samples were preserved in ethanol and 24 hours later transferred to tubes containing silica. Samples were stored at ambient temperature for up to six months before further processing. One additional sample consisted of dry skin tissue from a museum specimen (sample PC3: Papio cynocephalus, North-east bank of Lake Rukwa, Tanzania, coll. no. 03-74959, collected in 1902, Hum- boldt Museum, Berlin, Germany). Blood samples from other papionin genera were obtained from German zoos (Theropithecus gelada, Duisburg; Lophocebus aterrimus, Berlin; Cercocebus chrysogaster, Wuppertal; Mandrillus sphinx, Rostock). Laboratory methods DNA from fecal, tissue and blood samples was extracted with the DNeasy and Stool Mini Kit from Qiagen following the supplier’s recommendations. For Rungwecebus, sequence data from eight loci, including three mitochondrial (12SrRNA, COI, COII), one X chromosomal (Xq13.3), one Y chromosomal (TSPY) and three autosomal loci (a 1,3-GT, LPA, CD4) were available. To amplify respective fragments in baboons and other papionin species, PCR conditions and primers were identical to those used to amplify the loci from Rungwecebus [2,6] and other primates [41– 44] (Table S3). The Xq13.3 and TSPY sequences were each generated via three overlapping PCR products [2,6]. The results of the PCR amplifications were checked on agarose gels. PCR products were cleaned with the Qiagen PCR Purification Kit and subsequently sequenced on an ABI 3730xl sequencer using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). Sequences were deposited in GenBank (Tables S1, S2). To prevent contamination, laboratory procedures followed recommended, standard protocols [37,45–48]. Specifically, DNA extraction, PCR, PCR purification and sequencing were performed in separate laboratories and repeated after several months, while always only one individual per species was tested. Finally, all PCR reactions were performed with negative (HPLC-purified water) controls. Statistical methods To obtain a comprehensive overview of the phylogenetic position of Rungwecebus among papionins, further orthologous sequences from related taxa deposited at GenBank were included in our study (Tables S1, S2). Each of the five nuclear data sets comprised 11 sequences, representing all seven papionin genera and the five recognized baboon species. Each of the three mitochondrial data sets contained 33 sequences, which represent the seven papionin genera including baboons from most of their geographic range. The macaque sequences (Macaca mulatta or M. sylvanus) were used as an outgroup. Sequences were easily aligned by hand, because few or no indels were present. Due to the low number of polymorphic sites in the nuclear data sets, point mutations, deletions and/or insertions were individually inspected. For phylogenetic tree reconstructions, gaps and poorly aligned positions were manually removed. The sizes of the different alignments and the number of excluded indels are presented in Table S3. Calculations were performed for each locus separately as well as for concatenated nuclear and mitochondrial data sets. Trees were constructed with maximum-parsimony (MP) and neighbor-joining (NJ) algorithms as implemented in PAUP 4.0b10 [49] as well as with maximum-likelihood (ML) and Bayesian algorithms, using the programs GARLI 0.951 [50] and MrBayes 3.1.2 [51,52], respectively. For MP analyses, all characters were treated as unordered and equally weighted throughout. A heuristic search was performed with the tree-bisection-reconnection (TBR) algorithm with random addition of sequences. The maximum number of trees was set to 100. NJ, ML and Bayesian trees were Phylogeny of Rungwecebus constructed with the respective best-fitting models as selected under the Akaike information criterion with MODELTEST 3.7 [53] (Table S3). NJ and ML trees from the concatenated nuclear and mitochondrial data sets were analyzed with the TrN+I and TrN+I+G models, respectively. Bayesian analyses for the combined data set were performed in a partitioned framework, allowing locus- specific parameter estimation. Relative support of internal nodes was performed by bootstrap analyses with 1,000 (MP, NJ) or 500 replications (ML). In GARLI, only the model specifications settings were adjusted according to the respective data set, while all other settings were left at their default value. ML majority-rule consensus trees were calculated in PAUP. For Bayesian analyses, four Monte Carlo Markov Chains with the default temperature of 0.1 were used. Four repetitions were run for 10,000,000 generations with tree and parameter sampling occurring every 100 generations. The first 25% of samples were discarded as burnin, leaving 75,001 trees per run. Posterior probabilities for each split were calculated from the posterior density of trees. To evaluate the reliability of the depicted phylogenetic position of Rungwecebus, alternative tree topologies were evaluated with the KH [7] and SH [8] tests with full optimization and 1,000 bootstrap replications in PAUP. Hypothetical sister group relationships of 1) Rungwecebus to Papio, Lophocebus, Theropithecus and Cercocebus+Mandrillus or 2) that Rungwecebus is nested within Papio were tested. Due to the low number of variable sites in the nuclear data set, divergence ages were estimated only for mitochondrial data. Therefore, further outgroup taxa were included in the data set (Table S1). After removal of additional indel positions, the final alignment for divergence age estimations comprised 1,479 bp. A Bayesian MCMC method, which employs a relaxed molecular clock approach [54], as implemented in BEAST v1.4.6 [55], was used. We assumed a relaxed lognormal model of lineage variation and a Yule prior for branching rates. Data were partitioned by manually editing the XML file and by applying the respective best- fitting models and parameters [53]. As calibrations we used the divergence between human and chimpanzee, which has been dated at 6–7 mya [56,57], the divergence between Papio and Theropithecus, which is estimated at 3.5–4.0 mya [58,59 and references therein], and the spilt between hominoids and cercopithecoids, which is estimated at 23 mya [for discussion of fossil data see [60]. Instead of hardbounded calibration points, we used the published dates as a normal distribution prior for the respective node. For C1 (Pan/Homo) this translates into a normal distribution with a mean of 6.5 mya and a standard deviation of 0.3 mya, for C2 (Papio/Theropithecus) into a mean of 3.75 mya and a standard deviation of 0.3 ma (95% credibility interval: 3.0– 4.5 mya) and for C3 into a mean of 23.0 mya and a standard deviation of 2.5 ma. Four replicates were run for 10 million generations with tree and parameter sampling occurring every 100 generations. The adequacy of a 10% burnin and convergence of all parameters was assessed by visual inspection of the trace of the parameters across generations using the software TRACER v1.3 [61]. Subsequently, the sampling distributions of four independent replicates were combined and downsampled using the software LogCombiner v1.4.6 and the resulting 10,000 samples summa- rized and visualized using the software TreeAnnotator v1.4.6 and FigTree v1.1.1 [62]. The first two programs are part of the BEAST package [55]. Supporting Information Figure S1 50% majority rule consensus tree (cladogram) based on COI sequences. Numbers on nodes represent bootstrap or PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4859