an expanded plastid phylogeny of marsilea with emphasis on north american species
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An Expanded Plastid Phylogeny of Marsilea with Emphasis onNorth American SpeciesAuthor(s): W. Mark Whitten, Colette C. Jacono, and Nathalie S. NagalingumSource: American Fern Journal, 102(2):114-135. 2012.Published By: The American Fern SocietyDOI: http://dx.doi.org/10.1640/0002-8444-102.2.114URL: http://www.bioone.org/doi/full/10.1640/0002-8444-102.2.114
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An Expanded Plastid Phylogeny of Marsilea withEmphasis on North American Species
W. MARK WHITTEN
Florida Museum of Natural History, 385 Dickinson Hall, Gainesville, FL 32611-7800, email:
COLETTE C. JACONO
Center for Aquatic and Invasive Plants, University of Florida, 7922 NW 71st Street, Gainesville,
FL 32653-3701, email: [email protected]
NATHALIE S. NAGALINGUM
Royal Botanic Garden Sydney, National Herbarium of New South Wales, Mrs Macquaries Road,
Sydney, NSW, Australia, 2000, email: [email protected]
ABSTRACT.—Ferns of the genus Marsilea (water clover) are potentially invasive aquatic and wetland
plants. They are difficult to identify to species because of subtle diagnostic characters, the sterile
condition of many specimens, and unresolved taxonomic problems. We sequenced four plastid
regions (rbcL, rps4, rps4-trnS spacer, and trnL-F spacer) from 223 accessions across ca. 38 species.
Our goals were to: 1) attempt to identify problematic Marsilea specimens from the southeastern
U.S., and 2) assess species delimitation using molecular data. Florida specimens previously
identified as M. aff. oligospora do not match true M. oligospora (native to the western USA), and
might represent an undescribed native species. The molecular data fail to resolve many species as
monophyletic within the New World Marsilea section Nodorhizae. The data reveal two strongly
supported clades within section Nodorhizae: 1) A western U.S. /Mexican clade; and 2) A U.S. Gulf
coastal plain/Florida/Caribbean clade. This DNA/morphology discordance suggests that these taxa
either may have hybridized extensively or that the number of Marsilea species within these clades
may be overestimated. Either case warrants the addition of nuclear data sets and reevaluation of the
species boundaries within the genus.
KEY WORDS.—Marsilea, phylogenetics, plastid, species delimitations
Marsilea L. (ca. 50 spp.) occur worldwide as two ecological types: 1) trueaquatic species with glabrous leaves and fleshy rhizomes that inhabit morepermanent water bodies, and 2) semi-aquatic species with hairy leaves andtough, fibrous rhizomes that prefer fluctuating wetland habitats and prevailthrough seasonal extremes in wet and dry periods (Jacono and Johnson, 2006).Marsilea have few dependable morphological characters on which to basespecies-level identifications. Phenotypic plasticity is widespread, and sporo-carps, which contain many characters used for species delimitation, arecommonly absent in field populations. Because identification of Marsileabased upon morphology is so difficult, molecular data might provide morereliable tools for identification.
The impetus for this study was an applied resource management need toclarify the identity of three western North American species of Marsilea inFlorida (Jacono and Johnson, 2006). Marsilea vestita Hook. & Grev. and M.macropoda Engelm. ex A. Braun have been regarded as introduced to eastern
American Fern Journal 102(2):114–135 (2012)
North America based on their disjunct and widely scattered populations atruderal sites in Gulf coastal Alabama and Florida. A third species, centered onthree central Florida counties, was tentatively identified as M. aff. oligosporaGoodd. (Jacono and Johnson, 2006) based on sporocarp morphology; however,Marsilea oligospora is a semi-aquatic North American species otherwiseendemic to the northern fringe of the Great Basin. Variation was noted betweenthe Florida and the Great Basin material and it was difficult for the authors tospeculate how a geographically restricted plant with no known economicvalue might have become established in central Florida over 100 years ago. Thegreat difference in climate between northwestern U.S. and Florida added toour suspicion that these were two different taxa. These Florida M. aff.oligospora were first collected in the early 1890s near Eustis, Florida, and theirdetermination has vacillated from M. vestita, an introduction from the westernU.S. (Ward and Hall, 1976) to M. ancylopoda A.Braun, a rare and potentiallyextinct native species (FNA, 1993).
Here we use DNA sequences of four plastid regions (rbcL, rps4, the rps4-trnSspacer, and the trnL-F spacer) to expand upon the recent molecular phylogenyof Marsilea (Nagalingum et al., 2007), using a greater sampling of NorthAmerican specimens. Our first objective was to determine the status of theFlorida plants assigned to M. aff. oligospora. We surveyed all knownpopulations of Marsilea within Florida and compared them to all U.S.,Mexican, and Caribbean species, as well as Marsilea species common in theaquatic plant trade that are established in the southeastern U.S. These data willprovide a baseline for evaluating M. aff. oligospora in Florida and fordistinguishing future introductions of Marsilea. Our second objective is toassess species monophyly using multiple accessions of each species,particularly for the North American specimens assigned to Marsilea sect.Nodorhizae.
MATERIALS AND METHODS
Thirty-three samples were included from Nagalingum et al. (2007), and aredistinguished by the GenBank prefix DQ; the remainder were generated in thisstudy (Table 1). Because Florida collections of M. oligospora were hypothe-sized to be introductions from the western U.S. (Jacono and Johnson, 2006), weincluded as many specimens as possible from western states. Species notpresent in the Nagalingum et al. (2007) study include M. coromandelinaWilld., M. costulifera D.L.Jones, M. crenulata Desv., M. deflexa A.Braun, M.exarata A.Braun, M. fournieri C.Chr., M. hirsuta R.Br., M. mexicana A.Braun,M. mucronata A.Braun, M. scalaripes D.M. Johnson, M. tenuifolia Engelm. exKunze, and M. uncinata A.Braun.
Samples were taken from herbarium specimens. Leaf samples (ca. 25 mm2)were ground using a tissue mill and extracted using a modified version of the23 CTAB procedure of Doyle and Doyle (1987) with exclusion of beta-mercaptoethanol and inclusion of 5 units of proteinase K. Primers for rbcLwere designed to allow amplification and sequencing in two overlapping
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 115
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116 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
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.WHITTEN ET AL.: MARSILEA PHYLOGENETICS 117
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ora
070
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33976
FJ5
33884
FJ5
34067
AS
UG
.F
erg
uso
n118
Mexic
o:
Ch
ihu
ah
ua
071
Mars
ilea
mexic
an
aA
.B
rau
nH
Q631081
HQ
631273
FJ5
34056
AS
UJ.
Her
nan
dez
s.n
.M
exic
o:
Zacate
cas
072
Mars
ilea
fou
rnie
riC
.C
hr.
FJ5
33952
FJ5
33860
FJ5
34045
AS
UJ.
Reb
man
&C
.D
avis
1684
Mexic
o:
Baja
Cali
forn
iaS
ur
073
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34021
FJ5
33928
FJ5
34114
AS
UE
.W
ise
1716
US
A:
CA
:S
an
Lu
isO
bis
bo
074
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34022
FJ5
33929
FJ5
34115
AS
UR
.W
ort
hin
gton
21894
US
A:
NM
:L
un
a
075
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34023
FJ5
33930
FJ5
34116
AS
UD
.K
eil
18024
US
A:
CA
:S
an
Lu
isO
bis
bo
076
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34024
FJ5
33931
FJ5
34117
AS
UL
.M
cG
ill
7203
US
A:
AZ
:C
och
ise
077
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631082
HQ
631274
FJ5
34118
AS
UW
.T
.Jo
hn
son
s.n
.U
SA
:A
Z:
Cocon
ino
078
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34025
FJ5
33932
FJ5
34119
AS
UG
.M
arr
s-S
mit
h1211
US
A:
AZ
:C
och
ise
079
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
34026
FJ5
33933
FJ5
34120
AS
UD
.D
am
rel
627-A
US
A:
AZ
:G
ila
080
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
34027
FJ5
33934
FJ5
34121
AS
UT
.W
righ
t&
M.
Bak
er
93-1
02
US
A:
AZ
:Y
au
pai
081
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631083
HQ
631275
FJ5
34068
AS
UJ.
Coll
ins
s.n
.U
SA
:A
Z:
Coch
ise
082
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33977
FJ5
33885
FJ5
34069
AS
UE
.L
eh
to24541
US
A:
AZ
:C
ocon
ino
083
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33978
FJ5
33886
FJ5
34070
AS
UM
.B
aker
8595
US
A:
AZ
:C
ocon
ino
084
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33979
FJ5
33887
FJ5
34071
AS
UL
.M
cG
ill
6860
US
A:
AZ
:Coch
ise
085
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33980
FJ5
33888
FJ5
34072
AS
UM
.W
ind
ham
0114D
US
A:
AZ
:C
ocon
ino
086
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33981
FJ5
33889
FJ5
34073
AS
UD
.J.
Pin
kava
et
al.
s.n
.U
SA
:A
Z:C
ocon
ino
087
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34006
FJ5
33914
FJ5
34099
VP
IR
.D.
Th
om
as
114754
US
A:
LA
:C
ald
well
088
Mars
ilea
mu
tica
Mett
.F
J533992
FJ5
33900
FJ5
34085
VP
IM
.R
obin
ett
esn
US
A:
VA
:P
atr
ick
089
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34029
FJ5
33936
FJ5
34123
IDC
.R.
Bjo
rk6868
US
A:
ID:
Nez
Perc
e
090
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34030
FJ5
33937
FJ5
34124
IDC
.R.
Bjo
rks.
n.
US
A:
ID:
Nez
Perc
e
091
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34031
FJ5
33938
FJ5
34125
IDA
.T
ieh
m15267
US
A:
NV
:W
ash
oe
TA
BL
E1.
Con
tin
ued
.
118 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
DN
A
sam
ple
nu
mber
Tax
on
rbcL
trn
L-F
rps4
Herb
ari
um
of
dep
osi
tion
Vou
ch
er
Locali
ty
092
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34032
FJ5
33939
FJ5
34126
IDC
.R.
Bjo
rk2404
US
A:
OR
:M
alh
eur
093
Mars
ilea
oli
gosp
ora
Good
d.
FJ5
34000
FJ5
33908
FJ5
34094
IDC
.R.
Bjo
rk3916
US
A:
ID:
Ow
yh
ee
094
Mars
ilea
oli
gosp
ora
Good
d.
FJ5
34001
FJ5
33909
FJ5
34095
IDF
.D.
Joh
nso
nsn
US
A:
ID:
Idah
o
095
Mars
ilea
poly
carp
aH
ook.
&G
rev.
FJ5
34002
FJ5
33910
FJ5
34096
MO
S.R
.H
all
&L
.P
hil
lip
e28868
Dom
inic
a:S
t.A
nd
rew
s
096
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34033
FJ5
33940
FJ5
34127
UT
CN
.&
P.
Holm
gre
nU
SA
:N
V:
Elk
o
097
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34034
FJ5
33941
FJ5
34128
UT
CA
.T
ieh
m14569
US
A:
NV
:E
lko
098
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34035
FJ5
33942
FJ5
34129
UT
CR
.J.
Sh
aw
3708
US
A:
UT
:M
illa
rd
099
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34036
FJ5
33943
FJ5
34130
UT
CW
eber
&W
ittm
an
n18558
US
A:
CO
:B
aca
100
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34037
FJ5
33944
FJ5
34131
UT
CR
.J.
&M
.S
haw
4986
US
A:
MT
:L
ake
101
Mars
ilea
min
uta
L.
FJ5
33973
FJ5
33881
FJ5
34064
MIS
SA
.R.
Dia
mon
d14269
US
A:
AL
:P
ike
102
Mars
ilea
min
uta
L.
FJ5
33974
FJ5
33882
FJ5
34065
MIC
HD
.M.
Joh
nso
n800
Tri
nid
ad:N
ariv
aC
oca
l
103
Mars
ilea
defl
exa
A.
Bra
un
FJ5
33951
FJ5
33859
FJ5
34044
MIC
HD
.M.
Joh
nso
n794
Ven
ezu
ela
:G
uari
co
104
Mars
ilea
an
cylo
pod
aA
.B
rau
nF
J533949
FJ5
33856
FJ5
34042
MIC
HD
.M.
Joh
nso
n773
Arg
en
tin
a:
Corr
ien
tes
105
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631084
FJ5
33857
HQ
631177
MIC
HE
.L
ott
&A
.S
an
ders
3987
Mexic
o:
Jali
sco:
Qu
em
aro
106
Mars
ilea
min
uta
L.
FJ5
33975
FJ5
33883
FJ5
34066
MIC
HM
.D
yer
173
Nig
eria
:K
an
o:
Ach
aL
afi
a
107
Mars
ilea
poly
carp
aH
ook.
&G
rev.
FJ5
34003
FJ5
33911
HQ
631178
MIC
HW
.W
agn
er
Pu
erto
Ric
o:
Loiz
a
108
Mars
ilea
poly
carp
aH
ook.
&G
rev.
FJ5
34004
FJ5
33912
FJ5
34097
MIC
HD
.M.
Joh
nso
n793
Ven
ezu
ela
:A
pu
re
109
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
FJ5
33982
FJ5
33890
FJ5
34074
MIC
HN
.Mu
rray
&D
.M.J
oh
nso
n1404
Mexic
o:
Ch
iap
as
110
Mars
ilea
vest
ita
Hook.
&G
rev.
FJ5
34038
FJ5
33945
FJ5
34132
MIC
HB
.E
rtte
ret
al.
8131
US
A:C
A:C
on
tra
Cost
a
111
Mars
ilea
macro
pod
aE
nge
lm.
ex
A.
Bra
un
FJ5
33964
FJ5
33872
FJ5
34055
MIC
HD
.M.
Joh
nso
n721
US
A:
TX
:A
ran
sas
112
Mars
ilea
coro
man
deli
na
Wil
ld.
FJ5
33950
FJ5
33858
FJ5
34043
MIC
HM
.D
yer
172
Nig
eria
:K
an
o:
Ach
aL
afi
a
116
Mars
ilea
mu
tica
Mett
.H
Q631085
FJ5
33946
FJ5
34133
VP
IR
.P
age
s.n
.U
SA
:V
A:
Han
over
119
Mars
ilea
nash
iiU
nd
erw
.H
Q631086
HQ
631276
HQ
631179
FL
AS
R.J
.A
bbott
8678
US
A:
FL
:D
ad
e
121
Mars
ilea
qu
ad
rifo
lia
L.
HQ
631087
HQ
631277
HQ
631180
FL
AS
C.
Jacon
o622
US
A:
PA
:B
rad
ford
122
Mars
ilea
poly
carp
aH
ook.
&G
rev.
HQ
631088
HQ
631278
HQ
631181
FL
AS
R.
Dre
ssle
r6004
Pan
am
a:P
an
am
a
TA
BL
E1.
Con
tin
ued
.WHITTEN ET AL.: MARSILEA PHYLOGENETICS 119
DN
A
sam
ple
nu
mber
Taxon
rbcL
trn
L-F
rps4
Herb
ariu
m
of
dep
osi
tion
Vou
ch
er
Locali
ty
124
Mars
ilea
cost
uli
fera
D.L
.Jo
nes
HQ
631089
HQ
631279
HQ
631182
FL
AS
M.
Wh
itte
n3756
Au
stra
lia:
NS
W
125
Mars
ilea
vil
losa
Kau
lf.
HQ
631090
HQ
631280
HQ
631183
BIS
HM
ari
an
Ch
au
002
US
A:
HI:
Oah
u
127
Mars
ilea
vil
losa
Kau
lf.
HQ
631091
HQ
631281
HQ
631184
BIS
HM
ari
an
Ch
au
029
US
A:
HI:
Oah
u
128
Mars
ilea
vil
losa
Kau
lf.
HQ
631092
HQ
631282
HQ
631185
BIS
HM
ari
an
Ch
au
022
US
A:
HI:
Molo
ka’
i
129
Mars
ilea
vil
losa
Kau
lf.
HQ
631093
HQ
631283
HQ
631186
BIS
HM
ari
an
Ch
au
015
US
A:
HI:
Molo
ka’
i
130
Mars
ilea
an
gu
stif
oli
aR
.B
r.H
Q631094
HQ
631284
HQ
631187
ME
LI.
D.
Cow
ie9345
Au
stra
lia:
NT
131
Mars
ilea
cre
nata
C.
Pre
slH
Q631095
HQ
631285
HQ
631188
ME
LG
.W
igh
tman
1458
Au
stra
lia:
NT
132
Mars
ilea
cost
uli
fera
D.L
.Jo
nes
HQ
631096
HQ
631286
HQ
631189
ME
LI.
Tan
kard
s.n
.A
ust
rali
a:
UC
133
Mars
ilea
exara
taA
.B
rau
nH
Q631097
HQ
631287
HQ
631190
ME
LP
.K.
Latz
11823
Au
stra
lia:
NT
134
Mars
ilea
exara
taA
.B
rau
nH
Q631098
HQ
631288
HQ
631191
ME
LM
.E.
Tru
dgen
15576
Au
stra
lia:
WA
135
Mars
ilea
exara
taA
.B
rau
nH
Q631099
HQ
631289
HQ
631192
ME
LP
.I.
Fors
ter
20421
Au
stra
lia:
QL
D
136
Mars
ilea
hir
suta
R.
Br.
HQ
631100
HQ
631290
HQ
631193
ME
LK
.A
lcock
s.n
.A
ust
rali
a:
SA
137
Mars
ilea
hir
suta
R.
Br.
HQ
631101
HQ
631291
HQ
631194
ME
LP
.I.
Fors
ter
20681
Au
stra
lia:
QL
D
138
Mars
ilea
hir
suta
R.
Br.
HQ
631102
HQ
631292
HQ
631195
ME
LI.
D.
Cow
ie9590
Au
stra
lia:
NT
139
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631103
HQ
631293
HQ
631196
CA
SM
.L.
Arr
egu
in589
Mexic
o
141
Mars
ilea
min
uta
L.
HQ
631104
HQ
631294
HQ
631197
CA
SD
.M.
Joh
nso
n800
Tri
nid
ad:N
ariv
aC
oca
l
144
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631105
HQ
631295
HQ
631198
CA
SE
.J.
Lott
3987
wit
h
A.C
.S
an
ders
Mexic
o:
Jali
sco:
Mp
io
La
Hu
ert
a
145
Mars
ilea
mu
cron
ata
A.
Bra
un
HQ
631106
HQ
631296
HQ
631199
CA
SB
.E
rtte
r3894
wit
hJ.
Str
ach
anU
SA
:C
A:
Plu
man
s
146
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631107
HQ
631297
HQ
631200
CA
SA
.D
ay
83-5
9U
SA
:C
A:
Merc
ed
147
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631108
HQ
631298
HQ
631201
CA
SL
.A
hart
4404
US
A:
CA
:S
utt
er
148
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631109
HQ
631299
HQ
631202
CA
SA
.T
ieh
m11938
US
A:
NV
:E
lko
149
Mars
ilea
qu
ad
rifo
lia
L.
na
HQ
631300
HQ
631203
CA
SB
.B
arth
olo
mew
et
al.
Gu
izh
ou
Bot.
Exp
ed
.2380
Ch
ina:
Gu
izh
ou
150
Mars
ilea
poly
carp
aH
ook.
&G
rev.
na
HQ
631301
HQ
631204
CA
SW
.H.
Wagn
er
82018
Pu
ert
oR
ico:
Loiz
a
151
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631110
HQ
631302
HQ
631205
CA
SJ.
T.
How
ell
36949
US
A:
CA
:P
lum
as
152
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631111
HQ
631303
HQ
631206
CA
SA
.T
ieh
m13199
wit
hG
.S
ch
oolc
raft
US
A:
NV
:W
ash
oe
TA
BL
E1.
Con
tin
ued
.
120 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
DN
A
sam
ple
nu
mber
Taxon
rbcL
trn
L-F
rps4
Her
bari
um
of
dep
osi
tion
Vou
ch
erL
ocali
ty
154
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631112
HQ
631304
HQ
631207
JEP
SV
.H.
Osw
ald
&L
.A
hart
9591
US
A:
CA
:M
od
oc
155
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631113
HQ
631305
HQ
631208
JEP
SV
.H.
Osw
ald
&L
.A
hart
9597
US
A:
CA
:M
od
oc
156
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631114
HQ
631306
HQ
631209
JEP
SL
.A
hart
13088
US
A:
CA
:B
utt
e
157
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631115
HQ
631307
HQ
631210
JEP
SL
.A
hart
14483
US
A:
CA
:B
utt
e
158
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631116
HQ
631308
HQ
631211
JEP
SL
.A
hart
14579
US
A:
CA
:Y
uba
159
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631117
HQ
631309
HQ
631212
JEP
SL
.A
hart
9320
US
A:
CA
:P
lum
as
160
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631118
HQ
631310
HQ
631213
UC
B.
Ert
ter
9696
US
A:
CA
:A
mad
or
161
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631119
HQ
631311
HQ
631214
UC
V.H
.O
swal
d&
L.
Ah
art
4837
US
A:
CA
:B
utt
e
162
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631120
HQ
631312
HQ
631215
UC
V.H
.O
swal
d&
L.
Ah
art
5126
US
A:
CA
:L
ass
en
165
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631121
HQ
631313
HQ
631216
UC
R.
B.
Hayw
ard
et
al.
56
Ven
ezu
ela
:Z
uli
a
167
Mars
ilea
cost
uli
fera
D.L
.Jo
nes
HQ
631122
HQ
631314
HQ
631217
UC
R.G
.C
oven
yet
al.
12653
Au
stra
lia:
NS
W
169
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631123
HQ
631315
HQ
631218
UC
D.M
.Jo
hn
son
769
Arg
en
tin
a:
Corr
ien
tes
170
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631124
HQ
631316
HQ
631219
UC
E.J
.L
ott
3987
wit
h
A.C
.S
an
ders
Mexic
o:
Jali
sco
171
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631125
HQ
631317
HQ
631220
UC
A.C
.S
an
ders
et
al.
13527
Mexic
o:
Son
ora
172
Mars
ilea
qu
ad
rifo
lia
L.
HQ
631126
HQ
631318
HQ
631221
UC
B.
Dic
kore
13374
Pakis
tan
173
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631127
HQ
631319
HQ
631222
UC
A.
Tie
hm
12601
US
A:
NV
:W
ash
oe
174
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631128
HQ
631320
HQ
631223
UC
M.
Leh
nert
745
Boli
via
:T
ari
ja
175
Mars
ilea
defl
exa
A.
Bra
un
HQ
631129
HQ
631321
HQ
631224
UC
R.
Ru
ed
a&
R.
Dolm
us
1221
Nic
ara
gua
176
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631130
HQ
631322
HQ
631225
CIC
YJ.
L.
Tap
iaet
al.
1597
Mexic
o:
Yu
cat
an
177
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631131
HQ
631323
HQ
631226
F(U
S)
S.
Lla
tas
&Q
uir
oz
2401
Peru
:S
an
Nic
ola
s
179
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631132
HQ
631324
HQ
631227
US
J.P
rusk
i3743
US
A:
LA
:O
rlean
s
182
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631133
HQ
631325
HQ
631228
US
H.
van
der
Werf
f8708
US
A:
CA
:S
an
Die
go
186
Mars
ilea
exara
taA
.B
rau
nH
Q631134
HQ
631326
HQ
631229
US
A.
Fad
en
1/9
1A
ust
rali
a:
QL
D
187
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631135
HQ
631327
HQ
631230
QC
AT
ern
eu
s&
Gon
zale
z347
Ec
uad
or:
Imb
ab
ura
:
Lago
San
Pablo
188
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631136
HQ
631328
HQ
631231
GH
R.
Mora
n28429
Mex
ico:B
aja
Cal
iforn
ia
189
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631137
HQ
631329
HQ
631232
GH
P.H
.R
aven
16601
US
A:
CA
:S
tan
isla
us
190
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631138
HQ
631330
HQ
631233
GH
G.
Car
nevali
et
al.
6740
Mexic
o:
Yu
cat
an
TA
BL
E1.
Con
tin
ued
.WHITTEN ET AL.: MARSILEA PHYLOGENETICS 121
DN
A
sam
ple
nu
mber
Taxon
rbcL
trn
L-F
rps4
Herb
ariu
m
of
dep
osi
tion
Vou
ch
er
Locali
ty
192
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631139
HQ
631331
na
GH
V.
Soli
sN
effa
001
Arg
en
tin
a:
Corr
ien
tes
194
Mars
ilea
ten
uif
oli
aE
nge
lm.
ex
Ku
nze
HQ
631140
HQ
631332
HQ
631235
OW
UD
.M.
Joh
nso
n2124
US
A:
TX
:M
aso
n
195
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631141
HQ
631333
HQ
631236
OW
UD
.M.
Joh
nso
n2123
US
A:
LA
:L
aS
all
e
Pari
sh
196
Mars
ilea
an
cylo
pod
aA
.B
rau
nH
Q631142
HQ
631334
HQ
631237
NY
N.A
.Mu
rray
&D
.M.J
oh
nso
n1458
Mexic
o:
Nayar
it
197
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631143
HQ
631335
HQ
631238
NY
W.T
.B
ark
er1836
US
A:
KS
:K
iow
a
199
Mars
ilea
oli
gosp
ora
Good
d.
HQ
631144
HQ
631336
HQ
631239
NY
D.
Lytj
en
131
US
A:
OR
:M
alh
eur
200
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631145
HQ
631337
HQ
631240
NY
K.T
horn
e&
S.G
ood
rich
3550
US
A:
UT
:U
inta
h
201
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631146
HQ
631338
HQ
631241
NY
A.
Tie
hm
11766
US
A:
NV
:P
ers
hin
g
203
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631147
HQ
631339
HQ
631242
NY
N.H
.&
P.K
.H
olm
gre
n9585
US
A:
OR
:L
ake
204
Mars
ilea
poly
carp
aH
ook.
&G
rev.
HQ
631148
HQ
631340
HQ
631243
NY
M.B
ou
dri
e&
S.G
on
zale
z4191
Fre
nch
Gu
yan
a
205
Mars
ilea
min
uta
L.
HQ
631149
HQ
631341
HQ
631244
NY
D.M
.Jo
hn
son
798
Tri
nid
ad
207
Mars
ilea
exara
taA
.B
rau
nH
Q631150
HQ
631342
HQ
631245
NY
F.J
.B
ad
man
6949
Au
stra
lia:
SA
208
Mars
ilea
hir
suta
R.
Br.
HQ
631151
HQ
631343
HQ
631246
NY
C.R
.M
ich
ell
&D
.S.C
alli
ss994
Au
stra
lia:
NT
211
Mars
ilea
cre
nu
lata
Desv
.H
Q631152
HQ
631344
HQ
631247
NY
J.E
.M
ad
sen
6013
Bu
rkin
aF
aso:O
ud
alan
216
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631153
HQ
631345
HQ
631248
NY
L.
Ah
art
2376
US
A:
CA
:B
utt
e
217
Mars
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vest
ita
Hook.
&G
rev.
HQ
631154
HQ
631346
HQ
631249
NY
N.H
.&
P.K
.H
olm
gre
n14745
US
A:
NV
:E
lko
220
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631155
HQ
631347
HQ
631250
NY
R.
Mora
n28429
Mexic
o:
BC
222
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631156
HQ
631348
HQ
631251
NY
Bis
seet
al.
32886
Cu
ba
224
Mars
ilea
un
cin
ata
A.
Bra
un
HQ
631157
HQ
631349
HQ
631252
NY
S.W
.L
eon
ard
et
al.
8495
US
A:
FL
:F
ran
kli
n
225
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631158
HQ
631350
HQ
631253
NY
A.
Tay
e4957
US
A:
UT
:P
iute
227
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
HQ
631159
HQ
631351
HQ
631254
NY
R.
McV
au
gh
16917
Mexic
o:
Jali
sco
230
Mars
ilea
defl
exa
A.
Bra
un
HQ
631160
HQ
631352
HQ
631255
NY
J.T
.M
ickel
2856
Cost
aR
ica:
Gu
anac
aste
231
Mars
ilea
defl
exa
A.
Bra
un
HQ
631161
HQ
631353
HQ
631256
NY
R.
McV
au
gh
19287
Mexic
o:
Nayar
it
233
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631162
HQ
631354
HQ
631257
TX
W.R
.C
arr
25313
US
A:
TX
:M
cM
ull
en
234
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631163
HQ
631355
HQ
631258
TX
L.L
.H
anse
n5163
US
A:
TX
:C
ory
ell
235
Mars
ilea
macro
pod
aE
nge
lm.
ex
A.
Bra
un
HQ
631164
HQ
631356
HQ
631259
TX
W.R
.C
arr
24546
US
A:
TX
:G
oli
ad
239
Mars
ilea
ep
hip
pio
carp
aA
lsto
nH
Q631165
HQ
631357
HQ
631260
AA
PR
E99559
Nam
ibia
TA
BL
E1.
Con
tin
ued
.
122 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
TA
BL
E1.
Con
tin
ued
.
DN
A
sam
ple
nu
mber
Taxon
rbcL
trn
L-F
rps4
Herb
ariu
m
of
dep
osi
tion
Vou
ch
er
Locali
ty
240
Mars
ilea
cre
nata
C.
Pre
slH
Q631166
HQ
631358
HQ
631261
FL
AS
Wh
itte
n3765
Th
ail
an
d
241
Mars
ilea
scala
rip
es
D.M
.Jo
hn
son
HQ
631167
HQ
631359
HQ
631262
FL
AS
Wh
itte
n3766
Th
ail
an
d
242
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631168
HQ
631360
HQ
631263
ME
XU
R.S
.F
elg
er85-5
88
Mexic
o:
Son
ora
243
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631169
HQ
631361
HQ
631264
ME
XU
R.S
.F
elg
er01-7
46
Mexic
o:
Son
ora
244
Mars
ilea
vest
ita
Hook.
&G
rev.
HQ
631170
HQ
631362
HQ
631265
ME
XU
R.S
.F
elg
er85-1
118
Mexic
o:
Son
ora
256
Mars
ilea
aegy
pti
ca
Wil
ld.
DQ
643291
DQ
643359
DQ
536323
BM
Sm
ith
3623
Nam
ibia
257
Mars
ilea
an
cylo
pod
aA
.B
rau
nD
Q643292
DQ
643360
DQ
536324
FP
ryer
et
al.
963
Pu
ert
oR
ico
258
Mars
ilea
an
gu
stif
oli
aR
.B
r.D
Q643293
DQ
643361
DQ
536325
UC
Hosh
izaki
1250
Au
stra
lia
259
Mars
ilea
botr
yocarp
aB
alla
rdD
Q643294
DQ
643362
DQ
536326
UC
Fad
en
s.n
.K
en
ya
260
Mars
ilea
cap
en
sis
A.
Bra
un
DQ
643295
DQ
643363
DQ
536327
DU
KE
Ram
berg
s.n
.B
ots
wan
a
261
Mars
ilea
cre
nata
C.
Pre
slD
Q643296
DQ
643364
DQ
536328
DU
KE
Kato
J-38
Ind
on
esi
a
262
Mars
ilea
cre
nata
C.
Pre
slD
Q643297
DQ
643365
DQ
536329
DU
KE
Kato
s.n
.T
hail
an
d
263
Mars
ilea
cro
top
hora
D.M
.Jo
hn
son
DQ
643298
na
DQ
536330
HR
itte
ret
al.
4561
Boli
via
264
Mars
ilea
dis
tort
aA
.B
rau
nn
an
aD
Q536331
BM
Korn
as
6271
Nig
eri
a
265
Mars
ilea
dru
mm
on
dii
A.
Bra
un
DQ
643299
DQ
643366
DQ
536332
UC
Hosh
izaki
577
Au
stra
lia
266
Mars
ilea
ep
hip
pio
carp
aA
lsto
nD
Q643300
na
na
UC
Ch
ase
2255
Zim
babw
e
267
Mars
ilea
fad
enia
na
Lau
nert
DQ
643301
DQ
643367
DQ
536333
US
Evan
san
dM
aik
weki
55
Ken
ya
268
Mars
ilea
fari
nosa
Lau
nert
DQ
643302
DQ
643368
DQ
536334
US
Fad
en
70/9
02
Ken
ya
269
Mars
ilea
gib
ba
A.
Bra
un
DQ
643303
DQ
643369
DQ
536335
US
Fad
en
an
dN
g’w
en
o87/3
3K
en
ya
270
Mars
ilea
macro
carp
aC
.P
resl
DQ
643304
DQ
643370
DQ
536336
UC
Hosh
izaki
236
Sou
thA
fric
a
271
Mars
ilea
macr
op
od
aE
ngelm
.ex
A.
Bra
un
DQ
643305
DQ
643371
DQ
536337
DU
KE
Hosh
izaki
1458
US
A:
TX
272
Mars
ilea
min
uta
L.
DQ
643306
DQ
643372
DQ
536338
DU
KE
Sh
imozon
os.
n.
Bu
rma
273
Mars
ilea
min
uta
L.
DQ
643307
na
DQ
536339
DU
KE
Raj
esh
87938
Ind
ia
274
Mars
ilea
min
uta
L.
DQ
643308
DQ
643373
DQ
536340
DU
KE
Hosh
izaki
237
Afr
ica
275
Mars
ilea
moll
isB
.L.
Rob.
&F
ern
ald
na
na
DQ
536341
FJo
hn
son
s.n
.M
exic
o
276
Mars
ilea
mu
tica
Mett
.D
Q643309
DQ
643374
DQ
536342
DU
KE
Hosh
izaki
840
New
Cal
ed
on
ia
277
Mars
ilea
nash
iiU
nd
erw
.D
Q643311
DQ
643376
DQ
536344
DU
KE
Corr
ell
s.n
.W
est
Ind
ies
278
Mars
ilea
nash
iiU
nd
erw
.D
Q643310
DQ
643375
DQ
536343
FC
orr
ell
46631
Tu
rks
&C
aic
os
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 123
DN
A
sam
ple
nu
mber
Taxon
rbcL
trn
L-F
rps4
Herb
ariu
m
of
dep
osi
tion
Vou
ch
er
Locali
ty
279
Mars
ilea
nu
bic
avar.
gym
nocarp
a(A
.B
rau
n)
Lau
nert
DQ
643312
na
DQ
536345
BM
Sm
ith
1988
Bots
wan
a
280
Mars
ilea
nu
bic
avar.
gym
nocarp
a(A
.B
rau
n)
Lau
nert
DQ
643313
na
DQ
536346
BM
Korn
as
6379
Nig
eri
a
281
Mars
ilea
oli
gosp
ora
Good
d.
DQ
643314
DQ
643377
DQ
536347
UC
Tie
hm
13199
US
A:
NV
282
Mars
ilea
poly
carp
aH
ook.
&G
rev.
DQ
643315
DQ
643378
DQ
536348
DU
KE
Pry
er
960
Pu
ert
oR
ico
283
Mars
ilea
qu
ad
rifo
lia
L.
DQ
643316
DQ
643379
DQ
536349
DU
KE
An
no
s.n
.Ja
pan
284
Mars
ilea
sch
elp
ean
aL
au
nert
DQ
643317
DQ
643380
DQ
536350
DU
KE
Hon
shiz
aki
742
Sou
thA
fric
a
285
Mars
ilea
vera
Lau
nert
DQ
643318
na
DQ
536351
BM
Bu
rrow
s3716
Bots
wan
a
286
Mars
ilea
vest
ita
Hook.
&G
rev.
DQ
643319
DQ
643381
DQ
536352
US
How
ell
47460
US
A:
CA
287
Mars
ilea
vil
lifo
lia
Bre
m.
&O
berm
.
ex
Als
ton
&S
ch
elp
e
DQ
643320
na
DQ
536353
BM
Han
sen
3232
Bots
wan
a
288
Mars
ilea
vil
losa
Kau
lf.
DQ
643321
DQ
643382
DQ
536354
US
Degen
er
9049
US
A:
HI
289
Pil
ula
ria
am
eri
can
aA
.B
rau
nD
Q643288
DQ
643383
DQ
536355
DU
KE
Pry
er
978
US
A:
GA
TA
BL
E1.
Con
tin
ued
.
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FIG. 1. (A–C). A single randomly-chosen shortest tree from maximum parsimony analysis of
Marsilea combined plastid DNA data matrix (rbcL, rps4, rps4-trnS spacer, and trnL-F spacer).
Branch lengths are indicated by scale bars (except for longer branches of Fig. 1a); bootstrap support
is indicated by branch thickness/grayscale. Nodes that collapse in the strict consensus are marked
with a black dot. Tree length 5 743; consistency index (CI) 5 0.80; retention index (RI) 5 0.96.
Major clades are labeled A–L; the informal clade names (groups and subgroups) correspond to
those used in Nagalingum et al. (2007).
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 125
pieces, facilitating amplification from degraded total DNAs. Primers for rbcLare: rbcLF ATGTCACCACAAACAGAGACTAAAGC; rbcL intF TGAGAACG-TAAACTCCCAACCATTCA; rbcL intR CTGTCTATCGATAACAGCATGCAT;and rbcLR GCAGCAGCTAGTTCCGGGCTCCA. The rps4 exon and the adjacent
FIG. 1 Continued.
126 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
rps4-trnS spacer were amplified in one piece using the primers rps4FATGTCCCGTTATCGAGGACCT and rps4R TACCGAGGGTTCGAATC; prob-lematic samples were amplified in two pieces using the internal primers rps4intF TGCCAAACGAGAATCTATGG and rps4 intR CGATGGGTTGT-TAGTTGTTAG. Primers for the trnL-F spacer (primers E&F) were those ofNagalingum et al. (2007). All amplifications utilized Sigma Jumpstart Taqpolymerase and reagents (Sigma-Aldrich, Inc., St. Louis, MO, USA) in 25 mlreactions with 3.0 mM MgCl2. Thermocycler conditions were: 94 uC for
FIG. 1 Continued.
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 127
3 minutes followed by 37 cycles of 94 uC for 30 s, 56 uC for 30 s, 72 uC for 2 min,with a final extension of 3 min at 72 uC. Problematic taxa were amplified usingPhusion polymerase (New England Biolabs, Ipswitch, MA, USA) according tomanufacturer’s protocols. PCR products were sequenced in both directionsusing the Big Dye Terminator reagents on an 3130 automated sequencerfollowing manufacturer’s protocols (Applied Biosystems, Inc.). Electrophero-grams were edited and assembled using Sequencher 4.10 (GeneCodes Inc.,Ann Arbor, MI, USA), and the resulting sequences were aligned manuallyusing SE-AL (Rambaut, 1996). All sequences were deposited in GenBank(Table 1). A 25 bp portion of the rps4-trnS spacer contained a homopolymerregion of ambiguous alignment; this region was excluded from analyses. Weanalyzed the data using maximum parsimony rather than maximum likelihoodbecause the number of steps in the resulting trees more clearly represents thenumber of base pair differences among accessions. Analyses were performedusing PAUP* version 4.0 b10 (Swofford, 2003) with Fitch parsimony (equalweights, unordered characters, ACCTRAN optimization and gaps treated asmissing data). Heuristic searches consisted of 1000 random taxon additionreplicates of subtree-pruning-regrafting (SPR) and ‘‘keeping multiple trees’’(MULTREES) with the number of trees limited to 10 per replicate to minimizeextensive swapping on islands with many suboptimal trees; 10,000 shortesttrees were saved. Support was estimated by 1000 bootstrap (BS) replicates,saving only 5 trees per replicate and ten trees per bootstrap replicate. The datamatrix is available from the senior author or at ftp://ftp.flmnh.ufl.edu/Public/Marsilea/.
RESULTS
In total, our dataset comprised 2629 characters for a total of 223 ingroupaccessions, plus Pilularia americana A.Braun. We used existing sequence datafor 33 accessions from 26 species and newly sequenced data for an additional190 accessions from 12 species (Table 1).
Figure 1 (a, b, c) presents a single randomly-chosen maximum parsimony(MP) phylogram out of 10,000 shortest trees saved. Tree length 5 743;consistency index (CI) 5 0.80; retention index (RI) 5 0.96; ACCTRANoptimization. BS values are indicated by line thickness and shading of branches.
The DNA data revealed that several specimens sampled in this study weremisdetermined (based upon their anomalous placement in the tree andreexamination of the voucher specimens). DNA data were especially effectivein clarifying the identification of sterile specimens of both North Americanand introduced origin.
The cladogram is distinguished by a basal dichotomy separating twostrongly supported clades, earlier designated informally as Groups I and II(Schneider and Pryer, 2001; Nagalingum et al., 2007). Group I comprisesinformal subgroups ‘‘mutica’’/A and ‘‘clemys’’/B, and Group II includessubgroups ‘‘capensis’’, ‘‘macrocarpa’’, ‘‘nubica’’, ‘‘marsilea I–III’’, and ‘‘no-dorhizae I–IV’’, here designated Clades C through H, respectively. Clades A
128 AMERICAN FERN JOURNAL: VOLUME 102 NUMBER 2 (2012)
and C–H are Old World (Launert, 1968) and Clades A–G have glabrous leaves.Clade H includes hairy-leafed species from Australia. Clades I, J, K, and L areNew World, have hairy leaves typical of the semi-aquatic ecotype, and includethe majority of the specimens sequenced in this study. These latter four cladesare united by high BS support into a single clade that corresponds to Johnson’sMarsilea sect. Nodorhizae (Johnson, 1986; Nagalingum et al., 2007), whichincludes six species (plus many names that Johnson synonymized).
Clade A is monotypic, consisting only of M. mutica Mett. It is clearly distinctfrom all other taxa in terms of DNA sequence and morphology, with its two-toned leaflets and petioles inflated at the apex to function as air bladders forfloating leaves. This species has elliptical sporocarps that lack a transversevein, are borne at the base of the petiole, and are either solitary or in clusters of2–4 on branched pedicels. Indigenous to Australia and New Caledonia, M.mutica may be the most popular species in the water garden trade. Thesoutheastern U.S. specimens plus one from Oklahoma are genetically distinctfrom specimens from Arizona and Virginia, a result suggestive of at least twodistinct geographic origins for material introduced into the U.S.
Clade B includes several species that share the distinctive feature of linearrows of globose sporocarps borne on the petiole and a transverse sporocarpveining; this clade corresponds to Marsilea sect. Clemys (Johnson, 1986, 1988).The inclusion of M. scalaripes and M. deflexa in this clade confirms theirhypothesized placement in the clemys subgroup (Nagalingum et al., 2007).However, these plastid data do not resolve the sampled taxa into monophyleticspecies. There are two well-supported (between 90–100% BS) clades, both ofwhich include samples of M. polycarpa Hook. & Grev. and M. deflexa. The non-monophyly of species in this clade and Johnson’s (1986) putative designation ofhybrids of these species may warrant a reexamination of determinations of thesespecimens and/or species concepts. Sample #175 from Nicaragua is sterile andits determination as M. deflexa is tentative.
Clade C contains five African species: M. capensis A.Braun, M. gibbaA.Braun, M. crenulata Desv., M. distorta, and M. coromandelina, which asdescribed by Launert (1968) are all of the glabrous leaflet type. Although thisclade is strongly supported (100% BS), the plastid data fail to fully resolverelationships among these species.
Clade D contains eight African species: M. schelpeana Launert, M. aegypticaWilld., M. botryocarpa Ballard, M. ephippiocarpa Alston, M. farinosa Launert,and M. macrocarpa C.Presl, and partial plastid data also place M. vera Launertand M. villifolia Brem. & Oberm. ex Alston & Schelpe in this clade. In contrastto Clade C, all eight species of Clade D are of the hairy leaflet type (Launert,1968).
Clade E consists of two samples of M. nubica A.Braun, a glabrous speciesfrom Africa that forms abundant colonies (Launert, 1968).
Clade F consists entirely of M. quadrifolia L., the type species of the genus,the only glabrous species from a cool-temperate climate, and a protectedspecies in Europe. Four accessions from different continents, both native andintroduced in range, display little sequence variation.
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 129
Clade G is moderately supported (84% BS) and includes a single accessionof the African species M. fadeniana Launert, several Asian accessions of M.crenata C.Presl, and numerous accessions of M. minuta L., including severalfrom introduced populations in the southeastern U.S. and Trinidad. The M.crenata – M. minuta complex is one of the largest and most variable groupswithin the genus (Launert, 1968). Earlier molecular data showed that M.crenata was nested within M. minuta (Nagalingum et al., 2007), and theaddition of more accessions provides additional evidence that the two taxa arelikely conspecific. Three samples from Trinidad (introduced) form amoderately supported clade with samples from Kenya and Nigeria. A singleaccession (#138) originally determined as M. hirsuta was probably misde-termined, but was not available for examination.
Clade H includes Australian hairy-leaved species: M. drummondii A.Braun,M. exarata, M. hirsuta R.Br., M. angustifolia R.Br., and M. costulifera. There areseveral subclades resolved, but only one has high (90–100%) bootstrapsupport. None of the species within this clade are resolved as monophyletic.DNA data fail to distinguish M. hirsuta from M. angustifolia. Morphologically,M. angustifolia differs from M. hirsuta in having smaller and more elongatedleaves and smaller sporocarps (Aston, 1973). These characters, however, aretypically considered insufficient for species distinction within the genus(Launert, 1968). This clade includes a single specimen (#131) determined asM. crenata; it is probably misdetermined, as all other specimens of M. crenatafall in Clade G.
The majority of the specimens sampled are in Clades I, J, K, and L; theseform a highly supported group that include all species native to North andSouth America. Species within each clade are poorly resolved due to lowsequence divergence. Both clades K and L include members of a complex ofmainly North American species related to M. vestita Hook. & Grev. and M.oligospora. Although they receive moderate to high bootstrap support, cladesK and L correlate strongly with geographic origin (K5U.S. Gulf coastal plain,Yucatan, Mexico, and the northern Caribbean; L5 Mexico, western U.S., andHawaii), but not with accepted species concepts.
Clade I consists primarily of accessions of M. mollis B.L.Rob. & Fernald fromnorth central Mexico, Arizona, and one from Bolivia. One specimen fromZacatecas, Mexico is determined as M. mexicana; the molecular data do notdistinguish it from M. mollis.
Clade J has partially resolved but unsupported internal structure andincludes M. aff. oligospora from Florida, M. ancylopoda from west-centralMexico, Puerto Rico and northeastern Argentina, plus one sterile sample(#187) originally determined as M. mollis from Andean Ecuador (Lago SanPablo). Johnson (1986) cited three sterile collections of M. mollis from thissame lake and suggested that many sterile Andean collections above 1500 mare probably referable to M. mollis. Our molecular data indicate theseEcuadorian collections are not M. mollis, but instead belong to this clade thatincludes M. ancylopoda.
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Sample #38 (M. vestita from Louisiana) is sister to all other taxa in this cladein the strict consensus of all trees; its anomalous placement caused us toresequence this specimen, but the second sequence was identical to the first.
Clade K includes specimens of M. vestita, M. macropoda, and one of M.uncinata from the Gulf coastal plain of the southeastern U.S., together withseveral accessions of M. nashii from Yucatan and the northern Caribbean.Johnson (1986) regarded M. uncinata as a synonym of M. vestita, butconsidered M. nashii to be a valid species distinguished by its stronglynodding sporocarps (vs. slightly nodding to ascending in M. vestita), a featurewhich we have found to vary greatly across and within species, presumably inresponse to the microenvironment under which sporocarps develop. Themolecular data provide no resolution within this clade.
Clade L consists mostly of specimens of M. vestita and M. oligospora fromcentral Texas through the western United States and northwestern Mexico,plus a specimen of M. mucronata A.Braun from California, which Johnson(1986) regarded as a synonym of M. vestita. It also includes two specimens ofM. ancylopoda from Venezuela and Peru, but they are not resolved as sistertaxa. The clade also includes several accessions of M. villosa Kaulf., anendangered Hawaiian endemic, which form a weakly supported clade with M.vestita and M. fournieri, both from Baja California, Mexico. Johnson (1986)considered M. fournieri C.Chr. to be a small-leaved form of M. vestita. This treeis consistent with the hypothesis that M. villosa arose via long-distancedispersal of M. vestita from western Mexico to the dry lowlands of Moloka’i,Ni’ihau, and O’ahu where seasonal flooding of shallow depressions offersrestricted habitats (Wester, 1994). This clade also includes samples of M.oligospora from northern California and Idaho; the type locality of this speciesis in Wyoming (see discussion of M. aff. oligospora in Florida in clade J). Theten samples of M. oligospora are not monophyletic and are scatteredthroughout this clade, but without resolution or support.
DISCUSSION
Evaluation of Florida Marsilea aff. oligospora
Based on our phylogenetic trees, the eight accessions of M. aff. oligosporafrom central Florida (samples 30–37) fall within Clade J; these plants form aweakly supported clade distinct from all others and are sister to M. ancylopodafrom Mexico and Argentina. These eight plants also share a four basepairinsertion in the trnL-F spacer that is absent in all other Marsilea; this indel isan unambiguous synapomorphy that distinguishes these Florida plants. Jaconoand Johnson (2006) tentatively identified these populations as M. aff.oligospora, although noting subtle morphological differences from westernU.S. M. oligospora, and they regarded the Florida populations as introductionsfrom the western U.S. Our data contradict their hypothesis; ‘‘true’’ M.oligospora (e.g., samples 93 and 94, from near the type locality in the westernU.S.; Jackson Hole, Wyoming) fall in clade L, and our data clearly distinguish
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 131
the Florida populations from all other taxa. The molecular data indicate thatthese Florida populations are nested within M. ancylopoda from Mexico,Puerto Rico, Argentina, and Ecuador (based on current sampling).
According to Johnson’s (1986) morphological concept of M. ancylopoda, thespecies includes considerable variation in sporocarp morphology, but thesporocarps always lack a superior tooth. The Florida populations of M. aff.oligospora (sensu Jacono and Johnson, 2006) bear sporocarps with prominenttooth. The presence of toothed and toothless taxa together in Clade J indicatethat this character may be homoplasious and may not provide reliablecharacters for diagnosis of species, at least within this species complex.
The data show that central Florida specimens of M. aff. oligospora (samples30–37) are distinct from all other sampled Marsilea and might represent anundescribed species or a morphological and molecular variant of M.ancylopoda. We are unable to match them with Marsilea from any othergeographic locality. Our sampling of the Caribbean, Central America, andnorthern South America is poor, and more extensive sampling might provide amatch for the Florida populations. The type of M. ancylopoda is from coastalarid lowlands just north of the Gulf of Guayaquil, Ecuador. Future studiesshould include material from the type locality. Although our sampling doesnot include material from the type locality of M. ancylopoda, it does includePeruvian material from similar low-lying habitats along the arid west coastalstrip of South America. This specimen (#177, Llatas & Quiroz 2401), is inClade L where it groups weakly with M. vestita from the desert regions of NewMexico and Arizona. Additional sampling from low elevation neotropicallocalities is also needed to seek matches for M. ancylopoda from west-centralMexico and northeastern Argentina, as included in Clade J of this study. Untilfurther sampling yields a match for the Florida plants, we suggest that thepopulations should be regarded as endemic and given protected status byvegetation managers until its status as native or alien is resolved moredefinitively.
Evaluation of Morphological Species Concepts in Marsilea Section Nodorhizae
These plastid data provide an independent dataset with which to evaluatemorphological species concepts in Marsilea, especially for the North Americanspecies that were heavily sampled. The failure of the plastid data to resolvespecimens into clades that correspond to morphospecies is most obvious inMarsilea sect. Nodorhizae (M. oligospora, M. mollis, M. villosa, M. vestita, M.macropoda, M. nashii, and M. ancylopoda). Instead, plastid data group theseseven species into four distinct clades with strong geographic structure thatcorrespond to climactic and habitat zones: Clade L includes western NorthAmerican accessions from ephemeral ponds in arid climates; Clade K includesplants from humid, seasonally influenced low elevation floodplains and wetdepressions of the Gulf coastal plain, Florida, and the northern Caribbean;Clade I consists only of M. mollis from Arizona to Bolivia; Clade J includes M.ancylopoda (from Mexico and Argentina), the central Florida material (M. aff.
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ancylopoda) and nearby Puerto Rico, plus a geographically disparate accessionfrom the montane highlands of north central Ecuador and an aberrant sterilespecimen from Louisiana (#038).
The incongruence of these plastid trees and the currently accepted species ofMarsilea may have several explanations, which we discuss below. Extensivehybridization among Marsilea species might have led to chloroplast capture ofa single plastid type among many species resulting in plastid trees that do notaccurately reflect phylogenetic relationships. Johnson (1986) cited severalspecimens as putative interspecific hybrids, based solely on interpretation ofsubtle morphological characters. To our knowledge, no one has createdartificial Marsilea hybrids, nor used molecular data to demonstrate theparental origin of putative hybrids. Additionally, the non-monophyly ofspecies may be due to incomplete lineage sorting. However, we did notexamine the individual gene trees to determine if this could be the cause ofnon-monophyly.
The absence of monophyletic species may also be due to the presence ofcryptic species. This is exemplified by our finding that the plants originallyidentified as M. aff. oligospora are a potentially undescribed species (seeabove). These plants display subtle morphological differences compared to allother known Marsilea, and molecular data indicate that they have a uniquemolecular signature as well. Therefore, it is possible that through more intensesampling and reassessment of morphology, the non-monophyletic species mayreveal the presence of underlying cryptic species.
Through our analyses we discovered several accessions that were misiden-tified, and it is possible that some of polyphyletic species are due toidentification errors. However, given the extent of polyphyletic species (andthat many specimens were annotated by D.M. Johnson), we suggest that this isunlikely.
A final explanation for failure of the existing alpha-taxonomy could be aninflated number of species within Marsilea sect. Nodorhizae (clades J, K, L).Many species of Marsilea are based upon subtle morphological traits that arephenotypically plastic or that represent homoplasious local adaptations toenvironmental conditions. We found that plant size, leaflet size, extent ofleaflet hairiness, the angle and extent of sporocarp nodding, and the curvatureof the peduncle demonstrated variability that might preclude their taxonomicutility for species delimitation.
It was beyond the scope of this project to re-examine all of the specimensused in this study. However, we suggest that future work include re-examination of the morphology of multiple accessions within a phylogeneticframework to ascertain the reliability of the existing characters for speciesdelimitation and to determine if cryptic species are present. There is also theneed for more extensive sampling and sequencing of more variable plastidregions, to be contrasted with nuclear gene data sets, which will provide abetter framework to settle questions of hybridization and incomplete lineagesorting as well as provide greater resolution and support for the relationshipamong species.
WHITTEN ET AL.: MARSILEA PHYLOGENETICS 133
Conclusions
Using the extensively sampled phylogeny, we found that Florida plantsearlier identified as M. aff. oligospora possess a unique molecular signature (aninsertion in the trnL-F spacer) but morphological characters that distinguish itfrom other taxa in M. sect. Nodorhizae are subtle and require more detailedanalyses (Jacono and Johnson, 2006). It is possible that these plants representan undescribed, cryptic species endemic to Florida, or a geographicallyrestricted variant of an existing species. Our plastid trees reveal the same majorclades as the previous study by Nagalingum et al. (2007). Although ourincreased taxon sampling reveals no conflicts, many species are not resolvedas monophyletic within these informally named clades. We were unable todetermine if this is due to hybridization, incomplete lineage sorting,misidentification of specimens, the presence of cryptic species, and/orinappropriate morphological characters for species delimitation—the presentdata are inadequate to resolve these large taxonomic questions. We advise thatthe existing alpha-taxonomic classification and circumscription of species inMarsilea, especially M. sect. Nodorhizae, should be treated with caution.
ACKNOWLEDGMENTS
This work was funded by Florida Dept. of Environmental Protection Bureau of Invasive Plant
Management Grant #6770 and by The St. Johns Water Management District Contract #74607. We
thank curators of many herbaria for permission to destructively sample specimens; we are
especially grateful to Kent Perkins (FLAS) for management of loans. We thank Marian Chau, Robert
Gibson, and Siriporn Zungsontiporn for specimens.
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