Figure 1.
Phylogenetic analysis of 25 concatenated collagen α1 (I) and α2 (I) sequences taken from the Ensembl Genome Browser.
Maximum Likelihood analyses produce a phylogenetic signal in good agreement with previously published genomic data (e.g. [13]–[14]). Branch lengths are reported as the mean number of changes per site, bootstrap support is labelled and chicken (Gallus gallus) used as the outgroup.
Figure 2.
MALDI-ToF-MS spectra of collagen digests from the sub-fossil ‘malagasy aardvark’ Plesiorycteropus and modern specimens.
The peptide mass fingerprints (PMFs) show that the quality of the collagen extracted from the fossil is as good as from modern specimens (from top to bottom; aardvark (Orycteropus afer), Plesiorycteropus sp., rock hyrax (Procavia capensis) and greater hedgehog tenrec (Setifer setosus)). Peaks marked α and β represent some of the most variable homologous peptides manually sequenced (Fig. 3 & Fig. S2). A greater similarity between peptide m/z values can be seen between Plesiorycteropus and tenrec than any other species sampled.
Figure 3.
Example tandem MS spectra of three homologous peptides.
The spectra are from peptide COL1A2495–509 (peak ‘α’ in Fig. 2) with precursor (MH+) ions at (from top to bottom) m/z 1453 (elephants, pangolin anteater and tamandua), 1463 (hyrax and aardvark) and 1477 (armadillos and all afrosoricids including the extinct Plesiorycteropus). Labeling of fragment ions (inset) follows [37], underline represents hydroxylation site, red text highlights amino acids that differ from the inset (elephant, anteater & pangolin) sequence. Isobaric amino acids (I/L) were estimated based on conserved DNA sequence data where possible; for sites that have more than one isobaric amino acid present, the most abundant was used (see Supplementary Information).
Figure 4.
Phylogenetic analyses of Plesiorycteropus collagen (I) sequences obtained by LC-MS in comparison to previously postulated closest relatives.
The results are from A) Maximum Likelihood (rooted to Gallus; only bootstrap support >50 is shown) and B) Neighbor-Net analysis in SplitzTree4. Plesiorycteropus consistently forms a monophyletic group with the tenrecs within the order Tenrecoidea, forming a sister group to the aardvark (tubulidentates), rock hyrax, and the proboscideans. Silhouette denotes sequences obtained from LC-MS data; scale shows average amino acid substitutions per site.