Computational protocol: Molecular phylogeny and taxonomic revision of the sportive lemurs (Lepilemur, Primates)

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Protocol publication

[…] DNA from the biopsies was extracted using a standard proteinase K digestion, followed by a phenol chloroform extraction [] with minor modifications [], or isolated with the QIAamp DNA Mini Kit as recommended by the supplier.The complete mitochondrial cytochrome b gene was amplified via PCR using the oligonucleotide primers CYT-LEP-L: 5'-AATGATATGAAAAACCATCGTTGTA-3' and CYT-LEP-H: 5'-GGCTTACAAGGCCGGGGTAA-3'. Standard, wax-mediated hot-start PCRs were carried out for 40 cycles, each with a denaturation step at 94°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 90 s, followed by a final extension step at 72°C for 5 min. Aliquots of the PCR amplifications were checked by agarose gel electrophoresis. Subsequently, PCR products were cleaned using the Qiagen PCR Purification Kit and sequenced on an ABI 3100-Avant sequencer using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems), primers as indicated above and the internal primers CYT-LEP-L400: 5'-TGAGGACAAATATCATTCTGAGG-3' and CYT-LEP-H545: 5'-TGGAGTGCGAAGAATCGGGT-3'. The respective sequences were deposited in GenBank and are available under the accession numbers DQ108990-DQ109034 and DQ234881-DQ234900.Sequences were easily aligned by eye due to the lack of insertions or deletions, and were checked for their potential to be correctly transcribed in order to eliminate data set contaminations with pseudogenes. For a comprehensive evaluation of the sequence data, we expanded our data set with orthologous sequences, already deposited at GenBank, from two L. ruficaudatus and one L. dorsalis. As outgroup for phylogenetic tree reconstructions, we selected Phaner furcifer because it displays the most similar orthologous sequence of all Malagasy lemurs to Lepilemur []. Further details about analysed individuals and sequences are summarized in Figure and .Uncorrected pairwise differences within and between species and major populations were calculated with PAUP 4.0b10 [] and DnaSP 3.52 [].A population aggregation analysis (PAA) was performed according to the diagnostic character framework described in []. Accordingly, fixed nucleotide characters provide the unit for which aggregation of taxonomic units occurs. For diagnosis, attributes whose fixed unique states unite a group (populations, species), to the exclusion of other groups, are considered characters. Polymorphic attributes, or traits, are indicative of population frequency differences. To identify diagnostic sites, sequences were imported into MacClade 3.0 [].Phylogenetic tree reconstructions were carried out with the maximum-parsimony (MP), neighbor-joining (NJ) and maximum-likelihood (ML) algorithms as implemented in PAUP or TREEPUZZLE 5.0 []. For MP analyses, all characters were treated as unordered and equally weighted throughout. A heuristic search was performed with the maximum number of trees set to 100. NJ and ML trees were constructed with the TVM + I (= 0.5156) + Γ (= 2.2387) model of sequence evolution as it was selected as best-fitting model with MODELTEST 3.06 [], as well as with standard models. Relative support of internal nodes was performed by bootstrap analyses with 1,000 replications (MP, NJ), or by the quartet puzzling support values on the basis of 10,000 puzzling steps (ML). […]

Pipeline specifications

Software tools PAUP*, DnaSP, MacClade, ModelTest-NG
Application Phylogenetics
Organisms Leopoldamys edwardsi, Lepilemur ruficaudatus