Dingoes are culturally and ecologically important free-living canids whose ancestors arrived in Australia over 3,000 B.P., likely transported by seafaring people. However, the early history of dingoes in Australia—including the number of founding populations and their routes of introduction—remains uncertain. This uncertainty arises partly from the complex and poorly understood relationship between modern dingoes and New Guinea singing dogs, and suspicions that post-Colonial hybridization has introduced recent domestic dog ancestry into the genomes of many wild dingo populations. In this study, we analyzed genome-wide data from nine ancient dingo specimens ranging in age from 400 to 2,746 y old, predating the introduction of domestic dogs to Australia by European colonists. We uncovered evidence that the continent-wide population structure observed in modern dingo populations had already emerged several thousand years ago. We also detected excess allele sharing between New Guinea singing dogs and ancient dingoes from coastal New South Wales (NSW) compared to ancient dingoes from southern Australia, irrespective of any post-Colonial hybrid ancestry in the genomes of modern individuals. Our results are consistent with several demographic scenarios, including a scenario where the ancestry of dingoes from the east coast of Australia results from at least two waves of migration from source populations with varying affinities to New Guinea singing dogs. We also contribute to the growing body of evidence that modern dingoes derive little genomic ancestry from post-Colonial hybridization with other domestic dog lineages, instead descending primarily from ancient canids introduced to Sahul thousands of years ago.

The enzymatic breakdown and regulation of food passage through the vertebrate antral stomach and pyloric sphincter (antropyloric region) is a trait conserved over 450 million years. Development of the structures involved is underpinned by a highly conserved signalling pathway involving the hedgehog, bone morphogenetic protein and Wingless/Int-1 (Wnt) protein families. Monotremes are one of the few vertebrate lineages where acid-based digestion has been lost, and this is consistent with the lack of genes for hydrochloric acid secretion and gastric enzymes in the genomes of the platypus ( Ornithorhynchus anatinus ) and short-beaked echidna ( Tachyglossus aculeatus ) . Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genomes (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway constituents are generally conserved, but surprisingly, NK3 homeobox 2 ( Nkx3.2 ) was pseudogenized in both platypus and echidna. We speculate that the unique sequence evolution of Grem1 and Bmp4 sequences in the echidna lineage may correlate with their pyloric-like restriction and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

The stomach has been a highly conserved organ throughout vertebrate evolution; however, there are now over 20 lineages composed of monotremes, lungfish and teleost fish displaying a secondary loss of stomach function and morphology. This “agastric phenotype” has evolved convergently and is typified by a loss of gastric glands and gastric acid secretion and a near-to-complete loss of storage capacity of the stomach. All agastric species have lost the genes for gastric enzymes (Pga and Pgc) and proton pump subunits (Atp4a and Atp4b), and gastrin (Gast) has been lost in monotremes. As a key gastric hormone, the conservation of gastrin has not yet been investigated in the lungfish or agastric teleosts, and it is unclear how the loss of gastrin affects the evolution and selection of the native receptor (Cckbr), gastrin-releasing peptide (Grp) and gastrin-releasing peptide receptor (Grpr) in vertebrates. Furthermore, there are still many genes implicated in gastric development and function which have yet to be associated with the agastric phenotype. We analysed the evolution, selection and conservation of the gastrin pathway and a novel gastric gene repertoire (Gkn1, Gkn2, Tff1, Tff2, Vsig1 and Anxa10) to determine the correlation with the agastric phenotype. We found that the loss of gastrin or its associated genes does not correlate with the agastric phenotype, and their conservation is due to multiple pleiotropic roles throughout vertebrate evolution. We found a loss of the gastric gene repertoire in the agastric phenotype, except in the echidna, which retained several genes (Gkn1, Tff2 and Vsig1). Our findings suggest that the gastrin physiological pathway evolved differently in pleiotropic roles throughout vertebrate evolution and support the convergent evolution of the agastric phenotype through shared independent gene-loss events.

Development of the vertebrate antral stomach and pyloric sphincter (antropyloric region) – involved in enzymatic breakdown and thoroughfare of food - is underpinned by a highly conserved developmental pathway involving the hedgehog, bone morphogenetic protein (BMP) and Wingless/Int-1 (Wnt) protein families. Monotremes are a unique lineage where acid-based digestion has been lost, and this correlates with a lack of genes for gastric acid and enzymes in the genomes of the platypus ( Ornithorhynchus anatinus ) and short-beaked echidna ( Tachyglossus aculeatus ). Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genome sequences (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway is generally conserved but, NK3 homeobox 2 ( Nkx3.2 ) was pseudogenised in both platypus and echidna. We speculate that pyloric-like restriction in the echidna may correlate with independent evolution of Grem1 and Bmp4 sequences, and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

Independent and convergent gene loss, such as the loss of gastric enzymes ( Pga , Pgc ) and proton pump subunits ( Atp4a , Atp4b ) in monotremes and polyphyletic teleost species have been associated with the agastric phenotype – stomachs lacking gross morphology, gastric glands and acid secretion. The loss of the key gastric hormone gastrin ( Gast ) has been observed in monotremes, but not corroborated in agastric teleosts. Furthermore, it is unclear how this loss affects the evolution and selection of the native receptor ( Cckbr ), gastrin releasing peptide ( Grp ) and receptor ( Grpr ), or if there are further gene losses associated with the agastric phenotype. We investigated the evolution, selection and conservation of the gastrin physiological pathway and novel gastric genes in a broad range of gastric and agastric vertebrates. Our analyses showed no correlation between the losses of Gast and Grpr with the agastric phenotype in vertebrate lineages. We identified genes implicated in gastric mucosal maintenance and development ( Gkn1 , Gkn2 , Tff1 , Tff2 , Vsig1 , Anxa10 ) that are lost in agastric species except the echidna, which retained some of them ( Gkn1 , Tff2 , Vsig1 ). Our findings demonstrate a convergent repertoire of gastric genes lost in agastric species, partial retention of gastric functionality in the echidna, and the diversified roles of gastrin pathway constituents in vertebrate lineages.