Post on 01-Feb-2023
Abstract Genus Paspalum L. comprises approx-
imately 400 species worldwide and about 220 in
Brazil. Paspalum is ecologically and economically
important, and has been very useful as pasture.
Traditionally, taxonomists use informal groups,
most of which described by Agnes Chase in 1929.
Some groups present a problematic circumscrip-
tion, this is the case of the Linearia and Notata
groups. This work uses a phylogenetic approach to
study these groups and related species. DNA
sequences from ITS of nuclear rRNA, from chlo-
roplast intergenic spacer psbA-trnH and chloro-
plast trnL intron were used to perform the
analyses. The informal groups studied were con-
sidered highly artificial, being the representatives
from several informal groups splitted throughout
the trees. Only a small core of species from Notata
group could be accepted as a good formal clade.
Keywords Notata Æ Linearia Æ Paspalum L. ÆPoaceae Æ phylogeny
Introduction
Genus Paspalum L. belongs to the grass family
(Poaceae) and presents annual and perennial
species, distributed around the tropics and sub-
tropics of both hemispheres. About 400 species
are described for this genus (Chase 1929; Oliveira
and Valls 2002). In Brazil, there are not studies
with detailed information about the total number
of species, nevertheless Valls and Pozzobon
(1987) estimated the occurrence of about 220
species.
Paspalum presents several species with poten-
tial forage value, species which are sometimes
known as weedy, other are used as ornamental
plants in green gardens, parks, soccer fields and one
species is used as cereal in India, P. scrobiculatum
L., called ‘‘Kodo’’ (Clayton and Renvoize 1986).
In Brazil, the genus is dominant in many native
herbaceous communities and constitutes the
major part of available forage. Paspalum notatum
Fluegge is the most common species of the genus in
Southern Brazil and is widely used as forage and as
an ornamental plant.
In 1929, Agnes Chase divided the species
occurring in North America into 25 groups,
without a formal taxonomic category (informal
L. Essi (&)Programa de Pos-graduacao em Botanica,Universidade Federal do Rio Grande do Sul, Av.Bento Goncalves, 9500, Campus do Vale da UFRGS,Predio 43433. Bairro Agronomia, Porto AlegreRS 91501-970, Brazile-mail: liliana_essi@yahoo.com.br
T. T. de Souza-ChiesDepartamento de Botanica, Programa dePos-graduacao em Botanica, Universidade Federal doRio Grande do Sul, Porto Alegre, RS 91501-970,Brazil
Genet Resour Crop Evol (2007) 54:779–791
DOI 10.1007/s10722-006-9148-7
123
ORIGINAL PAPER
Phylogeny of Linearia and Notata groups of Paspalum L.(Poaceae, Panicoideae, Paniceae) and related species
Liliana Essi Æ Tatiana Teixeira de Souza-Chies
Received: 5 December 2005 / Accepted: 27 March 2006 / Published online: 7 December 2006� Springer Science+Business Media, Inc. 2006
groups). These species were grouped according to
their habit, inflorescence type, spikelet form and
dimensions, pilosity and foliar width. These
informal groups proposed by Chase (1929)
became widely used by agrostologists, with the
addition of new groups proposed or adapted to
Chase’s groups, according to regional floras.
Actually, there is a lack of infrageneric divi-
sions to adequately describe the morphological
diversity in Paspalum, and the use of informal
groups attenuates this fact. However, the place-
ment of many taxa in these informal groups is still
controversial and some groups seem to be highly
artificial. A good example of this situation is seen
in the Linearia and Notata groups.
The Linearia group, as originally proposed
(Chase 1929), included only one species, P. lineare
Trin. Studies of other regional floras added to this
group species supposedly related to P. lineare
(Swallen 1967; Filgueiras 1982), or associated it to
species previously cited to other groups, especially
to Notata (Valls 1987; Oliveira and Valls 2002).
The Notata group (Chase 1929) initially in-
cluded five species. Barreto (1974) and Canto-
Dorow et al. (1996) expanded the original con-
cept, concerning the species from Rio Grande do
Sul state (Brazil), and accepting nine species.
Barreto (1974) also described a new group, El-
liptica, constituted only by P. ellipticum Doell, a
species included by Chase (unpubl. mss.) in
Lineares (=Linearia) and which, years later, Valls
(1987) pointed out as related to the Notata group.
Oliveira and Valls (2002) called attention to a
morphological continuity between Notata and
Linearia, emphasizing the condition of P. crom-
yorhizon Trin. ex Doell, P. ionanthum Chase and
P. dedeccae Quarın as species with intermediate
morphology between both groups. The same
authors also emphasized the morphological rela-
tionships among some Linearia species and spe-
cies belonging to other groups. For instance,
P. pallens Swallen was associated to P. vaginatum
Sw. (Disticha group), species from Recta group
were associated with Linearia, based on vegeta-
tive characters, and P. ovale Nees, included by
Chase in ‘‘Lineares’’ (unpubl. mss.), and its allies,
were suggested to be better placed in the Ovalia
group sensu Barreto (1974). Oliveira and Valls
(2002) emphasized the heterogeneity of the
Linearia group and the possible obtention of a
single more homogeneous group, if the discrepant
species, such as P. pallens, and species which
could be included in other groups, like P. ovale,
were excluded. The concept of a single group
including Linearia and Notata was adopted by
Zuloaga et al. (2004) in their recent monograph
on the Notata group. In this work, the authors
accept a group called Notata including major
species of Linearia according to the concept of
Oliveira and Valls (2002), but maintaining
P. pallens in this species complex.
The situations mentioned above illustrate
several controversies concerning the circum-
scription of Linearia and Notata groups, and their
monophyly is questioned. Table 1 shows a syn-
thesis of the placement of the species analyzed in
the present work, in informal groups, as proposed
by different authors.
Aliscioni (2002) evaluated the monophyly of
many informal groups of Paspalum, including a
small number of species belonging to Notata and
Linearia. This study was based on the anatomy of
leaves and exomorphological characters, and the
author concluded that major informal groups are
artificial, including Notata and Linearia. Consid-
ering the controversies around morphological
characters, many of them highly affected by the
environment or differentially classified by every
author, a contribution with characters of a dif-
ferent nature is needed.
Different DNA fragments have been used as
source of characters for phylogenetic analysis.
Outstanding among them are the Internal Tran-
scribed Spacers (ITS) located between the nuclear
rRNA genes, the chloroplast spacer psbA-trnH
and the chloroplast intron from trnL gene. The
first one has been widely used in plant phyloge-
nies, due to its high nucleotide substitution rates
(Baldwin 1992; Baldwin et al. 1995) and to its
power to elucidate relationships at the infragen-
eric level in plants of several families, including
Poaceae (Grebenstein et al. 1998; Catalan and
Olmstead 2000; Guo et al. 2002; Baumel et al.
2002). The second fragment was pointed out by
Sang et al. (1997) as phylogenetically informative,
and in Paspalum presented an adequate length for
direct sequencing (about 600 base pairs). The last
fragment has also been widely used to elucidate
780 Genet Resour Crop Evol (2007) 54:779–791
123
phylogenetic relationships among taxa from sev-
eral plant families (Stedje 1998; Doust and Kellog
2002; Goldblatt et al. 2002). The chloroplast
fragments are generally more conserved than ITS
and, for this reason, they have usually been com-
bined with other DNA fragments and/or with
characters of another nature.
In this work, a molecular approach was used in
order to evaluate the monophyly of Linearia,
Notata and related groups such as Disticha, Recta
and Ovalia, and to establish the phylogenetic
relationships among Paspalum species that be-
long to these groups, looking for clades in this
complex that could be assigned a formal category.
The data were evaluated using three methods of
phylogenetic inference: unweighted parsimony
(maximum parsimony or MP), maximum likeli-
hood (ML) and Bayesian estimation (B), as de-
scribed bellow.
Materials and methods
Plant material and laboratory methods
The 31 species included in this study are listed in
Table 2. Five Axonopus species constituted the
outgroup, and four Paspalum species belonging to
Table 1 Placement of the Paspalum species included in this work in the informal groups, according to different authors.
Species Informal groups according to the literature
P. almum Chase Notata (Chase 1933 ; Barreto 1974; Canto-Dorow et al. 1996), Alma(Quarın 1974; Valls 1987; Zuloaga et al. 2004)
P. barretoi Canto-Dorow, Valls etLonghi-Wagner
Notata (Canto-Dorow et al. 1995)
P. conduplicatum Canto-Dorow, Vallset Longhi-Wagner
Notata (Canto-Dorow et al. 1995; Zuloaga et al. 2004)
P. cromyorhizon Trin. ex Doell Notata (Barreto 1974; Canto-Dorow et al. 1996; Zuloaga et al. 2004)P. dedeccae Quarın Linearia (Oliveira and Valls 2002), Notata (Zuloaga et al. 2004)P. ellipticum Doell Linearia (as Lineares in Chase, unpublished; Oliveira and Valls 2002),
Elliptica (Barreto 1974), Notata (Zuloaga et al. 2004)P. equitans Mez Fasciculata (Barreto 1974)P. filifolium Nees ex Steud. Linearia (Oliveira and Valls 2002), Notata (Zuloaga et al. 2004)P. flaccidum Nees Recta (Chase, unpubl. mss.; Zuloaga et al. 2004)P. intermedium Munro ex Morong et
BrittonQuadrifaria (Barreto 1974)
P. ionanthum Chase Notata (Canto-Dorow et al. 1996; Zuloaga et al. 2004)P. juergensii Hack. Paniculata (Barreto 1974)P. lineare Trin. Linearia (Chase 1929; Oliveira and Valls 2002), Notata (Zuloaga et al.
2004)P. maculosum Trin. Notata (Barreto 1974; Canto-Dorow et al. 1996; Zuloaga et al. 2004),
Maculosa (Chase, unpubl. mss.)P. minus E. Fourn. Notata (Chase 1929; Barreto 1974; Canto-Dorow et al. 1996; Zuloaga et al.
2004)P. notatum Fluegge Notata (Chase 1929; Barreto 1974; Canto-Dorow et al. 1996; Zuloaga et al.
2004)P. nummularium Chase ex Sendulski et
A. G. BurmanNotata (Zuloaga et al. 2004)
P. ovale Nees ex Steud. Linearia (as Lineares in Chase, unpubl. mss.), Ovalia (Barreto 1974;Zuloaga et al. 2004)
P. pallens Swallen Linearia (Swallen 1967; Oliveira and Valls 2002), Notata (Zuloaga et al.2004)
P. pauciciliatum (Parodi) Herter Dilatata (Valls and Pozzobon 1987) Livida (Barreto 1974)P. pumilum Nees Notata (Canto-Dorow et al. 1996; Zuloaga et al. 2004)P. ramboi Barreto Notata (Barreto 1974; Canto-Dorow et al. 1996; Zuloaga et al. 2004)P. subciliatum Chase Notata (Chase 1929; Zuloaga et al. 2004)P. urvillei Steud. Dilatata (Barreto 1974)P. vaginatum Sw. Disticha (Chase 1929; Barreto 1974)
Genet Resour Crop Evol (2007) 54:779–791 781
123
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782 Genet Resour Crop Evol (2007) 54:779–791
123
Ta
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Genet Resour Crop Evol (2007) 54:779–791 783
123
other informal groups not related to the complex
were arbitrarily chosen to test the Linearia/Notata
monophyly hypothesis. These species are consid-
ered well defined in Chase’s groups until the
present, clearly distinguished from the rest of
the ingroup, but not far enough to constitute
the outgroup: P. urvillei Steud., P. pauciciliatum
(Parodi) Herter, P. intermedium Munro ex
Morong & Britton and P. juergensii Hack. Genus
Axonopus was chosen to form the outgroup
because of its close morphological relationship
with Paspalum and, at the same time, because
of its clear and well-defined circumscription,
compared with the ingroup species. From the 21
species cited by Zuloaga et al. (2004) to Notata
group, 16 are included in this study, and other six
species belonging to closely related groups, or al-
ready cited by other authors to Notata or Linearia,
are also included. The sampling covered the main
controversies concerning the circumscription of
Linearia and Notata cited by Oliveira and Valls
(2002). All species included occur in Brazil.
The total DNA was isolated following a modi-
fied CTAB method (Doyle and Doyle 1987), from
dried or herbarium material. Voucher information
are listed in Table 2. The fragments were ampli-
fied using the primers described by Desfeux and
Lejeune (1996) to ITS, primers psbAF and trnHR
described by Sang et al. (1997) to psbA-trnH
spacer, and the primers C and D described by
Taberlet et al. (1991) to chloroplast intron from
trnL gene. The Polymerase Chain Reaction
(PCR) was performed following the recommen-
dations of the authors, with minor modifications.
The amplified material was purified with enzy-
matic pre-treatment (Shrimp alkaline phospha-
tase and exonuclease I, Amersham Biosciences),
and directly sequenced using the same primers
from PCR to chloroplast fragments and internal
primers to the ITS, also described by Desfeux and
Lejeune (1996). In the case of ITS, some of the
species were manually sequenced with chain ter-
minators labeled with 33P and Kit ‘‘Thermo
Sequenase’’. The other fragments and the ITS
region from the rest of the sampling were
sequenced using the automatic sequencers ‘‘ABI
Prism 310’’ (Perkin Elmer), ‘‘Mega Bace 1000’’
(Amersham-Pharmacia-Biotech) or ‘‘ABI Prism
3100’’ (Applied Biosystems). The ITS fragments
were sequenced at least twice, and the chloroplast
fragments were sequenced with forward and
backward primers. Ambiguous bases in the eletr-
opherograms were coded as ‘‘N’’. The sequences
obtained were stored in the GENBANK (http://
www.ncbi.nlm.nih.gov). All fragments were
sequenced to all species included in the study,
except the psbA-trnH spacer, which is not avail-
able for P. barretoi Canto-Dorow, Valls and
Longhi-Wagner and P. ovale.
Data analysis
The sequence alignments were made using the
software Clustal·1.81 (Thompson et al. 1997) and
manually refined with the help of the software
GeneDoc (Nicholas et al. 1997). The phyloge-
netic analyses were performed with the softwares
PAUP*, version 4.0b10 (Swofford 2002) and
Mr. Bayes, version 3 (Huesenbeck and Ronquist
2001). The analyses were made considering a
nuclear and a chloroplast data matrix (ITS or
psbA-trnH and trnL). ITS1 and ITS2 were always
analyzed together, as a single data collection. The
phylogenetic trees were calculated using three
methods: Maximum Parsimony (MP), Bayesian
(B) and Maximum Likelihood (ML). MP and ML
were performed with the PAUP* software, and B
was performed with Mr. Bayes software.
For MP analysis, heuristic searches with TBR
(tree bisection-reconnection), MULPARS option
and 1000 random-addition replications of heuristic
search and simple taxon addition, were used to all
trees saved option. The MAXTREES option was
selected, with auto-increase of 100. In the case of
the chloroplast matrix, the MAXTREES option
was set as 5000 trees due to the high number of
equally parsimonious trees. The relative propor-
tion of homoplasious data in the MP analysis was
estimated through Consistency and Retention
Indexes (CI and RI) available at PAUP* tools.
The heterogeneity among markers was assessed
using the partition homogeneity test (Farris et al.
1995), with 100 interactions, as implemented in
PAUP*. The G1 statistics (Hillis 1991) were
calculated through the generation of 10,000 ran-
dom trees by PAUP*. The tree-score frequency
distributions were ordenized by PAUP* as histo-
grams with 20 classes, showing the values to G1.
784 Genet Resour Crop Evol (2007) 54:779–791
123
Asymmetric histograms left inclinated and nega-
tive G1 values indicate that the trees generated
are unlikely to be produced by random, what
means that the fragments evaluated are phyloge-
netically informative. The Bootstrap test (Fel-
senstein 1985) was also performed with 1000
replications, heuristic search and simple taxon
addition, saving only the best trees, and the Bre-
mer support (Bremer 1988) was performed with
the Autodecay software, version 4.0.2’68k (Eri-
ksson 1999).
Nucleotide substitution models for ML and B
were chosen according to the best models selected
using the LTR criterion by software MODEL-
TEST 3.06 (Posada and Crandall 1998)-model
K80 + G to ITS matrix, model HKY85 + G to
chloroplast matrix and model TrN + I + G to the
combined matrix. ML analysis was performed with
PAUP* through heuristic search, as-is option,
TBR branch swapping and MULPARS option. B
analysis was performed with Mr. Bayes, through
1,000,000 generations.
Results and discussion
The ITS1 sequences presented lengths from
208 bp for Paspalum pallens and Axonopus
compressus (Sw.) P. Beauv., to 220 bp for P.
ionanthum and P. dedeccae, and the ITS2 length
varied between 207 bp for P. barretoi, and 220 bp
for P. cromyorhizon and A. brasiliensis (Spreng.)
Kuhlm. The total matrix length was 502 bp. When
single gaps were treated as missing data, the
matrix presented 269 conserved characters, 106
variable parsimony non-informative (autapomor-
phies) and 127 characters that were variable and
parsimony-informative. Considering gaps as fifth
base, the number of parsimony-informative
characters grew to 185. The average CG content
was 0.58.
The psbA-trnH spacer presented lengths vary-
ing from 534 bp for A. compressus, to 547 bp for
P. urvillei, and the CG content was 0.37 on the
average, similar to the CG content of the trnL
intron, which presented an average content of 0.34.
This intron presented lengths from 565 bp for
P. filifolium Nees ex Steud., to 590 bp for A. siccus
(Nees) Kuhlm. The whole chloroplast dataset was
1176 bp long, but with a low number of parsimony-
informative characters, 57 characters treating gaps
as fifth state character, and only 36 considering
gaps as missing data. The number of variable non-
informative characters was 142, treating gaps as
fifth base, and 64 treating gaps as missing data,
showing a high percentage of autapomorphic data.
The G1 statistic (Hillis 1991) for ITS matrix
was )1.635807, and for chloroplast matrix it was
)1.821921, presenting a phylogenetic signal good
enough for both datasets, although other statisti-
cal tests presented above pointed to a high degree
of conservation for the chloroplast matrix.
The measure of incongruence between the two
matrices, obtained through the Partition Homo-
geneity Test, rejected the null hypothesis, with a
strong significance level (P value = 0.01). This
clear incongruence between the chloroplast and
the nuclear dataset supported the decision to not
proceed with a combined analysis.
Statistics and phylogenetic trees
The phylogenetic trees inferred through ITS
(Fig. 1) and chloroplast (Fig. 2) showed quite
different groups and statistical values. The chlo-
roplast matrix provided a great number of possible
trees, and it was necessary to set a maximum
number of trees to be saved in MP analysis (5000).
The number of equally parsimonious trees was
always high for the chloroplast dataset, resulting
in highly polytomized consensus trees. It is also
interesting to take a look at the index used to
estimate homoplasy level. The chloroplast matrix
showed the lowest level of homoplasious data.
When treating single gaps as missing data,
CI and RI were the best (CI = 0.91, CI excluding
non-informative characters = 0.81, RI = 0.93),
but when gaps were included as fifth base,
the number presented a significant decrease
(CI = 0.85, CI excluding non-informative charac-
ters = 0.65, RI = 0.80). The index for ITS also
presented a significant decrease when including
gaps (from an RI = 0.71 to an RI = 0.60, for
instance), denoting that the MP analysis including
gaps could be not reliable enough. For this reason,
only trees produced with single gaps treated as
missing data are shown, although some results
with gaps as fifth base are commented.
Genet Resour Crop Evol (2007) 54:779–791 785
123
The trees inferred though ML presented
topologies similar to the B analyses, with minor
differences. In general, the groups supported by
bootstrap in MP analysis appeared in all other
phylogenetic analyses of the same dataset. Groups
not supported appeared in different positions
according to the phylogenetic method performed.
Groups that are not supported by any statistical
test and with an unstable position are not going to
be emphasized during the discussion. For instance,
it is possible to take a look at the behavior of
P. pallens in the trees. Considering the ITS matrix
and MP analyses, this species is placed within a
Notata clade, if treating gaps as fifth base. Treat-
ing gaps as missing data, its position changed, and
it was placed with P. almum Chase, as it can be
seen in Fig. 1. But considering a chloroplast ma-
trix and ML analysis, it was placed at the base of
the clade constituted by P. dedeccae, P. lineare, P.
notatum and P. vaginatum. Considering the
instability and lack of support of these relation-
ships, there is no reliable conclusion about its
phylogenetic relationships.
The position of P. chromyorhizon is always far
enough from P. notatum, against the possibility
of being considered a variety of P. notatum, as
Fig. 1 Consensus tree (majority rule values above thebranches) from 917 most parsimonious trees, with 442steps, inferred through ITS matrix, gaps treated as missingdata. Matrix with 127 parsimony-informative characters.
Support values included under the branches. D = decayvalues. B = bootstrap values. CI = 0.71, CI excluding non-informative characters = 0.60, IR = 0.70
786 Genet Resour Crop Evol (2007) 54:779–791
123
accepted by Smith and Wasshausen (1982). Also,
P. dedeccae, P. filifolium and P. ionanthum,
indicated by Oliveira and Valls (2002) as mor-
phological links between Notata and Linearia
groups, due to the continuity of their leaf shapes,
appeared always in distinct clades, showing that
these morphological affinities do not have the
same pattern at the molecular level.
From the ITS clades (Fig. 1) as a whole, it is
possible to highlight three structured clades. Clade
1 includes only species from the Notata group,
plus P. flaccidum Nees. Clade 2 includes species
from many informal groups, especially Linearia.
The clade 3 groups species representing the Con-
trol Group plus P. ionanthum and P. ovale.
Observing the distribution of the informal groups
in the tree, it can be seen that there is no mono-
phyletic group corresponding to Notata, Linearia
or both together, although a small part of Notata
could be considered monophyletic, when it in-
cludes P. flaccidum. This behavior agrees with the
work of Ferreira and Souza-Chies (2005), based
on RFLP data, but not with the total evidence
analysis including morphological data, which
Fig. 2 Consensus tree (majority rule values above thebranches) from 5000 most parsimonious trees, with 117steps, inferred through chloroplast matrix, gaps treated asmissing data. Matrix with 36 parsimony-informative
characters. Support values included under the branches.D = decay values. B = bootstrap values. CI = 0.91, CIexcluding non-informative characters = 0.81, IR = 0.93
Genet Resour Crop Evol (2007) 54:779–791 787
123
support the hypothesis of a single group formed by
Linearia plus Notata together (Zuloaga et al.
2004; Souza-Chies et al. 2006). Excluding P. flac-
cidum, the seven other species are in fact very
similar morphologically, so the clade maybe de-
serves some formal treatment.
Clade 2 is also very frequent in the analyses,
especially the subclade including P. equitans Mez,
P. ramboi Barreto and P. filifolium. The two
samples of P. ellipticum represent two distinct
biotypes, previously considered as different spe-
cies, but recently made synonymous (Oliveira and
Valls 2002). They are closely related, but curi-
ously do not present bootstrap support.
Clade 3, formed mostly by the species of other
informal groups morphogically well resolved, also
includes one species from the Ovale group and
another from the Notata group, showing that
neither species of those groups are far enough
from the complex, from the molecular standpoint.
In the chloroplast analysis (Fig. 2), other dis-
tinct clades appear. Clade B links P. barretoi and
P. nummularium Chase ex Sendulski et A.G.
Burman, both belonging to clade 1 from ITS
analysis, but not linked together in this clade.
Although their ITS sequences are very distinct,
their trnL intron sequences are almost identical,
differing in only one base pair. Clade D links
species not linked by ITS: P. dedeccae, P. lineare,
P. vaginatum and P. notatum. They belong to
different groups proposed by Chase and are
morphologically very distinct. Clade F links P.
subciliatum Chase and P. maculosum Trin. Both
belong to Notata, although P. maculosum pre-
sented an uncertain position in the ITS analysis
and Chase (unpublish. mss.) preferred to transfer
this species to the new Maculosa group.
The species P. almum appeared always far
from other Notata species. This species differs
from almost all species of Paspalum concerning
its chromosome number, which has a basic num-
ber of six instead of 10. On the other hand, Souza-
Chies et al. (2006) presented this species included
in the Linearia + Notata clade, a relationship
probably due mostly to the exomorphological
data.
Many authors have published chromosome
counts for species from several groups of Pas-
palum (e.g. Quarın and Burson 1983, 1991;
Quarın et al. 1984; Quarın and Norrmann 1987;
Norrmann et al. 1989; Pozzobon and Valls 1997;
Takayama et al. 1998; Pozzobon et al. 2000; and
Pagliarini et al. 2001), pointing out the occur-
rence of diploid, triploid, tetraploid, pentaploid,
hexaploid and also octoploid species. Many of
these species are possibly of hybrid origin, such as
the case of P. dilatatum Poir., which presents
three genomes, II, JJ and XX, that could be
donated by plants belonging to different groups,
like Quadrifaria and Paniculata (Casa et al. 2002,
Miz and Souza-Chies 2006). The occurrence of
hybridization in the genus can be an element that
makes it difficult to establish phylogenetic rela-
tionships. Natural hybrids can bring together
species that actually have a very different phy-
logeny. When a chloroplast fragment has unipa-
rental origin (as in the case of major
angiosperms), and it presents enough informative
sites, it is possible to recognize hybridization
cases, by comparisons between phylogenies pro-
duced by another molecular marker which is
inherited only from the opposite parent or has
biparental origin.
To avoid the distortions that could be pro-
duced by possible hybrid species, diploid speci-
mens were preferred for this work, whenever
possible (for chromosome counts, see Table 1).
Any further doubts could be checked contrasting
the phylogenies from chloroplast and nuclear
sources.
Differences between the ITS and chloroplast
trees topologies could be attributed to the effect
of some species of hybrid origin, but the poor
resolution of the chloroplast trees does not allow
for a definitive conclusion about the possible hy-
brids and parents, although some comments could
be made concerning chromosome numbers.
Paspalum ramboi, placed at clade 1 in the ITS
tree, which groups major Notata specimens,
appears linked to P. conduplicatum (clade C) in
the chloroplast tree. Both are the only hexaploid
Paspalum species included in the analyses. Clade
E in the chloroplast tree links plants from dif-
ferent clades of the ITS analysis, which, however,
share the same chromosome number: 2n = 20.
The clades A, F and G link species with different
chromosome numbers. Further details on the
relationships between major species included in
788 Genet Resour Crop Evol (2007) 54:779–791
123
clades A and G are being studied in our labora-
tory.
Conclusion
The three phylogenetic methods produced similar
trees, if only branches with good support or high
frequency in majority rule consensus are consid-
ered. Polytomized regions in strict consensus
presented different topologies when shown in
majority consensus from MP, B and ML. Since
MP trees were submitted to more statistical tests
than ML and B, they were the first choice to
synthesize the results obtained.
From the clades with high frequency, the most
similar to a previously circumscribed informal
group was clade 1 from ITS analyses, which also
occurred in combined analyses (not shown) but
excluding P. pallens. Another clade whose species
were already pointed out as related, if excluding P.
ovale, is clade 3. In general, representatives from
several informal groups are spread throughout the
trees, and only clade 1 (Notata group sensu stricto)
could be accepted as a good formal clade, due to its
high frequency in different analyses, to the good
support of some pairs, and to the morphological
similarities already observed by other authors.
The genus as a whole has complex reproduc-
tion and hybridization mechanisms, with sexual
and or apomitic plants, and this intricate web is
certainly being reflected in these molecular
markers. The unresolved groups can be better
studied in future analyses, considering the genus
as a whole, which is pointed out as paraphyletic
(Duvall et al. 2001; Giussani et al. 2001; Aliscioni
2002). The addition of species from other mor-
phological groups and the increase of informative
characters (molecular, morphological and cyto-
genetic) should improve the resolution and sup-
port of such groups, and bring new insights on the
relationships among species of this rich and
complex genus.
Acknowledgements We thank Drs. Jose F. M. Valls andRegina C. de Oliveira for the plant accessions and infor-mation supplied, Drs. Sandro Bonatto, Maurıcio R. Bogoand Giancarlo Pasquali for the use of sequencing facilities,and anonymous reviewers for suggestions. We also thankthe Genetics Department of Universidade Federal do Rio
Grande do Sul as a whole, for the welcome and theexchange of experiences, with a special thank to Dr.Alessandra Selbach. FAPERGS and CNPq, Brazil, sup-ported this work.
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