Secrets of weird, wonderful Tassie tiger revealed.

A newly available genome sequence for the extinct Tasmanian tiger, or thylacine (Thylacinus cynocephalus), is providing details on convergent phenotypes found in thylacines and canids, while exonerating humans as the source of population declines and genetic diversity losses linked to the animal’s extinction.

“Demographic analysis indicated a long-term decline in genetic diversity starting well before the arrival of humans in Australia,” senior author and University of Melbourne researcher Andrew Pask and his co-authors wrote for a study appearing in Nature Ecology & Evolution today.

Pask and his colleagues from Australia, Germany, and the US sequenced a thylacine pouch young specimen preserved in alcohol for more than 100 years. With this new sequence, they explored thylacine relationships with other marsupials, as well as the genetic diversity and population patterns that preempted its decline and extinction. The latter analysis suggests thylacine populations waned significantly between 70,000 and 120,000 years ago — before humans started making their way to Australia an estimated 50,000 or more years ago.

The sequence data also made it possible to take a look at the genetic basis of thylacine features resembling those found in modern day representatives from the eutherian canid lineage, which includes domestic dogs, wolves, and coyotes. Canids split from the thylacine lineage some 160 million years ago. And the team’s new findings hint that the thylacine — a dingo doppelganger with stripes, a long, smooth tail, and a pouch for developing young — may have reached canid convergence via regulatory changes.

“In spite of their extraordinary phenotypic convergence,” Pask and his co-authors explained, “comparative genomic analyses demonstrated that amino acid homoplasies between the thylacine and canids are largely consistent with neutral evolution.”

The last known thylacine died in captivity in the mid-1930s, and researchers have been systematically piecing its genetics back together over the last few decades. In 1992, for example, a Southern Illinois University team published a phylogenetic analysis based on thylacine mitochondrial cytochrome b gene sequences, while an international team led by Pennsylvania State University researchers reported the broader thylacine mitochondrial genome in Genome Research in 2009.

Pask was part of a team that revived a bit of thylacine sequence containing a suspected collagen transcription enhancer in transgenic mice for a PLOS One paper published in 2008.

For this latest thylacine genomic analysis, Pask and his team used Illumina HiSeq 2000 and NextSeq 5500 instruments to sequence 300- to 600-base pair DNA fragments extracted from a 108-year-old, alcohol-preserved pouch young thylacine sample. After weeding out contaminating reads through mapping to microbial and fungal genomes, they were left with apparent thylacine reads to align to a related Tasmanian devil genome.

The team noted that the thylacine reads had roughly 43-fold average coverage when mapped to the Tasmanian devil genome. It also checked the reads against the tammar wallaby and gray short-tailed opossum genomes to see how well these marsupial sequences coincided with the new thylacine genome.

Based on a phylogenetic analysis based on repeat elements and other informative markers in the genome, the researchers found evidence of a basal position for thylacine in an order called Dasyuromorphia, which contains the Tasmanian devil and other carnivorous marsupials.

From there, the team went on to perform detailed analysis of Tasmanian tiger demography, canid convergence, and selection. Despite levels of physical convergence that were unusual for such long-diverged lineages, it did not detect positive selection involving orthologous genes in the thylacine and canid representatives such as the wolf, red fox, arctic fox, coyote, or golden jackal.

Most convergent selection took place in the thylacine lineage over roughly 6 million years, the authors estimated, noting that “positive selection has not targeted orthologous genes more frequently in the thylacine and canids than in non-convergent species with similar divergence times.”

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