Isozymic and Chromosomal Evidence for the Allotetraploid Origin of Gymnocarpium Dryopteris (Dryopteridaceae) Author(S): Kathleen M

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Isozymic and Chromosomal Evidence for the Allotetraploid Origin of Gymnocarpium dryopteris (Dryopteridaceae) Author(s): Kathleen M. Pryer and Christopher H. Haufler Source: Systematic Botany, Vol. 18, No. 1 (Jan. - Mar., 1993), pp. 150-172 Published by: American Society of Plant Taxonomists Stable URL: http://www.jstor.org/stable/2419795 Accessed: 15-05-2015 17:24 UTC

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This content downloaded from 152.3.102.242 on Fri, 15 May 2015 17:24:29 UTC All use subject to JSTOR Terms and Conditions SystematicBotany (1993), 18(1). pp 150-172 ? Copyright1993 by the American Society of Plant Taxonomists

Isozymic and Chromosomal Evidence for the Allotetraploid Origin of Gymnocarpiumdryopteris (Dryopteridaceae)

KATHLEEN M. PRYER1 Botany Division, Canadian Museum of Nature, P.O. Box 3443, Station D, Ottawa, Ontario KlP 6P4, Canada

CHRISTOPHER H. HAUFLER Department of Botany,Haworth Hall, Universityof Kansas, Lawrence, Kansas 66045-2106 Present address: Department of Botany,Duke University, Biological Sciences Building, P.O. Box 90339, Durham, North Carolina 27708-0339

ABSTRACT. A critical examination of isozyme, chromosomal, and morphological charactersin subspecies formerlyincluded in Gymnocarpiumdryopteris demonstrated that three sexual taxa can be distinguished. We recognize these taxa as distinctspecies: the widespread, fertileallotetraploid G. dryopteris,with one genome derived fromthe western diploid G. disjunctumand the other fromG. appalachianum sp. nov., a previously undetected eastern North American diploid, which is described and illustratedhere and forwhich we reporta chromosomenumber of 2n = 80. Population comparisons of allele frequencies between G. disjunctumand G. appalachianumyielded an average Nei's genetic identityvalue (I) of 0.274. A wide-ranging assemblage of putatively triploid plants with both sterile,malformed spores and large, round spores capable of germinationis believed to representthe backcrosses G. disjunctumx dryopterisand G. appalachianumx dryopteris.The name G. x brittonianum comb. nov. is applied here to G. disjunctumx dryopteris.A key to fertilespecies, species descriptionsand illustrations,distribution maps, and habitat notes are included.

GymnocarpiumNewman (Dryopteridaceae)has Kamchatka,and Sakhalin Island (Kato and Iwa- been regarded as comprisingsix species native tsuki 1983). It has larger,tripinnate fronds and to the temperateregions of the northernhemi- is diploid with 2n = 80 (V. Sorsa 1966; Taylor sphere (Tryon and Tryon 1982). However, de- and Mulligan 1968; Wagner 1966). A putative spite a recentseries of systematicstudies (Pryer triploidhybrid, G. dryopterissubsp. x brittonian- 1981; Pryerand Britton1983; Pryeret al. 1983; um Sarvela, was described by Sarvela (1980) to Sarvela 1978, 1980; Sarvela et al. 1981; P. Sorsa include plants that are morphologically inter- 1980),the taxonomyof this group has remained mediate between these two subspecies and pro- contentious.One of the more vexing problems duce malformedspores. has been the status of the taxa that have been It had been presumed thatG. dryopterissubsp. included within G. dryopteris(L.) Newman. disjunctumgave rise to subsp. dryopteristhrough Plants of this species usually have been sepa- autopolyploidy because of the very subtle dif- rated into two infraspecifictaxa (Wagner 1966), ferences in their vegetative morphology,and most recently treated as subspecies (Sarvela the close similarityof their perispore patterns 1978). Gymnocarpiumdryopteris subsp. dryopteris (Pryer and Britton1983) and chromatographic has relatively small, bipinnate fronds, and is profiles(Pryer et al. 1983). However, if subsp. tetraploidwith 2n = 160 (Britton1953; Manton dryopteriswere an autopolyploid,one would ex- 1950; Pryer1981; Sorsa 1958; Vida 1963; Wagner pect some multivalentassociations during the 1963). This taxon is widely distributedin North firstdivision of meiosis (Jackson and Casey America, Europe, and Asia (Hulten and Fries 1982). All chromosome preparations of subsp. 1986; Jalas and Suominen 1972; Kato and Iwa- dryopterisconsistently revealed 80 bivalents tsuki 1983). Gymnocarpiumdryopteris subsp. dis- (pers. obs.), which raised two alternatives.Ei- junctum (Rupr.) Sarvela is restricted to the ther subsp. dryopterisis an autopolyploid that northwest coast of North America, southern has evolved a pairing control mechanism,thus 150

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concealing its origin (Jackson1982), or it is an metersapart to increase the likelihood thatdif- allopolyploid and has arisen throughinterspe- ferentindividuals would be collected. Because cifichybridization. of the potential for clonal replication of indi- The insights provided by data fromenzyme vidual genotypes within a population, Table 1 electrophoresisin tracing the origins of poly- lists both the number of leaf samples (ramets) ploid fernsare well-known(Haufler 1985b, 1987; obtained and the numberof differentgenotypes Werth1989). The numberof isozymes and their (genets) identified at each site. Fronds were patternsof variabilityfrequently allow one to storedin plasticbags and keptrefrigerated until distinguish between auto- and allopolyploids electrophoresiswas conducted. A total of 374 (Bryanand Soltis 1987; Crawford1985; Haufler individual sporophyteswas examined. et al. 1985). The electrophoreticprofile of an Enzyme Electrophoresis. All sporophytes autopolyploid taxon should show a subset of were surveyed for electrophoreticallydetect- the isozymes found in the diploid progenitor able enzyme variation using 12% starch gels. (Crawfordand Smith 1984; Gastony 1988; Soltis Small portions of fresh leaf material were and Rieseberg 1986). In contrast,an allopoly- ground in phosphate grinding buffersolution ploid should manifestfixed heterozygous (non- (Haufler 1985a) and the extractwas absorbed segregating)banding patternsfor a majorityof into wicks of Whatman 3MM chromatography enzymes with the component bands corre- paper. The wicks were then frozen at - 80?C sponding to additivityof the isozymes derived (Ranker and Schnabel 1986) until theywere in- fromtwo diploid progenitorspecies (Haufler et serted into gels. The following enzymes were al. 1990; Roose and Gottlieb 1976; Werth et al. resolved: aspartate aminotransferase (AAT), 1985). hexokinase (HK), isocitrate dehydrogenase This paper reports on electrophoretic,cyto- (IDH), leucine aminopeptidase (LAP), malate genetic,and morphologicalinvestigations of the dehydrogenase (MDH), phosphoglucoisomer- taxa included in G. dryopteris.The primaryob- ase (PGI), phosphoglucomutase (PGM), jectivesof thisstudy were to determinewhether 6-phosphogluconate dehydrogenase (6PGD), subsp. dryopteriswas derived through auto- or shikimate dehydrogenase (SkDH), and triose- allopolyploidy and, if allopolyploidy is in- phosphate isomerase (TPI). Clear band patterns volved, to discover the identity of its second were expressed for PGI, PGM, and TPI using diploid progenitor.As detailed below, our re- gel and electrode buffersystem 6 (Soltis et al. sults indicate that G. dryopterissubsp. dryopteris 1983). The modifiedgel-electrode buffer system is an allotetraploidbetween G. dryopterissubsp. 8 (Haufler 1985a) was used to resolve LAP, HK, disjunctumand a previouslyundescribed eastern and AAT. The enzymes SkDH, IDH, 6PGD, diploid taxon. We herein circumscribeG. dryop- and MDH were assayed using the morpholine teristo include only the tetraploid plants, and gel-electrodebuffer system (Werth 1985) at pH recognize two diploid species, G. disjunctum 7.0. The enzymes aconitase (ACON) and aldol- (Rupr.) Ching (western North America) and G. ase (ALD) were examined,but clear bands were appalachianum,newly described below (eastern not expressed consistently.Standard staining NorthAmerica). The putativetriploid backcross protocolswere followed (Soltis et al. 1983). Nei's between G. dryopterisand G. disjunctumis raised genetic identityvalues (I) were calculated using to specificstatus as G. x brittonianum. LYNSPROG provided by M. D. Loveless (Col- lege of Wooster,Ohio). Cytology. Leaf material with young spo- MATERIALS AND METHODS rangia undergoing meiosis was collected and Field Work. Plants of the G. dryopteriscom- fixedin Farmer'ssolution (absolute ethanol and plex were collected in the summersof 1987 and glacial acetic acid, 3:1) and stored at about 4?C. 1988 from42 localitiesover a broad range (Table Spore mothercells were stained and squashed 1); these collections provided the material for followingthe technique of Haufleret al. (1985). enzyme electrophoreticanalysis. At each local- Photographs of chromosome squashes were ity an average sample of 10 sporophytes was taken with Kodak Technical Pan film using a taken (range = 1-25). Due to the rhizomatous Nikon AFM camera on a Zeiss phase contrast natureof Gymnocarpium, itspopulations are made microscope. up of clones. Leaf samples were taken several Spore Measurements. Spores were mount-

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TABLE 1. Collection data forpopulations of the Gymnocarpiumdryopteris complex sampled forelectrophoretic analyses.All vouchersat CAN unless otherwisenoted. APP = G. appalachianum,BRI = G. x brittonianum,DIS = G. disjunctum,DRY = G. dryopteris.Number of ramets= 4 of frondsobtained fromeach site; numberof genets = 4 of differentgenotypes identifiedat each site.

Number of Number of Taxon ramets genets Location U.S.A.: APP 1 1 North Carolina: Ashe Co., BluffMtn. (no voucher) APP 2 1 Pennsylvania: Bedford Co., Wolfsburg,3 Jul 1988, Pryeret al. 940 APP 11 1 Virginia: Highland Co., Lantz Mtn., 4 Jul 1988, Pryeret al. 942 APP 9 2 Virginia: Madison/Page cos., Hawksbill Mtn., 6 Jul 1988, Pryeret al. 945 APP 11 2 Virginia: Page Co., Bush Mtn., 5 Jul 1988, Pryeret al. 944 APP 15 3 Virginia: Page Co., Stony Man Mtn., 6 Jul 1988, Pryeret al. 948 (type locality) APP 4 3 West Virginia: Hampshire Co., Ice Mtn., 11 Jun 1987, Morse 9486 APP 13 5 West Virginia: Hampshire Co., Ice Mtn., 7 Jul 1988, Pryeret al. 949 CANADA: BRI 14 1 Ontario: PrescottCo., Bourget (site 1), 6 Jun 1987, Pryer& Jarvis900; 28 May 1988, Pryer& Jarvis935 BRI 10 1 Ontario: PrescottCo., Bourget (site 2), 6 Jun 1987, Pryer& Jarvis901 BRI 8 1 Ontario: Wellington Co., Belwood, 8 Jun 1987, Pryeret al. 903 (type locality) U.S.A.: DIS 1 1 Alaska: Juneau,West Glacier Trail, 6 Jun 1988, Brodoet al. s.n. DIS 6 5 Alaska: Baranof Island (no voucher) DIS 25 14 Oregon: Linn Co., Tombstone Prairie (no voucher) DIS 21 8 Oregon: Yamhill Co., Carlton, 20 Jun 1987, Alverson905 DIS 10 4 Washington: King Co., Deception Creek Trail, 7 Jun 1987, Arnotet al. s.n. (WTU) DIS 12 4 Washington: King Co., Deception Creek Trail (no voucher) DIS 12 7 Washington:Yakima Co., Mt. Aix, Sept 1987, Alversons.n. (no voucher) CANADA: DIS 8 3 BritishColumbia: Kootenay District,Rossland, 30 Aug 1987, Ceska and Ogilvie23074 (V) DIS 10 2 BritishColumbia: New WestminsterDistrict, Mt. Seymour, 27 Jun 1987, Mehrhoffs.n. DIS 23 12 BritishColumbia: Vancouver Island, Cathedral Grove, 22 Aug 1987, Crins7417 (UBC) U.S.A.: DRY 15 4 Alaska: Fairbanks,24 Jun 1987, Batten87-1 (ALA) DRY 2 2 Arizona: Coconino Co., Dane Canyon, 17 Aug 1987, Boucher500 (ASC) DRY 9 2 Michigan: Marquette Co., Ishpeming, 20 Aug 1986, Windham& Ranker 878 (UT) DRY 10 4 Minnesota: Lake Co., GooseberryFalls, 13 Jun 1987, Pryer& Klein929 DRY 3 1 New Hampshire: GranthamCo., along shores of Eastman Lake, 18 Jul 1988, Hauflers.n. (KANU)

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TABLE 1. Continued.

Number of Number of Taxon ramets genets Location

DRY 5 1 Pennsylvania: Lackawanna Co., Roaring Brook, 2 Jul 1988, Pryer& Klein939 DRY 8 3 Vermont:Chittenden Co., Burlington(no voucher) DRY 11 2 Wisconsin: Manitowoc Co., Point Beach Ridges, 21 Jun 1987, Taylor s.n. DRY 3 2 Wisconsin: Polk Co., InterstateState Park, 21 Aug 1986, Windham& Ranker882 (UT) CANADA: DRY 2 2 Ontario: Algoma District,Batchawanna Falls, 18 Aug 1986, Windham& Ranker867 (UT) DRY 11 5 Ontario: Algoma District,Lafoe Creek, 9 Jun 1987, Pryer& Klein906 DRY 9 5 Ontario: Algoma District,Magpie Falls, 10 Jun 1987, Pryer& Klein909 DRY 3 2 Ontario: Frontenac Co., Ompah, 16 Aug 1986, Pryeret al. s.n. DRY 10 2 Ontario: Ottawa-CarletonRegional Municipality,Albion Rd., 5 Jun 1988, Pryer& Klein 937 DRY 3 1 Ontario: ParrySound District,Blackstone Lake Rd., 24 May 1987, Brit- ton 11310 DRY 10 2 Ontario: Thunder Bay District,Kakabeka Falls, 12 Jun 1987, Pryer& Klein920 DRY 6 4 Ontario: Thunder Bay District,Keemle Lake, 11 Jun 1987, Pryer& Klein910 DRY 10 4 Ontario: Thunder Bay District,Mt. McKay, 13 Jun 1987, Pryer& Klein925 DRY 9 3 Ontario: Thunder Bay District,Pass Lake, 12 Jun 1987, Pryer& Klein916 DRY 5 3 Ontario: Thunder Bay District,Red Rock, 11 Jun 1987, Pryer& Klein913 JAPAN: DRY 4 2 Niniu, Shimukappu Village, Ifutsu-gun,Kamifawa Pref. (no voucher) ed in Hoyer's medium on glass slides and mea- RESULTS AND DISCUSSION sured at 400 x using a filarocular micrometer on a Leitz Dialux 22 microscope. Between 20 Origin of Tetraploid G. dryopteris. Ten en- and 30 maturespores per sporophytewere mea- zyme systemsencoded by 12 putative gene loci sured along theirlongest dimension to the out- were resolved. Isozyme patterns were consis- er exospore walls. Measurements did not in- tentwith the hypothesisthat G. disjunctumwas clude the irregularfolds of the perispore. one of the diploid progenitorsof the tetraploid. Distributions. More than 3500 herbarium However, G. dryopterisshowed a large number specimens were examined fromA, CAN, CM, of "orphan" isozymes (sensu Werth 1989) that COLO, DAO, DUKE, FARM, GH, H, IA, ISTC, could not be attributedto G. disjunctum.This LE, LKHD, LSP, LYN, MICH, MIN, NEBC, patternwas evident at 7 of the 12 loci examined NFLD, OAC, ORE, OS, QFA, QK, RM, SASK, duringthis study. In PGI, forexample, the faster SDU, SFS, SLU, TRT, UBC, UNM, US, V, VDB, migratingisozymes found in G. dryopterishad VPI, VT, WAT, WILLI, WIN, WIS, WS, WTU, the same relative electrophoreticmobility as WVA. Data fromthese specimens were used to those found in G. disjunctum;however, bands plot species distributions. with the same mobilityas the slower migrating

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A lated and well-known diploid taxon and an unknown diploid parent (Cryptogramma,Alver- son 1988; Hemionitis,Ranker 1988; Polypodium, PGI Bryan and Soltis 1987). The key to discovering the "missing" parental genome in Gymnocarpiumwas the correlation DRYOPTERIS DISJUNCTUM between ploidy level and spore size. Tetraploid __ ~~~~~B G. dryopterisconsistently exhibited a mean spore length of 36 j,m, whereas diploid G. disjunctum had a mean spore length of 29.5 ,um(Pryer and Britton 1983). The differencein mean spore TPI lengthsbetween plants of the two ploidy levels was statisticallysignificant, and the range of means showed no overlap. This allowed us to use spores fromherbarium specimens to iden- DRYOPTERIS DISJUNCTUM tifypossible diploid populations. FIG. 1. Representative electrophoretic banding patterns of Gymnocarpiumdryopteris and G. disjunc- A surveyof spore sizes revealed a concentra- tum. A. PGI variation at loci 1 and 2. B. TPI vari- tion of small-spored populations in the Appa- ation at loci 1 and 2. Note the "orphan" bands in G. lachian Mountains of Pennsylvania, West Vir- dryopterisin both PGI and TPI. ginia, and Virginia. Chromosomal preparations of spore mothercells at diakinesisof plants from = bands of the tetraploid were absent in G. dis- Ice Mt., West Virginia, revealed 2n 80 (Fig. junctum(Fig. 1A). Similarly,in TPI, bands cor- 2). Because this was the firstreport of a diploid responding to the same mobilityas the fastest count for the genus in eastern North America, and slowest migratingisozymes in G. dryopteris itwas reasonableto hypothesizethat these plants were not observed in G. disjunctum(Fig. 1B). could be a new taxon,and representthe missing These data suggest that G. dryopteriscould be parental genome of tetraploidG. dryopteris.Ac- an allotetraploidresulting from hybridization cording to the working hypothesis, the "or- between G. disjunctumand a previously unde- phan" isozymes observed in tetraploidG. dryop- tecteddiploid. These resultsparallel those of a terisshould be found in the newly discovered numberof other recent studies, in which elec- diploid, herein described as G. appalachianum. TPI-2 trophoreticevidence for the allopolyploid ori- At the dimericPGI-2 and loci, G. dryopteris gin of tetraploidferns implicates a closely re- exhibited a fixed heterozygous pattern com- posed of a fastand a slow band, each with the same relativemobility as the homozygousbands found in G. disjunctumand G. appalachianum,respectively(Figs. 3A and 3C). In the monomeric enzyme SkDH, three differentallozymes were found in the diploid populations (Fig. 3B). The fixed heterozygous banding pattern observed in G. dryopterisat the SkDH locus was again additive of its putative diploid progenitors:the 6.'~~~~~~~~~~~~~~~~~~~1 fastband had the same relative mobilityas the homozygous band found in G. appalachianum, and the slow band had a mobilitycorresponding to the slow band in heterozygous G. disjunctum.This patternwas repeated at each of its seven fixed-heterozygousloci, strongly sug- gestingthat G. dryopterisis an allotetraploidthat FIG. 2. Chromosomal preparation from spore originatedfollowing hybridization between the mothercell of Gymnocarpiumappalachianum at diakinesis with 40 bivalents. Voucher: West Virginia, diploids G. disjunctumand G. appalachianum. Hampshire Co., Ice Mtn., 6 Jun 1988, Morse 9578, site Various lines of evidence suggest that for- 8 (CAN). x 2000. mation of the allotetraploid, G. dryopteris,was

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TABLE 2. Allele frequencies for polymorphic loci in Gymnocarpiumdisjunctum and G. appalachianum.Pre- ~~~~~~PGI-2 sumed loci and alleles foreach enzyme are numbered consecutively proceeding from anode to cathode. DIS DRY APP DRY DIS Numbers in parentheses indicate the sample sizes.

G. G. disjunctum appalachianum B Locus Allele (77) (16) Pgi-2 1 0.4545 0.0000 SkDH 2 0.0649 0.0000 3 0.4805 0.0000 DI8 DRY APP DRY DIS 4 0.0000 0.1563 5 0.0000 0.2500 6 0.0000 0.2188 C 7 0.0000 0.3750 Tpi-2 1 0.0065 0.0000 _A ' TPI-2 2 0.9935 0.0000 3 0.0000 0.5938 DIS DRY APP DRY DIS 4 0.0000 0.4063 Tpi-l 1 0.0000 1.0000 FIG. 3. Representativeelectrophoretic banding 2 0.2532 0.0000 patternsof Gymnocarpiumdisjunctum (DIS), G. dryop- 3 0.7468 0.0000 tens(DRY), andG. appalachianum(APP). A. PGI-2. B. Lap 1 SkDH. C. TPI-2. Notehere that the "orphan" bands 0.0000 1.0000 shownin Figure1 forPGI and TPI have the same 2 0.5584 0.0000 relativemobility as thehomozygous bands in G. ap- 3 0.0065 0.0000 palachianum,indicating the allopolyploid origin of G. 4 0.0455 0.0000 5 0.3896 0.0000 dryopteris. Hk 1 0.0000 0.4375 2 0.9156 0.5000 not recent. Comparisons of allele frequencies 3 0.0000 0.0625 4 0.0844 0.0000 (Table 2) between G. disjunctumand G. appala- Skdh 2 0.7338 0.0000 chianumyielded an average Nei's genetic iden- 3 0.0000 0.9688 tityvalue (I) of 0.274, a figurethat approximates 4 0.2662 0.0000 the 0.33 average calculated forcongeneric fern 6 0.0000 0.0313 species (Soltis and Soltis 1989). This low value Idh 1 0.0000 0.6875 suggeststhat the diploids are distinctand prob- 2 0.0779 0.0000 ably old taxa, particularlywhen compared to 3 0.0000 0.3125 the average value of 0.67 reported for conge- 4 0.9156 0.0000 neric angiosperm species (Crawford1983; Gott- 5 0.0065 0.0000 lieb 1981). The allotetraploid,G. dryopteris,de- 6Pgd-2 1 0.1169 0.0000 2 0.8636 rived fromthese two diploids, currentlyhas a 1.0000 3 0.0195 0.0000 broad circumboreal distribution well beyond Mdh-5 1 0.0130 0.0000 the ranges of its diploid progenitors.It also un- 2 0.0390 0.0000 dergoes normal chromosome pairing behavior 3 0.0000 1.0000 at-meiosis, without the formationof multiva- 4 0.0065 0.0000 lents. The hybridization event leading to the 5 0.9416 0.0000 origin of the allotetraploid possibly dates back Mdh-2 1 0.0065 0.0000 to the Pleistocene,when the geographical rang- 2 0.9740 1.0000 es of northernspecies were contractedand dis- 3 0.0195 0.0000 placed southwardas a resultof dramaticchanges in climate (Davis 1983). Such distributional changes no doubt separated the previouslycon- case of the diploid progenitorsof G. dryopteris. tiguous ranges of many species, while forcing Following hybridizationand polyploidization, other formerlyallopatric taxa into sympatry. G. dryopterisachieved meioticstability (i.e., con- This latter process may have occurred in the sistentbivalent formation)and dispersed to oc-

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cupy its present extensive range. As is charac- interpretationof and variabilityin the number teristic of many allopolyploids (Ferris 1984; of trivalents,bivalents, and univalents in a giv- Haufler et al. 1990; Roose and Gottlieb 1976; en preparation.In spite of these difficulties,in- Werthet al. 1985), G. dryopterismay have had a terpretationsof all squashes were consistent higher fitnessin a wider range of habitats and with a triploid compliment of 2n = 120 (Figs. thus have been able to exceed the distributional 4c, d; Pryer1981). Unfortunately,we could not limits of either diploid parent. Populations of use chromosome squashes to identifythe ge- G. disjunctumand G. appalachianumare presently nomes involved. believed to be completely allopatric. At fixed heterozygous loci, enzyme profiles Sterile Triploid Backcrosses. In 1980, Sar- for triploid backcrosses between G. disjunctum vela described the hybridG. dryopterissubsp. x and G. dryopteris,or between G. appalachianum brittonianum(new combinationG. x brittonianumand G. dryopteris,would not necessarily differ made herein), which he regarded as a sterile from the enzyme profiles for tetraploid G. triploid backcross between G. dryopterisand G. dryopteris.The triploids would likely be "un- disjunctum(then regarded as a subspecies). This balanced," i.e., a single "dose" of one genome hybrid was characterized by fronds morpho- vs. two "doses" of the otheralternative genome. logically intermediate between its two pre- However, because of numerous confounding sumed parents, malformed spores, a peculiar possibilities,dosage effectsas visualized through distributionwith plants in eastern and western isozyme electrophoreticmethods are not reli- NorthAmerica but not in the centerof the con- able forverification of the genomic constitution tinent,and a putative triploid count from the of individuals. type locality [count based on Pryer 1981; note Two featuresof the isozyme profilesof plants that the caption to fig. 3 in Pryer (1981) was identifiedhere as G. x brittonianumare relevant mislabelled and should read "Pryer375, Wel- to interpretingthe origin of these triploidback- lington Co., West Garafraxa Township, Bel- crosses. First,mechanisms of inheritanceguar- wood, Ontario - type locality."]. antee that when the triploid exhibits a three- In the contextof the present understanding banded patternfor a given monomericenzyme, of the origin of G. dryopterisas presentedherein, only two of the allozymes could have identical these putative triploids merit furtherinvesti- mobilities to bands found in the allotetraploid gation. There may have been ample opportu- parent, G. dryopteris.Even if the allotetraploid nity for secondary contact between the wide- had four differentallozymes, two at each of its rangingallotetraploid, G. dryopteris,and each of loci, aftermeiosis it could only pass on two to its diploid parents,G. appalachianumand G. dis- the triploidhybrid. One of these two allozymes junctum.This may have resultedin two different in the tetraploidwould correspondin mobility triploid backcrosses,G. appalachianumx dryop- to a band found in its diploid progenitor G. terisand G. disjunctumx dryopteris,which would appalachianum,whereas the other would corre- have the genome combinationsAAD and ADD, spond in mobilityto a band found in the diploid respectively. Were the widespread, putative G. disjunctum.The thirdallozyme in the triploid triploid plants derived from hybridization would have the same mobilityas a band found events between G. disjunctumand G. dryopteris, in one or the other diploid progenitor,i.e., G. or between G. appalachianumand G. dryopteris, disjunctumor G. appalachianum.If each of the or both? Plants of G. x brittonianumfrom the progenitordiploids of allotetraploid G. dryop- type locality in southwesternOntario, as well terishad unique bands forsuch an enzyme, one as plants fromother localities that correspond could preciselydetermine the parentageof trip- morphologicallyto G. x brittonianum,have se- loid backcrosses carryingthree allozymes. For vere meiotic irregularities(Fig. 4). Numerous example, G. x brittonianumplants fromthe type lagging chromosomes were observed at late localityexhibit a three-bandedpattern in SkDH. anaphase I (Fig. 4a), many of which form mi- Two of the threebands are identical in mobility cronuclei at teleophase II (Fig. 4b). These cells to bands found in G. disjunctum,but not found give rise to aneuploid spores that tend to be in G. appalachianum(Fig. 5B). Using this knowl- malformed and abortive (Pryer and Britton edge, we can statewith some certaintythat these 1983). Exact chromosomecounts fromthese hy- triploids resulted fromhybridization between brid plants were difficultto obtain due to the G. dryopterisand G. disjunctum.

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t. ~ ~ ~ ~ ~ ~ .

S.7"

tA ~~~~~~~~~~~~~~A

FIG. 4. Chromosomal preparations from spore mother cells of plants of Gymnocarpiumx brittonianum. x 2000. a. Late anaphase I, showing numerous lagging univalents. b. Telophase II, showing micronuclei formedby fusionof those univalentsthat were not incorporatedinto functionalnuclei. c. Diakinesis, showing a triploid compliment of 120 chromosomes. d. Interpretationof diakinesis preparation shown in c: solid shapes = univalents (27), stippled shapes = bivalents (36), open shapes = trivalents(7), for a total of 120 chromosomes. Voucher for4a and 4b = Ontario: Wellington Co., West GarafraxaTownship, Belwood (type locality),8 Jun 1987, Pryeret al. 902 (CAN). Voucher for4C and 4D = Ontario: PrescottCo., Plantagenet Twp., Bourget,30 May 1989, Pryer& Bristow951 (CAN).

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G. dryoptenssubsp. disjunctum I G. dryoptenssubsp. dryoptens A DD DDDD (2 x) (4x) PGI-2 G. dryoptenssubsp. x bttonianum A. DDD APP DRY DIS DIS SRI APP A. ~~~~~~~~~(3x )

__B~~~~~

G. appalachianum - G. dryoptens G. dsjunctum AA AADD DD SkDH (2x) (4x) (2x)

APP DRY DIS DIS BRI APP G. appalachianumx dryoptens G. dsjunctumx dryoptens FIG. 5. Representativeelectrophoretic banding AAD = x bnttonianum patterns of Gymnocarpiumappalachianum (APP), G. (3 x) ADD dryopteris(DRY), G. disjunctum(DIS), and G x brittoni- B. (3 x) anum(BRI). A. PGI-2. B. SkDH. FIG.6. Presumedrelationships among members of the Gymnocarpiumdryopteris complex. A. Based on Sarvela(1980), Pryer (1981) and Pryeret al. (1983). B. A second observationstrengthens this asser- Basedon presentstudy. tion. For most enzymes, the bands observed in G. dryopterisand G. x brittonianumhad identical mobilities. However, some of the bands ob- lachianumin the G. x brittonianumbackcross, as served in G. x brittonianumhad mobilities that there were no triploid plants fromthe type lo- were never observed in G. dryopteris,but were calitywith three-bandedpatterns in which two identicalto those presentin extantpopulations of the three allozymes were characteristicof G. of G. disjunctum.For example, at PGI-2, the fast appalachianum.Furthermore, there were no iso- band present in G. x brittonianum,which was zymes observed in extant plants of G. appala- absent in G. dryopteris,had the same relative chianumthat had mobilitiesthat were unique to mobilityas one of the two homozygous bands it and thatwere not also presentin G. dryopteris. found in populations of G. disjunctum;the fast Based on preliminarymorphological and geo- band present in G. dryopterishad the same mo- graphical data, however, we believe that ad- bilityas the other homozygous band known in ditional isozyme studies, using a wider sam- G. disjunctum(Fig. 5A). For SkDH, the slow and pling from throughout the range of abortive- intermediatebands in G. x brittonianumwere of spored plants, are likely to yield evidence for the same relative mobility as the two bands backcrosshybrids between G. appalachianumand found in G. dryopteris;however, the fast band G. dryopteris. in G. x brittonianum,which was absent in G. Sterile triploidplants are not restrictedto ar- dryopteris,had a mobilitycorresponding to the eas where the tetraploid overlaps with either fast band in heterozygous G. disjunctum(Fig. diploid. The wide distributionof triploidscould 5B). This fastband was common in extantpop- be explained if they resulted fromseveral sep- ulations of G. disjunctum(0.7338; see Table 2). arate evolutionaryevents involving both long- On the other hand, the slow band observed in lived triploidpopulations thatoriginated when both G. dryopterisand G. x brittonianum,which tetraploid and diploid species were sympatric corresponded in mobilityto the slow band in and survived as rhizomes over long periods of heterozygous G. disjunctum,was observed at time, and more recent hybridizationsby long much lower frequencies in extant populations distance spore dispersal or "remote control" (to of G. disjunctumthan the alternative allozyme use the terminologyof Wagner 1943). There is (0.2662, see Table 2). The presence of bands in some limited evidence of fertilityamong the G. x brittonianumfor PGI-2 and SkDH, with the triploids.The spores produced by these plants same mobilityas bands found in extantG. dis- are of two types:sterile, malformed, black spores junctum,but not observed in G. dryopteris,sup- with very exaggerated perispores and large, portsthe idea that the triploid plants fromthe round spores with extensive reticulate peri- typelocality of G. x brittonianuminvolve a back- spores (Pryerand Britton1983) thatare capable cross between G. dryopterisand G. disjunctum. of germination(Pryer 1981). Ifsuch large spores There was no electrophoreticevidence in our produce gametophytescapable of reproducing sample to suggest the involvement of G. appa- apomictically,this presumably would help to

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TABLE 3. Comparison of sexual membersof the Gymnocarpiumdryopteris complex. Superscriptnumbers and lettersrefer to frondterms depicted in Figure 7.

Characters G appalachianum G disjunctum G dryopteris Condition of bas- Pinnate-pinnatifidor pin- Pinnate-pinnatifidand Pinnatifidand sessile2A al basiscopic natifidand often sessile2A with basal with basal basiscopic pinnules2 of stalked2B,if sessile2A, basiscopic pinnulets6 pinnulets6 equaling proximal with basal basiscopic usually longer than, or slightlylonger pinnael pinnulets6 always though sometimes than adjacent basi- shorterthan adjacent equaling or shorter scopic pinnulets7 basiscopic pinnulets7 than, adjacent basiscopic pinnulets7 Condition of sec- Often stalked8B,if Sessile8A with basal basi- Sessile8A with basal basi- ond pinnae8 sessile8A,with basal scopic pinnules9 equal- scopic pinnules9 basiscopic pinnules9 ing or exceeding about as long as ad- shorterthan adjacent length of adjacent basi- jacent basiscopic basiscopic pinnules10, scopic pinnules1o and pinnules1o and and equaling basal ac- markedlylonger than about equaling basal roscopic pinnulesll, the basal acroscopic acroscopic lattershorter than adja- pinnulesll, the latter pinnulesll, the latter cent acroscopic distinctlyshorter than nearly as long as ad- pinnules12 adjacent acroscopic jacent acroscopic pinnules12 or absent pinnules12 Condition of Sessile with basal basiscop- Sessile with basal basiscop- Sessile with basal basi- third ic pinnules14 shorter ic pinnules14 equaling scopic pinnules14 pinnae13 than adjacent basiscopic adjacent basiscopic about as long as adja- pinnules15 and equaling pinnules15 and longer cent basiscopic basal acroscopic pin- than basal acroscopic pinnules15 and also nules 6, the lattershort- pinnules 6, the latter about as long as basal er than adjacent acros- distinctlyshorter than acroscopic pinnules16, copic pinnules17 adjacent acroscopic the latternearly as pinnules17 long as adjacent acroscopic pinnules17 Margins of ulti- Crenate to entire,with en- Slightly pinnatifidto cre- Crenate to entire,with mate seg- tire,rounded tips nate, often with crenu- entire,rounded tips ments of late, acute tips proximal pinnae Mean exposure 27-29-31 27-29.5-31 34-36.2-39 length (,um) Chromosome 2n = 80 2n = 80 2n = 160 number

explain how otherwise sterile triploid hybrids senting independent evolutionarylineages de- could have such a broad range. The biology of serve species names, but only if morphological these remarkabletriploid plants meritsfurther characters,however subtle,can be found to dif- investigation. ferentiatethem. Based on morphology,the three sexual taxa in the G. dryopteriscomplex can be distinguished using a combination of features TAXONOMIC CONCLUSIONS (see Table 3, as well as key and descriptions We agree with Paris et al. (1989) thata system below). Because hybridizationbetween the tet- of classificationshould reflectas closely as pos- raploid and either diploid would produce ster- sible the phylogeneticrelationships of the taxa ile triploidbackcrosses, and because the genetic under study, and that cryptic species repre- identitybetween the two diploids is so low (av-

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CY)

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C~~~~~~~ 0 C C ~~~~~~~~~~~~~~ C~~C5- 5- CC C CC C E ( ) U- 0 o0 0 0 0~o c on C .= 0- 0 0- 0- L-0 Q O 0 C E 8CZo a) CZ a) o Z CZ CZ Z ) C ~~~~~Cc~~~~~~~~~~~~~) - C C a)C a) ) CZ a)~~~~) C 0 C C CZ C CZ C:C ~~~C ( E~ C C C - C Z 0- --CC CZ -~~~CZ 0- -~~~ -~~'Z-Fz L-~- o -

-oE - C2 0- CC C C 0 C Z 0 C o C 0 CZ -0 L- - CZ Z - C CZCl) ~~~~~S-L 0 ) c~~~~~~~~~~Z D-a)' = 1 0 C C -1 - -~j3 C CZC C C: 1 CC (t) C C 0 C 0 -o CZ 0 )0 .E 0CZ 0 0 CZ 0 0 CZ) C C CZ CZ CZT CZ C a)~~~~~~~

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This content downloaded from 152.3.102.242 on Fri, 15 May 2015 17:24:29 UTC All use subject to JSTOR Terms and Conditions 1993] PRYER & HAUFLER: GYMNOCARPIUM DRYOPTERIS 161 erage I = 0.274), we feel that these taxa should 2. Second pair of pinnae sessile8Aor stalked8B be recognized as distinctspecies. Although iso- (rare); basal basiscopic pinnules of proxi- zymicallydistinct, members of the G. dryopteris mal pinnae sessile2A. complexare morphologicallysimilar species dif- 3. Second pair of pinnae sessile8Awith basal pinnules9&l unequal in length ferentiatedby minor characters. (basiscopic9markedly longer); thirdpair Figure 7 illustratespast and currenthypoth- of pinnae sessile13 with basal eses of relationships among members of the pinnulesl4&16 unequal in length complex. Sarvela (1980) and Pryeret al. (1983) (basiscopic14 longer); blades large (8-24 hypothesized that G. disjunctumgave rise to G. cm long) ...... 2. G. disjunctum dryopteristhrough autopolyploidy, and that G. 3. Second pair of pinnae rarely stalked8B; x brittonianumwas a sterile triploid backcross when sessile8A,with basal pinnules9& between them (Fig. 6A). As currentlydefined, ? equal in length (basiscopic9 however, the G. dryopteriscomplex is thought acroscopic 1); third pair of pinnae to include two divergent diploids, G. appala- sessile13with basal pinnulesl4&16 ? equal in length acroscopic16); chianumand G. disjunctum,a fertile allotetra- (basiscopic14 blades small (3-14 cm long). of two ploid, G. dryopteris,and an assemblage 4. Sessile basal basiscopic pinnules of different,abortive-spored triploid backcrosses, proximal pinnae2Awith basal basi- G. appalachianumx dryopterisand G. disjunctum scopic pinnulets6 ? equal in length x dryopteris(Fig. 6B). The name G. x brittonian- to adjacent basiscopic pinnulets7; um is applied here to the latterhybrid combi- second pinnae almostalways sessile8A nation (see below). with basal pinnules9&ll ? equal in The following key will permitidentification lengthto adjacentpinnulesl0&12; third of mostmature specimens, especially those pos- pinnae sessile13 with basal pin- sessing mature,fertile fronds (with sori). As is nulesl4&16 ? equal in length to adjacent pinnulesl5&17; spores 34-39 ,um truefor other members of the Dryopteridaceae, long . 3. G. dryopteris best characters for distinguishing species the 4. Sessile basal basiscopic pinnules of include features of the basal pinnules of the proximal pinnae2Awith basal basi- proximal pinnae. The sterile hybrids are mor- scopic pinnulets6shorter than adja- phological intermediatesbetween their paren- cent basiscopic pinnulets7; second tal species and are very difficultto identifyus- pinnae sessile8A with basal ing strictlyvegetative features. The presence of pinnules9&ll shorter than adjacent mostlyabortive spores is the mostreliable char- pinnulesl &12, or second pinnae acter for their identification.In order to facili- stalked8B(rare); thirdpinnae sessile13 tate identification,important terms pertaining with basal pinnulesl4&16 shorterthan adjacent pinnulesl5&17;spores 27-31 to the frondare schematicallydepicted in Fig- long ...... 1. G. appalachianum 7 the Am ure and are cross-referencedthroughout 1. Spores mostly malformed,irregular in shape, taxonomic treatment with small superscript oftenwith larger,round spores present .... numbers and letters...... 4. G. x brittonianum [or G. appalachianumx dryopteris;see comments KEY TO NORTH AMERICAN MEMBERS OF THE under G. x brittonianum] GYMNOCARPIUM DRYOPTERIS COMPLEX (N.B. Superscriptnumbers and lettersrefer to frondterms depicted in Fig. 7) 1. Gymnocarpium appalachianum Pryer & 1. Sporesreniform and uniformin size and shape. Haufler, sp. nov. (Fig. 8).-TYPE: U.S.A., 2. Second pair of pinnae and basal basiscopic Virginia,Page Co., Shenandoah Natl. Park, pinnulesof proximal pinnae stalked81&2B Stony Man Mt., growing among green- ...... 1. G. appalachianum stone outcrops on NW-facing slope about

FIG. 7. Schematic illustrationexplaining pertinentfrond terminology used in the taxonomictreatment of Gymnocarpium.Arrows indicate stalked pinnules and pinna. The numbersand lettersrepresenting frond terms are cross-referencedas small superscriptsthroughout the taxonomic treatment.

This content downloaded from 152.3.102.242 on Fri, 15 May 2015 17:24:29 UTC All use subject to JSTOR Terms and Conditions FIG. 8. Gymnocarpium appalachianum. A. Frond at right: i) second pinnae are stalkedIB, and ii) proximal pinnael have stalked basal basiscopic pinnuleSIB. Fronld at left: i) second pinnae are sessile8A with basal basiscopic pinnules9 shorter than adjacent basiscopic pinnules'? and equaling basal acroscopic pinnules"l, the latter shorter than adjacent acroscopic pinnules"2, and ii) proximal pinnael have sessile basal basiscopic pinnuleSIA with basal basiscopic pinnuletS6 shorter than adjacent basiscopic pinnulets7 . Based on holotype: Pryeret al. 948 (US). B. Morphological "extreme" in plants of G. appalachianum:i) second pinnae are stalked,B, and ii) proximal pinnael have stalked basal basiscopic and acroscopic pinnuleSIB64B. Based on Windham 81-31 (TTT). Bar li-nes = 1 cm.

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30 yds fromsummit, 6 Jul1988, Pryer, Klein, Pinnae basales pinnulis basalibus basiscopicis and Morse 948 (holotype: US!, drawing in instructae,plerumque stipitatisaut si sessiles Fig. 8 of this paper; isotypes: CAN!, CM!, subpinnulis basiscopicis brevioribusquam sub- GH!, US!, VPI!, WVA!).-PARATYPES: North pinnulis basalibus basiscopicis secundis. Pinnae Carolina, Ashe Co., BluffMt., near summit basales secundae plerumque stipitataeaut si ses- on N slope, 9 Jul 1967, Bozemanet al. 10678 siles pinnulis basalibus basiscopicis brevioribus (A, CM, DAO, FARM, LYN, MIN, VPI, quam pinnulis basalibus basiscopicis secundis WILLI, WVA). Ohio, Wayne Co., 25 Jun et aequantibus pinnulas basales acroscopicas, 1908, Hopkins s.n. (OS-4 sheets). pinnulae basales acroscopicae breviores quam Pennsylvania, Bedford Co., 23/4mi SSW of pinnulis basalibus acroscopicis secundis. Spo- Hyndman, 16 Jun 1948, Berkheimer9803 rae reniformes,27-31 Am longae, testaceae. (CM); 5 mi S of Hyndman, 5 May 1951, Chromosomatumnumerus 2n = 80. Buker s.n. (CM); Wolfsburg, Raystown Rhizomes 0.5-1.5 mm in diameter,with scales Branchof JuniataRiver, 3 Jul 1988, Pryeret 1.5-3.0 mm long. Fertilefronds usually 10-32 cm al. 940 (CAN). Virginia, Bath Co., Big Al- tall. Stipes 6-20 cm long with scales up to 6 mm leghanyMt., about 12 mi NE ofRimel, WVA, long. Blades 4-12 cm long, bipinnate-pinnatifid 6 Jun 1941, Henry2918 (VPI-2 sheets); on or tripinnate-pinnatifid.Pinnae with entire, NW side of Warm Springs Mt., 0.5 mi SW rounded tips. Proximal pinnael 3-10 cm long, of Sandy Gap, 29 Jun 1975, Stevens10886 with basal basiscopic pinnules either stalked2B (FARM, VPI); Greene Co., Bush Mt., 0.9 mi and pinnate-pinnatifidor pinnatifid,or sessile2A S of Bootens Gap on shady N-facing out- and pinnate-pinnatifidor pinnatifid,if the lat- crop, 25 Jun 1972, Wieboldtet al. 1033 ter, basal basiscopic pinnulets6always shorter (FARM); Highland Co., W side of Lantz Mt., than adjacent basiscopic pinnulets7;second bas- Rt. 642, 25 Jun1961, Freer 2598 (GH, LYN- al basiscopic pinnules3sometimes stalked, if ses- 2 sheets,US, VPI, WILLI); Lantz Mt., along sile, with basal basiscopic pinnuletsshorter than roadside on W side of summit,4 Jul 1988, adjacent basiscopic pinnulets; basal acroscopic Pryeret al. 942 (CAN); Madison/Page cos., pinnules sometimes stalked4B,if sessile4A,with Hawksbill Mt., westerlycliff top trail near basal basiscopic pinnulets shorterthan adjacent summit,6 Jul 1988, Pryeret al. 945 (CAN); basiscopic pinnulets. Second pinnae usually Nelson Co., Three Ridges, 1/4mi due N of stalked8B,if sessile8A, with basal basiscopic summit on rocky N-facing slope, 22 Aug pinnules9 shorter than adjacent basiscopic 1976, Wieboldt2645 (WILLI); Page Co., Bush pinnules10, and equaling basal acroscopic Mt., 0.9 mi S of Bootens Gap along Skyline pinnules11,the lattershorter than adjacent ac- Drive, 5 Jul 1988, Pryeret al. 944 (CAN); roscopic pinnules12; pinnules oftenwith entire, Hawksbill Mt., 23 Jun 1947, BrittonB215 rounded tips. Third pinnae sometimesstalked, (OAC); Hawksbill Mt.,along side trailfrom if sessile13, with basal basiscopic pinnules14 the W, 15 Jul1945, Walker 3682 (US); Neigh- shorterthan adjacent basiscopic pinnules15, and bor Mt.,by ridgecresttrail 1 mi E ofsummit, equaling or shorter than basal acroscopic 24 Aug 1975,Stevens 11563 (VPI); StonyMan pinnules6, the latter equaling or shorterthan Mt., SE of Luray, 24 Aug 1927, Wherry& adjacent acroscopic pinnules7. Ultimate seg- Pennell s.n. (VPI), Windham81-31 (UT); ments of the lower pinnae oblong, entire to Rockingham Co., Tomahawk Mt., near crenate,with entire,rounded tips. Spores reni- Rawley Springs, on rocky N-facing slope, form,27-31 Am long. Diploid 2n = 80 (Fig. 2). 3 Jun1972, Stevens 5006 (VPI); Slate Springs Mt., on N face near Flagpole Knob, 13 Jul Distribution.Restricted to the southern Ap- 1969,Stevens 1203 (FARM, VPI); WarrenCo., palachian region of the United States (Fig. 9): Marshall Mt., N end near summit, 3 Jun North Carolina, Ohio, Pennsylvania, Virginia, 1953, Hunnewells.n. (VPI). West Virginia, and West Virginia. Hampshire Co., Ice Mt., 11 Jun 1987,Morse Habitat. Maple-birch-hemlock(Acer-Betula- 9486 (CAN, possibly mixed collection); 6 Tsuga)woods on mountain slopes and summits, Jun 1988, Morse 9578, site 8 (CAN); 7 Jul on moistsandstone talus and scree, talus slopes 1988, Pryeret al. 949 (CAN). with cold air seepage (algific). 200-1400 m.

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0 1000

FIG. 9. North American distributionsof Gymnocarpiumappalachianum (triangles) and G. disjunctum(dots); the type localities of these species in Virginia and Alaska, respectively,are indicated by arrows.

Gymnocarpiumx heterosporumW. H. Wagner 2. GYMNOCARPIUMDISJUNCTUM (Rupr.) Ching, Jr.,a putative triploidhybrid known only from Acta Phytotax.Sin. 10: 304. 1965 (Fig. 10).- one locality in Pennsylvania that is now extir- Polypodium dryopterisL. var. disjunctum pated, is thought to have been the result of a Rupr., Distr. Crypt. Vasc. Ross. 52. 1845 unique hybridization event between G. appa- (',yPolypodium disjunctum').-Polypodium dis- lachianumand the glandular,limestone oak fern junctum(Rupr.) Schur, Oesterr. Bot. Z. 8: G. robertianum(Hoffm.) Newm. (Pryer 1992). 193. 1858. -Phegopterisdryopteris (L.) Fee var.

FIG. 10. Gymnocarpiumdisjunctum. A. Frondshowing i) sessile second pinnae8A withbasal basiscopicpinnules9 equaling adjacent basiscopic pinnules10and markedly longer than basal acroscopic pinnules11,the latter distinctlyshorter than adjacent acroscopic pinnules12, and ii) proximal pinnael with basal basiscopic pinnules that are pinnate-pinnatifidand sessile2A with basal basiscopic pinnulets6 equaling adjacent basiscopic pinnulets7. B-C: Morphological variation in plants of G. disjunctum. B. Proximal pinnael with sessile basal basiscopic pinnules2Awith basal basiscopic pinnulets6longer than adjacent basiscopic pinnulets7. C. i) Prox- imal pinnael with sessile basal basiscopic pinnules2Awith basal basiscopic pinnulets6shorter than adjacent basiscopic pinnulets7,and ii) second pinnae sessile8A with basal acroscopic pinnules11absent. Based on Alverson s.n. (CAN 531499), Calder & Savile 12265 (DAO), Ulmer614 (DUKE), Joness.n. (US 855738). Bar lines = 1 cm.

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disjuncta(Rupr.) Trel. in Harriman, Harri- jacent acroscopic pinnules'2; pinnule tips often man Alaska Expedition 5: 382. 1904.- crenulate,obtuse. Third pinnae usually sessile13 Dryopterislinnaeana C. Chr. var. disjuncta with basal basiscopic pinnules"4longer than or (Rupr.) Fomin in N. Busch,Fl. Sibir. Orient. equaling adjacent basiscopic pinnules'5and also Extr.5: 79. 1930.-Gymnocarpiumdryopteris longer than basal acroscopic pinnules6, the lat- (L.) Newman var. disjunctum(Rupr.) Ching, ter shorterthan adjacent acroscopic pinnules"7. Contr. Biol. Lab. Chin. Assoc. Advancem. Ultimatesegments of the lower pinnae oblong, Sci., Sect. Bot. 9: 41. 1933.-Dryopterisdis- crenate to slightly pinnatifid,with crenulate, juncta (Rupr.) C. Morton, Rhodora 43: 217. acute tips.Spores reniform,27-31 ,m long. Dip- 1941.-Carpogymniadisjuncta (Rupr.) A. Love loid 2n = 80 (V. Sorsa 1966; Taylorand Mulligan & D. Love, Taxon 16: 191. 1967.-Gymno- 1968; Wagner 1966). carpiumdryopteris subsp. disjunctum(Rupr.) Distribution.Sakhalin Island, southernKam- Sarvela, Ann. Bot. Fenn. 15: 103. 1978.- chatka; western coast of North America from TYPE: U.S.A., Alaska, Sitka, "Sitcha. Poly- Alaska to Oregon and east to Montana and podiumcalcareum Sm. Bongard Voy. Sitch. southwesternAlberta (Fig. 9). Dr. Mertens" [lectotype here designated: Habitat. Shaded, rocky slopes and ravines, LE!, photo CAN!; isolectotype:"Sitcha. Dr. mixed coniferouswoods, moiststream and creek Mertens" LE!, photo CAN!; syntype:"Sit- banks. 0-2400 m. cha. Specimina minuta sterilia. Polypodii calcarei.Diff. a P. Dryopteri.Stipite paleacea. 3. GYMNOCARPIUM DRYOPTERIS (L.) Newman, Dr. Fischer.1840" (foursmall sterilefronds) Phytologist4: 371. 1851 (Fig. 11A).-Poly- LE!]. podiumdryopteris L., Sp. P1. 2: 1093. 1753.- Rhizomes 1-3 mm in diameter,with scales 2- Polypodiumpulchellum (Salisb., Prodr. Stirp. 4 mm long. Fertilefronds usually 20-68 cm tall. Chap. Allerton404.1796, nom. illeg., ICBN Stipes 12-44 cm long with scales up to 6 mm Art. 63.1.-Polystichum dryopteris (L.) Roth, long. Blades 8-24 cm long, tripinnate-pinnati- Arch. Bot. (Leipzig) 2: 106. 1799.-Nephro- fid. Pinnae with acuminate tips. Proximal dium dryopteris(L.) Michx., Fl. Bor.-Amer. pinnael 5-18 cm long, with basal basiscopic pin- 2: 270. 1803.-Lastrea dryopteris(L.) Bory, nules sessile2A,pinnate-pinnatifid (with basal Dict. Class. Hist. Nat. 9: 233. 1826.-Aspidi- pinnulets,and sometimessecond and thirdbas- um dryopteris(L.) Baumg., Enum. Stirp. al pinnulets, not joined), and with basal basi- Transsilv. 4: 29. 1846.-Polypodium dryop- scopic pinnulets6 usually longer (sometimes terisvar. glabrumNeilr., Fl. Wien 6. 1846 equaling or shorter) than adjacent basiscopic ('av').-Phegopterisdryopteris (L.) F1e, Mem. pinnulets7; second basal basiscopic pinnules foug. 5. Gen. Filic. 243. 1852.-Polypodium sessile3with basal basiscopic pinnulets usually dryopterisvar. genuinumLedeb., Fl. Ross. 4: longer than or equaling adjacent basiscopic pin- 509. 1853 ('a'), non rite publ., ICBN Art. nulets; basal acroscopic pinnules sessile4Awith 24.3.-Polypodium triangulareDulac, Fl. basal basiscopic pinnulets usually longer than Hautes-Pyrenees31. 1867,nom. illeg.,ICBN or equaling adjacent basiscopic pinnulets. Sec- Art. 63.1, non L. (1774).-Phegopteristrian- ond pinnae usually sessile8Awith basal basi- gularisSt. Lag. in Cariot, Etude Fl., 8th ed., scopic pinnules9 longer than or equaling adja- 2: 964. 1889. nom. illeg., ICBN Art. 63.1.- centbasiscopic pinnules10,and markedlylonger Dryopterislinnaeana C. Chr.,Index Filic. 275. than basal acroscopic pinnules11,the latterab- 1905.-Dryopteris pulchella Hayek, Fl. sent (uncommon) or distinctlyshorter than ad- Steiermark1: 39. 1908, nom. illeg., ICBN

FIG. 1 1. Gymnocarpiumdryopteris and G. x brittonianum.A. G. dryopteris:frond has i) sessile second pinnae8A with basal basiscopic pinnules9equaling adjacentbasiscopic pinnules10and equaling basal acroscopicpinnules11, the latter almost as long as adjacent acroscopic pinnules12, and ii) proximal pinnael with basal basiscopic pinnules thatare pinnatifidand sessile2-with basal basiscopic pinnulets6equaling adjacentbasiscopic pinnulets7. Based on Pryer& Klein920 (CAN), Clausen& Trapido2830 (MIN), Deane s.n. (MIN 486807). B. G. x brittonianum: i) sessile second pinnae8A with basal basiscopic pinnules9 equaling adjacent basiscopic pinnules10and conspicuously longer than basal acroscopic pinnules11,the lattershorter than adjacent acroscopic pinnules12,and

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ii) proximal pinnael with basal basiscopic pinnules that are pinnate-pinnatifidand sessile2A Mature, fertile frondsof G. x brittonianumhave malformed,as well as largeand roundspores. Based on Dickinson& Kan 363 (CAN). Bar lines = 1 cm.

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Art. 63.1.-Dryopteris triangularisHerter, cent basiscopic pinnules'0, and about equaling Bull. Herb. Boissierser. 2, 8: 797. 1908,nom. basal acroscopic pinnules", the latterequaling illeg., ICBN Art.63.1 -Dryopterisdryopteris or slightly shorter than adjacent acroscopic (L.) Christ,Bull. Acad. Int. Geogr. Bot. 201: pinnules'2; pinnule tips often entire,rounded. 151. 1909, non ritepubl., ICBN Art. 23.4.- Third pinnae sessile'3 with basal basiscopic Thelypterisdryopteris (L.) Sloss. in Rydb.,Fl. pinnules'4 as long as adjacent basiscopic RockyMts. 1:1044. 1917.-Dryopteris pumila pinnulesl5 and equaling basal acroscopic Krecz. in Grossheim,Fl. Kavk., 2nd ed., 1: pinnules6, the latterequaling or slightlyshort- 19. 1939, nom. illeg., ICBN Art.63.1.-Cur- er than adjacent acroscopic pinnules"7.Ultimate raniadryopteris (L.) Wherry,Bartonia 21: 15. segmentsof the lower pinnae oblong, entireto 1942.-Carpogymniadryopteris (L.) A. Love crenate,with entire,rounded tips. Spores reni- & D. Love, Univ. Colorado Stud., Ser. Biol. form,34-39 ,m long. Tetraploid,2n = 160 (Brit- 24: 8. 1966.-TYPE: "Filix querna Bauh. Filix ton 1953; Manton 1950; Pryer 1981; V. Sorsa arborea Tragi. Eichelfarn.Baumfarn. In Lu- 1958; Vida 1963; Wagner 1963). satia, Bohemia, Dania." Burser specimen Distribution. Circumboreal (Fig. 12). XX.32(lectotype, designated in McNeill and Throughout northern and central Europe; Pryer 1985: UPS; microfiche!,photo in fig. northernAsia to China and Japan; Greenland; 1 of McNeill and Pryer 1985). temperate North America, Alaska to New- Polypodiumdryopteris var. erectumG. Laws., Ed- foundland, southwards to Arizona and Penn- inburghNew Philos. J.19: 109. 1864. -Phe- sylvania. gopterisdryopteris f. erecta(G. Laws.) Broun, Habitat. Commonly found in cool, conifer- Index N. Amer. Ferns 134. 1938.-Dryop- ous and mixed woods, and at base of shale talus terisdisjuncta (Rupr.) C. Morton f. erecta(G. slopes. 0-3000 m. Laws.) Roland, Proc. Nova Scotian Inst. Sci. Two syntypesfor the name Polypodiumdryop- 20: 92. 1941.-TYPE: Canada, Ontario, terisvar. erectumwere located at E. One was Kingston, Collins Bay, "Collins's Bay. Po- labeled "Polypodiumdryopteris a" and the other lypodiumdryopteris var. rigidium,"18 Jun "Polypodiumdryopteris var. rigidium."The first 1861, G.W. Lawsons.n. (lectotype here des- specimen is most likely what Lawson referred ignated: E, photo CAN!; see comment be- to as the "normal form,"and the second what low). he had in mind forvar. erectum.His change in Phegopterisdryopteris f. interruptaJewell, Fern varietal epithet was possibly due to his real- Bull. 16: 86. 1908.-TYPE: none located; not ization that Hooker had already published a P. at A, GH, or MAINE. dryopterisvar. rigidiumin 1832. Other species included in Gymnocarpiumin Rhizomes 0.5-1.5 mm in diameter,with scales NorthAmerica are Gymnocarpiumrobertianum and 1-4 mm long. Fertile fronds usually 12-42 cm G. jessoense(Koidz.) Koidz. subsp. parvulumSar- tall. Stipes 9-28 cm long with scales up to 6 mm vela, two morphologically distinct tetraploids long. Blades 3-14 cm long, bipinnate-pinnati- (Sarvela et al. 1981) that differmost conspicu- fid.Pinnae with entire,rounded tips. Proximal ously from those species in the G. dryopteris pinnael 2-12 cm long, with basal basiscopic pin- complex by having an indument of minute (0.1 nules usually sessile2A,pinnatifid (with basal mm) glands on their leaves. Pryer (1990) pre- pinnulets confluentwith adjacent pinnulets) or sentsa tabularcomparison of the morphological rarely pinnate-pinnatifid,and with basal basi- and ecological attributesof G. dryopteriswith scopic pinnulets6often equaling or longer than those of G. robertianumand G. jessoensesubsp. adjacent basiscopic pinnulets7;second basal ba- parvulum.Hybrids between the glabrous spe- siscopic pinnules sessile3 with basal basiscopic cies, and also between the glabrous and glan- pinnulets equaling or longer than adjacent basi- dular species, have played a significantrole in scopic pinnulets; basal acroscopic pinnules obscuring species boundaries in Gymnocarpium. sessile4Awith basal basiscopic pinnulets longer than or equaling adjacent basiscopic pinnulets. 4. Gymnocarpium x brittonianum (Sarvela) Second pinnae usually sessile8Awith basal basi- Pryer & Haufler, comb. et stat. nov. (Fig. scopic pinnules9 longer than or equaling adja- 11B).-Gymnocarpiumdryopteris subsp. x

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FIG. 12. North American distributionof Gymnocarpiumdryopteris.

brittonianumSarvela, Ann. Bot.Fenn. 17: 292. jacent basiscopic pinnulets; basal acroscopic 1980.-TYPE: Canada, Ontario, Wellington pinnules sessile4Awith basal basiscopic pinnu- Co., in moist cedar and yellow birch glade, lets usually equaling adjacent basiscopic pin- 21 July1978, D.M. Brittonand A. Anderson nulets. Second pinnae sessile8Awith basal basi- 6860 (holotype: H!; isotype: OAC!). scopic pinnules9 equaling adjacent basiscopic pinnules10,and usually conspicuously longer Rhizomes 1-2 mm in diameter,with scales 1- than basal acroscopic pinnules11,the latterdis- 4 mm long. Fertilefronds usually 14-60 cm tall. tinctly shorter than adjacent acroscopic Stipes 10-40 cm long with scales up to 6 mm pinnules12; pinnule tips oftencrenulate, obtuse. long. Blades 4-20 cm long, usually tripinnate- Third pinnae usually sessile13 with basal basi- pinnatifid, sometimes bipinnate-pinnatifid. scopic pinnules14 equaling adjacent basiscopic Pinnae with acuminate tips. Proximal pinnael pinnules15 and sometimes slightlylonger than 3-16 cm long, with basal basiscopic pinnules basal acroscopic pinnules16,the latter usually sessile2A,pinnate-pinnatifid or sometimes pin- slightly shorter than adjacent acroscopic natifid, and with basal basiscopic pinnulets6 pinnules7. Ultimatesegments of the lower pin- usually equaling adjacent basiscopic pinnulets7; nae of large blades oblong, crenate to slightly second basal basiscopic pinnules sessile3 with pinnatifid,with crenulate, acute tips; those of basal basiscopic pinnulets usually equaling ad- small blades oblong, entire to crenate,with en-

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SO.~~~~~~~~~~~~~~~~~~~~~~o

o~~~~~~~Cm 100 q 0 3

0 . I

FIG. 13. North American distributionof plants with malformed spores in the Gymnocarpiumdryopteris complex. The type locality of G. x brittonianumis indicated by an arrow in Ontario.

tire, rounded tips. Spores of two types: mal- disjunctumwas the diploid involved in these formed, black spores with very exaggerated triploidbackcrosses. We cannot rule out a con- perispores and large, round spores with exten- tributionfrom G. appalachianumin other parts sive reticulate perispores. Triploid, 2n = 120 of the range of triploidplants and, in fact,con- (Fig. 4; Pryer 1981). sider it likely. However, only one name can Distribution.Western North America: Alaska correctlyapply to any particular hybrid for- to Colorado; eastern North America: Atlantic mula; by implication,two differentcrosses can- Provinces to Ontario and southward. Precise not bear the same name (ICBN Article H.4.1). boundaries of range not well-understood (Fig. We thereforeapply the name G. x brittonianum 13, in part). to the G. disjunctumx dryopteriscombination Habitat. Rich, moist to wet mixed woods, (Fig. 6B). According to ICBN Article H.5.1, the wooded streambanksand creekbeds, montane appropriaterank of a hybridis that of the pos- forests.0-2400 m. tulated parent taxa. Consequently, the rank of The statementof putative parentage in Sar- this sterile hybrid is elevated here to that of vela's (1980) diagnosis does not play a role in species. The hybrid G. appalachianumx dryop- determiningthe application of the name here. terisis not assigned a binomial name, pending Based on our morphological and electropho- firmidentification of hybridswith this parent- reticobservations of materialfrom the type lo- age. cality of G. x brittonianum,it is clear that G. The frondsof the type specimen of G. x brit-

This content downloaded from 152.3.102.242 on Fri, 15 May 2015 17:24:29 UTC All use subject to JSTOR Terms and Conditions 1993] PRYER & HAUFLER: GYMNOCARPIUM DRYOPTERIS 171 tonianumand of othercollections examined from trations,Barbara Kobolak forcomputerizing the spec- the type locality stronglyresemble those of G. imen label data and preparinga complete list of spec- and Lewis Johnsonfor disjunctum(cf. Fig. 1lB), having blades that are imens verifiedin this study, preparingthe distributionmaps. Financial assistance tripinnateand usually with the following large, provided by grants awarded by the Research Advi- a) proximal morphological characteristics: soryCouncil of the Canadian Museum of Nature and pinnael with basal basiscopic pinnules that are the Pteridological Section of the Botanical Society of sessile2A and pinnate-pinnatifid, b) second America (Edgar T. WherryAward) are gratefullyac- pinnae that are sessile8Awith basal basiscopic knowledged. pinnules9that are usually conspicuously longer CITED than basal acroscopic pinnules11,the latterbe- LITERATURE ing shorter than the adjacent acroscopic ALVERSON,E. R. 1988. Biosystematicsof NorthAmer- pinnules12 and oftenwith crenulate,acute tips, ican parsley ferns,Cryptogramma (Adiantaceae). and c) the ultimate segments of the proximal American Journal of Botany 75: 136-137. Ab- pinnae of large blades tend toward crenate to stract. slightlypinnatifid. BRITTON,D. M. 1953. Chromosome studies on ferns. Hybrid plants that conformto this morpho- American Journalof Botany 40: 575-583. A. and D. E. SOLTIS. 1987. Electrophoretic logical description occur not only at the type BRYAN,F. evidence of allopolyploidy in the fernPolypodium locality,but throughouta substantialpart of the virginianum.Systematic Botany 12: 553-561. for abortive-spored North American range CRAWFORD,D. J. 1983. Phylogenetic and systematic plants in the G. dryopteriscomplex (Fig. 13). A inferencefrom electrophoretic studies. Pp. 257- detailed investigation of the morphology and 287 in Isozymesin plantgenetics and breeding,Part distributionrange of G. appalachianumx dryop- A, eds. S. D. Tanksley and T. J. Orton. Amster- teriswill be the subject of futurestudy. dam: Elsevier. . 1985. Electrophoreticdata and plant speci- ACKNOWLEDGMENTS.We are gratefulto George W. ation. SystematicBotany 10: 405-416. Argus, Fransois Lutzoni, Lucinda McDade, John and E. B. SMITH. 1984. Allozyme divergence McNeill, and Michael Windham forcritical comments and intraspecificvariation in Coreopsisgrandiflora on various drafts of the manuscript. John McNeill (Compositae). SystematicBotany 9: 219-225. provided additional assistance with nomenclatural DAVIS,M. B. 1983. Quaternaryhistory of deciduous difficulties.Alan R. Smith and Daniel J. Crawford forestsof easternNorth America and Europe. An- gave valuable comments in review. The Latin diag- nals of the Missouri Botanical Garden 70: 550- nosis for G. appalachianumwas kindly translatedby 563. William L. Culberson. JannisKlein and LarryMorse FERRIS,S. D. 1984. Tetraploidyand the evolution of were energeticand cheerfulfield companions and we the catostomid fishes. Pp. 55-93 in Evolutionary thank them for their assistance and encouragement. geneticsof fishes, ed. B. J.Turner. New York: Ple- LarryMorse is especially thanked for suggesting Ice num. Mountain and making several trips there to collect GASTONY,G. J. 1988. The Pellaea glabella complex: materialfor us; this site was the key to our discovery. Electrophoreticevidence for the derivations of We thank JianweiLi forhis great care in successfully the agamosporous taxa and a revised taxonomy. obtaining chromosome preparations at diakinesis of American Fern Journal78: 44-67. plants of G. x brittonianum.We are very gratefulto GOTTLIEB,L. D. 1981. Electrophoreticevidence and Edward Alverson, Alan Batten, David Barrington, plant populations. Progressin Phytochemistry7: Valerie Bristow,Donald Britton,Irwin Brodo, Elroy 1-46. Burnett,Adolf Ceska, William Crins, Allison Cusick, HAUFLER,C. H. 1985a. Enzymevariability and modes JohnKunsman, Loyal Mehrhoff,Mary Muller, David of evolution in Bommeria.Systematic Botany 10: Murray, David Snyder, Carl Taylor, Alan Weakley, 92-104. Michael Windham, and Anna Zeigler for helping us . 1985b. Pteridophyteevolutionary biology: to obtainadditional population samples.We also thank The electrophoreticapproach. Proceedings of the Daniel F. Brunton for his helpful comments on the Royal Society of Edinburgh 86B: 315-323. key and descriptions.Laboratory assistance was very 1987. Electrophoresisis modifyingour con- ably provided by Michael Windham, Jannis Klein, cepts of evolution in homosporous pterido- and Eric Rabe. We are gratefulto the curatorsof the phytes.American Journalof Botany 74: 953-966. various institutions(see Materials and Methods) for , M. D. WINDHAM, D. M. BRITTON, and S. J. the specimen loans and to the U.S. National Park ROBINSON. 1985. Triploidy and its evolutionary Service for providing a collecting permit for Shen- significance in Cystopterisprotrusa. Canadian andoah National Park.Special thanksare due to Susan Journalof Botany 63: 1855-1863. Laurie-Bourque for so skillfullypreparing the illus- 1 , and T. A. RANKER.1990. Biosystemat-

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ic analysis of the Cystopteristennesseensis (Dryop- SARVELA,J. 1978. A synopsis of the ferngenus Gym- teridaceae) complex. Annals of the Missouri Bo- nocarpium.Annales Botanici Fennici 15: 101-106. tanical Garden 77: 314-329. . 1980. Gymnocarpiumhybrids from Canada HULTEN, E. and M. FRIES. 1986. Atlas of NorthEuro- and Alaska. Annales Botanici Fennici 17: 292- pean vascularplants north of theTropic of Cancer.I. 295. Konigstein: Koeltz ScientificBooks. , D. M. BRITTON,and K. M. PRYER. 1981. Stud- JACKSON, R. C. 1982. Polyploidy and diploidy: New ies on the Gymnocarprumrobertianum complex in perspectiveson chromosomepairing and its evo- North America. Rhodora 83: 421-431. lutionaryimplications. American Journalof Bot- SOLTIS, D. E. and L. H. RIESEBERG. 1986. Autopoly- any 69: 1512-1523. ploidy in Tolmiea menziesii(Saxifragaceae): Ge- and J. CASEY. 1982. Cytogeneticanalyses of netic insights from enzyme electrophoresis. autopolyploids: Models and methods for trip- American Journalof Botany 73: 310-318. loids to octoploids. American Journalof Botany and P. S. SOLTIS. 1989. Polyploidy, breeding 69: 487-501. systems,and genetic differentiationin homospo- JALAS, J. and J. SUOMINEN, eds. 1972. Atlas Florae rous pteridophytes.Pp. 241-258 in Isozymesin Europaeae;Distribution of vascularplants in Europe. plant biology,eds. D. E. Soltis and P. S. Soltis. 1. Pteridophyta(Psilotaceae to Azollaceae). Helsinki: Portland: Dioscorides Press. The Committeefor Mapping the Flora of Europe , C. H. HAUFLER,D. C. DARROW, and G. J. and Societas Biologica Fennica Vanamo. GASTONY. 1983. Starch gel electrophoresis of KATO, M. and K. IWATSUKI. 1983. Phytogeographic ferns:A compilation of grindingbuffers, gel and relationships of pteridophytesbetween temper- electrode buffers,and staining schedules. Amer- ate North America and Japan.Annals of the Mis- ican Fern Journal73: 9-27. souri Botanical Garden 70: 724-733. SORSA,P. 1980. Spore morphologyof the ferngenus MANTON, I. 1950. Problemsof cytologyand evolution Gymnocarpiumand its relations to the taxonomy. in thePteridophyta. Cambridge: Cambridge Univ. Annales Botanici Fennici 17: 86-90. Press. SORSA, V. 1958. Chromosome studies on Finnish McNEILL, J. and K. M. PRYER. 1985. The status and Pteridophyta.I. Hereditas 44: 541-546. typificationof Phegopteris and Gymnocarpium.Tax- 1966. A diploid Gymnocarpiumdryopteris (L.) on 34: 136-143. Newman in Alaska. Nature 210: 1375. PARIS, C. A., F. S. WAGNER, and W. H. WAGNER, JR. TAYLOR, R. L. and G. A. MULLIGAN. 1968. Floraof the 1989. Crypticspecies, species delimitation,and Queen CharlotteIslands. Part 2. Cytologicalaspects taxonomic practice in the homosporous ferns. of thevascular plants. Ottawa: Departmentof Ag- American Fern Journal79: 46-54. ricultureMonograph 4. PRYER, K. M. 1981. Systematicstudies in the genus TRYON,R. M. and A. F. TRYON. 1982. Ferns and allied GymnocarpiumNewm. in North America. M.Sc. plants,with special referenceto tropicalAmerica. thesis, Universityof Guelph, Guelph, Ontario. New York: Springer-Verlag. . 1990. The limestone oak fern: New to the VIDA, G. 1963. A Dryopterisnemzetseg (sensu lato) floraof Manitoba. Blue Jay48: 192-195. szisztematikaja.Botanikai K6zlemenyek 50: 125- 1992. The status of Gymnocarpiumheterospo- 133. rumand G. robertianumin Pennsylvania. Ameri- WAGNER, W. H., JR. 1943. Hybridization by remote can Fern Journal82: 34-39. control.American Fern Journal33: 71-73. and D. M. BRITTON. 1983. Spore studies in 1963. A biosystematicsurvey of United States the genus Gymnocarpium.Canadian Journal of ferns-Preliminary abstract. American Fern Botany 61: 377-388. Journal53: 1-16. and J.McNEILL. 1983. A numerical 1966. New data on NorthAmerican oak ferns, analysis of chromatographicprofiles in North Gymocarpium.Rhodora 68: 121-138. American taxa of the fern genus Gymnocarpium. WERTH, C. R. 1985. Implementing an isozyme lab- Canadian Journalof Botany 61: 2592-2602. oratoryat a field station.Virginia Journalof Sci- RANKER, T. A. 1988. The origin of the tetraploid ence 36: 53-76. Hemionitispinnatifida. American Journal of Botany 1989. The use of isozyme data forinferring 75: 142. Abstract. ancestryof polyploid pteridophytes.Biochemical and A. F. SCHNABEL. 1986. Allozymic and Systematicsand Ecology 17: 117-130. morphological evidence for a progenitor-deriv- , S. I. GUTTMAN, and W. H. ESHBAUGH. 1985. ative pair in Camassia(Liliaceae). SystematicBot- Electrophoreticevidence of reticulateevolution any 11: 433-445. in the Appalachian Aspleniumcomplex. System- ROOSE, M. L. and L. D. GOTTLIEB. 1976. Genetic and atic Botany 10: 184-192. biochemical consequences of polyploidy in Tragopogon.Evolution 30: 818-830.

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