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Department of Botany, University of Kansas, Lawrence, Kansas 66045
Received for publication December 18, 1997. Accepted for publication October 13, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Key Words: Botrychium • breeding systems • isozymes • Ophioglossaceae • polyploids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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; Tryon and Tryon, 1982
; Wagner and Wagner, 1993
). Using morphological features, Clausen (1938)
recognized only six species in subgenus Botrychium, whereas W. H. and F. S. Wagner and others have raised that number to 
24 species based on a combination of morphological and cytological studies (F. S. Wagner, 1993
; Wagner and Wagner, 1981
, 1983a
, b
, 1986
, 1990a
, b
, 1994
; Wagner et al., 1984
; Farrar and Johnson-Groh, 1991
; see Table 1, Figs. 1–2
).
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). Cryptic species were defined by Stebbins (1950
, p. 193) as "...population systems which were believed to belong to the same species until genetic evidence showed the existence of isolating mechanisms separating them." Cryptic species are characterized by the following criteria (Paris, Wagner, and Wagner, 1989
): (1) poor morphological differentiation; (2) distinct evolutionary lineages that are reproductively isolated; and (3) historic misinterpretation as members of a single species.
Many species of subgenus Botrychium satisfy these criteria. First, they are notoriously difficult to differentiate without considerable field experience. Second, detection of ploidy level differences and sterile hybrids (Wagner, 1980
, 1991
; Wagner et al., 1984
; Wagner, Wagner, and Beitel, 1985
; Wagner and Wagner, 1988
) has provided evidence of reproductive isolation and hybridization. Third, the majority of recently recognized Botrychium species were once considered to be minor morphological variants within single species. For example, B. minganense, B. spathulatum, and B. ascendens were all considered insignificant variants of B. lunaria.
As emphasized by Stebbins (1950)
and Paris, Wagner, and Wagner (1989)
, cryptic species must be genetically distinct or they cannot be considered natural evolutionary units. Molecular features such as isozymes can provide markers to help substantiate the genetic distinctness of putative cryptic species (Good, 1989
). In ferns, isozyme studies have led to the recognition of new "cryptic" species in the Adiantum pedatum complex (Paris and Windham, 1988
; Paris, 1991
), Gymnocarpium (Pryer and Haufler, 1993
), and Polypodium (Haufler and Windham, 1991
). In each of these cases, isozymic characterization supported hypotheses that diploid progenitors of tetraploid species were distinct species, rather than infraspecific variants (i.e., Adiantum aleuticum (Ruprecht) Paris
, Gymnocarpium appalachianum Pryer and Haufler
, and Polypodium appalachianum Haufler and Windham
).
In this study, we used isozyme variation to evaluate species and sectional concepts within subgenus Botrychium and compared the results of these analyses to those of morphological and rbcL studies. These isozyme data provided evidence for (1) assessing relationships among diploid species, (2) characterizing the breeding system(s) of diploid species, (3) identifying possible diploid progenitors of polyploid species, and (4) evaluating hypotheses concerning classification of subgenus Botrychium species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Isozyme banding patterns were obtained using starch gel electrophoresis for the following enzymes: aspartate amino transferase (AAT), diaphorase (DIA), fructose 1,6-bis-phosphatase (F1-6), glyceraldehyde 3-phosphate dehydrogenase (G3PDH), isocitrate dehydrogenase (IDH), leucine amino peptidase (LAP), malate dehydrogenase (MDH) phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6PGD), shikimate dehydrogenase (SKDH), and triosephosphate isomerase (TPI). Band resolution was best for TPI, PGM, and GOT on system 6 of Soltis et al. (1983)
, for PGI, LAP, IDH, and DIA on system 8 (Haufler, 1985
), for G3PDH, F1-6, 6PGD, MDH, and SKDH on either system 11 (Haufler, 1985
) or a morpholine/citrate system (Werth, 1985
). Of the 12 enzyme systems surveyed, five yielded consistent, interpretable banding patterns for most populations (TPI, PGI, 6PGD, MDH, and DIA). Data from the other seven loci (Aat, F1-6, G3pdh, Idh, Lap, Pgm, and Skdh) were not included in the analyses. Photographs were taken of all gels using Kodak Technical Pan film and a red filter. Genetic interpretations of banding patterns assumed that subunit structure and cellular compartmentalization in Botrychium are similar to those of other plants (Gottlieb, 1982
). Loci and alleles were numbered sequentially from the most anodal to the most cathodal (e.g., Mdh-1, Mdh-2, etc.). Loci are reported using standard numbers (e.g., 2, 3), whereas alleles are denoted by superscripts (e.g., 1,1). All band profiles presented in Table 3 reflect the presumed ploidy of individual sporophytes. Thus, diploids are shown with two alleles (e.g., 2,2) at a single locus, whereas tetraploids have four alleles (e.g., 2,2;3,3), two at each of the two duplicated loci, and hexaploids have six alleles across three loci (e.g., 2,2;3,3;4,4). However, in the single hexaploid species, B. pseudopinnatum Wagner, banding patterns were identical to those seen in tetraploids and the genetic constitution of the two alleles presumably encoded at a third locus could not be inferred and are reported in Table 3 as (?,?). Homozygotes (Fig. 3) have two copies of the same allele (1,1), whereas heterozygotes (Fig. 4) have different alleles (1,3). Allelic heterozygosity (1,3) segregates during meiosis, whereas fixed interlocus heterozygosity (1,1;3,3) does not. When heterozygosity was fixed (i.e., all sporophytes sampled revealed the same multiple-banded pattern; Fig. 4) in a species known to be polyploid, it was assumed that the heterozygous patterns resulted from duplicate gene expression rather than allelic heterozygosity at a single locus. Because Botrychium gametophytes are difficult to culture (Whittier, 1981
), studies of allelic segregation to verify the interpretation of banding patterns were not attempted.
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). Botrychium lineare was the only species included in this study for which ploidy was not known. The banding patterns of individuals of B. lineare observed on the isozyme gels were typical of diploids (i.e., no fixed heterozygosity at any locus) and B. lineare was assumed to be diploid for the purpose of data analysis. BIOSYS-L was used to calculate five statistics of genetic variation (Tables 4–6): P (the percentage loci polymorphic), A (the mean number of alleles per locus), H (the mean expected heterozygosity), I (Nei's [1978]
genetic identity), and D (Nei's [1978]
genetic distance). UPGMA cluster analyses were performed on isozyme profiles of all diploid populations.
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) polyploid banding patterns were analyzed by reconstructing ancestral diploid genotypes. Ranker et al. (1989)
and Werth, Hilu, and Langner (1994)
compared isozyme patterns of diploids and polyploids by reconstructing two hypothetical, ancestral diploid genotypes from tetraploid isozyme patterns. The hypothetical diploid genotypes were analyzed with diploid isozyme profiles in UPGMA cluster analyses to establish genetic affinities and generate hypotheses of polyploid origins.
Before reconstruction of ancestral isozyme profiles from polyploid banding patterns, it was necessary to establish isozyme profiles for the diploid species surveyed (Table 8). To simplify computations, we selected the most common isozyme profile(s) observed in each diploid species. However, we excluded diploid populations with patterns of fixed heterozygosity because they may have been of hybrid origin (see below). All populations were standardized to a sample size of ten individuals to minimize the influence of sample size on the cluster analyses.
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and Werth, Hilu, and Langner (1994)
, we separated isozyme patterns from each polyploid into a "C" or chloroplast parental genotype and an "N" or nonchloroplast parental genotype (Table 9). Although maternal inheritance of the cpDNA has not been demonstrated in subgenus Botrychium, Gastony and Yatskievych (1992)
showed that in cheilanthoid ferns inheritance of the cpDNA is maternal. Hauk (1995)
found that polyploid subgenus Botrychium species possessed a single rbcL gene sequence and that the sequence is similar or identical to that of a single diploid. Thus, the probable chloroplast parent of many subgenus Botrychium polyploids was identified. In most cases, the isozyme profile of the diploid identified as the chloroplast parent by rbcL data served as the basis for reconstruction of hypothetical, ancestral genotypes for each polyploid (see the Appendix).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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Pgi
Two banding regions were present (Pgi-1 and Pgi-2; Figs. 3–4), but the more anodal region (Pgi-1) did not yield consistently interpretable bands for all taxa and was not included in the analyses. Five alleles were observed in Pgi-2 among the 16 species surveyed. Pgi-2 was monomorphic within six species [B. lunaria (L.) Swartz, B. crenulatum Wagner, and B. pinnatum St. John contained Pgi-24; B. spathulatum Wagner, B. pumicola Coville, and B. lineare had Pgi-23]. One of two different alleles was fixed in populations of B. ascendens Wagner (Pgi-24 or 1) and B. simplex (Pgi-23 or 4). Pgi-23,3:5,5 and 2,2:5,5 heterozygotes were present in B. hesperium, and B. acuminatum, whereas B. matricariifolium contained Pgi-21,1:5,5 and 2,2:5,5 heterozygotes. Pgi-22,2:5,5 heterozygotes were observed but not fixed in B. lanceolatum, whereas Pgi-22,2:5,5 was fixed in B. pedunculosum and B. echo. Botrychium pseudopinnatum was the only species with a fixed Pgi-22,2:4,4 pattern. Botrychium minganense was the most variable species and the only taxon that possessed all five Pgi-2 alleles. Most populations were fixed for either Pgi-2 3,3;3,3, Pgi-24,4:4,4 or Pgi-25,5:5,5. However, population 2 of B. minganense was uniformly heterozygous (Pgi-22,2;4,4) and populations 1 and 10 contained both homozygotes (Pgi-21,1:1,1 or 4,4:4,4) and heterozygotes (Pgi-21,1:4,4).
Tpi
Other studies have demonstrated that Tpi is compartmentalized in plant cells (Gottlieb, 1982
: Gastony and Darrow, 1983
), and we interpreted the two banding regions present in subgenus Botrychium Tpi patterns as separate loci, the more anodal region designated Tpi-1, and the more cathodal set Tpi-2. In contrast to nearly all other pteridophytes, subgenus Botrychium species had simple banding patterns. There was no evidence of either duplications or post-translational modifications of either Tpi locus (see Gastony, 1988
; Hickey, Guttman, and Eshbaugh, 1989
). However, Watano and Sahashi (1992)
reported multiple Tpi banding patterns in four species of Botrychium subgenus Sceptridium and interpreted the bands as duplicated loci. The absence of multiple Tpi banding patterns in subgenus Botrychium may indicate the reversal of a pleisiomorphic Tpi duplication common to other pteridophytes. Tpi-1 had three alleles and was the only locus that showed no infraspecific variability. Tpi-11 was present for B. simplex, and Tpi-13 was observed in B. lunaria and B. pumicola. All other species were monomorphic for Tpi-12.
Most taxa were monomorphic for Tpi-22 (B. lanceolatum, B. matricariifolium, B. echo, B. hesperium, B. acuminatum, B. pedunculosum, B. pinnatum, B. pseudopinnatum, B. pumicola, B. crenulatum, and B. lineare), but Tpi-21 was present in B. simplex. Six populations of B. lunaria were monomorphic for Tpi-23 and population 5 was heterozygous (Tpi-22,3). Most individuals of B. minganense were Tpi-22,2;3,3 heterozygotes, although a few Tpi-23,3;3,3 and/or 2,2;2,2 homozygotes were observed in populations 3, 4, 6, and 8. Populations 5, 7, 9, and 11 of B. minganense were fixed for Tpi-22,2;2,2.
Dia
There were several regions of Dia activity on the gels, although the most anodal set of bands (Dia-1) was the only one that was resolved consistently. Two alleles were detected. Dia-12 was monomorphic in nine taxa (B. lunaria, B. crenulatum, B. ascendens, B. spathulatum, B. matricariifolium, B. acuminatum, B. pseudopinnatum, B. pumicola, and B. lineare). Ten of the 11 populations of B. minganense were fixed for Dia-12,2;2,2; Dia-13 was observed in population 1 of B. minganense and was heterozygous (Dia-11,1;3,3). Depending on ploidy, Dia-11,2 or Dia-11,1;2,2 heterozygotes were present in B. lanceolatum, B. echo, B. hesperium, B. pedunculosum, and B. pinnatum. Five populations (four of B. lanceolatum and one of B. simplex) were monomorphic for Dia-11,1, but the allele was never monomorphic across all populations of a single species.
Mdh
Mdh had several regions of activity on the gels, but only the most anodal set of bands was consistently interpretable (Mdh-1). Three alleles were distributed among the 16 species surveyed. Mdh-11 was monomorphic in six species (B. simplex, B. pumicola, B. crenulatum, B. lineare, B. spathulatum, and B. echo). The most variable species was B. minganense, which possessed individuals monomorphic for each of the three Mdh-1 alleles. The majority of B. minganense populations had fixed Mdh-11,1;3,3 patterns, and population 10 was fixed for Mdh-11,1;2,2. Botrychium matricariifolium, B. acuminatum, and B. pseudopinnatum had invariant Mdh-11,1;2,2 patterns. Six of seven populations of B. lanceolatum were monomorphic for Mdh-12, and Mdh-11,2 was fixed in population 2. Five of seven populations of B. lunaria were monomorphic for Mdh-13; populations 4 and 5 had fixed Mdh-11,3 patterns. One population each of B. hesperium and B. pinnatum was fixed for Mdh-12,2;2,2, and the second population of each showed fixed Mdh-11,1;2,2 patterns. Of the three B. ascendens populations, one had a Mdh-11,1;3,3 pattern, and the other two were fixed for Mdh-11,1;2,2.
6Pgd
Two 6Pgd banding regions have been reported in many taxa (Gottlieb, 1982
). In Botrychium, however, only the more cathodal of the two areas (6Pgd–2) produced consistently interpretable bands. Four alleles were detected across all species. Four taxa were monomorphic: B. pumicola and B. lineare for 6Pgd–21, B. pedunculosum for 6Pgd–23, and B. crenulatum for 6Pgd–24. Botrychium simplex was homozygous (6Pgd–21,1) in all populations except population 1, which was heterozygous (6Pgd–21,2). Botrychium matricariifolium, B. echo, B. hesperium, and B. acuminatum were heterozygous for 6Pgd–21,1;2,2. Botrychium ascendens, B. spathulatum, and B. pseudopinnatum were fixed for 6Pgd–21,1;4,4. 6Pgd–23,3;4,4 patterns were found in only one species, B. pinnatum. Botrychium minganense was heterozygous (6Pgd–21,1;4,4) in all but population 5 where it was homozygous (6Pgd–21,1;1,1). Five populations of B. lanceolatum showed fixed 6Pgd–22,2 patterns and two populations had 6Pgd–21,2 patterns. Botrychium lunaria was homozygous (6Pgd–24,4) in five populations and heterozygous (6Pgd–21,2) in two.
Statistics of genetic variation
Across all species there was a mean of 3.33 alleles per locus. Within species, however, there was a mean of 1.30 alleles per locus (diploids = 1.08, tetraploids = 1.45, hexaploid = 1.50, unknown ploidy = 1.00). Mean heterozygosity per locus (H) for diploids was 3.5%, for tetraploids 21.6%, for the hexaploid B. pseudopinnatum 28.6%, and for B. lineare (uncertain ploidy) 0% (Table 4). The percentage of loci polymorphic (P) was 7.33 for diploids, 43.01 for tetraploids, 50.0 for the hexaploid, and 0.0 for B. lineare (Table 4).
Infraspecific values of Nei's unbiased genetic identity and distance were calculated among 21 populations of four diploid species: five populations of B. simplex, seven of B. lunaria, seven of B. lanceolatum, and two of B. crenulatum (Table 5). These diploids represented three of the sections proposed by Wagner and Wagner (Table 1). Infraspecific genetic identity values ranged from 0.67 to 1.00 with a mean of 0.912. Interspecific values were computed, based on mean allele frequencies, from the same database as infraspecific comparisons, except for the addition of single populations of B. lineare and B. pumicola (Table 6). The values for interspecific genetic identity ranged from 0.045 to 0.833 with a mean of 0.409.
Chi-square analysis for goodness-of-fit to Hardy-Weinberg equilibrium was calculated for the genotype frequencies (Pgi-2) observed within population 4 of B. simplex and populations 1, 2, and 7 of B. lanceolatum (Table 7). All chi-square values were significant (P < 0.05) when compared to the critical value, and the null hypothesis of random mating was rejected.
UPGMA cluster analysis of interpopulational genetic identities produced a dendrogram of diploid population relationships (Fig. 5). Populations of B. lanceolatum, B. lunaria, B. crenulatum, and B. simplex associated most strongly with other populations of the same species. Populations of B. lanceolatum had low genetic identities with all other populations included in the analysis. Populations of B. crenulatum clustered more closely to populations of B. lunaria than to populations of any other species. The single populations of B. lineare and B. pumicola were more similar to each other than to populations of any other species.
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genetic identities were conducted on the reconstructed hypothetical diploid genotypes plus the diploid isozyme profiles. A single analysis of all 73 diploid and hypothetical diploid genotypes was not conducted because BIOSYS-L permits simultaneous analysis of only 60 populations. To circumvent this limitation, we conducted four separate analyses: (1) diploids plus hypothetical genotypes from B. spathulatum and B. ascendens, (2) diploids plus hypothetical genotypes from B. minganense, (3) diploids plus hypothetical genotypes from B. matricariifolium, B. acuminatum, B. hesperium, and B. echo, (4) diploids plus hypothetical genotypes from B. pedunculosum, B. pinnatum, and B. pseudopinnatum (Tables 8, 9).
Reconstructed genotypes from the tetraploids B. spathulatum and B. ascendens were included in the same cluster analysis (Fig. 6) because they belonged to the same rbcL subclade (Hauk, 1995
). The B. ascendens 1 N genotype was identical to profiles of B. crenulatum populations. The B. ascendens 2 and 3 N genotypes grouped closely to B. crenulatum profiles. All three B. ascendens C genotypes clustered and formed a group independent of any sampled diploid. The B. spathulatum C genotype associated with B. lineare, and the N genotype grouped closest to the B. ascendens N genotypes and the B. crenulatum profiles.
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) and were included in the same cluster analysis (Fig. 8). The hypothetical B. matricariifolium, B. echo, and B. hesperium C genotypes were identical to the B. lanceolatum isozyme profiles. The two B. acuminatum C genotypes clustered separately from, but closely related to, the B. lanceolatum profiles. The N genotypes clustered as four distinct groups: (1) the four B. matricariifolium NA genotypes, (2) the four B. matricariifolium NB genotypes plus the B. echo 1 and B. acuminatum 2 N genotypes, (3) the B. acuminatum 1 and B. hesperium 1 N genotypes plus the B. lineare profile, and (4) the B. hesperium 2 N genotype.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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). In pteridophytes, mean genetic identities for conspecific populations range from 0.780 to 0.996 (Soltis and Soltis, 1990
) and the mean for diploid Botrychium species (0.912) fell within this range (Table 5). The populations sampled of the diploid species represented a substantial portion of the geographic distribution of each species, yet genetic identities did not always correlate with geographic distance between populations. For example, in B. simplex, the genetic identity between two populations from Oregon (4 and 5) was 0.833, whereas population 4 was allelically identical to population 3 from Michigan. Gene flow between the Oregon (4) and Michigan (3) populations is not a likely explanation for the high genetic identity detected. Reasons for the lack of correlation between diploid isozyme profiles and geographic distribution are unclear.
Breeding system(s) of diploid subgenus Botrychium species
Based on accumulated data from isozyme studies (e.g., Hamrick, Linhart, and Mitton, 1979
; Hamrick and Godt, 1989
), inbreeding species usually have most of their isozymic variation partitioned among populations, whereas outcrossers generally have most isozymic variation contained within individual populations. Following this line of reasoning, the relatively high mean value for genetic identity among diploid subgenus Botrychium populations indicated a tendency toward outcrossing. However, in diploid subgenus Botrychium species, little or no isozyme variation was observed within populations (Table 3); only four of 23 diploid populations possessed more than a single genotype. With such a small amount of infraspecific variation, it is possible to have high genetic identities between populations and still have most of the total genetic variation partitioned among populations (rather than contained within them). Thus, the limited data available on within-population structuring of genetic variation in these diploids could not exclude either inbreeding or outcrossing as the predominant breeding system of subgenus Botrychium.
Although low amounts of allozyme variation were detected within subgenus Botrychium populations, four diploid populations could be used to evaluate reproductive modes. Population 6 of B. simplex and populations 1, 2, and 7 of B. lanceolatum were the only diploid populations that contained more than one allele at any locus without exhibiting fixed heterozygosity (Table 3). For example, in population 6 of B. simplex, 24 of 30 individuals were homozygous for one Pgi-2 allele, four were homozygous for a second, and two individuals were heterozygous. A chi-square analysis of these data (Table 7) yielded a highly significant value (P < 0.001). These data did not support the null hypothesis of random mating but rather indicated that nonrandom mating (in this case a high frequency of inbreeding) is predominant in this population. Populations 1, 2, and 7 of B. lanceolatum exhibited similar nonrandom mating patterns (Table 7).
Data from four populations of two species cannot be extrapolated to characterize the breeding systems of an entire subgenus of species, but the information obtained from the B. simplex and B. lanceolatum populations substantiates other evidence concerning breeding systems in Botrychium. McCauley, Whittier, and Reilly (1985)
and Watano and Sahashi (1992)
reported inbreeding in diploid species of the closely related subgenus Sceptridium, and Soltis and Soltis (1986)
concluded that B. virginianum (subgenus Osmundopteris) is predominantly inbreeding. Although interspecific hybrids between species of subgenus Botrychium are documented (Wagner, 1980
, 1991
; Wagner et al., 1984
; Wagner, Wagner, and Beitel, 1985
; Wagner and Wagner, 1988
), individuals of several species typically grow sympatrically and yet hybridization appears to be rare. All Botrychium species have subterranean gametophytes that may hinder sperm transfer (Tryon and Tryon, 1982
) and reduce the opportunity for outcrossing. Therefore, all available evidence is consistent with inbreeding as the most probable breeding system operating in Botrychium.
Interspecific comparisons for populations of diploid species
Interspecific values of Nei's (1978)
genetic identity for six diploid species ranged from 0.045 to 0.833 with a mean of 0.409 (Table 6). These genetic identities were relatively low when compared to conspecific values (Table 5) and indicated that considerable genetic differentiation has occurred among the diploids (Fig. 5). Gottlieb (1981)
and Crawford (1983)
reported a mean genetic identity of 0.67 for congeneric angiosperm species, whereas congeneric ferns have much lower values, averaging 0.33 (Soltis and Soltis, 1990
). The Botrychium mean (0.409) was consistent with typical values for pteridophytes. Assuming a constant rate of mutation, the low genetic identity values suggested that diploid Botrychium species are older than many angiosperm species and similar in age to most extant fern species.
Among some diploid species, interrelationships suggested by isozymes correspond to those based on rbcL analysis (Hauk, 1995
). In the isozyme analysis (Fig. 5), the species pair B. lunaria and B. crenulatum is more similar to the group composed of B. simplex, B. pumicola, and B. lineare than either group is to B. lanceolatum. Similarly the rbcL tree (Fig. 2) placed the "lunaria" and "simplex-campestre" clades as sister groups and the "lanceolatum" clade as the earliest diverging lineage of subgenus Botrychium. Morphology supports the position of B. lanceolatum as sister to all other species of subgenus Botrychium; the leaves of B. lanceolatum are ternate, a condition that occurs within the closely related subgenera Sceptridium and Osmundopteris, whereas all other subgenus Botrychium species exhibit a presumably more derived pinnate leaf construction. Concordance of isozymic, DNA, and morphological data indicates that the "lanceolatum" clade was the earliest diverging lineage of subgenus Botrychium and that the "lunaria" and "simplex-campestre" clades are more derived species assemblages.
Levels of genetic identity among species, however, did not always correlate with morphological similarity, or with hypotheses of relationships suggested by rbcL sequence analysis. Botrychium lineare and B. pumicola, for example, aligned with different subclades in rbcL analyses (Hauk, 1995
) and are morphologically distinct, but had the highest isozymic interspecific genetic identity value (0.833) among the species sampled, differing only at Tpi-1. In contrast, B. simplex and B. pumicola, which are similar morphologically and possess identical rbcL sequences, had a mean genetic identity of only 0.589. Similarly, the formerly conspecific B. crenulatum and B. lunaria have identical rbcL sequences, yet the two species had a genetic identity value of 0.535. Species with low genetic identity values and identical rbcL sequences may have divergent nuclear genotypes but share a common chloroplast ancestor, possibly as a result of introgression (Rieseberg, Beckstrom-Sternberg, and Doan, 1990
; Rieseberg and Brunsfeld, 1991
) or lineage sorting (Doyle, Doyle, and Brown, 1990
).
Diploid/polyploid relationships
Isozymic patterns among polyploid species were not easily interpretable because overlapping of isozyme profiles among polyploid species was prevalent. Undoubtedly this is a consequence of some diploid species (i.e., B. lanceolatum) contributing to the origin of several polyploids (Fig. 1). Separating polyploid isozyme patterns into hypothetical diploid genotypes and analyzing these hypothetical genotypes with diploid isozyme profiles provide one method of unraveling reticulate relationships among morphologically cryptic species. Furthermore, this method may be the only currently available means of simultaneously analyzing diploid and polyploid isozyme data.
The origins of B. spathulatum and B. ascendens
Morphological characters indicate that B. spathulatum is an allopolyploid derivative of the diploids B. lunaria and B. campestre (Fig. 1), and rbcL data identified B. campestre as the chloroplast parent of B. spathulatum (Fig. 2). Botrychium campestre (not surveyed isozymically) and B. lineare (surveyed) are sister species based on both morphological (Wagner and Wagner, 1994
) and rbcL data (Hauk, 1995
). Although sister species frequently do not possess identical isozyme profiles (e.g., B. lunaria and B. crenulatum), at every isozyme locus examined, B. spathulatum C genotype possessed all alleles found in B. lineare. If B. campestre is the chloroplast parent of B. spathulatum, the lack of isozyme divergence between the B. spathulatum C genotype and B. lineare suggests that B. campestre and B. lineare possess similar or identical isozyme profiles.
Isozyme data did not lend support to the hypothesis that B. lunaria is the nonchloroplast parent of B. spathulatum. Confusion between polyploid derivatives of B. lunaria and B. crenulatum is possible because the two species have similar fan-shaped pinnae and were once considered conspecific. However, B. lunaria is generally larger, more robust, and a darker green than the smaller, more delicate, yellow-green B. crenulatum. Some populations of B. spathulatum strongly resemble B. lunaria morphologically, whereas other populations more closely approximate B. campestre in form (W. D. Hauk, personal observation); population 1 of B. spathulatum is smaller and more delicate than populations of B. spathulatum collected near Marathon, Ontario. As currently circumscribed, B. spathulatum may represent a taxon composed of allopolyploid derivatives of B. campestre x B. lunaria and B. campestre x B. crenulatum. This hypothesis is consistent with the morphological variation present in B. spathulatum and should be tested. Additional sampling of B. spathulatum populations may reveal populations that possess the diagnostic alleles of B. lunaria.
Botrychium ascendens is hypothesized to have originated as an allotetraploid involving B. montanum and B. crenulatum (Fig. 1), although it did not possess the rbcL sequence of either putative diploid parent (Fig. 2). Similarly, the three B. ascendens C genotypes did not associate with any diploid surveyed isozymically (Fig. 6). Thus, both rbcL and isozyme evidence indicate that an unsampled and perhaps unknown diploid contributed its chloroplast genome to B. ascendens. Clustering of the three B. ascendens N genotypes with B. crenulatum is consistent with the hypothesis that B. crenulatum is the source of the B. ascendens N genotype.
The origin of B. minganense
The most isozymically variable taxon sampled was B. minganense, a tetraploid species distributed across northern and western regions of North America (Wagner and Wagner, 1993
). Traditional morphologically based hypotheses implicate the diploids B. lunaria and B. pallidum as progenitors of B. minganense (Fig. 1). However, only one of these putative ancestors, B. lunaria, was available for isozyme analysis. Despite the apparent close relationship between B. minganense and B. lunaria, no population of B. minganense possessed the marker allele Tpi-13 of B. lunaria (Fig. 11). However, all populations of B. minganense (except IP) possessed a complement of isozyme bands consistent with B. crenulatum ancestry (Fig. 11), and B. crenulatum appears to be the source of the B. minganense N genotype.
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; Werth, Gutman, and Eshbaugh, 1985
).
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A single population (5) of B. minganense from Independence Pass, Colorado, did not exhibit the interlocus heterozygosity observed in all other B. minganense populations and possessed simple isozyme patterns more typical of diploid species. Individuals of population 5 were indistinguishable morphologically from other collections of B. minganense, yet the isozyme profile of population 5 clustered with B. lineare (Fig. 7). Population 5 was the most isozymically divergent of all the B. minganense populations sampled. The distinctive isozymic profile of population 5 supported, in part, the hypothesis that there may be hidden geographic variants within B. minganense (W. H. Wagner, Jr., personal communication), although the absence of rbcL sequence variation between eastern and western populations of B. minganense did not support the hypothesis of undetected cryptic species (Hauk, 1995
).
The origins of B. acuminatum, B. hesperium, B. matricariifolium, and B. echo
The diploid B. lanceolatum is thought to be one diploid progenitor of the tetraploids B. matricariifolium, B. acuminatum, B. hesperium, and B. echo (Fig. 1), and rbcL data indicate that B. lanceolatum is the chloroplast parent of each polyploid (Hauk, 1995
). Isozyme data provided further support for this relationship because B. matricariifolium, B. acuminatum, B. hesperium, and B. echo shared Pgi-25 with B. lanceolatum (Fig. 12), and the C genotypes of each polyploid clustered closely with B. lanceolatum population profiles (Fig. 8). However, clustering of the N genotypes from these four tetraploids (Fig. 8) did not correlate well with current hypotheses of species boundaries. The lack of association between N genotypes from different populations of B. hesperium (or B. acuminatum) suggested that this species arose from more than one hybridization event. The clustering of N genotypes from B. matricariifolium, B. echo, and B. acuminatum (Fig. 8) did not lend support to the hypothesis that each of these three tetraploids arose from a different nonchloroplast parent species (Fig. 1). Furthermore, the complex relationships among these N genotypes did not resolve the nonchloroplast parentage of B. matricariifolium, B. acuminatum, B. hesperium, or B. echo.
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). However, these three polyploids did not possess isozyme profiles consistent with B. lanceolatum ancestry. For example, neither B. pinnatum nor B. pseudopinnatum possessed Pgi-25 (Fig. 12), an allele found only in B. lanceolatum and known polyploid derivatives of B. lanceolatum (B. matricariifolium, B. hesperium, B. echo, and B. acuminatum). Likewise, B. pedunculosum was homozygous for 6Pgd–23, an allele not detected in B. lanceolatum (or any other diploid surveyed). Consequently, the C genotypes of B. pedunculosum, B. pinnatum, and B. pseudopinnatum did not cluster closely with populations of B. lanceolatum (Fig. 9). The isozyme contribution of B. lanceolatum to these polyploids may have been obscured by amino acid substitutions in critical isozyme alleles. If this hypothesis is correct, at least three separate substitutions in B. lanceolatum isozyme alleles must have occurred: (1) the modification of Pgi-25 to Pgi-24 in B. pinnatum (presumably B. pseudopinnatum inherited this modification from B. pinnatum), (2) the conversion of 6Pgd–22 to 6Pgd–24 in B. pinnatum, and (3) the alteration of 6Pgd–22 to 6Pgd–23 in B. pedunculosum. Other equally parsimonious scenarios may explain the origin of these isozyme alleles, but simple inheritance of isozyme alleles from diploid progenitor directly to polyploid derivative cannot explain the isozyme composition of B. pinnatum, B. pseudopinnatum, and B. pedunculosum. Unfortunately, Dia-1, Mdh-1, Tpi-1, and Tpi-2 were not variable enough to provide further evidence concerning the uncertain origins of these three polyploid species.
The ages of B. pedunculosum, B. pinnatum, and B. pseudopinnatum are not known, although the absence of rbcL sequence divergence suggested that these polyploids were of relatively recent origin, certainly more recent than divergence among the primary diploids, B. lanceolatum, B. lunaria, B. simplex, and B. campestre (Hauk, 1995
). The absence of critical B. lanceolatum marker alleles in B. pinnatum, B. pseudopinnatum, and B. pedunculosum may indicate that these three polyploid species are of more ancient origin than the polyploids that retained the B. lanceolatum isozyme alleles (B. matricariifolium, B. hesperium, B. acuminatum, and B. echo). Clear patterns of relationship in rbcL data and the lack of clear patterns in isozyme data provide circumstantial evidence that isozymes are evolving more rapidly than rbcL sequences in these subgenus Botrychium polyploids.
Infraspecific variation for populations of polyploid species
More isozymic variation was detected within polyploid species than within diploid species, and infraspecific isozyme variation in polyploids ranged from almost none in B. pedunculosum to the relatively high level seen in B. minganense (Table 3). Presuming that the C and N hypothetical diploid genotypes are a true representation of the variation present in each polyploid, it is possible to estimate levels of infraspecific variation in polyploids by assessing independently the variation present in the hypothetical diploid genotypes (Werth, Hilu, and Langner, 1994
).
Infraspecific isozyme variation in hypothetical diploid genotypes was greater than that observed among diploid populations (Table 10). Several hypotheses can be developed to explain higher infraspecific isozymic variation among populations of polyploid species than among populations of diploid species: (1) polyploid species may be the result of several independent hybridization events that captured and preserved genetic diversity no longer present in the putative progenitor diploids, (2) extinct diploid progenitors of extant polyploids may have contained more isozymic variability than extant diploids, (3) polyploid subgenus Botrychium species may have diverged isozymically more rapidly than diploids, (4) founder effects may be exaggerated in polyploids relative to diploids due to inbreeding, (5) polyploids, by nature of their fixed heterozygosity, preserve alleles that would have been selected against if homozygous in a diploid individual. In the last case, extant diploids are products of a higher degree of natural selection than a diploid genome "hidden" (by heterozygosity) from such selective forces in a polyploid. Although these data neither refute nor support any of the hypotheses, the observed patterns of infraspecific variation between diploids and polyploids suggest that some mechanism is preserving (or promoting) isozyme variation in the polyploids.
Polyploid banding patterns in diploid species
Diploid subgenus Botrychium species typically showed monomorphic isozymic banding patterns, whereas polyploid taxa frequently showed fixed patterns of heterozygosity. The allelic variation present in diploids should segregate during meiosis and the fixed heterozygosity of polyploids should not. However, apparent fixed heterozygosity was present at some loci in certain populations of the diploids B. lanceolatum and B. lunaria. In other diploid species heterozygotes were rare and never fixed (Table 3). Analysis of hypothetical, ancestral genotypes from populations 1 and 2 of B. lanceolatum and populations 4 and 5 of B. lunaria (Fig. 10) showed that the anomalous alleles may have originated from hybridization with the diploids B. crenulatum, B. lineare, or B. pumicola. Several hypotheses may explain the fixed heterozygosity detected in these populations of B. lanceolatum and B. lunaria: (1) the "diploid" populations with fixed heterozygosity may be allopolyploid, (2) hybridization or introgression may have introduced new alleles into these diploid populations, (3) these populations may contain individuals with duplicated gene loci, or (4) the small sample sizes of these populations may misrepresent the true distribution of alleles. Further investigations will be necessary to evaluate these hypotheses.
Anomalous gene banding patterns in polyploids
In addition to detecting duplicate banding patterns in diploid species, some banding patterns typical of diploids were observed in certain polyploids. Botrychium pedunculosum is a tetraploid species that had homozygous banding patterns at five of six loci; Pgi-2 was the only locus that showed fixed heterozygosity. Because the putative parents of B. pedunculosum (B. lanceolatum and B. montanum; Fig. 1) belong to different rbcL clades (Fig. 2) and members of the "lanceolatum" and "simplex" clades have different isozyme profiles, it is reasonable to assume that B. pedunculosum should possess heterozygous banding patterns additive of the two presumed ancestors. However, the absence of heterozygosity in B. pedunculosum did not support this line of reasoning.
Equally puzzling are the occasional homozygous banding patterns observed in a few individuals of polyploid populations otherwise fixed for two alleles. For example, in population 2 of tetraploid B. matricariifolium, 41 individuals showed fixed heterozygosity between Pgi-21 and 5, and three between Pgi-22 and 5. However, three individuals from this population had homozygous banding patterns. Additional examples of anomalous homozygous banding patterns in polyploid populations are found in B. minganense where single Tpi-1 alleles were found in otherwise fixed heterozygous populations (3, 4, 6, and 8). Similar observations were made for Pgi-2 in population 10 of B. minganense and population 2 of B. hesperium.
Explanations for these enigmatic banding patterns include reciprocal gene silencing (Werth and Windham, 1991
) and homoeologous chromosome pairing (Klekowski, 1979
), but neither accounts for the absence of Pgi-21,1;1,1 homozygotes in population 2 of B. matricariifolium (Table 3). This population contained 41 Pgi-21,1;5,5 heterozygotes, three Pgi-22,2;5,5 heterozygotes, two Pgi-22,2;2,2 homozygotes, and a single Pgi-25,5;5,5 homozygote. It is unlikely that sampling was insufficient to detect the Pgi-21,1;1,1 genotype; only five plants in this population possessed Pgi-22 and yet two of these had homozygous patterns. Selection against this genotype may explain the absence of Pgi-21,1;1,1 homozygotes, but without further data such explanations are speculative.
Molecular evidence for species discrimination
The relatively distinct morphological characters of the diploids B. lanceolatum, B. lunaria, and B. simplex led Wagner and Wagner (Table 1) to establish different sections for each within subgenus Botrychium, as well as a fourth section based on the tetraploid species B. matricariifolium. The majority of new species identified and classified by Wagner and Wagner are putative allopolyploids whose interspecific distinctions are subtler than those of the diploids. Many of these polyploids resembled one of the type members of the sections, and all but a few species were assigned to sections.
Analysis of rbcL sequences provided the first molecular test of the Wagner and Wagner morphologically based classification. Relationships among the three primary diploid subgenus Botrychium species were largely consistent in the two classifications, but a new clade composed of the diploids B. campestre and B. lineare emerged in the rbcL analysis (Fig. 2). Differences in the placement of polyploid species in the rbcL classification relative to the Wagner and Wagner classification were primarily the result of interpreting the presumably uniparental inheritance of the chloroplast genome vs. biparentally inherited morphological characters (Hauk, 1995
). Three groups of polyploids were identified in the rbcL analysis: (1) polyploids associated with the diploid B. lanceolatum, (2) polyploids associated with the diploid B. campestre, and (3) polyploids that did not associate with any diploid sampled for rbcL (Fig. 2).
Isozyme data substantiated some hypotheses of relationship suggested by rbcL data. For example, the rbcL phylogeny identified Section Campestre members B. campestre, B. lineare, and B. spathulatum as sister to members of Section Simplex (Fig. 2). UPGMA cluster analyses of isozyme data aligned B. lineare and B. spathulatum with members of Section Simplex (B. pumicola and B. simplex; Fig. 5). In fact, B. lineare shared five of six isozyme loci with B. spathulatum, and these two species had higher genetic identities than any other species pair.
Some relationships left unresolved in the rbcL analysis were clarified by isozyme data. Identical rbcL sequences indicated a close relationship between the diploids B. lunaria and B. crenulatum, and isozyme data provided the first molecular evidence of differentiation between the two species. However, other members of Wagner and Wagner Section Lunaria did not associate with B. lunaria and B. crenulatum in the rbcL analysis (Fig. 2). Botrychium minganense, B. spathulatum, and B. ascendens did not possess the rbcL sequence of B. lunaria and were not chloroplast descendants of B. lunaria (or B. crenulatum). Isozymes were useful for ascertaining which lineages contributed to the origin of these three polyploids because the diagnostic alleles of Section Lunaria (Pgi-24, Mdh-13, and 6Pgd–24) were found in populations of B. minganense, B. ascendens, B. pinnatum, and B. pseudopinnatum.
Although Wagner and Wagner established Sections Lanceolatum and Matricariifolium, B. lanceolatum, the only member of Section Lanceolatum, is one progenitor of all the polyploids assigned to Section Matricariifolium (Fig. 2); all seven polyploids assigned to Section Matricariifolium had rbcL sequences identical to that of B. lanceolatum. Shared isozyme banding patterns established B. lanceolatum as closely allied to species of section Matricariifolium and provided no rationale to retain the two sections as distinct.
One of the most controversial questions surrounding the Wagner and Wagner classification is whether all of the polyploids of Section Matricariifolium represent distinct species. Although stable morphological characters exist to distinguish these species, rbcL sequence analysis revealed no differentiation among species of the "lanceolatum" clade (Fig. 2). Isozyme data, however, provided the first molecular characters for distinguishing some Section Matricariifolium polyploids. For example, populations of B. pinnatum and B. pseudopinnatum were the only members of Section Matricariifolium lacking Pgi-25, a marker allele for B. lanceolatum. Because B. pinnatum, B. pseudopinnatum, and B. lanceolatum have the same rbcL sequence, it is likely that Pgi-25 has been lost in the two tetraploids, although these species share a common chloroplast ancestor. If hexaploid B. pseudopinnatum arose from within the B. pinnatum lineage, the loss of Pgi-25 probably occurred first in the B. pinnatum lineage and was passed on to B. pseudopinnatum through hybridization with an undetermined diploid species.
The allele Pgi-21 was a marker for B. matricariifolium, distinguishing it from nearly all other members of Section Matricariifolium. However, not all individuals within populations of B. matricariifolium possessed Pgi-21, and it appears that two isozymically different groups of B. matricariifolium exist, one that is isozymically distinct from B. acuminatum and another that is not. These isozyme data are consistent with the hypothesis of Wagner (1993)
that B. acuminatum originated as a "metaspecies" from within the ranks of B. matricariifolium.
Among other species of Section Matricariifolium, only B. pedunculosum had distinctive isozymic markers. This species was fixed for 6Pgd–23, an allele found as a polymorphism in only one other species, B. pinnatum. The presence of 6Pgd–23 in both B. pedunculosum and B. pinnatum is problematic because no diploid sampled in this study possessed the allele. It is possible that the 6Pgd–23 bands in B. pedunculosum and B. pinnatum are not homologous and that differences could be detected by the use of alternative buffer systems. The three remaining species of Section Matricariifolium, B. hesperium, B. echo, and B. acuminatum, had nearly identical isozymic profiles and none possessed unique alleles. At this time no molecular evidence, from isozymes or rbcL sequences, can distinguish these species, and morphological characters provide the only reliable features for delimiting these closely related polyploids.
Summary of conclusions
Members of subgenus Botrychium have less infraspecific variability than many other taxa that have been studied isozymically. The average number of alleles per locus within species was 1.30 compared to 1.64 for the closely related subgenus Sceptridium (Watano and Sahashi, 1992
) and 2.69 for angiosperms (Gottlieb, 1981
). For both diploid and polyploid species, the paucity of variation may be explained in at least two ways: (1) within some species, not enough populations were sampled to survey adequately the genetic variation, (2) infraspecific genetic variation may have been lost as a result of selective and/or random events, (3) too few isozyme loci were surveyed to yield a robust assessment of genetic diversity. Inadequate sampling may have contributed to low estimates for infraspecific variability for six species included in this study (B. spathulatum, B. pseudopinnatum, B. pedunculosum, B. echo, B. pumicola, and B. lineare), as only one population of each was obtained (Table 2). Seven other species (B. ascendens, B. hesperium, B. acuminatum, B. pinnatum, B. matricariifolium, and B. crenulatum) may possess substantial amounts of undetected variation as only two to four populations were sampled (Larsen and Marx, 1981). However, between six and 11 populations for each of the remaining four species (B. lanceolatum, B. lunaria, B. simplex, and B. minganense) were sampled across broad geographic areas. Estimates of genetic variation for the more widely sampled species tended to be higher than for the less sampled species. For example, 11 populations of tetraploid B. minganense had nine different genotypes, whereas two populations of tetraploid B. pinnatum had only two genotypes. The lack of autapomorphic alleles among the species of subgenus Botrychium indicated that sampling additional populations might yield more infraspecific variability than interspecific differentiation. Although examining other enzyme systems would seem appropriate, minimal variability was observed in seven additional enzymes (see Materials and Methods) that were not consistently resolved across all populations of all species and that were therefore not included in the present analysis and discussion. If these additional loci had been included in statistics of genetic variation, the values reported for H, A, and P would almost certainly have been even lower.
The lack of interspecific allozymic differentiation, especially among some polyploid species, suggests that: (1) members of subgenus Botrychium have originated recently (Crawford, Stuessy, and Silva O., 1987
), (2) variation may be internally constrained and may have little relation to the age of the species, or (3) some taxa may not deserve status as species. Ophioglossaceae are considered an ancient lineage (Bierhorst, 1971
; Bower, 1926
; Clausen, 1938
). Fossil specimens of extinct B. wightonii from Alberta established the presence of modern Botrychium species in the earliest Tertiary and perhaps the Paleocene (Rothwell and Stockey, 1989
). Although both isozymic and rbcL sequence comparisons indicate that the diploid species we surveyed are distinct and possibly ancient lineages, hybridization among these diploids has occurred. The age of polyploid Botrychium species is less certain, but clearly they are younger than most of the diploids. Whether all currently recognized species of Botrychium should be retained is a difficult and controversial question. The exhaustive field research conducted by Wagner and Wagner indicates that stable morphological criteria exist to distinguish these species. Studies of rbcL and isozymic variation have supported the differentiation of certain species, but relationships among other species remain equivocal. In the absence of more definitive data, we present this investigation as another step in the continued examination of species and species concepts in Botrychium subgenus Botrychium.
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APPENDIX |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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FOOTNOTES |
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2 Author for correspondence, current address: Department of Biology, Denison University, Granville, OH 43023.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIXLITERATURE CITED |
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