TITLE: BEAST v1.X tutorial: Mammalian timetree
AUTHORS: kawaoso 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">A Bayesian inference (BI) tree is constructed using the software BEAST v.1X (Bayesian Evolutionary Analysis Sampling Trees; Suchard et al., 2018; released on 10th June, 2018), running BEAUti, BEAST, TreeAnnotator, and FigTree, in ascending order.Before operating the BEAST software, the BEAGLE Library must be downloaded. Platform software is BEAST, and the calculation time was more than 2 hours.</div><style>
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?. General flow
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							A Bayesian inference (BI) tree is constructed using the software BEAST v.1X (Bayesian Evolutionary Analysis Sampling Trees; Suchard et al., 2018; released on 10th June, 2018), running BEAUti, BEAST, TreeAnnotator, and FigTree, in ascending order.Before operating the BEAST software, the BEAGLE Library must be downloaded. Platform software is BEAST, and the calculation time was more than 2 hours. 5 hours were needed for the present analysis.
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							BEAST v1.X has an important function of calibration in the associated software of BEAUti (Bayesian Evolutionary Analysis Utility). The calibration protocol of BEAUti is to input a time of the most recent common ancestor (tMRCA = minimum age) of the ingroup species, either by fossil calibration or by the geological event calibration. Using BEAST v1.X, without fixing maximum age constraint, we can simply build a reliably-dated phyologeny.
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							Another important function is combined gene analysis, but we applied whole mitochondrial sequence data from GenBank/DDBJ. The alighment was by ClustalW in MEGA X (Stecher et al., 2020). Gapped areas of non-protein cording regions were excluded, and the consequent protein cording sequence data have totally 10,112 bp.
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							Calibrations points are shown on the consequent BI tree, and these dates were input in “Priors” in BEAUti; they are summarized below. See references in our paper.
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							Calibration point A: Fossil chimpanzee was reported from the East African Rift, and dated by the Ar-Ar method as 0.545 ± 0.003 Ma (McBrearty and Jablonski, 2005).
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							Calibration point B: Fossil gorilla was recently reported also from the East African Rift, and combined with the Ar-Ar method and megneto-stratigraphy, reliably dated as 8.072± 0.8 Ma（Katoh et al., 2016）.
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							Benton et al. (2015) reviewed many calibration data available for animals, and the following mammalian calibration points and dates are addressed by their proposal, except for calibration point K that we ourselves offered. Ages are after International Chronostratigraphic Chart by International Commission on Stratigraphy, 2013.
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							Calibration point C: Crown Hominoidea (86) by Benton et al. (2015). Fossil euprimate was found from Pakistan, and considered to be the Serravallian stage (12.72 ± 1.1 Ma).
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							Calibration point D: Crown Catarrhini (Cercopithecoidea + Hylobatidae + Hominoidea) (84) by Benton et al. (2015). The Nsungwe Formation, Tanzania, contains these mammalian fossils, and dated by U-Pb method as 24.93 ± 0.49 Ma (Roberts et al., 2010; Stevens et al. 2013).
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							Calibration point E: Crown primates (81) by Benton et al. (2015). Fossil euprimate was found from Morocco,and considered to be the Thanetian stage (57.6 ± 1.6 Ma).
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							Calibration point F: Crown Euarchontoglires (74) by Benton et al. (2015). Fossil Paromomys was found from Montana, USA, and considered to be the Danian stage (63.8 ± 2.2 Ma).
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							Calibration point G: Crown Eulipotyphla (68) by Benton et al. (2015). Fossil Erinaceidae was found from Montana, USA,and considered to be the Danian stage (63.8 ± 2.2 Ma).
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							Calibration point H: The common ancestor of leporids (rabbits and hares) and ochotonids (pikas) (76) by Benton et al. (2015). The Indian fossil horizon was considered to be the Yepresian stage (51.96 ± 4.6 Ma) based on the foraminifer zone (Rose et al., 2008).
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							Calibration point I: Crown Rodentia (81) by Benton et al. (2015). Fossil Paramys was found from Montana, USA,and considered to be the Thanetian stage (57.6 ± 1.6 Ma).
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							Calibration point J: Intra-Murinae, Divergence of Mus from Rattus (79) by Benton et al. (2015). The fossil horizon, Pakistan, was stratigraphically, magneto-stratigraphically, oxygen isotopically dated at 10.4 Ma (Barry et a., 2002).
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							Calibration point K: Orohippus was reported from the Green River Formation, USA (Grande, 1980). Ar-Ar dating applied to the silicic tuff within the formation yields ages of 53.5 - 48.5 Ma (weighted average of 51.25 ± 0.31 Ma; Smith et al., 2003). Although Orohippus was evolved from equids such as Eohippus, we used above date for the calibration. Bat fossils, as well as primate fossils, were also reported from the Green River Formation (Grande, 1980), and bat is included in our timetree (Fig. 1).
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							Calibration point L: Crown Carnivora (69) by Benton et al. (2015). The fossil was from the Canadian Duchesnian with radiometric age (Robinson et al., 2004) and estimated at 37.8 ± 0.5 Ma.
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							Calibration point M: Crown Artiodactyla (70) by Benton et al. (2015). The fossil horizon, Pakistan, was correlated to the planktonic foraminifer zone, and 53.25 ± 0.75 Ma (Bajpai and Gingerich, 1998).
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							Calibration point N: Divergence of Bovinae (cow) and Antilopinae (sheep)(73) by Benton et al. (2015). The fossil horizon in the Siwalik Deposits is the Burdigalian stage(18.205 ± 2.235 Ma) based on the paleomagnetic chronology (Johnson et al., 1985).
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							Calibration point O: Crown Xenarthra (62) by Benton et al. (2015). Fossil Xenarthra was found from Brazil,and considered to be the Thanetian stage (57.6 ± 1.6 Ma).
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							Calibration point P: Crown Afrotheria (63) by Benton et al. (2015). Fossil Eritherium was found from Morocco,and considered to be the Thanetian stage (57.6 ± 1.6 Ma).
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							Calibration point Q: Crown Marsupialia (58) by Benton et al. (2015). Fossil Djarthia, Australidelphia, was found from Murgon, Australia,and considered to be the Yepresian stage (51.96 ± 4.6 Ma).
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							Calibration point R: Crown Theria (59) by Benton et al. (2015). Juramaia sinensis from Liaoning, Northeastern China, with an Ar-Ar age of 160.7 ± 0.4 Ma corresponding to the Oxfordian stage (Luo et al., 2011).
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							Calibration point S: Crown Mammalia (57) by Benton et al. (2015). Ambondro mahabo from Madagascar is placed within Monotremata,and considered to be the Bathonian stage (162.7 ± 1.1 Ma).
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?. Prepare .txt files of whole mitochondrial data.
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?. Partitions: Loading .text file is by using the plus button.
?. Taxa: Loading of taxa as ingroup was by using the plus button.
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							The left screen: Taxon Set (monophyletic boxes were checked for all in the present case, and stem box were checked in case by case but for Erinaceidae, Theria, and Prototheria), and the right screen: Included Taxa.
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?. Sites: Substitution Model: HKY (Hasegawa, Kishino and Yano) model.
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?. Clocks: Clock Type: Uncorrected relaxed clock, Relaxed Distribution: Lognormal.
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							Uncorrelated relaxed clocks allow each branch of a phylogenetic tree to have its own evolutionary rate under log-normal distribution, and the node rate is the rate median of three branches (Drummond et al., 2006).
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?. Trees: Tree Prior: Speciation: Yule Process.
?. Priors: tmrca (time of MRCA) was input from the calibration point date noted above as Prior Distribution: Normal, and the Mean and Standard deviation.
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?. MCMC: Length of chain: 10,000,000. Default setting.
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?. The consequent tree was drawn by FigTree v1.4.2, for that, the tree files were input into TreeAnnotator.
?. FigTreeThe 95% highest posterior density for confidence intervals of ages can be output in FigTree, but not shown in BI tree to avoid confusion. In FigTree, posterior probability (“posterior”), posterior age (“Node ages”), and “rate median” (not constant) can be output.
?. Adobe Illustrator