Where are the glacial refugia in Europe? Evidence from pteridophytes

Biological Journal of the Linnean Society (1999), 66: 23–37. With 3 figures Article ID: bijl 1998.0257, available online at http://www.idealibrary.com on

Where are the glacial refugia in Europe? Evidence from pteridophytes JOHANNES C. VOGEL1∗, FREDERICK J. RUMSEY1, J. JAKOB SCHNELLER2, JOHN A. BARRETT3 AND MARY GIBBY1 1

Conservation Biology Laboratory, Department of Botany, The Natural History Museum, Cromwell Road, London SW7 5BD; 2Institut fu¨r Systematische Botanik, Universita¨t Zu¨rich, Zollikerstr. 107, 8008 Zu¨rich, Switzerland; 3Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH Received 2 February 1998; accepted for publication 23 May 1998

In this paper we demonstrate that, by investigating polyploid complexes in Asplenium, it is possible to locate the areas in Europe that are southern glacial refugia, and are likely to have been so since the beginning of the Pleistocene during the consecutive cold and warm periods in Europe. Identification and conservation of these specific areas that serve as safe havens for plants, and perhaps animals, is of paramount importance for the maintenance of European biodiversity because Man’s activities are resulting in an ever-increasing loss of natural habitats and putting diversity at risk. The genus Asplenium in Europe comprises some 50 taxa: half of these are diploid while the other half are polyploids derived from the diploids. All aspleniums in Europe are (small) rock ferns with high substrate specificity. Today, most of mainland Europe, Scandinavia and the British Isles has been colonized by polyploid Asplenium species, while the diploids that gave rise to these polyploids are distributed around (and more or less confined to) the Mediterranean Basin. In the tetraploids genetic variation is partitioned mostly between sites, whereas diploids show a high degree of genetic variation both within and between sites. The tetraploid taxa seem capable of single spore colonization via intragametophytic selfing, but the diploid taxa appear to be predominantly outbreeding. For most diploids at least two gametophytes, produced by different spores, have to be present to achieve fertilization and subsequent sporophyte formation for the successful colonization of a new site. This results in a slower rate of colonization. The formation of auto- and allopolyploid taxa from diploid communities appears to have been a recurrent and common feature in Europe. Minority cytotype exclusion is likely to prevent the establishment of tetraploids within the diploid communities, but spores from tetraploids can establish populations outside the diploid communities. The differences between colonization abilities of tetraploid and ancestral diploid taxa, resulting from their different breeding systems, has prevented the merging and mingling of their ranges and led to the establishment of contact/ hybrid zones. This has resulted in the restriction of diploid populations to ancient glacial refugia and the colonization of the rest of Europe by polyploids. Mapping the current distribution of these diploid communities and comparing the genetic diversity within and between outbreeding diploid Asplenium taxa allows us to define the area, age and historical biogeography of these refugia and to assess their importance for present day genetic and species diversity in Europe.  1999 The Linnean Society of London

∗ Corresponding author. Email: [email protected].

1

0024–4066/99/010023+15 $30.00/0

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 1999 The Linnean Society of London

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J. C. VOGEL ET AL.

ADDITIONAL KEY WORDS—glacial refugia – Asplenium – rock ferns – genetic diversity – polyploidy – reproductive biology – historical biogeography. CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . Reproductive biology and breeding systems in homosporous ferns . . . Biogeography and ecology of Asplenium in Europe . . . . . . . . . A comparison of species diversity of diploid Asplenium in relation to putative glacial refugia of trees . . . . . . . . . . . . . . . . . Genetic diversity in different Asplenium taxa in previously glaciated areas and refugia . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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24 25 26

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29

. . . .

30 34 35 35

. . . .

INTRODUCTION

The present distributions of animal and plant species are determined not only by current suitable climatic and biotic conditions but also by historic events. Large parts of Europe have been directly influenced by the last Weichselian glaciation, and current distributions of organisms still reflect colonization into these areas after the retreat of the glaciers. Hewitt (1993, 1996) reviewed evidence derived from pollen analysis, fossil insect remains and the distribution of hybrid zones in order to reconstruct postglacial colonization patterns and to explain the frequent geographic subdivision in the genetic structure of many species. He also discussed the problem of higher genetic diversity in southern parts of Europe (mainly around the Mediterranean Basin) and the relatively low genetic diversity in northern Europe. This partitioning of genetic diversity is explained by the hypothesis that colonization happened only from the northern fringes of refugial areas (leading edge colonization)—the southern populations in such refugia are prevented from participating because they are blocked by the northern, expanding populations. Such a process would lead to only a small fraction of the overall genetic diversity in refugia being dispersed into the areas vacated by the retreat of glaciers, with the bulk of genetic variability remaining in situ in refugial areas. In this paper we shall explore whether evidence from the investigation of the genetic variability of pteridophytes provides further support for this hypothesis (Hewitt, 1993, 1996). We shall further demonstrate that, by investigating polyploid complexes in Asplenium and other pteridophytes, it is possible to locate the areas mainly in southern Europe that are safe havens for genetic and species diversity, and are likely to have been so since the beginning of the Pleistocene during the consecutive cold and warm periods in Europe. In the last 130 years pteridophytes in Europe have been studied extensively, making them one of the best known and researched groups of organisms on a continental scale. A wide spectrum of information on their distribution, ecology and biosystematics is available (Luerssen, 1889; Manton, 1950; Hegi, 1984; Prelli & Boudrie, 1992; Page, 1997). The genus Asplenium has received particular attention (Lovis, 1977; Reichstein, 1981, 1984) and comprises some 50 taxa in Europe, half of which are diploid, while the other half are polyploids derived from the diploids. All of these taxa are (small) rock ferns with high substrate specificity, and hence have special habitat requirements on natural rock faces. In the last few thousand years a small number of taxa have colonized

GLACIAL REFUGIA

25

walls, quarries and other man-made structures as substitute habitats. Unlike other vascular plants, ferns disperse as haploid spores and the sporophyte and gametophyte generations are independent. Spores are well adapted for long-range dispersal and consequently can reach scattered rock outcrops. A further adaptation is that some taxa can colonize a new site with a single spore via an extreme form of selfing, intragametophytic selfing. The extreme leptokurtic nature of spore dispersal (95% of spores fall within 1–10 m from the parent plant; Schneller, 1975) combined with intragametophytic selfing may produce an extreme form of colonization by the ‘leading edge’ process proposed by Hewitt (1993, 1996). From a comprehensive biosystematic study into the genus Asplenium in Europe, using allozyme variation and chloroplast DNA (cpDNA) markers (Vogel, 1995; Vogel, unpubl.), it became evident that there was a strong partitioning of genetic diversity within and between diploid and polyploid species in relation to biogeography. This has led us to test the possibilities that: (i) correlations exist between genetic variability, reproductive biology and the current distributions of diploid and polyploid taxa and (ii) the presence of specific taxa of Asplenium in mountainous areas close to the Mediterranean Sea might indicate glacial refugia and safe havens for genetic and species diversity in Europe. While it is widely acknowledged that refugia must have existed in the Iberian Peninsula, southern Italy, the Balkans and the Caucasus (Huntley & Birks, 1983; Bennett et al., 1991; Birks & Line 1993; Taberlet et al., 1998) the exact locations of these refugia are not known, nor is information available about the role of these areas for the maintenance of biodiversity in Europe today.

REPRODUCTIVE BIOLOGY AND BREEDING SYSTEMS IN HOMOSPOROUS FERNS

Homosporous ferns have an alternation of generations with the mature diploid sporophyte forming haploid spores after meiosis. These spores germinate into haploid short-lived gametophytes which bear the sex organs, antheridia and archegonia. Fertilization occurs on the gametophyte and the sporophyte emerges from the, now redundant, gametophyte. Three breeding systems can be found in homosporous ferns: outbreeding, inter- and intragametophytic selfing (Klekowski, 1979; Schneller, 1995). Intragametophytic selfing is a very extreme form of self-fertilization within a single haploid gametophyte, intergametophytic selfing is fertilization between gametophytes derived from spores from one plant, and outbreeding is fertilization between gametophytes derived from different plants. Allozyme studies have shown that most populations of diploid ferns are genetically variable and allele frequencies are in, or close to, Hardy–Weinberg equilibrium, indicating that outcrossing may be the common breeding system, while inbreeding is reported to be the predominant breeding system in polyploids (Soltis & Soltis, 1989, 1992; Masuyama & Watano, 1990). In outcrossing ferns, selfing may be prevented by structural, developmental and genetic systems. For example, Schneller (1979) found a strong correlation between sex, age and width of prothalli of Athyrium filix-femina, with small and young prothalli bearing predominantly male sex organs, whereas large and old prothalli bear predominantly female organs or were bisexual. In bisexual gametophytes the two sets of sex organs were divided by a sterile zone. These features have been reported to encourage intergametophytic fertilization and contribute to the reduction of inbreeding. Furthermore, antheridiogen systems have

26

J. C. VOGEL ET AL.

been reported that induce dark-germination of spores and antheridial formation in neighbouring gametophytes, which is another mechanism that may promote outbreeding (Schneller, 1979; Schneller et al., 1990). Outbreeding taxa accumulate deleterious recessive mutations as heterozygotes. On inbreeding, the presence of these mutations is manifest as ‘inbreeding depression’. Thus, inbreeding a previously outbred (random mating) taxon leads to an increase in the ‘genetic load’ in the inbred descendents. Deleterious recessive mutations cannot accumulate as heterozygotes in an inbreeding taxon because they will segregate as homozygotes in generations following their initial occurrence. The presence of recessive lethal and sublethal genes has been demonstrated experimentally for several taxa following intragametophytic selfing (Klekowski, 1979; Schneller, 1979; Masuyama, 1986). High genetic load on inbreeding has been associated with taxa in stable environments, while taxa capable of long-range dispersal and single spore colonization via intragametophytic selfing have been found to be free or nearly free from genetic load (Lloyd, 1974; Crist & Farrar, 1983). The amount of genetic load on selfing is correlated with the breeding system (Hedrick, 1987): species with a high genetic load tend to be outbreeders and species with low genetic load tend to be inbreeders. Segregational genetic load is strongly reduced in polyploids, and thus a change in breeding system from outbreeder to inbreeder could coincide with the abrupt speciation process of polyploidy, without substantially increasing the ‘genetic load’.

BIOGEOGRAPHY AND ECOLOGY OF ASPLENIUM IN EUROPE

The distribution and ecology of plants, and pteridophytes in particular, is very well studied in Europe ( Jalas & Suominen, 1972; Hegi, 1984; Prelli & Boudrie, 1992; Page, 1997). The occurrence of diploid and polyploid taxa within the genus Asplenium in Europe is roughly partitioned between the Mediterranean Basin and the other, more northern areas. In areas previously affected by glaciation polyploid taxa are predominant, both in species number and relative abundance while most diploid taxa are confined to the Mediterranean Basin. Of the 22 diploid taxa reported from around the Mediterranean Basin and Macaronesia (Table 1) only five have colonized central Europe, Scandinavia or the British Isles but up to 10 tetraploid taxa are recorded from the latter areas. Birks (1976) analysed the distribution of 144 species of pteridophytes in 65 areas within Europe by minimum-variance cluster and principal co-ordinate analysis. He delimited 15 floristic regions and 21 floristic elements amongst pteridophytes and discussed climate and historical causes as principal factors that influenced distribution. Pichi-Sermolli (1979, 1991) analysed the pteridophyte flora of the Mediterranean region and concluded that more taxa are of Tertiary than Quaternary origin; many of these taxa have close affinities with tropical and subtropical taxa and may have had their centres of origin in Asia and Africa. Most species are widely distributed around the Mediterranean, and only a few endemic taxa are known. From the presence of a high number of diploids and a small number of polyploids, PichiSermolli (1991) concluded that the Mediterranean pteridophyte flora is of ancient origin, despite the fact that the number of taxa indicates that it is impoverished. Areas around the Mediterranean (Iberian Peninsula, Italy, Balkans and Greece) have served as refugia during the warm and cold stages in the Pleistocene (Huntley

Huntley & Birks tree refugia

N

B

C

H

M

L

G

K

F

J

E

D

Number present in taxa area

5 5 5 10 7 13 7 12 7 11 11 12 12 12 7 4 3 4 4 5 4

scolopendrium X X X X X X X X X X X X X X X X X X X X X X X X X

X X X X

X X X X X X X X

X X X X X X X X

trichomanes subsp. trichomanes X X X

viride

X X

X

X X X

X

X X X X X X X

X

marinum

Number of areas in which taxon is present: 21 14 15 14

Canary Isles Madeira Azores Morocco Northern Iberian Peninsula Southern Iberian Peninsula Balearic Isles Maritime Alps Corsica/Sardinia Apuane Alps (+ northern Italy) Southern Italy/Sicily Austria/northern Balkans Southern Balkans Greece/Crete/western Turkey Northern Turkey/Caucasus French Atlantic coast South-western Ireland British Isles Southern Poland Central Europe Scandinavia

hemionitis

5

X

X X X X

anceps

3

X X X ?

onopteris sagittata X X X X X X X X X

X

X

X X X X

X

X X

X X X

obovatum

17 10 10

X X X X X X X X X X X X X X X X X

fontanum

9

X

X X X

X X X X X

seelosii

4

X

(X)

X ? X

petrachae subsp. bivalens

2

X X ?

hispanicum

2

X

X

inexpectans

6

X X X

X X X

cuneifolium

7

X X

X ? X X X X

fissum

5

X X X X

X

ruta-muraria subsp. dolomiticum

6

X X X X X

X

ceterach subsp. bivalens

5

X X X X X

jahandiezii

1

X

aegaeum

2

X X

bourgaei

1

X

1

X

septentrionale subsp. caucasicum

TABLE 1. Distribution of diploid taxa in the genus Asplenium (all taxa 2n =72) in Europe and adjacent regions. While the highest species diversity is recorded from around the Mediterranean Basin only very few species occur in previously glaciated areas such as Central Europe, Scandinavia and the British Isles. Many of the diploid taxa are involved in European polyploid taxa

GLACIAL REFUGIA 27

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J. C. VOGEL ET AL.

& Birks, 1983; Bennett et al., 1991; Birks & Line, 1993). Here taxa may have survived and it can be assumed that large areas of Europe (Scandinavia, the British Isles and Central Europe, north of the Alps) were colonized from refugia in the Mediterranean area following the retreat of the ice after the Weichselian glaciation. Taberlet et al. (1998) compared the phylogeography of four tree and six animal taxa in Europe to explore the most likely postglacial migration routes. Almost all species investigated in their study migrated out of the three potential southern refugia (Iberian Peninsula, Italy and the Balkans), but Norway spruce (Picea abies (L.) H. Carsten) must have survived in eastern refugia in the Dinaric Alps, the Carpathians and near the present-day area of Moscow (Huntley & Birks, 1983; Lagercrantz & Ryman, 1990). Further evidence for the role of colonization from the east into Scandinavia via (southern) Finland was reported for oak (Quercus sp.) by Ferris et al. (1998). The profuse production of wind dispersed propagules (=spores) allows pteridophytes to colonize suitable habitats over large geographical distances, including remote islands (Tryon, 1986). However, colonization may be limited as many species have a rather narrow ecological niche that is determined by the requirements of both the gametophyte and sporophyte stage. Of the c. 50 different taxa distinguished in the genus Asplenium in Europe (including the Macaronesian islands and northern Turkey) about two-thirds of the taxa are calcicole, one-third are calcifuge, and three taxa are confined to serpentine. Morphological comparison and cytological evidence have demonstrated that nearly all tetraploids are derived from diploid taxa currently occurring in Europe and Asia Minor (Manton, 1950; Lovis, 1977; Reichstein, 1984). In most cases, the diploid ancestral taxa and derived tetraploids have the same substrate specificity and habitat requirements. However, the current distributions of ancestral diploid taxa and derived tetraploid taxa do not always coincide. Twenty-two diploid taxa are currently recognized in Asplenium in Europe (Table 1) of which two, A. anceps Lowe ex Hooker & Grev. and A. hemionitis L., are confined to Macaronesia and hyper-oceanic enclaves in Portugal or on the Atlantic coast of Morocco. Three taxa—A. scolopendrium L., A. trichomanes L. subsp. trichomanes and A. viride Huds—are widespread throughout Europe, and the latter two are widespread in the Northern Hemisphere. Two taxa—A. marinum L. and A. cuneifolium Viv.—have a widespread but scattered distribution that is greatly affected by their very special ecological requirements, the former growing on sea cliffs and the latter being confined to serpentine outcrops. Three calcicole taxa—A. seelosii Leybold, A. fissum Kit. ex Willd. and A. viride—prefer alpine environments. The majority of diploid Asplenium taxa (A. aegaeum Lovis, Reichst. & W. Greuter, A. ceterach L. subsp. bivalens (D.E. Meyer) W. Greuter & Burdet, A. hispanicum (Cosson) W. Greuter & Burdet, A. jahandiezii (Litard.) Rouy, A. bourgaei Boiss. ex Milde, A. obovatum Viv. subsp. obovatum, A. septentrionale (L.) Hoffm. subsp. caucasicum Fraser-Jenk. & Lovis, A. rutamuraria L. subsp. dolomiticum Lovis & Reichst. and A. trichomanes L. subsp. inexpectans Lovis), are to be found in southern Europe close to the Mediterranean Basin. Many of these areas have been regarded as Pliocene/Pleistocene refugia (Huntley & Birks, 1983; Jermy, 1984; Cooper et al., 1995). Asplenium onopteris L. is widespread around the Mediterranean Basin but so far has not been reported from outside the putative glacial refugia. The Macaronesian Islands are probably poor in Asplenium taxa as they are geographically isolated, rather small in size and uniform in geological substrate, which further reduces the availability of suitable habitats for rock ferns. The putative refugia near the French Atlantic coast and in the south-west of Ireland

GLACIAL REFUGIA

29

are also depauperate in species, although both contain A. onopteris. Only three diploid taxa, A. jahandiezii, A. bourgaei, and probably A. anceps, could be described as palaeoendemics, while seven tetraploid taxa have been recorded from restricted areas, e.g. A. majoricum Litard., A. macedonicum Ku¨mmerle ( Jalas & Suominen, 1972; Reichstein, 1984), with the most extreme being A. presolanense (Mokry, Rasbach & Reichstein) J.C. Vogel & Rumsey, which is confined to one population with about 30 surviving plants in the northern Italian Alps (Mokry et al., 1986; Vogel et al., 1998b). Whether such tetraploid taxa can be described as apoendemics (=narrow endemic polyploid of local origin) according to the definition of Favarger & Contandriopoulos (1961), is questionable. Despite their narrow geographical range, they could be of considerable age, but be restricted rather by their special ecological requirements. All European endemic taxa in Asplenium can be found in the area south of the extent of the last ice-sheet. A COMPARISON OF SPECIES DIVERSITY OF DIPLOID ASPLENIUM IN RELATION TO PUTATIVE GLACIAL REFUGIA OF TREES

Extensive palynological, palaeobotanical and phylogeographic work has identified areas around the Mediterranean Basin which must have served as glacial refugia for trees in consecutive glaciation cycles (Huntley & Birks, 1983; Taberlet et al., 1998). The highest number of diploid Asplenium taxa in Europe are also found in the same areas. West (1977) suggested that some information on the location of refugia might be obtained by comparing pollen profiles from across a wide area and from following the direction of migration of various taxa. Huntley & Birks (1983) collated data from all suitable pollen sequences in Europe, and plotted the data at 500-year intervals for each taxon, especially for all distinguishable tree pollen types, during the last 13 000 years. They suggested that the area where each tree type first appeared during the post-glacial period should correspond broadly to the glacial refugium for that taxon (Fig. 1). These refugia always include mountainous areas which would have allowed altitudinal plant migration, especially for rock ferns. The possibility of altitudinal migration has been reported to be of paramount importance for tree survival in refugia (Bennett et al., 1991). Assuming an average altitudinal lapse rate of 1°C per 200 m (Webster & Streten, 1978; Rind & Peteet, 1985), it is likely that plant taxa could have survived cold- and warm-stage temperature changes in areas where the change is small relative to the available relief, e.g. in the Alps (up to over 4000 m), the Balkans (over 3,000 m) or northwest Greece (2500 m) (Tzedakis, 1993). Under the present climatic conditions and topography, rainfall and surface temperature probably have steeper gradients in the Balkans than anywhere in Europe; July temperatures range from 10°C to 25°C, January temperatures from −15°C to 5°C, and annual rainfall ranges between 750 and 2000 mm per year over distances of less than 100 km (Bennett et al., 1991). The data presented by Huntley and Birks (1983) and Birks and Line (1993) for the refugia of tree taxa are correlated with the distribution of diploid Asplenium taxa in these areas (Fig. 1). In this geographically coarse comparison a high number of diploid Asplenium taxa was observed in many regions that had served as refugia for trees; the highest species diversity is strongly correlated with these areas (Table 1 and Fig. 1), such as the southern Iberian Peninsula, the Maritime Alps, Italy, northern and southern Balkans as well as Greece. The highest number of diploid

J. C. VOGEL ET AL.

30

70°N

20°E

C

40°E

60°N

60°N

T: 1 A: 2 50°N A

T: 2 A: 4

50°N

N C

T: 3 A: 4

L

T: 1 A: 4

B

K

T: 8 (+1) A: 11

T: 4 A: 12

G

J

T: 4 A: 7

M

T: 12 (+3) A: 12 40°N

F

D

T: 3 A: 7

40°N E

T: 10 (+1) A: 12

T: 5 (+3) A: 13 0

T: 9 (+1) A: 11

H

T: 5 (+2) A: 12 20°E

Figure 1. Distribution of tree pollen taxa (T) and diploid Asplenium taxa (A) amongst areas of glacial refugia (after Huntley & Birks, 1983). Refugia deliminated by Huntley and Birks are lettered A–N and the number of tree taxa, identified with certainty from pollen sampling, in each are shown. This distribution is compared with the extant distribution of diploid Asplenium taxa. The detailed list of Asplenium taxa in each area is given in Table 1.

Asplenium taxa (13) was found in the southern Iberian Peninsula (Fig. 1, region E). However, the highest density of diploid taxa in relation to area is found in the Apuane Alps near Massa-Carrara in northern Italy (Fig. 1, south-west corner of region K ) and the region west of the Maritime Alps from Avignon in France east to the Italian border (Fig. 1, region J). GENETIC DIVERSITY IN DIFFERENT ASPLENIUM TAXA IN PREVIOUSLY GLACIATED AREAS AND REFUGIA

Investigation of the partitioning of genetic diversity within polyploid complexes in Asplenium has revealed that outbreeding diploid populations in putative glacial

GLACIAL REFUGIA

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Figure 2. The wall-rue, Asplenium ruta-muraria subsp. ruta-muraria (tetraploid cytotype), one of the most common ‘rock ferns’ in Europe. Its fronds are about 10 cm long. Saaletal near Blankenstein in Germany on diabase rocks (Photo: Vogel).

refugia have a high genetic variability within and between populations while their more widespread polyploid derivatives are comparatively genetically depauperate. A comprehensive biosystematic study into the genus Asplenium in Europe using allozyme variation and cpDNA markers (Vogel, 1995, unpubl.), has resolved a strong partitioning of genetic diversity within and between diploid and polyploid species in relation to biogeography. Results so far have demonstrated that genetic variation is partitioned mainly between the widespread populations in polyploids such as A. adulterinum Milde, A. adiantum-nigrum L., A. trichomanes subsp. pachyrachis (Christ) Lovis & Reichst. and subsp. quadrivalens D.E. Meyer emend. Lovis, and probably also in A. ceterach L. subsp. ceterach and A. ruta-muraria L. subsp. ruta-muraria. In contrast, some diploid taxa show high genetic variation within populations. This correlation of genetic variation and population distribution can be used to locate centres of genetic diversity and discontinuities in the distribution of breeding systems which can help identify glacial refugia. Three polyploid complexes—A. ruta-muraria, A. trichomanes and A. ceterach—appear to be informative to test this hypothesis; so far we have investigated the first two. Some 8000 individual plants from several hundred populations from all over Europe have been investigated using horizontal starch gel electrophoresis. Taxa in the genus Asplenium can be stained for 13 enzyme systems which reveal between 18 and 23 loci. The detailed methods are described in Vogel et al. (1996, 1998a,b). A summary of results from two polyploid complexes, A. rutamuraria and A. trichomanes are presented here, as examples. Asplenium ruta-muraria, the wall-rue (Fig. 2), is certainly one of the most common ‘rock-ferns’ in Europe. The natural habitat of A. ruta-muraria is calcareous rock faces, but it has been able to expand its range by colonizing mortar in man-made walls.

J. C. VOGEL ET AL.

32

A

65°

30°

2° 70° 18° 12°

18°

12°

26° 30° 36° 42° 48° 54°

60°

66° 72°

36°

60° 65°

55°

55°

50°

50°

40° 45° 30°W

40°

40°

35°

6°

36°

6°

0°

12°

18°

30°

36°

96

B ?

A. aegaeum A. ruta-muraria subsp. dolomiticum

Figure 3. A, distribution of tetraploid Asplenium ruta-muraria subsp. ruta-muraria in Europe ( Jalas & Suominen, 1972).B, distribution of diploid Asplenium ruta-muraria subsp. dolomiticum (Χ) and Asplenium aegaeum (Μ) in Europe (after Brownsey, 1976). Localities of Asplenium ruta-muraria subsp. dolomiticum without cytological confirmation are indicated ‘?’. The distribution of Asplenium ruta-muraria subsp. dolomiticum coincides with glacial refugia.

Cytological investigations have revealed that the widespread and common cytotype A. ruta-muraria subsp. ruta-muraria (Fig. 3A) is an autotetraploid (2n=144) derived from the rare diploid taxon, A. ruta-muraria subsp. dolomiticum (2n=72) (Lovis, 1964; Lovis & Reichstein, 1964) (Fig. 3B). Both the diploid and tetraploid cytotypes are calcicole, light-demanding plants. Asplenium ruta-muraria must be an extremely stresstolerant plant as its habitats are subject to great fluctuations of temperature and moisture. The width of cracks in the substrate is important for the colonization and

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early establishment of the fern (Suter & Schneller, 1986). Freeze-thaw cycles can produce narrow cracks in mortar and this could explain why A. ruta-muraria subsp. ruta-muraria is so successful, and common, on man-made walls.The highest number of young plants occur in cracks of up to 2 mm in width, while no plants occur in cracks wider than 9 mm. Asplenium ruta-muraria subsp. ruta-muraria is common throughout Europe (Fig. 3A) and extends from the Near Orient through temperate Asia to the Himalaya and Japan. It can be speculated that A. ruta-muraria subsp. ruta-muraria has dramatically expanded its habitat range in Europe, with the spread of human stone-dwellings during the last 2000 years. In contrast the diploid taxon A. ruta-muraria subsp. dolomiticum has a scattered distribution (Fig. 3B) and appears to be confined to natural rock faces. It was discovered in northern Italy on dolomite rocks near Trentino (Lovis & Reichstein, 1964), and has since been reported only from a few localities in Europe that are recognized as centres of relict survival, such as the Gorge du Verdon, France (Bouharmont, 1972; Prelli & Boudrie, 1992), Apuane Alps (Marchetti, 1992; Ferrarini & Marchetti, 1994), the Italian Dolomites, south-west Austria and Slovenia (Melzer, 1987, 1990; Melzer & Bregant, 1989; Hartl et al., 1992), Bosnia (Vrbas-valley north of Jajce; Brownsey, 1976) and Bulgaria ( Jeßen, 1991). Reports on the occurrence of A. ruta-muraria subsp. dolomiticum in Afghanistan (Fraser-Jenkins, 1992), Bavaria (Reichstein, 1981) and southern Italy (Pichi-Sermolli, 1991) await cytological confirmation. Populations of the tetraploid taxon on natural rock faces, e.g. in Hessen or in Rheinland-Pfalz (both Germany), show little genetic polymorphism (up to three loci with two alleles each) whereas, in contrast, two populations of diploid A. ruta-muraria (n=15 and n=25) in the Gorge du Verdon in southern France showed variation with a high number of alleles at nearly all loci: seven alleles in PGI-2, four alleles in IDH and PGM-1, three alleles in AAT-1, HEX, TPI-2, PGM-2, SkDH, ACP and 6-PGD-2 and two alleles in GDH, ALD, DIA-1 and DIA-2 as well as two phenotypes in MDH-2 (Vogel, unpubl.). This, to our knowledge, is the highest allelic variability ever discovered in ferns. In Europe, two diploid taxa are reported in A. trichomanes: subsp. trichomanes, a widespread calcifuge, and subsp. inexpectans, a rare calcicole. Investigation of the genetic variability in A. trichomanes subsp. trichomanes has revealed that, in Europe, this taxon is variable only at four loci, with two alleles each. Of the 15 populations studied, variation was present only in five populations, the other ten populations being monomorphic. Among the genetically variable populations, the most variable (with two variable loci) has a mean fixation index of F=0.885 (n=44) (Vogel, unpubl.). Howard et al. (1994) reported a significant deficiency of heterozygotes in North American populations of A. trichomanes subsp. trichomanes. The two results support the hypothesis that A. trichomanes subsp. trichomanes, in most of its range, is predominantly an inbreeding taxon and, therefore, capable of single spore colonization and long-range disperal. This could explain its widespread distribution in Europe. In contrast, all eight populations of A. trichomanes subsp. inexpectans investigated from putative refugia in southern France and eastern Austria are genetically variable with genotype frequencies at, or near, Hardy–Weinberg equilibrium. Furthermore, 14 loci in A. trichomanes subsp. inexpectans show allelic variation (Vogel, unpubl.). The observations are compatible with A. trichomanes subsp. inexpectans being an outbreeding taxon, and therefore restricted in its ability to colonize. While the two diploid sister taxa, the calcifuge A. trichomanes subsp. trichomanes and the calcicole A. trichomanes subsp. inexpectans, do not compete for the same habitat, several tetraploid taxa in

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the A. trichomanes complex, viz. subsp. quadrivalens, subsp. hastatum (Christ) S. Jeßen and subsp. pachyrachis, are widespread and common throughout Europe on limestone. In these tetraploid taxa genetic variation is partitioned mainly between populations, indicating colonization via single spores, and investigation of the population dynamics of A. trichomanes subsp. pachyrachis suggests it to be an inbreeding taxon (Vogel, unpubl.).

DISCUSSION

From our survey of allozyme diversity in European Asplenium conducted so far, it is evident that the amount of genetic variation in diploid outbreeding taxa occupying areas considered to be glacial refugia is greater than that within both diploid and tetraploid conspecific taxa that occur in areas that became available for colonization only after the retreat of the glaciers about 12 000 years ago. The ‘obligate’ outbreeding behaviour of most diploid taxa does not allow for single spore colonization of distant, suitable, safe sites for gametophyte development and subsequent sporophyte formation—two spores have to germinate within millimetres or a few centimetres of one another to permit successful fertilization and the establishment of a sporophyte. However, tetraploid derivatives are capable of intragametophytic selfing (Schneller & Holderegger, 1996), and are able to colonize rapidly and exploit new habitats. While a gametophyte from a diplospore derived from restitution during sporogenesis or somatic polyploidy will most likely be crossfertilized by the majority (diploid) cytotype within established diploid communities, such a spore taken away by wind, and landing outside such communities, will be immediately able to establish its own populations outside the range of the diploids. Bouharmont (1972) produced artificial tetraploids via apospory from a diploid plant of A. ruta-muraria subsp. dolomiticum from the Gorges du Verdon. These autotetraploid plants showed not only different degrees of meiotic irregularity including quadrivalents, trivalents, bi- and univalents, but also cells with nearly regular meiosis showing up to 63 bivalents (full pairing would be 72 bivalents). This observation by Bouharmont allows us to envisage a scenario whereby a diploid taxon produces diplospores at a low frequency, and, by intragametophytic selfing, tetraploids can be produced which cannot establish themselves within the diploid population. Felber (1991) modelled the establishment of a tetraploid cytotype in a diploid population in relation to the fitness of the cytotypes and found that the conditions for the establishment of a polyploid were quite restrictive. He concluded that, in nature, only genetical or environmental changes, a high and constant immigration of polyploids, or changes within small populations leading to a substantial increase of 2n gametes would allow the spread of a polyploid. However, diplospores dispersed from the diploid communities into new areas, perhaps vacated by glacial retreat, would give rise to tetraploids that were free, both ecologically and sexually, from competition from the diploid ancestor/parent. Among these pioneer colonizing tetraploids, there would be variation in the degree of meiotic irregularity and those able to produce chromosomally balanced meiotic products (spores) at a reasonable frequency, would be favoured by Natural Selection and establish new colonies. At the same time minority cytotype exclusion may prevent the tetraploid taxon from invading the diploid communities (Felber, 1991; Dijk & Barx-Schotman, 1997), and,

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perhaps, driving the diploids to extinction. Under these conditions, the ancient distribution of the relictual diploid taxon is preserved, leading to the appearance of contact/hybrid zones. Such contact/hybrid zones are most likely to be found in polyploid complexes where the diploid and polyploid taxon have overlapping ecological requirements, e.g. in the A. ruta-muraria, A. ceterach and A. trichomanes complexes. The great number of polyploid complexes within the three genera Asplenium, Cheilanthes and Dryopteris in Europe, allow us to examine this phenomenon. The identification of contact/ hybrid zones, and the measure of overall genetic diversity in different diploid taxa within, and between, local areas will not only allow us to delimit long-term refugia, but also to compare the different regions in southern Europe, Macaronesia and Asia Minor to assess their importance for the survival of biodiversity in Europe. A measure of the time since a diploid became established in an area, and began to accumulate (private) mutations, can be obtained from the genetic variation present in extant populations. This will inevitably vary between taxa and refugia. However, relative estimates could be obtained from an array of potentially refugial diploids such as A. ruta-muraria subsp. dolomiticum, A. ceterach subsp. bivalens, A. trichomanes subsp. inexpectans and A. septentrionale subsp. caucasicum that grow together in different refugia in Austria, northern Turkey/Caucasus, former Yugoslavia, Bulgaria, southern France and the Apuane Alps. The differences in genetic variation, in combination with discontinuity of breeding systems, substrate specificity and the different distribution patterns of diploids and derived polyploids, have the potential to make Asplenium a model organism for attempting to locate glacial refugia and to reconstruct postglacial migration routes in Europe. Seed plants disperse with diploid propagules. Therefore they are much more ‘mobile’ than obligate outbreeding diploid Asplenium with a very short-lived sexual stage. Thus, while seed plants can migrate in a general area (migratory relicts), to escape adverse conditions, the choice for diploid Asplenium, would be in situ long term survival during consecutive warm and cold stages since, perhaps, the Tertiary, or extinction.

ACKNOWLEDGEMENTS

This research is supported by grants from the Natural History Museum Research Fund and by the TOTAL Foundation. We would like to thank Prof. Richard Abbott, Prof. John Birks and Prof. Godfrey Hewitt for their very valuable comments and constructive criticisms on the manuscript.

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