Phylogenetic Relationships and Divergence Times of the Family Araucariaceae Based on the DNA Sequences of Eight Genes

Chinese Science Bulletin

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 Univer- sity, 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 Wol- lemia and the two tribes Araucaria and Eutacta of the genus Araucaria, the divergence time of Arauca- riaceae 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. Statis- tical 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 origin and divergence of Araucariaceae can be helpful the living conifers. Its extant species are mainly distrib- for better understanding the geological climate change uted in tropical and subtropical zones of the southern of the paleocontinent. hemisphere, including Australia, New Zealand, New Received January 16, 2009; accepted March 27, 2009 Guinea, New Caledonia and other South Pacific Islands, doi: 10.1007/s11434-009-0373-2 †Corresponding author (email: [email protected]) as well as South America. The fossil records indicate Supported by the National Infrastructure of Natural Resources for Science and that this family had a wide distribution both in the Technology (Grant No. 2005DKA21403), MOST Basic Science and Technology (Grant No. 2007FY110100), Shanghai Leading Academic Discipline Project (Grant southern and northern hemispheres during the Mesozoic No. B111), Shanghai Science and Technology Committee (Grant No. 07XD14025), era, and gradually extinct in the northern hemisphere the Graduate Students Innovation Foundation of Fudan University (Grant No. EYH1322098) and National Science Fund for Fostering Talents in Basic Science after the Late Cretaceous. Therefore, investigation of the (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

The family Araucariaceae consists of 41 species and 1 effects of the eight genes on the phylogenetic recon-

subspecies in three genera, i.e., Araucaria, Agathis, and struction and estimation of the divergence times of the ARTICLES Wollemia [1], among which Wollemia is a monotypic ge- family were also discussed. nus discovered in 1994[2,3], dubbed “a living dinosaur on earth”, because the fossil records proved that Wollemia 1 Materials and methods [4] occurred in the Cretaceous period . When W. nobilis 1.1 Materials was found, only 26 adults and 14 juveniles existed in the W. nobilis was provided by Wollemi Pine International Wollemi National Park in New South Wales, Australia. Pty Ltd. and Araucaria araucana was provided by Ham- Since it shares the morphological characteristics of burg Botanical Garden, Germany. Other samples used in Araucaria and Agathis[5], the W. nobilis cone scale re- this study, including 7 species of Araucariaceae, two sembles that of the tribe Eutacta of Araucaria, and the species of Podocarpaceae, one species of Cephalotax- winged seeds are free from the cone scale like Agathis[6]. aceae, one species of Taxaceae, one species of Sciado- Recently, molecular phylogenetics has provided a pityaceae, two species of Taxodiaceae, two species of new method to identify the controversial phylogenetic Cupressaceae, and two species of Pinaceae, were col- relationships among the 3 genera of the family Arauca- [7] lected from the South China Botanical Garden. Voucher riaceae. Setoguchi et al. constructed a phylogenetic specimens were stored in the Institute of Biodiversity tree of W. nobilis, 10 Agathis species and 19 Araucaria Science at Fudan University. Fresh leaves were immedi- species based on the chloroplast rbcL gene. In this tree, [8,9] ately dried by silica gel after collection for further DNA which has been supported by many paleontologists , W. nobilis lies in the basal position of the Araucariaceae, extraction and sequencing. and Agathis is sister to Araucaria. However, a number 1.2 DNA extraction [10,11] of authors disagreed with this conclusion . They Total DNA were extracted using CTAB method[19]. believed that Agathis is sister to Wollemia, and Arauca- 1.3 Amplification and sequencing ria is a basal lineage of the Araucariaceae. There are two main reasons for the incongruence. One is about differ- Primer sequences were synthesized according to refer- [14,20–22] ent outgroups selected for constructing the phylogenetic ences or self-designed. The total PCR reaction tree. The class Coniferopsida was regarded as mono- volume was 50 μL, containing 1 μL DNA template, 1.5 [11,12] phyletic in some studies , while in other studies, mmol/L MgCl2, 0.2 mmol/L dNTPs, 1×PCR buffer, 1 paraphyletic[13,14]. Previous studies indicated that the μmol/L of each primer, and 2.5 U Taq DNA polymerase. Pinaceae, Sciadopityaceae, Podocarpaceae, or Cupres- The PCR cycling parameters were 1 cycle of 5 min at saceae was regarded as the closest group to the family 94℃ for denaturation; 35 cycles of 0.5 min at 94℃, 0.5 Araucariaceae[13,15–18]. In the work of Setoguchi et al.[7], min of 45－56℃ for primer annealing and 1.5－2 min the families Taxodiaceae, Cupressaceae and Podocar- at 72℃ for primer extension; 1 cycle of 7 min at 72℃. paceae were chosen as outgroups, while in other work, PCR products were isolated by electrophoresis in 1.2% the outgroups were from Coniferopsida, even Gymno- agarose gel and purified by gel extraction kit, cloned to [10,11] spermae . Another reason is about the different pMD19-T vector and sequenced by an ABI 377 DNA genes used in phylogenetic analyses. In previous studies, automated sequencer. The GenBank accession numbers a phylogenetic tree was generated with a single gene, yet of the DNA sequences used in this study are listed in MOLECULAR REVOLUTION a gene tree may have different topology from other gene Table 1. trees. Therefore, we collected the samples of Araucari- 1.4 Sequence analyses and statistical tests aceae worldwide and conducted a combined phylogenetic analysis based on the DNA sequences of nuclear Multiple sequence alignment of DNA sequences from 15 18S and 26S rRNA sequences, chloroplast 16S rRNA, genera in Coniferopsida (9 families) containing 8 genes rbcL, matK and rps4 genes, and mitochondrial coxI and was obtained by using CLUSTAL X[23]. The best model atp1 genes in this study. The fossil records of Wollemia parameters were estimated with Modeltest 3.7[24]. Sin- and two tribes Araucaria and Eutacta of the genus gle-gene maximum likelihood (ML) trees and the com- Araucaria were used as calibrations for dating the di- bined ML tree were constructed by using PHYML[25], vergence times of the Araucariaceae. In addition, the with 1000 bootstrap replicates.

Liu N et al. Chinese Science Bulletin | August 2009 | vol. 54 | no. 15 2649

Table 1 DNA sequences used in this study and their GenBank accession numbers Family Taxon 18S 26S rbcL matK rps4 cp16S coxI atp1

Araucariaceae Agathis alba EU164976b) EU165000b) EU164984b) EU164995b) EU165030b) EU164989b) EU165023b) EU165017b)

Araucariaceae Agathis dammara EU164977b) EU165001b) U96477 EU164996b) EU165031b) EU164987b) EU165024b) EU165016b)

Araucariaceae Agathis robusta EU164978b) EU165002b) AF249665 AF456371 EU165032b) EU164988b) EU165025b) EU165018b)

Araucariaceae Araucaria araucana FJ179543 FJ179544 AF249664 AF456373 FJ179545 DQ629437 FJ179546 FJ179547

Araucariaceae Araucaria bidwillii EU164979b) EU165003b) ABU87751 EU164997b) EU165033b) EU164993b) EU165026b) EU165022b)

Araucariaceae Araucaria angustifolia EU164980b) EU165004b) EU164985b) EU164998b) EU165034b) EU164994b) EU165027b) EU165021b)

Araucariaceae Araucaria cunninghamii EU164981b) EU165005b) EU164986b) EU164999b) EU165035b) EU164990b) EU165028b) EU165020b)

Araucariaceae Araucaria heterophylla EU164982b) EU165006b) U96462 AF456374 AY188260 EU164991b) AF020558 AF209104

Araucariaceae Wollemia nobilis EU164983b) EU165007b) U87757 AF456377 EU165036b) EU164992b) EU165029b) EU165019b)

Phyllocladaceae Phyllocladusa) U87300 EU161339b) AF249654 AF280997 AY188258 DQ629439 AF020573 DQ646219

Podocarpaceae Nageiaa) D16447 EU161315b) AF249648 AB023990 AY188248 EU161544b) DQ478773b) EU161400b)

Podocarpaceae Podocarpusa) U87301 DQ008664 AF307931 AF228111 AY188252 EU161550b) AF020575 AF197620

Cupressaceae Chamaecyparisa) EF053165 EU161307b) L12570 AB030132 AY188283 EU161522b) EF053140 EU161379b)

Cupressaceae Juniperusa) D38243 EU161311b) AF119182 AB030136 AY188279 U24586 AF020567 AF209106

Pinaceae Abiesa) X79407 EU161347b) X58391 AF143441 AY188224 EU161508 b) AF020556 DQ646224

Pinaceae Piceaa) DQ987890b) EU161351b) X58136 AB161012 AY188226 EU161546b) DQ987880b) EU161403b)

Cephalotaxaceae Cephalotaxusa) D38241 EU161354b) AF456387 AF457108 AY188264 EU161520b) AF020559 DQ646222

Taxaceae Taxus a) DQ478811b) EU161462b) AF456388 DQ478791b) X84145 EU161474b) AF020579 DQ646221

Taxodiaceae Cryptomeriaa) D85304 EU161341b) AJ621937 AB030117 AY188272 EU161523b) DQ478768b) EU161380b)

Taxodiaceae Metasequoia glyptostroboides EF053174 EU161306b) AJ235805 AF152203 EF053201 EU161540b) AF020569 AF197619

Sciadopityaceae Sciadopitys verticillata D85292 EU161318b) L25753 AB023994 AY188262 EU161554b) AF020576 EU161409b) a) Sequences come from different species of the same genus; b) sequenced in this study.

Among these trees, all species in the Araucariaceae 1.5 Estimation of divergence times were clustered into a clade, while Phyllocladus and Po- Fossil calibration is required for the estimation of diver- ARTICLES docarpaceae were clustered into another clade and gence times. The earliest Wollemia-like fossil was Phyllocladus lay in the basal position. These two main Agathis jurassica[4] at the Late Jurassic. However, clades became sister groups, with the bootstrap supports Chambers et al.[6] indicated this leaf fossil required fur- between 80% and 100%, except atp1 gene with a rela- ther confirmation. The Araucariaceae macrofossils tively low (56%) bootstrap support (Figure 1). It indi- which Chambers et al.[6] discovered in the Strzelecki cated that Phyllocladus and Podocarpaceae are the clos- Group (Barremian-Aptian) in Australia shared similar est group to the Araucariaceae and therefore, we selected cone scales with Araucaria and winged seeds with Phyllocladus as outgroup to Araucariaceae. The con- Agathis, which were consistent with the characteristics struction of eight single gene trees and the combined of Wollemia. [26] tree of Araucariaceae were made by PAUP 4.0 , with The fossil records of Wollemia (118 Ma)[6] and the 100 bootstrap replicates. 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.

2 Results

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. Figure 1 Combined ML tree of the Coniferopsida. In the rbcL tree, the three genera were clustered into one

clade respectively, and Agahis was sister to Wollemia Statistical tests were performed on different gene with a low (61%) bootstrap support. With a high resolu- trees with CONSEL[27]. The phylogenetic relationships tion, species in each genus were clustered into a single among Araucariaceae can be grouped into three topologies: ((Agathis, Wollemia), Araucaria), (Agathis, (Wol- clade in the matK and rps4 trees. Agathis and Wollemia lemia, Araucaria)), and (Wollemia, (Agathis, Araucaria)). formed a strongly supported sister clade, with 98% and MOLECULAR REVOLUTION The best trees of each topology were obtained through 93% bootstrap support respectively. Wollemia lay in the exhaustive search using PAUP 4.0. The site-wise log- basal clade of the Araucariaceae in the atp1 tree and likelihoods for the candidate trees were calculated and Araucaria was sister to Agathis with 100% bootstrap introduced into CONSEL, then AU (approximately un- support. Agathis was sister to Wollemia in the coxI tree biased test), NP (non-scaled bootstrap probability test), with 92% bootstrap support. The combined tree had the BP (bootstrap probability test), PP (Bayesian posterior best resolution among all the trees, in which each genus probability test), KH (Kishino-Hasegawa test), SH was clustered into a single clade respectively with 100% (Shimodaira-Hasegawa test), WKH (weighted Kishino- bootstrap support. Agathis and Wollemia formed a Hasegawa test), and WSH (weighted Shimodaira- strongly supported clade, with 98% bootstrap support Hasegawa test) test were performed. (Table 2).

Liu N et al. Chinese Science Bulletin | August 2009 | vol. 54 | no. 15 2651

Table 2 Statistical test results of each gene tree AU NP BP PP KH SH WKH WSH Genes Phylogenya) test test test test test test test test 18S a 0.151 0.198 0.189 0.019 0.187 0.19 0.187 0.315 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 26S 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 atp1 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 coxI 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 matK 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 c 0.009 0.007 0.006 3.00E-05 0.031 0.031 0.031 0.077 rbcL 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 rps4 a 0.998 0.952 0.977 0.999 0.939 0.939 0.939 0.939 b 0.002 0.048 0.023 3.00E-04 0.061 0.061 0.061 0.061 c 0.002 0.048 0.023 3.00E-04 0.061 0.061 0.061 0.061 Combine 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 Araucari- aceae 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 Figure 2 Combined ML tree of the Araucariaceae. C1, Calibration are also listed in Table 3. The results obtained from point of Wollemia, 118 Ma; C2, calibration point of the tribe Arauca- Beast were similar to the ones from MCMCTREE, while ria, 98.9－142.0 Ma; C3, calibration point of the tribe Eutacta, 98.9 r8s and Baseml using different models could not pro- －142.0 Ma; T1, (308 ± 53) Ma; T2, (246 ± 47) Ma; T3, (61 ± 15) Ma.

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Table 3 Estimation of the divergence times of the Araucariaceae using 8 genes (Ma) Species Software Algorithm 18S 26S cp16S matK rbcL rps4 coxI atp1 Combined

Araucariaceae Paml4.1 Bayesian 320±57 160±24 164±48 338±53 256±64 327±57 352±49 340±55 308±53

BEASTa) Bayesian 297±1.43 150±1.64 154±1.87 308±1.77 250±1.31 308±1.45 329±1.89 326±1.55 285±1.62

r8s NPRS 349±78 154±64 135±99 303±83 300±124 267±89 389±76 506±222 323±68

Paml4.1 Local Clock 313±420 137±266 118±1 148±58 118±1 221±115 809±425 410±370 158±38

Paml4.1 Globe Clock 318±145 129±20 118±1 261±56 134±23 195±71 485±144 152±29 /

Araucaria Paml4.1 Bayesian 237±54 119±11 137±27 264±55 203±49 271±56 281±58 271±59 246±47

BEASTa) Bayesian 211±1.47 119±1.34 124±1.64 258±1.32 185±1.21 257±1.63 267±1.55 246±1.35 232±1.17

r8s NPRS 220±54 122±58 135±66 227±39 193±47 267±40 310±43 239±123 225±47

Paml4.1 Local Clock 273±991 101±408 118±1 71.8±36 61±12 181±152 693±495 321±322 93±32

Paml4.1 Globe Clock 215±122 91±23 118±1 152±45 85±24 146±78 341±120 101±32 /

Agathis Paml4.1 Bayesian 69±25 105±11 76±25 65±22 64±19 65±27 81±32 57±36 61±15

BEASTa) Bayesian 62±0.54 88.3±0.56 65±0.46 59±0.47 50±0.27 53±0.60 76±0.61 49±0.22 56±0.34

r8s NPRS 84±26 93±39 52±25 89±22 36±20 37±15 105±27 23±13 39±24

Paml4.1 Local Clock 29±22 92±15 45±21 35±14 25±8 28.5 46±26 0 38±6

Paml4.1 Globe Clock 35±24 91±15 45±20 41±12 34±9 28±20 34±18 0 /

a) The results calculated by Beast software present as X ± SE , while others present as X ± SE .

duce reliable results. Araucariaceae was dated to be (308 ± 53) Ma; based on low resolution or unreliable gene trees, the divergence 3 Discussion times were much closer. For example, the divergence

times from the 26S, cp16S and rbcL genes were (160 ± Our study used 8 genes from nuclear, chloroplast and 24), (164 ± 48) and (256 ± 64) Ma, respectively. On the mitochondria to construct the phylogenetic trees of the contrary, from other genes, the times were all dated to be Araucariaceae. Combined analysis strongly supported 300 MYA and were close to each other. It can be con- that Wollemia was sister to Agathis, that is, the phyloge- cluded that genes which constructed high resolution and netic relationships among the three genera in the family reliable phylogeny trees should be used to estimate the Araucariaceae are ((Wollemia, Agathis), Araucaria), divergence times. A number of programs such as PAML, while the result of ref. [7] might be caused by the wrong Beast and r8s can be used for estimating the divergence outgroup and low resolution of the rbcL gene tree. times, though they often yield different results. Our According to the results of statistical tests for the sin- study shows that MCMCTREE and Beast using Bayes- gle-gene trees and the combined tree, cp16S, rbcL and ian MCMC algorithm yield reasonable and similar re- coxI genes most supported the tree topology ((Wollemia, sults. Agathis), Araucaria), but did not reject either ((Agathis, In this study, the divergence time of the family Arau- Araucaria), Wollemia) or ((Araucaria, Wollemia), cariaceae, estimated by MCMCTREE, was calculated to Agathis); by contrast, the results of the statistical tests on [30–32] be (308 ± 53) Ma (Late Carboniferous) , but most of the 26S, matK, rps4 and atp1 genes are statistically sig- the Araucariaceae fossil records were in Triassic (205－ nificant. For example, the statistical P value of three 250 Ma). One reason for this mismatch is that the fossil topologies of the rps4 trees was 0.998, 0.002 and 0.002, records usually lag behind the divergence time. The respectively, indicating that either ((Agathis, Araucaria), Araucariaceae is derived from primitive conifers, which Wollemia) or ((Wollemia, Araucaria), Agathis) can be appeared from Carboniferous and had a broad distribu- rejected but ((Agathis, Wollemia), Araucaria) can be tion after Permian. Moreover, the fossils indicate that the accepted. Statistical analyses on the 26S and matK trees primitive conifers are too similar to the Araucariaceae to yield similar conclusion. However, the atp1 tree is in be distinguished from each other. As a result, the fossil contradiction with the above trees. The topology woods from Permian are often named “Araucarioxylon”. ((Agathis, Araucaria), Wollemia) was accepted, which Therefore, the Araucariaceae derived from the Late needs to be further studied. Considering the low resolu- Carboniferous was possible. Our study indicated the tion of the 26S tree, matK and rps4 genes are most suit- Araucariaceae derived from Late Carboniferous but not able for constructing the phylogenetic trees of the family Triassic, and provided a clue for estimating the diver- Araucariaceae. gence times of other gymnosperms. The divergence times estimated by using the eight genes respectively are different from each other. Based We would like to thank Prof. Masami Hasegawa for his comments and on the combined tree, the time of origin of the family suggestions.

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