Japan. Ph.D. thesis, Department of Botany, University of Adelaide, compepey Paumer, T. C. 1927. A Chinese Isoetes. Amer. Fern J. 17:111-113. Picc, K. B. 1992. voltion of Isoétalean lycopsids. Ann. Missouri Bot. Gard. 79:589-612 RETALLACK, G. J. 1997. 7 Triassic origin of Isoétes and quillwort evolutionary ielbciion. Sworrorp, D. L. 2002. sea Phylogenetic analysis using parsimony (* and other methods). Sinauer, Sunderland Massachusetts, U TaKAMIYA, M., M. WaTANABE and O. Kanji. 1997. Biosystematic studies on the genus Isoetes (soetaceae) i in i IV. Morphology and anatomy of sporophytes, phytogeography and axonomy. Acta Phytotax. Geobot. 48:89-121. Mois M. 200 nye sinensis var. sinensis in Korea (Isoetaceae: Pteridophyta). Fern Gaz. 16:1 Taytor, W. C. and R. i ong 1992. Habitat, evolution and speciation in Isoétes. Ann. Missouri TAYLOR, W. C., N. T. ies D. M. Brirton, R. J. Hickey and D. F. BRUNTON. 1993. Isoetaceae, pp. 64— 75, in FNA Editorial Committee, eds. Flora of North America, North of Mexico, Volume 2. Oxford University Press, New Yor Trois, A. 2001. ag genus Isoétes L. (I aeuglivta Isoét ) thesis of } logical data. Webbia J Urpatscu, L. E., nt L. BALDwin and M. J. Donocuue. 2000. Phylogeny of the coneflowers and relatives (Heliantheae: Asteraceae) based on nuclear rDNA internal transcribed spacer (ITS) sequences and chloroplast DNA restriction site data. Syst. Bot. 25:539-5 Wane, Q. F., X Liu, W. C. Taytor and Z. R. H ee oe yungutensis (Isoetaceae), a new basic diploid ct from China. Novon os 587- APPENDIX Genbank accession numbers for the new Isoétes DNA sequences used in this manuscript. Genbank Species accession number Nuclear ribosomal ITS region sequences I. brevicula AY641098 I. hypsophila AY641099 I. kirkii AY641100 I. taiwanensis AY641101 I. yunguiensis AY641102 LEAFY second intron homolog sequences I. brevicula AY641103 I. hypsophila AY641104 I. kirkii AY641105 I. taiwanensis AY641106 nguiensis AY641107 1 sinensis (I. yunguiensis type clone) AY641108 I. sinensis (I. taiwanensis type clone) AY641109 American Fern Journal 94(4):206—206 (2004) Referees for 2004 All papers submitted to the journal are peer reviewed. Members of the editorial board and the Society, as well as additional scientists in cognate areas, do these reviews on a voluntary basis. It is continued success. The American Fern Society and I extend our thanks to the following reviewers for their assistance, diligence, and patience in the year 2004 PRESTON ALDRICH WarREN HAUK ERIC SNYDER MICHAEL BARKER Davip B. LELLINGER MICHAEL SUNDUE W. L. CuHlou NicHOLAS MONEY JACK TESSIER Davip CONANT Ross Mo RONNIE VIANE LD R JAMES JAMES E. WATKINS GERALD GASTONY VALERIE PENCE DEAN P. WHITTIER Gary GREER THOMAS RANKER MICHAEL WINDHAM HoOwaARD GRIFFITHS PAUL RUSSELL GEORGE YATSKIEVYCH CHRISTOPHER HAUFLER A. SALINO PETER ZIKA Index to Volume 94 A Comparison of Useful Pteridophytes Between Two Amerindian Groups from Amazo- nian Bolivia and Ecuador, 39 A Contribution to the Gametophyte Morphol- fe) 43 A New Species of Adiantum sonata from hailand, 77 A Taxonomic Study of the Fern Genus Arach- niodes Blume (Dryopteridaceae) from i 163 Acrorumohra, 163 sig Abi 126, 127, 130, 132; australis, Pe canine folidlosas Adiantum, 40, 162; ne en 80; cap- ie ct 80, 99, 162; caudatum, 80; igan 2 reniforme, 99; thongthamii (spec. nov.), 77, 77-80 Aeonium spp., 99 Afropteris, 126, 130, 135, 137; 140; kl At-Hampanl, S. H. and S. L. Buair. Influence of Copper on Selected Physiological Re- sponses in heap ati minima and Its Po- tentia n Copper Remediation, 47 Alsophila, 44; peat Anogramma, 126-128, 130, 132, 135, 137; chaerophylla, 129-131, 133, 134, 136; “ain cue sae 129-131, 133, 134, 136; Api rele 99 Aquilegia eS 159 Arabis lyrata, papers ek ped Sect. Amoenae, 163, 6 Sect. Arachniodes, Group cet os henryi, 167, 171, 173-175, 178, 179; Sect. Arachniodes, he oe nippon- fed; 167; 171; 176; hniodes, Group Arachniodes ee 171, 79; 167; austro-yunnanensis, 1 ; bai- seensis, 167, 172; basipinnata, 167; calcar- da ta, 166; dimorphophylla, 165, 173; elevata, P73, 176: pec: 173; ; erythrosora, 173; exilis, 164, 173, 174; falcata, 173; fengii, 173; lat oe a festi- na, 173: hei 172, 173; foliosa, 166; yep tein 173; Potortndedans 173, 179; gigantea, 173; gijiangensis, 173, 1 gizushanensis, 174, 178; globisora, 165, 166, 168, 174, 176, 178; gongshanensis, 174, 178; gradata, 174; grossa, 174; guang- ingshanensis, 175: infus lanceolata, 175; leuconeura, A 5 liyangensis, 175; longipinna, 175; lusha- nensis, 175; lushuiensis, 175, 178; magua- nensis, 174, 176; maoshanensis, 176; menglianensis, 174, 176; mengziensis, sporadosora, ubamoena, 179; 78, 179; suijiangensis, 167 179; tibetana, 178, 179; tiendongensis, 177 yunnanensis, 178, 180; yungiensis, 180; ziyunshanensis, 177,18 ARAGON, C. F. and E. PaNncua. Spore — rime Diff erent Storage Conditions in r Rupicolous Asplenium L. nee 28 eso canariensis, 99 —— azori u 4, 87— —100, 102, ia 110; aureum ar. pee ium, ae azomanes, 114; AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) azoricum, eles azoricum X A. onop- 116, pti 101, 102; ruta-muraria. rivalens, 113-116, 123; unilaterale, 89, 9 Buplenian ceterach and A. octoploideum on the Canary Islands ial acca, Pter- hyta, 81 Asplenium ruta-muraria L. in Iowa, 157 Aster azureus, 158; sericeus, Athyrium, 143; filix-femina, 1-8, 36; filix- emina var. asplenoides, Aulacomnium palustre, 156 Austrogramme, 126-140; decipiens, 129-136; pore Bank of Ferns in a Gallery Forest of — Ecological Station of Panga, Uberlan- - MG, Brazil, 57 LAIR, s. L. (see S. H. AL-HAMDANI) Blechnum brasiliense, 64; fragile, 70 Bolbitis nicotianifolia, 41 Bommeria, 162 serine lunaria, 156; oo 155, 6; pallidum, 155, 156; simplex, 155, 156 Potschim aga Newly eran in aine, 15 ee Sie hie 15 py, T. F. and D. Horton. eerie ruta- muraria L. in nen: 157 INDEX TO VOLUME 94 Campyloneurum, 9, 11, 12, 16, 26, 40; aglao- 42; ineumenaense, 9, 11, 13; 14, 17, 23; Carex richardsonii, 158; umbellata, 158 id 120 ae bivaens 84; officinarum ssp. offici- Giadontives muanelien, 99; pulchella, 99 hromosome Behavior ladonia spp., Co sah cee 128, 130; fraxinea, 126—136; jap , 138 bates "langsdorffi, 61-6 CG ell Costaricia wercklea ER) Crates, 44; amazonica, 42; delgadii, 36, 42; lasiosora, 42; pungens, 39, 40, 42, Cystopteris pulbifern, 159 ale Daphne gnidium, 99 Davallia canariens Dennstaedtia, 89, a ene a Diplazium nipponicum, Distribution, Ecology and Cytology of Asple- nium azoricum Lovis, Rasbach & Reich- stein oo Pteridophyta) and Its Hybrids Doritis spp., 80 Dryopteris, 143, 161, 163; michelii, 176 —67 ; giganteum, 43, 44 Erica, 121; azorica, 116 209 Eriosorus, 126-128, 131, 132, 135, 137; flexuo- sus, pe ye 133, 134, 136; insignis, — 7 34, 136; rufescens, 129, E ae E. Grup Rameate eas 61-67; eeaune 61-67 uphorbia maculata, 159 Festuca filifo on. rubra, 1 ILMAN, : rychium pallidum Newly Discord in oe ne, 1 Giupice, G. ese ia M. PINeIRO, M. eae andl G. Erra. Spore bloehdlngy of the oe from Northwestern rgentin Oana seekecnatte: 120 Greer, G. K. and D. Curry. Pheromonal Inter- actions Riche Cordate Gametophytes of the Lady Fern, Athyrium filix-femina, 1 ears 162 He Z., H. W ANG, J. Li, Q. Ye and W. C. Taytor. e Fern Genus Awichntodes Blume Tie peuinwuas from eo 163 Hemionitis, 1 62 Hieracium iets 156; —— 156 T B. (see W. C. TayLor) 205; japonica, 190; kirkii, 196, 198-202, 205; 1 oda, 200; nuttallii, 200; olympica, 200; orcuttii, 200; pantii, 193; pseudojaponica, 190; sinensis, 183-195, 203 sis var. core a yunguiensis, 183, 19 193, 196-203, 210 Jamesonia, 126-128, 131, 132, 135, 137, 142; alstonii, 129, 130, 133, 134, 136; imbricata, 129, 130, 133, 134, 136; rubricaulis, 120 Lastrea coniifolia, 172 rus azorica, 99 LeksHas, A. R. (see W. C. Taytor) Liatris cylindrica, 158 Liu X. (see W. C. prebiea Lomariopsis japure 43 Loxsoma soe acenla 162; cunninghamii, 162 Luehea divaricata, 3, 65-67 oa japonicum, 6; venustum, 64 ci Comparison of Used Pterido- phytes between Two Amerindian Groups mazonian Bolivia and Ecuador, 39 Microgramma, 9, 15, 20, 25, 26; fuscopunctata, 43, xXmortoniana, 15; persicariifolia, 60, 61; squamulosa, 9, 13, 14, Micromeria Mimulus guttatus, 5 Minuartia pened 159 Monanthes laxiflora, 99 M C. and R. V. Russe... The Occurrence a Selva Biological Station, Costa MokreELLI, M. A. (see G. E. Grupice) Napier, N. S. (see W. C. TAyLor) aria, 159 Nephopteris, 126, 127, 128 AT, ee wo2; “Laz, T40* japonicum, 129-134 36 Osmunda, 36 PajARON, S. (see C. J. VAN DEN HEEDE) Pancua, E. (see C. F. ARAGON) Pancua, E. (see C. J. VAN DEN HEEDE) Parietaria aegis 159 a, O13; 18, 25, 26; fillicula, 9, 13- : it oranense, 9, 13—15, 18; venturi, 9, 13-15, AMERICAN FERN JOURNAL: VOLUME 94 NUMBER 4 (2004) Pellaea atropurpurea, 157; glabella, 157, 159 het sp., z-Garcia, B. and A. Menpoza-Ruiz. A Con- pop goin to the Gametophyte Morphology and Development in Several Species of Thely tate, Thelypteridaceae, 143 ersea indica, 99 pipers , 163 Pheromonal Interaction Among Cordate Game- toph of the Lady Fern, Athyrium filix- mina, Phlebodium, 9, 13, 17, 20, 26; areolatum, 57, 60, 64, 65; pseudoaureum, 9, 13, 14, 17, Phylogenetic pram of Isoétes (Isoéta- eae) in a as Revealed by Nucleotide Share of the Nuclear Ribosomal ee sala — the Second Intron of a LEAF molog Phylogenet ain of the Subfamily itido folie Sian a 196 as M. (see G IUDICE) Pinus canariensis, 99 Pittosporum, 124; undulatum, 116, 120-122, 124 Pityrogramma, 127, 128, 130, 135, 137, 140; i he 129-131, 133, 134, 136; calomelanos var. oo 60, 64, 65, 67; trifoli we 64, 65, Be peons longispina, ii papillifolia, 120; , 120; virginica, 120 Pleopeltis, 9, 13, 8, 20, 25, 26; macrocarpa, 9, Polybotrya, 40: sae ey 43; osmundacea, Sista 9) 10; 12, “19 22, 23; 25, 26% argentinum, 9, 12, 14, 19, 21, 22, 25; podum, 9, 12). 14; 19; 22. 23) 25: cambricum ssp. macaronesicum, 99; sol ; Oy 12, j ; 21, 23, 25: hirsutissimun, 9; lasiopus, 9, 12, 14, 19, 21, 23, 25, 26; loriceum, 9, 12, 14, 19, 21, 24, 25; squalidum, 9, 12, 14, 1 tweedianum, 9, 12, 14, 19, 21, 25; sil ger, 36 Polystichopsis, 164 Polystichum, 100, 163; acrostichoides, 5; ama bile var. chinense, 172; di rer’ lum, 173; globisorum, 174; henryi, 174; nigrospinos 176; nipponicum, 176; m pciaetinaone 17; setiferum, - sim en var. jus, 16 peeves piliferum, 156 INDEX TO VOLUME 94 Potamogeton pectinatus, Nomenclatural + in Adian- eritte ufum, 60 Pteridaceae Subf. Cheilanthoideae, 127; subf. aenitidoideae, 126, 127, 137, 140; subf. Pteridoideae, ia ani aquilinum, 7, 36, 67; aquilinum var. achnoideum, 64, 65 panes a 435; 140; pratied, 138, 161; fauriei, 138; multifida, 126, 128-137; quadriaur- ita, 126, 128-137; vittata, 36, 60, 64, 65, 67, 159, 161 Pterozonium, 126, 127, 131, 132, 135, 137, 140; losor 4 RanaL, M. Bark Spore Bank of Ferns in a Gallery Forest of the eo ae bi of Panga, Uberlandia — MG, Brazil, 5 Rassacu, H. (see F. Sires Rhinanthus crista-galli, 156 Rhododendron canadense, 156 S, rn ast 17 Rumsey, F., S. Seon H. ScHAFER and H bach & Reichstein 7-5 RACALDO, P. Pitoeeuulic Relation- ships of the — Taenitidoideae, Perdacene, 12 SCHAFER, H. (see Aaa chizachyrium scoparium, 158, 159 spc denticulata, 99; exaltata, 43; geni- 44; omega 44; siamensis, 80 Spirodela peek Spore es ato of tha Polypodiaceae from Northwestern Argentina, Spore Viability under Different Storage Con- nae in Four Rupicolous Asplenium L. Sporbols paar 0H 158; neglectus, 159 SUSKATHAN, w Species of Adiantum om Prick me etic 126, 127, 131, 132, 135, 137, 140; grande, Taenitis, 126, 127, 131, interrupta, 129, 130, 133, 134, 241 Tapirira sania 61-63, 65-67 Taraxacum syptiades m, 156 TAYLor, W. C. BH HZ TayLor, W. C., . LEKsHasS, Q. F. me Li, NS. ae and S. B. Hoor. bag lo- ylo genetic Relationships of Isoétes (Isoéta- ceae) in China as Reveale Region and the Second Intron of a LE. Homolog, 196 Terminalia brasiliensis, 60 Terpsichore lehmanniana, 70; semihirsuta, 70 occurrence of Trichomanes godmanii (Hy- menophyllaceae) on Welfia georgii (Are- soar at fo La Selva Biological Station, a Rica, 70 Thlyptos = 61, 66-68, 143, 148, 150, 151; ubg. Am 66; ithe var. agneeraren, 144; wetwlione, tetragona, 144, 145, 147-150; .piedrensis, 145 Tmesipteris, 70 achypteris, 162 Trichomanes Sect. Didymoglossum, 74-76; an- onesie 74, 75; capillaceum, 70; god- 1 manil, 70-76; venosum, 16 Trifolium a sear ae — re ia 156; myrtilloides, 56 s-idae Vallisneria sali Sea) ee “| PajARON, E. PaNGcua, and A Vitexin 7-O- Deeninnaice: a New Flavonoid from Pteris vittata, 159 Vittaria-type, 147, 149, 151 F. - e W. C. TAyYLor) a ©): Welfia georgii, 70-76 gaa ph hea a5 YE Q. (see HE : : ms a 7 ne = at a 7 _ on aM ; ae 7 oa nm aa _ te mary © aibeaet j a : a ie : oe on - - 7 Pe ior. a a4 - 7. © ‘ 7 ao > a I : yy ” : 7 a - - we - iar a ‘eo ; : at ee ote? 4 eu ‘ cs . ne! a a - ea! ¥ id, sei ay eel " a 7 = ee saga tates ae LO 3 1753 00321 Hu INFORMATION FOR AUTHUKDS ———___ Authors are encouraged to submit manuscripts pertinent to pteridology for pub- lication in the American Fern Journal. Manuscripts should be sent to the Editor. Acceptance of papers for publication depends on merit as judged by two or more referees. 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