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From European Priority Species to Invasive Weed: Marsilea azorica (Marsileaceae)
is a Misidentified Alien
Author(s) :Hanno Schaefer, Mark A. Carine, and Fred J. Rumsey
Source: Systematic Botany, 36(4):845-853. 2011.
Published By: The American Society of Plant Taxonomists
URL: http://www.bioone.org/doi/full/10.1600/036364411X604868
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Systematic Botany (2011), 36(4): pp. 845–853
© Copyright 2011 by the American Society of Plant Taxonomists
DOI 10.1600/036364411X604868
845
The clover ferns or waterclovers, Marsilea L., are a genus
of 45–50 aquatic, heterosporous fern species ( Nagalingum
et al. 2007 ). Together with the pillworts, Pilularia L. (five spe-
cies), and the monotypic Regnellidium Lindm., they are placed
in the family Marsileaceae, which is the sister group to the
Salviniaceae ( Johnson 1986 ; Kubitzki 1990 ; Nagalingum et al.
2007 ; Nagalingum et al. 2008 ). Waterclovers are most diverse
in tropical and subtropical Africa and America, where they
grow in seasonal and permanent ponds, and in shore regions
of larger fresh water bodies ( Launert 1968 ; Johnson 1986 ).
Marsilea species are easily recognized by their clover-like
leaves, which are composed of four leaflets on a long peti-
ole. Most species show extreme morphological plasticity in
their vegetative organs and sterile material is often impos-
sible to identify based on morphology alone ( Launert 1968 ).
The current classification is instead based on morphology
of the sporocarps, long-lived, drought-resistant reproduc-
tive structures produced near or at the petiole base ( Launert
1968 ; Johnson 1986 ; Kubitzki 1990 ). The sporocarps are the
primary dispersal and dormancy units in Marsileaceae and
release their spores only in water, where they germinate rap-
idly ( Schneider and Pryer 2002 ). Even though these sporo-
carps suggest Marsileaceae is well adapted to long-distance
dispersal by waterfowl, a population-level study in the
Mediterranean region found extremely reduced gene flow
between distant or close populations of the clover fern M. stri-
gosa Willd., pointing to a low dispersal ability, at least in that
species ( Vitalis et al. 2002 ).
In the middle-Atlantic islands, Marsilea has been recorded
from the Cape Verde archipelago, the Canary Islands, and
the Azores. In the Cape Verdes, the African species Marsilea
coromandelina Willd. is listed as native on Branco island
( Arechavaleta et al. 2005 ). In the Canarian archipelago, the
genus was known from a single locality at Arucas on Gran
Canaria. Initially identified as M. quadrifolia ( Webb and
Berthelot 1840 ), the Arucas species was identified by most
later authors to be M. diffusa A. Br. (= M. minuta L.; Milde
1867 ; Christ 1888 ; Burchard 1929 ; Kunkel 1971 ), with Braun
(1870) regarding it as a distinct form (f. canariensis ). However,
Hansen (1972) considered it to be M. quadrifolia and some
doubt therefore exists over the identity of the Canarian plant.
The plant was last collected from a wetland area at Arucas in
1897 ( O. Gelert s. n. , Arucas, 22 April 1897, (C) [sterile speci-
men]). More recently, the genus has been considered an intro-
duction to the archipelago ( Izquierdo et al. 2004 ), although
evidence for this is unclear. Complete loss of the wetland area
on Gran Canaria, as already noted by Burchard (1929) , means
it is likely that the genus is now extinct in the Canaries.
In contrast to the situation in the Canaries, Marsilea was
not mentioned in any of the 19th century plant invento-
ries for the Azores ( Seubert and Hochstetter 1843 ; Seubert
1844 ; Drouet 1866 ; Watson 1870 ; Trelease 1897 ; Gandoger
1899 ). Clover ferns are also missing in all Azores plant lists
based on fieldwork from the first 70 yr of the 20th century
( Christ 1906 ; Druce 1911 ; Gonçalves da Cunha and Sobrinho
1938 ; Palhinha 1943 ; Palhinha et al. 1946 ; Tardieu-Blot 1946 ;
Dansereau 1961 ; Palhinha 1966 ; Vasconcellos de Carvalho
1968 ; Sjögren 1973). The first collection from the Azores was
made on Terceira Island in the central group of the archipel-
ago by I. Botelho Gonçalves, an inspector of the local forest
department ( Gonçalves 3259 , 12.7.1971 (BM)). It was identified
as M. quadrifolia , and reported as such in Atlas Flora Europaea
( Jalas and Suominen 1972 ). At the same time, the plant was
independently discovered and the find published by Hansen
(1972 , 1973 ), who found the plants “abundant in mud-bottom
of a small temporary lake or pond near Pico de Bagacina
(about 600 m),” which remains the only known Marsilea
population in the Azores. Hansen identified the plants as
M. strigosa Willd. and, commenting on the record in Jalas and
Suominen (1972) , said “[it] may very well be the same plant
and the same locality, yet its identity as M. strigosa is quite
clear!” In 1983, based on examination of an herbarium speci-
men in Copenhagen ( Hansen 308 , 4 July 1972 (C)), E. Launert
concluded that the Azorean material belonged to an unde-
scribed species closely related to the western European
M. strigosa, but differing in sporocarp morphology. Conse-
quently, it was described as Marsilea azorica Launert & Paiva
( Launert and Paiva 1983 ; our Fig. 1 ).
Even though it is restricted to a single, small, roadside pond
in a heavily grazed pasture, the Azorean Marsilea population
From European Priority Species to Invasive Weed: Marsilea azorica (Marsileaceae) is
a Misidentified Alien
Hanno Schaefer , 1,2, 4 Mark A. Carine , 3 and Fred J. Rumsey 3
1 Department of Organismic and Evolutionary Biology, Harvard University, 22 Divinity Avenue,
Cambridge, Massachusetts 02138, U. S. A.
2 CITA-A, Azorean Biodiversity Group, University of the Azores, Terra Chã, 9700-851 Angra do Heroísmo, Portugal.
3 Department of Botany, The Natural History Museum, Cromwell Road, London, SW7 5BD, U. K.
4 Author for correspondence (hschaef@fas.harvard.edu)
Communicating Editor: Allan J. Bornstein
Abstract— The clover fern Marsilea azorica was described in 1983 from the isolated Azores archipelago in the northern Atlantic, where it is
restricted to a single roadside pond. Thought to be an extremely local endemic, it was subsequently listed as a conservation priority species for
the Azores, Macaronesia, and Europe, included as ‘critically endangered’ on the IUCN red list, and as ‘strictly protected’ species by the Bern
convention and the European Union’s habitats directive. However, we present morphological and molecular data ( rbcL gene, rps4 gene, rps4-
trnS spacer and trnL-trnF spacer sequences), which demonstrate that M. azorica is conspecific with M. hirsuta, a species native to Australia, but
widely cultivated and locally invasive in the southern U. S. A. Based on our DNA data, we conclude that these plants are most likely a recent
introduction to the Azores from Florida. We recommend removal of Azorean Marsilea from conservation priority lists. While there is no evi-
dence that the small existing population threatens native species, further spread in the Azores should be prevented.
Keywords— Azores , Azorean endemic , DNA barcoding , invasive ferns , molecular phylogenetics.
846 SYSTEMATIC BOTANY [Volume 36
has remained stable since the 1970s. Marsilea azorica was
classified as a conservation priority species in the Azores,
Macaronesia, and Europe ( Martín et al. 2008 ) and the popula-
tion is managed by the local conservation agency. In a recently
published ‘taxonomically unbiased’ approach to prioritize
conservation resources in Macaronesia, it reached the highest
scoring of all organisms included in the ranking ( Martín et al.
2010 ). It was listed as ‘critically endangered’ in the IUCN red
list ( IUCN 2010 ) and was included in Appendix 1 of the ‘Bern
convention on the conservation of European wildlife and nat-
ural habitats’ and Annex 2 of the ‘Habitats Directive’ (Council
Directive 92/43/EEC on the conservation of natural habitats
and of wild fauna and flora) as a strictly protected species
( Council of Europe 2010 ).
In contrast to the considerable effort focused on the conser-
vation of M. azorica, its taxonomy and relationships have been
largely neglected and all recent treatments of the Azorean
flora and vegetation (e.g. Dias 1989 , 1996 ; Hansen and
Sunding 1993 ; Schaefer 2002 , 2003 , 2005a , b ; Silva et al. 2010 )
have adopted the view of Launert and Paiva (1983) without
a critical re-evaluation of the plant. Nevertheless, its late dis-
covery, disturbed habitat, and highly localized distribution
have seemed suspicious to many. In this paper, we use DNA
data, in combination with a morphological reassessment of
M. azorica, to determine its status and specifically to establish
whether or not it is a genetically and morphologically distinct
entity endemic to the Azores that deserves special protection
and management.
Materials and Methods
Sampling for Molecular Analysis and DNA Extraction— We included
57 accessions of 39 species of Marsileaceae. Fourteen sequences for acces-
sions of M. aegyptiaca Willd., M. azorica , M. hirsuta , and M. strigosa were
generated for this study and deposited in Genbank (accession numbers
HQ 728338–48; Appendix 2). The remaining sequences, produced by
Nagalingum et al. (2007 , 2008 ) and Whitten and Jacono (2009) , were down-
loaded from Genbank. Trees were rooted with Pilularia and Regnellidium
based on Pryer (1999) . DNA isolation from silica-dried leaves and her-
barium material, amplification, and sequencing of the rbcL , rps4 , rps4-trnS
and trnL - trnF regions (all from the chloroplast) followed the protocol in
Nagalingum et al. (2007) . Primer sequences and PCR conditions are as in
Whitten and Jacono (2009) .
Sequence Alignment and Phylogenetic Analyses— Sequences were
edited using Geneious pro 5.0.4 ( Drummond et al. 2010 ), and aligned
with MAFFT ( Katoh et al. 2005 ). The final alignments were checked in
MacClade 4.08 ( Maddison and Maddison 2005 ). There were no ambigu-
ously aligned regions and therefore nothing had to be excluded from sub-
sequent analyses.
Equally weighted maximum parsimony (MP) analyses for matri-
ces of nucleotides were conducted using PAUP 4.0b10 ( Swofford 2002 ).
The search strategy involved 100 random addition replicates with TBR
Fig 1. Marsilea hirsuta (= M. azorica ), Terceira, Azores (Portugal). A. habit (scale bar 10 mm). B, C. sporocarps (scale bars 1 mm); photos 1A:
H. Schaefer; 1B, C: F. Rumsey).
2011] SCHAEFER ET AL.: MARSILEA AZORICA IS A MISIDENTIFIED 847
branch swapping, saving all optimal trees. Gaps were treated as missing
data. To assess node support, parsimony bootstrap analyses ( Felsenstein
1985 ) were performed using 1,000 replicate heuristic searches, each with
10 random addition replicates and TBR branch swapping, saving all opti-
mal trees. We detected no statistically supported topological conflicts
between the data sets for the individual markers and therefore combined
all sequences in one alignment used in all subsequent analyses.
Maximum likelihood (ML; Felsenstein 1973 ) tree searches and ML
bootstrap searches ( Felsenstein 1985 ) for the individual and combined
data sets were performed using RAxML-HPC2 vs. 7.2.6 ( Stamatakis
et al. 2008 ) on the CIPRES cluster ( Miller et al. 2009 ). Based on the Akaike
information criterion ( Akaike 1974 ) and the Bayesian information cri-
terion ( Schwarz 1978 ) as implemented in jModeltest ( Posada 2008 ), we
selected the GTR + Γ model (six general time-reversible substitution rates,
assuming gamma rate heterogeneity), with model parameters estimated
over the duration of specified runs. Bayesian Markov chain Monte Carlo
(MCMC) inference ( Yang and Rannala 1997 ) used the GTR + Γ model (with
the default four rate categories) plus a proportion of invariable sites, and
relied on MrBayes 3.1.2 ( Huelsenbeck and Ronquist 2001 ) on the CIPRES
cluster ( Miller et al. 2009 ). The MCMC runs started from independent ran-
dom trees, were repeated twice, and extended for ten million generations,
with trees sampled every 100th generation. We used the default priors in
MrBayes, namely a flat Dirichlet prior for the relative nucleotide frequen-
cies and rate parameters, a discrete uniform prior for topologies, and an
exponential distribution (mean 1.0) for the gamma-shape parameter and
branch lengths. Convergence of the Bayesian analyses was assessed by
checking that final likelihoods and majority rule topologies in different
runs were similar, that the standard deviations of split frequencies were
< 0.01, and by examining the plot of generation number versus log prob-
ability of the data.
The data matrix and trees have been deposited in TreeBASE (study
number 11196).
Morphology—Marsilea azorica was studied in the field during several
visits to Terceira in 1994, 1998, 2000, 2005, 2008, and 2010. Herbarium spec-
imens of Marsilea from the Azores and other origins were studied at BM
and GH (Appendix 1). While we screened through all available Marsilea
material at the two herbaria, we focused especially on comparisons of
the Azorean taxon with the following: (i) specimens of M. quadrifolia and
M. strigosa/M. batardae because of previous determinations; (ii) M. minuta
specimens from the Canaries and elsewhere to exclude the possibility that
the same taxon occurs in both archipelagos, (iii) genetically similar spe-
cies from Australia as revealed here by our molecular phylogenetic analy-
ses: M. hirsuta R. Br., M. drummondii A. Braun, M. exarata A. Braun (incl.
M. paradoxa Diels), and M. angustifolia R. Br. We have not seen or sequenced
material of the Australian endemics M. costulifera D. L. Jones, M. latzii
D. L. Jones, and M. cryptocarpa Albr. & Chinnock, which are all uncom-
mon. However, based on descriptions in Entwisle (1993) , Jones (1998) , and
Albrecht and Chinnock (2008) , they are close to M. hirsuta and compari-
sons with the Azorean material were therefore made using the published
descriptions of these taxa.
Our examination of morphology ( Table 1 ) encompassed both leaflet
and sporocarp traits. In general, vegetative features, especially leaflet size
and shape, vary among Marsilea species but also within a species ( Johnson
1986 ). The presence, structure, abundance, and persistence of an indu-
mentum may also vary. The sporocarps may be borne singly or in clusters
(although poor production by taxa that normally produce multiple sporo-
carps may cause confusion), and may be sessile or more usually pedicel-
late. The characters of the pedicel, notably its branching or lack thereof,
seem to be informative. The sporocarp itself provides many characters:
shape, size, surface detail (ribbing), and the number and relative size of
the so-called teeth on its dorsal surface would appear to be among the
most reliable and regularly used taxonomic discriminants.
Results
Phylogenetic Analyses— The aligned matrix comprised
2,532 nucleotides with a total of 5.3% gaps or missing data in
the combined alignment and 525 variable characters, 404 of
them parsimony informative. Maximum parsimony, ML, and
Bayesian analyses resulted in highly congruent topologies,
so we only show the ML tree ( Fig. 2 ). We recover six highly
supported clades, one comprising M. mutica and a number
of other Neotropical species with branched pedicels corre-
sponding to ‘group I’ of Nagalingum et al. (2007) , and five
other clades with usually unbranched pedicels correspond-
ing to ‘group II’ of these authors. The latter group consists
of five well-supported clades and a few species ( M. quadrifo-
lia , M. strigosa , and M. nubica ) placed among them with rela-
tively poor support. One of those five clades, the Neotropical
M. mollis clade, is sister to the remaining African, Asian, and
Australian clades (even though it appears to be a polytomy in
Fig. 2 because of the short branches).
The rbcL , rps4 , rps4-trnS , and trnL-trnF sequences for the
Azorean Marsilea are completely identical to the sequences
of two M. hirsuta accessions from invasive populations in
Florida, U. S. A., and a native population in New South Wales,
Australia ( Fig. 2 ). Together with other accessions of invasive
M. hirsuta from southern U. S. A., they are nested in a well-
supported clade of Australian species (‘ M. hirsuta clade’ in
Fig. 2 ; 92% likelihood bootstrap (BS), 0.99 Bayesian posterior
probability (PP)).
Morphology— The Azorean Marsilea forms dense mats in
shallow water, creeping with relatively robust rhizomes of ca.
1 mm diameter, with slightly hairy nodes and internode dis-
tances between 5–62 mm. The leaves are on petioles 12–125 mm
long, and the four leaflets are fresh green, obovate-cuneate,
entire, 8–24 × 6–22 mm, densely hairy when young, later
almost glabrous. The sporocarps are solitary or in groups of
2–3 at the base of the petioles, but not adnate to them, on rela-
tively robust, unbranched pedicels that are as long as or up
to twice as long as the sporocarp (< 5 mm), erect, recurved,
and densely hairy with multicellular, uniseriate hairs. The
sporocarp is 3.8–4.8 × 2.9–3.8 mm, oblong-ellipsoid, with six
to eight faint lateral ridges when young. Each sporocarp con-
tains six to eight sori on each side. There are two teeth on the
sporocarp dorsal wall; the lower tooth is prominent, conical,
and slightly truncate; the upper tooth is also well-developed,
broadly conical, obtuse, and elliptic in outline.
The Azorean plant is clearly different from both M. quadrifo-
lia and M. strigosa / M. batardae in the degree of pubescence of
the leaflets on both surfaces and the possession of two, equal-
sized sporocarp teeth ( Table 1 ). Leaflet pubescence, together
with the larger sporocarps, also distinguish the Azorean
plants from M. minuta.
All members of the ‘ M. hirsuta ’ clade in our phylogenetic
tree ( Fig. 2 ), plus the three unsequenced Australian endem-
ics ( M. costulifera , M. cryptocarpa , and M. latzii ), share the fea-
ture of pubescent leaves ( Table 1 ). However, only M. hirsuta
possesses the two, equal-sized sporocarp teeth evident in
Azorean plants, and using our morphological traits ( Table 1 )
the Azorean plants and M. hirsuta are indistinguishable, sug-
gesting that the two are conspecific.
Habitat— The only Marsilea population in the Azores is
restricted to a small roadside pond in a pasture at ca. 460 m,
where it grows together with Agrostis stolonifera L., Cyperus
eragrostis Willd., Cyperus longus L., Eleocharis multicaulis (Sm.)
Desv., Galium palustre L.,
Hydrocotyle vulgaris L., Isolepis flu-
itans (L.) R. Br., Juncus articulatus L., J. bulbosus L., J. effusus
L., Mentha suaveolens Ehrh., Myosotis ramosissima Rochel
ex Schult., Polygonum hydropiperoides Michx., Potamogeton
polygonifolius Pourr., Ranunculus flammula L., R. repens L.,
Rumex conglomeratus Murray, and Trifolium repens L.
Discussion
The Taxonomic Status of ‘M. azorica’— Morphological
and molecular data both indicate that M. azorica is conspecific
848 SYSTEMATIC BOTANY [Volume 36
Table 1. Morphological comparison of selected Marsilea species based on herbarium material (Appendix 1) and literature ( Johnson 1986 ; Paiva 1986 ; Akeroyd 1993 ; Entwistle 1993 ; Jones 1998 ; Jacono and
Johnson 2006 ; Albrecht and Chinnock 2008 ).
Marsilea
azorica
Launert & Paiva
Marsilea
strigosa
Willd.
Marsilea
batardae
Launert Marsilea
quadrifolia L.
Marsilea
minuta
L. (incl. M.
crenata C.Presl) Marsilea hirsuta
R. Br.
Marsilea
drummondii
A. Braun
Marsilea exarata
A. Braun
(incl. M.
paradoxa Diels)
Marsilea
angustifolia
R. Br.
Marsilea
latzii
D. L. Jones
Marsilea
costulifera
D. L. Jones
Marsilea
cryptocarpa
Albr. &
Chinnock
Range Portugal,
Azores
Western
Europe,
Southwest
Russia
Iberian
peninsula
Eurasia,
Africa,
introduced
in the
Americas
Africa, Asia,
Australia,
introduced
in the
Americas
Australia,
wide spread,
introduced
in North
America
Australia,
wide-
spread
Australia,
wide-
spread
Australia,
Northern
Territory &
Western
Australia
Australia,
Northern
Territory,
Tanami
Desert
Australia,
wide-
spread
Australia,
Northern
Territory &
South
Australia
Leaflet
shape
obovate-
cuneate
obdeltate-
cuneate
obdeltate-
cuneate
obovate-
cuneate
broadly
obovate-
cuneate
obovate-
cuneate
broadly
obovate-
cuneate
oblanceolate-
cuneate
narrowly
cuneate
(broadly)
cuneate
oblanceolate-
cuneate
obdeltate
Leaflet
pubescence
hairy
becoming
glabrescent
glabrous to
sparsely
hairy
glabrescent glabrous or
with few
hairs only
glabrous to
sparsely
hairy
hairy to
glabrous
hairy glabrescent glabrescent glabrous to
hairy
hairy (densely)
hairy
Leaflet size 8–24 ×
6–22 mm
2–15(-30) ×
2–15(-25) mm
2–8(-11) ×
2–7(-9) mm
7–30 ×
6–31 mm
12–20 ×
8–21 mm
5–21 ×
5–21 mm
5–39 ×
3–32 mm
5–13 ×
2–8 mm
7–20 ×
3–5 mm
1–12 ×
1–5 mm
4–12 ×
1–5(-8) mm
8–35 ×
8–35 mm
Sporo-carp
shape
oblong-
ellipsoid
oblong-
ellipsoid
oblong-
ellipsoid
oblong-
ellipsoid
oblong-
ellipsoid
oblong-
ellipsoid
oblong oblong-
ellipsoid
oblong-
ellipsoid
ellipsoid oblong-
ellipsoid
oblong-
ellipsoid
Sporo-carp
size
3.8–4.8 ×
2.9–3.8 mm
2.5–4.7 ×
3–4 mm
3–4.5 ×
2.5–4.5 mm
4.0–5.6 ×
3.1–4.0 mm
2.6–4.1 ×
<2.0 mm
3–7 × 4–6 mm (4)-8–9 ×
6–9 mm
4–5 × 3–5 mm 5–5.5 ×
3–5 mm
3–4 ×
2–4 mm
3–5 × 2–4 mm 2–3 ×
2–3 mm
Sporo-carp
surface
not or faintly
ribbed
not or faintly
ribbed
not or faintly
ribbed
not ribbed not ribbed not or faintly
ribbed
faintly to
distinctly
ribbed
ribbed indistinctly
ribbed
distinctly
ribbed
distinctly
ribbed
faintly
ribbed
Sporo-carp
teeth
2, equal
in size
1, rarely a
second
smaller
tooth
1, rarely a
second
smaller
tooth
0 or upper
tooth
present,
very
small
2, equal in
size or
lower
tooth
absent
2, equal
in size
2, upper
tooth
< lower
tooth
2, the lower ±
recurved
1, rarely a
second
smaller
tooth
1, base of
stalk
forming
second
tooth like
structure
1, rarely a
second
smaller
tooth
0 or 1–2
slight
protuberances
Sporo-carp
stalk
(pedicel)
< 5 mm,
unbranched
< 1–2.5 mm,
unbranched
2–6(-8) mm,
unbranched
3–16 mm,
often
branched
2.6–6 mm,
some-
times
branched
1.5–6 mm,
unbranched
10–60(-100) mm,
unbranched
1–4 mm,
unbranched
1–3 mm,
unbranched
4–6 mm,
unbranched
< 5 mm,
unbranched
1–3 mm,
unbranched
Grouping of
sporo-
carps
solitary or
clustered
(1–3)
mostly
solitary
mostly
solitary
clustered
2–3(-4),
rarely
solitary
Usually
paired,
solitary or
clustered
(1–4)
solitary or
clustered
solitary or
clustered
solitary clustered solitary or
in small
clusters
clustered 6–12 in a
dense
row at
base of
stipe
2011] SCHAEFER ET AL.: MARSILEA AZORICA IS A MISIDENTIFIED 849
Fig. 2. Maximum likelihood phylogram of combined rbcL , rps4 , rps4-trnS , and trnL-F sequences (2,532 aligned nucleotides) analyzed under the
GTR + Γ model. Likelihood bootstrap support values ≥ 60% are indicated above branches at each node, Bayesian posterior probability values > 0.95 below
the branches. Arrow points to position of Marsilea azorica in M. hirsuta clade (photographs by Andreea Filip, Jenny Hsu, Armin Jagel, Forest and Kim Starr,
and Barbara Quinn (top to bottom)).
850 SYSTEMATIC BOTANY [Volume 36
with the variable Australian species M. hirsuta . By virtue
of a suite of pedicel and sporocarp characters ( Table 1 ),
the Azorean population of Marsilea is clearly distinct from
M. strigosa with which it was compared by Launert and Paiva
(1983) , and the few European ( Akeroyd 1993 ) and signifi-
cantly greater number of African Marsilea species ( Launert
1984 , not in our overview table). Of the geographically closest
taxa, arguably the greatest resemblance is with the Canarian
taxon (most likely M. minuta ), which shares its stature, widely
creeping growth form, and the presence of two definite teeth
on the sporocarp. However, M. minuta is less densely seri-
ceous in both foliage and particularly the sporocarp, and the
pedicels are not entirely free.
Specimens of M. hirsuta examined at BM and GH show
considerable variability in growth form and stature. Smaller
leaved, sub-caespitose plants look different, but other larger,
laxer expressions are indistinguishable in habit from typical
Azorean material.
It is not unreasonable to suppose that Launert and Paiva
(1983) did not make detailed comparisons with the geograph-
ically remote Australasian taxa, which were not known to be
invasive elsewhere at that time, and this may be why the obvi-
ous similarities to M. hirsuta were overlooked when describ-
ing M. azorica as new. However, in light of the results reported
here, it is clear that M. azorica should be treated as a synonym
of M. hirsuta.
Marsilea hirsuta was described by Brown (1810) based on
material he collected in 1802 on the Flanders expedition to
Australia. Jones (1998) indicated that the syntypes of this spe-
cies were at BM. All three ( R. Brown s. n., Iter Austral. 138–140 )
possess sporocarps. The specimen R. Brown s. n., Iter Austral.
139 was collected from Broad Sound and his diary entry for
15 September 1802 indicates that this was the first time he
encountered this plant (given as M. quadrifolia both in the
diary entry and initially on the label; Vallance et al. 2001 ). This
specimen not only agrees well with the protologue and has all
of the important features necessary for identification, but also
is the best localized and with the most supporting additional
literature information. It is therefore selected as lectotype for
M. hirsuta and we propose the following synonymy:
M. hirsuta R. Br., Prodr. Fl. Nov. Holland. 167. 1810.—TYPE:
AUSTRALIA. Broad Sound in pratis depressis [sestris]
temporibus inundatis, R. Brown , Iter Austral. 139 , (lecto-
type, here designated: BM!).
M. azorica Launert & Paiva, Iconogr. Selecta Fl. Azor. 2:
159. 1983.—TYPE: PORTUGAL. Terceira, Azores, A.
Hansen 308 (holotype: C [photograph!]; isotype: LISI
[photograph!]).
Marsilea hirsuta in the Azores— Given that M. azorica is
conspecific with M. hirsuta, it should clearly be removed from
all conservation priority lists and the IUCN red list. Within
the Azores, the relatively recent discovery of M. hirsuta sug-
gests that it may be the result of recent human introduction
and it is therefore most appropriate to consider it as an intro-
duced species in the archipelago.
Marsilea hirsuta is invasive in Florida, southeastern U. S. A.
( Jacono and Johnson 2006 ; Whitten and Jacono 2009 ). Indeed,
it is one of several species of the genus that are invasive in
tropical Asia and North and South America: M. minuta and
M. quadrifolia are rice field weeds in Asia ( Ampong-Nyarko
and De Datta 1991 ), and M. minuta is also invasive in some
places in Brazil and Trinidad ( Johnson 1986 ). In the U. S. A.,
M. minuta , M. quadrifolia , and
M. mutica are invasive in natural
and disturbed wetland habitats from New England to Florida
( Jacono and Johnson 2006 ). Even though there is no evidence
that M. hirsuta is spreading in the Azores, the potential to
become invasive and a threat to endemic shallow water spe-
cies such as the endangered quillwort, Isoëtes azorica Durieu,
should be recognized.
How M. hirsuta was transported to the Azores remains
unclear. Given that DNA sequences are identical to those
obtained from two populations in Florida, it is plausible that
the Azorean plants were not introduced from within its native
range in Australia, but came from Florida ( Fig. 3 ). Marsilea
hirsuta is a popular ornamental plant for fish tanks and
ponds, and is commonly traded via the internet. In Florida,
it was introduced with the horticultural trade before becom-
ing locally invasive in natural habitats ( Jacono and Johnson
2006 ; Whitten and Jacono 2009 ). The horticultural trade, how-
ever, seems an unlikely source of introduction into the Azores
since M. hirsuta is not on sale in the few Azorean nurseries
and was not observed in any park or private garden during
any of our visits (but it might be present in private fish tanks).
Furthermore, introduction to the Azores must have happened
before the 1970s (there was already a population covering
several square meters of ground, when it was first recorded
in 1970), well before M. hirsuta was ‘discovered’ for the horti-
cultural industry. However, one of us (FR) recently found the
related water fern Salvinia molesta D. Mitch as an introduced
ornamental in an artificial pond on Flores Island, Azores, so
even if it seems unlikely, intentional introduction for orna-
ment followed by spread to the actual habitat remains a possi-
bility. Introduction by vagrant waterfowl blown to the Azores
from the southeastern U. S. A. is another possible, but not
very likely, explanation for its dispersal to the Azores. Several
species of American ducks, waders, and herons or egrets are
reported every year from the Azores and they might carry
diaspores of water plants attached to their feet or plumage as
was recently shown in a study of migrating waterfowl on the
European mainland ( Figuerola and Green 2002 ). However,
those vagrants are usually observed on the bigger and undis-
turbed lakes or wetland areas in other parts of the archipel-
ago and not on small ponds next to busy roads. Finally, an
obvious connection between Florida and the Azores is the
U. S. A. airbase on Terceira Island: introduction of sporocarps
attached to the tyres of machines or vehicles brought from the
U. S. A. to Terceira is also possible.
The case of M. azorica is similar to that of Diplazium allor-
gei Tardieu, which was described as an Azorean endemic in
1938. Some 40 yr later it was shown to be conspecific with
Deparia petersenii (Kunze) M. Kato, an introduced species of
Asian origin ( Sledge 1975 , 1977 ). In other groups, however,
recent molecular data suggest that current species circum-
scriptions may be under-estimating the levels of endemic
diversity ( Schaefer et al. 2011 ). The status of many other taxa
in the Azorean flora remains unclear. Selaginella kraussiana
(Kunze) A. Braun, for example, was thought to be an invasive
introduced from the African continent ( Schaefer 2003 ), until
its native status was confirmed through sediment core analy-
ses ( Van Leeuwen et al. 2005 ).
Even the most sophisticated conservation priority ranking
schemes, like the one developed by Martín et al. (2010) , will
fail if there is no solid taxonomic basis. If we want to avoid
directing precious conservation resources to misidentified
alien species, we urgently need a comprehensive and detailed
2011] SCHAEFER ET AL.: MARSILEA AZORICA IS A MISIDENTIFIED 851
reassessment of the taxonomy and status of the Azorean flora
based on approaches that combine molecular and morpho-
logical data.
Acknowledgments. The authors are grateful to W. Mark Whitten for
advice and comments, to David Mabberley and David Moore for assis-
tance with the lectotypification of M. hirsuta , to O. Ryding for images
of Marsilea material in C, to Katy Jones for information concerning the
isotype of M. azorica in LISI, and to the Azorean Direcção Regional do
Ambiente for issuing collection permits.
Literature Cited
Akaike, H . 1974 . A new look at the statistical model identification . IEEE
Transactions on Automatic Control 19: 716 – 723 .
Akeroyd, J. R . 1993 . Marsilea L . Pp. 31 – 32 in Flora Europaea , Vol. 1 , 2nd ed .:
Psilotaceae to Platanaceae, eds. T. G. Tutin , N. A. Burges , A. O. Chater ,
J. R. Edmondson , V. H. Heywood , D. M. Moore , D. H. Valentine , S. M.
Walters , and D. A. Webb . Cambridge, U. K. : Cambridge University
Press .
Albrecht, D. E. and R. J. Chinnock . 2008 . A new species of Marsilea
(Marsileaceae) from arid Australia . Journal of the Adelaide Botanic
Gardens 22: 1 – 4 .
Ampong-Nyarko, K. and S. K. De Datta . 1991 . Rice weeds of worldwide
importance . Pp. 7 – 39 in A handbook for weed control in rice . Manila,
Philippines : International Rice Research Institute .
Arechavaleta, M., N. Zurita , M. C. Marrero , and J. L. Martín (eds.). 2005 .
Lista preliminar de especies silvestres de Cabo Verde (hongos, plantas y ani-
males terrestres) . La Laguna, Spain : Consejería de Medio Ambiente y
Ordenación Territorial, Gobierno de Canarias .
Braun, A . 1870 . Neuere Untersuchungen über die Gattungen Marsilia
und Pilularia . Monatsbericht der Königlich-Preussischen Akademie der
Wissenschaften zu Berlin 1870: 653 – 753 .
Brown, R . 1810 . Prodromus Florae Novae Hollandiae et Insulae Van Diemen
exhibens characteres plantarum quas annis 1802–1805 . Vol. 1 . London :
R. Taylor .
Burchard, O . 1929 . Beiträge zur Ökologie und Biologie der Kananarenpflanzen.
Bibliotheca Botanica 98 . Stuttgart : Schweizerbart .
Christ, D . 1906 . Filices Azoricae. Leg. Dr. Bruno Carreiro . Bulletin de
l’Académie Internationale de Géographie Botanique 16: 153 – 160 .
Christ, H . 1888 . Spicilegium canariense . Englers Botanisches Jahrbuch
9: 156 .
Council of Europe . 2010 . Council Directive 92/43/EEC of 21 May 1992
on the conservation of natural habitats and of wild fauna and
flora .
Dansereau, P . 1961 . Études Macaronésiennes: I. Géographie des cryptog-
ames vasculaires . Agronomia Lusitana 23: 151 – 181 .
Dias, E. M . 1989 . Flora e vegetação endemica na ilha Terceira. M. S. thesis .
Angra do Heroísmo : Universidade dos Açores .
Dias, E. M . 1996 . Vegetação natural dos Açores. Ecologia e sintaxonomia das
florestas naturais. Ph. D. thesis . Angra do Heroísmo : Universidade dos
Açores .
Drouet, M. H . 1866 . Catalogue de la flore des îles Açores précédé de
l’itinéraire d’un voyage dans cet archipel . Mémoires de la Société
Académique d’Agriculture des Sciences, Arts et Belles- Lettres du
Département de l’Aube . 30: 81 – 233 .
Druce, G . 1911 . Plants of the Azores . Le Journal de Botanique 49: 23 – 28 .
Drummond, A. J., B. Ashton , S. Buxton , M. Cheung , A. Cooper , J. Heled ,
M. Kearse , R. Moir , S. Stones-Havas , S. Sturrock , T. Thierer , and A.
Wilson . 2010 . Geneious , version 5.0.4. http://www.geneious.com.
Entwisle, T. J . 1993 . Marsilea . Pp. 54 – 57 in Flora of Victoria , eds. N. G. Walsh
and T. J. Entwisle . Melbourne : Inkata Press .
Felsenstein, J . 1973 . Maximum likelihood and minimum-steps methods
for estimating evolutionary trees from data on discrete characters .
Systematic Zoology 22: 240 – 249 .
Felsenstein, J . 1985 . Confidence limits on phylogenies: an approach using
the bootstrap .
Evolution 39: 783 – 791 .
Figuerola, J. and A. J. Green . 2002 . How frequent is external transport of
seeds and invertebrate eggs by waterbirds? A study in Doñana, SW
Spain . Archiv fuer Hydrobiologie 155: 557 – 565 .
Gandoger, M. M . 1899 . Plantes nouvelles pour les îles Açores . Bulletin de la
Société Botanique de France 46: 252 – 258 .
Gonçalves da Cunha, A. and L. G. Sobrinho . 1938 . Estudos botanicos no
Arquipélago dos Açores . Revista da Faculdade de Ciências (Universidade
de Lisboa), 2
a sér . 1: 177 – 220 .
Hansen, A . 1972 . A note on the genus Marsilea in the Canary Islands .
Cuadernos de Botánica Canaria 16: 9 – 10 .
Hansen, A . 1973 . Contributions to the flora of the Azores - III (especially
Terceira) . Anuário da Sociedade Broteriana 39: 25 – 38 .
Hansen, A. and P. Sunding . 1993 . Flora of Macaronesia. Checklist of vascu-
lar plants . 4th ed . Sommerfeltia 17: 1 – 295 .
Fig. 3. Overview map showing most likely scenario for origin of the Azorean Marsilea population: native to Australia (1), primary introduction to
southeastern U. S. A. (2), and from there a further introduction to the Azores (3).
852 SYSTEMATIC BOTANY [Volume 36
Huelsenbeck, J. P. and F. Ronquist . 2001 . MRBAYES: Bayesian inference of
phylogeny . Bioinformatics 17: 754 – 755 .
IUCN . 2010 . IUCN red list of threatened species . Version 2010.2. available
at www.iucnredlist.org [accessed Nov. 2010].
Izquierdo, I., J. L. Martín , N. Zurita , and M. Arechavaleta , eds. 2004 . Lista
de especies silvestres de Canarias (hongos, plantas, y animales terrestres) .
La Laguna, Spain : Consejería de Medio Ambiente y Ordenación
Territorial, Gobierno de Canarias .
Jacono, C. and D. M. Johnson . 2006 . Water-clover ferns, Marsilea , in the
southeastern United States . Castanea 71: 1 – 14 .
Jalas, J. and J. Suominen . 1972 . Atlas Florae Europaeae: distribution of vascular
plants in Europe vol. 1 , map no. 143. Helsinki : Committee for Mapping
the Flora of Europe .
Johnson, D. M . 1986 . Systematics of the New World species of Marsilea
(Marsileaceae) . Systematic Botany Monographs 11: 1 – 87 .
Jones, D. L . 1998 . Marsileaceae . Pp. 166 – 172 in Flora of Australia: Ferns,
Gymnosperms and allied groups vol. 48 , ed. P. M. McCarthy . Melbourne :
ABRS/CSIRO Australia .
Katoh, K., K. Kuma , H. Toh , and T. Miyata . 2005 . MAFFT vs. 5: improve-
ment in accuracy of multiple sequence alignment . Nucleic Acids
Research 33: 511 – 518 .
Kubitzki, K . 1990 . Pteridophytes and gymnosperms . Pp. 1 – 404 in The families
and genera of vascular plants vol. 1 , eds. K. U. Kramer and P. S. Green .
Berlin : Springer-Verlag .
Kunkel, G . 1971 . Lista revisada de los pteridofitos de las Islas Canarias .
Cuadernos de Botánica Canaria 13: 24 .
Launert, E . 1968 . A monographic survey of the genus Marsilea Linnaeus.
I. The species of Africa and Madagascar . Senckenbergiana Biologica 49:
273 – 315 .
Launert, E . 1984 . A revised key to and new records of African species of
the genus Marsilea. Garcia de Orta - Série de Botânica 6: 119 – 140 .
Launert, E. and J. Paiva . 1983 . Marsilea azorica . Pp. 159 – 162 in Iconographia
selecta florae Azoricae vol. 1 (2) , eds. A. Fernandes and R. Fernandes
Batarda . Coimbra, Portugal : Conimbriga .
Maddison, W. P. and D. R. Maddison . 2005 . MacClade: analysis of phylogeny
and character evolution, version 4.08 . Sunderland : Sinauer Associates .
Martín, J. L., M. Arechavaleta , P. A. V. Borges , and B. F. Faria (eds.).
2008 . Top 100. Las 100 especies amenazadas prioritarias de gestión en la
región Europea biogeográfica de la Macaronesia . Santa Cruz de Tenerife :
Consejería de Medio Ambiente y Ordenación Territorial, Gobierno
de Canarias .
Martín, J. L., P. Cardoso , M. Arechavaleta , P. A. V. Borges , B. F. Faria ,
C. Abreu , A. F. Aguiar , J. A. Carvalho , A. C. Costa , R. T. Cunha , F.
M. Fernandes , R. Gabriel , R. Jardim , C. Lobo , A. M. F. Martins ,
P. Oliveira , P. Rodrigues , L. Silva , D. Teixeira , I. R. Amorim , N.
Homem , B. Martins , M. Martins , and E. Mendonça . 2010 . Using
taxonomically unbiased criteria to prioritize resource allocation for
oceanic island species conservation . Biodiversity and Conservation
19: 1659 – 1682 .
Milde, J . 1867 . Filices Europae et Atlantidis . Lipsiae : A. Felix .
Miller, M. A., M. T. Holder , R. Vos , P. E. Midford , T. Liebowitz , L. Chan , P.
Hoover , and T. Warnow . 2009 . The CIPRES Portals . CIPRES . http://
www.phylo.org/sub_sections/portal.
Nagalingum, N. S., H. Schneider , and K. M. Pryer . 2007 . Molecular phy-
logenetic relationships and morphological evolution in the hetero-
sporous fern genus Marsilea . Systematic Botany 32: 16 – 25 .
Nagalingum, N. S., M. D. Nowak , and K. M. Pryer . 2008 . Assessing phylo-
genetic relationships in extant heterosporous ferns (Salviniales), with
a focus on Pilularia and Salvinia . Botanical Journal of the Linnean Society
157: 673 – 685 .
Palhinha, R . 1943 . Pteridófitos do Arquipélago dos Açores . Boletim da
Sociedade Broteriana 17: 215 – 249 .
Palhinha, R . 1966 . Catálogo das plantas vasculares dos Açores . Lisboa :
Sociedade de Estudos Acorianos Afonso Chaves .
Palhinha, R., A. Gonçalves da Cunha , and L. G. Sobrinho . 1946 . Plantas
vasculares da ilha Terceira . Açoreana 4: 1 – 77 .
Paiva, J . 1986 . Marsilea L . Pp. 66 – 69 in Flora Iberica , vol. I . Madrid : Real
Jardín Botánico, CSIC .
Posada, D . 2008 . jModelTest: Phylogenetic model averaging . Molecular
Biology and Evolution 25: 1253 – 1256 .
Pryer, K. M . 1999 . Phylogeny of marsileaceous ferns and relationships
of the fossil Hydropteris pinnata reconsidered . International Journal of
Plant Sciences 160: 931 – 954 .
Schaefer, H . 2002 . Flora of the Azores. A fieldguide . Weikersheim : Margraf
Publishers/Backhuys Publishers .
Schaefer, H . 2003 . Chorology and diversity of the Azorean flora. Dissertationes
Botanicae 374 . Stuttgart : J. Cramer .
Schaefer, H . 2005a . Flora of the Azores. A fieldguide . Ed. 2 . Weikersheim :
Margraf Publishers/Backhuys Publishers .
Schaefer, H . 2005b . Endemic vascular plants of the Azores: an updated list .
Hoppea 66: 275 – 283 .
Schaefer, H., M. Moura , M. G. Belo Maciel , L. Silva , F. J. Rumsey , and M. A.
Carine . 2011 . The Linnean shortfall in oceanic island biogeography:
a case study in the Azores . Journal of Biogeography 38: 1345 – 1355 .
DOI: 10.1111/j.1365-2699.2011.02494.x.
Schneider, H. and K. M. Pryer . 2002 . Structure and function of spores in
the aquatic heterosporous fern family Marsileaceae . International
Journal of Plant Sciences 163: 485 – 505 .
Schwarz, G . 1978 . Estimating the dimension of a model . Annals of Statistics
6: 461 – 464 .
Seubert, M . 1844 . Flora Azorica quam ex collectionibus schedisque Hochstetteri
patris et filii elaboravit . Bonnae : Apud A. Marcum .
Seubert, M. and C. Hochstetter . 1843 . Übersicht der Flora der azorischen
Inseln . Archiv für Naturgeschichte 9: 1 – 24 .
Silva, L., M. Moura , H. Schaefer , F. Rumsey , and E. F. Dias . 2010 . Vascular
plants (Tracheobionta) . Pp. 117 – 146 in A list of the terrestrial and marine
biota from the Azores , eds. P. A. V. Borges , A. Costa , R. Cunha , R.
Gabriel , V. Gonçalves , A. F. Martins , I. Melo , M. Parente , P. Raposeiro ,
P. Rodrigues , R. S. Santos , L. Silva , P. Vieira , and V. Vieira . Cascais :
Princípia, Sjögren, E. A. 1973. Recent changes in the vascular flora
and vegetation of the Azores islands. Memórias da Sociedade Broteriana
22: 5–453 .
Sledge, W. A . 1975 . Lunathyrium in the Azores . The Fern Gazette 11: 81 – 85 .
Sledge, W. A . 1977 . Lunathyrium in the Azores; a postscript . The Fern
Gazette 11: 331 – 336 .
Stamatakis, A., P. Hoover , and J. Rougemont . 2008 . A rapid bootstrap algo-
rithm for the RAxML web-servers . Systematic Biology 75: 758 – 771 .
Swofford, D. L . 2002 . PAUP* Phylogenetic analysis using parsimony (*and
other methods) 4.0b10 . Sunderland : Sinauer Associates .
Tardieu-Blot, M . 1946 . Sur la flore ptéridologique des îles atlantiques .
Mémoires de la Société de Biogéographie 8: 325 – 347 .
Trelease, W . 1897 . Botanical observations on the Azores . Annual Report
Missouri Botanical Gardens 1897: 77 – 220 .
Vallance, T. G., D. T. Moore , and E. W. Groves . 2001 . Nature’s investigator:
the diary of Robert Brown in Australia, 1801–1805 . Canberra : Australian
Biological Resources Study, Flora .
Van Leeuwen, J. F. N., H. Schaefer , W. O. Van der Knaap , T. Rittenour , S.
Björck , and B. Ammann . 2005 . Native or introduced? Fossil pollen
and spores may say. An example from the Azores Islands . Neobiota
6: 27 – 34 .
Vasconcellos de Carvalho, J . 1968 . Pteridófitas de Portugal continental e ilhas
adjacentes . Lisboa : Fundação Calouste Gulbenkian .
Vitalis, R., M. Riba , B. Colas , P. Grillas , and I. Olivieri . 2002 . Multilocus
genetic structure at contrasted spatial scales of the endangered water
fern Marsilea strigosa Willd. (Marsileaceae, Pteridophyta) . American
Journal of Botany 89: 1142 – 1155 .
Watson, H. C . 1870 . Botany of the Azores . Pp. 113 – 288 in Natural history
of the Azores or Western Islands , ed. F. du Cane Godman . London :
J. Van Voorst .
Webb, P. B. and S. Berthelot . 1840 . Histoire naturelle des iles canaries, Tom. 3,
pt.2, Phytographia Canariensis, Sectio. 2 . Paris : Béthune .
Whitten, W. M. and C. C. Jacono . 2009 . Molecular identification of invasive
water-clover ferns: final report for a contract from the Florida depart-
ment of environmental protection . Available at: http://www.flmnh.
ufl.edu/herbarium/research/barcoding/dep-marsilea1-report.htm
[accessed Nov. 2010].
Yang, Z. and B. Rannala . 1997 . Bayesian phylogenetic inference using
DNA sequences: a Markov Chain Monte Carlo method . Molecular
Biology and Evolution 14: 717 – 724 .
Appendix 1. Representative Specimens Examined for Morphological
Analysis (determination of all specimens confirmed by the authors).
Marsilea aegyptiaca Willd. EGYPT. Zagazig, L. Boulos s. n. (GH).
RUSSIA. Astrachanj, Volga Delta, Sagalaev et al. s. n. (GH). Marsilea angus-
tifolia R. Br. AUSTRALIA. New South Wales, Bethungra, Cootamundra
district, K. Mair P2022 (GH); Royal Botanic Gardens Sydney (cult. mate-
rial from Bethungra), A. Rodd P9410 (GH). Marsilea azorica Launert &
Paiva. PORTUGAL. Azores, Terceira, [ I. ] B [ otelho ] Gonçalves 3259 (BM);
Terceira, M. G. Belo Maciel et al. (BM; AZU); Terceira, H. Schaefer 2008/923
(M). Marsilea burchellii (Kunze) A. Braun. SOUTH AFRICA. Cape
Province, E. A. Schelpe 4552 (GH). Marsilea drummondii A. Braun.
AUSTRALIA. New South Wales, Narrabri, J. L. Boorman s. n. (GH);
Queensland, Rifle Creek, southwest of Port Douglas, C. B. Hellquist 15117
2011] SCHAEFER ET AL.: MARSILEA AZORICA IS A MISIDENTIFIED 853
(GH). Marsilea ephippiocarpa Alston. NAMIBIA. West of Otavi, L. Smook
10000 (GH). Marsilea exarata A. Braun. AUSTRALIA. Queensland,
A. Fraser 333 (GH); New South Wales, Mundi Mundi station near Broken
Hill, E. F. Constable P237 (GH). Marsilea hirsuta R. Br. AUSTRALIA.
Iter Australiense 1802-5, R. Brown 138-140 (BM); New South Wales,
E. F. Constable 4069A (BM); New South Wales, A. & R. Tryon 7317 (GH);
Northern Territory, Prof. Sir Baldwin Spencer s. n. (BM); Pickanjinnie Creek,
E. H. Belson (BM); Queensland (cultivated at Botanical Garden of Berlin,
Germany), A. Braun 15, 16, 17, 18, 19 (GH); Queensland, N. Michael 1430
(GH); Queensland, M. S. Clemens s. n. (GH); Queensland, L. J. Brass 19752
(GH); Victoria, W. Watts 1458 (GH); Queensland, Proserpine, river bed,
N. Michael 1430 (BM, GH); Western Australia far eastern section, R. H.
Kuchel 259 (BM); Western Australia, C. B. Hellquist 15103 (GH). Marsilea
macrocarpa Presl. KENYA. Kajiado district, road to Masai Lodge, R. B. &
A. J. Faden 77/295 (GH). SOUTH AFRICA. Cape Colony, pr. Grahamstown,
R. Schlechter 2612 (GH); Cape Province, Komgha, E. A. Schelpe 5025 (GH).
Marsilea minuta L. (incl. M. crenata Presl). AUSTRALIA. Queensland,
Townsville, K. Kennedy & M. Tindale P7967 (GH); Queensland, Inkerman,
S. E. Stephens P2924 (GH). MALAYSIA: Perak, B. E. G. Molesworth-
Allen 4714 & 4516 (GH); fields near Kodianq, B. E. G. Molesworth-Allen
2819 (GH). PAPUA NEW GUINEA. Western district, near Bula village,
R. Pullen 7031 (GH). PHILIPPINES. Luzon, Bayninan, D. R. Mendoza &
Buwaya 76795 (GH). SENEGAL. R. Berhaut 4926 (GH) [as M. diffusa ].
SPAIN. Canary Islands, Gran Canaria, E. Bourgeau Plantae Canarienses
1168 (BM, GH) [as M. quadrifolia L., the BM specimen redet. M. minuta
by E. Launert, 1968 ]; Gran Canaria, Arucas, O. Gelert s. n. (C, photograph
seen) [as M. quadrifolia L.]. Marsilea mutica Mett. AUSTRALIA. New
South Wales, St. Albans Lagoon, E. F. Constable P8312 (GH); New South
Wales, Emu Plains, E. F. Constable NSW 9770 (GH); Sullivan’s creek, A.C.T.,
J. Pulley 1218 (GH); New South Wales, Coorabong, E. F. Constable P8142
(GH). Marsilea quadrifolia L. FRANCE. Montbison (Loire), Le Grand s. n.
(GH). HUNGARY. Holt Körös prope pagum Vesztö, Borbás s. n. (GH).
INDONESIA. Java, Horsfield 174 (GH). ROMANIA: Muntenia, Vlasca
district, I. Prodan s. n. (GH). RUSSIA. Astrachanj, Volga Delta, G. Malutin
s. n. (GH). Marsilea polycarpa Hook. & Grev. FRENCH POLYNESIA.
Society Islands, Borabora, Turapuo H. St. John 17414 (GH). Marsilea
schelpeana Launert. SOUTH AFRICA. Cape Province, Zwartskops River
valley, R. Anderson 28 (GH). Marsilea strigosa Willd. ALGERIA. Oran,
Mare du Djebel-Santo, Clauson s. n. (GH) [as M. pubescens Ten.]. FRANCE.
Dept. Hérault, J. de Vichet 81 (GH) [as M. pubescens Ten.]; B. Lugardon s. n.
(GH); Agde, pr. Montpellier, A. Braun s. n. (BM). MOROCCO. Oued Issen,
E. Jahandiez 298 (BM). RUSSIA. Astrachanj, Volga Delta, N. Dessiatoff
741 (GH); Saratow, Sarepta, A. Becker s. n. (GH). SPAIN. Islas Baleares,
Menorca, N. of Mahon, C. Jermy 18172 (BM). Marsilea villosa Kaulf.
U. S. A. Hawai’i, Oahu, F. R. Fosberg 9704 (GH); Oahu, D. F. Topping
3409 & 3518 (GH); West of Mokio, Molokai, O. Degener & H. Wiebke 3215
(GH); Oahu, H. L. Lyon s. n. (GH).
Appendix 2. Material sequenced with GenBank accession number
(rbcL, trnL-trnF, rps4 + rps4-trnS ).
Marsilea aegyptiaca . Russia, Sagalaev et al. s. n. (GH) (HQ728338, HQ
728342, HQ728346); Marsilea azorica . Portugal, Azores, H. Schaefer 2008/923
(M) (HQ728339, HQ728343, HQ728347); Marsilea hirsuta . Australia, A. &
R. Tryon 7317 (GH) (HQ728340, HQ728344, HQ728348); Marsilea strigosa .
Russia, N. Dessiatoff 741 (GH) (HQ728341, HQ728345, —).
