When Genomics Is Not Enough: Experimental Evidence for a Decrease in LINE-1 Activity During the Evolution of Australian Marsupials.

The autonomous transposable element LINE-1 is a highly abundant element that makes up between 15% and 20% of therian mammal genomes. Since their origin before the divergence of marsupials and placental mammals, LINE-1 elements have contributed actively to the genome landscape. A previous in silico screen of the Tasmanian devil genome revealed a lack of functional coding LINE-1 sequences. In this study we present the results of an in vitro analysis from a partial LINE-1 reverse transcriptase coding sequence in five marsupial species. Our experimental screen supports the in silico findings of the genome-wide degradation of LINE-1 sequences in the Tasmanian devil, and identifies a high frequency of degraded LINE-1 sequences in other Australian marsupials. The comparison between the experimentally obtained LINE-1 sequences and reference genome assemblies suggests that conclusions from in silico analyses of retrotransposition activity can be influenced by incomplete genome assemblies from short reads.

Gallus S ，Lammers F ，Nilsson MA 《Genome Biology and Evolution》

Evolutionary histories of transposable elements in the genome of the largest living marsupial carnivore, the Tasmanian devil.

The largest living carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is the sole survivor of a lineage originating about 12 Ma. We set out to investigate the spectrum of transposable elements found in the Tasmanian devil genome, the first high-coverage genome of an Australian marsupial. Marsupial genomes have been shown to have the highest amount of transposable elements among vertebrates. We analyzed the horizontally transmitted DNA transposons OC1 and hAT-1_MEu in the Tasmanian devil genome. OC1 is present in all carnivorous marsupials, while having a very limited distribution among the remaining Australian marsupial orders. In contrast, hAT-1_MEu is present in all Australian marsupial orders, and has so far only been identified in a few placental mammals. We screened 158 introns for phylogenetically informative retrotransposons in the order Dasyuromorphia, and found that the youngest SINE (Short INterspersed Element), WSINE1, is no longer active in the subfamily Dasyuridae. The lack of detectable WSINE1 activity in this group may be due to a retrotransposon inactivation event approximately 30 Ma. We found that the Tasmanian devil genome contains a relatively low number of continuous full-length LINE-1 (Long INterspersed Element 1, L1) retrotransposons compared with the opossum genome. Furthermore, all L1 elements in the Tasmanian devil appeared to be nonfunctional. Hidden Markov Model approaches suggested that other potential sources of functional reverse transcriptase are absent from the genome. We discuss the issues associated with assembling long, highly similar L1 copies from short read Illumina data and describe how assembly artifacts can potentially lead to erroneous conclusions.

Gallus S ，Hallström BM ，Kumar V ，Dodt WG ，Janke A ，Schumann GG ，Nilsson MA ... -  《-》

Expansion of CORE-SINEs in the genome of the Tasmanian devil.

Nilsson MA ，Janke A ，Murchison EP ，Ning Z ，Hallström BM ... -  《BMC GENOMICS》

Disentangling the relationship of the Australian marsupial orders using retrotransposon and evolutionary network analyses.

The ancestors to the Australian marsupials entered Australia around 60 (54-72) Ma from Antarctica, and radiated into the four living orders Peramelemorphia, Dasyuromorphia, Diprotodontia, and Notoryctemorphia. The relationship between the four Australian marsupial orders has been a long-standing question, because different phylogenetic studies have not been able to consistently reconstruct the same topology. Initial in silico analysis of the Tasmanian devil genome and experimental screening in the seven marsupial orders revealed 20 informative transposable element insertions for resolving the inter- and intraordinal relationships of Australian and South American orders. However, the retrotransposon insertions support three conflicting topologies regarding Peramelemorphia, Dasyuromorphia, and Notoryctemorphia, indicating that the split between the three orders may be best understood as a network. This finding is supported by a phylogenetic reanalysis of nuclear gene sequences, using a consensus network approach that allows depicting hidden phylogenetic conflict, otherwise lost when forcing the data into a bifurcating tree. The consensus network analysis agrees with the transposable element analysis in that all possible topologies regarding Peramelemorphia, Dasyuromorphia, and Notoryctemorphia in a rooted four-taxon topology are equally well supported. In addition, retrotransposon insertion data support the South American order Didelphimorphia being the sistergroup to all other living marsupial orders. The four Australian orders originated within 3 Myr at the Cretaceous-Paleogene boundary. The rapid divergences left conflicting phylogenetic information in the genome possibly generated by incomplete lineage sorting or introgressive hybridization, leaving the relationship among Australian marsupial orders unresolvable as a bifurcating process millions of years later.

Gallus S ，Janke A ，Kumar V ，Nilsson MA ... -  《Genome Biology and Evolution》

Evolutionary inventions and continuity of CORE-SINEs in mammals.

We characterized short interspersed elements (SINEs), of the CORE-suprafamily in egg-laying (monotremes), pouched (marsupials) and placental mammals. Five families of these repeats distinguished by the presence of distinct LINE-related 3'-segments shared tRNA-like promoter and the central core region. The putative active elements were reconstructed from the alignment of genomic repeats representing molecular fossils of sequences that amplified in the past and since then underwent multiple mutations. Their mode of proliferation by retroposition was indicated by the presence of: (1) internal RNA PolIII promoter; (2) simple sequence repeated tail; (3) direct repeats; and (4) subfamilies recording the evolution of elements. The copy number of CORE-SINEs in placental genomes was estimated at about 300,000; they were highly divergent and apparently ceased to amplify before radiation of these lineages. On the other hand, among almost half a million fossil elements present in marsupials and monotremes, the youngest subfamilies could still be retropositionally active. CORE-SINEs terminate in sequence repeats of a few nucleotides similar to their 3'-segment LINE-homologues, CR1, L2 and Bov-B. These three LINE elements fall into clades distinct from that of L1 elements which, similar to their co-amplifying SINEs, end in a poly(A) tail. We propose a model in which new CORE-families, with distinct 3'-segments, are created at the RNA level due to template switching between LINE and CORE-RNA during reverse transcription. The proposed mechanism suggests that such an adaptation to the changing amplification machinery facilitated the survival and prosperity of CORE-elements over long evolutionary periods in different lineages.

Gilbert N ，Labuda D 《JOURNAL OF MOLECULAR BIOLOGY》