Original publication date: 12/13/2011
Edited for grammar and syntax: 07/22/2019
Previous analysis of Mammalian phylogenies has made heavy use of nuclear 18S or 28S rDNA genes, but more recently large numbers of other protein-coding genes have also been incorporated (Meredith, 2011; Science, VOL 334, pg. 521-524). Some work has been done looking at the efficacy of using the complete mitochondrial genome (MTCG) of eutherian mammals to resolve deep relationships (Kjer, 2007; BMC Evolutionary Biology, doi:10.1186/1471-2148-7-8). However, the evaluation of an updated, complete MTCG phylogeny of the mammals (including two species of Mammuth, one Mammut (Mastadon), and two hominins, all extinct) could be useful for determining the efficacy of complete mitochondrial genomes at resolving both deep and shallow relationships, within mammals. All of the 308 mammal and eight other amniote MTCGs were downloaded from GenBank in FASTA format. In order to obtain the genomic DNA of all 13 protein-coding MT genes from those MTCGs, as well as two rRNA genes, a series of BioPerl scripts were developed. The position of each gene in the MTCGs was first estimated using BLAST and then extracted out using a BioPerl script. All protein-coding genes were aligned by protein in MEGA, and then the corresponding nucleotide alignment was inferred. The two rDNA genes were aligned to reference alignments using the MAFFT alignment tool, which takes the secondary structure of RNA into consideration. The final alignment contained 14,958 nucleotides, 10,430 of which were parsimony informative. Maximum likelihood (ML) trees were constructed using GARLI, of which 44 were combined in a 50% consensus tree for topological analysis. Further phylogenetic analysis was conducted on a GARLI-optimized version of the best found. Overall tree topology matched well with previous estimates of mammalian phylogeny based on nuclear and morphological data sets.
Previous studies have shown that large, combined data sets of either complete mitochondrial genomes (Kjer, 2007) or many protein-coding genes (Meredith, 2011), produce complete, well-resolved phylogenetic trees in line with current taxonomic efforts (Springer et al. 2001). However, efforts using complete mitochondrial genomes (MTCG's) have been constrained to only a limited number of taxa. Here, a method is explored in which more comprehensive taxon sampling (308 mammals) compared to 78 Eutherians (Kjer, 2007) and 164 mammals (Meredith, 2011) is used to test the effectiveness of an MTCG tree.
One interesting group of mammal is the arboreal group of gliders called Dermoptera, or flying lemurs, whose extant representatives consist of only two species; Galeopterus variegatus and Cynocephalus volans. Both species are endemic to South East Asia and the Philippines, respectively. Traditionally flying lemurs have been thought to be more closely related to bats than to primates. However, recent molecular evidence has shown them to be the closest living ancestor of primates (Meredith, 2011) and in some cases, nested within primates (Kjer, 2007). Here, the relationship of flying lemurs regarding primates is explored similarly to Kjer (2007).
All 13 protein-coding genes and two ribosomal (rDNA) genes were identified in target MTCG's using query sequences, selected arbitrarily from the Homo sapiens MTCG, against a BLAST database of 308 mammalian MTCG's. BioPerl scripts were used to extract and compile orthologs into FASTA files for alignment. Sequences for the 13 protein-coding genes were aligned by amino acid using MUSCLE. A small subsample of rDNA sequences (3) was selected and aligned in MAFFT to aid in the creation of meaningful rDNA alignments. The initial MAFFT alignment was locked in place, and the remaining 305 mammal rDNA sequences were aligned and constrained to the existing gaps using CLUSTALW.
Alignments of each gene (15 in total) were all concatenated and combined into a single FASTA file and imported into GARLI (https://molevol.mbl.edu/index.php/Garli_wiki) for phylogenetic analysis. Best Maximum Likelihood (ML) tree search was allowed to continue for 44 search replicates until the best tree was found (-1474019.37098 lnL). Initial bootstrap analysis was done using a Windows 7 64-bit version of GARLI with a bootstrap replicate sample size of 51 and constrained rate parameters. Additional bootstrap analysis was done using locally compiled Linux (Ubuntu 11.04, 64-bit) GARLI binaries on the same machine wherein 106 bootstrap replicates were obtained, and rates and parameters were allowed to evolve optimal values.
Consensus trees were constructed for each of the three obtained tree data sets (ML search replicates, n = 51; bootstrap 1; n = 51, bootstrap 2, n = 106) and compared to existing complete mammal phylogenetic trees to determine overall congruence. Prototherian, Metatherian, and Eutehrian clades were all strongly supported with 100% bootstrap support under all three consensus methods (Fig. 2). Euarchontoglires, containing both primates and rodents, was weakly supported as a monophyletic clade with only 78% support under bootstrap method 1, and 61% support under method 2. Lagomorphs and primates both retained 100% bootstrap supported, whereas Rodentia only achieved a maximum of 90% support. However, all of the rodent superfamilies achieved 100% bootstrap support. Laurasiatheria, Xenarthra, and Afrotheria all had 100% support as monophyletic clades.
Flying lemurs (Cynocephalus volans) were found to be nested within the monophyletic primate clade and were found to be a sister taxon to the superorder Simiiformes on the best ML tree, putting them more closely related to apes and monkeys than both tarsiers and true lemurs. However, support for flying lemurs at this position is low with only 71% bootstrap support (Fig. 2).
The positioning of flying lemurs as nested within primates coincides well with Kjer et. al. (2007), who also used MTCG's to assess mammal relationships. This is in opposition to the positioning of Meredith et. al. (2011), which puts both flying lemur species as a sister taxon to all primates. This suggests a difference in the evolutionary rate of the mitochondrial and nuclear genes. If nuclear, protein-coding genes are evolving quicker in flying lemurs than their mitochondrial counterpart, as suggested by their highly divergent morphological features; this could explain the pattern we see.
Kjer, M Karl ( 2007), Site specific rates of mitochondrial genomes and the phylogeny of eutheria, BMC Evolutionary Biology, doi:10.1186/1471-2148-7-8
Meredith, Robert ( 2011), Impacts of the cretaceous terrestrial revolution and KPg extinction on mammal diversification, Science, VOL 334, pg. 521-524
Springer MS (2001), Mitochondrial versus nuclear gene sequences in deep-level mammalian phylogeny reconstruction, Mol Biol Evol, 18:132-143.
Figure 1. Consensus tree (95%) of 51 GARLI ML search replicates.
Figure 2. Subset of the consensus tree (50%) of 106 bootstrap replicates of the best ML tree. Shown here are only the primates and their nearest neighbors, the Lagormorphs. Galeopterus variegatus, the Sunda flying lemur, is nested within primates with BS support of 71%.