Genetic Evidence for Allopolyploidy in the Neotropical Fern Hemionitis Pinnatifida (Adiantaceae) and the Reconstruction of an Ancestral Genome Author(S): Thomas A

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Genetic Evidence for Allopolyploidy in the Neotropical Fern Hemionitis pinnatifida (Adiantaceae) and the Reconstruction of an Ancestral Genome Author(s): Thomas A. Ranker, Christopher H. Haufler, Pamela S. Soltis, Douglas E. Soltis Source: Systematic Botany, Vol. 14, No. 4 (Oct. - Dec., 1989), pp. 439-447 Published by: American Society of Plant Taxonomists Stable URL: http://www.jstor.org/stable/2418989 Accessed: 16/05/2010 14:49

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Genetic Evidence for Allopolyploidy in the Neotropical Fern Hemionitis pinnatifida (Adiantaceae) and the Reconstruction of an Ancestral Genome

THOMAS A. RANKER1and CHRISTOPHERH. HAUFLER Department of Botany, University of Kansas, Lawrence, Kansas66045 Present address:Ifawaiian EvolutionaryBiology Program, University of Hawaii, Honolulu, Hawaii 96822

PAMELA S. SOLTIS and DOUGLAS E. SOLTIS Department of Botany, Washington State University, Pullman, Washington 99164

ABSTRACT. Genetic evidence from enzyme electrophoresisand restrictionsite analyses of cpDNA was employed to explore the origin of the tetraploidHemionitis pinnatifida by determining the role of diploid species of Hemionitisin contributingto the tetraploidgenome. The resultsclearly supported the hypothesis that H. pinnatifidais an allotetraploidwith H. palmataas one of its diploid progenitors. The other diploid parent was not identified among the existing species of Hemionitisand is either extinct or exceedingly rare. Genetic identity analyses compared the extant diploid genomes with the divergent diploid genomes combined in tetraploids.One of these divergent genomes was shown to be a good approximationof that of H. palmata,and the other is a reconstructionof that of the missing diploid parent.These analyses demonstratedthat the missing ancestorwas genetically more similar to H. palmatathan either of these species is to the other diploid species surveyed. Such greater genetic similarity may have been important in allowing hybridization between the two diploid progenitors of H. pinnatifida.The data also suggest that tetraploid individuals may have been synthesized de novo at least five times among the three populations surveyed.

Many species of homosporous ferns are al- pear "fixed" in all individuals of a given pop lotetraploid in origin, having arisen through ulation or species (Roose and Gottlieb 1976). chromosome doubling preceding or following Fixation of heterozygosity is maintained by di- hybridization between two distinct diploid pro- somic inheritance resulting from preferential genitors (e.g., Manton 1950; Shivas 1961a, 1961b; pairing of homologous chromosomes from each Wagner 1954; Wagner and Wagner 1980; Walk- parental taxon (Manton 1950). er 1979). Such hybrids usually can be recog- Hemionitis pinnatifidaBaker is a tetraploid (2n nized by their morphological intermediacy for = 60 11;Ranker, unpubl. data; Smith and Mickel characters at which two known diploids are di- 1977) distributed from southern Mexico to Cos- vergent. Recent studies have employed enzyme ta Rica. Smith and Mickel (1977) suggested that electrophoresis to test hypotheses of allopoly- H. pinnatifida arose as a cross between diploid ploidy and reticulate relationships in several H. palmata L. (2n = 30 II; Ranker, unpubl. data; genera of ferns, including Asplenium (Gastony Smith and Mickel 1977; Walker 1966, 1985) and 1986; Werth et al. 1985a, 1985b), Cystopteris an undiscovered diploid form of H. pinnatifida. (Haufler 1985b), Polypodium (Bryan and Soltis Evidence for this was based on meiotic pairing 1987), and Polystichum (Soltis et al. 1987). These behavior in a naturally occurring triploid hy- studies have shown that diploid taxa often have brid (H. palmata x pinnatifida), which was re- unique alleles that distinguish them from con- ported as 2n = 30 II + 30 I, with putatively generic relatives. The combination of marker homologous chromosomes from the H. palmata alleles in hybrid-derived species provides strong genome forming bivalents (Smith and Mickel evidence for ascertaining the parentage of such 1977). Subsequent observations of leaf and rhi- taxa. In allopolyploids, diploid genomic marker zome macromorphology, SEM analyses of leaf alleles typically are combined to produce het- trichomes (Ranker 1987, unpubl. data), and SEM erozygous isozymic patterns. These usually ap- analyses of spore wall morphology and ontog- 439 440 SYSTEMATIC BOTANY [Volume 14

TABLE 1. Approximate locations of populations eny (Ranker 1989) supported the hypothesis that sampled. Vouchers of all collections deposited in H. palmata is one of the parents of the tetraploid. KANU and UC. Population abbreviations are in quo- Those data also suggested that the other rec- tation marks. ognized diploid species of Hemionitis [H. levyi Fourn., H. rufa (L.) Sw., and H. tomentosa (Lam.) Hemionitis pinnatifida: Raddi] are not progenitors of H. pinnatifida. MEXICO. Est. Oaxaca: "SG," Hwy 131 ca. 53 km N This study employs genetic evidence from of Hwy 200 at Pto. Escondido, 26 Dec 1983, Ranker enzyme electrophoresis and restriction et al. 728; "ZAN," Hwy 190, between km posts site 78 and 79, E of Santo Domingo Zanatepec, just analyses of chloroplast DNA (cpDNA) to de- W of Gral. Pascual Fuentes, 15 Jul 1985, Ranker termine the role of H. palmata in contributing & Yatskievych809; "TAP," Hwy 190, between km to the origin of the tetraploid, H. pinnatifida. In posts 7 and 8 at bridge E of junction with Hwy addition, we explore the genetic affinities of a 200, 15 Jul 1985, Ranker & Yatskievych 812. missing diploid ancestor by subtracting the iso- Hemionitis palmata: zymic profile of H. palmata from that of the tetraploid and, thus, reconstruct a MEXICO. Est. Oaxaca: "PASG," Hwy 131 ca. 53 km portion of the N of Hwy 200 at Pto. Escondido, 26 Dec 1983, missing genome for use in genetic identity anal- Ranker et al. 727. yses. JAMAICA. Portland Parish: "J845," Rd Bi, 7 km S MATERIALS AND METHODS of Buff Bay, 23 Nov 1985, Ranker& Trapp845. St. Andrew Parish: "J849," 0.3 km S of Gordon Town Enzyme Electrophoresis. Natural popula- on rd to Guava Ridge, 25 Nov 1985, Ranker & tions of Hemionitis pinnatifida, H. palmata, H. le- Trapp849; "J852," ca. 0.4 km upstream from Gor- vyi, and H. rufa were analyzed electrophoreti- don Town on banks of Hope River, 27 Nov 1985, cally [table 1; because the latter two species are Ranker & Trapp 852; "J853," Junction of rd from not likely candidates for parents of the Gordon Town with Yallahs River, 17 Nov 1985, tetra- Ranker & Trapp 853. Clarendon Parish: "J861," ploid, electrophoretic results will not be pre- ca. 2.7 km from Crofts Hill towards Arthur's Seat, sented in detail, but will only be summarized 30 Nov 1985, Ranker & Trapp 861; "J862," ca. 1.2 in the form of genetic identity analyses (Ranker km from Crofts Hill towards Arthur's Seat, 30 1987)]. All populations were surveyed for elec- Nov 1985, Ranker & Trapp 862. trophoretically detectable enzyme variation us- COSTA RICA. Prov. San Jose: "CRRG,"Banks of Rio ing 12.4% to 12.8% starch gels. The extraction General near bridge of Interamerican Hwy (C.R. procedures were as described in Ranker and Hwy #2), 5 Dec 1984, Ranker 778. Prov. Here- Werth (1986). Both sporophytes and laboratory- Arboretum of Finca la dia: "CRLS," Selva, OTS cultured gametophytes were employed in the field station near Pto. Viejo, 11 Dec 1984, Ranker analyses. Enzymes were surveyed using gel and et al. 793c. electrode buffer systems 6, 8, and 11 (Soltis et Hemionitis levyi ("Hele" indicates the summation of al. 1983). System 8 was modified as described both populations): in Haufler (1985a); for system 11, the gel buffer COSTA RICA. Prov. Alajuela: Service road to Planta was prepared at 0.009 M and 0.011 M histidine- de 3 Dec Ranker Hidroelectrica Ntro. Amo, 1984, HCI. The morpholine-citrate buffer system (here & Gomez-P. 777. abbreviated MC) of Odrzykoski and Gottlieb MEXICO. Est. Oaxaca: Hwy 190, between km posts 7 and 8 at bridge E of junction with Hwy 200, 15 (1984) also was employed but at pH 7.5 and pH Jul 1985, Ranker & Yatskievych 813. 8.0 [pH adjusted by titrating with N-(3-amino- propyl)-morpholine]. Two or three different sets Hemionitis rufa ("Heru" indicates the summation of all of electrophoretic conditions were employed for populations): most enzymes to increase the probability of cor- COSTA RICA. Prov. San Jose: Banks of Rio General rectly assessing the identity of alleles at a locus near bridge of Interamerican Hwy (C.R. Hwy #2), 5 Dec 1984, Ranker 779. among species. The following nine enzymes JAMAICA. St. Andrew Parish: Along Hope River, were assayed using staining procedures de- W of Gordon Town near Blue Mtn. Inn, 26 Nov scribed by Werth (1985), except where noted 1985, Ranker & Trapp 850. Clarendon Parish: (enzyme abbreviation and buffer systems in pa- Near Arthur's Seat ca. 5 km W of Crofts Hill, 30 rentheses): aspartate aminotransferase (AAT Nov 1985, Ranker & Trapp 858; ca. 1.2 km from [after Soltis et al. 1983]; 6, 8), aldolase (ALD; 6, Crofts Hill towards Arthur's Seat, 30 Nov 1985, 11), hexokinase (HK; 8), isocitrate dehydroge- Ranker & Trapp 863. nase (IDH; 11, MC), leucine aminopeptidase 1989] RANKER ET AL.: HEMIONITIS 441

(LAP; 8), phosphoglucomutase (PGM; 6, 11, MC), TABLE 2. Allelic frequencies assigned to recon- phosphoglucose isomerase (PGI; 6, 8), shiki- structed, ancestral diploid genomes of H. pinnatifida mate dehydrogenase (SKDH; 11, MC), and tri- populations. N = allozymically most similar to H. osephosphate isomerase (TPI; 6, 8). Loci and palmata;F = allozymically most dissimilar to H. palmata.See text for further alleles for a given enzyme were numbered se- explanation.Population abbreviations as in table 1. quentially beginning with the most anodal. Al- leles are denoted by superscripts; the number- Ancestral genome ing scheme employed accounts for all alleles Locus Allele ZAN-N ZAN-F TAP-N TAP-F SG-N SG-F detected among the species of Hemionitis, Gym- Pgi-2 4 0.5 0.5 nopteris, and Bommeria(Ranker 1987). 6 0.24 1.0 Genetic identity values (I; Nei 1972) were cal- 12 0.5 0.5 0.76 1.0 1.0 culated from allozymic frequencies for all pair- Skdh 1 1.0 1.0 wise comparisons of H. pinnatifida populations 4 1.0 a using FORTRAN program provided by M. D. 7 1.0 1.0 1.0 Loveless (College of Wooster, Wooster, Ohio, Hk 8 0.5 0.5 U.S.A.). To assess critically the genetic affinities 0.5 0.5 0.5 0.5 12 0.5 0.5 0.5 0.5 0.5 0.5 of the diploid genomes combined in the tetraploid, two hypothetical, ancestral diploid ge- Idh 13 1.0 1.0 0.12 1.0 1.0 nomes were reconstructed from each tetraploid 15 1.0 0.88 population such that one was allozymically more Aat 1 0.5 0.5 0.5 0.5 similar (abbreviated "N," for "near") and the 4 0.5 0.5 0.5 0.5 other was allozymically more dissimilar (ab- 5 1.0 1.0 breviated "F," for "far") to the putative pro- Lap 4 0.5 0.5 genitor H. palmata (table 2). The N genomes and 7 1.0 1.0 the F genomes are presumed to represent the 11 1.0 1.0 0.5 0.5 contributions of H. palmata and the other diploid ancestor, respectively. The F genome was constructed, therefore, by subtracting the known contribution of H. palmata from the isozymic Because the Jamaican populations of H. pal- profile of H. pinnatifida. Both parental genomes mata had diverged very little from each other were scored as fixed (i.e., frequency of 1.0) for (I = 0.91; Ranker 1987, unpubl. data), identity alleles at those loci observed to be monomor- values were averaged for these populations phic across all populations of H. pinnatifidaand ("Hepa-JAM" in table 4) when compared to all H. palmata (Ald-2, Tpi-1, Tpi-2, and Pgm-2). At others. Values were similarly averaged within loci where H. palmata accounted for one allele species for the populations of H. levyi ("Hele") of a fixed heterozygous pattern in a tetraploid and H. rufa ("Heru"). population, the N genome was scored as fixed Restriction Site Analyses of cpDNA. De- for that allele and the F genome was scored as tailed analyses of structure and variability fixed for the alternate allele. Where both alleles among the cpDNA genomes of the species of in a tetraploid population were found in H. Hemionitis will be reported elsewhere (Ranker, palmata (e.g., Lap, table 3) and where both alleles Soltis, and Soltis, unpubl. data). Our primary were similarly absent (e.g., Aat, table 3), each interest in the present study was to compare allele was scored at a frequency of 0.5 in each restriction fragment patterns from cpDNA of ancestral genome. Tetraploid population TAP H. pinnatifida to those from its putative parent (table 3) contained both heterozygotes and H. palmata. Restriction site data were obtained, homozygotes (the latter for only one of the two as described below, from what is typically the alleles expressed in the heterozygote) at Pgi-2 most variable portion of the cpDNA genome, and Idh (see RESULTS).At Pgi-2 it was assumed the large single-copy region (e.g., Palmer 1985). that the TAP-N (table 2) genome was polymor- Plants of H. pinnatifida (population ZAN), H. phic, containing two alleles, one discovered and palmata (population PASG), and their putative the other not discovered in sampled popula- triploid backcross (the latter generously sup- tions of H. palmata. At Idh it was assumed that plied by John Mickel; see Smith and Mickel the TAP-F genome was polymorphic, sharing 1977) were collected in Mexico (table 1) and one allele with H. palmata (table 2). maintained in cultivation. Because fresh leaf 442 SYSTEMATICBOTANY [Volume 14

TABLE 3. Allelic frequencies at polymorphic loci in populations of H. pinnatifidaand H. palmata. N = sample size/population/locus. Population abbreviations as in table 1.

Population H. pinnatifida H. palmata Locus Allele ZAN TAP SG PASG J845 J849 J852 J853 J861 J862 CRRG CRLS Pgi-2 4 0.50 6 0.12 0.50 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 9 1.00 12 0.50 0.88 0.50 N 8 17 7 29 50 39 23 26 17 18 49 81 Skdh 1 0.50 0.50 4 0.50 7 0.50 0.50 0.50 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N 9 17 7 29 50 39 23 26 17 18 49 81 Hk 8 0.50 0.50 0.50 0.33 0.95 0.96 0.94 1.00 0.28 1.00 0.02 12 0.50 0.50 0.50 1.00 0.67 0.05 0.04 0.06 0.72 0.98 N 8 17 7 18 24 21 23 26 17 18 49 81 Idh 13 0.50 0.56 1.00 0.86 1.00 0.23 0.20 0.40 1.00 1.00 1.00 1.00 15 0.50 0.44 17 0.14 0.77 0.80 0.60 N 8 17 7 29 50 39 23 26 17 18 49 81 Aat 1 0.50 0.50 4 0.50 0.50 5 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 N 2 2 2 25 50 39 23 26 17 18 49 81 Lap 4 0.50 0.96 0.98 0.58 0.50 0.23 0.97 1.00 7 0.50 0.50 11 0.50 0.50 0.50 0.04 0.02 0.42 0.50 0.77 0.03 0.14 1.00 15 0.86 N 8 17 7 27 50 20 23 26 17 18 7 5

material was not available for H. rufa, gameto- nomes of lettuce (Jansen and Palmer 1987) and phytes were cultured (following Windham et petunia (Palmer et al. 1983) were employed in al. 1986) from spores obtained from the CRRG filter hybridizations (following Palmer 1986). collection (table 1). Total DNA was isolated from Petunia cpDNA probes were used in place of about 1.0 g of freshly collected leaves or ga- the 22-kb inversion present in lettuce (see Jan- metophytes following the miniprep procedure sen and Palmer 1987). All probes were kindly of Saghai-Maroof et al. (1984) as modified by provided by R. Jansen and J. Palmer. Doyle and Doyle (1987), except that our procedure stopped after their step 8 (resuspension RESULTS of DNA pellet in ammonium acetate-EDTA buffer). The DNA's were digested with the fol- Enzyme Electrophoresis. Ten putative loci lowing eight restriction endonucleases follow- were scored. The more anodal of the two staining the specifications of the suppliers: BanlI, ing regions of TPI always appeared as a three- BglII,EcoRI, EcoRV, HindlIl, HpaII, Sacl, and SaclI. banded pattern, even in extracts from single Fragments were separated on 1.0%agarose gels, gametophytes (Ranker 1987, unpubl. data). Be- denatured, and bidirectionally blotted onto ny- cause no variability was detected within a lon membranes (ZETABIND, Cuno Laboratory species, we treated this pattern as representa- Products). Radioactively labeled probes from the tive of a single locus (Tpi-1). Although the pres- large single-copy regions of the cpDNA ge- ence of a duplicated locus cannot be excluded, 1989] RANKER ET AL.: HEMIONITIS 443

TABLE 4. Nei's genetic identities for all pairs of reconstructed, ancestral diploid genomes and diploid species of Hemionitis. Hepa = H. palmata. Hepa-JAM, Hele, and Heru = average I values for H. palmata from Jamaica, H. levyi, and H. rufa, respectively. Other abbreviations as in tables 1 and 2.

Hepa- Hepa- Hepa- Hepa- ZAN-N ZAN-F TAP-N TAP-F SG-N SG-F PASG JAM CRRG CRLS Hele Heru

ZAN-N - 0.70 0.97 0.68 0.70 0.67 0.67 0.70 0.69 0.77 0.37 0.32 ZAN-F - 0.67 0.97 0.50 0.57 0.47 0.49 0.47 0.47 0.27 0.32 TAP-N - 0.71 0.73 0.70 0.70 0.73 0.79 0.80 0.37 0.32 TAP-F - 0.51 0.61 0.48 0.50 0.48 0.49 0.28 0.32 SG-N - 0.80 0.94 0.95 0.77 0.87 0.40 0.42 SG-F - 0.74 0.75 0.67 0.67 0.40 0.42 Hepa-PASG - 0.92 0.70 0.90 0.44 0.38 Hepa-JAM - 0.79 0.85 0.36 0.44 Hepa-CRRG - 0.72 0.30 0.49 Hepa-CRLS - 0.40 0.39 Hele - 0.18 Heru

either interpretation is consistent with our con- tions were Hk where both alleles in the tetra- clusions herein. ploid were also found in H. palmata and Lap The three populations of H. pinnatifida sur- where both alleles in population SG of H. pin- veyed were monomorphic for Ald-23, Tpi-14, natifidawere found in the diploid (table 3). Nei- Tpi-24, and Pgm-2". Population SG was mono- ther of the two alleles fixed at Aat in popula- morphic for Idhl" (fig. 4) and Aat5. All three tions ZAN and TAP of H. pinnatifidawas detected populations exhibited fixed heterozygosity for in H. palmata(or any other species; Ranker 1987). Skdhl"7or Skdh4'7(fig. 5), Lap715'or Lap411'(fig. Genetic Identity Analyses of Reconstructed 2), and Hk8112(fig. 3; table 3). Additional fixed Parental Genomes. Pair-wise genetic identi- heterozygosity was detected in population ZAN ties are presented in table 4 for the N and F for Pgi-24/'2,Idh""', and Aat"4, population TAP reconstructed, ancestral genomes of each tet- for Aat"4, and population SG for Pgi-26/12 (fig. raploid population and for the diploid species 1). Population TAP was polymorphic at Pgi-2 H. palmata, H. levyi, and H. rufa. These results and Idh (table 3) with homozygotes and het- can be summarized as follows: 1) The two an- erozygotes at both loci. Alleles comprising het- cestral genomes (N and F) reconstructed from erozygous patterns did not segregate among each tetraploid population were generally more gametophytic progeny when individual ga- similar to each other (I = 0.66, range 0.50 to metophytes were assayed electrophoretically 0.80) than were diploid species of Hemionitis to (Ranker 1987, unpubl. data). A total of 20 alleles one another (I = 0.30, range 0.10 to 0.50). 2) was detected across the 10 loci surveyed, eight Both N and F genomes were more similar to H. of which were unique to H. pinnatifida. palmata (7= 0.77 and 0.56, respectively) than Genetic identity values among the three pop- either was to H. levyi (I = 0.38 and 0.32, re- ulations were as follows: ZAN vs. TAP, 0.97; spectively) or to H. rufa (I = 0.36 for both). 3) ZAN vs. SG, 0.72; and TAP vs. SG, 0.76. The mean N genome identity to H. palmata (I The nine populations of H. palmata were = 0.77, range 0.67 to 0.95) was within the range monomorphic for the same alleles at those four of intraspecific identity values among popula- loci for which all three populations of the tet- tions of H. palmata (I = 0.86, range 0.70 to 1.00). raploid were monomorphic (Ald-23, Tpi-14, Tpi- In contrast, the mean N genome identities to 24, and Pgm-2"5). In addition, the two species H. levyi (I = 0.38, range 0.37 to 0.40) and to H. shared alleles at the other loci (table 3). Alleles rufa (I = 0.36, range 0.32 to 0.42) were much found in H. palmata usually accounted for at lower than intraspecific comparisons of popu- least one of the alleles in the fixed heterozygous lations of those species (I = 0.97 and 0.73, re- patterns of the tetraploid with the alternate al- spectively). 4) The mean F genome identity to leles unique to the latter (figs. 1-3, 5). Excep- H. palmata (I = 0.56, range 0.47 to 0.75) was 444 SYSTEMATIC BOTANY [Volume 14

linear portion of the genome examined (esti- mated for each enzyme as the number of frag-

6- ments observed plus one, to account for the extra site at the end of the linear fragment). 12-__ Hemionitisrufa shared only 20 fragments (out of 108 total) with the other species and, therefore, 40 restriction sites of approximately 116 assayed for this species. Several differences between H. rufa and the other species could easily be ex- plained via simple restriction site mutations. For example, H. rufa contained two BglII (1.3 kb + 3.5 kb) and two EcoRV (3.0 kb + 4.3 kb) fragments, each pair of which was equal in size to single fragments found in the other species 4_~~76 3 (4.8 kb and 7.3 kb, respectively; figs. 6, 7). These differences represent restriction site losses or gains; the actual direction is unknown because 13-__ no attempt was made to polarize the mutations. Most other differences were not as easily inter- 4 preted, and small length mutations appeared 17-wp common. A complete list of fragment sizes is available from the first author.

4- . I:K 5 DISCUSSION The distribution of alleles shared between H. FIGS.1-5. Photographsof gels demonstratingsome pinnatifida and H. palmata strongly supports an of the allozymes detected in Hemionitispinnatifida and allopolyploid origin of the tetraploid, with H. H. palmata. 1-3. H. pinnatifida individuals (popula- palmata as one diploid progenitor. As with mor- tion SG), to left of arrows, exhibiting fixed hetero- phological observations (Ranker 1987, 1989), the one allele with H. in- zygosity and sharing palmata electrophoretic data suggest that no other rec- dividuals (population PASG), to right of arrows. 1. ognized diploid species in Hemionitis contrib- PGI. 2. LAP. 3. HK. 4, 5. H. pinnatifida individuals (population SG), to right of arrows, H. palmataindi- uted to the origin of H. pinnatifida. Following viduals (population PASG),to left of arrows.4. IDH. this hypothesis, H. palmata would have contrib- 5. SKDH. uted one allele per locus and the other diploid parent would have contributed the alternate allele in fixed heterozygotes. Fixed heterozygous higher than typical genetic identities among patterns that lack a known H. palmataallele (e.g., the three diploid species of Hemionitisexamined at Aat for populations ZAN and TAP, table 3) (I = 0.30, range 0.10 to 0.50). The mean F ge- could be due to an allele not detected from this nome identities to H. levyi (F = 0.32, range 0.27 diploid or to what was a relatively rare allele to 0.40) and to H. rufa (F = 0.36, range 0.32 to that has been lost due to genetic drift, founder 0.42), however, were similar to other interspe- effect, or natural selection. Alternatively, such cific comparisons and almost identical to the N alleles could have arisen in the tetraploid genome pair-wise identities to each of these two through mutation subsequent to hybridization species. and polyploidization, becoming fixed through Restriction Site Analyses of cpDNA. Restric- inbreeding. tion enzyme digests of DNA from Hemionitis Because of differences in isozyme profiles, palmata, H. pinnatifida, and their putative trip- tetraploid individuals may have been synthe- loid backcross produced identical cpDNA frag- sized de novo at least five times: a minimum of ments for each enzyme when hybridized with once each in populations ZAN and SG, and at each probe (figs. 7-9). For those three taxa we least three times in population TAP. Because detected a total of 132 restriction sites in the populations ZAN and SG expressed no intra- 1989] RANKSR ET AL.: HEMONrrTS 445

0 ~~~~0 0 0 R P R P 3x Pi P 3X Pi P 3x Pi 4.8- 73 - 7 3.8- 3.5- ~~~~~~~~~~2.5- 4.3- 3.0- 3.3-

1.3- 0.97- ~

BgIl Eco RV Sac 11 EcoRI

FIGs.6-9. Autoradiographsof Southern Blots. R = Hemioitisrufa. P = H. palmata.3x = H. palmatax H. pinnatifida.Pi = H. pinnatifida.Numbers at left indicate approximatesizes of DNA fragmentsin kilobase pairs. Restrictionenzymes used to digest HemionitisDNA are listed at base of each figure. Probesto which fragments hybridized: 6, 7. Petunia PstI, 9.2 kb. 8, 9. Lettuce Sacl, 7.7 kb.

populational variability,there is no evidence to per heterozygous locus, resulting in polyploid suggest multiple origins of tetraploid individ- offspring expressing two or more alleles per uals withinthese populations but each popula- polymorphic locus. Multiple genotypes ob- tion had a unique combination of alleles across served in an allotetraploid species could arise loci. The sample from population TAP, how- following a single hybridizationevent between ever, did express allozymic variationamong in- such heterozygous, diploid gametes. Subse- dividuals in that there were homozygotes and quent segregationand recombinationfrom such heterozygotes at both Idh and Pgi-2 that com- complex patterns would produce variability bined to form three distinct genotypes: Idh'3115 among offspring. If the allotetraploid H. pin- + Pgi-26112,Idh13'15 + Pgi-212112,and Idh13'13+ Pgi- natifidaoriginated via a single hybridization 26/12. Each genotype may have arisen from a event, we would need to make some unlikely separate hybridization event. These observa- assumptionsabout levels of heterozygosity and tions aresimilar to the dataof Werthet al. (1985b) polymorphism present in the individual par- in Asplenium,Haufler and Soltis (1986) in Cys- ents. For a single hybridization to account for topteris, and Soltis et al. (1987) in Polystichum the observed genotypic variation among and demonstrating multiple origins of allopoly- within tetraploid populations, the H. palmata ploids. These multiple-origin hypotheses as- parent would have needed to have been het- sume that hybridizations occurred between erozygous at three or four of the six polymor- diploid species and that tetraploidization re- phic loci. Similarly,the other parentwould have sulted from the productionof unreduced spores needed to have been heterozygous at five or all in allodiploid hybrids [ClassII polyploidization of those loci. Given the rarityof heterozygosity of Harlan and deWet (1975)]. Under these cir- observed among extant populations of the dip- cumstances the original, hybridizing haploid loid species of Hemionitis(table 3; Ranker1987), gametes would each contribute only one allele the existence of such highly heterozygous in- per locus, even if the diploid parents were het- dividuals is improbable. Multiple origins erozygous. Whereas such a mechanism was through several independent hybridizations, demonstrated in allodiploid Asplenium ebenoides therefore, is more likely in the case of H. pin- Scott (Wagner and Whitmire 1957), Gastony natifida. (1986) has shown that unreduced spore pro- The genetic identity analyses of the recon- ductionmay precede hybridization in some cases structed,ancestral diploid genomes provide ad- [Class I polyploidization of Harlan and deWet ditional insights into the origin of H. pinnatifida (1975)]. Unreduced, diploid spores from het- and the genetic constitution of the undiscov- erozygous parentswould contributetwo alleles ered or extinct diploid parent. Our analyses 446 SYSTEMATIC BOTANY [Volume 14 support the hypothesis that H. palmata is most of H. pinnatifida. Given the generally great ge- likely one of the diploid progenitors of H. pin- netic divergence among diploid species of natifida. They also demonstrate that the N ge- Hemionitis (table 4), as evidenced by the allo- nome from population SG was derived from zymic studies of nuclear-encoded genes and the populations of H. palmata nearly genetically comparison of cpDNA restriction fragments identical to those sampled from Mexico and Ja- from H. rufa and H. palmata, it is unlikely that maica (I = 0.94 and 0.95, respectively; table 4). any other species contributed cpDNA to H. pin- Tetraploid population SG and the Mexican pop- natifida. These results are similar to those re- ulation of H. palmata (PASG) were located at the ported for other congeneric species of homo- same site (table 1), thus population SG may have sporous ferns in demonstrating that even closely originated at or near its present location. In related diploid species do not share a common contrast, the ancestral N genomes comprising chloroplast genome. For example, Stein et al. tetraploid populations ZAN and TAP were more (1986) showed that the three species of Osmunda divergent from sampled populations of H. pal- "differ by an extensive series of small deletions mata, although generally within the range of and insertions." Similarly, Yatskievych et al. pair-wise identities among the populations of (1988) found that diploid species within Pha- H. palmata. The putatively homologous constit- nerophlebia, Cyrtomium, and Polystichum pos- uent genomes of ZAN and TAP were nearly sessed distinctive restriction fragment patterns. genetically identical (i.e., I = 0.97 for both In conclusion, genetic data from allozymes ZAN-N vs. TAP-N and ZAN-F vs. TAP-F), in- support an allopolyploid origin of H. pinnatifida dicating that they may have arisen from the as hypothesized from chromosomal (Smith and same two, polymorphic, parental populations. Mickel 1977) and morphological information A wider sampling of populations in southern (Ranker 1987,1989). The evidence demonstrates Mexico and northern Central America may lo- that H. palmata and a rare or extinct diploid cate more probable ancestral populations of H. contributed to the nuclear genome of the tet- palmata, which contributed to ZAN and TAP. raploid. Genetic identity comparisons of the re- Perhaps the greatest value of reconstructing constructed, missing ancestral genome with the divergent genomes comprising the. tetra- those of extant diploids show the least genetic ploid is that it permits an estimation of the ge- divergence between the presumed parents of netic relatedness of the missing parental dip- the tetraploid. Analyses of cpDNA restriction loid to known diploid species of Hemionitis. fragments suggest that only H. palmata contrib- Identity values suggest that the missing species uted the chloroplast genome to H. pinnatifida. (as represented by the F genomes) is consid- ACKNOWLEDGMENTS. We thank Genie Trapp for = erably more closely related to H. palmata (I field assistancein Jamaicaand for providing helpful 0.56) than either of these two species is to H. commentson the manuscript,Mike Windhamfor field levyi or H. rufa (table 4). Such relatedness may assistancein Mexico and discussions on genetic iden- have been an important factor in allowing in- tity analyses, and Luis D. Gomez and Marla Spivak terspecific hybridization (Stebbins 1980). for field assistance in Costa Rica. We are grateful to That the reconstructed, hypothetical ge- two anonymous reviewers for providing valuable nomes represent reasonable approximations of suggestions which led to improvementsin the manu- the allozymic constitution of actual diploid script. This research was supported in part by NSF species is suggested by comparing the identity GrantNo. BSR-8514431to CHH and TAR,NSF Grant No. BSR-8620444to and a from the Uni- values of the N genomes to H. levyi and H. rufa PSS, grant versitv of KansasEndowment Association to TAR. with those calculated for H. palmata (i.e., that genome which the N genomes are presumed to LITERATURE CITED represent). The mean identity values for the N genomes to H. levyi and H. rufa (0.38 and 0.36, BRYAN, F. A. and D. E. SOLTIS. 1987. Electrophoretic respectively) were nearly identical to those for evidence for allopolyploidy in the fern Polypo- H. palmata (0.35 and 0.39, respectively). dium virginianum.Syst. Bot. 12:553-561. DOYLE,J. J. and J. L. DOYLE. 1987. A rapid DNA Because H. and H. have palmata pinnatifida isolation procedure for small quantities of fresh identical restriction fragment profiles for the leaf tissue. Phytochem. Bull. 19:11-15. large single-copy region of the chloroplast ge- GASTONY,G. J. 1986. Electrophoretic evidence for nome, restriction site data clearly support the the origin of fern species by unreduced spores. hypothesis that H. palmata is one of the parents Amer. J. Bot. 73:1563-1569. 1989] RANKER ET AL.: HEMIONITIS 447

HARLAN, J. R. and J. M. J. DEWET. 1975. On 0. Winge taxonomy of species of Polypodiumin Europe and and a prayer: The origins of polyploidy. Bot. Rev. America. II. Taxonomy. J. Linn. Soc., Bot. 58:27- (Lancaster) 41:361-390. 38. HAUFLER,C. H. 1985a. Enzyme variability and modes SMITH,A. R. and J. T. MICKEL. 1977. Chromosome of evolution in Bommeria. J. Arnold Arbor. 60: counts for Mexican ferns. Brittonia 29:391-398. 445-476. SOLTIS,D. E., C. H. HAUFLER,D. C. DARROW,and G. . 1985b. Pteridophyte evolutionary biology: J. GASTONY.1983. Starch gel electrophoresis of The electrophoretic approach. Proc. Roy. Soc. ferns: A compilation of grinding buffers, gel and Edinburgh 86B:315-323. electrode buffers, and staining schedules. Amer. and D. E. SOLTIS. 1986. Genetic evidence sug- Fern J. 73:9-27. gests that homosporous ferns with high chro- SOLTIS,P. S., D. E. SOLTIs,and E. R. ALVERSON.1987. mosome numbers are diploid. Proc. Natl. Acad. Electrophoretic and morphological confirmation Sci. U.S.A. 83:4389-4393. of interspecific hybridization between Polysti- JANSEN, R. K. and J. D. PALMER. 1987. Chloroplast chum kruckebergiiand P. munitum. Amer. Fern J. DNA from lettuce and Barnadesia (Asteraceae): 77:42-49. Structure, gene localization, and characterization STEBBINS,G. L. 1980. Polyploidy in plants: Unsolved of a large inversion. Curr. Genetics 11:553-564. problems. Pp. 495-520 in Polyploidy: Biologicalrel- MANTON, I. 1950. Problems of cytology and evolution evance, ed. W. H. Lewis. New York: Plenum Press. in the Pteridophyta. Cambridge: Cambridge Univ. STEIN, D. B., J. D. PALMER,and W. F. THOMPSON.1986. Press. Structural evolution and flip-flop recombination NEI, M. 1972. Genetic distance between populations. of chloroplast DNA in the fern genus Osmunda. Amer. Naturalist 106:283-293. Curr. Genetics 10:835-841. ODRZYKOSKI,I. J. and L. D. GOTTLIEB. 1984. Dupli- WAGNER,W. H., JR. 1954. Reticulate evolution in the cations of genes coding 6-phosphogluconate de- Appalachian aspleniums. Evolution 8:103-118. hydrogenase in Clarkia (Onagraceae) and their and F. S. WAGNER. 1980. Polyploidy in pteri- phylogenetic implications. Syst. Bot. 9:479-489. dophytes. Pp. 199-214 in Polyploidy:Biological rel- PALMER, J. D. 1985. Comparative organization of evance, ed. W. H. Lewis. New York: Plenum Press. chloroplast genomes. Annual Rev. Genet. 19:325- and R. S. WHITMIRE. 1957. Spontaneous pro- 354. duction of a morphologically distinct, fertile al- . 1986. Isolation and structural analysis of lopolyploid by a sterile diploid of Asplenium ebe- chloroplast DNA. Meth. Enzymol. 118:167-186. noides. Bull. Torrey Bot. Club 84:79-89. , C. R. SHIELDS, D. B. COHEN, and T. J. ORTON. WALKER,T. G. 1966. A cytotaxonomic survey of the 1983. Chloroplast DNA evolution and the origin pteridophytes of Jamaica. Trans. Roy. Soc. Edin- of amphidiploid Brassica species. Theor. Appl. burgh 66:169-237. Genet. 65:181-189. - . 1979. The cytogenetics of ferns. Pp. 87-132 RANKER, T. A. 1987. Experimental systematics and in The experimentalbiology of ferns, ed. A. F. Dyer. population biology of the fern genera Hemionitis London: Academic Press. and Gymnopteriswith reference to Bommeria.Ph.D. 1985. Cytotaxonomic studies of the ferns of dissertation, University of Kansas, Lawrence. Trinidad. 2. The cytology and taxonomic impli- 1989. Spore morphology and generic delim- cations. Bull. Brit. Mus. (Nat. Hist.), Bot. 13:149- itation of neotropical Hemionitis,Gymnopteris, and 249. Bommeria(Adiantaceae). Amer. J. Bot. 76:297-306. WERTH,C. R. 1985. Implementing an isozyme lab- and C. R. WERTH. 1986. Active enzymes from oratory at a field station. Virginia J. Sci. 36:53- herbarium specimens: Electrophoresis as an af- 76. terthought. Amer. Fern J. 76:102-113. , S. I. GUTTMAN, and W. H. ESHBAUGH. 1985a. ROOSE,M. L. and L. D. GOTTLIEB. 1976. Genetic and Electrophoretic evidence of reticulate evolution biochemical consequences of polyploidy in in the Appalachian Asplenium complex. Syst. Bot. Tragopogon.Evolution 30:818-830. 10:184-192. SAGHAI-MAROOF,M. A., K. M. SOLIMAN, R. A. JORGEN- and . 1985b. Recurring origins SEN, and R. W. ALLARD. 1984. Ribosomal DNA of allopolyploid species in Asplenium. Science 228: spacer-length polymorphisms in barley: Men- 731-733. delian inheritance, chromosomal location, and WINDHAM,M. D., P. G. WOLF,and T. A. RANKER.1986. population dynamics. Proc. Natl. Acad. Sci. U.S.A. Factors affecting prolonged spore viability in 81:8014-8018. herbarium collections of three species of Pellaea. SHIVAS, M. G. 1961a. Contributions to the cytology Amer. Fern J. 76:141-148. and taxonomy of species of Polypodiumin Europe YATSKIEVYCH,G., D. B. STEIN, and G. J. GASTONY. 1988. and America. I. Cytology. J. Linn. Soc., Bot. 58: Chloroplast DNA evolution and systematics of 13-25. Phanerophlebia(Dryopteridaceae) and related fern . 1961b. Contributions to the cytology and genera. Proc. Natl. Acad. Sci. U.S.A. 85:2589-2593.