Proc. Nati. Acad. Sci. USA Vol. 85, pp. 2589-2593, April 1988 Botany Chloroplast DNA evolution and systematics of Phanerophlebia (Dryopteridaceae) and related fern genera (restriction site variation/plant taxonomy/Polystichum/Cyrtomium) GEORGE YATSKIEVYCH*t, DIANA B. STEINt, AND GERALD J. GASTONY* *Department of Biology, Indiana University, Bloomington, IN 47405; and tDepartment of Biological Sciences, Mount Holyoke College, South Hadley, MA 01075 Communicated by Warren H. Wagner, Jr., December 21, 1987

ABSTRACT Restriction site variation in chloroplast DNA neotropical in distribution. Cyrtomium, in contrast, is pri- was examined in the neotropical fern genus Phanerophiebia marily Asiatic in distribution and contains ca. 15 taxa. and in selected species of the related Asiatic genus Cyrtomium Species relationships within each genus remain poorly un- and the cosmopolitan progenitor of these two, Polystichum. A derstood, and evolutionary affinities between the two groups total of 103 restriction site mutations was identified; these were are currently controversial (16). Two competing hypotheses used to construct phylogenetic networks and trees based on have been presented to explain the presence of Old and New Wagner and Dollo parsimony and Fitch-Margoliash distance World taxa of strikingly similar structures: (i) Phanerophle- algorithms. The analyses provided evidence that Phanerophie- bia and Cyrtomium evolved independently from different bia did not arise from Cyrtomium. Both genera are convergent ancestral stocks within Polystichum on different continents descendants from different progenitor groups in Polystichum, and developed similar structural features convergently (17). and Asiatic Cyrtomium is more closely related to temperate (ii) The two groups share direct ancestry; Cyrtomium species New World Polystichum than it is to neotropical Phanerophk- evolved from an Asiatic ancestor, and the American taxa bia. Reticulate venation, previously considered an important (submerged in Cyrtomium) represent a transoceanic disjunc- taxonomic character for infrageneric classification in Phaner- tional event (18). Structurally, the two segregates differ ophiebia, most likely evolved independently twice within the primarily in details of frond dissection and venation, the genus. Diploid maternal progenitors are suggested for two of evolutionary importance of which is difficult to assess, and four tetraploid species analyzed, and species-level distinctions the overall phenotypic similarity of the species obscures for two closely related taxa of Phanerophlebia are questioned. relationships between them. We undertook a study of chloroplast DNA variation as The chloroplast genome has been shown to possess great part of general systematic studies centering on the species of potential as a tool for the assessment of evolutionary rela- Phanerophlebia, in the hope that analysis of restriction site tionships among plants. Physical and gene mapping and/or mutations would clarify some poorly understood aspects of comparative analysis of restriction endonuclease site muta- the following topics: (i) What are the interrelationships tions have been used by many workers to study problems at among the taxa of Phanerophlebia? Information from this both specific and generic levels in various plant groups independent and highly conserved data source should help to (1-10). The latter approach is particularly well suited for assess the relative importance of the structural differences studies of evolutionary divergence within species com- currently used in species circumscriptions, provide a mea- plexes, and the highly conserved nature of chloroplast DNA sure of primary divergence among the diploid taxa, and and relatively low levels of homoplasy facilitate the con- suggest a potential maternal ancestor for each of the tetra- struction of relatively unambiguous phylogenies using cla- ploids (inheritance of the chloroplast genome has been distic analysis. determined to be uniparental in Polystichum; D.B.S. and D. To date, relatively few studies have been published on the Barrington, unpublished data). (ii) Are Cyrtomium and Phan- chloroplast DNA of pteridophytes. Most of these have erophlebia monophyletic; i.e., do the Asian and American aimed primarily at characterization of the genome and species groups share direct common ancestry (with subse- general structural comparisons of fern chloroplast DNA to quent transoceanic disjunction) from a single Polystichum that of gymnosperms or angiosperms (11-14). Exceptions ancestor, or are they convergent derivatives from different are ongoing research on evolution of the chloroplast ge- Old and New World ancestral stocks within Polystichum? nomes of Osmunda species (14, 15) and a recent study on (iii) What are the levels of point-mutational (site change) divergence between chloroplast DNAs of two Polystichum variation within the large and complex fern genus Polysti- species (D. and P. Soltis, personal communication). In this chum? A detailed investigation of this topic was not the paper we present a phylogenetic analysis of restriction site intent of this work, but comparisons among a small sample mutations among chloroplast DNAs of species in the genus of species showing different distributional patterns should Phanerophlebia and of selected taxa in the closely related provide preliminary information for further research on this fern genera Cyrtomium and Polystichum. subject. Polystichum Roth (Dryopteridaceae; Dryopterideae) com- prises a large and variable alliance of about 160 species and is nearly cosmopolitan in distribution (16). Most pteridolo- MATERIALS AND METHODS gists currently recognize several segregate genera, of which The 17 samples studied are listed in Table 1 and include all the structurally similar Phanerophlebia Presl and Cyrto- extant species ofPhanerophlebia. Two additional taxa found mium Presl contain the most species. Phanerophlebia, the in some of the recent literature (19) were not studied: focus of this study, comprises ca. 9 taxa and is primarily Phanerophlebia hautensis C. Chr., a Haitian endemic not recollected since the early 1900s, is now presumed to be The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" tPresent address: Missouri Botanical Garden, P.O. Box 299, St. in accordance with 18 U.S.C. §1734 solely to indicate this fact. Louis, MO 63166.

Downloaded by guest on September 28, 2021 2589 2590 Botany: Yatskievych et al. Proc. Natl. Acad. Sci. USA 85 (1988) Table 1. Samples used in this study Sample no. Species name Source of sample Phanerophlebia 1 *Ph. auriculata Underw. United States: Arizona (Yatskievych 83-161) 2 Ph. juglandifolia (Willd.) J. Smith Mexico: Chiapas (Yatskievych et al. 85-182) 3 *Ph. juglandifolia (Willd.) J. Smith Costa Rica: San Jose (Yatskievych and McCrary 86-13) 4 Ph. macrosora (Baker) Underw. Mexico: Oaxaca (Yatskievych and Gastony 86-329) 5 Ph. nobilis (Schlecht. & Cham.) Presl Mexico: Mexico (Yatskievych et al. 85-211) 6 *Ph. pumila (Mart. & Gal.) Fee Mexico: Oaxaca (Yatskievych et al. 85-209) 7 Ph. remotispora Fourn. Mexico: Veracruz (Yatskievych and Wollenweber 83-158) 8 Ph. umbonata Underw. Mexico: Nuevo Le6n (Yatskievych and Wollenweber 83-87) Cyrtomium 9 C. caryotideum (Hook. & Grev.) Presl United States: Hawaii, Oahu (Terry et al. in 1984) 10 C. falcatum (L. f.) Presl United States: South Carolina (Yatskievych and McCrary 83-184) 11 C. fortunei J. Smith Japan: Honshu, Shiga (Mitsuta in 1984) 12 C. macrophyllum (Makino) Tagawa Japan: Honshu, Shiga (Mitsuta in 1984) Polystichum 13 Po. acrostichoides (Michx.) Schott United States: Massachusetts (Sprague in 1986) 14 Po. imbricans (D. C. Eaton) D. H. Wagner United States: California (Yatskievych and McCrary 84-119) ssp. curtum (Ewan) D. H. Wagner 15 Po. lonchitis (L.) Roth United States: Arizona (Yatskievych and Windham 85-302) 16 Po. munitum (Kaulf.) Presl United States: California (Yatskievych and McCrary 84-131) 17 *Po. scopulinum (D. C. Eaton) Maxon United States: Arizona (Yatskievych and Windham 85-297) Asterisks indicate tetraploid Phanerophlebia and Polystichum taxa. Pressed vouchers of all samples are accessioned at the Deam Herbarium of Indiana University (IND) and include more precise locality data. extinct, whereas Phanerophlebia lindenii Fourn. of southern icut, Storrs). Because the large single-copy region of the Mexico has recently been designated a minor structural Lactuca chloroplast genome contains a 22-kilobase (kb) variant of the more common Ph. pumila (G.Y., unpublished inversion in comparison with the primitive genome organi- data). We also examined selected species of Cyrtomium and zation found in most other land plants (3), the three probes Polystichum for comparative purposes. Systematic relation- containing this portion of the genome were replaced with ships in these two genera are poorly understood, and taxa in three comparable fragments of Petunia (provided by J. these groups were chosen primarily on availability of suffi- Palmer, University of Michigan, Ann Arbor), to preserve cient plant material. The five Polystichum species chosen colinearity in our analyses. Labeling of probes with 32P by exhibit three distributional patterns: eastern United States nick-translation and filter hybridizations was done as de- (Po. acrostichoides), western United States (Po. imbricans, scribed (20, 23). Groups of 1-3 adjacent fragments (9.0-18.8 Po. munitum, Po. scopulinum), and circumboreal (Po. lon- total kb per group) were simultaneously hybridized to the chitis), though all are temperate, rather than neotropical, filters in eight sets sequentially around the circular genome. species. All species possess simply pinnate fronds, as in the Hybridizations were done at 55°C for 48 hr to allow adequate other two genera, except for Po. scopulinum, which has binding of heterologous probes and were visualized by more complex frond dissection. The sample set included autoradiography. A control lane ofpurified chloroplast DNA three tetraploid Phanerophlebia species and one tetraploid ofPh. remotispora on each gel was used to monitor potential Polystichum species (Table 1), all sexually reproducing nonspecific binding (none of which occurred). The reusable ferns. All four Cyrtomium species studied are naturally filters were treated with 0.4 M NaOH at 40°C between occurring apomictic polyploids of uncertain origin. successive hybridizations to remove previously bound Extractions were done on either fresh field-collected probes. fronds or on fronds of greenhouse-grown material trans- Data were analyzed using three different strategies. The planted from field populations. Amounts of leaf material computer program "Phylogenetic Analysis Using Parsi- available for extraction were limited, and yields of purified mony" (PAUP, version 2.4) developed by D. Swofford (Illi- chloroplast DNA from greenhouse-grown ferns are quite nois Natural History Survey, Urbana) was used for cladistic low. Chloroplast DNA was therefore only purified from a analysis of restriction site mutations. The "branch and single robust accession ofPh. remotispora as described (20), bound" option of this program was used to find the shortest modified by adding 10%o PEG 4000 to all buffers (12). Total phylogenetic networks by the Wagner parsimony algorithm; DNA was purified from all 17 samples as described above i.e., those requiring the least reversals and parallelisms to but without the sucrose gradient step. Total DNAs were account for the observed restriction fragment changes. Re- further purified by phenol and ether extractions (21). Re- sults were treated as unrooted networks rather than as striction endonucleases were purchased from Bethesda Re- rooted trees because a suitable outgroup for this study was search Laboratories and New England Biolabs, and diges- unavailable (see Results and Discussion). Site mutations tions were done according to suppliers' instructions. Elec- were also analyzed cladistically using the "Phylogeny Infer- trophoresis was on 0.7% horizontal agarose gels in 100 mM ence Package" computer program (PHYLIP, version 3.0) Tris HCl, pH 8.1/12.5 mM NaOAc/1 mM EDTA. Gels were developed by J. Felsenstein, University of Washington, run at 40-70 mA overnight until the bromphenol blue dye Seattle). The DOLMETRO option was used to find multiple markers had migrated 19 cm. The separated DNA fragments equally parsimonious trees by the Dollo parsimony algo- were then transferred to Zetabind nylon filters (AMF- rithm. Dollo parsimony discriminates against convergent site CUNO, Laboratory Products, Meriden, CT) (22). Filters gains (statistically improbable events) and favors convergent were baked at 80°C for 1 hr under vacuum. site losses, whereas Wagner parsimony treats these two Cloned chloroplast DNA fragments of Lactuca were used types of convergent site mutations as equally probable (24). as heterologous probes of our filter-bound restriction frag- The proportion of substitutions per nucleotide position, p ments (clones supplied by R. Jansen, University of Connect- (25), a measure of overall sequence divergence was calcu- Downloaded by guest on September 28, 2021 Botany: Yatskievych et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2591 lated for all pairs of samples in the data set. These values similar to divergence values for less-closely related species were analyzed using PHYLIP'S FITCH option, which con- pairs in some previously cited angiosperm-based studies. structs unrooted networks using the Fitch-Margoliash dis- Phylogenetic Inferences. Of the 103 total restriction site tance algorithm (26). changes documented, 57 were shared by two or more samples and thus informative for phylogenetic analysis; the RESULTS AND DISCUSSION remaining 46 changes were unique to individual taxa. The PAUP program was able to find six equally parsimonious, Approximately 305 restriction sites were surveyed for each shortest networks that differed from each other only in of the 17 chloroplast DNAs, or a total of 2.1% of each relative placement of the four Cyrtomium taxa with respect chloroplast genome (ca. 140 kb, based on calculations from to each other (Fig. 1). These networks each required 134 our restriction digests). Samples differed, both within and steps, or 31 extra mutations, to group the samples most between genera, in a variety of minor size changes (<500 parsimoniously. The 31 additional steps involved 11 extra base pairs each), which are not further discussed here. A site gains, 11 extra site losses, 6 gain/loss reversions, and 3 detailed summary of the total 103 restriction site mutations loss/gain reversions for a total rate of parallelisms or con- in the data set will be published elsewhere (27). At least vergences of 30.1%. This overall value is six to fifteen times some samples contained restriction site changes for all 13 of as great as interspecific levels calculated for some angio- the enzymes used, with the lowest incidence of mutation sperm studies cited above and is surprisingly large for data present in the inverted-repeat portions of the genomes. The derived from chloroplast DNA analysis. Such high rates of number of restriction site differences between species pairs parallelisms or reversals can be partially explained by the is indicated in Table 2. fact that we were examining variation at the generic as well Genetic Distance Measures. The proportion of substitu- as specific levels and suggest that the three involved genera tions per nucleotide position, p (25), estimated from the have diverged relatively long ago, in spite of the relatively proportion of site differences between any pair of the 17 low intergeneric divergence values here calculated (Table 2). sample DNAs, was significantly different at interspecific and When asked for "nearly most parsimonious" networks intergeneric levels of analysis (Table 2). Values of diver- only one step longer than the shortest (134-step) trees, the gence ranged from 0 to 0.0107 for species pairs within any of PAUP program found a bewildering array of 101 additional the three genera, with an average of 0.0063. Compared with networks of 135 steps each. These networks differed in data from available studies involving angiosperms, these branch lengths and species relationships for some samples divergence levels are similar to those found in Pennisetum within each genus, but with one exception retained the three (1), Lycopersicon (7), and some comparisons in Brassica (5), genera as distinct lineages. Approximately a fifth of the generally less than other comparisons in Brassica (5) and networks involved some arrangement that grouped Po. maximum divergence levels in Linum (2) and Clarkia (8, 9), imbricans with the Cyrtomium lineage and separate from the and greater than those for closely related species of Lisian- other Polystichum samples. This result agrees with the thius (10) and Pisum (4). Values for comparisons of species relatively low levels of intergeneric sequence divergence pairs of Phanerophlebia/Cyrtomium ranged from 0.0160 to cited above, which also suggest that Cyrtomium is more 0.0209, those for pairs of Phanerophlebia/Polystichum closely related to Polystichum than is Phanerophlebia, but ranged from 0.0137 to 0.0191, and those for pairs of Cyrto- casts doubt on the apparently great age of each genus as mium/Polystichum ranged from 0.0107 to 0.0154, with aver- suggested by levels of parallelisms or convergences. age sequence-divergence values of 0.0181, 0.0156, and Dollo parsimony analysis resulted in three equally parsi- 0.0136, respectively. These averages are two to three times monious trees, again differing from one another only in as large as the average divergence among species pairs position among the Cyrtomium species. These trees were within each genus but are only half as large as divergence topologically similar to the networks generated by Wagner values calculated for Lactuca/Barnadesia (3), far smaller parsimony (Fig. 1), but differed in three ways: (i) Po. than those for three legume genera (6), and are, in fact, scopulinum and Po. lonchitis diverged from the same node Table 2. Sequence differences among chloroplast DNAs from Phanerophlebia, Cyrtomium, and Polystichum species Phanerophlebia Cyrtomium Polystichum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 - 7 17 9 3 7 1 9 31 36 29 29 28 27 28 28 28 2 0.39 - 0 10 17 17 12 16 33 34 32 31 27 28 33 28 26 3 0.96 0.00 - 10 17 17 12 16 33 34 32 31 27 28 33 28 26 4 0.50 0.56 0.56 - 7 11 8 12 31 34 30 28 26 25 27 25 27 5 0.16 0.96 0.96 0.39 - 8 2 10 28 35 30 28 27 27 27 24 27 6 0.39 0.96 0.96 0.61 0.44 - 6 8 30 33 28 28 27 24 28 26 31 7 0.05 0.67 0.67 0.44 0.11 0.33 - 8 28 35 30 27 27 27 28 29 26 8 0.50 0.90 0.90 0.67 0.56 0.44 0.44 - 35 35 32 33 25 27 26 27 29 9 1.79 1.91 1.91 1.79 1.60 1.73 1.60 2.03 - 14 10 9 25 23 27 23 22 10 2.09 1.97 1.97 1.97 2.03 1.91 2.03 2.03 0.78 - 14 12 23 22 25 24 26 11 1.67 1.85 1.85 1.73 1.73 1.60 1.73 1.85 0.56 0.78 - 7 24 22 26 25 26 12 1.67 1.79 1.79 1.60 1.60 1.60 1.54 1.91 0.50 0.67 0.39 - 26 19 25 24 22 13 1.60 1.54 1.54 1.48 1.54 1.54 1.54 1.43 1.43 1.31 1.37 1.48 - 16 14 10 15 14 1.54 1.60 1.60 1.43 1.54 1.37 1.54 1.54 1.31 1.25 1.25 1.07 0.90 - 14 15 18 15 1.60 1.91 1.91 1.54 1.54 1.60 1.60 1.48 1.54 1.43 1.48 1.43 0.78 0.78 - 14 16 16 1.60 1.60 1.60 1.43 1.37 1.48 1.67 1.54 1.31 1.37 1.43 1.37 0.56 0.84 0.78 - 19 17 1.60 1.48 1.48 1.54 1.54 1.79 1.43 1.67 1.25 1.48 1.48 1.25 0.84 1.01 0.90 1.07 The number of restriction site mutations between any two chloroplast DNAs appears in the upper right portion of the matrix. Total number of restriction sites surveyed per species was ± 305. Values of divergence (25) appear as 100 x p in the lower left portion of the matrix. Samples are numbered as in Table 1. Downloaded by guest on September 28, 2021 2592 Botany: Yatskievych et al. Proc. Natl. Acad. Sci. USA 85 (1988) States and were until relatively recently regarded as conspe- cific. Our chloroplast DNA data agree with similar observa- tions of D. Soltis and P. Soltis (personal communication) and LI .. caryotideum confirm the significance of structural details of rhizome anatomy and vestiture emphasized in recent classifications of this species complex (28). In contrast, in all three analyses Po. munitum is evolutionarily closest to Po. acrostichoides of the eastern United States, a not unexpected result based on structural similarities. A more detailed examination of a larger number of Polystichum species would help elucidate imbricans the presumed vicariant event that resulted in the evolution of closely related species in the eastern and western United States from some, perhaps circumboreal, ancestor. Within Phanerophlebia, the two most closely related -lonchitis diploid taxa are Ph. nobilis and Ph. remotispora, the DNAs Polystichum of which differed at only two restriction sites (Table 2). These two widely distributed Mexican taxa share broad auriculata (4x) sympatry and are structurally indistinguishable, except for remotispora reticulate venation in the latter. The presence or absence of nobilis regular marginal reticulations (as opposed to free vein-ends) pumila (4x) has universally been regarded by taxonomists as a funda- umbonata mental character in the classification of Phanerophlebia _ macrosora species (16). Recent studies, however, have indicated that the two reticulate-veined species, Ph. remotispora and Ph. juglandifolia, are quite distinct structurally, cytologically, and biochemically (G.Y., unpublished data). These results, 0 5 10 15 20 25 30 together with those from the present chloroplast DNA mutations (steps) analysis, in which these species are widely separated within the genus (Fig. 1), suggest that reticulate venation has FIG. 1. Unrooted Wagner parsimony network, redrawn from the evolved independently in diploid and tetraploid Ph. jugland- output of the PAUP computer program. Six equally parsimonious ifolia and in Ph. remotispora. The very low level of shortest networks of 134 steps each were generated, different from chloro- one another in relative placement of species within the Cyrtomium plast DNA divergence is consistent with arguments from lineage (data not shown). Dollo parsimony and Fitch-Margoliash structure and biogeography that Ph. remotispora and Ph. distance algorithms resulted in three equally parsimonious shortest nobilis might best be regarded as conspecific varieties. trees and a single unrooted network, respectively, that were all Restriction analysis was able to locate potential diploid topologically similar to the diagrammed network (see text for progenitors for only two of the four sexual tetraploid taxa in discussion of differences). the study set. Po. scopulinum differs by at least 15 chloro- plast DNA mutations from any other species studied (Table and only later from each other, rather than at separate 2), including 7 autoapomorphies. Po. scopulinum differs by adjacent nodes, within the Polystichum lineage; (ii) Ph. 18 mutations from Po. imbricans, which had been implicated pumila and Ph. umbonata diverged at separate adjacent as one of its parents (28) and is relatively remote from this nodes, rather than together, within the Phanerophlebia species in its placement on the most parsimonious tree (Fig. lineage; (iii) Ph. juglandifolia and Ph. macrosora diverged 1). Unfortunately, material of Polystichum lemmonji together, rather than at separate adjacent nodes within the Underw., the other suggested parental taxon, was unavail- Phanerophlebia lineage. The longest branch of the trees able for study. separated the Phanerophlebia and Cyrtomium lineages, as Because all extant species of Phanerophlebia were sam- was also true with the Wagner parsimony networks (Fig. 1). pled in this study, our failure to identify a potential parental The shortest Dollo parsimony trees required 43 extra steps taxon for Ph. pumila was unexpected. Based on sequence- (site losses) to account for the data. This equates to 29 extra divergence values (Table 2), this free-veined tetraploid convergent site losses over the 14 statistically improbable shares closest affinity with net-veined Ph. remotispora, from extra site gains or loss/gains present in the shortest Wagner which it differs by six site mutations, including one auto- parsimony networks. apomorphy. A more likely progenitor is free-veined Ph. The Fitch-Margoliash distance analysis resulted in a sin- nobilis, which differs by eight total site mutations and is gle unrooted network that was also topologically similar to probably conspecific with Ph. remotispora (see above). In those generated by the Wagner parsimony method. As with the most parsimonious Wagner networks Ph. pumila is the Dollo parsimony trees, this network differed from the grouped with Ph. umbonata, an unlikely parent based on Wagner trees (Fig. 1) in minor relative branch lengths and in biogeographic and structural characters. To account for the joint divergence of Ph. juglandifolia and Ph. macrosora divergence of this unusual tetraploid from potential diploid within the Phanerophlebia lineage. In the Polystichum lin- progenitors in the study set are two explanations; (i) the eage, Po. scopulinum branched at one node below the Po. hybridization event involving extant diploid progenitors of lonchitis branch, rather than at one node above it. As in the Ph. pumila occurred sufficiently long ago that its chloroplast previous two analyses, the longest branch was that separat- genome has had time to accumulate the observed site ing the Phanerophlebia and Cyrtomium lineages. mutations; and (ii) the maternal progenitor of Ph. pumila is Species Level Systematics. Although our phylogenetic anal- extinct and therefore was not sampled. ysis could not resolve relationships among the species of In contrast, the other two tetraploid Phanerophlebia spe- Cyrtomium, several interesting patterns were apparent cies were readily linked to diploid taxa. The chloroplast within the other two genera, as seen in Fig. 1. Po. imbricans DNA of tetraploid Ph. juglandifolia was exactly identical at diverges basally with respect to the other Polystichum taxa, all restriction sites to that of diploid Ph. juglandifolia. including the morphologically very similar Po. munitum. Tetraploid Ph. auriculata differed by only a single autoapo- These two taxa share broad sympatry in the western United morphy from Ph. remotispora and by only two additional Downloaded by guest on September 28, 2021 Botany: Yatskievych et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2593

mutations from Ph. nobilis. These two potential parents are study have diverged a relatively long time ago, in spite of the probably conspecific (see above), and the node separating fact that levels of sequence divergence calculated for species Ph. auriculata and its potential maternal progenitors from pairs between genera in this study were found to be lower other Phanerophlebia species is a trichotomy in the Wagner than those in similar intergeneric studies of various angio- networks generated by the PAUP program (Fig. 1), as well as sperm groups. in the trees generated by Dollo parsimony methods. Generic Level Systematics. The relatively low levels of Our research benefited greatly from Lilian Hsu's advice and sequence divergence between intergeneric species pairs rel- encouragement. We also appreciate the assistance of Jeffrey Palmer and Robert Jansen, who provided clones for this study and helped ative to similar values for generic level analyses in angio- with both laboratory techniques and data analysis. Joseph Felsen- sperms (see above) were surprising and cast some doubt on stein kindly clarified some aspects of the algorithms used in PHYLIP, the validity of recognizing Cyrtomium and Phanerophlebia while Warren Kovach showed great patience in helping with com- as genera distinct from Polystichum. The present study puter analysis. Portions of this research were funded by National shows, however, that based on chloroplast DNA restriction Science Foundation Grant for Improving Doctoral Research BSR site mutations, the derivatives, Phanerophlebia and Cyrto- 86-01305 (G.Y., D.B.S., and G.J.G.) and National Science Founda- mium, do not share direct common ancestry from a single tion Grant BSR 85-16666 (G.J.G.). Funding by a William and Flora progenitor within Polystichum. These two groups show less Hewlett Foundation Grant of Research Corporation to D.B.S. is affinity to each other than either does to the taxa sampled in also gratefully acknowledged, as are an Indiana University Alumni ancestral genus. Our data clearly reject the Off-Campus Research Fellowship and grants-in-aid from the Indiana the presumed Academy of Science, Indiana University Graduate School and hypothesis that a putative transoceanic disjunctional event Department of Biology, and the National Academy of Sciences accounts for the present-day similarities between Phaner- Joseph Henry Fund (administered by Sigma Xi, the Scientific ophlebia and Cyrtomium. Therefore, if these derivatives Research Society) (G.Y.). should continue to be recognized as taxonomically distinct from Polystichum, they should both be maintained as sepa- 1. Clegg, M. T., Rawson, J. R. Y. & Thomas, K. (1984) Genetics rate genera showing convergent morphology. This study 106, 449-461. cannot directly answer whether these genera should both be 2. Coates, D. & Cullis, C. A. (1987) Am. J. Bot. 74, 260-268. taxonomically reincorporated into Polystichum because the 3. Jansen, R. K. & Palmer, J. D. (1987) Curr. Genet. 11, 553-564. Polystichum species sampled apparently do not account for 4. Palmer, J. D., Jorgensen, R. A. & Thompson, W. F. (1985) amounts of the restriction site variation within Genetics 109, 195-213. significant 5. Palmer, J. D., Shields, C. R., Cohen, D. B. & Orton, T. J. genus. above, levels of nucleotide this large As noted (1983) Theor. Appl. Genet. 65, 181-189. divergence for intergeneric species pairs here are generally 6. Palmer, J. D., Singh, G. P. & Pillay, D. T. N. (1983) Mol. Gen. lower than for similar generic level studies involving angio- Genet. 190, 13-19. sperms; yet, these intergeneric divergence levels are signif- 7. Palmer, J. D. & Zamir, D. (1982) Proc. Natl. Acad. Sci. USA icantly higher than for species pairs within each genus (Table 79, 5006-5010. 2). For the present, we prefer to retain the three lineages as 8. Sytsma, K. J. & Gottlieb, L. D. (1986) Proc. Natl. Acad. Sci. distinct genera until further studies resolve questions of USA 83, 5554-5557. evolution within Polystichum on a worldwide scale. 9. Sytsma, K. J. & Gottlieb, L. D. (1986) Evolution 40, 1248-1261. General Conclusions. Previous studies involving compara- 10. Sytsma, K. J. & Schaal, B. A. (1985) Evolution 39, 594-608. site mutations in various angio- 11. Herrmann, R. G., Palta, H. K. & Kowallik, K. V. (1980) Planta tive analysis of restriction 148, 319-327. sperms have emphasized the advantages of chloroplast DNA 12. Palmer, J. D. & Stein, D. B. (1982) Curr. Genet. 5, 165-170. for constructing evolutionary inferences. The present study 13. Palmer, J. D. & Stein, D. B. (1986) Curr. Genet. 10, 823-833. documented higher levels of homoplasy and somewhat 14. Stein, D. B., Palmer, J. D. & Thompson, W. F. (1986) Curr. greater resultant numbers of equally parsimonious trees than Genet. 10, 835-841. did preceding reports; our resolution of species level diver- 15. Stein, D. B. (1985) Proc. R. Soc. Edinburgh Sect. B 86, 283- gence was therefore slightly hampered. Our somewhat arbi- 288. trary choice ofPolystichum species for comparative analysis 16. Tryon, R. M. & Tryon, A. F. (1982) Ferns and Allied Plants to these problems as may have our inability to (Springer, New York), pp. 510-515 and 524-533. contributed 41-52. to our sample species. More 17. Christensen, C. (1930) Am. Fern J. 20, find an adequate outgroup 18. Copeland, E. B. (1947) Genera Filicum (Chronica Botanica, detailed general taxonomic knowledge of species limits and Waltham, MA), pp. 110-111. infrageneric classification in Polystichum on a worldwide 19. Smith, A. R. (1981) in Flora of Chiapas, ed. Breedlove, D. E. basis than presently available is a prerequisite for proper (California Acad. Sci., San Francisco), Part 2, pp. 166-167. selection of species from this large and variable group for 20. Palmer, J. D. (1982) Nucleic Acids Res. 10, 1593-1605. expanding this research. 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular We were unable to completely analyze the data for some Cloning (Cold Spring Harbor Lab., Cold Spring Harbor, NY), restriction enzymes because of the complex patterns of pp. 458-460. variation present. Several independent mutations involving 22. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. of DNA in different taxa, which could not 23. Palmer, J. D. (1986) Methods Enzymol. 118, 167-186. the same stretch 24. DeBry, R. W. & Slade, N. A. (1985) Syst. Zool. 34, 21-34. all be scored conveniently, were seen in a minority of our 25. Nei, M. & Li, W.-H. (1979) Proc. Natl. Acad. Sci. USA 76, blots. To this extent, our sequence-divergence values prob- 5269-5273. ably represent underestimates of the actual evolutionary 26. Felsenstein, J. (1984) Evolution 38, 16-24. divergence between both species and genera. Such problems 27. Stein, D. B., Yatskievych, G. & Gastony, G. J., Biochem. Syst. of overlapping independent mutations and high levels of Ecol., in press. homoplasy suggest that the evolutionary lineages under 28. Wagner, D. H. (1979) Pteridologia 1, 1-64. Downloaded by guest on September 28, 2021