Reunion of Australasian Possums by Shared SINE Insertions

Abstract

Although first posited to be of a single origin, the two superfamilies of phalangeriform marsupial possums (Phalangeroidea: brushtail possums and cuscuses and Petauroidea: possums and gliders) have long been considered, based on multiple sequencing studies, to have evolved from two separate origins. However, previous data from these sequence analyses suggested a variety of conflicting trees. Therefore, we reinvestigated these relationships by screening $\sim$200,000 orthologous short interspersed element (SINE) loci across the newly available whole-genome sequences of phalangeriform species and their relatives. Compared to sequence data, SINE presence/absence patterns are evolutionarily almost neutral molecular markers of the phylogenetic history of species. Their random and highly complex genomic insertion ensures their virtually homoplasy-free nature and enables one to compare hundreds of shared unique orthologous events to determine the true species tree. Here, we identify 106 highly reliable phylogenetic SINE markers whose presence/absence patterns within multiple Australasian possum genomes unexpectedly provide the first significant evidence for the reunification of Australasian possums into one monophyletic group. Together, our findings indicate that nucleotide homoplasy and ancestral incomplete lineage sorting have most likely driven the conflicting signal distributions seen in previous sequence-based studies. [Ancestral incomplete lineage sorting; possum genomes; possum monophyly; retrophylogenomics; SINE presence/absence.].

Figure 1.

Comparison of SINE presence/absence and classical DNA sequence analyses. Left: The common brushtail possum and the Leadbeater’s possum share a diagnostic SINE (black ball) at an orthologous genomic position (pictured as gray area). The SINE was inherited from a shared common ancestor of Phalangeroidea plus Petauroidea and is absent (dashes) in Macropodiformes and all outgroup representatives. Right: The classical DNA sequence analysis compares orthologous conserved sequences that can be variably exposed to homoplasy depending on selection and drift. Both marker systems are equally exposed to hemiplasy as a result of ILS.

Figure 2.

Compilation of phylogenetic signals for presence/absence SINE markers, their flanking sequence regions, and a random genome sequence data set from 11,532 loci. Sixty-one SINE insertions (black ball) provide significant evidence to merge Phalangeroidea and Petauroidea (KKSC test, ). Sequence flanks of the three sets of conflicting SINE insertions (61/26/19) imply considerable support for the respective topologies of the SINEs. The 11,532 randomly selected sequence loci (each 2500 nt) revealed two conflicting tree topologies depending on the method applied (MP vs. ML). The IQ-TREE 2 gene concordance factor (gCF) and site concordance factor (sCF) for flanking and the 11,532 random sequences are shown below the bootstrap results. Bootstrap values are represented in bold.

Figure 3.

Phylogenetic reconstructions of informative SINE presence/absence loci. a) SplitsTree analysis. Sixty-one SINE loci (black ball) are mapped on the common branch of Phalangeroidea and Petauroidea, a branch substantiated by 100 bootstrap support. b) Bayesian inference of the presence/absence loci revealed a common origin for Phalangeroidea and Petauroidea by 100 bootstrap resampling.