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Pteridology |
Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland
Received for publication March 7, 2006. Accepted for publication April 10, 2007.
ABSTRACT
Genetic variability of Athyrium filix-femina populations was evaluated with regard to phenotypic, allozyme, and RAPD variation in 20 Swiss populations along five altitudinal gradients at four different elevations in the northern Swiss Alps. Additionally, allozyme and phenotypic variations in one Italian and two Spanish populations were compared with the variation in the Swiss populations. We hypothesized that there will be statistically significant genetic differences among populations of different altitudes and sites. The results showed no substantial correlation between genetic variation and phenotypic variation among Swiss populations. These results imply that outbreeding and effective gene exchange (long-distance spore dispersal) are the keys to population structure in this fern species, and as a consequence, phenotypic plasticity is assumed to be favored. This contrasts with results found in similar studies of herbaceous flowering plants where genetic adaptation to gradients like altitude is common. However, when data from the more distant Italian and Spanish populations of A. filix-femina were included, significant variation was detected.
Key Words: altitudinal gradient • gene flow • genetic variability • long-distance dispersal • phenotypic variation • population structure • pteridophyte
Examination of genetic similarities among populations within a species is crucial for a better understanding of evolutionary processes and the nature of species. Breeding behavior and life history peculiarities play an important role in shaping the genetic composition of populations. Genetic drift, limited gene flow, and the mode of reproduction are essential to creating genetic differentiation among or within populations (Hedrick, 2000
). Many plant species grow in a range of different habitats and have developed adaptive strategies suited to the particular habitat (Coyne and Orr, 2004
). Of the numerous investigations on genetically based population differentiation along altitudinal gradients (e.g., Clausen et al., 1940
; Clausen, 1951
; Gurevitch, 1988
; Aradhya et al., 1993
; Wen and Hsiao, 2001
; Reisch et al., 2003
), the majority have addressed genetic variation in gymnosperms and angiosperms, while little is known about the partitioning of genetic diversity along altitudinal gradients in Pteridophyta. Sciaretta et al. (2005) recently studied the genetic structure of Athyrium filix-femina subsp. asplenioides (Michx.) Farwell (Woodsiaceae) along an altitudinal gradient in the southern Appalachians. Most previous studies on the genetic structure of fern populations have focused on general patterns (Haufler, 1987
; Soltis and Soltis, 1987
, 1988
, 1990
), and most are based on enzyme studies. More recently molecular techniques such as RAPD (Wang et al., 2004
), inter simple sequence repeat (ISSR) (Camacho and Liston, 2001
), AFLP (Woodhead et al., 2005
), and microsatellites (Pryor et al., 2001
) have been applied. Investigations using mainly allozyme markers have shown that many fern species are outbred, frequently characterized by a Hardy–Weinberg equilibrium within populations (Soltis and Soltis, 1990
), and mostly have a low genetic distance across wide geographic ranges (Soltis et al., 1988
; Sciaretta et al., 2005
). Breeding system and spore dispersal have been suggested to constrain mechanisms of reproductive isolation and may explain why ferns have lower rates of endemism than angiosperms (Sciaretta et al., 2005
and literature therein).
The diploid (2n = 40) fern species Athyrium filix-femina (L.) Roth sensu stricto is widely distributed in Europe and Northern Asia. It belongs to the species complex A. filix-femina sensu lato that has a circum-north temperate distribution (Hulten, 1961
). In our study, we chose 20 populations from four elevations in each of five Swiss regions. Our aim was to compare characteristics of phenotypic, allozymic, and RAPD markers, all of which are based on genetic variation. We hypothesized that environmental differences along an altitudinal gradient generate genetically determined adaptations in A. filix-femina populations, and we expected genetic and phenotypic diversity to be partitioned along altitudinal gradients. For comparison, but using only allozymes and phenotypes, we included three distant populations from Italy and Spain,
Similar studies on gymnosperms or angiosperms have shown that plants growing along altitudinal gradients or growing under different conditions are characterized by fixed, locally adapted phenotypes (Turesson, 1922
; Claussen et al., 1940
; Claussen, 1951
). In more recent studies, these adaptations were shown to have a genetic background (e.g., Linhart and Grant, 1996
; Briggs and Walters, 1997
; Hufford and Mazer, 2003
). We asked the following questions: To what degree are populations from different elevations phenotypically and genetically variable? Is there a general pattern of variability among populations from different altitudes? To what degree do geographic distances between populations correlate with genetic differences? Are levels of variation the same when considering phenotype frequencies, enzyme patterns, and RAPD markers?
MATERIALS AND METHODS
Plants used
Athyrium filix-femina is a member of a common circum-temperate species complex and occurs in a wide range of mesic to humid habitats from lowlands to alpine areas. It is especially common within forests but can also grow in alpine grasslands, where it is partially protected in niches among large stones. Earlier studies have shown that the European Athyrium filix-femina is characterized by outbreeding (Schneller, 1979
, 2004
).
Study sites
In 1999, between 22 and 26 plants were collected from each of 20, well-developed populations in seven regions on the northern slopes of the Alps. Four of the regions were connected valleys (Urnerboden/Glarnertal and Meiental/Urnertal) and were combined into two sampling regions (Table 1). Four populations at four different altitudinal ranges of 1750–1800 m (
= 1780 m), 1400–1450 m ( = 1424 m), 810-1170 m ( = 946 m), and 450–520 m ( = 490 m) a.s.l. were sampled from each region (Table 1). Locations of the 20 populations are given in Fig. 1. The distances from these Swiss populations to the Italian and Spanish populations were ca. 360 km and 1300 km, respectively. Swiss plants were excavated and transferred into a garden bed in the Botanic Garden of Zurich, where they have been cultivated since 1999. Within the garden bed, they were randomly arranged into three subpopulations. For the three Spanish and Italian populations, live parts of leaves were sampled, and only allozyme and phenotypic variation were investigated.
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). The frequency of each phenotype was calculated under the assumption that separate regulatory genes are responsible for each character. The following characteristics were determined: (1) Petiole and rachis color: green or red (anthocyanin). The suggestion that this character is regulated by two alleles, R (dominant) and r (recessive) (Andersson-Kottö, 1931
) was recently confirmed experimentally (J. Schneller, unpublished data). Individuals that are homozygous dominant (RR) or heterozygous (Rr) for petiole and rachis color develop a red stem, whereas those that are homozygous recessive for stem color (rr) produce green petioles and rachises. (2) Petiole and rachis hairs: distribution of unicellular hairs. Individuals with the allelic combinations HH and Hh produce hairs on the adaxial and abaxial side of the petiole and rachis, whereas individuals with two recessive alleles for hair production (hh) produce hairs only on the adaxial side of the petiole and on part of the rachis. Dominance of the H allele was revealed by J. Schneller (unpublished data). (3) Petiole and rachis stripe. Individuals with the assumed allelic combination SS and Ss have a red stripe on the adaxial side, whereas individuals with two recessive alleles do not have a stripe. This character is also observable on individuals with the RR phenotype. Eight possible phenotypes (rhs, rHs, rhS, rHS, Rhs, RHs, RhS, RHS) were distinguished in A. filix-femina, with allelic frequencies shown in Table 1.
Allozyme analysis
Pinnae of living plants were collected and kept for a maximum of 6 d at 4°C. One to two pinnules were ground and extracted in 100 µl Tris-HCl-4% PVP buffer (Soltis et al., 1983
; Schneller and Scheffrahn, 1989
). Part of each extract (2–3 µl) was added to a slotted foil on an agarose gel (Schneller and Scheffrahn, 1989
). Three enzyme systems were examined for variation: glucose-6-phosphate isomerase (GPI; EC 5.3.1.9), isocitrate dehydrogenase (IDH; EC 1.1.1.41), and phosphoglucomutase (PGM; EC 5.4.2.2.). These systems (with 14 alleles in total) were used because they have provided sufficient variation in previous studies (J. Schneller, unpublished data). An agar overlay technique was used for staining the agarose gels. Substrate ingredients of each system, as cited by Wendel and Weeden (1989)
, were dissolved in 3 mL of buffer, and 0.3 g of agar was boiled in 27 mL buffer. Before staining, the agar solution was cooled to 35°C, mixed with the corresonding substrate, 3 µL MTT, and 2 µL PMS. The mixture was poured into a frame, placed on the gel, and developed at 37°C in the dark.
RAPD analysis
Two to three pinnules of fresh leaves from plants in the Botanic Garden of Zürich were collected and dried in silica gel. For DNA extraction, we used the Qiagen DNeasy MiniKit following the manufacturer's protocol, but the incubation time was extended by 15 min. Optimized reactions were prepared using 12.5 µL each of 2 mM MgCl2, 0.1 mM dNTP's, 0.05 U Taq DNA polymerase with 1.5 mM supplied polymerase buffer (Amersham Pharmacia Biotech), and 0.2 µM oligonucleotide RAPD primers (Operon Technologies, Alameda, CA), and ca. 3 ng template DNA in sterilized double-distilled water.
Thermal cycling was performed with the following Genius thermocycler (Techne TC-412, Staffordshire, UK) profile: an initial denaturation (3 min at 94°C), followed by 40 cycles of denaturation (30 s, 94°C), primer annealing (60 s, 39°C), and extension (90 s, 72°C), with a final extension of 5 min (72°C). Amplification products were separated in 1.5% agarose gels in Tris-EDTA-acetic acid (TAE) buffer at 65 V for 4 h and stained with ethidium bromide. Images of the gels were captured using the MultiGenius System (Syngene, Cambridge, UK). Amplification products were analysed using the program GeneSnap 4.00 (Syngene, 1993–2000).
Forty-two primers from Operon Technologies (kits A–D) were screened using 23 individuals, one or two from each population. A total of 33 primers yielded amplification products, five of these had uniform patterns. Patterns were tested with repeated amplifications and varying MgCl2 and DNA concentrations. Eight primers (A10, A19, B11, B15, C07, D07, D13, D20) reliably yielded 60 amplification products and were used in the investigation. Amplification products were treated as phenotypes, with each band position representing a character with presence/absence states (1 or 0).
Statistical analysis
Bootstrap values of the three assumed morphological characters, allozyme data, and RAPD banding phenotypes were calculated using Biosys-2 (Swofford and Selander, 1997
) and TFPGA software (Miller, 1997
) and presented in UPGMA trees. Neighbor joining analyses using Biosys-2 and Phylip 3.6 (Felsenstein, 2004
) revealed largely unresolved trees. Fixation indices and gene diversity were calculated from allozyme results as described by Goudet (1995)
. An AMOVA analysis of RAPD data was carried out using the software Arlequin, version 2.0 (Schneider et al., 2000
). The original data set containing 381 different multilocus banding patterns was transformed into a squared Euclidean distance matrix, using the program NTSYS-pc2.1 (Rohlf, 2000
) and then analyzed using Arlequin. NTSYS-pc2.1 was also used to calculate Mantel values.
RESULTS
Phenotypic analysis
The sample size for the phenotypic analyses is shown in Table 1. The investigation revealed no statistically significant correlation among the five Swiss regions, elevations, or 20 populations. In contrast, the populations from Italy and Spain segregated from the Swiss sites (Fig. 2). The Italian population differed in the frequency of the red petiole of rhachis phenotype (R-) and the presence of a red stripe (S-); both Spanish populations had only the green phenotypes (rr, ss) for both traits.
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Species growing along altitudinal gradients can be exposed to both abrupt and gradual differences in environmental conditions over relatively short distances. Plant species distributed across a range of altitudes can adapt differently to the local conditions. They may adapt to changing conditions either by developing considerable phenotypic plasticity, genetic variation, or a combination of both. A plethora of investigations has shown that clinal variation along altitudinal or latitudinal gradients has a predominantly genetic background (Linhart and Grant, 1996; Briggs and Walters, 1997
; Hufford and Mazer, 2003
, and references therein).
The extent of phenotypic plasticity and its correlation to genetic variability has been studied repeatedly (Bradshaw, 1965
; Scheiner, 1993
; DeWitt et al., 1998
; Sultan, 2003
). Although a few studies on flowering plants demonstrate low genetic differentiation among populations along an altitudinal, most of these studies were carried out on tree species (Aradhya et al., 1993
; Oyama et al., 1993
; Konnert, 1996
; Leonardi and Menozzi, 1996
; Lewandowski et al., 1997
; Hilfiker et al., 2004
).
Earlier investigations have shown that most fern populations have high genetic variability within and considerable genetic similarity among populations (Haufler, 1987
, and references therein). However, the number of populations and collection areas have differed tremendously (Haufler, 1987
, 2002
; Soltis and Soltis, 1999
). Sciaretta et al. (2005)
recently described the relationship between population distance and altitude with genetic variability in Athyrium filix-femina subsp. asplenioides. They showed that to revealing genetic differentiation among populations for fern species requires much larger sample sizes than for most higher plants. To quantify genetic variation in European members of the species complex of Athyrium filix-femina, we studied Swiss populations from five regions and at four altitudes. For comparison, one Italian and two Spanish populations were included. Our results show that genetic (allozyme and RAPD) and genetically regulated phenotypic variability in A. filix-femina is mainly found within populations. Genetically regulated phenotypic variability has also been shown in an earlier investigation on different populations using autocorrelation analysis (Schneller, 2004
). The differentiation among Swiss populations for all three markers (phenotypes, allozyme, and RAPD) is small and does not correlate with an altitudinal gradient or with the distance among regions. Populations from different regions and altitudes clustered together (Figs. 2, 3, 4), in agreement with the investigation of Sciaretta et al. (2005)
on A. filix-femina subsp. asplenioides. The greatest geographical distance between two Swiss populations was nearly 100 km. The altitude differed by about 1400 m. Very low differentiation between populations in spite of substantial geographical distance was also supported for the North American A. filix-femina subsp. asplenioides populations, which also have very low differentiation between populations (Sciaretta et al., 2005
).
Considering the substantial environmental differences at varying elevations, our result is unusual. Whether other parts of the genome responsible for ecophysiological features have similar patterns or are locally adapted remains an open question.
At broader geographical scales, our analyses revealed an increase in genetic distance with increasing geographic distance. The population from Italy (Alpe Apuani) differed less from the Swiss populations than did the more distant Spanish populations. This suggests a possible, more general, genetic structure of the species Athyrium filix-femina. More detailed investigations are necessary to determine if this represents the more general genetic structure of the species A. filix-femina.
Given that A. filix-femina is outcrossing (Schneller, 1979
), the observed distribution of genetic variation can be explained by long-distance spore dispersal and extensive gene flow. Long-distance spore dispersal and extensive gene flow are characteristic of many homosporous fern species that outcross and produce large numbers of small, wind dispersed spores (Soltis and Soltis, 1990
; Sciaretta et al., 2005
). Therefore, local genetic adaptations to strong differences in ecological conditions across an altitudinal range are less probable because of effective gene exchange. If this is true for the whole genome, it may be argued that selection for phenotypic plasticity allows A. filix-femina (and similar outbreeding fern species) to colonize a broad ecological niche. This characteristic cannot be easily reconciled with the hypothesis of genetic adaptation to environmental differences along altitudinal gradients (Coyne and Orr, 2004
). One explanation may be that our investigation is based mainly on neutral genes, which regulate allozymes and, eventually, phenotypes. Our investigation does show, however, that genetic variability of populations, breeding system, and spore dispersal are all linked. Although not detected in our study, genes responsible for adaptation to environmental conditions may exist and may be locally selected. This would then lead to ecotypes in the sense of Turesson (1922)
or Claussen et al. (1940)
.
We find similar patterns of genetic variation in many wind-pollinated tree species such as Fagus sylvatica (Konnert, 1996
; Gömöry et al., 2003
; Wang, 2004
) and Abies alba (Liepelt et al., 2002
) where effective gene flow consequently leads to genetic similarities among distant populations.
Genetic adaptation has to be assumed, when comparing populations over a larger distribution area, such as Italy (Alpe Apuani) or Spain in the current study. In this case isolating mechanisms due to limits of spore dispersal become evident.
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1 The authors thank Prof. E. Conti for providing financial support, A. Humphreys for reading and corrections, and M. Schneller for help collecting the ferns in their natural habitat. 
2 Author for correspondence (e-mail: schnell{at}systbot.unizh.ch
)
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