Computational protocol: Clouded leopard phylogeny revisited: support for species recognition and population division between Borneo and Sumatra

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Protocol publication

[…] We used a 426 bp portion of a central conserved region within the D-loop of the control region [] in addition to two mtDNA (ATPase-8 and Cyt-b) genes []. The control region primers were modified by Janecka JE (pers. comm.) [(F) CTC AAC TAT CCG AAA GAG CTT] and [(R) CCT GTG GAA CAT TAG GAA TT]. In total we amplified 900 bp of mtDNA.PCR reactions were performed in a final volume of 25 μl containing 2.5 μl MolTaq 10x PCR Buffer, 2 mM MgCl2, 0.2 mM dNTPs, 1 μM of each primer, 2 units of MolTaq polymerase (Molzym GmbH, Bremen, Germany) and 2 μl of genomic DNA. PCR reactions were performed in an Eppendorf Mastercycler (Eppendorf GmbH, Wesseling-Berzdorf, Germany), with an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, elongation at 72°C for 45 s, and were completed with a final elongation step at 72°C for 10 min. PCR products were purified by ultra filtration through Montage TM filter devices (Millipore GmbH, Schwalbach, Germany) and sent to Seqlab (Seqlab Laboratories, Göttingen, Germany) for sequencing. Sequences were edited, assembled and aligned using ClustalW [] implemented in BioEdit (Version [], before being exported to PAUP (Version 4.0b10) [] for phylogenetic analysis. Sequences from each of the mtDNA fragments were concatenated into a 900 bp sequences, because results from separate analysis of each gene fragment showed identical topologies [] and mitochondrial genes usually do not recombine []. Phylogenetic relationships among haplotypes were estimated using minimum evolution (ME), maximum likelihood (ML) and maximum parsimony (MP) [,]. We used Kimura 2-parameter distance with neighbor-joining (NJ) algorithm followed by tree-bisection reconnection branch-swapping procedure (TBR) for the ME analysis. MP trees were conducted using a heuristic search, with 10 random taxon addition replicates and TBR branch swapping. The ML approach was performed using the HKY85 model []. Each phylogenetic tree was rooted with the domestic cat sequence. Reliability of all trees was tested with bootstrap values by 1000 replicates of heuristic search and TBR branch swapping.The approximate age of separation between N. nebulosa and N. diardi was estimated using LINTREE []. A neighbor-joining tree [] was generated with Kimura 2-parameter γ-corrected distances [] using the combined 900 bp mtDNA sequence. The molecular clock test implemented in LINTREE [] showed that the sequences did not deviate significantly from the rate constancy test (p > 0.05). The coalescence point between clouded leopards and the Panthera genus, being 6.37 MYA based upon a comprehensive analysis of nuclear gene sequences and multiple fossil dates [], was chosen to be the calibration point for this study. We used a range of two standard errors to calculate a 95 % confidence interval. [...] We used 10 felid dinucleotide microsatellite primers (FCA 8, FCA 45, FCA 77, FCA82, FCA 105, FCA 126, FCA 132, FCA 144, FCA 261, FCA 310) [], which were already used in a previous study on clouded leopards []. Those microsatellites are located on 8 felid autosomes and all of them are at least 5 centimorgans apart from each other []. In addition to those ten microsatellite loci of known allele size ranges for clouded leopards, we applied 8 microsatellites of unknown allele sizes for clouded leopards, FCA 23 and FCA 43 [], and HDZ 3, HDZ 57, HDZ 64, HDZ 89, HDZ817, HDZ 859 []. PCR amplifications were performed in a final reaction volume of 10 μl utilizing described methods [,]. The IR-dye-labeled PCR products were diluted and analyzed on a LI-COR 4300 DNA-Analyser (LI-COR Bioscience GmbH, Bad Homburg, Germany). Data were collected and analyzed using Saga Generation 2 (Version 3.2.1).To test their performance as population genetic markers, all microsatellites were tested for deviations from linkage disequilibrium (LD) using GENEPOP on the web version 3.4 []. Measures of microsatellite genetic variation in terms of observed and expected heterozygosities were estimated with Arlequin 3.1 [].Pairwise genetic distance among clouded leopards and the two outgroup species was estimated with two microsatellite genetic distance estimators: the proportion of shared alleles (Dps) and the kinship coefficient (Dkf) with the [1 – ps/kf] option in MICROSAT []. Phylogenetic NJ-trees were constructed from the Dps and Dkf distance matrixes using NEIGHBOR (included in PHYLIP version 3.66) []. Bootstrap values for 1000 bootstrap replicates in MICROSAT were calculated using CONSENSE TREE (included in PHYLIP version 3.66) []. Trees were drawn using the program TREEVIEW (version 1.6.6) []. [...] A Bayesian clustering method as implemented in BAPS [-] was used to infer population structure based on multilocus microsatellite genotype data. This program estimates the hidden population substructure by testing whether the allele frequencies between populations are significantly different. A major advantage compared to most other methods is that the number of populations is treated here as an unknown parameter that can be estimated from the dataset. We performed 10 independent runs of clustering of individuals with the microsatellite genotypes to ensure homogenous results. In all ten runs we obtained similar results (data not shown). After the clustering of individuals by their allele frequencies the results were used to perform an admixture analysis. We used 500 iterations and a number of 1000 reference individuals per population each with 20 iterations. The estimated admixture coefficient for an individual in each cluster q (maximum = 1) was used as a measure of correct assignments. The Bayesian p-value tells the proportion of reference individuals simulated from the population in which the individual was originally clustered having the admixture coefficient to the cluster smaller than or equal to the individual []. Individuals having p-values larger than 0.05 are by default considered as having "non-significant" evidence for admixture []. […]

Pipeline specifications

Software tools Clustal W, BioEdit, PAUP*, Genepop, Arlequin, TreeViewX, BAPS
Applications Phylogenetics, Population genetic analysis
Organisms Neofelis nebulosa
Chemicals Nucleotides