Computational protocol: The ancient evolutionary origins of Scleractinia revealed by azooxanthellate corals

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[…] For large specimens, whole mesenteries were dissected out (with forceps) prior to extraction, whereas for smaller specimens an entire system (including skeleton) was extracted and immersed in the lysis buffer. Genomic DNA was extracted using the DNeasy Tissue Kit (QIAGEN). DNA concentrations were determined using a Nanodrop 1000 (Thermo Scientific) prior to Polymerase Chain Reaction (PCR) amplification under the following conditions:(i) 16S rDNA - the primers developed by Le Goff-Vitry et al. [] (LP16SF 5' -TTGACCGGTATGAATGGTGT and LP16SR 5' -TCCCCAGGGTAACTTTTATC) were used to amplify a fragment whose size varied between 280 and 420 bp. Reactions were carried out in a total volume of 50 μl, and contained 0.2 mM dNTPs, 1.5 mM MgCl2, 1 mM of each primer, 1.5 units of Taq polymerase (Fisher Biotec - Australia) and 125 ng of template. The PCR protocol used was: an initial denaturation step (95°C for 5 min), then 35 cycles of 30 s at 94°C, 30 s at 50°C, and 45 s at 72°C, followed by 10 min at 72°C.(ii) COX1 - the universal primers developed by Folmer et al. [] (LCO1 490 5' -GGTCAACAAATCATAAAGATATTGG and HCO2 198 5' -TAAACTTCAGGGTGACCAAAAAATCA) were used to amplify a fragment whose size varied between 690 and 710 bp. Reactions were carried out as described by Folmer et al. []: 95°C for 1 min, then 35 cycles of 30 s at 95°C, 30 s at 40°C, and 90 s at 72°C, followed by 10 min at 72°C.(iii) 12S rDNA - the primers developed by Chen and Yu [] (ANTMT12SF 5'-AGCCACACTTTCACTGAAACAAGG and ANTMT12SR 5'-GTTCCCYYWCYCTYACYATGTTACGAC) were used to amplify a fragment whose size varied between 800 and 920 bp. Reactions were carried out in 50 μl, with 0.2 mM dNTPs, 1.5 mM MgCl2, 1 mM of each primer, 1.5 units of Taq polymerase (Fisher Biotec - Australia), and 125 ng of template. PCR conditions were: 95°C for 4 min, followed by 4 cycles of 30 s at 94°C, 60 s at 50°C, 120 s at 72°C, and 30 cycles of 30 s at 94°C, 60 s at 55°C, 120 s at 72°C and then 4 min at 72°C.(iv) 28S rDNA - the primers developed by Medina et al. [] (28S.F63sq 5'-AATAAGCGGAGGAAAAGAAAC and 28S.R635sq 5'-GGTCCGTGTTTCAAGACGG) were used to amplify a fragment of approximately 750 bp. Reactions were carried out using the Advantage2 PCR kit (Clontech) with 100 ng of template, and following manufacturer's protocol. PCR conditions were: 95°C for 5 min, then 30 cycles of 30 s at 94°C, 60 s at 54°C, 90 s at 72°C, followed by 5 min at 72°C.When amplification reactions based on Taq polymerase did not yield product, amplification was carried out using the Clontech Advantage-2 Kit (with the same template and primer concentrations, and under the same PCR protocol). PCR reactions were performed using a Bio-Rad DNA engine (Peltier Thermal Cycler). PCR products were purified using Mo-Bio Ultra Clean (PCR Clean Up) spin columns, and subjected to direct (Sanger) sequencing at Macrogen (South Korea).Two different approaches were tested using sequences determined here and others retrieved from GenBank (see Additional file ). The first approach included representatives of all hexacorallian orders but Ceriantharia and was intended to validate the Scleractinia monophyly. For this purpose, four single gene phylogenies all rooted with Octocorallia were constructed. The second approach, used herein for time divergence between scleractinian groups (Basal, Complex, and Robust groups), was based on concatenated sequences of the ribosomal genes16S rDNA and 28S rDNA, and included a broad range of scleractinian representatives. Alignments for both approaches were performed for each gene separately using ClustalW (EBI) and manually edited using JalView version 8.0 [].Alignments for the first approach were individually tested for substitution saturation [] using DAMBE [], which indicated little saturation for COX1 and 28S rDNA sequences (i.e. Iss. significantly lower than Iss.c), but higher levels of saturation for the 16S and 12S rDNAs (Iss. higher than Iss.c). Saturation related to mitochondrial ribosomal genes was induced by their respective fast evolving regions. This phenomenon was particularly evident because sequences from distant Anthozoa representatives were included in these alignments. To improve the phylogenetic signal, the most rapidly evolving regions were excluded from the alignment, resulting in a sharply decrease in saturation levels. The final alignments used in the first approach consisted of 298 positions for the16S rDNA, 599 positions for COX1, 631 positions for 12S rDNA, and 709 positions for the 28S rDNA. For each marker, appropriate models of nucleotide substitution were determined by the hierarchical likelihood ratio test implemented in MrModeltest []. Phylogenetic analyses were performed using PhyML [] for maximum likelihood (ML) and MrBayes (version 3.1.2) [] for Bayesian Inference (BI). The maximum likelihood analyses were performed under the GTR model with a non-parametric Shimodaira-Hasegawa-like procedure. For the Bayesian inference, two runs each of 10 million generations were calculated for each marker with topologies saved at each 1000 generations, with the average standard deviation of split frequencies between runs of each marker converging to or less than 0.01. The first quarter of the 10000 saved topologies were discarded as burnin, and the remaining used to calculate posterior probabilities (Figure ).The final alignment that based the second approach contained concatenated 16S rDNA and 28S rDNA sequences (without excluding the fast evolving regions) from 121 scleractinians and 1 corallimorpharian, totalling 1334 bp. This alignment was also tested for substitution saturation, which indicated good phylogenetic signal. ML phylogenetic analyses were performed as described above. However, instead of Shimodaira-Hasegawa-like statistical support, they were performed under the Chi-square and 100 bootstrap replicates.To estimate divergence times for gardineriids/micrabaciids and other scleractinians, we applied a relaxed-clock (uncorrelated lognormal) Bayesian Markov chain Monte Carlo method as implemented in BEAST (version 1.4.8) []. This method allows nucleotide substitution rates to vary between lineages and incorporates phylogenetic uncertainty by sampling phylogenies and parameter estimates in proportion to their posterior probability. Additionally, Yule process was chosen as tree prior, and the prior distribution of divergence of each calibrated node was set as normal with standard deviation of 3.5. Hierarchical likelihood ratio tests led to the adoption of the General Time Reversible model with a proportion of invariant sites and gamma distributed rate heterogeneity (GTR+I+Γ) as the most appropriate evolutionary model for the molecular clock analyses. One run of 10 million generations was calculated with topologies and other parameters saved at each 1000 generations. A quarter of the 10000 saved topologies were discarded as burnin, and the remaining used to calculate posterior probabilities and node ages (Figure ). Additionally, phylogenetic reconstruction from the same alignment was also calculated on MrBayes in two MCMC runs of 10 million generations each with topologies sampled every 1000 generations. Average standard deviation of split frequencies between runs was less than 0.01. The first quarter of the 10000 sampled topologies were discarded as burnin, and the remaining used to calculate posterior probabilities. The resulting topology was consistent with the one calculated using BEAST (data not shown).For the calibration of the molecular clock, stringent constraints were applied based on fossils that can be unambiguously assigned to extant clades and whose unique skeletal characters can be unequivocally recognized in fossil coralla. Nodes used for the calibration were: (A) the appearance of Caryophyllia (ca. 160 Ma), based on the Late Jurassic (Oxfordian) species C. simplex and C. suevica [,]. Both species have well-developed "true" pali present in one crown before the penultimate cycle of septa, fascicular columella composed of several twisted laths and septothecal walls, characters which together occur only in fossil and extant representatives of this genus []; (B) The divergence of the Dendrophylliidae (ca. 127 Ma), corresponding to the first occurrence of solitary Palaeopsammia (Barremian) []. The first appearance of colonial dendrophylliids (Blastozopsammia) in the Albian (ca. 100 Ma) [] is consistent with the earlier origin of solitary genera. Skeletal synapomorphies of dendrophylliids include the Pourtalès plan of septal arrangement and the presence of a synapticulothecate wall []; and (C) the origin of Flabellum (ca. 77.5 Ma), based on the earliest known record of the genus (F. fresnoense) from the Late Cretaceous (Coniacian; ?early Maastrichtian, based on foraminiferal assemblage) []. Unequivocal Flabellum fossils are also known from the Late Cretaceous (?Campanian, Maastrichtian) of Seymour Island (F. anderssoni) [] and Late Cretaceous (Maastrichtian) of Western Australia (Flabellum miriaensis) []. Flabellum is clearly distinguishable based on the following unique combination of characters: the marginothecal wall is present throughout ontogeny, lack of pali/paliform lobes and scale-like microtexture of septa (sometimes preserved in the fossil record). Monophyly was enforced in the case of the dendrophylliids (calibration node B), but not for nodes A or C. […]

Pipeline specifications

Software tools Clustal W, Jalview, DAMBE, MrModelTest, PhyML, MrBayes
Application Phylogenetics