Chinese Science Bulletin © 2009
SCIENCE IN CHINA PRESS
Springer
Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes LIU Nian1, ZHU Yong1, WEI ZongXian2, CHEN Jie1, WANG QingBiao1, JIAN ShuGuang4, ZHOU DangWei5, SHI Jing1, YANG Yong6 & ZHONG Yang1,3† 1
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China; 2 Lushan Botanical Garden, Chinese Academy of Sciences, Lushan 332900, China; 3 Shanghai Center for Bioinformation Technology, Shanghai 200235, China; 4 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China; 5 Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; 6 Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
Araucariaceae is one of the most primitive families of the living conifers, and its phylogenetic relationships and divergence times are critically important issues. The DNA sequences of 8 genes, i.e., nuclear ribosomal 18S and 26S rRNA, chloroplast 16S rRNA, rbcL, matK and rps4, and mitochondrial coxI and atp1, obtained from this study and GenBank were used for constructing the molecular phylogenetic trees of Araucariaceae, indicating that the phylogenetic relationships among the three genera of this family should be ((Wollemia, Agathis), Araucaria). On the basis of the fossil calibrations of Wollemia and the two tribes Araucaria and Eutacta of the genus Araucaria, the divergence time of Araucariaceae was estimated to be (308 ± 53) million years ago, that is, the origin of the family was in the Late Carboniferous rather than Triassic as a traditional view. With the same gene combination, the divergence times of the genera Araucaria and Agathis were (246 ± 47) and (61 ± 15) Ma, respectively. Statistical analyses on the phylogenetic trees generated by using different genes and comparisons of the divergence times estimated by using those genes suggested that the chloroplast matK and rps4 genes are most suitable for investigating the phylogenetic relationships and divergence times of the family Araucariaceae. Araucariaceae, nrDNA 18S rRNA, nrDNA 26S rRNA, cpDNA 16S rRNA, cpDNA rbcL, cpDNA matK, cpDNA rps4, mtDNA coxl, mtDNA atp1, phylogeny, divergence time
Araucariaceae is one of the most primitive families of the living conifers. Its extant species are mainly distributed in tropical and subtropical zones of the southern hemisphere, including Australia, New Zealand, New Guinea, New Caledonia and other South Pacific Islands, as well as South America. The fossil records indicate that this family had a wide distribution both in the southern and northern hemispheres during the Mesozoic era, and gradually extinct in the northern hemisphere after the Late Cretaceous. Therefore, investigation of the
origin and divergence of Araucariaceae can be helpful for better understanding the geological climate change of the paleocontinent. Received January 16, 2009; accepted March 27, 2009 doi: 10.1007/s11434-009-0373-2 † Corresponding author (email:
[email protected]) Supported by the National Infrastructure of Natural Resources for Science and Technology (Grant No. 2005DKA21403), MOST Basic Science and Technology (Grant No. 2007FY110100), Shanghai Leading Academic Discipline Project (Grant No. B111), Shanghai Science and Technology Committee (Grant No. 07XD14025), the Graduate Students Innovation Foundation of Fudan University (Grant No. EYH1322098) and National Science Fund for Fostering Talents in Basic Science (Grant No. J0630643)
Citation: Liu N, Zhu Y, Wei Z X, et al. Phylogenetic relationships and divergence times of the family Araucariaceae based on the DNA sequences of eight genes. Chinese Sci Bull, 2009, 54: 2648―2655, doi: 10.1007/s11434-009-0373-2
ARTICLES
effects of the eight genes on the phylogenetic reconstruction and estimation of the divergence times of the family were also discussed.
1 Materials and methods 1.1 Materials W. nobilis was provided by Wollemi Pine International Pty Ltd. and Araucaria araucana was provided by Hamburg Botanical Garden, Germany. Other samples used in this study, including 7 species of Araucariaceae, two species of Podocarpaceae, one species of Cephalotaxaceae, one species of Taxaceae, one species of Sciadopityaceae, two species of Taxodiaceae, two species of Cupressaceae, and two species of Pinaceae, were collected from the South China Botanical Garden. Voucher specimens were stored in the Institute of Biodiversity Science at Fudan University. Fresh leaves were immediately dried by silica gel after collection for further DNA extraction and sequencing. 1.2 DNA extraction Total DNA were extracted using CTAB method[19]. 1.3 Amplification and sequencing Primer sequences were synthesized according to references[14,20–22] or self-designed. The total PCR reaction volume was 50 μL, containing 1 μL DNA template, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 1×PCR buffer, 1 μmol/L of each primer, and 2.5 U Taq DNA polymerase. The PCR cycling parameters were 1 cycle of 5 min at 94℃ for denaturation; 35 cycles of 0.5 min at 94℃, 0.5 min of 45-56℃ for primer annealing and 1.5-2 min at 72℃ for primer extension; 1 cycle of 7 min at 72℃. PCR products were isolated by electrophoresis in 1.2% agarose gel and purified by gel extraction kit, cloned to pMD19-T vector and sequenced by an ABI 377 DNA automated sequencer. The GenBank accession numbers of the DNA sequences used in this study are listed in Table 1. 1.4 Sequence analyses and statistical tests Multiple sequence alignment of DNA sequences from 15 genera in Coniferopsida (9 families) containing 8 genes was obtained by using CLUSTAL X[23]. The best model parameters were estimated with Modeltest 3.7[24]. Single-gene maximum likelihood (ML) trees and the combined ML tree were constructed by using PHYML[25], with 1000 bootstrap replicates.
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MOLECULAR REVOLUTION
The family Araucariaceae consists of 41 species and 1 subspecies in three genera, i.e., Araucaria, Agathis, and Wollemia[1], among which Wollemia is a monotypic genus discovered in 1994[2,3], dubbed “a living dinosaur on earth”, because the fossil records proved that Wollemia occurred in the Cretaceous period[4]. When W. nobilis was found, only 26 adults and 14 juveniles existed in the Wollemi National Park in New South Wales, Australia. Since it shares the morphological characteristics of Araucaria and Agathis[5], the W. nobilis cone scale resembles that of the tribe Eutacta of Araucaria, and the winged seeds are free from the cone scale like Agathis[6]. Recently, molecular phylogenetics has provided a new method to identify the controversial phylogenetic relationships among the 3 genera of the family Araucariaceae. Setoguchi et al.[7] constructed a phylogenetic tree of W. nobilis, 10 Agathis species and 19 Araucaria species based on the chloroplast rbcL gene. In this tree, which has been supported by many paleontologists[8,9], W. nobilis lies in the basal position of the Araucariaceae, and Agathis is sister to Araucaria. However, a number of authors disagreed with this conclusion[10,11]. They believed that Agathis is sister to Wollemia, and Araucaria is a basal lineage of the Araucariaceae. There are two main reasons for the incongruence. One is about different outgroups selected for constructing the phylogenetic tree. The class Coniferopsida was regarded as monophyletic in some studies[11,12], while in other studies, paraphyletic[13,14]. Previous studies indicated that the Pinaceae, Sciadopityaceae, Podocarpaceae, or Cupressaceae was regarded as the closest group to the family Araucariaceae[13,15–18]. In the work of Setoguchi et al.[7], the families Taxodiaceae, Cupressaceae and Podocarpaceae were chosen as outgroups, while in other work, the outgroups were from Coniferopsida, even Gymnospermae[10,11]. Another reason is about the different genes used in phylogenetic analyses. In previous studies, a phylogenetic tree was generated with a single gene, yet a gene tree may have different topology from other gene trees. Therefore, we collected the samples of Araucariaceae worldwide and conducted a combined phylogenetic analysis based on the DNA sequences of nuclear 18S and 26S rRNA sequences, chloroplast 16S rRNA, rbcL, matK and rps4 genes, and mitochondrial coxI and atp1 genes in this study. The fossil records of Wollemia and two tribes Araucaria and Eutacta of the genus Araucaria were used as calibrations for dating the divergence times of the Araucariaceae. In addition, the
Araucaria bidwillii
Araucaria angustifolia
Araucaria cunninghamii
Araucaria heterophylla
Wollemia nobilis
Araucariaceae
Araucariaceae
Araucariaceae
Araucariaceae
Araucariaceae
Chamaecyparis
Juniperusa)
Cupressaceae
Cupressaceae
b)
b)
b) b)
Cryptomeriaa)
Metasequoia glyptostroboides
Sciadopitys verticillata
Taxodiaceae
Taxodiaceae
Sciadopityaceae
D85292
EF053174
D85304
DQ478811
D38241 b)
b)
EU161318
L25753
AJ235805
EU161306b) b)
AJ621937
AF456388
AF456387
X58136
X58391
AF119182
L12570
AF307931
AF249648
AF249654
U87757
U96462
EU164986b)
EU164985
b)
ABU87751
AF249664
AF249665
U96477
EU161341b)
b)
EU161462
EU161354
b)
b)
EU161351
EU161347
b)
EU161311b)
EU161307
b)
DQ008664
EU161315
EU161339
b)
EU165007
EU165006
rbcL EU164984b)
a) Sequences come from different species of the same genus; b) sequenced in this study.
Taxus
Taxaceae
a)
Cephalotaxus
Cephalotaxaceae
DQ987890
Picea
Pinaceae
X79407
Abies
Pinaceae
a)
D38243
EF053165
U87301
D16447
U87300
EU164983
EU164982
b)
EU165005b)
EU164981b)
EU165004
b)
EU164980
EU165003b) b)
FJ179544
EU165002b)
EU164978b)
EU164979b)
EU165001b)
EU164977b)
FJ179543
26S EU165000b)
18S EU164976b)
a)
a)
Podocarpus
Podocarpaceae
a)
Nageia
Podocarpaceae
a)
a)
Araucaria araucana
Araucariaceae
Phyllocladus
Agathis robusta
Araucariaceae
Phyllocladaceae
Agathis dammara
Araucariaceae
a)
Agathis alba
Taxon
DNA sequences used in this study and their GenBank accession numbers
Araucariaceae
Family
Table 1 matK
AB023994
AF152203
AB030117
DQ478791
AF457108
AB161012
AF143441
AB030136
AB030132
AF228111
AB023990
AF280997
AF456377
AF456374
b)
EU164999b)
EU164998
b)
EU164997b)
AF456373
AF456371
EU164996b)
EU164995b)
rps4
b)
AY188262
EF053201
AY188272
X84145
AY188264
AY188226
AY188224
AY188279
AY188283
AY188252
AY188248
AY188258
EU165036
AY188260
EU165035b)
EU165034
b)
EU165033b)
FJ179545
EU165032b)
EU165031b)
EU165030b)
cp16S
b)
b)
b)
b)
b)
b)
EU161554b)
EU161540b)
EU161523b)
EU161474
EU161520
EU161546
EU161508
U24586
EU161522
EU161550
EU161544
b)
DQ629439
b)
EU164992
EU164991
b)
EU164990b)
EU164994
b)
EU164993b)
DQ629437
EU164988b)
EU164987b)
EU164989b)
coxI
b)
AF020576
AF020569
DQ478768b)
AF020579
AF020559
DQ987880
AF020556
AF020567
EF053140
AF020575
DQ478773
AF020573 b)
b)
EU165029
AF020558
EU165028b)
EU165027
b)
EU165026b)
FJ179546
EU165025b)
EU165024b)
EU165023b)
atp1
EU161409b)
AF197619
EU161380b)
DQ646221
DQ646222
EU161403b)
DQ646224
AF209106
EU161379b)
AF197620
EU161400b)
DQ646219
EU165019b)
AF209104
EU165020b)
EU165021b)
EU165022b)
FJ179547
EU165018b)
EU165016b)
EU165017b)
1.5 Estimation of divergence times Fossil calibration is required for the estimation of divergence times. The earliest Wollemia-like fossil was Agathis jurassica[4] at the Late Jurassic. However, Chambers et al.[6] indicated this leaf fossil required further confirmation. The Araucariaceae macrofossils which Chambers et al.[6] discovered in the Strzelecki Group (Barremian-Aptian) in Australia shared similar cone scales with Araucaria and winged seeds with Agathis, which were consistent with the characteristics of Wollemia. The fossil records of Wollemia (118 Ma)[6] and the two tribes Araucaria and Eutacta of Araucaria (98.9- 142.0 Ma)[28] were used as the calibration points to date the divergence times. The divergence times were estimated by using MCMCTREE in PAML 4.1[29]. Beast1.41, r8s1.71, and Baseml in PAML 4.1 were also used to calculate the divergence times as control.
ARTICLES
Among these trees, all species in the Araucariaceae were clustered into a clade, while Phyllocladus and Podocarpaceae were clustered into another clade and Phyllocladus lay in the basal position. These two main clades became sister groups, with the bootstrap supports between 80% and 100%, except atp1 gene with a relatively low (56%) bootstrap support (Figure 1). It indicated that Phyllocladus and Podocarpaceae are the closest group to the Araucariaceae and therefore, we selected Phyllocladus as outgroup to Araucariaceae. The construction of eight single gene trees and the combined tree of Araucariaceae were made by PAUP 4.0[26], with 100 bootstrap replicates.
Figure 1
Combined ML tree of the Coniferopsida.
Statistical tests were performed on different gene trees with CONSEL[27]. The phylogenetic relationships among Araucariaceae can be grouped into three topologies: ((Agathis, Wollemia), Araucaria), (Agathis, (Wollemia, Araucaria)), and (Wollemia, (Agathis, Araucaria)). The best trees of each topology were obtained through exhaustive search using PAUP 4.0. The site-wise loglikelihoods for the candidate trees were calculated and introduced into CONSEL, then AU (approximately unbiased test), NP (non-scaled bootstrap probability test), BP (bootstrap probability test), PP (Bayesian posterior probability test), KH (Kishino-Hasegawa test), SH (Shimodaira-Hasegawa test), WKH (weighted KishinoHasegawa test), and WSH (weighted ShimodairaHasegawa test) test were performed.
The single-gene trees of the 9 species in three genera of the family Araucariaceae were constructed first. Among these trees, Wollemia and Araucaria were clustered in a clade in the 18S tree with 69% bootstrap support. In the 26S tree, Wollemia and Agathis were clustered in a clade with 90% bootstrap support. However, Araucaria had low resolution with many parallel branches. The cpDNA 16S also had a low resolution. Except the species in Agathis were clustered into one clade with 90% bootstrap support, the other species formed parallel branches. In the rbcL tree, the three genera were clustered into one clade respectively, and Agahis was sister to Wollemia with a low (61%) bootstrap support. With a high resolution, species in each genus were clustered into a single clade in the matK and rps4 trees. Agathis and Wollemia formed a strongly supported sister clade, with 98% and 93% bootstrap support respectively. Wollemia lay in the basal clade of the Araucariaceae in the atp1 tree and Araucaria was sister to Agathis with 100% bootstrap support. Agathis was sister to Wollemia in the coxI tree with 92% bootstrap support. The combined tree had the best resolution among all the trees, in which each genus was clustered into a single clade respectively with 100% bootstrap support. Agathis and Wollemia formed a strongly supported clade, with 98% bootstrap support (Table 2).
Liu N et al. Chinese Science Bulletin | August 2009 | vol. 54 | no. 15
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MOLECULAR REVOLUTION
2 Results
Table 2
Statistical test results of each gene tree
Genes 18S
26S
atp1
coxI
matK
rbcL
rps4
Combine
Phylogenya)
AU test
NP test
BP test
PP test
KH test
SH test
WKH test
WSH test 0.315
a
0.151
0.198
0.189
0.019
0.187
0.19
0.187
b
0.907
0.782
0.795
0.977
0.813
0.875
0.813
0.855
c
0.016
0.047
0.016
0.005
0.104
0.112
0.104
0.195
a
0.978
0.937
0.939
0.999
0.917
0.917
0.917
0.917
b
0.022
0.063
0.04
4.00E-04
0.083
0.083
0.083
0.083
c
0.022
0.063
0.023
4.00E-04
0.083
0.083
0.083
0.083
a
0.006
0.008
0.008
4.00E-06
0.02
0.02
0.02
0.02
b
0.006
0.008
0
4.00E-06
0.02
0.02
0.02
0.02
c
0.994
0.992
0.992
1
0.98
0.98
0.98
0.98
a
0.879
0.85
0.851
0.962
0.856
0.901
0.856
0.892
b
0.064
0.021
0.02
0.012
0.08
0.082
0.08
0.159
c
0.174
0.129
0.129
0.026
0.144
0.144
0.144
0.239
a
0.974
0.967
0.967
1
0.957
0.966
0.957
0.964
b
0.046
0.034
0.028
6.00E-05
0.043
0.043
0.043
0.046 0.077
c
0.009
0.007
0.006
3.00E-05
0.031
0.031
0.031
a
0.788
0.644
0.646
0.611
0.74
0.77
0.74
0.77
b
0.273
0.184
0.182
0.197
0.26
0.276
0.26
0.461
c
0.265
0.175
0.172
0.192
0.259
0.275
0.259
0.449
a
0.998
0.952
0.977
0.999
0.939
0.939
0.939
0.939 0.061
b
0.002
0.048
0.023
3.00E-04
0.061
0.061
0.061
c
0.002
0.048
0.023
3.00E-04
0.061
0.061
0.061
0.061
a
0.979
0.978
0.978
1
0.974
0.987
0.974
0.981
b
0.025
0.022
0.022
4.00E-08
0.026
0.026
0.026
0.043
c
0.002
4.00E-04
2.00E-04
7.00E-10
0.005
0.005
0.005
0.011
a) The rbcL gene tree could not be classified into the tree topologies below due to its low resolution, therefore statistical test was not performed. A row with bold font indicates the results of the statistical test on the consensus ML tree: a, ((Agathis, Wollemia), Araucaria); b, (Agathis, (Wollemia, Araucaria)); c, (Wollemia, (Agathis, Araucaria)).
Due to low resolution, the cpDNA 16S tree could not be classified into the three tree topologies and therefore was excluded for the statistical tests. Among the tests, 18S, rbcL and coxI had two or more tree topologies with AU P >0.1, while 26S, atp1, matK and rps4 had only one tree topology with AU P >0.95, and the other two tree topologies with AU P <0.05 (Table 2). Other statistical tests had similar results with the AU test. On the basis of the two calibrations, the divergence times of the family Araucariaceae, and genera Araucaria and Agathis calculated by MCMCTREE from PAML v4.1 were 308 ± 53, 246 ± 47 and 61 ± 15 Ma, respectively (Figure 2). The divergence times of the Araucariaceae calculated by using single-gene and combined-gene trees are listed in Table 3. The divergence times of the Araucariaceae estimated by using Beast, r8s and Baseml with different models are also listed in Table 3. The results obtained from Beast were similar to the ones from MCMCTREE, while r8s and Baseml using different models could not pro2652
Figure 2 Combined ML tree of the Araucariaceae. C1, Calibration point of Wollemia, 118 Ma; C2, calibration point of the tribe Araucaria, 98.9-142.0 Ma; C3, calibration point of the tribe Eutacta, 98.9 -142.0 Ma; T1, (308 ± 53) Ma; T2, (246 ± 47) Ma; T3, (61 ± 15) Ma.
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Bayesian
a)
Bayesian
Bayesian
NPRS
Local Clock
Globe Clock
r8s
Paml4.1
Paml4.1
BEAST
Bayesian
Globe Clock
Paml4.1
a)
Local Clock
Paml4.1
Paml4.1
NPRS
r8s
BEAST
Bayesian
Paml4.1
a)
Local Clock
Globe Clock
Paml4.1
Paml4.1
NPRS
r8s
BEAST
Bayesian
Paml4.1
Algorithm
35±24
29±22
84±26
62±0.54
69±25
215±122
273±991
220±54
211±1.47
237±54
318±145
313±420
349±78
297±1.43
320±57
18S
91±15
92±15
93±39
88.3±0.56
105±11
91±23
101±408
122±58
119±1.34
119±11
129±20
137±266
154±64
150±1.64
160±24
26S
45±20
45±21
52±25
65±0.46
76±25
118±1
118±1
135±66
124±1.64
137±27
118±1
118±1
135±99
154±1.87
164±48
cp16S
a) The results calculated by Beast software present as X ± SE , while others present as X ± SE .
Agathis
Araucaria
Araucariaceae
Software
Estimation of the divergence times of the Araucariaceae using 8 genes (Ma)
Species
Table 3
41±12
35±14
89±22
59±0.47
65±22
152±45
71.8±36
227±39
258±1.32
264±55
261±56
148±58
303±83
308±1.77
338±53
matK
34±9
25±8
36±20
50±0.27
64±19
85±24
61±12
193±47
185±1.21
203±49
134±23
118±1
300±124
250±1.31
256±64
rbcL
28±20
28.5
37±15
53±0.60
65±27
146±78
181±152
267±40
257±1.63
271±56
195±71
221±115
267±89
308±1.45
327±57
rps4
34±18
46±26
105±27
76±0.61
81±32
341±120
693±495
310±43
267±1.55
281±58
485±144
809±425
389±76
329±1.89
352±49
coxI
0
0
23±13
49±0.22
57±36
101±32
321±322
239±123
246±1.35
271±59
152±29
410±370
506±222
326±1.55
340±55
atp1
/
38±6
39±24
56±0.34
61±15
/
93±32
225±47
232±1.17
246±47
/
158±38
323±68
285±1.62
308±53
Combined
duce reliable results.
3 Discussion Our study used 8 genes from nuclear, chloroplast and mitochondria to construct the phylogenetic trees of the Araucariaceae. Combined analysis strongly supported that Wollemia was sister to Agathis, that is, the phylogenetic relationships among the three genera in the family Araucariaceae are ((Wollemia, Agathis), Araucaria), while the result of ref. [7] might be caused by the wrong outgroup and low resolution of the rbcL gene tree. According to the results of statistical tests for the single-gene trees and the combined tree, cp16S, rbcL and coxI genes most supported the tree topology ((Wollemia, Agathis), Araucaria), but did not reject either ((Agathis, Araucaria), Wollemia) or ((Araucaria, Wollemia), Agathis); by contrast, the results of the statistical tests on the 26S, matK, rps4 and atp1 genes are statistically significant. For example, the statistical P value of three topologies of the rps4 trees was 0.998, 0.002 and 0.002, respectively, indicating that either ((Agathis, Araucaria), Wollemia) or ((Wollemia, Araucaria), Agathis) can be rejected but ((Agathis, Wollemia), Araucaria) can be accepted. Statistical analyses on the 26S and matK trees yield similar conclusion. However, the atp1 tree is in contradiction with the above trees. The topology ((Agathis, Araucaria), Wollemia) was accepted, which needs to be further studied. Considering the low resolution of the 26S tree, matK and rps4 genes are most suitable for constructing the phylogenetic trees of the family Araucariaceae. The divergence times estimated by using the eight genes respectively are different from each other. Based on the combined tree, the time of origin of the family 1 2
We would like to thank Prof. Masami Hasegawa for his comments and suggestions.
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