Asplenium ceterach and A. octoploideum on the Canary Islands (Aspleniaceae, Pteridophyta)

Article (PDF Available) in American Fern Journal 94(Apr 2004):81-111 · January 2009 with 512 Reads

DOI: 10.1640/0002-8444(2004)094[0081:ACAAOO]2.0.CO;2

Isozyme and plastid DNA analysis prove that true A. ceterach occurs on the Canary Islands, in addition to A. aureum and an octoploid taxon. Combining morphological and cytological observations leads to correct determination, but the exospore length alone also allows reliable identification of these Canarian species. Our allozyme data suggest that the Canarian A. ceterach population is not completely genetically isolated from the European ones. The holotype of Ceterach aureum var. parvifolium, formerly regarded as an octoploid taxon, proved to be A. ceterach, leaving the octoploid without a correct name. The recently described A. octoploideum shows monomorphic, presumably fixed heterozygosity for a combination of the patterns seen in A. ceterach and A. aureum at four loci (Aat, Skdh, Me, and Pgi-2) confirming its allo-octoploid nature. It most probably originated by chromosome doubling in a tetraploid hybrid between A. aureum and A. ceterach or via the union of their unreduced gametes. Pgi-2 indicates multiple origins of the allo-octoploid, implicating recurrent gene flow from tetraploids to octoploids.

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American

Fern

/ournal

94(2):81_111

(2oo4)

Asplenium

ceterach

and

octoploideum

the

canary

Islands

(Aspleniaceae,

pteridophyta)

Canolrrun

VnN

DEN

HEEDE

Pteridological

section,

Department

Biology,

Ghent

Universitv,

K.L.

Ledeganckstraat

3S,

8-9000

Ghent,

Belgium

Snrurraco

Pnlanórl

and

Evrr.ra

parucua

Departamento

Biología

Vegetal

Facultad

Biología,

Universidad

Complutense,

E-2g040

Madrid,

Spain

Roxnln

L.L.

VrnNrl

Pteridological

Section,

Department

Biology,

Ghent

University,

K.L.

Ledeganckstraat

3S,

8-9000

Ghent,

Belgium

AsstRact'-Isozyme

and

plastid

DNA

analysis

prove

that

true

ceterach

occurs

the

canary

Islands'

addition

á.

aureum

and

óctoploid

taxon.

combining

morphological

and

cytological

observations

leads

correct

determination,

but

the

exospore

lerïgth

alone

also

allows

reliable

identification

these

canarian

species.

our

allozyme

data

suggestihat

the

canarian

á.

ceteroch

population

not

completely

genetically

isolated

frtm

the

n,rroiË"r,

ones.

The

holotype

ceterach

oureum

var.

paruifofium'

formerly

rágarded

octoploid

taxon,

proved

á.

ceterach,leaving

the

octoploid

without

a correct

name.

The

recently

described

octoploideum

shows

monomorphic,

presumably

fixed

heterozygosity

for

combinaiion

the patterns

seen

á.

ceteroch

and

á'

oureum

at four

loci

(áoÍ,

skdh,

ti",

uÁd

Pgi-2)confirming

its

allo-octoploid

nature.

most

probably

originated

chromosome

doubling

Ltraploid

hybrid

between

aureum

and.

ceterach

via the

union

their

unreduced

gu-u1"r.

Pgi-2indicatËs

multiple

origins

the

allo-

octoploid,

implicating

recurrent

gene

flow

froÀ

tetraploiás

octoploids.

Asplenium

subgenus

CeÍerach (Willd.)

Bir

eÍ

o1.

a small

group

about

nine

fern

taxa

within

the

large

(729

species),

subcosmopohàn

genus

Asplenium

(Kramer

and

Viane,

1990).

rËis

subgenus

contains

xerophytic

rock

ferns

with

the

dorsal

side

the

lamina

denóly

covered

with

reddish-

brown

scales

paleae).

van

den

heed

ol.

(2003)

háve

shown

that

the

grorrp

must

restricted

the

Eurasian

and

Macaronesian

species.

Ever

since

the

description

Asplenium

aureum

Cav.

from

Tenerife

Cavanilles

[taor),

has

been

uncleai

how

many "

ceterach.,

species

are

extant

the

canarian

Archipelago,

and

whether

the

,,Éuropean,,

àeterachL.(syn.:

Ceterach

officinarum

Willd.)

occurs

Macaronesia (Table

1).

This

confusion

was

caused

the

lack

distinctive

characters

disti.rgr,ish

both

,pu"i"r.

cavanilles

(raor)

and

Bory

st.

Vincent (1802)

mentioied

only

the

much

larger

size

aureum

compared

that

ceteracrl.

Willdáo* (1810)

introduced

the

concept

of "toothed

scales"

diagnostic

feature,

whereas

Milde

(1865)

claimed

that

"Cuticularstreifen"

(cuticul-ar

lines

ridges

the

periclinal

cell

walls

the

scales)

could

used

distinguish

oureum

from

ar,tro.

for

correspondence.

82 AMERICAN FERN

IOURNAL:

VOLUME

NUMBER 2

(2004)

TasLE

1. Taxa covered by names found in the literature. Abbreviations

used: A.

cet.:A. ceterach,

aur.:

aureum,

A. lol.: A. lolegnamense, A.

par.:"

parvifofium

sensu Vida and Reichst,"

A. octoploideum. I: the filled symbol

indicates that the taxon was included in this author's

concept of the species mentioned

column 2; tr: symbol

indicates that this taxon was included in

the author's concept of the species mentioned in column

2 prior

its formal description.

Taxa included in

this

name

Literature reference Name used or

published

A. cet. A. aur. A.Iol.

"A. paL"

Borv de St. Vincent 1802

ceterach

Linnaeus 1753

Cavanilles

1801

Desvaux 1827

Moore 1857

Lowe, E.

1858

Kuhn 1868

Sauer 1.880

Luerssen 1889

Schneider 1892

Christ

1897

Burchard 1929

Chevalier 1935

Copeland to+z

Manton 1950

Romariz L953

Dansereau 1961

Fabbri 1965

Kunkel tgos

A. ceterach

aureum

A. Iatifolium

C. aurea

C. aurcum

C. officinarum

C. officinorum

A. ceterach var. oureum

C. aureum

C. officinarum

C. aureum

C. officinarum

C. auteum

C. officinarum

C. aurcum

C. officinarum

C. aureum

C. officianarum

C. aureum

C. officinarum

C, aureum

C. aureum

C. officinarum

A. ceterach

var.

oureum

C. aureum

C. officinarum

C. aureum

C. officinarum

var.

aureum

ITDtr

Itr

tr!

r!!

von

Buch 1.828

["1825"]*

C. aureum

Hooker 1860, 1861 A. ceterach

Bolle 1864

Milde 1866b. 1867a,b C. aurcum

rfl

rtn

Tardieu-Blot 1946 C. officinarum

Itr

ITtr

l!Lems 1958. 1960 C. aureum

Benl and Kunkel tg0z C. aureumvar. ouÍeum

C. aureum

var. parvifolium I

Lid 1e67

Hansen 1969

aureum

C. aureum tr

Benl and Sventenius 1970 C. aureumvaÍ. oureum

C. aureum

var. parvifolium I

VAN DEN HEEDE ET

AL.: CANARIAN ASPLENIUM CETERACH GROUP

Tnels 1. Continued.

Taxa included in this name

Literature reference Name used or

published

Kunkel tgzt

Hansen and Sunding 1979

Reichstein 1984

Bir et al. tggs

Manton

et al. tgg6

Gibby and Lovis 1989

Ormonde 1990

Viane

and

Reichstein

1992

Griffiths 1997

Hoshizaki

and Moran 2001

C. aureum var. oureum

oureum var. parvifolium

C. oureum var.

oureum

C. aureum

var. parvifolium

C. aureum

var. ou.reum

C. aureum var.

parvifolium

C. offinarum

A. aureum

ceterach ssp. ceterach

C. aureum var. aureum

C. aureum

var. parvifolium

C. lolegnamense

C. aureum vat. aureum

C. aureum var. madeirense

C. aureum

var. parvifolium

parvifolium

A. lolegnamense

aureum

A. ceterach

C. aureum

C. officinarum

According

to Stafleu and Cowan

(1976)

this book was only

published

after 28 May 1828, it is not

clear whether Link or von Buch made the combination

"C.

aureum", which is, in

anv case,

antedated by Desvaux

(1.827).

its continental counterpart,

though

soon

(Milde

1866a,

1866b, L867a,

1867b) cast doubt

the utility

of this character.

early as May 1,866, Milde

admitted that

"die

Cuticularstreifen,

welche

die Spreuschuppen

von

oureum

stets

zeigen, fand

ich nun auch an exemplaren, die sich von

officinarum

nicht

unterscheiden liessen." Finally, he came to the

conclusion

that the character was useless to

discriminate

Á.

ceterach

from A.

aureum

[Milde,

1867b). Nevertheless, Bornmiiller

(Plantae

exsicc. Canarienses-l901),

and

Benl

and Kunkel

(rgOz)

heavily relied

on this character to

recognize

taxa.

Since

1.970,

chromosome numbers were used to distinguish

species

this

group

(T.

Reichstein, pers.

comm.), and morphological characters

became

less

important

(e.9.,

Bir

et aL.,1985; Manton et aL.,1986; Gibby and Lovis, 19Bg).

In 1967, Benl

and

Kunkel

considered all Canarian plants that looked like

ceterach to be a dwarfed variety of A. aureum. Unfortunately,

their

variety

Ceterach oureum

(Cav.)

Desv. var. parvifolium Benl

and G.Kunkel was

published without

cytological information. In March

'1,967,

T. Reichstein

collected

living

ceterach'

on Gran Canaria, and sent material for

chromosome

counts

Vida. In 1970,

these plants were found to

octoploid, but because good

cytological

photographs

were lacking the results

were not published

(T.

Reichstein,

pers.

comm.). From then onwards,

but

without studying

the type of

A. parvifolium

(Benl

and G.Kunkel) Vida and

AMERICAN FERN

JOURNAL:

VOLUME 94 NUMBER 2

(2004)

Reichst., octoploid status

was attributed to

it. To

clarify

the origin of

Á.

parvifoliltm, a hybridization

program was started by G.

Vida in Budapest;

results were

partly published in Manton et al.

(1986).

Meanwhile,

Reichstein

had informed many

pteridologists about the putative allo-octoploid

nature of. A. parvifolium and briefly

mentioned

it in Hegi

(1984).

date two

cytologically different endemic species

are generally accepted to

occur on the Canary

Islands: A. aureum and

Á. parvifolium. Asplenium

aureum

was found to be tetraploid by

Manton

(1950).

Vida

and

Reichstein

(Vida,

1.972; Viane and Reichstein,

1,992)

suggested

A. oureum to be

allotetraploid,

which was confirmed by ITS analysis

(Van

den heede et al.,

2003). The

name A. parvifolium was used for the allo-octoploid

that

prob-

ably

formed by chromosome doubling of

the tetraploid hybrid between

Á.

oureum and Á. ceterach

(Vida,

1,972; Viane and Reichstein,

1.992). After 1.970,

all small Canarian

plants that looked like A. ceteracft

were

considered

to be

a) A.parvifolium and b)

octoploid. According to

Manton ef a1.

(rge0)

"C.

officinarum

is not positively

recorded from Macaronesia, but its

former

presence, at least in the Canaries,

suggested

by the morphology of some

representatives of C. oureum sens.

IaÍ. from

these

islands."

Within

ceterach sensu

1oÍo three cytotypes are

known,

and

according to

the

Biological Species Concept

(Mayr,

'1.942,2000;

see

review in King, 1993),

autopolyploids

should be considered separate species because

they

produce

sterile hybrids with their

parents from which they are reproductively

isolated.

Diploid

javorkeanum

Vida

[Syn.:

Á. ceterach ssp. biva]ens

(D.E.

M"y.)

Greuter and Burdet; C. officinarum

Willd. ssp. bivalens D.E.

Mey.l is known

from Albania,

Bulgaria,

Croatia, Greece,

Hungary, Italy, Romania, and

Slovenia

(Vida,

1963; Reichstein, 1984), and should be

looked for in northern

Algeria

and Turkey, because the triploid

hybrid A.

xmantoniaeYáróczy

and

Vida was

found

there

(Greuter,

1980; Viane et aL.,1996).

Tetraploid

(Manton,

L950; Vida,

1963)

ceteracft

[Syn.:

C. officinorum Willd. ssp. officinarum]

is supposed to

have originated

via

chromosome doubling

in Á.

javorkeanum;

its autopoly-

ploid

status

was confirmed cytologically by

Rasbach et aI.

(1987).

The

autotetraploid

the

more common species, occurring throughout

Europe

(see

maps in

falas

and Suominen,

'1,972;

Pichi Sermolli,

'1.979;

Reichstein,

1984),

southwestern

Asia and the

western Himalayas. Asplenium ceterach

more rare in northern Africa

(Jahandiez

and Maire, 1931; Maire,

1.952;

Quezel

and Santa,1962; Siddiqi,

19Bg),

but extends

into Eritrea and Somalia

(Viane

eÍ

o/., 1996), the

Arabian Peninsula

(Collenette,

19Bb), and

Yemen

(Christ,

1900;

Wood, 1997). The autohexaploid

cyprium

Viane and Van den

heede

(Syn.:

A. ceteracft ssp. cyprium Viane)

was

described

from Cyprus

(Van

den

heede

and Viane, 2OO2; Viane and Van den heede,

2OO2),

and

is also known from

Greece and Sicily

(Viane

et aL.,1996; Van den heede et al.,

2oo2).

For

the biosystematic

revision of ï}ire Ceterac.h

group

(Van

den

heede, 2003),

field trips were organized to study the

Macaronesian representatives. Plants

that we could not distinguish

from

the

European A. ceterach were tentatively

called A. parvifolium, and assumed to be octoploid.

To our great surprise many

of them turned out

to be tetraploid.

VAN DEN HEEDE ET AL.:

CANARIAN

ASPLENIUM

CETENACH GROUP

order to clear up the

ceterach-A.

parvifolium muddle

on the Canarian

Archipelago, we

studied type material, and cytologically checked 145 samples

from Gran Canaria, La Palma, and Tenerife. Because the

type

of A. parvifolium

turned

out to be

Á.

ceterach, the octoploid taxon

needed

new name

and

was

described as Á. octoploideum Viane and Van den heede

(Van

den heede and

Viane,2OO2).

Because

electrophoretic analysis of

isozymes has

been successfully used

studies of reticulate

complexes of

Pteridophyta

(Werth

et aI.,

1985a, L985b;

Werth, 1991; Haufler

et aI.,

1995)

and

has

been applied at

population

and

species

levels

(see

Haufler, 1985b, 1997), we

tried this method together

with

DNA

sequencing, to determine whether true A. ceteracft

grows

on the Canary

Islands. A combination

morphological,

cytological, and biogeographical

data and isozyme markers can determine whether taxa are auto- or allo-

polyploid

(Crawford,

1985; Haufler, 1985b; Bryan and Soltis, 1987; Weeden

and Wendel, tg8g;

Crawford, 1990; Pryer and Haufler, 1993). An overview

of the literature about DNA

sequencing

in Pteridophyta is given in Van

den

heede et aI.

(2003).

Marnzunl RNr MnrHous

Between April 1995

and

May 1999, field

trips were organized to three Canary

Islands from which A. parvifolium was known in

the literature: Gran Canaria,

La Palma

and Tenerife. From 145

specimens,

fronds with ripe

spores

were

collected

by C.V. and

R.V,

and ecological notes were made. Voucher

information

(Appendix

1, 2, and

is given only for

specimens

from which

we were

able to raise

progeny

and obtain cytological data.

The

following localities are

shown

in Fig. 1:

Gran Canaria, S of Moya,

"Los

Tilos" Reserve, W exposed slopes of

Barranco

del

Laurel,

degraded

laurel

forest, in fissures of volcanic rocks;

28'05'03'N, L5o35'28"w,6oo m

alt.

2) Gran Canaria, 4 km from

junction

Tejeda-San

Mateo-Las Mesas, E

exposed

slopes of

"El

Nieblo"

Nature Reserve, in fissures of volcanic

rocks; 2B"O'1,'03"N, 15'36',07"W,

L550 m alt.

3) Gran Canaria, lava field near

Cueva Corcho, along road GC110 from

Artenara

to Valleseco, 4 km NW

junction

Artenara-Valleseco-Tejeda,

fissures

volcanic

rocks; 1350 m

alt.

Gran Canaria, 900 m S of Valsendero, W exposed cliff

sides of

narrow gully

with laurel

forest remnants;28"O2'48"N,

'1,5"34'27"W,

900

alt.

Palma, c. 3 km E of Tijarafe, Pinar Lomo

del Hornoi 28"42'N, 1,7o55'W,

1.140 m alt.

6) La Palma,

S of Gallegos,

Barranco

Lomo de los Machines, Laurel forest

of tunnel El Envetadero,

E exposed slope; 2B.4B'N, 17o50'W,

390 m alt.

La Palma, volcanic rocks

above

roadside

to Fuencaliente, S

Monte

Luna; 28"3l',N,

L7o49',W, 71,O m

alt.

Palma, footpath

Monte

Luna

in Pinar S of Flores, in fissures

volcanic rocks; 28"31,'N, 17"49'W,

810 m alt.

Tenerife

Palma

Gran Canaria

AMERICAN FERN

IOURNAL:

VOLUME

NUMBER 2

(2004)

Frc. 1. Map of the western Canary Islands with localities of

voucher specimens

(see

also

Appendix 1, 2, and 3).

9) La Palma, along track to Pinar de la

Virgen

junction

with track to Caldera

Los Arreboles, in fissures of volcanic rocks;

28"30'N, 17o50'W,

g2O

m alt.

10) La

Palma,

along track to

Refugio de Tigalate from

Zona Recreativa Fuente

de los Roques, above

"Malpais"

Monte de Luna, in

fissures of

volcanic rocks; 28"3'1,'N, L7o49'W,

'Lo7o

alt.

L1) La Palma, Caldera de Taburiente, track

from La Cumbrecita to

Hoyo

los

Pinos, Pinar in Barranco de

la Faya, in fi.ssures of volcanic rocks;

28o43'N,

17050'w, 1.200 m alt.

1,2) La Palma, Iava

field 2.5 km E

EI Paso church,

in fissures

volcanic

rocks; 28"38'N,

17"51,'W,

800

alt.

13) La Palma, Iava field

El Paso, NE

Montaía Las Moraditas,

in fissures

volcanic rocks; 28o39'N, 17o50'W, 800

alt.

14) Tenerife, along road from

Vilaflor

Pico del Teide, ca. 8.1

km from

junction

Vilaflor-Santiago del Teide-La Orotava,

under disc-like, SW

exposed volcanic rocks;

2B'10'55'N, 16o39'L7uW,1850 m alt.

15) Tenerife, Barranco de

las

Gambuesas

above Arafo,

exposed

slopes;

2Bo2O'29"N, L6o26'Oz"w, 710 m alt.

16) Tenerife, Barranco del Espigon de

Tea, NE exposed slopes; 28"20'44"N,

16"26'33"W,

825

m alt.

17) Tenerife, Montafla de

la Hoya, ridge

S of

Las Manchas, above

Ermita

Santa Angel del Guardo,

in fissures

volcanic rocks;

28o16'34'N,

16"48'05"w, L',l-20

alt.

18) Tenerife,

Teno, Barranco head between Tierra del

Trigo and Ruigomez,

along track above Tierra del

Trigo, 1.8 km NW of Ruigomez,

exposed

basaltic slopes; 29o2o'48'N, L6"48'28"W,800

m alt.

VAN DEN HEEDE

ET AL.: CANARIAN ASPLENIUM CETERACH

GROUP

Tenerife,

Pinar above Vilaflor,

Bandes

de Chasna,

c. 2.5

km NNW of

Vilaflor,

exposed,

in fissures of volcanic

(phonolite)

rocks; z8oL0'48"N,

16"38'36"w, L880

alt.

Tenerife, Pinar above Vilaflor,

exposed

slopes of small

Barranco, in

fissures of volcanic

rocks; 28"1,0'48'N, L6o38'39"W,

1900 m alt.

Tenerife,

Barranco

Ia Piedra Cumplida above

(NW)

Arafo;

28"2'1.'L7"N,

1,6'26'05'W. 900

alt.

Tenerife, volcanic outcrop along

footpath

between

Santiago del

Teide and

Arguavo, SE of

El Retamar, between

Montana

la Hoya and

La Hoya;

28oL6'N,

L6o48"w,

920

m alt.

Tenerife, lava field

Montafra de las Flores,

"Vuelta

Grande",

along

track

from El Portillo del Rastrojo to

Llanos del Hospital;

2B.LB"N,

16o45'W, \41.o m alt.

24) Tenerife, Chio Street, direction Cafradas,

Restaurant

"De

Evora".

Vouchers listed in Appendix 1, 2 and 3, are deposited

in the

personal

herbarium of

Viane and Van den heede

(including

the T. Reichstein

herbarium),

with

duplicates

GENT.

Between 1,992 and 2001, R.V., C.V., and

W. Bennert

gathered

additional

material in Europe, Madeira, and

Turkey

(Appendix

Voucher information

about 108 Cypriot samples is

published in Van den heede et al.

(zOOZ).

Our

Iiving European and

Macaronesian Asplenium subg. Ceterach collection

contained

rp to 550 specimens.

AII material for this study

has

been

cultivated in Ghent University

Botanical

Garden

(Belgium).

Spores

were sown on agar-solidified

medium containing

a nutrient solution recommended by

Dyer

(1979).

The cultures were stored

continuous

light

room

temperature.

After formation of

mature gametophytes,

distilled

water was

added to

achieve fertilization. If necessary,

prothallia were

transplanted onto

fresh

agar.

Young sporophytes were

planted individually in

pots kept in a temperate

greenhouse

(minimum

temperature 1.2"C).

air- and

water-permeable soil mixture was required

for

these

xerophytic rock ferns.

Full-grown maturity was reached after approximately two

years.

For

chromosome counts,

immature spore mother cells

were fixed in the field,

in the greenhouse, using freshly prepared 3:1 absolute ethanol:glacial

acetic

acid,

and

stored

at freezing-temperature until

required. Acetocarmine squash

preparations were made as described by

Heitz

('1.925,1950)

and Manton

(rgso).

Photographs

were

taken

with

an Olympus

BH2 phase contrast microscope.

Preparations were made

permanent

by dehydrating cover

slip and slide in

graded mixtures of acetic acid and absolute ethanol,

followed

mounting in

Euparal

(T.

Walker

and

H. Rasbach, pers. comm.). All permanent

preparations

are kept in the Pteridological Section of the

Department

Biology at Ghent

University. Sixteen cytologically checked

plants

(five

tetraploids

identified as

A. aureum, seven tetraploids

identified

"A,

parvifolium sensu Benl," and

four

octoploids,

(Appendix

L, 2, 3) were used as standards to compare the

nuclear DNA content of the

remaining

specimens

by a flow cytometer

(Partec

PA-1), using the manufacturer's

protocol

(Partec

GmbH,

Mrinster,

1e)

20)

21)

22)

23)

AMERICAN FERN

IOURNAL:

VOLUME e4 NUMBER

(2004)

Nordrein-Westfalen, Germany).

Both

nuclei extraction solution

and DAPI

staining

dilution were

provided by Partec

(Germany).

Methods

for making

permanent

epidermis

preparations and for measuring

stomatal

guard

cells

and spores, are described

Viane

(1990,

1992).

For

exospore

measurements untreated,

fresh spores

mounted in DePeX

were used.

Spore

size is unaffected by

DePeX,

whereas in some other

mounting media,

e.g.,

glycerin-gelatin

(Ormonde,

1990), spores expand

by 5-L5

The values of

microcharacters are

extracted from our

regularly updated database,

presently

containing 110 different

specimens of the

European

"A,

ceteracft"

group,

and

specimens of the

Macaronesian

aurelrm"

group

(raw

data available

upon

request).

Only

vigorously growing plants

were included

the

allozyme study.

Fresh

leaves from

105

Canarian

(Appendix

'1.,

2, 3) and

22O European and

Turkish

specimens

(Appendix

a) were

collected

in the

greenhouse, where the

ferns

were growing under the same

conditions. Sporulating

fronds of similar

age

were wrapped

in wet tissue, stored

in plastic bags, and

kept refrigerated at

4'C

for maximum 0.5-2

days

(until

extraction).

Polyacrylamide

gel electrophoresis

(PAGE)

was

performed by C.V. at the

laboratory of

"General

Botany and Nature

Management"

of the Free University

Brussels

(Belgium).

Starch

gel

electro-

phoresis

(SGE)

was done by

S.P. and E.P.

the

"Departamento

de Biologia

Vegetal I" of the Universidad

Complutense

in Madrid

(Spain).

All specimens

from the Canarian

Archipelago

were

analysed

by starch

gel

electrophoresis.

Equal amounts of tissue and

extraction buffer

were used to obtain

uniform

concentrations of extracts.

Cooling

(+'C)

was applied during

both

homogeni-

zation and electrophoresis.

Acquaah

(fgg2)

was

consulted

for the

Enzyme

Commission

(E.C.)

numbers.

PAGE

procedures mentioned

in Triest

(1989)

and

Van den heede et al,

(zOoz)

were used,

whereas SGE

protocols followed Soltis et

al.

(1983)

and

Haufler

(1985a).

preliminary survey

19 enzyme systems

(G-3PDH,

G-6PD,

GDH,

IDH, MDH, ME, 6-PGD, SKDH,

SOD,

XDH, ACO, AAT,

HK, PGM,

B-EST,

LAP,

ALD,

PGI, and TPI)

were

checked

for

polymorphism. Because the

primary

goal

of this

isozyme research

was

test the hypothesis

that tetraploid

A. ceterach

occurs on the Canary

Islands

addition

to A. aureum

and related taxa,

it was

necessary to

identify unique

"marker"

alleles characterizing

each species

its

progenitors. Finally, only

five enzyme systems

were suitable:

aspartate

aminotransferase

(AAT:

GOT, E.C. 2.6.L.1), shikimate

dehydrogenase

(SkDH,

E.C. 1.1.'J-..25),

malic

enzyme

(ME,

E.C. L.1.1.4o),

phosphoglucose isomerase

(PGI,

E.C. 5.1.3.9), and

triosephosphate

isomerase

(TPI,

E.C. 5.3.1.1).

AII

pictures and dried

gels

are

kept in the

Pteridological Section of the

Department

of Biology at Ghent

University.

Band homologies

were determined by

running samples

side-by-side on the

same gel

(see

Haufler et oI., 1995).

Allelic variants

within loci

were

distinguished

from the products of different

loci by assuming

that

Ásplenium

enzymes

conformed to established

models of organellar

compartmentalization

(Gottlieb,'1.982;

Gastony

and

Darrow, L9B3; Soltis,

1986; Weeden and

Wendel,

19Bg). Presumed

loci were numbered sequentially,

with

the

most anodally

VAN DEN

HEEDE ET AL.: CANARIAN

ASPLENIUM CETERACH GROUP

(i.e.,

the

fastest

band) migrating

one designated

"1."

Similarly, different

alleles

of the same

gene

locus

(i.e.,

allozymes, Crawford, 1990) were

denoted

alphabetically with

the

most

anodal being

"o."

Sequencing work

was done by C.V. at the

Jodrell

Laboratory

in Kew

(United

Kingdom).

Nineteen cytologically

and isozymically interesting, vigorously

growing plants, including

three A.

aureum specimens

(CVí

64, CV670, CV712)

from

Gran Canaria, Tenerife, and La Palma,

one

putative

Á. ceterach from

Tenerife

(CVl87),

and one octoploid from La Palma

(CV709),

were selected to

generate

DNA

sequences from the

plastid

trnL-trnFintergenic spacer. European

material for

comparison included two Á.

javorkeanum

specimens from Italy

and

Slovenia

(CV14

and CVBSb),

two

Á.

ceterach samples from Italy

and Cyprus

(CV494

and CV225),

and a

hexaploid

Á. cyprium plant

(CV249)

from Cyprus

(see

Appendix

4). Sequences

of the closely related A. Iolegnamense

(Gibby

and

Lovis) Viane

from Madeira

(CV9Bí

and CV993), of the less related

dalhousiae Hook.

from Ethiopia and Pakistan

(CV3LB

and Tn7ffi4),

and of

the

distantly related A. nidusL.

(AF425118),

and

Á.

scolopendrium L. and

unilaterale Lam.

(R.

Cranfill, University of California, Berkeley,

California,

USA, unpublished

data)

were included

as outgroups. The

trnL-F sequence of

a species

Dennstaedtia

(R.

Cranfill,

unpublished data) was used to represent

group

basal

to the Aspleniaceae

(e.g.,

Bower,

'1,928;

Christensen, 1938;

Copeland,

'1.947;

Pichi

Sermolli, 1,977; Kramer

and Green, Lgg0; Hasebe et

aL,

1995; Pryer

et aL.,1995). Methods

are explained in Van

den

heede

et al.

(2003).

Rnsur,rs nrun PnrlrMrNARy DrscussroN

avoid prolixity, we have

combined both the results

and the

interpretation

of the isozyme phenotypes.

Chromosomes were

counted for 16 specimens

collected on Gran

Canaria,

Palma, and Tenerife. In

addition to five tetraploid A.

aureum

--72")

plants,

eleven

small specimens that we

could

not

distinguish from European

Á.

ceterach, were examined.

Seven of them turned out

to be tetraploid

72tt)

and

four

were octoploid, having

a meiotic chromosome number

of n

1,44rr

(Fig.

2). Meiosis in

all cells examined was regular,

showing only bivalents, and

giving no indication

about

the

polyploid

status of the

species.

This

agrees with

Lovis'

(tgzz)

statement that most

autopolyploid ferns possess

diploidized

meiosis

(only

bivalent formation), and

"that

the absence of multivalents

is no

valid

evidence

of allopolyploidy."

The counted

samples were used as standards

to determine the ploidy level

the remaining 146

specimens using

flow

cytometer. Results

are

given

Appendix

1., 2 and 3; localities

of cytologically checked material

are indicated

on the map of the

Canarian Archipelago

(Fig.

1).

May 1995

(nvilSs)

and 1997

(CV165-L70;

CVLB3-1B7), we

discovered

tetraploi

d A. ceterach specimens

on both Gran

Canaria and Tenerife

(see

Appendix 1

and 3).

The three

species

(A.

aureum,

the

"small

tetraploid",

and the octoploid)

cannot

always be distinguished

macromorphologically,

but can be identified

AMERICAN FERN

|OURNAL:

VOLUME e4 NUMBER 2

(2004)

art

*'ïl

itr

.ltt'

3.a

1ïL

lll

i,r"

Éfuàf:ii

{.'ga'g

tt4

$Jt

k^.*:dltr

lt&ffit

$:Í

.tï

É'trjf

t',

Ftc.2. Cytology showing spore mother cells in

first meiotic division. A, B:

photographs;

A', B':

explanatory diagrams

with bivalents in black.

A, A': A. ceterach

(CV

170b),

metaphase I showing n:72tt. B, B': A. octoploideum

(CV

188,

holotype), cell showingn:1,44tt. Scale bar: L0

pm.

(preparations,

photos and diagrams: C.V.).

by measuring exospore

length

(Table

Z). Stomatal

guard

cell length can only be

used to distinguish the

"small

tetraploid"

(+s

3.8

pm)

from

the octoploid

(sz

4.8

pm).

We found no differences in

perispore morphology,

stomatal

type,

or epidermal

cell pattern. Perispores

have

costato-cristate

folds with few

perforations,

stomates

are mesopolocytic, and epidermal cells

mostly sinuous.

Polyploidy factors

(Viane,

1986, 1990) in Á. ceterach are

P,"1,

exo:

t.ZS

(for

the

exospore) and Pcet,

sro:

1.L6

(for

the stomates),

and P"r.,

u*o:1.L8

and Pu,r.,

,1o:

1.07 in A. aureum. Using these

P-values, the theoretical spore and stomate sizes

calculated

for

the octoploid

are less than 1 s.d. different

from

their actual

means

(Table

2), thus supporting the proposed ancestry

(Viane,

1990).

We stress the

presence

of a small

indusium in all taxa; it can best be observed

epidermis preparations

(Viane,

rggO).

Our observations

show that the

(toothed)

margin and the

"cuticular

lines" of the scales, are unreliable

characters to

VAN

DEN HEEDE ET AL.:

CANARIAN ÁSPTEN/UM CETERACH GROUP

Taslr 2. Microcharacters

differentiating taxa within the á. auteum-ceterach group

on the Canary

Islands.

All measurements are based on cytologically checked

material. Additional information

about material and the number of measurements

is available from the authors.

Taxon

Ploidy

Mean exospore

length

s.d.

Mean

guard

cell

length

s.d.

A. aurem

A. ceterach

octoploideum

1.9

2.6

3.1

4.4

3.8

4.8

discriminate Á. oureum and relatives from A.

ceteracà. AII taxa have

scales

with

more or less dentate margins,

and

periclinal

cell walls with

without "cuticular

Iines". These "cuticular

stripes" are folds in

the

periclinal

cell wall

(FiS.

3), and

the

bigger the cell the more folds seem to

present. However,

A. aureum

scales

usually show numerous

folds, whereas A.

ceteracft

(from

its entire range

distribution) paleae possess

only few. In the octoploid

the number

folds is

usually intermediate

between that in Á. ouÍeum

and

Á.

ceterach.

Isozyme

analysis can be used to

determine

whether

taxa are auto-

allopolyploid.

The electrophoretic phenotype

of an autopolyploid

should

show a subset

of the

isozymes

present in its progenitor,

assuming no mutation

subsequent

to the origin of the

polyploid

(Weeden

and Wendel, 1989;

Crawford, 1990; Pryer and Haufler,

1993). An allopolyploid

should display

fixed heterozygous

(i.e.,

nonsegregating)

banding patterns for

many loci,

resulting

from

the combination of different parental genomes

(Gottlieb,

1982;

Werth, et aI. 1985b; Pryer

and Haufler, 1993; Soltis

and Soltis, 2000). Fixed

heterozygous

banding patterns differ from normal

heterozygous zymograms,

because the bands do not

segregate among

progeny

and remain fixed in

all

specimens.

Nineteen

enzyme systems were

tested in a

preliminary

survey. The

low

resolution

of ALD, and the smeared patterns

of XDH made both unusable. IDH,

FIc. 3. Folds

("cuticular

lines")

micrographs of laminal

scales

(CV

Bar: A:50

pm,

B:10

pm.

periclinal

cell

walls

of A. aurcum

157). A: scale margin with

several cells.

paleae.

Phase

contrast

B: single cell with folds.

AMERICAN

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VOLUME e4 NUMBER 2

(2004)

Iumlvv

b-r=:-

C--

genotypcs:

bbb bbbb aaanhbbb

atrcc aou

tere:

"srnall

tctraploif

'srnall

tetraploif

'octoploid'

atrann Á, awanr

Á. ceterach A- ceterach

distribution:

Canry

Islands

Canry lslands Cmry Islands Canry

lslsrds

Canry

lslands

Europe

Euope

Turlccy

Frc.

Diagrams

of electrophoretic

AAT

phenotypes,

showing 3 alleles

and

the

corresponding

genotypes,

as observed

the

European-Canarian Asplenium ceterach-aureum

group. Zymotype II

was also

found in

all samples

of A.

javorkeanum (bb)

and Á. cyprium

(bbbbbb)

examined.

MDH, ACO, and SOD yielded unclear

patterns with limited variation. G-6PD

and G-3PDH

gave inconsistent zymograms. Consequently, these

enzyme

systems were not retained.

To test our

hypothesis

that

in addition to

aureum and

the octoploid, true

A. ceterocft occurs

on the Canary Islands, the

following four enzyme systems

were

suitable:

AAT, SkDH, ME, and PGI.

These enzyme systems

yielded

reproducible, well

resolved

banding

patterns discriminating specimens

representing Á. oureum,

A. ceterach and the allo-octoploid

hybrid. These

enzymes, encoded by four

putative loci, were also used to

get

idea

of the

variation

of the

species.

AAT or GOT: this dimeric enzyme

was studied using both

PAGE

and SGE

(system

B of Haufler,

1985a).We

observed

only one activity

zone, which

agrees

with Gastony and

Darrow

(rggg),

who proved that this single enzyme

activity

is chloroplastic.

Most of the specimens studied

are homozygous, showing a single

well-

resolved AAT

band

(Fig.

4, zymotype II). This applies to all 50

Á.

javorkeanum

specimens

(Italy

and Slovenia),

most

(1.o2)

A. ceterach

plants

(Belgium,

Croatia, Cyprus,

France, Italy, Slovenia, Spain,

Turkey, and the United

Kingdom), 23

"small

tetraploids"

from the Canary

Islands, and all

(aa)

cyprium samples tested

(Van

den

heede

ol. 2OO2). Ten A. ceterach

individuals

from

Croatia,

France, Italy, and Slovenia, and two

"small

tetraploids" from the Canary Islands, showed

the rarer three-banded zymotype

I with skewed staining intensities, interpretable as

heterozygous for a dimer.

VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM

CETENACH GROUP

Corresponding

genotypes

are bb, bbbb,

or bbbbbb for zymotype

of the

homozygous

(diploid

to hexaploid)

plants, versus bbbc for zymotype

of the

heterozygous tetraploid specimens.

Identical banding

patterns in the Canarian

"small

tetraploid"

and in

European A. ceterach

plants

indicate that true

Á. ceterach is growing on the

Canary Islands. Because

neither European nor Canarian samples reveal unique

electrophoretic

phenotypes,

the Canarian

populations do not seem to be

ge-

netically

isolated. Though more sampling of

iavorkeanum

is needed,

these

preliminary results

(Fig.

4, zymotype II) seem to confirm the

autotetra-

ploidy

ceterach.

The a allele

(Fig.

4, zymotype

can

be used as a

"marker"

allele

characterizing

oureum.

Some plants, showing a single band corresponding

genotype ooao, are homozygous, whereas others,

with

a balanced three-

banded

zymogram corresponding to

genotype

aecc, are

heterozygous. To

explain zymotypes I and

IV,

an extra

genotype

is postulated and expected

in Á.

javorkeanum,

which was studied only on the basis of

Italian

and Slovenian

material.

All

54 octoploid

specimens

(from

Gran Canaria, La Palma, and

Tenerife)

show a monomorphic,

presumably fixed heterozygous

banding

pattern of

genotype aaaabbbb

(Fig.

4, zymotype III), which we postulate to be derived

from a combination of

zymotypes II and V

(Fig.

This would agree with the

suggestions of

Reichstein

(rge+)

and Viane and Reichstein

(rggz),

that the

Canarian octoploid is an allo-octoploid,

which originated either by chromo-

some doubling

in an unknown tetraploid hybrid between

oureum

(with

zymotype V) and A. ceteracft

(with

zymotype

II),

via

unreduced

gametes of

each species

(Fig.

9).

The fact

that,

in all the octoploids

(from

15 different

localities) only one AAT zymotype was detected can be explained by the

preponderance of the

"small

tetraploid" with zymotype

II. It may

also

reflect

incomplete sampling of the variation

present in

the octoploid.

SkDH:

resolution for

this

monomeric enzyme was superior on SGE

(system

of Weeden and Wendel,

19Bg). In

our study,

the enzyme was represented by

a single locus, which agrees

with

Gastony and

Darrow

(tgAg).

As expected for

a monomeric enzyme, homozygotes had a typical one-banded

pattern whereas

heterozygotes showed two or more bands.

We detected four alleles

the

European-Macaronesian Asplenium

ceterach-oureum

group. Two

of these,

o and b, were observed in Á. eureum,

whereas

and

d characterized

the

"Á.

ceterach"

group.

Although SkDH

was

polymorphic in Á.

ceterach

(Fig.

5), only

zymotyp e V

(cccd)

was found on the

Canary Islands. This two-banded

pattern with

unequal

staining intensities

forms also part of zymotype VI found for all 54 octoploid specimens.

Thus

the

octoploid

is monomorphic

and

presumably heterozygous for this locus,

showing a

four-banded zymogram

corresponding

to genotype aabbcccd. The

and b alleles are unique

"marker"

alleles for A. eureum, one of the

progenitors

of the octoploid. This

monomorphic pattern

showing

presumed

fixed heterozygosity seems to confirm the putative allo-octoploid origin of this

species

(Reichstein,

1984; Viane

and

Reichstein, 1.gg?). The cccd SkDH

AMERICAN FERN

|OURNAL:

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e4 NUMBER

(2004)

|ilIIIlvvvlvll

i'-

bi--

- - -

- -

Seootypes:

cccc ccdd

cMd

cccd oahlrccd

ubh

trn

(óbreviated):

-srnall4x' -oooploid-

.4. our.

jo.

Á. cet. A. cet.

A. cet.

.4. cet.

distribution:

Italy

Cyprus

ltaly

Crotia

Canarian

Archipelago

Slovcnia

France Spain

Slovenia

'-"Ó;á,iË"

ttaty Trrkey

Cyprus

Slovcnia

Italy

Ftc.

5. Diagrams

of electrophoretic

SkDH

phenotypes,

with

corresponding genotypes,

as observed

in the European{anarian

Asplenium ceterach-auÍeum group.

Zymotype

II was also found

some Á.

javorkeanum (genotype:

cc); zymotype V

was

present

all Á. cyprium

(genotype:

ccccdd)

samples

checked. The

Canarian small tetraploid

is abbreviated

as:

"small

4x."

genotype

(zymotype

of the

"small

tetraploids" is not

limited

to the

Canaries,

but was

also found in Á.

ceterach from

Croatia, Cyprus,

and ltaly.

These results

again

both

prove

the

occurrence of A.

ceterach on

the Canary Islands,

and the

fact

that the populations

in this Archipelago

are

not

genetically

isolated.

Three

additional zymotypes

were

detected in

continental Á.

ceterach:

a single-

banded

(cccc),

a balanced

two-banded

(ccdd),

and an

unbalanced

two-banded

pattern

(cddd).

The presence

of the unbalanced patterns

(cccd,

cddd) in

tetraploid A.

ceteroch

at Skdà can

be explained by

tetrasomic

inheritance

(see

discussion).

Diploid Á.

javorkeanum

from Italy

and

Slovenia showed

a single-

banded

pattern

of either genotype

(zymotype

II) or

(zymotype

I).

ME:

this tetrameric

enzyme was

studied only

by PAGE. The

single enzyme

activity visible

was

shown to be

cytosolic

by Gottlieb

(rggZ),

Gástony

and

Darrow

(rgag),

and Soltis

(rgao).

ME was monomorphic

in each

of the three species,

and thus

can be used

distinguish

them from each

other

(FiS.

6).

All

Á. ceterach

specimens

(Europe)

and

"small

tetraploids"

(Canary

Islands) were

heterozygous

showing

identical five-banded

zymogram, typical

for a tetrameric

enzyme controlled

one locus with

two alleles,

o and d. Heterozygous

A. aureum

was

characterized

five-banded pattern

controlled

by the same locus,

but with

two different

alleles,

b and c, and

corresponding

genotype

bbcc. All octoploid

plants

aadd

addd

dddd

getrotypcxr:

addd

tere:

"gnall4x"

caerach

distribution:

Canarian

Europe

Trukcy

Archipelago

ella

azeb

ffiB

::::-

bbbb

tÉc

aail

bcCC

bbbd

agr!,

bbcc

a,dld

accc

bbdd

bCCC

addd

cccc

bddd

Eod

oodd

cddd

dddd

VAN DEN HEEDE

AL.:

CANARIAN ASPLENIUM CETENACH GROUP

FIc. 6. Diagrams explaining the ME zymotypes showing 4

alleles,

with

corresponding

genotypes,

as observed in

the European-Canarian Asplenium ceterach-aureum

group.

For

each band four

Ietters represent the association

of subunits

(coded

alleles)

joined

form

this tetrameric

enzyme. The homotetramers in the

"hybrid" pattern

are in boldface. Zymotype III

confirms the

allopolyploid status

of the octoploid. Dotted lines

indicate

very faint bands. The Canarian small

tetraploid is abbreviated

"small

4x."

showed a complex zymogram, and conform to the expected hybrid phenotype

resulting from

the cross between A. ceteracft and A. aureum. The

"hybrid"

had

the four

parental

alleles,

and

since

ME is a

tetramer, each of the six

pairs

alleles

(ax

c, ax

bx c, bx d, cx

formed

three heterotetramers of

intermediate

mobility. Theoretically this results in

22-banded pattern

homotetramers

plus

18 heterotetramers,

makes

bands), but

because

twice two bands have

the

same mobility, a maximum

of 20 bands was visible

(Fig.

6).

The monomorphic and

presumably

fixed banding

pattern

of the

"hybrid"

zymogram is in agreement with

the

putative

allopolyploid

origin

the octoploid

(Reichstein,

1984; Viane

and Reichstein,

1992).

PGI: this

dimeric enzyme

was

studied by both PAGE and

SGE.

Because the

resolution

was much better with

SGE, all

results

shown

were

obtained using

starch gel electrophoresis

(system

6 of Soltis ef o/., tgag).

Two loci were present:

Pgi-I, most

probably

chloroplastic,

and

Pgi-2,

cyto-

Pgi-2

-aC

c-c.c

gcnotypes:

aacc

texe:

Á. ceterrch

distribution:

Italy

-cc-cc-cc

-Cd-CdrCd

-csFdd

-::

ccdd

cdde

ccccccdd

A. aureun

auream

"octoploid"

VVIVII

(rr

ru)

IIr)

aa_aa

-ad

-dd

aaccccdd

qaqadddd

"octoploid"

-aA

-CC

-cd

-dd

cccc

"small

4x"

Á. ceterrch

Canarian

Europe

Archipelago

AMERICAN

FERN

JOURNAL:

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e4 NUMBER

(2004)

FIc. 7.

Diagrams

of electrophoretic

Pgi-2

phenotypes,

showing

5 alleles,

with

corresponding

genotypes

and their

distribution,

as observed

in the

European{anarian

Asplenium

ceterach-

aureum

group.

Zymolype

was also

found in

most A.

javorkeanum

(cc)

samples.

Each

band is

indicated

two letters

representing

the association

subunits,

joined

to form this

dimeric

enzyme.

The

Canarian

small

tetraploid

is abbreviated

as "small

4x."

solic

(Gastony

and

Darrow,

L983;

Soltis,

1986).

Consistent

with

observations

on other

ferns

(Gastony

and

Gottlieb,

tg85;

Werth,

1991;

Haufler

al.,19gb;

Hauk

and Haufler,

1999),

resolution

the more

anodal locus

Pgi-1was

inferior

that of Pgi-2.

Because

Pgi-l

appears invariant

across

all

taxa, it will

not

discussed.

Among

the European

and

Macaronesian

samples

studied,

five

allozymes

were

observed

Pgi-2

(Fig.

7).

Although

the

continental

Á.

ceterach,

with

its

six different

zymotypes,

was

highly polymorphic

for this locus

(Van

den heede

et al., 2OOz),

only

a single

banding pattern

was

detected

for

the 22 "small

tetraploids"

from

the Canary

Islands,

corresponding

genotype

cccc.

This

widely

distributed

zymotype

was

also found

Á.

ceterach

specimens

from

Belgium,

Croatia,

France,

Italy,

Slovenia,

Spain,

and

the

United Kingdom.

obtained

two

electrophoretic

phenotypes

for

the 28 A.

oureum

plants,

with

corresponding

genotypes

ccdd

(ZS

specimens)

and

cdde

specimens).

The

octoploid

was

the most

variable

taxon in

the

Canarian

Archipelago,

showing

three

different

zymotypes

translated

into genotypes

(Fig.

ccccccdd

(zymotype

v),

oaccccdd

(zymotype

vI),

and

aaaadddd

(zymotype

vII).

zymo-

type V

(found

only

on La Palma)

most

probably

resulted

from

hybridization

VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH

GROUP

Tasl-e 3. Genbank

accession

numbers for trnL-trnF nucleotide

sequences of newly sequenced

Asplenium

specimens. CV and TR are abbreviations for Caroline Van den heede

and

Tadeus

Reichstein respectively. Localities are given in Appendix 4.

Species

Voucher

number Data of collection

GenBank accession

number

A. aureum

A. ceterach

A. cyprium

A. dalhousiae

javorkeanum

A. Iolegnamense

A. octoploideum

25 IluÍay 1.997

fan.

1999

Apr.

1999

27 May 1.997

fune

1997

17 Aug. 1998

1.2ltne

'1.997

|an.

1998

27 Aug. 1990

fuly

1996

30 Aug. 1996

29 May 2000

fune

2000

2 Apr. 1999

4Y160993

4Y160994

4Y160995

AY162333

4Y162334

4Y162335

l'Y1.62337

4Y161000

4Y161001

4Y162330

AY162331

AY160998

4Y160999

4Y161003

cv164

CV67O

CV712

cv187

CV225

CV494

CV249

CVS1B

Tn7ffi4

CV14

CVBs

CVgB5

cv993

CV7O9

between a

"small

tetraploid"

with genotype

cccc

and an Á. oureum with

genotype

ccdd,

which

are both abundantly

present on the

Canaries,

followed

by chromosome doubling, or via unreduced

gametes

of each species. Zymotype

though

presently known

only

from ltaly,

can

be used to explain zymotype VI,

which was found only on La Palma. Octoploids

with

this

genotype

(aaccccdd),

expressed three

homodimeric

bands

(Fig.

7, aa, cc, dd) plus three hetero-

dimeric bands

(ac,

ad, cd). More sampling is desirable and might detect other

genotypes

such as aocc

the

"small

tetraploid," as well as dddd

needed

explain zymotype VII from Gran Canaria and Tenerife.

Pgi-2

suggests

that the

formation of the allo-octoploid happened at

least

three times.

Because plastid DNA is uniparentally inherited,

discloses only the

maternal

Iineage

(Stein

and

Barrington, 1990; Gastony and Yatskievych, 1.992). GenBank

accession numbers for trnL-trnF nucleotide sequences of

newly sequenced

specimens are listed in Table 3.

Analysis

of the

plastid trnL-trnF intergenic

spacer sequences resulted in the clustering of

the

"small

tetraploid" from

Tenerife

(CV187),

A. ceterach

from Italy

and Cyprus,

and A. cyprium, with their

diploid ancestor

javorkeanum

(Fig.

B). We found no chloroplast

variation

(with

the exception of CV494) between

specimens sampled

from

the

Mediterranean

(Cyprus,

Italy, Slovenia) and

Tenerife. Asplenium

javorkeenum,

ceteracir, and

A. cyprium form a cluster of their own, different

from the

"Á.

oureum clade," which includes all the

aureum specimens,

Á. lolegnamense,

and the octoploid

(CV709)

from the Canaries.

Identical groups are obtained by

analysing rbcL

gene

sequences.

The position of A. Iolegnamense and the

octoploid,

the

plastid

trees, suggests

that Á. oureum acted as the

maternal

parent in

the

formation

the specimens used.

These molecular data

independently

prove

that

addition

to an octoploid species, true

Á.

ceterach

AMERICAN

FERN

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VOLUME e4 NUMBER 2

(2004)

aurewn

CVl64 Gran Canaria

A. aureun CV670

Tenerife

aurewnCVT|Z

La Palma

Iolegnamense

CV985

Madeira

I ole

gnamen

se CY 993 Madeira

octoploid CV709

Le Pelma

Á.

dolhousiae CV318

Ethiopia

A. dalhousiae

TR7634 Pakistan

anrkestwn

| 4

Slovenia

A.j anr

onan CV85

Italy

teÍa

ploid-

C_V l-87_Te ner ife

A, ceterachCVns Cyprus

'A.

celerach

CV49a

l-taly

A. cypriumcYz4g Cyprus

scolopendrium

A. nidus

unilaerale

hrutstaedlia

5 changes

FIc. B. Tree randomly selected from the 73 shortest trees of European-Canarian Asplenium

ceterach-aureum taxa and A. dalhousiae, resulting from

parsimony

analysis of our

1.4

trnL-trnF

intergenic

spacer sequences

(Table

3) and

trnL-trnF sequences of other

species available in

GenBank; length

:227

steps, CI:0.92, and RI:0.91. Based on rbcL evidence

(Hasebe

et al., 1995;

Pryer et al., 1995), the sequence

of the

distantly related DennstaedÍio was specified as

outgroup. Fitch branch lengths

(ACCTRAN

optimized) are shown above and

bootstrap percentages

(1000

replicates) below the branches. Other sequencing results are described extensively in

Van

den heede et al.

(2003).

the

"small

tetraploid")

is growing

on the Canary

Islands. Other sequencing

results are

described extensively

in Van den heede et al.

(2003).

Because Gran Canaria, La Palma, and Tenerife are of

volcanic

origin, these

epilithic ferns

mainly

grow

on rocks of basaltic types, like phonolites,

rhyolites,

trachytes and olivine basalts

(Page,

1979). Asplenium oureum

prefers

moister, shady

habitats

lower altitudes, whereas

the

"small

tetraploid"

and

octoploid plants share

exposed, drier habitats. However,

on both Gran Canaria and Tenerife,

only

single localities were found where

"small

tetraploids" and octoploids

grew

together

(loc.

2 and 19; see

Appendix

1 and 3). Though forty-five

specimens

from nine different localities on La

Palma were cytologically checked

(see

Appendix 2 and

Van

den heede and

Viane, unpublished

data),

could

not

detect any

"small

tetraploid"

specimen. We intensively looked for it in the field, especially in the higher

regions of La Palma. Whereas we discovered

"small

tetraploids" between 1500

and tg00 m altitude on Gran Canaria and Tenerife, the

highest

altitude

found ferns of the

Ceterach

group

La Palma

was near

1200 m.

We found Á.

oureum between 300 and

1000

m altitude

in valleys and

sheltered

ravines

("barrancos")

with remnants of

(degraded)

evergreen laurel

VAN DEN HEEDE ET AL.:

CANARIAN

ASPLENIUM

CETENACH GROUP

forest

dominated by broad-leaved

trees: Laurus azorica

(Seub.)

Franco, Persea

indica

(L.)

Spreng., Ocotea

foetens

(Aiton)

Berthel., Apollonias

barbujana

(Cav.)

Bornm., IIex canariensis Poir.

IIex

platyphyllo

Webb and Berthel., and

Arbutus

canariensis

Vieill.

(Bramwell

and

Bramwell, 1.974). Asplenium

oureum usually grows in humus-rich soils, often together with Adiantum

capillus-veneris L., A. reniforme L., Anogramma leptophylla

(L.)

Link,

Asplenium aethiopicum

(Burm.f)

Bech., A. hemionitis L., Cheilanthes

pulchella Bory

Willd., Davollia

canariensis

(L.)

Sm.,

Polypodium

cambricumL. ssp.

mocaronesicum

(A.E.

Bobrov)

Fraser-Jenk.,

and Selaginella

denticulafa

(L.)

Spring.

Asplenium

ceteracft and

octoploideum

were found in

the

natural pine

forests

("Pinar")

at 900-2000 m on Tenerife, and at 1200-1600 m on Gran

Canaria. The octoploid was observed on La Palma at 700-1200 m. The open

savannah-like vegetation is dominated by Prnus canariensis C. Sm. and a few

shrubs,

such as Adenocorpus

foliolosus

(Aiton)

DC., Cistus symphytifolius

Lam., Daphne

gnidium

L., Micromeria

species,

and

Rumex

lunaria L.

(Bramwell

and Bramwell, tgz+). Asplenium ceterach and the

octoploid

grow

in rock fissures,

often together

with Monanthes laxiflora

(DC.)

Bolle, Aeonium

species,

Asplenium aethiopicum, A. trichomones L., Anogrammo leptophylla,

Cheilanthes guanchico Bolle, C. pulchella, Cosentinia vellea

(Aiton)

Tod.,

Notholaeno morontae

(L.)

Desv. subsp. subcordata

(Cav.)

G. Kunkel, and

Polypodium

cambricum ssp. mocoronesicum. Where A. ceterach and A.

octoploideum grow together abundantly, we discovered their

sterile

hexaploid

hybrid, A.

Xchasmophilum

Van

den

heede

and

Viane

(Van

den

heede

and

Viane, 2OO2)

DrscussroN

In combination with morphological, cytological, and biogeographical data,

isozyme markers can determine whether taxa are auto- or allopolvploid

(Crawford,

1985; Haufler, 1985b; Bryan

and Soltis,

19B7; Weeden and Wendel,

1989;

Crawford, 1990; Gastony, 1990;

Pryer

and Haufler,

1993). Electrophoretic

analysis of isozymes is an ideal way to investigate the origin of allopolyploid

taxa because

parental

loci are expressed as stable marker bands in the

progeny

(Haufler,

1985b; Werth et al.,19B5b; Gastony, 1986). The

potential

of isozyme

data to clarify

relationships in fern

complexes

dependent upon the degree of

differentiation

among the ancestral genomes.

the

present

study,

four loci

(Aat,

Skdh,

Me,

and

Pgi-2)

showing a unique

set of bands characterizing

aureum and different

from

the banding

patterns

found in European Á. ceterach, proved adequate to disentangle the"Ceterech"

complex

the Canarian

Archipelago.

AII zymograms present in

the Canarian

"small

tetraploid",

were

also

observed in A. ceterach, and

confirm that

true Á.

ceterach

is growing

on the

Canary

Islands.

Moreover, this suggests an occasional spore flow from Europe

towards the Canaries. A flow in the opposite direction is less likely because the

western islands are dominated bv the northeast trade wind svstem.

Conse-

100

AMERICAN FERN

IOURNAL:

VOLUME e4 NUMBER 2

(2004)

quently,

the Canarian Á. ceterach

population

cannot be considered genetically

isolated. No local

zymotypes seem to have originated in this taxon in the

Canary

Islands, contrary to the situation on Cyprus, which is much

older than the

Canary Islands

(Van

den

heede

et al.,

2OO2). For

example, the unique Tpi-2

zymogram,

present in all Á. ceterach and Á. cyprium

specimens

from

Cyprus,

suggests the local origin of the Cypriot taxa

(Van

den heede et al.,2OO2).

All four loci

(Áof

Skdh, Me, and Pgi-2) of the Canarian octoploid show

monomorphic heterozygosity for

a combination of the

patterns

seen in Á.

ceterach

and

Á.

aureum. Our allozyme data confirm the allo-octoploid nature

of this species,

which most probably

originated by chromosome doubling in

a tetraploid hybrid between A. aureum and A. ceteracft

(Viane

and Reichstein,

1992). Theoretically,

though

less parsimoniously,

the formation of this taxon

could

also

happen

directly

via

the union of unreduced

(ax)

gametes

(on

gametophytes resulting from unreduced

spores) of both species.

All allozymes observed in the allo-octoploid were

electrophoretically

identical to

those

found in

the

parental

tetraploids.

However,

in some

octoploid samples from

Gran Canaria and

Tenerrfe, Pgi-2

expressed a zymotype

(corresponding

genotype

aaaadddd) resulting

from

the combination of two

undetected

genotypes

(aaaa)

in Á. ceterach and

(dddd)

in A.

aureum.

The

occurrence of this putative

"orphan"

genotype may reflect incomplete

sampling of the

variation present

in the tetraploids, or alternatively these

genotypes may no longer

present in

extant

ceteraclr and A. aurcum

specimens.

The variation in the allo-octoploid

seems

related to the mono-

polymorphism

(and

its abundance) in the parental tetraploids. Thus,

at the two

Ioci

(Skdft

and

Me)

showing a single octoploid

genotype,

only one

genotype

was observed in each of the Canarian parents. At Aat

two different

genotypes

were found for the Canarian A. ceteracft, though only a single zymotype was

detected for the octoploid. However, the A. ceteracft genotype not detected in

any

octoploid,

was found in

only ca.

1.Oo/o

of the

population.

On the other

hand,

this

may

also be the

result

limited

sampling of the

variation present

the octoploid.

The allo-octoploid species showed three different isozyme profiles at Pgi-2,

a locus that is

polymorphic

in its tetraploid

progenitors,

indicating that the

octoploid

probably

originated at least three times. According to Werlh et aI.

(1985a)

such

patterns

variation in

allopolyploids are almost certainly the

result of repeated allopolyploidizations involving pairs of different genotypes.

Thus,

each of the octoploid

zymotypes may have

arisen

from

a separate

hybridization

event.

The present

observations, demonstrating

multiple

origins

of allopolyploids, are similar to those of Werth et al.

(1985a,

b) for Asplenium,

Soltis et

aI.

(1987)

for Polystichum, and Ranker

al.

(rgag)

for Hemionitis.

Recurrent

origins of

the allo-octoploid

species

implicate

repeated gene

flow

from tetraploids to octoploids, and mean a continued gain of genetic

diversity

by the allopolyploid.

Our electrophoretic data also

provide

evidence for the operation

tetrasomic inheritance in natural populations of autotetraploid

(Rasbach

VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETENACH GROUP

oI.,

'1.987)

ceteroch.

Skdft,

for which

only

two allozymes were observed,

three types of

heterozygotes were present: balanced heterozygotes

(ccdd)

and

two types of unbalanced heterozygotes

(cccd,

cddd).

The presence

these

three types

tetraploid

Á. ceterach at Skdft is suggestive of the three

possible

classes of heterozygotes expected

an autotetraploid at a

locus having

two

alleles

(Weeden

and

Wendel rggg). Unbalanced staining activities indicate

multiple doses of

individual

alleles.

Tetrasomic inheritance with chromatidal

segregation explains

the arrays of homozygous, balanced heterozygous, and

unbalanced

heterozygous

banding

patterns

observed

in Á. ceterach

(Weeden

and

Wendel

tggg). Tetrasomic

inheritance implies that a chromosome

can

pair

with any of its three homologous chromosomes

(e.9.,

Soltis and

Rieseberg,

1986; Weeden

and

Wendel, tg8g;

Crawford,

1990),

and that

there is apparently

no strict

preferential

chromosome

pairing.

Consequently,

the present isozyme

analysis confirms the

autotetraploid status of A. ceterach, which was

cytologically

proven by Rasbach et al.

(1982).

The fact that isozyme studies

point

to tetrasomic

inheritance

and that

we found

only bivalents during

meiosis in autotetraploid

ceterach, suggests that both

processes

are

controlled by different

(sets

ofl

genes.

Similar unbalanced

patterns found in

allotetraploid

ferns

have been explained

also by segregating intralocus

heterozygosity

and

fixed interlocus heterozygosity

(Gastony,

1990).

We were

able to

prove,

isozyme and plastid DNA analysis, that in

addition to A. oureum and the octoploid, true A. ceteracft occurs on Gran

Canaria and Tenerife.

combination of

morphological and cytological

analysis leads to correct determination, but even the exospore

length

alone

allows reliable identification of the three Canarian species:

aureum

(32

1.9

pm),

A. ceteraclr

(gg

-r

2.6

pm),

and

the

octoploid

(qq

3.1

pm).

As mentioned in the introduction, Benl and Kunkel

(rg0z)

published

aureumvar.

paruifolium without cytological investigation. Plants collected in

1.967

T. Reichstein and G. Kunkel were found to be octoploid,

leading T.

Reichstein and other

European pteridologists to attribute octoploid status to Á.

parvifolium

(including

all small Canarian

"Ceterach"

specimens), but

without

having checked the holotype.

repeatedly visited the

type

locality of A. parvifolium, the Pinar above

Vilaflor

(Tenerife),

and found several taxa

(see

Appendix 3)

growing

together.

As soon as we were convinced that two kinds of

"small

Ceterach" species

were

growing

at the locus classicus

(and

on Gran Canaria),

decided

study the

holotype

(BenI

s.n.,

2611.211.966, M)

eureum

var. parvifolium. This

holotype consists of one single

plant.

We studied

its microcharacters

(see

also

Table 4) and found a mean exospore length

(gg

2.8

pm)

and

very few folds

("cuticular

lines") in the

scales

characteristic for true Á. ceterach. The values

for

the exospore and stomate

length

(38

-r

3.7

pm)

prove that the holotype was

not an octoploid, but a tetraploid

plant!

Consequently, C. eureum

var.

parvifolium Benl

and G.Kunkel

and A. parvifolium are synonyms of Á.

ceterach, and the octoploid had no correct name and was described as

Á.

octoploideum

(Van

den

heede and Viane, 2OO2). Asplenium octoploideum is

morphologically intermediate

between

Á.

eureum

and Á. ceterach, from which

101

1.O2

AMERICAN FERN

IOURNAL:

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e4 NUMBER 2

(2004)

Tneln 4. Comparison

of mean exospore length

(LEXO)

various

types to that of cytologically

checked vouchers

(see

Table 2).

Taxon Voucher,

status, and herbarium

LEXO

s.d.

(types)

LEXO

s.d.

(cytol.

checked)

aureum

ceterach

parvifolium

A. octoploideum

Broussonet

s. n., iso-:P

Hort.

Cliff.

Aspl.

4,lecto-: BM

Benl s. n., holo-: M

CV 188. holo-:

GENT

1.9

2.6

3.1

can be distinguished

its

different mean exospore length

(++

pm)

and

mean stomate length

(SZ

pm),

and its

octoploid chromosome number n

:'l.44rr

(Fig.

+B').

It is

endemic to the Canarian Archipelago

but presently

confirmed

only

(cytology)

for

Gran Canaria, La Palma,

and

Tenerife,

and to be expected

Gomera and El Hierro. In

addition to the holotype from

Gran Canaria

[ava

field near

Cueva Corcho, in fissures

volcanic

rocks, 1350 m

alt, 28th M"y

1997, Ieg. Van

den heede

qnd

Viane

CV lSS

(Holo-:

GENT, iso-: personal

herbarium

of Viane

and

Van

den heede)1,

the

following

collections

(paratypes)

were

also made

(for

localities see

Appendix 1, 2,

and 3): CV 171, CV 172,

173,

cv 1.75,

cv 176, cv 177,

179,

cv 672A+8,

cv 674, cv 686, cV 687,

cV 695,

708,

709,

cv 715,

716,

cv 717, cv 718,

719A+8,

cV 720,

72L,

cv 723,

724,

cv 725,

726,

cv 727, cV 729,

730A+8,

CV 731,

732,

733,

cv 734, cv 740,

cv 741, cv 742,

743,

cv 744, cv 745,

746,

747,

cv 748,

749,

cv 750,

751A+8,

cV 752, CV 754A+B+C,WB

22/93.

Many herbarium

specimens

still need to

inspected

before the ranges

the

"small

Canarian

Ceteracft" taxa

can be established. Literature references

and information

on herbarium labels

are often unreliable,

e.g., Bornmtiller

3094

(P),

labeled

as C. officinorum f.

typica

(cellulis

polearum

non striatis!)

collected on Gran

Canaria, has a mean

exospore length of

3.0

and

numerous

folds in the

scale cells: it is without

any doubt an

octoploid.

As far

as is known,

the octoploid is

endemic to the Canary Islands,

but

because Madeira

could also harbor

this species

(climatologically

and

topographically),

we studied

30 specimens from

five Madeiran localities.

However, all

of them turned

out to be hexaploid

and

were

identified

Á.

lolegnamense.

Both in

the Madeiran

and the Canarian Archipelago

the

northeast

trade wind prevails

during the year. This

phenomenon

may help

to explain

the restricted range

of several Macaronesian

taxa,

because most

propagules

fall into the Atlantic

Ocean. Even when

spores

occasionally reach

the African

continent,

the Western

Sahara and Mauritania

offer

appropriate

habitats for

these

ferns,

because

of their ultra

dry climate. The derivation

hypothesize

for the

allo-octoploid species is

presented in Fig.

It is generally

accepted

(e.g.,

Burchard,

'1.929;

Lems,

1960; Page, 1.923;

Bramwell

and Bramwell,

'1,974;

Page,

'1.977,

1,979)

that many

of the Canarian

ferns

are endemic relicts

of the Tertiary

fern flora

that existed in

southern

Europe during

the Miocene and Pliocene.

These ferns

form an important part

of the

original vegetation

of the Canary Islands,

especially

of the evergreen

-f

1.6

2.8

3.4

VAN DEN

HEEDE ET AL.: CANARIAN ASPLENIUM

CETERACH GROUP 103

hybnidization

followed

clromosome doubling

via unreduced

gametes

ceterach

JJJJ

A. aureum

JIXX

A. octoploideum

JJJJJJXX

FIc.

9. Scheme of relationships explaining the

origin of the Canarian Asplenium

octoploideum

based on

(micro)morphological,

cytological and molecular data. Each capital represents

one set of

36 ancestral chromosomes. The

genome

represents

javorkeanum.

Because molecular

studies

(Van

den heede et al., 2003) suggest that Á.

aureum

an allotetraploid, involving A.

javorkeanum

0])

and

"A.

semi-alrreum"

(XX,

unknown) as ancestors, its

genome

formula is

given

JfXX.

forests

(Page,

1,977). Unfortunately,

these habitats, if not

totally destroyed

today, are greatly

endangered by modern tourism

(building,

water supply).

Several

mountain

areas need further research,

and new species and hybrids

await description. Undoubtedly,

this Tertiary

(fern)

flora forms

irreplace-

able

genetic

resource that

should be conserved.

AcruowlnocMENTS

We are very

grateful

Triest

(Free

University, Brussels), M.

Chase

(lodrell

Laboratory, Royal

Botanic

Gardens Kew, UK), and R.

Johns

(K)

for

providing

laboratory facilities, and to the curators

of various herbaria

(BM,

M, P) for permission

to study type material. We

thank R. Cranfill for

providing

trnL-F sequences

some

outgroups; G. Van der Kinderen for his

technical assistance

(PAGE)

and for

cultivating sporophytes; D. Rosseel for his help

with

photography

and for

cultivating sporophytes. We

thank W. Bennert for extra material from Turkey,

and P.S. and D.E.

Soltis

for

discussions and encouragements, T. Ranker

and an anonymous reviewer for

constructive

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of Ghent Botanical

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kindly

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STRFLEU, F. A. and R. S. CowaN.

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and

conservation

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VRN oeN HEEDE, C.

2003.

biosystematic study of

Asplenium subgenus Ceterach

(Aspleniaceae,

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sequencing. PhD dissertation. Ghent University, Ghent,

Belgium.

VaNoeNHEEDE, C.]., S.Palanoru, E.

PaNcun

and

R. L. L. VIaNE. 2OO2. A new species and a new hybrid

Asplenium

(Aspleniaceae)

from Cyprus and evidence of their origin.

Belg.

Bot. 135:

92-'t1.6.

VRN nnN HEEDE,

and

R. L. L. VraNn. 2002. New species and new hybrids in Asplenium subgenus

Ceterach

(Aspleniaceae).

GEP News 9:1,4.

VaN nnN HEEDE, C.

I.,

R. L. L. VtaNr, and M. W. CHnse. 2003.

Phylogenetic

analysis

of Asplenium

subgenus Ceterach

(Pteridophyta:

Aspleniaceae) based on

plastid

and nuclear

ribosomal ITS

DNA

sequences.

Amer.

Bot. 90:481-495.

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1.986.

Taxonomical

Significance

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(Pteridophyta):

Some North American,

Macaronesian

and

European taxa. Pl. Syst. Evol. 1,53:77-1,O5.

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Taxonomia,

Biogeografia y Conservacion de Pteridofitos. IME,

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Mallorca,

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VTANE, R. L. L. and C.

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P.S.

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'1,o7

108

AMERICAN FERN

JOURNAL:

VOLUME

e4 NUMBER 2

(2004)

WERTH,

C. R., S. E. GurruRN,

and W. H. EsHsRucH.

1985b. Electrophoretic

evidence of reticulate

evolution

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Asplenium

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V, 1. G.C.

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WooD,

R. L

'l,gg7.

handbook of

the Yemen

/oro,

Royal

Botanic Gardens,

Kew.

Appnruux

1. Vouchers

from Gran

Canaria, with

corresponding taxa, locality

numbers, and

chromosome

numbers.

the abbreviation

for Caroline Van

heede.

The description

of the

localities

and

their

number

given

Material

and

Methods.

For samples

with chromosomes

counted,

the number

of bivalents is

given.

Counted

specimens served as standards

to determine

the

ploidy

level

(indicated

by 4x and

8x) by flow

cytometry. All specimens

were used for

isozyme

analvsis.

Voucher

number

Taxon Localitv

Date

of collection

Meiotic chromosome

number

(n),

ploidy

CV 157 A.

aureum

CV 158

A. aureum

CV 159

A. aureum

160 A. aureum

CV 161 A.

aureum

CV 162

A. aurcum

CV 163 A.

aureum

164 A. aureum

CV 165

A. ceterach

CV 166 A.

ceterach

CV 167

A. ceterach

CV L68

A. ceteruch

169

A. ceterach

CV 17Oa A.

ceterach

CV 17Ob

A. ceterach

CV 171 A.

octoploideum

CV 172 A.

octoploideum

173 A. octoploideum

175

A. octoploideum

CV 176 A.

octoploideum

CV 177

A. octoploideum

CV 179

A. octoploideum

188

A. octoploideum

CV 180

A. aureum

CV 181

A. aureum

CV 182 A.

aureum

25 May 1997

May

'I.997

25 May

1997

25 May 7997

25 May 1997

25 May

1997

25 May

1997

25 May 1997

May

1997

May 1997

25 May 1997

25 May 1.997

May 1.997

May

1997

26 May

1997

26 May 1997

28 May

1997

26 May 1997

Mav 1997

7ztr

72rr

72II

72rl

72rr

744rr

VAN DEN HEEDE ET AL.: CANARIAN ASPLENIUM CETERACH GROUP

109

Appnuux 2. Vouchers from La Palma, with corresponding taxa, locality numbers, and

chromosome numbers. CV is the abbreviation

for

Caroline

Van den heede. The description of

the localities and their number is

given

in Material and Methods. For samples

with

chromosomes

counted, the number of bivalents is

given.

Counted specimens served as standards

to determine the

ploidy

level

(indicated

by 4x and 8x) by flow cytometry. All specimens

were

used

for isozyme

analvsis.

Voucher

number Taxon Localitv Date of collection

Meiotic chromosome number

(n),

ploidy

708 A. octoploideum

709 A.

octoploideum

711 A.

aureum

CV 712 A. aureum

CV 713 A. aureum

CV 715 A. octoploideum

CV 716 A. octoploideum

CV 717 A. octoploideum

CV, 7L8 A. octoploideum

CV 719A A. octoploideum

CV 7198 A. octoploideum

CV 720 A. octoploideum

CV 721 A. octoploideum

CV 723 A. octoploideum

724 A.

octoploideum

CV 725 A. octoploideum

CV 726 A. octoploideum

CV 727 A. octoploideum

729 A. octoploideum

730A

A. octoploideum

CV 7308 A. octoploideum

CV 731 A. octoploideum

732 A,

octoploideum

CV 733 A. octoploideum

CV 734 A. octoploideum

740 A.

octoploideum

CV 741 A. octoploideum

CV 742 A. octoploideum

CV 743 A. octoploideum

CV 744 A. octoploideum

CV 745 A. octoploideum

746 A. octoploideum

CV 747 A. octoploideum

CV 748 A. octoploideum

CV 749 A.

octoploideum

750 A.

octoploideum

1.1.

'1.1.

1.1.

1.2

1,3

2 Apr. 1999

3 Apr. 1999

Apr. 1999

3 Apr. 1999

4 Apr. 1999

5 Apr. 1999

Apr. 1999

5 Apr.

1999

5 Apr. 1999

Apr. 1999

5 Apr. 1999

Apr. 1999

9 Apr. 1999

9 Apr.

1999

9 Apr. 1999

Apr. L999

'l,44rr

1.44tr

110

AMERICAN FERN

fOURNAL:

VOLUME

NUMBER

(2004)

AppsNInIx 3. Vouchers from Tenerife, with corresponding

taxa,

locality numbers,

and chromosome

numbers. CV, RV,

and

are abbreviations for Caroline Van den heede, Ronald Viane, and

Wilfried Bennert.

The description of the localities and their number is

given

in Material

and

Methods. For

samples with chromosome counted, the number of bivalents is

given.

Counted

specimens

served as standards to determine the

ploidy

level

(indicated

and 8x) by flow

cytometry. All specimens were

used

for isozyme

analysis.

Voucher number Taxon

Locality Date of collection n or

ploidy

613s

cv 183

cv 184

cv 185

186

187a

cv 187b

CV 1B7c

cv 6638

665

cv 666

cv 667

cv 668

cv 669

cv 670

cv 671

cv 672A

CV 6728

cv 674

cv 675

cv 676

cv 677

cv 678

cv 68s

cv 684

cv 686

cv 687

cv 69s

cv 696

cv 701

702A

cv 7028

704

705

cv 706

cv 707

cv 7s1A

CV 7518

CV 752

754A

cv 7s4B

cv 754c

22/9s

A. ceterach

ceterach

A. ceterach

A. ceteruch

A. ceterach

ceterach

A. ceterach

A. aureum

A. auteum

A. aureum

aureum

A. auteum

A. aureum

octoploideum

A. octoploideum

octoploideum

aureum

A. aureum

A. auteum

aureum

A. ceterach

A. octoploideum

octoploideum

A. ceterach

ceterach

A. ceterach

ceterach

A. ceterach

A. aureum

aureum

octoploideum

A. octoploideum

octoploideum

A. octoploideum

1.4

't7

1,7

2't

2',t

7 May 1995

27 May 7997

27 May 1.997

27 IN.4.ay 7997

27 May 1.997

27 May 7997

27 May 1.997

Jan.

1999

fan.

1999

Jan.

1999

fan.

L999

fan.

1999

fan.

1999

)an.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

L999

Jan.

1.999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

fan.

1999

lan.

1999

Jan.

1999

Jan.

1999

Jan.

1.999

Jan.

1999

Jan.

1999

Jan.

1999

10 Apr. 1999

15 Apr. 1993

72TI

72TT

72rr

72IT

72rr

'l.44rr

VAN

DEN

HEEDE

ET AL.:

CANARIAN

ASPLENIUM

CETERACH GROUP

771

Appexox

Alphabetical

list of material for comparison

used in this

study. CV, RV, TR, and WB

are

abbreviations

of C. Van

den heede, R.

Viane,

T. Reichstein,

and W. Bennert. Vouchers

are deposited

GENT and in our personal

herbarium at Ghent

University.

Additional

information

about locali-

ties

is available

from the first and the

last

author

(lienvdheede@hotmail.com;

ronnie.viane@

UGent.be).

Voucher information

about 108 Cypriot

samples

given

in Van

den heede

et al.

(Zooz).

Asplenium ceterach:

CV25b+c

CV25b, CVS0a+b,

CV31

CV36a+b,

CV37

cv78b

cv41,cv4

2b, cv44,cv48,

cv49b

cv64,

cv65, cv66,

cv67,

cV494

cv225

CV275a+b, CV276,

CV277

cv27g,

cv279

cv429,

cv430,

cv431

cv445,

cv448,

cV449

cv450, cv451.

cv657, cv658,

cV659

cv77s

cv774, cv775,

cv776

nv'900

WBlb+e/97

wB10d/s7

WBsc/97

WB12c+d/97

Asplenium

cyprium:

cv21.3

cv24e

Asplenium

dalhousiae

cv718

TR7634

Asplenium

jovorkeanum

cv7, cv4,

cví

CVTa+b+c,

CVSa+b+c

cv86,

cv87, cv88

cv89,

cv90, cv91,

cv92,

cv93

CVgqa+b,

CV95

cv404,

cv405

cv410

cv480

cv483,

cv4B4

cv504, cv506

Asplenium

lolegnamense

CV985

cv993

CV767,

CV768, CV769,

CV770, CV771

Spain,

Alava

Slovenia,

Kal

Croatia, Roó

Croatia, Bassania

Slovenia,

Korte

Italy,

Valle

della Marossa

Italy, Termine

di Roverano

Cyprus, Troodos

Mts., Chandria

Belgium,

Marcourt

Italy,

Cannero

Riviera

Crotia, Losinj

Italy,

Berceto

Italy,

Boio

Spain, Torrelodones

France,

Coulgens

France,

Paulmy

France,

NNE

of Montpellier,

La Pene

Turkey,

Karaoba

Turkey, Manisa

Turkey,

OkEular

Turkey,

Mugla

Cyprus, Troodos

Mts., Tsakistra-Vroiska

road

Cyprus,

Kyrenia Mts.,

Kyrenia-Kythrea

road

Ethiopia,

Harerge

Province, Asbe

Teferi

Pakistan,

Swat

Province, Ambela

Italy,

Stupizza

Slovenia,

1 km E

Baóa

towards Podbrdo

Italy,

Monte

Freddone, 730m

Italy,

Monte Freddone,

1320m

Italy,

Monte Freddone,

970m

Slovenia,

Bovec-Kobarid

road

Slovenia,

Ljubinj

Italy, N slope

of Pania

Secca

Italy,

Fosso di

Antona

Italy, E

slope of Monte

Corchia

Madeira,

SW slope of Pico

Ruivo

Madeira, N

of Serra de Agua

CV2S1a*b,

CV282, CV283,

CV285,

CV286

United

Kingdom, Wales,

Snowdonia

cv10,

cv11,

Cv12,

Cv74, Cv472,

Cv414

Slovenia, Baóa-valley,

KneÍa-Klavze

road

CV 2

0a+ b+ c+ d,

CV2 1 a+b+

c+ d+ e

Slovenia.

Matavun

CV81, CV82a+b,

CV83,

CVB4,

CVsSalb

ltaly, Arni

The discovery of Asplenium lolegnamense and A. ×chasmophilum (Aspleniaceae) on the Canary Islands and evidence of their origin

Article

Full-text available

Jan 2013

A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae)

Article

Jul 2019
CLADISTICS

Halophytic herbs of the Mediterranean basin: An alternative approach to health

Article

Feb 2018
FOOD CHEM TOXICOL

Wild native species are usually grown under severe and stressful conditions, while a special category includes halophytic species that are tolerant to high salinity levels. Native halophytes are valuable sources of bioactive molecules whose content is higher in saline than normal conditions, since the adaptation to salinity mechanisms involve apart from changes in physiological functions the biosynthesis of protectant molecules. These compounds include secondary metabolites with several beneficial health effects which have been known since ancient times and used for medicinal purposes. Recent trends in pharmaceutical industry suggest the use of natural compounds as alternative to synthetic ones, with native herbs being strong candidates for this purpose due to their increased and variable content in health promoting compounds. In this review, an introductory section about the importance of native herbs and halophyte species for traditional and modern medicine will be presented. A list of the most important halophytes of the Mediterranean basin will follow, with special focus on their chemical composition and their reported by clinical and ethnopharmacological studies health effects. The review concludes by suggesting future requirements and perspectives for further exploitation of these valuable species within the context of sustainability and climate change.

Genetic and genomic aspects of hybridization in ferns

Article

Oct 2016

Erin Sigel

The morphological and ecological intermediacy of hybrid taxa has long interested and challenged fern biologists, resulting in numerous systematic contributions focused on disentangling relationships within reticulate species complexes. From a genetic perspective, hybrid ferns are especially interesting because they represent the union of divergent parental genomes in unique evolutionary entities. This review summarizes advances in our knowledge of the genetic and genomic aspects of hybridization in ferns from the mid-20th century to the present. The different organismal products of hybridization, evolutionary aspects of additive and non-additive gene expression in allopolyploids, genetic and genomic mechanisms leading to gene silencing and loss, the roles of multiple origins and introgression for imparting genetic variation to hybrid fern taxa and their progenitors, and the utility of allopolyploid ferns to investigate mechanisms of genome evolution in the homosporous ferns are discussed. Comparisons are made to other plant lineages and important future research directions are highlighted, with the goal of stimulating additional research on hybrid ferns.

Dispersal, diversity and evolution of the Macaronesian cryptogamic floras

Article

Jan 2011

The Macaronesian region comprises the volcanic, oceanic archipelagos of the Cape Verdes, the Canaries, the Savage or Selvagem Islands, Madeira and the Azores, located in the Atlantic Ocean between 15 and 30°N (Fig. 14.1). In keeping with other volcanic oceanic archipelagos, Macaronesia is characterised by high levels of endemicity and recent years have seen the rapid development of novel hypotheses to explain aspects of the evolution of diversity in the region. The availability of checklists for the biota for each of the Macaronesian archipelagos (Izquierdo et al., 2004; Arechavaleta et al., 2005; Borges et al., 2005a, 2008) has led to the development of new hypotheses to explain diversity patterns in the flora in relation to abiotic factors (Emerson & Kolm, 2005; Whittaker et al., 2007, 2008; Borges & Hortal, 2009). Silvertown (2004; see also Silvertown et al., 2005; and for other views, Herben et al., 2005; Saunders & Gibson, 2005) reviewed the results of phylogenetic analyses and suggested that ‘niche pre-emption’ may explain the radiation of some groups but not others. Carine et al. (2010) also used phylogenetic data to demonstrate that angiosperm groups which showed island woodiness (i.e. the evolution of woodiness linked to the colonisation of islands) were typically more species-rich than groups with the same habit on both continent and islands and hypothesised that the evolution of woodiness promoted diversification. Data from checklists together with phylogenetic information led Carine and Schaefer (2010) to suggest that differences in the climatic histories of the Canaries and Azores may explain the marked differences observed in patterns of endemicity in the angiosperm floras of the two archipelagos.

Spatial pattern of multilocus phenotypes within a population of a tetraploid fern, Asplenium ceterach ssp. ceterach

Article

Jun 2011
COMMUNITY ECOL

We studied the intrapopulational genetic structure of a small rock fern subspecies Asplenium ceterach ssp. ceterach living in a fragmented habitat. The level and distribution of genetic variation are primarily affected by the colonization events, the reproductive properties of the species, and the short-distance (leptokurtic) dispersal of spores. These biological processes are at the same time strongly influenced by the geomorphological heterogeneity of the habitat. The individuals' genetic variation was detected as multilocus RAPD profile. The spatially constrained portion of genetic variance within subpopulations was studied by means of variograms, i.e., the average genetic dissimilarity between the pairs of organisms against the separation distance. The range of reduced dissimilarity was 11-13 m. Mantel test proved that subpopulations are isolated by distance. The connected local population patches may create a metapopulation structure. This study also surveyed the adequacy of RAPD products to detect the low level of genetic variation of a polyploid plant population.

Toward a monophyletic Notholaena (Pteridaceae): Resolving patterns of evolutionary convergence in xeric-adapted ferns

Article

Full-text available

Aug 2008
TAXON

Cheilanthoid ferns (Pteridaceae) are a diverse and ecologically important clade, unusual among ferns for their ability to colonize and diversify within xeric habitats. These extreme habitats are thought to drive the extensive evolutionary convergence, and thus morphological homoplasy, that has long thwarted a natural classification of cheilanthoid ferns. Here we present the first multigene phylogeny to focus on taxa traditionally assigned to the large genus Notholaena. New World taxa (Notholaena sensu Tryon) are only distantly related to species occurring in the Old World (Notholaena sensu Pichi Sermolli). The circumscription of Notholaena adopted in recent American floras is shown to be paraphyletic, with species usually assigned to Cheilanthes and Cheiloplecton nested within it. The position of Cheiloplecton is particularly surprising—given its well-developed false indusium and non-farinose blade, it is morphologically anomalous within the “notholaenoids. In addition to clarifying natural relationships, the phylogenetic hypothesis presented here helps to resolve outstanding nomenclatural issues and provides a basis for examining character evolution within this diverse, desert-adapted clade.

Additions to the bibliography for the pteridophyte flora of Macaronesia (2).

Article

Full-text available

Dec 2015

This paper presents a second update, including 62 new entries from the period between 2003 and 2015, to our pteridological bibliography published in Vieraea 25 (1996).

Two novel asplenium hybrids (Aspleniaceae: Pteridophyta) from Tenerife, Canary Islands

Article

Jan 2010

A plant closely resembling Asplenium hemionitis L. but with more dissected, lobed fronds was discovered during a trip to the Anaga mountains, Tenerife, Canary Islands in 2009. This was found to show almost complete spore abortion, indicating a hybrid origin. From the associated species and frond form we suggest the other parent to be A. onopteris L. This represents the first documented hybrid of the rather taxonomically isolated A. hemionitis. The hybrid, A. x tagananaense, is described and its distinguishing features given. A further novel Asplenium hybrid, photographed in 1995 but not subsequently refound, is identified as that between A. onopteris and A. aureum Cav. In the absence of a specimen it is not formally described but its distinctive features are illustrated and its occurrence reported.

Jesters, red queens, boomerangs and surfers: A molecular outlook on the diversity of the Canarian endemic flora

Chapter

Full-text available

Jan 2011

Juli Caujapé-Castells

The Canarian archipelago has been sampling biodiversity from other areas, possibly since shortly after the formation of its oldest islands in the Miocene (Fig. 12.1). The sources, and intensity, of such a prolonged sampling process have varied in different epochs, largely conditioning the origins, makeup and success of the colonising stocks (see Figs 12.1 and 12.2). Because of the bias inherent in any sampling, and the variations in extinction and colonisation rates through time and space, the current Canarian endemic flora is an imperfect representation of the historical floristic links between the archipelago and different geographic enclaves.

A multivariate morphological-anatomical analysis of the perispore in Aspleniaceae (Pteridophyta)

Thesis

Jan 1993

Ronald Viane

The Families and Genera of Vascular Plants. Vol. 1. Pteridophytes and Gymnosperms

Article

May 1991
TAXON

A Handbook of the Yemen Flora

Article

Jan 1999

AUTOPOLYPLOIDY IN TOLMIEA MENZIESII (SAXIFRAGACEAE): GENETIC INSIGHTS FROM ENZYME ELECTROPHORESIS

Article

Feb 1986
AM J BOT

Despite over 30 years of speculation, the genetic consequences of autopolyploid speciation are largely unknown. Evidence from several sources indicates that Tolmiea menziesii is one of the best documented examples of autopolyploidy in natural populations. As such, Tolmiea can serve as a model for providing insights into autopolyploid speciation. Data from enzyme electrophoresis indicate that tetrasomic inheritance operates in tetraploid Tolmiea. These data indicate that a chromosome can pair with any of its three homologous chromosomes. For all polymorphic loci, heterozygosity is substantially higher in tetraploid Tolmiea compared to the diploid cytotype. Furthermore, individual tetraploid plants can maintain as many as three or four alleles at a single locus. Enzyme multiplicity was also observed in tetraploid Tolmiea. For the dimeric enzyme PGI, individual tetraploid plants were identified at Pgi-2 that maintained three alleles, resulting in the production of six different enzymes. Although these genetic consequences of autopolyploidy had been predicted, they have not been previously demonstrated for an autotetraploid in nature. The increased heterozygosity and enzyme multiplicity observed may afford an autopolyploid greater genetic and biochemical versatility relative to its diploid progenitor. These findings suggest that tetrasomic inheritance provides a genetic avenue through which autopolyploid speciation can successfully occur.

ELECTROPHORETIC EVIDENCE FOR THE ORIGIN OF FERN SPECIES BY UNREDUCED SPORES

Article

Nov 1986
Am J Bot

Gerald J. Gastony

Allopolyploid speciation is well documented in the ferns, but data from enzyme electrophoresis have only recently shown that certain sexual and agamosporous taxa are autopolyploids. Autopolyploidy may arise through fertilization involving gametophytes from unreduced spores, a mechanism previously proposed to account for the origin of allopolyploid Asplenium plenum. This report assesses the ability of unreduced spores to function in the origin of polyploid fern species by using enzyme electrophoresis to test their hypothesized role in the origin of A. plenum. Six isozymes of the enzymes PGI, PGM, TPI, 6PGD, and LAP are species-specific for the taxa proposed as parental under two competing hypotheses for the origin of this species. Electrophoretic data reject the more conventional hypothesis involving simple hybridization and agree perfectly with expectations under the more complex hypothesized origin via unreduced spores. The mechanism whereby unreduced spores have functioned in this case is no different from that by which they would function in the origin of autopolyploid taxa and may be more common in the origin of fern species than previously suspected.

GENETIC EVIDENCE FOR DIPLOIDY IN EQUISETUM

Article

Jun 1986
Am J Bot

Douglas E Soltis

Sporophytes and gametophytes of Equisetum arvense, E. laevigatum, and E. telmateia were analyzed using enzyme electrophoresis to estimate isozyme number. Despite their uniformly high chromosome numbers (2n = 216), these three species exhibited isozyme numbers typical of diploid seed plants for the enzymes AAT, ADH, ALD, GDH, [NADP]IDH, LAP, MDH, [NADP]ME, PGI, PGM, SkDH, and 6PGDH. All three species exhibited an additional isozyme for TPI. There is, therefore, no genetic evidence for low base numbers such as x = 9 and x = 12 suggested for Equisetum. Intact chloroplasts were isolated from E. arvense and the chloroplast extract compared electrophoretically to whole plant extracts. The single enzymes observed for LAP, GDH, [NADP]IDH, and [NADP]ME were absent from the chloroplast extract. Isozymes AAT-1, ALD-2, MDH-3, PGI-1, PGM-2, SkDH-2, 6PGDH-2, TPI-2, and TPI-3 were active in the chloroplast fraction; 6PGDH-1, PGI-2, PGM-1, and TPI-1 were lacking from the chloroplast fraction and were considered cytosolic. Isozymes AAT-2, MDH-1, MDH-2, MDH-4, and SkDH-1 were also lacking from the chloroplast fraction but because AAT, MDH, and SkDH have been reported from several subcellular compartments, their localization is unknown. These findings indicate that isozymes in Equisetum species are subcellularly compartmentalized as has also been demonstrated for homosporous ferns, gymnosperms, and angiosperms.

A new Asplenium (Sectio Ceterach) species and the problem of the origin of Phyllitis hybrida (Milde) C. Christ

Article

Jan 1963

G. Vida

New ferns from Madeira

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Modes and Mechanisms of Speciation in Pteridophytes

Chapter

Jan 1997

Christopher H. Haufler

Curiosity about the origin and maintenance of organic diversity is one of the forces that has propelled the science of biology. From direct observation of the remarkable variety that surrounds and supports us as one of the millions of species on Earth to experiments designed to reproduce the circumstances that generate this variation, natural historians, population biologists, and systematists are driven to discover how and why there are so many species.

Les îles du Cap Vert. Flore de l'Archipel

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A. Chevalier

Asplenium ceterach and A. octoploideum on the Canary Islands (Aspleniaceae, Pteridophyta)

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