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© 1999 Macmillan Magazines Ltd
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Angiosperm phylogeny inferred
from multiple genes
as a tool for comparative biology
Pamela S. Soltis*, Douglas E. Soltis* & Mark W. Chase
†
* School of Biological Sciences, Washington State University, Pullman,
Washington 99164-4236, USA
†
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS,
UK
.................................. ......................... ......................... .................................................. ........
Comparative biology requires a firm phylogenetic foundation to
uncover and understand patterns of diversification and evaluate
hypotheses of the processes responsible for these patterns. In the
angiosperms, studies of diversification in floral form
1,2
, stamen
organization
3
, reproductive biology
4
, photosynthetic pathway
5
,
nitrogen-fixing symbioses
6
and life histories
7
have relied on
either explicit or implied phylogenetic trees. Furthermore, to
understand the evolution of specific genes and gene families,
evaluate the extent of conservation of plant genomes and make
proper sense of the huge volume of molecular genetic data
available for model organisms
8
such as Arabidopsis,
Antirrhinum, maize, rice and wheat, a phylogenetic perspective
is necessary. Here we report the results of parsimony analyses of
DNA sequences of the plastid genes rbcL and atpB and the nuclear
18S rDNA for 560 species of angiosperms and seven non-flowering
seed plants and show a well-resolved and well-supported phylo-
genetic tree for the angiosperms for use in comparative biology.
Efforts to infer angiosperm phylogeny have greatly improved our
understanding of the major lineages of flowering plants
9–14
.How-
ever, despite these advances, the phylogenetic trees inferred from
these studies are not completely congruent in the inter-relationships
portrayed among the major lineages (although the alternatives are
not strongly supported), and nearly all trees suffer from areas of
poor resolution and/or weak support, usually due to low levels of
divergence. In addition, molecular studies using hundreds of taxa
present difficult analytical problems; the large number of taxa
results in a huge number of possible trees
15
, increasing the
amount of time needed to conduct a thorough analysis and
decreasing the chances of finding the optimum tree(s). Phylogenetic
analyses of 500 rbcL sequences did not find the most parsimonious
trees
9
; further analyses of these data found slightly shorter trees
16,17
.
An analysis based on 18S rDNA
11
also probably failed to find the
most parsimonious trees, in spite of extensive analyses. Thus,
despite a decade of major improvements in our understanding of
angiosperm phylogeny, the picture is not yet complete.
The analytical issues involved in large-scale phylogenetic analyses
have been discussed
18,19
. Both simulations
20,21
and empirical studies
18
indicate that additional data can improve phylogenetic inferences
from large data sets. Analyses of angiosperm relationships on the
basis of gene sequences for rbcL, atpB and 18S rDNA for 190
angiosperm species and 3 outgroups showed increased resolution
and internal support (as measured by bootstrap values), and faster
run times when the data sets for these genes were combined rather
than analysed separately. These studies indicated that additional
data (that is, gene sequences) and taxa could improve inferences of
angiosperm phylogeny.
Our analysis, which is based on parsimony analyses of 4,733
aligned nucleotides, provides a well-resolved and well-supported
estimate of angiosperm phylogeny. Most of the major clades and
some of the smaller ones recovered in this analysis were also found
in previous studies
9,11–13
. However, contrary to previous analyses
based on data for one or two genes, all major clades and most of the
spine of the tree receive jackknife (JK) support equal to or greater
than 50%. Amborellaceae are the sister to all remaining angio-
sperms, consistent with results inferred from 18S rDNA
11
and atpB
13
alone; the JK value for the clade of all angiosperms except Amborella
is 65%. Nymphaeaceae (with a JK value of 100%) are then sister to
all remaining angiosperms (JK 72%), followed by a clade of
Austrobaileya, Illicium and Schisandra (JK for this clade of three
genera 100%). This same branching order was found with stronger
support in analyses based on five genes and nearly 9,000 base pairs
(bp) of sequence data
14
and is further corroborated by analyses of
letters to nature
402 NATURE | VOL 402 | 25 NOVEMBER 1999
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Table 1 Familiar taxa and the clades in which they are placed in the
phylogenetic tree shown in Fig. 1
Familiar taxa/model organisms Clade (Family or Order) Larger group
.............................................................................................................................................................................
Nymphaea (water lilies) Nymphaeaceae
Magnolia Magnoliales magnoliids
Orchidaceae (orchids) Asparagales monocots
Poaceae (grasses): Triticum, Oryza, Zea Poales monocots
Fabaceae (legumes) Fabales eurosid I
Rosaceae (rose family): Rosa, Malus Rosales eurosid I
Arabidopsis, Brassica Brassicales eurosid II
Dianthus (carnation), Spinacia Caryophyllales eudicots
Cornus (dogwoods) Cornales asterids
Hydrangea Cornales asterids
Lamiaceae (mints) Lamiales euasterid I
Antirrhinum Lamiales euasterid I
Solanum (potato, tomato) Solanales euasterid I
Nicotiana (tobacco) Solanales euasterid I
Petunia Solanales euasterid I
Apium (celery), Daucus (carrot) Apiales euasterid II
Helianthus (sunflower) Asterales eusterid II
.............................................................................................................................................................................
100
100
100
65
100
56
99
96
100
89
94
100
86
96
88
98
98
99
100
100
100
75
100
60
98
100
80
100
59
84
87
100
100
99
53
100
98
71
56
99
100
100
95
95
99
100
97
65
84
94
72
100
99
100
100
100
100
100
98
68
60
77
51
99
99
95
Fagales (9)
Cucurbitales (7)
Rosales (18)
Fabales (6)
Oxalidales (9)
eurosid I
eurosid II
rosids
euasterid II
euasterid I
asterids
magnoliids
gymnosperms
eudicots
97
Malpighiales (44)
Sapindales (13)
Malvales (16)
Brassicales (14)
Myrtales (10)
Geraniales (7)
Saxifragales (25)
Cornales (13)
Ericales (32)
Myrothamnaceae/Gunneraceae (2)
Berberidopsidales (2)
Santalales (7)
Caryophyllales (24)
Buxaceae/Didymelaceae (3)
Trochodendraceae (2)
Proteales (4)
Sabiaceae (2)
Ranunculales (19)
Ceratophyllales (1)
monocots
Winterales
Laurales (9)
Magnoliales (10)
Chloranthales (3)
Piperales (8)
Illiciaceae (1)
Schisandraceae (1)
Austrobaileyaceae (1)
Nymphaeaceae (1)
Amborellaceae (1)
Taxaceae (1)
Podocarpaceae (1)
Pinaceae (1)
Ginkgoaceae (1)
Ephedraceae (1)
Gnetaceae (1)
Welwitschiaceae (1)
Lamiales (34)
Solanales (10)
Gentianales (10)
Garryales (4)
Asterales (18)
Dipsacales (7)
Apiales (8)
Aquifoliales (5)
Crossosomatales (3)
100
55
Celastrales (8)
Zygophyllales
62
100
51
Figure 1 Summary of phylogenetic relationships for angiosperms inferred from analysis of
rbcL
,
atpB
and 18S rDNA sequences; the jackknife consensus tree (for groups receiving
.50% support) is shown. The shortest trees found were 45,100 steps. The number of
species in each clade is given in parentheses; not all 560 species occurred in a clade
portrayed in this summary tree. Jackknife support is given below branches.
© 1999 Macmillan Magazines Ltd
duplicate phytochrome genes
22
. The remaining angiosperms (JK
71%) form two major clades. One of these (JK 56%) consists of
Chloranthaceae, Magnoliales, Laurales, Winterales and Piperales, all
classified in subclass Magnoliidae
23
, and the monocots. Each of
these branches is strongly supported (JK $95%), but the relation-
ships among these clades have JK values of less than 50%.
Within the monocots, Acorus is sister to a clade containing all
other monocots (JK 99%). Alismatales are the next branching
monocots and sister to a large clade (JK 99%) that comprises six
main lineages: Petrosaviaceae, Dioscoreales, Pandanales, Liliales,
Asparagales and commelinoids. Although all but Asparagales (JK
56%) and commelinoids (JK 68%) have JK percentages greater than
80%, relationships among clades of monocots are poorly resolved
and/or weakly supported. The commelinoids, in turn, comprise
Arecales (palms), Poales (including grasses), Commelinales and
Zingiberales (gingers) as successive sister groups, although relation-
ships among these four clades are not strongly supported. Only 102
out of the roughly 65,000 species of monocots
24
were included in
this analysis; more extensive sampling of monocots and discussion
of relationships are given in ref. 25.
The second major clade above the first three basal branches
comprises Ceratophyllum as the weakly supported sister (JK 53%)
to the eudicots, that is, all remaining angiosperms. The eudicots (JK
99%) comprise a series of successively branching orders: Ranuncu-
lales, Proteales, Trochodendrales, Gunneraceae/Myrothamnaceae,
and a large clade of ‘core eudicots’ (JK 100%). This last clade
contains the majority of all angiosperm species. Major clades within
the core eudicots are Saxifragales as sister to the remaining rosids;
Caryophyllales; and asterids, which comprise Cornales, Ericales,
euasterids I and euasterids II. Model organisms in the rosids include
Arabidopsis, Brassica, Gossypium and legumes. Nitrogen-fixing
symbioses with nodulating bacteria arose within this clade and
are confined to a portion of the eurosid I subclade. Although this
‘nitrogen-fixing clade’ has been found in previous analyses
6,11,13
, this
is the first study to our knowledge to provide strong support for it.
Like the rosids, the asterid clade was recognized in previous
studies
9,11–13
, but without strong support. The model organisms
Antirrhinum, Nicotiana, Petunia, Solanum and Helianthus are found
in this large asterid clade. Familiar families found within each clade
are given in Table 1. All three major clades of core eudicots identified
here cut across subclass boundaries established in previous
classifications
23,24
. For the complete tree see Supplementary Infor-
mation.
Although some minor portions of the tree are weakly supported,
the first branches of angiosperm phylogeny, Amborella,Nym-
phaeales, Austrobaileya-Illicium-Schisandra (plus Trimenia
14
) now
seem clear
14,22
. The relationships among orders of magnoliids are
only weakly supported, as is the position of the monocots. Within
the eudicots, the branching order of the major clades is fairly clear,
but levels of support could be increased by additional data. Finally,
although the core eudicots form a clade (JK 100%), the relation-
ships among rosids, Caryophyllales, asterids and a number of
smaller clades lack substantial JK support. Additional data, both
molecular and morphological, could increase support for these
relationships.
Despite some areas of poor resolution and/or weak support, the
general structure of angiosperm phylogeny is now clear and well
supported. Using this framework, long-standing questions of plant
diversification can be addressed, and recent discoveries can be
placed in the proper historical context. For example, petals have
apparently arisen multiple times within the angiosperms, from
sepals in some cases to stamens in others
1,26,27
, but the phylogenetic
pattern of petal derivation is not clear. Using a phylogenetic tree
such as ours, it would be possible to assess the homology of ‘petals’
throughout the angiosperms and then to search for genetic and
developmental mechanisms of ‘petal’ formation
28
. Other aspects of
floral evolution, such as the origin, maintenance and diversification
of synorganized flowers
1
in the core eudicots and patterns of stamen
organization
3
, can also be addressed. Inferences of diversification of
other angiosperm characteristics, such as morphology, physiology,
ecology and genome structure, are now possible on a much more
robust basis than previously possible. Although large analyses of the
angiosperms have been reported
9,11–13
, this study provides both
greater resolution and stronger support for the relationships
inferred. The use of broad taxon sampling and many characters
limits spurious results due to homoplasy and unequal rates of
evolution among taxa and provides stronger support for relation-
ships because historical information from all three genes is com-
bined. Our tree, which employs three times the quantity of data used
to construct most other trees, provides a substantially more secure
foundation for inferring the evolution of key features in the
angiosperms.
M
Methods
Selection of species was designed to sample all major groups of angiosperms, using recent
classifications
23,24
and phylogenetic studies
11,13,16
as guides. Sequences of rbcL, atpB, and 18S
rDNA were amplified by polymerase chain reaction (PCR) and sequenced using an ABI
377 automated DNA sequencer. Details of sampling, DNA extraction, PCR amplification,
sequencing and alignment will be published elsewhere (D.E.S. et al., manuscript in
preparation). Voucher information, GenBank numbers and the aligned data matrix are
available at http://www.wsu.edu:8080/,soltilab/, http://www.kewgardens.org and
http://www.ucjeps.berkeley.edu/bryolab/greenplantpage.html. Heuristic parsimony
analyses were conducted using PAUP* 4.0 (ref. 29) and the ratchet (K. Nixon) with NONA
(P. Goloboff). Parsimony JK analyses
30
(1,200 replicates, eachwith ten random-entry order
replicates and branch swapping) were used to measure support for the topology. The
gymnosperms Ephedra, Gnetum, Welwitschia, Ginkgo, Pinus, Podocarpus and Taxus were
specified as the outgroup.
Received 6 September; accepted 19 October 1999.
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large DNA data sets for angiosperms. Syst. Biol. 47, 32–42 (1998).
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on the Comparative Biology of the Monocotyledons, Sydney, Australia, (eds Wilson, K. & Morrison, D.)
(CSIRO Press, Sydney, in the press).
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nature.com) or as paper copy from the London editorial office of Nature.
Acknowledgements
This work was supported in part by the NSF.
Correspondence and requests for material should be addressed to P.S.S.
(e-mail: psoltis@wsu.edu).
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The earliest angiosperms: evidence
from mitochondrial, plastid
and nuclear genomes
Yin-Long Qiu*, Jungho Lee*, Fabiana Bernasconi-Quadroni*,
Douglas E. Soltis
†
, Pamela S. Soltis
†
, Michael Zanis
†
,
Elizabeth A. Zimmer
‡
, Zhiduan Chen*§, Vincent Savolainenk,
Mark W. Chasek
* Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107,
8008 Zurich, Switzerland
†
School of Biological Sciences, Washington State University, Pullman,
Washington 99164-4236, USA
‡
Laboratory of Molecular Systematics, Smithsonian Institution,
Washington DC 20560, USA
k Jodrell Laboratory Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK
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Angiosperms have dominated the Earth’s vegetation since the
mid-Cretaceous (90 million years ago)
1
, providing much of our
food, fibre, medicine and timber, yet their origin and early
evolution have remained enigmatic for over a century
2–8
. One
part of the enigma lies in the difficulty of identifying the earliest
angiosperms; the other involves the uncertainty regarding the
sister group of angiosperms among extant and fossil gymno-
sperms. Here we report a phylogenetic analysis of DNA sequences
of five mitochondrial, plastid and nuclear genes (total aligned
length 8,733 base pairs), from all basal angiosperm and gymno-
sperm lineages (105 species, 103 genera and 63 families). Our
study demonstrates that Amborella, Nymphaeales and Illiciales-
Trimeniaceae-Austrobaileya represent the first stage of angio-
sperm evolution, with Amborella being sister to all other angio-
sperms. We also show that Gnetales are related to the conifers and
are not sister to the angiosperms, thus refuting the Anthophyte
Hypothesis
1
. These results have far-reaching implications for our
understanding of diversification, adaptation, genome evolution
and development of the angiosperms.
Difficulty in identifying the earliest angiosperms is the result of
three problems that characterize diversification of most major
clades. First, the great divergence between gymnosperms and
angiosperms makes assessment of character homology difficult
and thus renders the otherwise powerful outgroup-approach prob-
§ Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences,
Beijing, 100093, China.
lematic in morphological cladistic analyses
1,9
. Second, extinction,
which is partly responsible for this divergence, has almost certainly
occurred in both groups
1,10–16
, and highlights the need of extensive
taxon sampling when relying on the living diversity. Last, the fossil
evidence indicates that the early angiosperms went through an
explosive radiation
1,10–16
, which to resolve requires the sampling of
a large number of characters. Previous molecular analyses have
had some success in resolving relationships among basal angio-
sperms
17–21
; however, their results are only weakly supported, and
worse, are often contradictory because of evolutionary rate hetero-
geneity among lineages of the particular gene used, weak phyloge-
netic signal in single genes, and insufficient taxon sampling. From
both theoretical and empirical studies, it is becoming increasingly
clear that to address such a difficult issue as basal angiosperm
phylogeny, extensive sampling in both dimensions of taxa and
characters (genes) is necessary
22–24
.
We obtained sequences of five genes from all three plant genomes:
mitochondrial atp1 and matR, plastid atpB and rbcL and nuclear
18S rDNA. They encode products involved in energy metabolism,
carbohydrate synthesis and information processing. Thus, our
character sampling strategy of taking multiple genes of different
functions from all three genomes is designed to reduce homoplasy
generated by gene-, function- and genome-specific molecular
evolutionary phenomena such as rate heterogeneity, GC content
bias, RNA editing and protein structural constraints
25,26
. To opti-
mize the performance of phylogenetic methods in analysing com-
plex diversification patterns in early angiosperms
22,23
, we included
97 species, 95 genera and 55 families of basal angiosperms, essen-
tially sampling all living families
5,8,11,20,21
. Eight gymnosperms from
eight families were used as outgroups. The DNA sequences were
analysed with parsimony methods; bootstrap (BS) and jackknife
(JK) analyses were conducted to measure stability of phylogenetic
patterns.
The same single most parsimonious tree was found in each of
1,000 random taxon-addition replicates in the analysis (Fig. 1).
Amborella, a shrub of the monotypic New Caledonian family
Amborellaceae, is sister to all other angiosperms, which are strongly
(90% BS and 92% JK) supported as a monophyletic group. The next
diverging lineage corresponds to Nymphaeales, the water lilies;
its sister clade of the remaining angiosperms receives 98% BS and
99% JK support. The third clade consists of two small Australasian
families, Austrobaileyaceae and Trimeniaceae, and two small eastern
Asia-eastern North America disjunct families, Illiciaceae and Schi-
sandraceae (Illiciales). All remaining angiosperms (euangiosperms)
make up a strongly supported large clade (97% BS and 99% JK). The
relationships among lineages within euangiosperms are resolved in
the shortest tree but generally receive less than 50% BS support. All
major lineages, however, are strongly supported; these agree with
previous classifications
5,8,11
and results of cladistic analyses of mor-
phological and molecular data
9,20,21,27
. Among gymnosperms, two
gnetalean genera, Gnetum and Welwitschia, are not sister to angio-
sperms as suggested by the Anthophyte Hypothesis
1
, but fall close to
the conifers.
We observed one INDEL (insertion/deletion) in matR that
supports the basal position of Amborella, Nymphaeales and
Illiciales-Trimeniaceae-Austrobaileya (ANITA) in angiosperms: an
18-base-pair (bp) deletion in all euangiosperms but not in ANITA
or gymnosperms, some of which have 6–15-bp deletions (Fig. 2).
Although we cannot rule out the possibility that the sequence in the
INDEL region of ANITA and gymnosperms results from indepen-
dent insertions, two lines of evidence suggest that this scenario is
unlikely. The sequences are found in all three ANITA lineages and all
four gymnosperm lineages. Furthermore, there are identical or
similar codons shared by ANITA and gymnosperms in the INDEL.
Reconstruction of deep phylogenies using DNA sequences has
been plagued by problems caused by rate heterogeneity, weak
phylogenetic signal in single genes, insufficient taxon sampling,
