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Systematics |
Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense, E-28040 Madrid, Spain
Received for publication January 18, 2001. Accepted for publication April 17, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Aspleniaceae • Asplenium obovatum group • genetic relationships • isozymes • polyploid origin and evolution • Pteridophyta • systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Maire, 1952
; Salvo et al., 1992
) as subsp. numidicum (Trab.) Salvo and Cabezudo. Other authors treated (Becherer, 1935
) or suggested (Rumsey and Vogel, 1996
) A. obovatum subsp. numidicum as synonymous with Asplenium foreziense Legrand ex Hérib. Demiriz, Viane, and Reichstein (1990)
distinguished three varieties for the diploid subsp. obovatum: obovatum; deltoideum Demiriz, Viane and Reichst.; and protobillotii Demiriz, Viane and Reichst. Previouly, until that time, subsp. lanceolatum (= A. billotii F.W. Schultz) was believed to be an autotetraploid derived from the typical forms of subsp. obovatum (now var. obovatum), due to the observed meiotic behavior of its natural and artificial hybrids with other taxa (Vida, 1972
; Bouharmont, 1977a, b
; Badré et al., 1981
; Reichstein, 1981
; Sleep, 1983
).
Asplenium obovatum subsp. obovatum and A. obovatum subsp. lanceolatum differ morphologically (Fig. 1b, e). Morphological differences are unusual between autotetraploids and their diploid progenitors. Sleep (1983)
stated that A. obovatum subsp. lanceolatum "has nevertheless arisen by chromosome doubling from A. obovatum or a form with chromosomes homologous to it." This suggests the possibility that another taxon could be involved in the origin of the tetraploid.
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and Rasbach et al. (1990)
thought this was the ancestor, via autopolyploidy, of the tetraploid, and they named it var. protobillotii (Fig. 1c). Geographical distribution and ecological data for each taxon are provided in Table 1.
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). The electrophoretic profile of an autopolyploid taxon should show a subset of the isozymes found in the diploid progenitor. In contrast, an allopolyploid should manifest a banding pattern for a majority of isozymes with the component bands corresponding to addition of the alleles derived from two diploid species hypothesized as progenitors. In this case, since isozymes are codominant, the polyploid is expected to have a banding pattern that does not segregate among progeny and that remains stable or fixed in all individuals (Werth, Guttman, and Eshbaugh, 1985a
; Bryan and Soltis, 1987
; Soltis, Soltis, and Alverson, 1987
; Ranker et al., 1989
; Haufler, Windham, and Ranker, 1990
; Soltis, Soltis, and Wolf, 1991
; Werth, 1991
; Pryer and Haufler, 1993
; Haufler, Windham, and Rabe, 1995
). This paper reports on electrophoretic investigations of the taxa included in the A. obovatum group, as well as on cytological analysis of an artificial hybrid that we synthesized to confirm hypotheses derived from electrophoretic results. The objectives of this study were to analyze the genetic variability of the diploid taxa and their genetic relationships with the tetraploids and evaluate the present taxonomic treatment of the group.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1 wk until electrophoresis was conducted or were ground, the extract absorbed in Whatman no. 3 paper wicks, and the wicks stored frozen (–80°C) until they were used (between 1 and 6 mo). Multispore cultures on mineral agar (Dyer, 1979
) were established to obtain gametophytes to complement sporophytic electrophoretic analyses. When only dry specimens of a taxon were available, sporophytic banding patterns were obtained from newly formed sporophytes arisen from the gametophyte cultures or just from a mixture of gametophytes, as indicated in Table 2. Gametophytes were also produced to study segregation patterns of sporophytes in their resulting haploid gametophytes in order to interpret their enzymatic phenotypes; in this case, single gametophytes were used.
Starch gel electrophoresis was conducted following established methods (Soltis et al., 1983
). Triosephosphate isomerase (TPI) and phosphoglucoisomerase (PGI) resolved best on system 6 of Soltis et al. (1983)
; malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6PGD) and shikimate dehydrogenase (SDH) on system 2 of Wendel and Weeden (1989)
; and phosphoglucomutase (PGM) on system 1 of Wendel and Weeden (1989)
. The modified system 8 of Haufler (1985)
provided the best resolution of aspartate aminotransferase (AAT) and leucine aminopeptidase (LAP). Allelic variants within loci (allozymes) were distinguished from the products of different loci (isozymes). When more than one set of bands putatively coded by additional loci was observed for a particular enzyme, such sets of bands were numbered sequentially from anode to cathode. Presumed allelic variants within these loci were assigned alphabetically with the most anodal being "a." With bands identified and characterized genetically, statistical analyses of diploids were performed using the computer program BIOSYS-1 (Swofford and Selander, 1989
), and the following measures of genetic diversity were calculated for each population: proportion of polymorphic loci (p, 99% criterion), mean number of alleles per locus (A), and observed (Ho) and expected (He) mean individual heterozygosities. Nei's (1978)
unbiased genetic identity (I) were calculated for all pairwise population and taxa comparisons. A dendrogram for the taxa was constructed with these similarity data using the UPGMA method (Sneath and Sokal, 1973
). These computations were carried out also with the program BIOSYS-1. For the tetraploids, only the frequencies of allelic combinations were calculated.
Synthesis of artificial hybrids and their cytological analysis
Artificial hybrids between the diploids A. obovatum subsp. obovatum var. obovatum (individual VAR 7) and var. protobillotii (individual MIE 27) were obtained following the method proposed by Rasbach, Reichstein, and Viane (1994)
. For cytological analysis, leaf material with young sporangia was fixed in "Farmer's solution" (a 3 : 1 mixture of absolute ethanol to glacial acetic acid), stored at 4°C or –18°C at least 2 d, stained with Wittman's hematoxylyn, and squashed in diluted Hoyer's medium to make semipermanent slides.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For leucine aminopeptidase (LAP), all individuals of diploid subsp. obovatum showed a homozygous pattern, interpreted as locus Lap-1, but for different alleles (Lap-1a in var. protobillotii and Lap-1b in var. obovatum and var. deltoideum). An additive profile was found in all but one (HOY 20) individual of tetraploid subsp. lanceolatum and in subsp. numidicum; this pattern showed the two alleles present in the diploid subspecies. These alleles did not segregate in the gametophytic progeny of the tetraploid.
Gels stained for malate dehydrogenase (MDH) showed two regions of band activity. We interpreted the most anodal as locus Mdh-1, having a one-banded phenotype, the most frequent (Tables 3 and 4) corresponding to homozygous individuals for allele Mdh-1a, and a three-banded phenotype as expected for heterozygous in dimeric enzymes. The latter was only found in three individuals of diploid var. protobillotii (Table 3).
The second region predominantly showed two different patterns, one two-banded in the diploids and another four-banded in the tetraploids (Fig. 2a, b). The diploids have specific bands depending on the taxon. Since these two bands do not segregate in the gametophytic progeny of the diploids, we have interpreted each band as an isozyme encoded by a different locus (Mdh-2 and Mdh-3), all plants (except two individuals of var. protobillotii) being homozygous; both varieties obovatum and deltoideum had alleles Mdh-2a and Mdh-3a, whereas var. protobillotii had alleles Mdh-2b and Mdh-3b. The intensity of staining is stronger in Mdh-2 than in Mdh-3 (Fig. 2a, b).
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Only in two sporophytes of the diploid var. protobillotii (individuals MIE 75 and MIE 76) was a four-banded phenotype found, the same as in tetraploid plants. Gametophytes obtained from one of those individuals showed an allelic Mendelian segregation (1 : 1), which is consistent with the genetic interpretation given above.
Among tetraploids only two individuals from population CHE (defined in Table 2) and all individuals from Rodalquilar (AH92F-AH104F) were homozygous for loci Mdh-2 and Mdh-3, instead of a fixed heterozygous pattern, which was the phenotype found in the remaining individuals.
Two zones of band activity have been observed in phosphoglucoisomerase (PGI), corresponding to isozymes PGI-1 and PGI-2 encoded by two different loci (Pgi-1 and Pgi-2). The faster migrating locus (Pgi-1) stains weakly and is monomorphic. Pgi-2 showed variability only in a few individuals of diploid var. protobillotii and in the tetraploid subsp. lanceolatum from population MIE, expressing the bands of alleles Pgi-2a and Pgi-2b plus the band of the heterodimer; the remaining individuals of the group were homozygous for allele Pgi-2a (Tables 3, 4).
Analysis of the gametophytic progeny indicated Mendelian segregation (1 : 1) in the diploids, and there was no segregation at all in the tetraploids.
Phosphoglucomutase (PGM) zymograms showed a complex banding pattern. Two separate regions of band activity with different staining intensities were seen. For the most anodal region, diploid plants always had a two-banded pattern and tetraploid plants a four-banded pattern; for the second region, both diploids and tetraploids showed either a one- or a two-banded pattern (Fig. 3).
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1 : 1), whereas those from tetraploid plants did not segregate. Since PGM has typically two loci (Gottlieb, 1982
), we interpreted these results as the two regions of band activity corresponding to two different loci (Pgm-1 and Pgm-2) with the most anodal duplicated (Pgm-1 and Pgm-1'). Duplication for PGM has been reported before in Asplenium (Werth, Guttman, and Eshbaugh, 1985a
), as well as in other pteridophytes (Haufler, 1985
; Bryan and Soltis, 1987
) and spermatophytes (Rieseberg and Soltis, 1987
; Soltis, Soltis, and Gottlieb, 1987
). Each taxon showed different monomorphic allozymes in the duplicated locus Pgm-1. Variety protobillotii expressed alleles Pgm-1b and Pgm-1'b, whereas varieties obovatum and deltoideum expressed alleles Pgm-1a and Pgm-1'a, and these were additive in the tetraploids subsp. lanceolatum and subsp. numidicum (Fig. 3).
For the most slowly migrating isozyme there is a single coding locus with two alleles, Pgm-2a and Pgm-2b. Homozygous or heterozygous phenotypes have been detected regardless of the taxon or ploidy level (Fig. 3; Tables 3, 4). As indicated above, bands in the tetraploids do not segregate in the gametophytic progeny.
Gels stained for 6-phosphogluconic dehydrogenase (6PGD) showed two zones of band activity with different staining intensities. The fastest migrating zone, interpreted as locus 6Pgd-1, appears as very faint bands, but in some populations did not stain at all. When it was possible to visualize, as it is a dimeric enzyme, one-banded phenotypes and three-banded phenotypes were seen, as expected for homozygous and heterozygous individuals, respectively. All diploid individuals were homozygous; in var. protobillotii they were homozygous for allele 6Pgd-1a, and in var. obovatum and var. deltoideum they were homozygous for allele 6Pgd-1b (Table 3). Tetraploid subsp. lanceolatum showed homozygous phenotypes for either allele in 37.6% of the analyzed sporophytes, as well as a combination of both alleles in the remaining 62.4%. Due to the low staining intensity of this locus it was not possible to verify segregation in gametophytes derived from heterozygous tetraploids; however, in the tetraploids 6Pgd-1 showed addition of both the alleles found in diploid varieties obovatum and protobillotii.
For the slowest migrating zone, interpreted as locus 6Pgd-2, two different alleles have been detected: 6Pgd-2a and 6Pgd-2b. All individuals of the group were homozygous for allele 6Pgd-2a, except for individuals of subsp. lanceolatum from population CAB (Table 4), which showed both alleles added and did not segregate in the gametophytic progeny.
In summary, tetraploid subsp. lanceolatum has seven alleles exclusively in common with var. protobillotii, five with var. obovatum, and six alleles are shared with both diploid varieties.
Genetic diversity
Values obtained for parameters estimating genetic variability (A, p, Ho, and He) of diploid populations are given in Table 5. All individuals studied of var. obovatum and var. deltoideum were monomorphic at every scored locus, except population VAR, where PGM-2 was polymorphic (see also Table 3). The population of var. protobillotii showed higher levels of genetic variability.
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unbiased genetic identity (I) values for the populations of diploids and for the diploid taxa are given in Table 6 and Table 7, respectively. Identity values between all pairs of populations of var. obovatum ranged from 0.929 to 1 with an average of 0.973 over all pairwise comparisons, indicating no differences among them. Nei's unbiased genetic identity value between varieties obovatum and deltoideum was 0.983, higher than the mean values obtained from pairs of populations of var. obovatum. However, when comparing var. protobillotii with varieties obovatum and deltoideum, values obtained were 0.551 and 0.576, respectively. The dendrogram constructed from Nei's unbiased genetic identity values clearly depicted the relationships between taxa (Fig. 4); two distinct groups, one corresponding to varieties obovatum and deltoideum and a second corresponding to var. protobillotii, were evident in the resulting dendrogram.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Polymorphic loci Lap-1, Mdh-2, Mdh-3, Pgi-2, Pgm-2, and 6Pgd-2 in tetraploid individuals did not segregate among gametophytic progeny, a common fact in plants of allopolyploid origin (Soltis and Soltis, 1989, 1993
; Werth, 1989
); furthermore, analysis of segregation in gametophytes did not show evidence of tetrasomic inheritance in any case.
The band corresponding to allele 6Pgd-2b found in all individuals of population CAB of tetraploid subsp. lanceolatum has not been found in any of the sampled individuals representing either of its progenitor diploid lineages. The occurrence of this supposed "orphan" allele can be attributed to any of the three situations suggested by several authors (Werth, Guttman, and Eshbaugh, 1985b
; Werth, 1991
; Haufler, Windham, and Rabe, 1995
): (1) 6Pgd-2b allele of subsp. lanceolatum is rare in extant populations of var. obovatum and var. protobillotii and has not been discovered in the studied populations; (2) subsp. lanceolatum carries alleles that have been lost from extant populations of one of its progenitor diploid lineages; or (3) subsp. lanceolatum has evolved novel variants since its origin.
Cytological data could support that subsp. lanceolatum is an allotetraploid involving both diploid varieties obovatum and protobillotii. The cytological behavior of the natural triploid hybrids between subsp. lanceolatum and var. obovatum [A. obovatum Viv. nothosubsp. cyrnosardoum (Rasbach et al.) Rasbach et al. nothovar. cyrnosardoum], and between subsp. lanceolatum and var. protobillotii [A. obovatum Viv. nothosubsp. cyrnosardoum (Rasbach et al.) Rasbach et al. nothovar. ibericum Rasbach et al.] has been reported to be very similar at meiosis, producing a range of 0–3 trivalents, 31–36 bivalents, and 35–37 univalents for nothovar. cyrnosardoum (Rasbach, Vida, and Reichstein, 1981
), and 0–4 trivalents, 29–36 bivalents, and 31–35 univalents for nothovar. ibericum (Rasbach et al., 1990
). This cytological behavior is characteristic of triploid hybrids between an allotetraploid and either of its diploid parents (Reichstein, 1981
). For all natural and artificial hybrids studied involving subsp. lanceolatum in which three genomes of A. obovatum are present, the number of trivalents was lower than would be expected if subsp. lanceolatum was of autopolyploid origin (for a review, see Rasbach et al., 1991
; and Cubas and Sleep, 1994
).
As indicated above, cytological behavior of genomes of A. obovatum is concordant with the hypothesis that subsp. lanceolatum is an allotetraploid. However, in natural and synthetic hybrids involving subsp. lanceolatum and unrelated species in which only two genomes of subsp. obovatum are present, a high number (36) of bivalents corresponding to both sets of chromosomes contributed by subsp. lanceolatum have been observed, indicating that both genomes are able to recognize each other in such situations (for a review, see Cubas and Sleep, 1994
). In these cases, subsp. lanceolatum has a cytological behavior more characteristic of an autotetraploid. Closely related genomes of varieties obovatum and protobillotii observed in the artificial diploid hybrid and the existence of a genetic regulation of meiosis tending to avoid multivalent associations could account for the double cytological behavior seen in subsp. lanceolatum.
Given that the number of chromosome pairs that two genomes can form together constitutes a measure of their relationship (Lovis, 1973
), our results from the cytological analysis of the artificial diploid hybrid between varieties obovatum and protobillotii indicate substantial genomic homology; their genomes have become partially differentiated, but they have retained a high level of similarity. This situation has also been found in other complexes of Asplenium: A. ruta-muraria L. subsp. dolomiticum Lovis and Reichst. var. dolomiticum-var. eberlei (D. E. Meyer) Rasbach, K. Rasbach, Reichst. and Viane (Rasbach et al., 1992
); A. seelosii Leybold subsp. seelosii-subsp. glabrum (Litard. and Maire) Rothm. (Lovis, 1977
); A. trichomanes L. subsp. trichomanes-subsp. inexpectans Lovis (Lovis, 1977
), and A. trichomanes L. subsp. coriaceifolium Rasbach, K. Rasbach, Reichst. and Bennert-subsp. quadrivalens D. E. Meyer emend. Lovis (Rasbach et al., 1991
).
Taken together, our results suggest that A. obovatum subsp. lanceolatum is an allotetraploid derived from varieties obovatum and protobillotii of diploid A. obovatum subsp. obovatum.
Most individuals of tetraploid subsp. lanceolatum have a banding pattern for loci Lap-1, Mdh-2, Mdh-3, and 6Pgd-1 containing unique alleles of varieties obovatum and protobillotii together (Table 4). However, some individuals of subsp. lanceolatum showed the contribution of only one of the parents at these loci; some of these individuals lacked expression for the var. obovatum band, while others lacked expression for the var. protobillotii band (Table 4). These data suggest the possibility of gene silencing in subsp. lanceolatum that would substantiate the hypothesis proposing that fern phylogeny has involved repeated cycles of polyploidization followed by progressive genetic diploidization through gene silencing (Haufler, 1987
). Diploidization for each locus might be happening at a different pace: whereas for Lap-1 percentage of single allele expression is very low (0.6%), as is the case for Mdh-2 and Mdh-3 (6.9%), for 6Pgd-1 this rate is much higher (62.4%). However, these genetic differences could also be explained by a multiple origin of the tetraploid.
A wider geographical distribution range than its diploid parents (see Table 1) may suggest an old origin of the tetraploid subsp. lanceolatum. Stebbins (1971)
has observed that range is often a function of age in polyploid species. Old polyploids commonly exceed the range of their diploid progenitors, which may no longer occur in sympatry.
Our results can contribute to an evaluation of the present taxonomic treatment of the group. The mean values obtained for parameters of genetic variability in diploid taxa indicated a very low level of intra- and interpopulational genetic diversity in var. obovatum (Table 5). Populations BON and BEA for which a suitable number of individuals have been analyzed were monomorphic at every locus, sharing their alleles with the individuals of the remaining distant populations studied, included the one of var. deltoideum. Only in population VAR was a polymorphic locus (Pgm-2) found.
Variety protobillotii has higher levels of genetic diversity than var. obovatum, although they are also relatively low (A = 1.3, p = 28.6, Ho = 0.014, and He = 0.014) and are similar to the ones obtained for Botrychium virginianum (L.) Sw. (Soltis and Soltis, 1986
), Blechnum spicant (L.) Roth (Soltis and Soltis, 1988
), Equisetum arvense L. (Soltis, Soltis, and Noyes, 1988
), and Hemionitis palmata L. (Ranker, 1992
).
Lower genetic diversity in var. obovatum may be, at least in part, a consequence of the small sample size and number of populations included in this study. Because the two well-studied populations (BON and BEA) share the same small set of alleles and the remaining populations, although separated by several hundred kilometers, also showed the same set of alleles at all loci (except in Pgm-2 from population VAR), we infer that var. obovatum is genetically depauperate relative to the closely related var. protobillotii.
Steinecke and Bennert (1993)
obtained the same diploid hybrid that we did and reported normal production of spores; however in our case, a low percentage of spore mother cells fail to produce regular meiosis and hence resulting in viable spores. Herrero (1998)
observed that spores produced by normal sporangia were viable, but had a lower germination rate and slower gametophyte development than the nonhybrid diploids of this group under the same culture conditions, and the hybrid sporophytes also showed slower rates of growth than the other diploids under the same culture conditions. Viane et al. (1996)
claimed, based on morphology, that in Greek populations all three diploid varieties are sympatric and are interfertile, producing plants with intermediate morphologies. If our laboratory results on fertility and viability of these hybrid plants can be extrapolated to natural conditions, it seems it would be relatively easy to produce the hybrid, but hybrid sporophytes, as well as gametophytes derived from their viable spores, are not as healthy or vigorous as are nonhybrid diploid plants with which the hybrids would compete in such mixed populations.
On the other hand, no differences were found in the banding patterns of var. deltoideum and var. obovatum, nor were there banding patterns differences between subsp. numidicum and subsp. lanceolatum. The genetic similarity values in the diploid varieties range from 1 to 0.929, and their close relationship is revealed in the dendrogram (Fig. 4). This suggests that these pair of taxa are genetically identical. A morphological analysis of this group (Herrero, 1998
) revealed the existence of deltoid fronds in different specimens of all diploid and tetraploid taxa. Even individuals bearing both lanceolate and deltoid fronds were found. Thus, in our opinion, the character of deltoid fronds that separates var. deltoideum (see Demiriz, Viane, and Reichstein, 1990
; Viane et al., 1996
) does not have taxonomic value. Although sample size of var. deltoideum was small, the genetic similarity together with morphological characters suggest that var. deltoideum should be considered the same taxon as var. obovatum.
The other diploid taxon, var. protobillotii, has lower genetic similarity values with the obovatum–deltoideum unit (see dendrogram in Fig. 4) that range from 0.575 to 0.504 with an average of 0.551; this average is similar to the average of genetic identity values between other temperate sister pteridophyte species (Haufler, 1996
). Morphological differences found between this variety and var. obovatum (Demiriz, Viane, and Reichstein, 1990
; Rasbach et al., 1990
; Viane et al., 1996
; Herrero, 1998
) are consistent. These, together with our cytological data (discussed above), suggest that var. protobillotii is a distinct taxon.
No genetic similarity values have been calculated for the tetraploids. However, it can be deduced from the frequencies of allelic combinations shown in Table 4 that subsp. lanceolatum and subsp. numidicum do not show genetic differences. In the morphological study (Herrero, 1998
) no substantial differences were found. Thus, in our opinion, subsp. numidicum should be considered the same taxon as subsp. lanceolatum and not related at all to A. foreziense (Herrero, 1998
).
A practical taxonomic criterion for this group, similar to the one adopted by Rasbach et al. (1991)
and Bennert and Fischer (1993)
for European taxa of the A. trichomanes complex, would be to consider the diploids and the tetraploid of the A. obovatum group as subspecies. Since the genomes of the diploids in our case have become partially differentiated, we can consider the derived tetraploid subsp. lanceolatum as a segmental allopolyploid (Stebbins, 1947
; Lovis, 1977
) for which subspecific rank is convenient.
The A. obovatum group consists, in our opinion, of two diploid subspecies, obovatum and protobillotii, and their derived tetraploid subspecies lanceolatum (Fig. 6). We propose the new combination Asplenium obovatum Viv. subsp. protobillotii Herrero, Pajarón and Prada stat. et comb. nov. [A. obovatum Viv. subsp. obovatum var. protobillotii Demiriz, Viane and Reichst. in Candollea 45: 244. 1990 (basion.)].
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FOOTNOTES |
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2 Author for reprint requests, current address: Real Jardín Botánico, Plaza de Murillo 2, E-28014 Madrid, Spain (FAX: + 34 91 4200157; herrero{at}ma-rjb.csic.es
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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