Computational protocol: Land snails of Leptopoma Pfeiffer, 1847 in Sabah, Northern Borneo (Caenogastropoda: Cyclophoridae): an analysis of molecular phylogeny and geographical variations in shell form

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[…] In addition to the sequences collected from 17 specimens in this study, 16S rDNA and COI sequences of L. tigris, L. vitreum and an outgroup species—Cyclophorus formosensis (Nevill, 1882) from and —were obtained from GenBank (, Page 1: for informations of specimens). All the DNA sequences were aligned and checked manually using Bioedit v7.1.9 (). In order to find the best-fit model of substitution, jModelTest2 () as implemented in CIPRES portal () was performed based on corrected Akaike Information Criterion (AICc) for ITS-1 sequences, 16S rDNA sequences and each of the codon positions of COI sequences. Phylogenetic trees were estimated using Maximum likelihood (ML) and Bayesian Inference methods (BI) as implemented in CIPRES portal (). ML analysis was conducted using Raxml-HPC2 () with 1,000 rapid bootstraps. BI analysis was performed using MrBayes v3.2.3 (). This consisted of running four simultaneous chains for 100,000 generations and 10 sampling frequency. The first 250 trees were discarded as burn-in, while the rest were used to obtain the final consensus tree. [...] Phylogenetic signal analysis was used to investigate the relationship between phylogeny and morphology, with all the analyses done in the R statistical environment, version 3.1.3 (). The tips in the tree corresponding to juvenile specimens and outgroup taxa were excluded by using package ‘ape’ (). The final tree for phylogenetic signal analysis consisted of 14 adults of the three Leptopoma species, L. atricapillum, L. pellucidum, and L. sericatum. The six quantitative and three qualitative shell characters were mapped onto the tree by utilising package ‘phytools’. Phylogenetic signals for each of these nine shell characters were examined using maximum likelihood (λ) () and K (). The consensus tree was transformed into an ultrametric tree after which a lambda analysis was performed using the ‘chronopl’ function from the ‘ape’ package (). As a result, a chronogram was generated using penalised likelihood with an arbitrary lambda value of 0.1, the alternative model. A null model, the Leptopoma phylogenetic tree with λ = 0 (no phylogenetic signal), was generated using the ‘rescale’ function from the ‘geiger’ package (). The λ value of each shell character was estimated for both models using the ‘fitDiscrete’ function for three qualitative shell characters and ‘fitContinuous’ function for six quantitative shell characters in the ‘geiger’ package (). Likelihood scores for the alternative and null models were compared by performing a likelihood ratio test in order to examine the phylogenetic signal in each shell character, wherein Blomberg’s K was calculated using the ‘physig’ function from the ‘phytool’ package ((); R script in ). […]

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