Table Of Content95(1):1-21
•nal (2005)
Biogeography and
Insights into the Polyploid
New
Evolution of Zealand Asplenium from
DNA
Chloroplast Sequence Data
Leon 1
R. Perrie
BOTANICAL
MISSOURI
Allan Wilson Centre for Molecular Ecology & Evolution.
Molecular Massey
Institute of BioSciences, University,
New
Private Bag 11 222, Palmerston North, Zealand
MAY
2 3 2005
Brownsey
Patrick
J.
Museum New
of Zealand Te Papa Tongarewa,
GARDEN
New
Box
P.O. 467, Wellington. Zealand LIBRARY
sequences
b of the chloroplast trnL-trnF intergenic spacer
21 of the 22 indigenous Asplenium taxa presently recognized
f
sequences of the chloroplast rbcL gene were also obtained from
<
representative of the diversity found in the trnL-trnF intergenic spacer. Phylogenetic analyses of
New
these chloroplast sequence data indicate that the Asplenium species of Zealand are not
monophyletic. More Asplenium New
specifically, the species ipating hybridization
parti< in in
Zealand form
a closely related 'Austral' group, whereas the non-hybridizing species have closer
sub-groups are recognized, represented by the species A. bulbiferum, A. flaccidum, and A.
New
and many
lineages in Zealand, the distributions of the Asplenium species disjunct between
New
Zealand and elsewhere appear best explained by long-distance dispersal. The likely
Asplenium, with approximately 700 species worldwide, appears have
to a
complex and and
history of hybridization auto- allopolyploidy, and model
a
is
group for the study of fern evolution (e.g., Manton, 1950; Wagner, 1954; Lovis,
DNA
1977; Reichstein, 1981). Recent sequencing studies Murakami
(e.g., etal.,
1999a; Yatebe et al., 2001; Gastony and Johnson, 2001; Pinter et al, 2002; Van
den heede have shed on
et al, 2002, 2003) light the evolutionary history of
this
DNA
we
iconic group of ferns. Here provide chloroplast sequences for 21 of the
New
22 indigenous Asplenium taxa in Zealand (Table Brownsey and Smith-
1;
Dodsworth, Brownsey, where genus
2000; 2003), the often ecologically con-
is
10%
spicuous and comprises about of the indigenous fern
flora.
New
taxonomic Zealand Asplenium
Early studies of Hooker,
1855;
(e.g.,
Martin, 1920) indicated hybridization was rife and found the different 'kinds' to
New
grade together. In his account of Zealand Asplenium, Allan
(1961,
75)
p.
remarked that the species were "very ill-defined", and that while many species
appeared respond "markedly environmental
to to conditions there also
...[,] is
Museum New
1 Current address: of Zealand Te Papa Tongarewa, P.O.
Asplenium, togothei '.nth additional spin ies mc slud\ I'lobh levels obtained from Love
Iini.-i in
I ,
Dawson
ll'i'iob). rt ul. (200(1). I'inter ft (2002). Timid.- .m.i Ko\ 120021. ami Van den heede
til.
e
Mteinathr
ikon from Ogle (1087) Distribution details urn. ompiled from various sour, ami. ploidv
like lev.
!S
< ,
New
Sample
ns. localities are only provided for Zealand taxa. ol which duplicate samples are
i,
is. Herbarium vouchers are given loi samples from whii oovel sequence data was generated for this study. Sequent SB ol
Ii
fenced by the study that generated them. GenBank aci ession numbers are given for the tniL-trnF intergeni< spacer an
led in the analyses.
Indigenous
distribution:
trni,
trill-
New
iippriuli, uhitlliil (l.abill C.l'resl Australia, Zealand:
\.
)
subsp. iippniuliriiliiiiiin trrrrstn
|/1.
WELT
ulatum subsp.
I'204'l.'i
(Brownsev) Brownsov
i
Brownsey]
HVKLT
A. hiilhifiTiiin foist subsp. P20494
< I.
bulbi/mim
bulbiftirum [A.
A. biilbifunini subsp. i>rticilliniuni
Brownsey
(Colenso) ^mcillimu,
[A.
Colenso)
WITT
l'204'»!
WELT
Brownsey P20499
i e
WELT
llnlllbh.liumCM.
P20500
1
A
WELT
P20501
"WELT
l'2tr,02
WITT
l'20'.O
:
A A
WELT
.1 lionkrrinuuin CiiIimiso \nsii,,li,i. \,.n /.r.iland: Vunu-virki!. P20504 AY283212 *AY283229
I
B WELT
"Banks Peninsula P20505 "AY283213
hnnjnnphvUiin, Cilsr Sew .u.dnmic: Auckland WELT P20506 AY283214 AY283230
.1. /..•al.ind
Mow
Zealand endfMiiic: VVEI.T ('20507 AY283215
Ctistlitpciiiit
A A
Mew VWELT B
Zealand endemic:: Paihiii. P20508 *-*AY283218 AY28323
"WELT
North P20509
'T.ilnuT.stdii
A
WELT A
Zealand. South America: P20510 VJ,AY283217 "AY28323
\'rv\
A B
"WHIT
Haast P20511
Iiluli.
WELTP20512
\ustmliii. NewZeiiliiiid. AY283218
WELTP20515
AY283221
WELT
P20516 AY283222
WELTP20517
AY283223
Indigenous
dis
Niiu
/.ealiind
New
WELTP20519
Austra Guniea, AY283225 AY283237
lia,
Pacific Island
Vogel unpublished AF240658 AF240643
Hook,
siforme Grev. Murakami
et 1999a AB014709
et
al.,
Pinter 2002 AF240663 AF240648
et
al.,
\rinum Europe
L. AF240647
AF240(i(i2
Yatabe 2001
et
al., AH023r>()(l
Listed as A. cordatum AF525235 AF240050
group with sequences
A. phillipsianum
by Van den
Asia Murakami
AB014B91
et
al., i«)'in,i
sagittatum Bange
(DC.) Europe
Asia, Vogel unpublished/ AF240661 AF240646
.
Hymenasplenium
unilaterale (Lam.) Hayata
lined from analysis of 190 hybrid spe
Brownsey and more WELT.
(1977b) recent collections in Subspccii'ic taxa arc not
no doubt that hybridism plays an important part". However, Brownsey (1977a,
New
1977b) concluded that while hybridization amongst the species of
Zealand Asplenium was indeed relatively common, the spores of the hybrid
were and swarms were
plants aborted introgressant
virtually absent.
Brownsey (1977b) also noted that hybridization was unequally distributed,
some and
with taxa participating frequently others not Although geo-
at
all.
graphy and ecology influence the frequency of hybridisation, some taxa not
are
New
known
form hybrids Zealand despite frequent sympatric occurrence
to in
Asplenium
with congenerics. For instance, polyodon, despite frequent sym-
known
patry, not to hybridize with A. bulbiferum subsp. bulbiferum,
is
A. bulbiferum subsp. gracillimum, A. flaccidum subsp. flaccidum, or A.
oblongifolium. Similarly, A. trichomanes, despite frequent sympatry, not
is
known
hybridize with A. bulbiferum subsp. gracillimum, A. hookerianum,
to
or A.
lyallif.
A
shows and
crossing diagram the extent frequency of hybridization
(Fig.
1)
New
amongst Zealand Asplenium taxa. Brownsey (1977b) suggested that the
New
Asplenium taxa involved in hybridization in Zealand were more closely
related to one another, and constituted an 'Austral' group of Asplenium. Spe-
cies from this Austral group, comprising A. appendiculatum, A. bulbiferum,
6 AMERICAN FERN VOLUME NUMBER
JOURNAL:
95
(2005)
1
A. chathamense,
A. cimmeriorum,
A. flaccidum, A. hookerianum, lamp-
A.
rophyllum,
A. A.
lyallii, oblongifolium, A. obtusatum, A.
richardii, A.
scleroprium and A. shuttleworthianum, common New
are generally
in Zea-
and some
land,
of these species
or their close relatives also occur in southern
Australia, the southern and South
Pacific, America. There no
are diploid
New
Asplenium
species in Zealand (Dawson
et al, 2000), but amongst the
New
tetraploid Zealand
representatives of the Austral Asplenium
group,
Brownsey
(1977a, p.84) suggested the presence of "three natural groups of
closely related species
(1) the A. flaccidum aggregate with thick and rather
. . . :
leathery bipinnate fronds, the A. hookerianum complex
bulbiferum with
(2)
I
thin highly dissected and
fronds, the A. luciduml obtusatum complex
with
(3)
thick fleshy pinnate fronds" [Asplenium lucidum non N.L.Burman
G.Forst.
is
an name
but
earlier, illegitimate, for A. oblongifolium Colenso; Brownsey,
1979).
Brownsey
(1977b) considered Asplenium New
the
species not hybridizing
in
Zealand
have
to closer with groups
affinities other than the Austral group.
For
instance, A. polyodon widespread
is in the palaeo-tropics and has mor-
phological
similarities to species such A. cuneatum and
as A. aethiopicum.
Asplenium
trichomanes
widespread
is in the northern hemisphere. In Eu-
rope, diploid, tetraploid and hexaploid forms of this species are known, and
there good and
is cytological morphological
evidence
that these hybridize
naturally with
at least 13 other distinct species (Reichstein,
1981), in contrast
New
to the total absence of hybridization in Zealand. Asplenium folium
flabelli
also appears have
to northern hemisphere showing some morpho-
affinities,
logical resemblance The
A.
to viride. relationship of A. pauperequitum
less
is
Brownsey
and
clear. Jackson
(1984) suggested based on morphology,
that,
this
endemic
species was not closely any New
related to of the other Zealand
may
species, although have
it distant affinities with A. polyodon.
This mixture
of postulated
affinities suggests that the extant Asplenium taxa
New
in Zealand do common New
not share a Zealand and
ancestor that they
are not monophyletic.
Rather,
it suggests they are derived from several non-
New
Zealand
ancestors, implicating multiple, Asplenium
distinct origins of
in
New
New
Zealand.
multiple
If origins for Zealand Asplenium are indeed the
case, then of considerable when
biogeographic
interest these lineages
is ar-
Are New
rived. the
separate lineages Zealand Asplenium
of perhaps
old, in
accord with New
the separation of Zealand from the remainder Gondwana
of
about
80 million
years ago (McLoughlin, and
2001), therefore consistent with
a vicariant explanation? do
Or, they appear younger than 80
m.y.a., neces-
sitating a long-distance
dispersal explanation
for their origins?
New
Amongst
the Zealand members
of the Austral Asplenium group
there
are eight and
tetraploid nine octoploid taxa. Five of these octoploids are en-
New
demic
to Zealand, and from morphological and
ecological evidence they
have been
hypothesized have
to arisen via auto- or allopolyploidy (Brownsey,
1977b; Brownsey &
de
Lange,
1997).
To
conduct New
a phylogenetic
analysis of the Zealand Asplenium and
to
whether
test
their relationships indeed
are
reflected in hybridization frequency/
we DNA
ability, obtained sequence data from two While
chloroplast markers.
NEW
PERRIE BROWNSEY: ZEALAND
& ASPLENIUM CHLOROPLAST
SEQUENCE
7
may
the
chloroplast only be
inherited maternally, as has been reported by Vogel
European we
et al. (1998) for A. trichomanes,
anticipated
that a chloroplast
New
phylogeny
of Zealand Asplenium would
nevertheless provide important
insights into their origins, in terms of both the number
of origins and,
via
We
molecular
a clock approach,
their timing. hoped
also to identify the
chloroplast, or maternal, parent each
for of the Austral
octoploids.
The
trnL-trnF intergenic spacer was sequenced New
for every Zealand
Asplenium
taxon
presently recognized
(Table except A. trichomanes
subsp.
1),
quadrivalens which we know New
no
for of extant population
in Zealand.
We
did not include in our study Pleurosorus
rutifolius (R.Br.) Fee, another
member
New
of the Aspleniaceae indigenous
to Zealand, but phylogenetic
its
position
currently being
is investigated elsewhere (Johannes
Vogel,
pers.
comm.). Not
only does
the trnL-trnF
intergenic spacer amplify and
easily
consistently across a wide range of plants because
but, non-coding,
is
it
it
evolves and
relatively accumulates both
fast, base-pair and
substitutions
may
insertion-deletion
events be
('indel') that phylogenetically
informative.
It
one
is therefore of the best sequence markers
available detecting
for genetic
New
amongst
differences the Zealand Asplenium
taxa.
The was New
rbcL gene sequenced
for representative Zealand Asplenium
Although may
taxa. the rbcL gene evolve slower than
the trnL-trnF intergenic
spacer, does not accumulate indel which means
it events, the alignment
of
se-
quences from
different taxa
is easier. In addition, the large existing database
of
rbcL sequences Asplenium
for taxa from around the world allows placement
of
New
representative Zealand
taxa into a global framework.
Materials and Methods
Table details for each of the indigenous Asplenium
1 taxa currently
rec-
New
ognized from Zealand (Brownsey and
Smith-Dods
worth, 2000; Brownsey,
taxonomy
2003) the adopted
for this study, the natural
distribution, ploidy
and We
level, the location of the sample(s) analyzed. note some
that for of
these taxa there is debate as to whether they should be recognized
the
at
subspecifc (Brownsey,
1977a) or specific (Ogle, 1987) and provide
level, the
taxonomy where
alternative Herbarium
appropriate.
abbreviations
follow
Holmgren
et al. (1990).
DNA
was
from CTAB
extracted
silica-gel dried tissue using modified
a
pro-
PCR
tocol (Doyle & Doyle, 1990). was performed in 20 ^L volumes containing
mM
Q lOmM
IX pH
solution (Qiagen), Tris-HCl 50 1.5mM MgCl
8.8, KC1, 250
2
,
umol dNTPs, U DNA
pmol
10 of each primer, of Taq polymerase
1 (Qiagen),
and
approximately 50 ng DNA.
of template
The
complete sequence
for the trnL-trnF intergenic spacer was
obtained
from every Asplenium New
taxon indigenous Zealand Asplenium
to (except
trichomanes
subsp. quadrivalens; Table using the primers E and F
1) of
Taberlet and
et al. (1991) a thermocycling profile beginning with an
initial de-
naturation of 94°C for 2 minutes, followed by 38 cycles of 94°C minute,
for 1
58°C minute, and
for 72°C minute, with
1 for 1 a final extension of 72°C
for
VOLUME NUMBER
AMERICAN FERN JOURNAL:
95 (2005)
8 1
minutes. Duplicate samples of the geographically-widespread tetraploid
5
New
Zealand (A bulbiferum subsp. bulbiferum, A. hookerianum, A.
taxa in
flaccidum subsp. flaccidum, A. oblongifolium, and A. obtusatum subsp.
obtusatum), which are the most likely to be genetically variable, were analyzed
marker. Duplicate samples of the wide-
assess infra-taxon variation in this
to
were
gracillimum analyzed. Local-
spread octoploid A. bulbiferum subsp. also
GenBank numbers
herbarium voucher number, and accession are
ity details,
was
given Table Sequence the trnL-trnF intergenic spacer also ob-
in for
1.
tained from a cultivated plant of A. australasicum (J.Sm.) Hook. (Table
1).
New
Sequences for the rbcL gene were obtained for representative Zealand
New
species from each of the three Austral groups, each of the non- Austral
Asplenium
Zealand species, and a cultivated plant of australasicum (Table 1).
The external primers aF and cR of Hasebe er (1994) were used with a
al.
thermocycling beginning with an denaturation of 94°C for
profile initial
and
2 minutes, followed by 38 cycles of 94°C for 1 minute, 58°C for 1 minute,
The
72°C for minute, with a final extension of 72°C for 5 minutes. novel
1
(5'-AAGCCAAAATTAGGTCTATCTGC-3')
rbcLAsForward2
primers
internal
(5'-CCCAATTCTCTCGCAAAAACAG-3')
and rbcLAsReverse2 were used
where necessary obtain single amplification products and/or complete bi-
to
directional sequencing.
PCR CONCERT PCR
were Rapid
products purified using the Purification
PCR
System (Gibco BRL). The purified products were sequenced in both
A DNA
directions using an Applied Biosystems 73 Sequencing System and
3
PRISM™
the ABI Dye Terminate Cycle Sequencing Ready Reaction Kit
(Perkin Elmer).
GenBank
Additional trnL-trnF and rbcL sequences were obtained from
species representative of the genetic diversity previously reported in the
for
Aspleniaceae (Murakami et al, 1999a; Yatebe et al, 2001; Gastony and
Van den heede
Johnson, 2001; Pinter 2002; al, 2003). Details are
et et
al.,
provided in Table Only rbcL sequence was available for some of the selected
1.
and combined
species, preventing their inclusion in the trnL-trnF alone
analyses below).
(see
we
Murakami Hymenasple-
In this study follow (1995) in the recognition of
nium Hayata as a separate genus sister to Asplenium, and use H. unilateral,
for which both trnL-trnF and rbcL sequences are available from GenBank, as
and
the outgroup. However, the of outgroup choice (Adachi
to test effect
Hasegawa, 1995), the rbcL analyses were repeated with H. unilaterale replaced
(GenBank
with either H. hondoense (N. Murak. et Hatanaka) Nakaike
ABO14705)
or H. riparium (Liebm.) N. Murak. (ABO14708). Previous studies
Murakami Van
den heede these spe-
1999a; 2003) indicate
(e.g., et al., et al.,
known Hymenasplenium.
cies are representative of the diversity within
(Thompson
The
sequences for each marker were aligned with ClustalX 1.8
was
The alignment amongst sequences
et 1997). of indels the trnL-trnF
al.,
One
edited further with Se-Al vl.O (Rambaut, 1995). central region of the trnL-
{New
trnF intergenic spacer could not be unambiguously aligned between the
Zealand Asplenium + and remaining
Austral species A. australasicum} the
NEW
PERRIE & BROWNSEY: ZEALAND ASPLENIUM CHLOROPLAST SEQUENCE
9
was
species. excluded from analyses except those encompassing only
It all the
New
Zealand Austral species and A. australasicum. Analyses
of the trnL-trnF
sequences were performed with encompassing
sites indels either completely
excluded, or included and treated as missing data. In addition, analyses were
performed with marinum,
also the large indels of A. A. aethiopicum, A.
MATHF
trichomanes, and
A. hemionitis, the A. flaccidum group
(the indels)
and
included
treated as missing data, but with other indels excluded.
all
PAUP*
was
4.bl0 (Swofford, 2002) used perform
to heuristic search analyses
maximum
under parsimony
(MP; with
tree bisection-reconnection branch-
swapping, and 100 replicates of random sequence and maximum
addition),
likelihood (ML; with tree bisection-reconnection branch-swapping, and
ran-
dom ML
sequence addition). Appropriate models of evolution were
for
se-
lected using Modeltest v3.06 (Posada and Crandall, 1998) with the hierarchical
likelihood-ratio Analyses were conducted on and
test. the trnL-trnF rbcL data
and with them combined
sets separately, into single data
a set after testing for
incongruence using homogeneity implemented PAUP*
a partition test as in
was
4.bl0. Bootstrapping performed to assess confidence in the groups iden-
maximum =
maximum =
by parsimony
tified (n 1000) or likelihood
(n 100;
with the parameters estimated by Modeltest
fixed).
The
timing of divergences within Asplenium were estimated with
the pro-
gram r8s v.1.60 (Sanderson, 2002), which offers methods with the advantage
of allowing rates of evolution to vary throughout the and has been applied
tree, to
other pteridophyte groups Lycopodiaceae, Wikstrom and
Kenrick, 2001;
(e.g.,
Equisetum, Des Marais The used was
et al., 2003). tree for the r8s analysis that
maximum
estimated by likelihood. The r8s analysis was implemented under
penalised likelihood, using the Powell algorithm with
a log-scale pen-
alty function, and a smoothing value of 125 as determined by cross-validation.
Following the recommendation Sanderson more
of (2002), distant outgroups
GenBank
{Polystichum vestitum AF208395;
(G. Forst.) C. Presl, Thelypteris
acuminata Morton, D43919; Grammitis AF468198)
(Houtt.) tenella Kaulf.,
were used branch Hymenasplenium
to estimate the length of unilaterale
before being discarded prior to the r8s analysis. Confidence intervals (mean ±
standard deviation) were calculated through a bootstrapping procedure with 100
replicates (Sanderson and Doyle, 2001). The analysis was calibrated temporally
by common
assigning an age of 140 m.y. (million years) the most recent
to
ancestor of what appears to be the most distantly related extant components of
the Aspleniaceae, Hymenasplenium and Asplenium. This date follows Skog
who
(2001) reported Aspleniaceae from the early Cretaceous.
fossils
Only two
instances of trnL-trnF sequence variation within a taxon were
found. The two samples of Asplenium bulbiferum subsp. gracillimum
differ
at position 50. The two samples of A. hookerianum also vary position
at 50,
as well as at two other positions invariant in the other taxa investigated.
AMERICAN FERN VOLUME NUMBER
10 JOURNAL:
95
(2005)
1
Because and
of insertions deletions, the length of the trnL-trnF sequences
ranged between 238 and 398 base pairs. The trnL-trnF alignment, containing
indels, comprised 393 positions for the Austral group plus Asplenium
and
australasicum, 428 positions for all taxa with the central region that
Maximum
could not be unambiguously aligned excluded. parsimony
analysis
of all trnL-trnF sequences strongly supported recognition of an Austral clade,
with A. australasicum recovered as its sister (not shown). As depicted in Fig.
2,
MP
analysis of the trnL-trnF sequences of the Austral taxa, together with A.
australasicum as an outgroup, indicated three strongly supported sub-clades
within more
the Austral clade, or less corresponding the groups suggested
to
by Brownsey
(1977a; see above).
The 98%
Flaccidum clade, with bootstrap support includes Asplenium
(BS),
appendiculatum
subsp. appendiculatum, A. appendiculatum
subsp. mariti-
mum,
A. chathamense, A. flaccidum subsp. flaccidum, A. flaccidum subsp.
haurakiense, A. lamprophyllum, and A. shuttleworthianum. The Bulbiferum
98%
with
clade, BS, includes A. bulbiferum subsp. bulbiferum, A. bulbiferum
subsp. gracillimum, A. cimmeriorum, A. hookerianum, and The
A.
richardii.
Obtusatum 100%
with
clade, BS, includes A. A. oblongifolium, A.
lyallii,
obtusatum
subsp. northlandicum, A. obtusatum subsp. obtusatum, and A.
The
scleroprium. Bulbiferum and Flaccidum
clades supported
are as sister
85%
clades with BS. The inclusion/exclusion had minimal
of indel
sites
ef-
on
fect these principal
results.
Incorporation
of representatives from each
of the three Austral sub-clades
in analyses of rbcL sequences with a broader sample including species
set,
which
for trnL-trnF sequences were not supported
available, also recognition
The
of the Austral clade. rbcL sequences of A. oblongifolium and A. obtusatum
were
identical, so only the former was included The
in the analyses. alignment
of the rbcL sequences, with no
inference of indels necessary, comprised 1191
=
Using the model by + +
selected Modeltest (TrN
base frequency
G;
I
=
[0.2698, 0.2215, 0.2461, 0.2626]; rate matrix 6.1972, 11.4499];
[1, 1, 1,
= Gamma = ML
Invariable proportion
sites 0.5235; shape parameter
0.8450),
analysis selected a single tree with a -In likelihood score of 4426.3070. This
is
presented where
in Fig. bootstrap support
3, is also indicated. Eight trees of
=
= -
487 RC
steps (CI 0.624, RI 0.689, 0.430) are recovered under maximum
parsimony. The
consensus
of these equally most parsimonious
trees (not
shown)
maximum
is similar to the set of relationships depicted by the
like-
lihood
tree, except that the position of Asplenium antiquum within
the
Greater-nidus group
is unresolved, A. hemionitis and A. phillipsianum form
a clade that is sister to the remainder of the Marinum group, and A.
wrightii
is
part of the basal polytomy rather than sister to the remainder of Asplenium.
Four
major, monophyletic groups within Asplenium
this set of are evident,
which
communication we
for purposes here label as the Aethiopicum,
Marinum,
Ceterach-Phyllitis, and
Greater-nidus groups Asplenium
(Fig.
3).
wrightii does not appear to be closely related to any of these groups, and may
it
represent group
a fifth within Asplenium. Support for these four major groups
