Genetic diversity and phylogeography in two diploid ferns, Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens, in the western Mediterranean

Molecular Ecology (2009) 18, 4940–4954 doi: 10.1111/j.1365-294X.2009.04402.x

Genetic diversity and phylogeography in two diploidferns, Asplenium fontanum subsp. fontanum and A.petrarchae subsp. bivalens, in the western Mediterranean

H. V. HUNT,*† S . W. ANSELL,* S . J . RUSSELL,* H. SCHNEIDER* and J . C. VOGEL*

*Department of Botany, Natural History Museum, London SW7 5BD, UK, †Department of Genetics, University of Cambridge,

Downing Street, Cambridge CB2 3EH, UK

Corresponde

E-mail: hvh2

Abstract

Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens are diploid rock

ferns of limestone outcrops of the western Mediterranean region. Asplenium fontanumsubsp. fontanum occurs from Valencia through northeastern Spain to the Alpes-

Maritimes and Swiss Jura. Asplenium petrarchae subsp. bivalens occurs only on Majorca,

in Valencia and possibly in southern Spain. We analysed allozyme and chloroplast

genetic marker diversity in 75 populations of A. fontanum subsp. fontanum and 12

populations of A. petrarchae subsp. bivalens sampled from across their respective ranges.

The two species show similar levels of species and population genetic diversity to one

another and to other diploid European Asplenium taxa. Both are predominantly

outbreeding, as indicated by FIS = 0.108 and 0.167 respectively. Substantial between-

population differentiation results largely from differentiation between regions. Isolation

by distance operates over limited geographic ranges, up to 50 km. In A. fontanum subsp.

fontanum, the major geographical differentiation between Valencia and the rest of the

taxon range probably represents an ancient range fragmentation. A less pronounced

differentiation divides populations in the SW from those in the NE of the range, with

evidence for a biogeographic link between the eastern Pyrenees and southeastern France.

High diversity in the Pyrenees may either represent ancient population differentiation,

or a suture zone. In A. petrarchae subsp. bivalens, populations on Majorca exhibit a

subset of the genetic diversity present in Valencia, although the two regions are strongly

differentiated by differing allele frequencies. Dispersal from the mainland may have

founded Majorcan populations, although a role for in situ island survival cannot be

excluded.

Keywords: allozyme electrophoresis, Asplenium, chloroplast DNA, fern phylogeography, rock

fern

Received 18 June 2009; revision received 26 August 2009; accepted 15 September 2009

Introduction

The western Mediterranean is one of the most biodi-

verse regions in Europe (Garcıa-Barros et al. 2002). This

high biodiversity has been attributed to the habitat

diversity resulting from the wide range of climates,

topography and underlying geology, and the complex

climatic and vegetation history of the region (Rivas-

nce: Harriet V. Hunt, Fax: +44 (0)1223 339285;

[email protected]

Martınez et al. 1999; Ferrer-Castan & Vetaas 2005). Paly-

nological and phylogeographic studies have established

that a wide range of plants and animals maintained

populations in the Iberian Peninsula throughout the

Pleistocene glaciation cycles (Huntley & Birks 1983;

Taberlet et al. 1998; Gomez & Lunt 2006). The moun-

tainous topography likely allowed species to respond to

climatic change by migrating along altitudinal gradients

(Gomez & Lunt 2006) and promoted differentiation of

isolated populations: the areas of highest plant species-

richness and endemism within Iberia correspond to

� 2009 Blackwell Publishing Ltd

WE STERN MEDITERRANEAN ROCK FERNS 4 94 1

mountain ranges, in particular the Pyrenees and the

Sierra Nevada (Ferrer-Castan & Vetaas 2005). Some

gene flow continued in both directions across the Pyre-

nees throughout the Pleistocene: Iberian forest popula-

tions show less extreme genetic differentiation than

those in other major refugia (Petit et al. 2003).

Ferns are currently underrepresented in phylogeo-

graphic studies, although there is considerable interest

in their ecology and biodiversity in the Iberian Penin-

sula at the species level and above (Ferrer-Castan &

Vetaas 2005; Moreno Saiz & Lobo 2008). Asplenium, the

most species-rich fern genus (Smith et al. 2006) is an

excellent model for addressing questions of species evo-

lution and response to climate change at a pan-Euro-

pean scale. The distribution of the c. 50 species and

subspecies in Europe has been extensively studied, and

areas of greatest species-richness broadly correspond to

the glacial refugia identified by Huntley and Birks

(Vogel et al. 1999). A molecular phylogeny of the genus

is established (Schneider et al. 2004).

Here we analyse the geographic structuring of allo-

zyme and chloroplast DNA (cpDNA) diversity in two

Asplenium taxa, A. fontanum (L.) Bernh. subsp. fontanum

and A. petrarchae (Guerin) DC. subsp. bivalens (D.E.

Meyer) Lovis & Reichst., across their entire respective

ranges in southwestern Europe. Both taxa are diploids

(Lovis & Reichstein 1969; Reichstein & Schneller 1982),

and are nested in the same clade in Schneider et al.’s

(2004) phylogeny of asplenioid ferns. They grow in fis-

sures and crevices of (predominantly) natural limestone

outcrops; A. fontanum reaches higher altitudes (up to

1800 m) than A. petrarchae (up to 700 m; Ibars et al.

1999). Asplenium fontanum subsp. fontanum has a rela-

tively wide geographic range extending from Valencia

province through northeastern Spain and the Pyrenees,

to the Alpes-Maritimes and Swiss Jura, with scattered

populations in Central Europe (Fig. 1A; Jalas & Suomi-

nen 1972; Tutin et al. 1993; Prelli & Boudrie 2002). Asp-

lenium petrarchae subsp. bivalens has a much narrower

distribution, recorded only from the Spanish provinces

of Majorca, Valencia, Malaga (Serranıa de Ronda), Sevil-

la (Sierras Subbaeticas) and Cadiz (Sierra de Grazalema)

(Fig. 2A) (Ibars et al. 1999).

The ranges of A. fontanum subsp. fontanum and A.

petrarchae subsp. bivalens lie almost entirely to the south

of the Pleistocene ice sheet limits, with the exception of

those areas of the Alps and Pyrenees that were covered

by ice caps. Such distributions are typical for diploid

Asplenium taxa in Europe, and have been explained by

the competitive disadvantage of obligate outbreeding in

diploid taxa, compared with their derived polyploids,

in colonizing new habitat: polyploids can establish

populations with single-spore colonization followed by

intragametophytic selfing (Vogel et al. 1999). The exten-


sive distribution of an autotetraploid derived from

A. petrarchae subsp. bivalens, A. petrarchae subsp.

petrarchae, around the Mediterranean coasts of Spain

and France, and sporadically eastward to the Adriatic

and Greece (Tutin et al. 1993), supports this hypo-

thesis and may account for the restricted distribution of

the diploid cytotype.

Population genetic structure in plants is influenced

by various life-history traits including breeding system,

dispersal mechanism and life-form (Hamrick & Godt

1996). Reduced levels of genetic variation are expected

in rare plants, with small and ⁄ or fragmented popula-

tions, as a result of erosion by genetic drift (Gitzendan-

ner & Soltis 2000; Cole 2003). Here we address the

following questions: (1) Does the narrow endemic A.

petrarchae subsp. bivalens show reduced genetic diversity

compared with its widespread congener A. fontanum

subsp. fontanum? (2) How do the observed levels of

genetic diversity compare with those of other plants,

particularly other Asplenium? (3) What is the breeding

system and population structure of these taxa? (4) What

biogeographic regions do the patterns of diversity

suggest? and (5) What is the likely population history

of A. fontanum subsp. fontanum and A. petrarchae subsp.

bivalens?

Materials and methods

Plant collection

Populations of A. fontanum subsp. fontanum and A. petr-

archae subsp. bivalens were sampled throughout their

ranges. Where possible, at least 30 individuals from

each population were collected. Seventy five popula-

tions of A. fontanum subsp. fontanum (2026 plants) from

six geographic regions and 12 populations of A. petrar-

chae subsp. bivalens (621 plants) from two regions were

sampled (Table S1, Supporting information). We dis-

criminated between the diploid A. petrarchae subsp. biv-

alens and its derived autotetraploid A. petrarchae

(Guerin) DC. subsp. petrarchae using fixed heterozygos-

ity banding patterns on allozyme gels and correlation of

this data with spore size measurements and chromo-

some counts on a sub-sample of plants (Hunt 2004).

Allozymes

Allozyme variation was determined for all individuals

sampled. Proteins were extracted in a Tris-HCl (pH 7.5)

buffer (Soltis et al. 1983), and fractionated on 13.7% hy-

drolysed potato starch gels following established proto-

cols (Wendel & Weeden 1989; Vogel et al. 1999). Seven

enzymes were resolved on the lithium borate gel and

electrode buffer system 8 of Soltis et al. (1983): aconitate

B

A

C

Fig. 1 Geographical analysis of genetic variation in A. fontanum subsp. fontanum. (A) Distribution map of A. fontanum subsp. fonta-

num (blue), A. petrarchae subsp. bivalens (green), and the zone where both taxa co-occur (yellow). Proportion of assignation to gene-

pools for each sampled population under K3 Structure output (B), and neighbour-joining phenogram of Nei’s (1972) genetic

distances (Bi) showing bootstrap support values >70%. Populations referred to in the text are numbered. Distribution of chloroplast

haplotypes (C) and their minimum spanning network (Ci). Haplotype numbers are indicated in the network.

4942 H. V. HUN T ET AL.

hydratase (ACN, E.C. 4.2.1.3), acid phosphatase (ACP,

E.C. 3.1.3.2), aspartate aminotransferase (AAT, E.C.

2.6.1.1) leucine aminopeptidase (LAP, E.C. 3.4.11.1),

phosphoglucose isomerase (PGI, E.C. 5.3.1.9), phospho-

glucomutase (PGM, E.C. 5.4.2.2) and triose-phosphate

isomerase (TPI, E.C. 5.3.1.1). Six enzymes were resolved

using a morpholine citrate (pH 7.0–7.4) gel and elec-

trode buffer system (Wendel & Weeden 1989): 6-phos-


B

A

C

Fig. 2 Geographical analysis of genetic variation in A. petrarchae subsp. bivalens. (A) Distribution map of A. fontanum subsp. fontanum

(blue), A. petrarchae subsp. bivalens (green), and the zone where both taxa co-occur (yellow). Proportion of assignation to genepools

for each sampled population under K3 Structure output, with assignations to genepools 2 and 3 combined into a single value (B),

and neighbour-joining phenogram of Nei’s (1972) genetic distances (Bi) showing bootstrap support values >70%. Populations

referred to in the text are numbered. Distribution of chloroplast haplotypes (C) and their minimum spanning network (Ci). Haplo-

type numbers are indicated in the network.


phogluconate dehydrogenase (6-PGD, E.C. 1.1.1.44),

hexokinase (HEX, 2.7.1.1), isocitrate dehydrogenase

(IDH, E.C. 1.1.1.42), malate dehydrogenase (MDH, E.C.

1.1.1.37), PGM, shikimate dehydrogenase (SKDH,

1.1.1.25) and utp-glucose pyrophosphorylase (UGPP,

E.C. 2.7.9). Allozyme banding systems were interpreted

using known enzyme sub-structuring (Kephart 1990),

taking into account the results of previous Asplenium


allozyme studies (Vogel et al. 1999; Suter et al. 2000).

For enzymes with multiple loci, the most anodally

migrating isozyme was designated locus-1, except in

the case of one system (TPI) whose relative migration

rates were reversed between the two species in the

study. Correspondence of loci between A. fontanum

subsp. fontanum and A. petrarchae subsp. bivalens was

determined by the shared banding patterns in the


hybrid, A. x protomajoricum Pangua & Prada, and the

derived allotetraploid, A. majoricum Litard., of these

species (Hunt 2004). Alleles were labelled by their rela-

tive mobility to a high-frequency ‘100’ allele; identity of

alleles was determined by running plants from an

invariant marker population of A. majoricum on all gels.

The allozyme diversity statistics percentage of poly-

morphic loci by the 95% criterion (P95), number of

alleles (A), expected heterozygosity (HE), observed het-

erozygosity (HO) and inbreeding coefficient (f) were cal-

culated in GDA version 1.1 (Lewis & Zaykin 2001).

Effective number of alleles (Ae) was calculated in Pop-

Gene version 1.32 (Yeh et al. 1999). Population structure

and breeding system were analysed using ANOVA (Weir

& Cockerham 1984), providing estimators (f, F, Q-P,

Q-S) of Wright’s (1978) F-statistics FIS, FIT, FST, FCT.

These were calculated for all populations, and sepa-

rately for Valencian vs. non-Valencian samples. F-statis-

tics and associated 95% confidence intervals (obtained

by bootstrapping with 9999 replicates) were estimated

in GDA. Nei’s (1972) genetic distance was calculated

between all pairs of populations for each data set in

GDA. Neighbour-joining phenograms were constructed

in GDA, and bootstrap support (1000 replicates) was

obtained by resampling the allelic frequency data using

DISPAN (Ota 1993). Trees were manipulated in Dend-

roscope version 2.2 (Huson et al. 2007). Mantel tests for

isolation by distance were performed in the R package

(version 4.00, Casgrain & Legendre 2001) between

matrices of log10-transformed pairwise genetic distance

(Nei 1978) and pairwise geographic distance. A modi-

fied Mantel test was performed on matrices of pairwise

genetic similarity (Nei 1978) and distance classes to

determine if genetic isolation by distance was restricted

over a particular geographic range, applying a sequen-

tial Bonferroni correction. Pairwise geographic distances

were calculated from the population location coordi-

nates (Table S1, Supporting information), using the for-

mula at http://www.cpearson.com/excel/latlong.htm.

The number of homogenous gene pools (K) was exam-

ined by Bayesian clustering, as implemented by Structure

2.2 (Pritchard et al. 2000), following the admixture model

(Falush et al. 2003), with correlated alleles, with a priori

assignment of individuals to locations. Ten simulations

were performed for each K, with 1–6 K per dataset, utiliz-

ing 400 000 Markov-Chain Monte-Carlo (MCMC) calcu-

lations per simulation, plus 100 000 MCMC replicates for

burn-in period. Evaluation of the number of K followed

the method in Evanno et al. (2005).

Chloroplast DNA

Chloroplast DNA sequence variation was analysed in

209 individuals of A. fontanum subsp. fontanum from 21

populations (Table S2, Supporting information) and in

47 individuals of A. petrarchae subsp. bivalens from nine

populations (Table S3, Supporting information), i.e. in

approximately 10% of the total number of individuals

analysed for allozyme diversity in each taxon. DNA

extraction was as described in Schneider et al. (2004)

with minor modifications. In A. fontanum subsp. fonta-

num, we sequenced the rps4-trnS intergenic spacer (IGS)

using the primers described by Schneider et al. (2005).

In A. petrarchae subsp. bivalens, we sequenced the

trnLUAA–trnFGAA region, including the trnL intron and

the intergenic spacer (IGS) between trnL and trnF

(henceforth abbreviated as ‘trnL-F’), using the primers

Fern-1 (Trewick et al. 2002) and f (Taberlet et al. 1991).

Amplifications were performed according to Trewick

et al. (2002) and Schneider et al. (2004) with minor

modifications. PCR products were sequenced for both

strands using BigDye Terminator Kits (v. 1.1 Applied

Biosystems, ABI) and an ABI 3730 capillary DNA

sequencer.

Sequences were assembled using SeqMan version

3.56 (DNASTAR, Inc.), and aligned in MegAlign version

3.14 (DNASTAR, Inc.) with manual checking. Sequences

have been submitted to GenBank (accession

numbers EU239484-EU239496 and FJ456852-FJ456857;

Tables S4 and S5, Supporting information). Variable

mononucleotide repeat regions were excluded from the

alignments prior to haplotype determination, because of

the potential for sequence reading errors, or for homo-

plasy or ancient polymorphisms whose inclusion may

result in an ambiguous network. Haplotype assignation,

network construction and AMOVA were performed in

Arlequin version 3.11 (Excoffier et al. 2005).

Results

Asplenium fontanum subsp. fontanum

Allozyme data. Thirteen loci were resolved and scored

for A. fontanum subsp. fontanum. Two loci were mono-

morphic (ACP and TPI-1), and in total 61 alleles were

identified across the 13 loci. Thirty-four of these were

rare (global frequency <5%) and eleven alleles were pri-

vate to a single population. Most alleles private to a sin-

gle population or region were found in the Pyrenees,

Aragon or Alpes-Maritimes regions (Table S6, Support-

ing information). Species-level allelic richness at the 11

polymorphic loci ranged from 2 (MDH-2) to 12 (PGI-2).

Mean species-level diversity statistics across all 13 loci

were as follows: P95 0.615, A 4.692, Ae 1.691, HE 0.272,

HO 0.137.

Twenty-two out of the 75 populations sampled con-

tained fewer than 15 individuals. To reduce sampling

error effects, these populations were excluded for all


A

B

Fig. 3 Mean-population genetic diversity estimates for A. fon-

tanum subsp. fontanum (A) and A. petrarchae subsp. bivalens (B),

grouped by sampling region. Numbers of populations in each

region are shown in the key. Bars indicate standard errors of

the mean values.


subsequent tests, resulting in a dataset comprising 1898

individuals in 53 populations. Diversity statistics for

each population are shown in Table S7 (Supporting

information). Mean values across the 53 populations


HO 0.144 and f 0.108. Mean-population diversity was

substantially lower in Valencia and Switzerland than in

the four intervening regions, Aragon, Catalunya, Pyre-

nees and Alpes-Maritimes, which showed similar

mean-population diversity (Fig. 3A and Table S7, Sup-

porting information).

Hierarchical analysis (Table 1) confirmed significant

departure from Hardy–Weinberg equilibrium with

FIS = 0.108, 95% CI = 0.069–0.145. Strong inter-popula-

tion differentiation was observed (FST = 0.447, 95%

CI = 0.331–0.514), attributable primarily to differentia-

tion between the six geographic regions (FCT = 0.415,

95% CI = 0.285–0.500). The strongest differentiation was

seen between Valencian populations and those else-

where in the range (FCT = 0.552, 95% CI = 0.406–0.646).

Inter-regional differentiation reduced substantially

when Valencia was excluded (FCT = 0.059, 95%

CI = 0.019–0.100). Inter-population differentiation

within the three geographic population clusters

identified by Structure analysis (see below) was broadly

similar: cluster 1 (Alpes-Maritimes and eastern Pyre-

nees) – FST = 0.110 (95% CI = 0.087–0.125); cluster 2

(western Pyrenees and Aragon) – FST = 0.166 (95%

CI = 0.129–0.194); cluster 3 (Valencia) – FST = 0.140

(95% CI = 0.028–0.175). Mantel permutation tests

showed a weak relationship between geographical dis-

tance and genetic distance for the overall data set

(r2 = 0.008, P = 0.340). A modified Mantel test was per-

formed on the dataset excluding Valencian populations

to determine if genetic isolation by distance occurred

over a restricted geographic scale within the major por-

tion of the species’ range. A significant positive correla-

tion was detected over the first four of 15 distance

classes, covering distances up to 50 km (Fig. 4).

Modelling of the number of genetically homoge-

neous clusters in A. fontanum subsp. fontanum using

Structure gave a minimum probability with K = 1

()ln = 24019.9) and maximum probability with K = 5

()ln = 15376) when all 53 populations with N ‡ 15

were included. DK estimates (Fig. 5) supported K = 2,

which split the Valencian from non-Valencian popula-

tions. The model with K = 3 was also biogeographical-

ly meaningful. This model was supported by the

results of Structure analysis excluding Valencian popu-

lations, which gave a minimum probability with K = 1

()ln = 14360.1) and maximum probability with K = 6

()ln = 13114.6), and a DK maximum at K = 2 (Fig. 5).

We therefore propose a model for the whole geo-

graphic range of K = 3. This model additionally splits


the western Aragonese and western Pyrenean popula-

tions from those in the eastern Pyrenees and the Al-

pes-Maritimes (Fig. 1B). Structure plots indicate some

admixture between these two groups throughout the

extra-Valencian range, but especially in northeastern

Spain and Switzerland (Fig. 1B). Assignation of popu-

lations to genepools was highly stable among replicate

runs (Table S7, Supporting information). Neighbour-

joining phenograms showed clusters of populations

consistent with those identified by the Structure

results, with a strong genetic differentiation (bootstrap

value of 99%) separating Valencian from non-Valen-

cian populations, and the remaining populations

broadly separating into a northeastern and two south-

western clusters (Fig. 1Bi).

Ta

ble

1T

wo

-an

dth

ree-

lev

elh

iera

rch

ical

esti

mat

eso

fg

enet

icd

iffe

ren

tiat

ion

and

bre

edin

gco

effi

cien

ts.

All

ozy

me

dat

a:b

rack

ets

ind

icat

eth

e95%

con

fid

ence

inte

rval

s(9

999

bo

ot-

stra

pre

pli

cate

s).

Ch

loro

pla

sth

aplo

typ

ed

ata:

bra

cket

sin

dic

ate

pro

bab

ilit

yv

alu

es(1

000

per

mu

tati

on

s)

Reg

ion

All

ozy

me

dat

aC

hlo

rop

last

dat

a

Np

op

sF

IS(f

)F

IT(F

)F

ST

(h)

FC

T(h

P)

FS

C(h

S)

Np

op

sF

ST

FS

TF

CT

FS

C

Asp

len

ium

fon

tan

um

sub

sp.

fon

tan

um

Tw

o-l

evel

hie

rarc

hic

alan

aly

sis

To

tal

ran

ge

530.

108

0.50

70.

447

210.

513

0.50

4

(0.0

69–0

.145

)(0

.391

–0.5

76)

(0.3

31–0

.514

)(0

.000

)(0

.000

)

Val

enci

a(=

maj

ori

tyas

sig

nat

ion

tog

enep

oo

l3)

80.

099

0.22

50.

140

30.

222

0.22

2

(0.0

20–0

.347

)(0

.166

–0.3

79)

(0.0

28–0

.175

)(0

.082

)(0

.093

)

No

n-V

alen

cia

450.

113

0.26

40.

170

180.

399

0.30

8

(0.0

70–0

.154

)(0

.198

–0.3

06)

(0.1

16–0

.225

)(0

.000

)(0

.000

)

Po

pu

lati

on

sw

ith

maj

ori

ty

assi

gn

atio

nto

gen

epo

ol

1

270.

111

0.20

90.

110

(0.0

54–0

.163

)(0

.138

–0.2

65)

(0.0

87–0

.125

)

Po

pu

lati

on

sw

ith

maj

ori

ty

assi

gn

atio

nto

gen

epo

ol

2

180.

116

0.26

30.

166

(0.0

89–0

.147

)(0

.237

–0.2

75)

(0.1

29–0

.194

)

Th

ree-

lev

elh

iera

rch

ical

anal

ysi

s

[Val

enci

a]:[

all

oth

erre

gio

ns]

[8]:

[45]

0.10

80.

669

0.55

20.

629

[3]:

[18]

0.72

40.

598

0.31

4

(0.0

69–0

.145

)(0

.533

–0.7

37)

(0.4

06–0

.646

)(0

.486

–0.7

01)

(0.0

00)

(0.0

00)

(0.0

01)

[Val

enci

a]:[

Ara

go

n]:

[Cat

alu

ny

a]:

[Py

ren

ees]

:[A

lpes

-Mar

itim

es]:

[Sw

itze

rlan

d]

[8]:

[11]

:[3]

:

[14]

:[14

]:[3

]

0.10

80.

548

0.41

50.

493

[3]:

[4]:

[2]:

[5]:

[5]:

[2]

0.53

70.

459

0.14

5

(0.0

69–0

.145

)(0

.423

–0.6

19)

(0.2

85–0

.500

)(0

.368

–0.5

63)

(0.0

00)

(0.0

00)

(0.0

00)

[Ara

go

n]:

[Cat

alu

ny

a]:[

Py

ren

ees]

:

[Alp

es-M

arit

imes

]:[S

wit

zerl

and

]

[11]

:[3]

:[14

]:

[14]

:[3]

0.11

30.

275

0.05

90.

182

[4]:

[2]:

[5]:

[5]:

[2]

0.33

70.

229

0.14

0

(0.0

70–0

.154

)(0

.205

–0.3

21)

(0.0

19–0

.100

)(0

.122

–0.2

44)

(0.0

00)

(0.0

00)

(0.0

04)

Asp

len

ium

petr

arch

aesu

bsp

.bi

vale

ns

Tw

o-l

evel

hie

rarc

hic

alan

aly

sis

To

tal

ran

ge

120.

167

0.33

80.

205

(0.0

81–0

.259

)(0

.186

–0.5

01)

(0.1

08–0

.349

)

Maj

orc

a3

0.38

60.

405

0.03

1

()0.

053–

0.55

6)(0

.062

–0.5

38)

()0.

040–

0.11

0)

Val

enci

a9

0.16

20.

251

0.10

7

(0.0

75–0

.251

)(0

.128

–0.3

60)

(0.0

58–0

.145

)

[Maj

orc

a]:

[Val

enci

a](P

op

ula

tio

ns

mer

ged

)

0.68

30.

724

(0.0

00)

(0.0

00)

Th

ree-

lev

elh

iera

rch

ical

anal

ysi

s

[Maj

orc

a]:[

Val

enci

a][3

]:[9

]0.

167

0.52

40.

357

0.42

7

(0.0

81–0

.259

)(0

.275

–0.7

47)

(0.0

50–0

.628

)(0

.155

–0.6

67)



Fig. 4 Isolation by distance in A. fontanum subsp. fontanum.

Correlogram of Mantel RM values on the allozyme data set

excluding Valencian populations, geographical distance vs.

pairwise Nei’s (1978) genetic distance among 45 populations.

Filled dots indicate RM values significantly different from 0

after Bonferroni correction to P values at a = 0.05.ΔK800

1000

1200

1400

1600

600

400

200

0

Ln

(P

) D

–20 000

–15 000

–25 000

–30 000

–10 000

–5000

0

K1 2 3 4 5 6

Fig. 5 Modelling of number of genepools in A. fontanum

subsp. fontanum. Ln (P) D (means of 10 replicate runs) for all

populations N ‡ 15 (filled triangles) and excluding Valen-

cian populations (filled squares). Values of DK calculations

according to Evanno et al. (2005) for all populations N ‡ 15

(open triangles) and excluding Valencian populations (open

squares).


Chloroplast data. Eight variable sites were found in the

991-bp alignment of the rps4-trnS IGS. Additional varia-

tion in a poly-C microsatellite was excluded from the

analysis. Nine distinct haplotypes were found

(Table S4, Supporting information), related as shown in

the network in Fig. 1Ci. The distribution of haplotypes

among the samples is shown in Table S2 (Supporting

information) and Fig. 1C. The most common, haplotype

1 (overall frequency 45.1%), dominates in the Pyrenees


and northeastern Spain. Haplotype 4 also occurs at high

frequency (26.5%) and dominates in the Alpes-Mari-

times and Switzerland. These two common haplotypes

occur ‘reciprocally’ at low frequency away from their

main areas. The remaining seven haplotypes are rare

(overall frequencies 0.5–13.0%), although haplotype 6 is

locally common in Valencia, where its presence, along

with the related haplotype 5, differentiates these popu-

lations from those further north in Aragon and

Catalunya.

Hierarchical AMOVA (Table 1) showed significant dif-

ferentiation between populations both with conven-

tional F-statistics (FST = 0.513, P = 0.000) and using the

genetic distance matrix (FST = 0.504, P = 0.000). Differ-

entiation between populations within geographical

regions was relatively modest (FSC = 0.145, P = 0.000)

but strong differentiation was found between regions

(FCT = 0.459, P = 0.000). When Valencian populations

were excluded, significant differentiation between

regions remained but was reduced in extent

(FCT = 0.229, P = 0.000).

Asplenium petrarchae subsp. bivalens

Allozyme data. Fourteen loci were resolved and scored

in A. petrarchae subsp. bivalens, of which one (ACP) was

monomorphic. Forty seven alleles were identified across

the 14 loci, of which 24 were rare (global frequency

<5%). Twenty-eight alleles were found only in Valencia,

of which seven were private to a single population, four

to the population PET-19. No alleles private to Majorca

were found (Table S6, Supporting information).

Species-level allelic richness at the 13 polymorphic loci

ranged from 2 (HEX and MDH-2) to 6 (PGI-2).

Species-level diversity statistics across all 14 loci were:

P95 0.571, A 3.357, Ae 1.329, HE 0.182, HO 0.125.

All populations, even those with small numbers of

individuals, were retained for further analyses due to

the natural rarity of the species. Diversity statistics for

each population are given in Table S8 (Supporting

information). Mean values across the 12 populations


HO 0.099 and f 0.177. Majorcan populations were less

diverse than Valencian populations by all measures

analysed (Fig. 3B). Hierarchical analysis (Table 1) con-

firmed significant departure from Hardy–Weinberg

equilibrium (FIS = 0.167, 95% CI = 0.081–0.259). FIS was

considerably higher in Majorca, although with a wide

95% bootstrap confidence interval that includes zero

(0.386, 95% CI = )0.053–0.556), than in Valencia (0.162,

95% CI = 0.075–0.251). Moderately strong inter-popula-

tion differentiation was observed (FST = 0.205, 95%

CI = 0.108–0.349), primarily resulting from interregional

differentiation between Majorca and Valencia


(FCT = 0.357, 95% CI = 0.050–0.628). Interpopulation dif-

ferentiation within Majorca was not statistically signifi-

cant (FST = 0.031, 95% CI = -0.040–0.110) and was

modest within Valencia (FST = 0.107, 95% CI = 0.058–

0.145).

Bayesian analysis using Structure gave a minimum

probability with K = 1 ()ln = 5463.9) and maximum

probability with K = 5 ()ln = 4591.01), and DK calcula-

tions indicated an optimal model with three clusters

(Fig. 6). K = 3 splits the Majorcan from the Valencian

populations, the latter consisting of two clusters which

were extensively admixed among all populations. To

test this model, we repeated the analysis excluding the

Majorcan populations, whose high FIS (0.386) violates

the underlying assumption of Hardy–Weinberg equilib-

rium (HWE). Calculated probabilities ranged from a

minimum at K = 1 ()ln = 4717.6) to a maximum at

K = 4 ()ln = 4512.64).The obtained DK values were low

and gave no clear maximum (Fig. 6), providing no evi-

dence to indicate any sub-division of the Valencian

genepool. This pattern is consistent with the observed

low differentiation among Valencian populations

(FST = 0.107). We thus inferred the existence of two

genepools. Assignations of populations to these are

shown in Fig. 2B, in which we combined the propor-

tions assigned to clusters 2 and 3 under K = 3 for the

full dataset. This gives a predominantly Majorcan gene-

pool 1 and a predominantly Valencian genepool 2. This

interpretation is consistent with the neighbour-joining

phenogram (Fig. 2Bi), which supports the Valencian-

–6000

–5000

–4000

–3000

–2000

–1000

0

1 2 3 4 5 6 K

Ln

(P

)D

0

100

200

300

400

500

600

700

800

ΔK

Fig. 6 Modelling of number of genepools in A. petrarchae

subsp. bivalens. Ln (P) D (means of 10 replicate runs) for all

populations (filled squares) and excluding Majorcan popula-

tions (filled triangles). Values of DK calculations according to

Evanno et al. (2005) for all populations (open squares) and

excluding Majorcan populations (open triangles).

Majorcan split with very high bootstrap support (98%),

and gives only limited support for internal structure

within Valencia, with only the node joining populations

PET-20 and PET-33 having bootstrap support >70%.

Chloroplast data. Four polymorphic sites were found in

the 900bp alignment of the trnL-F region. Additional

variation involving indels was excluded from the analy-

sis. Five haplotypes were observed (Table S5, Support-

ing information), related as shown in the network given

in Fig. 2Ci. Their distribution among the samples is

shown in Table S3 (Supporting information) and

Fig. 2C. The most common haplotype, 4, occurs at a

frequency of 72.5% among Valencian samples, with the

remaining four haplotypes occurring at lower frequency

in this region. The seven Majorcan plants sequenced

were monomorphic for haplotype 1. An AMOVA, in

which all Majorcan plants were treated as one popula-

tion, and all Valencian plants as a second population,

found significant differentiation between Majorca and

Valencia both using haplotype frequencies only

(FST = 0.683, P = 0.000) and incorporating the genetic

distance matrix (FST = 0.724, P = 0.000) (Table 1).

Discussion

Ferns are currently underrepresented in studies of

genetic diversity and phylogeography, despite their

potential as models to explore the evolutionary

responses of plants to environmental change (Vogel

et al. 1999; Trewick et al. 2002; Shepherd et al. 2007).

Understanding these processes – including population

fragmentations and expansions, and formation and

establishment of new species – requires analysis of

populations across a taxon’s entire range. Most pub-

lished studies on fern intraspecific diversity (Werth

et al. 1985; Soltis & Soltis 1990; Ranker et al. 1994; Sch-

neller & Holderegger 1996; Suter et al. 2000; Herrero

et al. 2001; Quintanilla et al. 2007; Schneller & Liebst

2007) quantify genetic diversity from a small number of

populations and ⁄ or in a selected area of the species’

range. In the current study, we have achieved compre-

hensive sampling from across several hundred kilome-

tres for Asplenium fontanum subsp. fontanum and from

the two population clusters of A. petrarchae subsp. biva-

lens restricted to Majorca and Valencia. We did not find

any of the populations of the latter taxon reported from

southern Spain by Meyer (1964), despite extensive

searching; all populations collected from this region

were of the autotetraploid A. petrarchae subsp. petrarchae

(Hunt 2004). This discrepancy with our results may be

either the result of extinction in southern Spain or

reflect confusion of diploid and tetraploid cytotypes.

We have extended the known range of A. petrarchae



subsp. bivalens on Majorca beyond the single site

recorded by Bennert et al. (1990).

Levels of genetic diversity in plant species are

affected by life-history, population biology (Hamrick &

Godt 1996) and evolutionary history (Gitzendanner &

Soltis 2000). Thus, comparisons between taxa sharing

reproductive traits and that belong to the same evolu-

tionary lineage are the most meaningful. Within Euro-

pean Asplenium, (typically) outbreeding diploid taxa

harbour substantially higher levels of variation than

inbreeding polyploid populations, which can be

founded by just a single spore (Schneller & Holderegger

1996; Vogel et al. 1999; Suter et al. 2000). Comparison of

species- and population-level allozyme diversity in A.

fontanum subsp. fontanum and A. petrarchae subsp. biva-

lens with other diploid European Asplenium taxa

(Table S9, Supporting information) suggests that these

two taxa support intermediate levels of variation rela-

tive to their congeners.

A. fontanum subsp. fontanum has slightly higher diver-

sity estimates than A. petrarchae subsp. bivalens by all cri-

teria considered. FIS estimates were slightly higher in A.

petrarchae subsp. bivalens than in A. fontanum subsp. fon-

tanum. Estimates of FST and FCT indicated similar levels

of between-population and between-region differentia-

tion in both species. A paired-sample t-test on species-

level allelic richness across the twelve loci analysed for

both species gave t = 0.563; t0.05(1),11 = 1.796, indicating

no statistically significant difference in allelic richness

between A. fontanum subsp. fontanum and A. petrarchae

subsp. bivalens. Differences in loci analysed and popula-

tion size criteria precluded further interspecific compar-

ative analysis of either allozyme or cpDNA data.

Pairwise comparisons of rare vs. common plant cong-

eners have found greater species- and population-level

diversity in the widespread congener in 75–85% of

cases, although a substantial minority of pairs (15–25%)

did not follow this trend (Gitzendanner & Soltis 2000;

Cole 2003). The definition of ‘rarity’ varies between

studies (Rabinowitz 1981), encompassing one or more

of: small population sizes, narrow geographic ranges,

and fragmented populations. Rare species with large,

localized populations can maintain high levels of

genetic variation (Ellstrand & Elam 1993). Asplenium

fontanum subsp. fontanum and A. petrarchae subsp. biva-

lens differ primarily in range size. Both taxa have patch-

ily-distributed populations of variable (<10 to >100

individuals) but comparable size. Theoretical consider-

ations and empirical evidence therefore support the

conclusion that A. petrarchae subsp. bivalens is not

strongly genetically depauperate, corroborating the

findings of Schneller & Holderegger (1996) that small,

isolated fern populations can maintain genetic

variability.


FIS estimates from allozymes have been used to infer

breeding system in a number of fern species (Soltis &

Soltis 1989; Masuyama & Watamo 1990; Li & Haufler

1999; Vogel et al. 1999; Suter et al. 2000; Pryor et al.

2001). Our data show that both A. fontanum subsp. fon-

tanum and A. petrarchae subsp. bivalens are predomi-

nantly outbreeding. FIS was reduced when

subpopulations were considered separately (data not

shown), indicating spatial structuring within popula-

tions. Population substructuring has been demonstrated

in other rock fern species, e.g. Cheilanthes gracillima

(Soltis & Soltis 1989) and Pteris multifida (Murakami

et al. 1997) and explained by the patchy within-site

niche distribution typical of rock fern habitats (Soltis &

Soltis 1989; Pryor et al. 2001), and the strongly leptokur-

tic pattern of spore dispersal.

The strongest genetic differentiation in both taxa in

this study is between Valencia and the rest of their

respective ranges: a wide area from Aragon to the

Alpes-Maritimes in the case of A. fontanum subsp. fonta-

num, and the few populations on Majorca of A. petrar-

chae subsp. bivalens. This differentiation results from

distinct allozyme allele and haplotype frequencies, and

accounts for most of the total FST in each data set.

Genetic distinctiveness of Valencian populations has

also been noted in another seed-free land plant, the

moss Pleurochaete squarrosa (Grundmann et al. 2007).

Given the co-occurrence of A. fontanum subsp. fontanum

and A. petrarchae subsp. bivalens in Valencia, and the

recorded presence of their diploid hybrid A. x protoma-

joricum (Pangua et al. 1992; Perez Carro & Fernandez

Areces 1992), it is theoretically possible that back-

hybridisation and introgression could contribute a novel

set of alleles into one or other taxon. However, we con-

sider this scenario highly unlikely because the diploid

hybrid A. x protomajoricum shows very low fertility

(Pangua et al. 1992; Perez Carro & Fernandez Areces

1992). Side-by-side analysis of allozymes in both diploid

taxa and hybrid individuals also showed that very few

alleles are shared between A. fontanum subsp. fontanum

and A. petrarchae subsp. bivalens (Hunt 2004).

The genetic separation of Valencian populations is

likely to result from past or ongoing restricted gene

flow, and ⁄ or past allopatric fragmentation. Processes of

gene flow in ferns have been the subject of some debate.

van Zanten (1978) predicted that long-distance, wind-

mediated dispersal of spores would result in panmixia

and low genetic differentiation. The work of Soltis &

Soltis (1989) appeared to support this hypothesis, find-

ing low interpopulation differentiation in a range of

North American fern species, regardless of breeding

system. However, their FST estimates were derived from

a small number (£8) of populations for each species.

These results are consistent with our finding that,


although A. fontanum subsp. fontanum and A. petrarchae

subsp. bivalens showed high overall population differen-

tiation, FST within each geographic or genetically homo-

geneous cluster of populations is low. However, the

small residual FST within regions is significantly differ-

ent from zero, and the spatial autocorrelation analysis

for A. fontanum subsp. fontanum indicates that an isola-

tion-by-distance model with restricted gene flow

between populations operates over distances up to

50 km. This is consistent with the expectation that exten-

sive gene flow via spore dispersal will be limited in

obligate outbreeding fern taxa with small, patchily-

distributed populations. Over middle- and long-distance

ranges, dispersal events are random and probably rare.

It was not possible to test for isolation by distance in

A. petrarchae subsp. bivalens, because of the small num-

ber of sampled populations (reflecting the species’ rar-

ity) and the discontinuous range. However, F-statistic

estimates for partitioning of genetic diversity suggest

that comparable patterns of dispersal will operate as in

A. fontanum subsp. fontanum. The low diversity, moder-

ately high FIS and non-significant FST among Majorcan

populations, together with the observations that the Ma-

jorcan haplotype and allozyme diversity are a subset of

that found in Valencia, suggest a founder effect follow-

ing dispersal from the mainland. However, the reduced

diversity estimates may result at least in part from the

small population sizes, and the high FIS estimates

depend on variation at a small number of loci, and are

not significantly different from zero. Additionally, the

Majorcan populations are differentiated from those in

Valencia by the high frequency of alleles (at two loci,

AAT and PGM) that are extremely rare in Valencia.

This differentiation could suggest an ancient dispersal

from the mainland, allowing the evolution of new

alleles in situ followed by dispersal back to Valencia, or

that colonization of Majorca also involved gene flow

from other now-extinct mainland populations (e.g. in

southern Spain), or that relict populations have sur-

vived on Majorca, perhaps representing remains of a

more extensive Mediterranean distribution during the

Pleistocene that was fragmented by rising sea levels.

Further DNA sequence analysis of these populations is

required to discriminate between these hypotheses.

In A. fontanum subsp. fontanum, there is little allele

and haplotype sharing between Valencia and the rest of

the range, suggesting that these regions are not linked

by a past dispersal event, and ancient range fragmenta-

tion is a more likely scenario. Pleistocene climate

change drove cycles of population expansion and con-

traction within Iberia ⁄ southwestern Europe in many

organisms, which may have responded to climate

change by migrating up and down the mountainous

topography of this region (Gomez & Lunt 2006). The

limestone mountains in Valencia and those to the north

in Teruel and surrounding provinces, where A. fonta-

num subsp. fontanum is relatively common, belong to

two distinct mountain systems, the Baetic ranges and

the Iberian system respectively. These are linked by a

relatively narrow limestone ‘corridor’, in which A. fonta-

num occurs sporadically (Herrero-Borgonon et al. 1997).

This corridor partially coincides with relatively low ele-

vations under 500m (Rivas-Martınez et al. 1999), sug-

gesting that the present-day climatic conditions may be

unfavourable for A. fontanum, which prefers higher and

thus cooler altitudes. In glacial periods, cooler condi-

tions might have supported more suitable habitats in

this limestone corridor; post-glacial warming, combined

with edaphic restrictions, could have effected allopatric

fragmentation between populations in the Baetic and

Iberian mountain systems.

Outside Valencia, a further genetic split between pop-

ulations to the southwest and those to the north east is

supported by both allozyme and cpDNA data. This is

less pronounced than the separation of both these clus-

ters from Valencia, with extensive sharing of alleles and

haplotypes across a wide geographic range from Ara-

gon to Switzerland. The two most common haplotypes

(4 and 1) show centres of distribution in two of the tree

refugia identified by Huntley & Birks (1983), southeast-

ern France and northeastern Spain respectively. The

dominant haplotype (1) in northeastern Spain occupies

the central position in the topology and hence may rep-

resent the ancestral haplotype. Under a K = 3 model in

the Structure analysis of allozyme data, populations

with a high assignation probability to genepool 1 (pink)

cluster in southeastern France, and those strongly

assigned to genepool 2 (blue) cluster in the Sierra de Al-

barracın in Aragon. The allozyme data show a sharp

divide in the Pyrenees between the westernmost group

of populations in Ariege (predominantly genepool 2)

and the eastern cluster in the Pyrenees-Orientales (pre-

dominantly genepool 1). This pattern is less evident in

the cpDNA, where haplotype 1 dominates all sampled

Pyrenean populations, with haplotype 4 showing a

minority presence in three of the five populations,

including two in the western Pyrenees. The small num-

bers of samples analysed for cpDNA make it difficult to

interpret whether this discrepancy between marker

types is significant. In contrast to flowering plants, the

haploid spore is the only significant unit of dispersal in

ferns, with male gametes being dispersed over at most

a few cm; hence, nuclear and chloroplast genetic pat-

terns would be expected to concur. However, we did

find haplotype 4 at 50% frequency in population 72 in

Catalunya, consistent with the allozyme genepool assig-

nations for this population showing admixture between

genepools 1 and 2.



The geographic division between genepools 1 and 2

may be explained either by a scenario of expansions from

relict populations in southeastern France and Aragon

respectively, meeting at a suture zone that runs through

the Pyrenees and extends down the Spanish eastern sea-

board, or by a scenario of long-term survival of diverse

Pyrenean populations, isolated from one another by the

extreme topology, which then expanded to the southeast

and north west respectively. ‘Refugia within refugia’ in

the Pyrenees have been identified for a range of other

plant and animal species (Gomez & Lunt 2006). The Pyre-

nees also show a high level of endemicity (Garcıa-Barros

et al. 2002), suggesting long-term in situ survival of

organisms, either in exposed nunatak zones or in perigla-

cial regions around the ice sheets (Segarra-Moragues

et al. 2007). However, none of the A. fontanum subsp. fon-

tanum populations in Aragon, Catalunya, the Pyrenees or

the Alpes-Maritimes show characteristics of recently-

established populations. The Pyrenees have the greatest

allelic-richness, largely due to the presence of a number

of rare alleles, but the two populations from Mont Caro

in Catalunya (71 and 72) have the highest expected het-

erozygosity and effective allele number. High genetic

diversity may indicate long-term population survival

(Hewitt 1996), or represent suture zones between

recently-established populations sourced from multiple

refugia: Petit et al. (2003) found the highest intrapopula-

tion cpDNA haplotype diversity for 22 European tree

and shrub species to the north of the Alps, in regions col-

onized postglacially. In Fagus sylvatica, high isozyme alle-

lic richness corresponded with glacial refugia identified

from the pollen record, but highest expected heterozy-

gosity occurred in areas remote from these refugia

(Comps et al. 2001). Refugia may also be identified by

private alleles (Gomez & Lunt 2006). We observed the

greatest number of private alleles in A. fontanum subsp.

fontanum in the Pyrenees, both as a region and in individ-

ual populations. Aragonese and French populations also

exhibited some private alleles at notable frequencies. In

summary, the high diversity and presence of rare alleles

across a wide zone from Aragon to southeastern France

suggests that, although it is not possible to determine the

underlying process separating genepools 1 and 2, this is

likely to represent an ancient phylogeographic pattern.

Further analysis of DNA sequence markers is needed to

further explore the evolutionary relationship between the

two genepools.

The phylogeographic link we have found between the

eastern Pyrenees and southeastern France echoes bioge-

ographic connections between the Pyrenees and the

southwestern Alps that have been suggested by phylog-

eographic data for the plants Anthyllis montana and Prit-

zelago alpina and the butterflies Erebia cassioides and

Erebia epiphron (Kropf et al. 2002, 2003; Martin et al.


2002; Schmitt et al. 2006). A disjunction exists in the

present-day distribution of A. fontanum subsp. fontanum

in France, separating the Pyrenean and Alpes-Maritimes

population clusters. This probably reflects the lack of

suitable habitat in the low-lying area around the Canal

du Midi and the Rhone delta. Population migration via

both coastal and inland routes would have been possi-

ble if populations belonging to genepool 1 formed part

of a continuous distribution during periods of lower sea

level during the Quaternary, extending across what is

now the Golfe de Lion. The patchy modern-day distri-

bution of A. fontanum subsp. fontanum in southwestern

Europe may reflect the closure of some of those routes

by rising sea levels, and ⁄ or by landscape changes.

The ranges of A. fontanum subsp. fontanum and A.

petrarchae subsp. bivalens mean that standard models of

south European ‘glacial refugia’ with postglacial recolo-

nisation of more northerly regions are not particularly

applicable to these taxa. However, the Swiss popula-

tions exhibit a subset of the alleles and chlorotypes

found elsewhere in the range, suggesting a more recent

population expansion into this region. The presence of

two Swiss populations (51 and 84) with >50% allocation

to genepool 2 indicates that this recolonisation may have

involved dispersal from populations several hundred

km to the southwest.

Acknowledgements

HVH is grateful for support from a Natural Environment

Research Council PhD studentship, St John’s College, Cam-

bridge, and the Department of Genetics, University of

Cambridge, and for advice from the late Professor Michael

Majerus. Thanks to Michael Grundmann and Cecılia Duraes in

the lab, Cameron Petrie and Mim Bower for assistance with

graphics, and many colleagues who collected samples.

References

Bennert HW, Rasbach H, Rasbach K (1990) Asplenium petrarchae

(Guerin) DC. subsp. bivalens und Asplenium x helii

nothosubsp. calobrense - Neufunde auf der Insel Mallorca.

Farnblatter, 21, 15–26.

Casgrain P, Legendre P (2001) The R-Package for Multivariant

and Spatial Analysis. Version 4.00 (d6). Departement de

sciences biologiques, Universite de Montreal, Montreal.

Available at http:fas.umontreal.ca ⁄ BIOL ⁄ legendre ⁄ .Cole CT (2003) Genetic variation in rare and common plants.

Annual Review of Ecology and Systematics, 34, 213–237.

Comps B, Gomory D, Letouzey J, Thiebaut B, Petit RJ (2001)

Diverging trends between heterozygosity and allelic richness

during postglacial colonization in the European beech.

Genetics, 157, 389–397.

Ellstrand NC, Elam DR (1993) Population genetic consequences

of small population size: Implications for plant conservation.

Annual Review of Ecology and Systematics, 24, 217–242.


Evanno G, Regnaut S, Goudet J (2005) Detecting the number of

clusters of individuals using the software STRUCTURE: a

simulation study. Molecular Ecology, 14, 2611–2620.

Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: An

integrated software package for population genetics data

analysis. Evolutionary Bioinformatics Online, 1, 47–50.

Falush D, Stephens M, Pritchard JK (2003) Inference of

population structure using multilocus genotype data: linked

loci and correlated allele frequencies. Genetics, 164, 1567–1587.

Ferrer-Castan D, Vetaas OR (2005) Pteridophyte richness,

climate and topography in the Iberian Peninsula: comparing

spatial and nonspatial models of richness patterns. Global

Ecology and Biogeography, 14, 155–165.

Garcıa-Barros E, Gurrea P, Lucianez MJ et al. (2002) Parsimony

analysis of endemicity and its application to animal and

plant geographical distributions in the Ibero-Balearic region

(western Mediterranean). Journal of Biogeography, 29, 109–124.

Gitzendanner MA, Soltis PS (2000) Patterns of genetic variation

in rare and widespread plant congeners. American Journal of

Botany, 87, 783–792.

Gomez A, Lunt DH (2006) Refugia within refugia: patterns of

phylogeographic concordance within the Iberian Peninsula.

In:Phylogeography in Southern European Refugia: Evolutionary

Perspectives on the Origins and Conservation of Biodiversity (eds

Weiss S, Ferrand N). pp. 155–188, Kluwer Academic

Publishers, Dordrecht.

Grundmann M, Ansell SW, Russell SJ, Koch MA, Vogel JC

(2007) Genetic structure of the widespread and common

Mediterranean bryophyte Pleurochaete squarrosa (Brid.) Lindb.

(Pottiaceae) – evidence from nuclear and plastidic DNA

sequence variation and allozymes. Molecular Ecology, 16, 709–

722.

Hamrick JL, Godt MJW (1996) Effect of life-history traits on

genetic diversity in plant species. Philosophical Transactions of

the Royal Society of London: B series, 351, 1291–1298.

Herrero A, Pajaron S, Prada C (2001) Isozyme variation and

genetic relationships among taxa in the Asplenium obovatum

group (Aspleniaceae, Pteridophyta). American Journal of

Botany, 88, 2040–2050.

Herrero-Borgonon JJ, Martınez-Solis I, Estrelles E, Ibars AM

(1997) Avance al atlas pteridologico de la comunidad

Valenciana. Flora Montiberica, 7, 72–86.

Hewitt GM (1996) Some genetic consequences of ice ages, and

their role in divergence and speciation. Biological Journal of

the Linnean Society, 58, 247–276.

Hunt HV (2004) Evolution in the Asplenium majoricum Complex.

PhD thesis, University of Cambridge, Cambridge.

Huntley B, Birks HJB (1983) An Atlas of Past and Present Pollen

Maps for Europe: 0-13000 Years ago. Cambridge University

Press, Cambridge.

Huson DH, Richter DC, Rausch C et al. (2007) Dendroscope –

An interactive viewer for large phylogenetic trees. BMC

Bioinformatics, 8, 460.

Ibars AM, Herrero-Borgonon JJ, Estrelles E, Martınez I (1999)

Helechos de la Comunidad Valenciana. Conselleria de Medio

Ambiente, Valencia, Generalitat Valenciana.

Jalas J, Suominen J (1972) Atlas Florae Europeae. 1. Pteridophyta.

Helsinki University Press, Helsinki.

Kephart SR (1990) Starch gel electrophoresis of plant isozymes:

a comparative analysis of techniques. American Journal of

Botany, 77, 693–712.

Kropf M, Kadereit JW, Comes HP (2002) Late Quaternary

distributional stasis in the submediterranean mountain plant

Anthyllis montana L. (Fabaceae) inferred from ITS sequences

and amplified fragment length polymorphism markers.

Molecular Ecology, 11, 447–463.

Kropf M, Kadereit JW, Comes HP (2003) Differential cycles of

range contraction and expansion in European high mountain

plants during the Late Quaternary: insights from Pritzelago

alpina (L.) O. Kuntze (Brassicaceae). Molecular Ecology, 12,

931–949.

Lewis PO, Zaykin D (2001) Genetic Data Analysis: computer

program for the analysis of allelic data. Version 1.0 (d16c).

http://lewis.eeb.uconn.edu/lewishome/software.html.

Li JW, Haufler CH (1999) Genetic variation, breeding systems,

and patterns of diversification in Hawaiian Polypodium

(Polypodiaceae). Systematic Botany, 24, 339–355.

Lovis JD, Reichstein T (1969) Der Farnbastard Asplenium x

orellii hybr. nov. = Asplenium majoricum Litard. x A.

trichomanes L. subsp. quadrivalens D.E. Meyer und die

Abstammung von A. majoricum. Berichte der Schweizerischen

Botanischen Gesellschaft, 79, 335–345.

Martin J-F, Gilles A, Lotscher M, Descimon H (2002)

Phylogenetics and differentiation among the western taxa of

the Erebia tyndarus group (Lepidoptera: Nymphalidae).

Biological Journal of the Linnean Society, 75, 319–332.

Masuyama S, Watamo Y (1990) Trend for inbreeding in

polyploid pteridophytes. Plant Species Biology, 5, 13–17.

Meyer DE (1964) Uber neue und seltene Asplenien Europas. 3

Mitteilung. Berichte der Deutschen Botanischen Gesellschaft, 77,

3–13.

Moreno Saiz JC, Lobo J (2008) Iberian–Balearic fern regions

and their explanatory variables. Plant Ecology, 198, 149–167.

Murakami N, Nishiyama T, Satoh H, Suzuki T (1997) Marked

spatial genetic structure in three populations of a weedy

fern, Pteris multifida Poir., and reestimation of its selfing rate.

Plant Species Biology, 12, 97–106.

Nei M (1972) Genetic distance between populations. American

Naturalist, 106, 283–292.

Nei M (1978) Estimation of average heterozygosity and genetic

distance from a small number of individuals. Genetics, 89,

583–590.

Ota T (1993) DISPAN: a computer program for the genetic

distance and phylogenetic analysis. http://

www.bio.psu.edu/people/faculty/nei/lab/software.htm.

Pangua E, Prada C, Pajaron S, Salvo E (1992) A new Asplenium

hybrid from Valencia (Spain) related to A. majoricum Litard.

Botanical Journal of the Linnean Society, 108, 1–13.

Perez Carro FJ, Fernandez Areces MP (1992) Asplenium x

protomajoricum hybr. nov. (A. fontanum x A. petrarchae subsp.

bivalens) y precisiones corologicas sobre A. majoricum en el

Levante espanol. Anales Jardın Botanico de Madrid, 49, 187–194.

Petit RJ, Aguinagalde I, de Beaulieu J-L et al. (2003) Glacial

refugia: hotspots but not melting pots of genetic diversity.

Science, 300, 1563–1565.

Prelli R, Boudrie M (2002) Les Fougeres et Plantes Allies de France

et D’Europe Occidentale. Editions Lechevalier, Paris.

Pritchard JK, Stephens M, Donelly P (2000) Inference of

population structure using multilocus genotype data.

Genetics, 155, 945–959.

Pryor KV, Young JE, Rumsey FJ et al. (2001) Diversity, genetic

structure and evidence of outcrossing in British populations



of the rock fern Adiantum capillus-veneris using

microsatellites. Molecular Ecology, 10, 1881–1894.

Quintanilla L, Pajaron S, Pangua E, Amigo J (2007) Allozyme

variation in the sympatric ferns Culcita macrocarpa and

Woodwardia radicans at the northern extreme of their ranges.

Plant Systematics and Evolution, 263, 135–144.

Rabinowitz D (1981) Seven forms of rarity. In:The Biological

Aspects of Rare Plant Conservation (ed Synge H). pp. 205–218,

Wiley, Chichester.

Ranker TA, Floyd SK, Trapp PG (1994) Multiple colonisations

of Asplenium adiantum-nigrum onto the Hawaiian

archipelago. Evolution, 48, 1364–1370.

Reichstein T, Schneller J (1982) Asplenium pseudofontanum

Kossinsky (Aspleniaceae, Pteridophyta). Studies in Asplenium

for ‘Flora Iranica’. 3. Candollea 37, 117–128.

Rivas-Martınez S, Loidi Arregui J, Costa Talens M, Dıaz

Gonzalez TE, Penas Merino A (1999) Iter Ibericum A.D.

MIM. Itinera Geobotanica, 13, 5–347.

Schmitt T, Hewitt GM, Muller P (2006) Disjunct distributions

during glacial and interglacial periods in mountain

butterflies: Erebia epiphron as an example. Journal of

Evolutionary Biology, 19, 108–113.

Schneider H, Russell SJ, Cox CJ et al. (2004) Chloroplast

phylogeny of Asplenioid ferns based on rbcL and trnL-F

spacer sequences (Polypodiidae, Aspleniaceae) and its

implications for biogeography. Systematic Botany, 29, 260–274.

Schneider H, Ranker TA, Russell SJ et al. (2005) Origin of the

endemic fern genus Diellia coincides with the renewal of

Hawaiian terrestrial life in the Miocene. Proceedings of the

Royal Society B, 272, 455–460.

Schneller JJ, Holderegger R (1996) Genetic variation in small,

isolated fern populations. Journal of Vegetation Science, 7, 113–

120.

Schneller J, Liebst B (2007) Patterns of variation of a common

fern (Athyrium filix-femina; Woodsiaceae): population

structure along and between altitudinal gradients. American

Journal of Botany, 94, 965–971.

Segarra-Moragues JG, Palop-Esteban M, Gonzalez-Candelas F,

Catalan P (2007) Nunatak survival vs. tabula rasa in the

Central Pyrenees: a study on the endemic plant species

Bordera pyrenaica (Dioscoreaceae). Journal of Biogeography, 34,

1893–1906.

Shepherd LD, Perrie LR, Brownsey PJ (2007) Fire and ice:

volcanic and glacial impacts on the phylogeography of the

New Zealand forest fern Asplenium hookerianum. Molecular

Ecology, 16, 4536–4549.

Smith AR, Pryer KM, Schuettpelz E, Korall P, Schneider H,

Wulf PG (2006) A classification for extant ferns. Taxon, 55,

705–731.

Soltis DE, Soltis PS (1989) Polyploidy, breeding systems, and

genetic differentiation in homosporous pteridophytes.

In:Isozymes in Plant Biology (eds Soltis DE, Soltis PS). pp.

241–258, Dioscorides Press, Portland, Oregon.

Soltis PS, Soltis DE (1990) Genetic variation within and among

populations of ferns. American Fern Journal, 80, 161–172.

Soltis DE, Haufler CH, Darrow DC, Gastony GJ (1983) Starch

gel electrophoresis of ferns: a compilation of grinding

buffers, gel and electrode buffers, and staining schedules.

American Fern Journal, 73, 9–27.

Suter M, Schneller JJ, Vogel JC (2000) Investigations into the

genetic variation, population structure, and breeding systems


of the fern Asplenium trichomanes subsp. quadrivalens.

International Journal of Plant Sciences, 161, 233–244.

Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal

primers for amplification of three non-coding regions of

chloroplast DNA. Plant Molecular Biology, 17, 1105–1109.

Taberlet P, Fumagalli L, Wust-Saucy A-G, Cosson J-F (1998)

Comparative phylogeny and postglacial colonization routes

in Europe. Molecular Ecology, 7, 453–464.

Trewick SA, Morgan-Richards M, Russell SJ et al. (2002)

Polyploidy, phylogeography and Pleistocene refugia of the

rockfern Asplenium ceterach: evidence from chloroplast DNA.

Molecular Ecology, 11, 2003–2012.

Tutin TG, Heywood VH, Burges NA, Valentine DH, Moore

DM (1993) Flora Europaea. Volume 1, Psilotaceae to Platanaceae.

2nd edn. Cambridge University Press, Cambridge.

Vogel JC, Rumsey FJ, Schneller JJ, Barrett JA, Gibby M (1999)

Where are the glacial refugia in Europe? Evidence from

pteridophytes. Biological Journal of the Linnean Society, 66, 23–37.

Weir BS, Cockerham CC (1984) Estimating F-statistics for the

analysis of population structure. Evolution, 38, 1358–1370.

Wendel JF, Weeden NF (1989) Visualization and interpretation

of plant isozymes. In:Isozymes in Plant Biology (eds Soltis DE,

Soltis PS). pp. 5–45, Dioscorides Press, Portland, Oregon.

Werth CR, Guttman SI, Eshbaugh WI (1985) Electrophoretic

evidence of reticulate evolution in the Appalachian

Asplenium complex. Systematic Botany, 10, 184–192.

Wright S (1978) Evolution and Genetics of Populations, Volume 4.

Variability Within and Among Natural Populations. University

of Chicago Press, Chicago.

Yeh FC, Yang R, Boyle T (1999) POPGENE. Microsoft

Windowbased Freeware for Population Genetic Analysis. Version

1.32. Centre for International Forestry Research, University

of Alberta. Available at http://www.ualberta.ca/~fyeh/.

van Zanten BO (1978) Experimental studies of trans-oceanic long-

range dispersal of moss spores in the Southern Hemisphere.

Journal of the Hatton Biological Laboratory, 44, 455–482.

SWA, SJR, HS and JCV are researchers in the Botany Depart-

ment at the Natural History Museum, studying the evolution

of genetic diversity of ferns and other land plants in the

spatial-temporal context of Europe in the last 5 Ma. HVH was

a PhD student in this group with interests in spatial patterns

of plant genetic diversity, phylogeography, and polyploid spe-

ciation. She is now working on these topics on domesticated

cereals at the McDonald Institute for Archaeological Research,

University of Cambridge.

Supporting information

Additional supporting information may be found in the online

version of this article.

Table S1 Locality information for populations of A. fontanum

subsp. fontanum and A. petrarchae subsp. bivalens sampled for

this study. NR = information not recorded

Table S2 Chloroplast rps4 – trnS IGS haplotype data for A. fon-

tanum subsp. fontanum. Gene diversity statistics for each region

are shown


Table S3 Chloroplast trnL-F IGS haplotype data for A. petrar-

chae subsp. bivalens

Table S4 Definition of rps4 – trnS IGS haplotypes in A. fontanum

subsp. fontanum and associated GenBank accession numbers

Table S5 Definition of trnL-F haplotypes in A. petrarchae subsp.

bivalens and associated GenBank accession numbers

Table S6 Allozyme alleles observed in A. fontanum subsp. fon-

tanum and A. petrarchae subsp. bivalens. Enzyme abbreviations

follow Wendel & Weeden (1989)

Table S7 Allozyme diversity statistics and Structure genepool

assignation proportions under K = 3 model for populations of

A. fontanum subsp. fontanum. Abbreviations: n, number of indi-

viduals; P, proportion of polymorphic loci; A, number of alleles

per locus; Ap, number of polymorphic alleles per locus; Ae,

effective number of alleles per locus; HE, expected heterozygos-

ity; HO, observed heterozygosity; f, inbreeding coefficient. 95%

criterion used for determining polymorphic loci. All values

correct to 3 d.p.

Table S8 Allozyme diversity statistics and Structure genepool

assignation proportions under K = 3 model for populations of

A. petrarchae subsp. bivalens. Assignations to clusters 2 and 3

were summed to give the yellow cluster proportion shown in

Fig. 2B. Abbreviations: n, number of individuals; P, proportion

of polymorphic loci; A, number of alleles per locus; Ap, num-

ber of polymorphic alleles per locus; Ae, effective number of

alleles per locus; HE, expected heterozygosity; HO, observed

heterozygosity; f, inbreeding coefficient. 95% criterion used for

determining polymorphic loci. All values correct to 3 d.p.

Table S9 Comparison of genetic diversity measures in A. fonta-

num subsp. fontanum and A. petrarchae subsp. bivalens with

those of other diploid European Asplenium taxa. n ⁄ a = data not

available. Unpublished data for A. ceterach subsp. bivalens, A.

trichomanes subsp. inexpectans, A. viride and A. hemionitis from

JC Vogel and C Duraes. Species information: A. hemionitis is a

Macaronesian ⁄ SW European Atlantic fringe taxon of putative

Tertiary origin. A. ceterach subsp. bivalens has a scattered east-

ern Mediterranean ⁄ Balkan distribution. Asplenium trichomanes

subsp. inexpectans has a main range in south central Europe,

with scattered southern Mediterranean populations. Asplenium

viride is a widespread Alpine taxon also found in North Amer-

ica and Asia. Diversity measures are based on a very similar

set of allozyme loci in all taxa

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting information supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.


Documents

Genetic diversity and phylogeography in two diploid ferns, Asplenium fontanum subsp. fontanum and A. petrarchae subsp. bivalens, in the western Mediterranean