Adaptation and divergence of the Eastern Yellow Robin by mitonuclear interactions

A project of PART – the Persistence and Adaptation Research Team at the School of Biological Sciences, Monash University, Melbourne, Australia

The team contributing to this project

Ethics and permits

This research is conducted under relevant ethics, wildlife and bird-banding permissions

The main issue: Are there two differently adapted forms of Eastern Yellow Robin?

The biggest questions in evolutionary biology involve how new forms of life arise. This is also of central interest in conservation biology, because it determines what biological diversity exists, and how it persists and adapts.

Our previous research indicates that the Eastern Yellow Robin may be on its way to becoming two differently adapted lineages: Eastern Yellow Robins inland of the Great Dividing Range are genetically very different to those on the coastal side in ways that are likely to be ecologically important, and they do not interbreed freely and successfully.

We want to understand the differences between these two previously unrecognized forms of the species. Are they differently adapted to local environments such as temperature, moisture and food? When they do interbreed, what are their offspring like? Do the two forms have different songs or colours, and if so, do they recognize them and change their mating patterns accordingly?

This work is important for understanding the Eastern Yellow Robin, but it also serves as an exciting example of the underlying principles of adaptation and speciation that are likely to apply to many types of birds and other animals.


Eastern Yellow Robin Eopsaltria australis is a small passerine bird native to eastern Australia, a member of the Australo-Papuan robin family Petroicidae. Recently we discovered two unexpected divergent genetic lineages within this species (shown on the map to the right). One lineage (red dots) is found inland in warm, dry, variable environments west of the Great Dividing Range (a low-elevation mountain range) in southern part of the species range, while the other (blue dots) occurs in cooler, moister, more stable environments coastally of the Great Dividing Range. While distinct over a large part of their distribution, the two forms naturally meet, admix and interbreed to a limited extent where they come into contact.

The genetic differences between the two lineages are different depending on which of their two genomes we look at. Most animals, including birds, have two genomes – the small mitochondrial genome (inside mitochondria - the ‘powerhouses’ of cells), and the large nuclear genome comprising the main chromosomes of the cell. Mitochondrial genomes are inherited only from mothers, whereas nuclear genomes are inherited from both parents.

Detailed genetic analysis of Eastern Yellow Robins revealed that while there are no apparent physical barriers preventing gene flow between the two mitochondrial types, there is almost no gene flow in mitochondrial genes between the two lineages shown on the map above. This means that very few or no females move and successfully breed between the coastal and inland areas. In contrast, there is some flow of nuclear genes, which tells us that some males manage to meet and breed successfully with females of the other lineage.

These contrasting patterns between mitochondrial and nuclear genomes indicate that there is strong natural selection on females that prevent them and their genes from moving between the two environments (detailed support for this is given in Pavlova et al. 2013). Investigations of complete mitochondrial genome sequences suggested that the two mitochondrial DNA lineages diverged because they were under different pressures of natural selection (Morales et al. 2015).

But the plot thickens! Mitochondrial DNA genes provide the genetic code for 13 proteins: these interact directly with the proteins from ~80 nuclear genes to do a task that is essential to any animal – making energy available by using the machinery of the mitochondrion. The details of this can impact many aspects of the biology of a species, because it affects how they respond to temperature, how much food they need, and many other attributes. Remarkably, we have discovered that the two lineages do not just differ at their mitochondrial genomes, but also at a small subset of nuclear genes biased towards ones that have mitochondrial functions (Morales et al. 2016, 2017b).

Some suspicious nuclear genes! Most of the nuclear genome is not very different between the two lineages. But one genome region is hugely different – marked ‘island of divergence’ on this image. A notably high proportion of these genes are known to have functions in the mitochondrion. Data from Morales et al. (2016).

A note for birders: no, the two lineages are not the well-recognized subspecies! The yellow-rumped northern subspecies has representatives of each mito-nuclear lineage, and so does the southern green-rumped subspecies (Morales et al. 2017a).

Eastern Yellow Robin plumage colour variation:

Credits: Clockwise starting top left:

Eopsaltria australis chrysorrhoa at Lake Tinaroo, Atherton Tablelands, Queensland. Photo by Graham Winterflood

E. a. chrysorrhoa near Dorrigo, New South Wales. Photo by Rohan Clarke

E. a. australis from South Gippsland, Victoria. Photo by Chris Tzaros

E. a. australis from box-ironbark forest, Victoria. Photo by Chris Tzaros

How to measure the colour of a bird? We use a sensitive spectrophotometer to measure light reflected from a bird, including ultraviolet colours than humans cannot see.

Hernán Morales with an Eastern Yellow Robin, and three graphics from his PhD research concerned with colouration of the birds: left - the 19 parts on each robin for which Hernán measured colour; centre - an example of the colour variation in sample of different individual birds; right - an example of the spectrum of amounts of light of different wavelength reflected by a patch of feathers.

The project

Focussing on an area where the two lineages meet (around Newstead, near Castlemaine in central Victoria), we are investigating the biological differences between the two lineages: are they on the path to evolving two differently adapted forms, and if so, why?

Approximately along the edge of the Great Dividing Range throughout eastern Australia, the two forms of Eastern Yellow Robin meet with limited interbreeding. They occur together even at fine spatial scales. This map shows inland (red) and coastal (blue) birds co-existing even at only a few metres apart.

All of our work so far suggests that the two lineages of Eastern Yellow Robins are genetically different in ways that make their metabolisms different. If true, that would be expected to have downstream consequences for many aspects of biology – preferred environment, ability to withstand high heat or to function in the cold, food requirements, and more. It is also possible that the two lineages have evolved to recognize each other and choose mates that will help them produce offspring that are suited to the place they live in.

In this projects we will integrate genomic, physiological and behavioural approaches to investigate many questions, including:

(1) Do individuals of different with constrasting mitochondrial + nuclear genetic combinations have different metabolisms?

(2) If so, are the two mito-nuclear lineages adapted to different environments?

(3) Are there differences in characteristics linked to metabolism - such as colour, song or other behaviours - between the mito-nuclear lineages?

(4) If so, do birds of the same mito-nuclear lineage choose their mates to be of a compatible type with themselves?

  • By song variation?

  • By colour variation?

(5) Are hybrids less fit (robust, long-lived, reproductively successful) than the pure lineages? If so, is the effect sex-specific as predicted by Haldane’s rule (a fundamental rule in biology that predicts which sex of hybrid will be less fit)?

(6) How are nuclear genes associated with mitochondria and metabolic adaptations distributed within the eastern yellow robin nuclear chromosomes? Our data so far show that there is one particularly large concentration of genes in a specific part of one chromosome. This has important implications for how a set of metabolic attributes would be passed from parent to offspring.

We will extend this framework to examining the situation in other birds. We have developed an approach that should be useable on most species (Sunnucks et al. 2017) - summarized in the graphic below.

The next big surprise from this little bird - Neo-sex chromosomes! It turns out that much of chromosome 1A, which is inherited in the regular autosomal fashion in all other birds studied so far, in Eastern Yellow Robins is inherited in a sex-specific fashion. This means there is a female-only version of the chromosome and a two-sex version. This might be the key to understanding the female-biased selection we inferred previously (Gan et al. 2019). What if any role do neo-sex chromosomes play in local adaptation and restriction of gene flow, and what if any connection is there with mitochondrial-nuclear interactions?

Answering all these questions is important for understanding adaptation, speciation, biodiversity patterns and predictions under climate change.

A depiction our research scheme, which could also be applied to other species (Sunnucks et al. 2017).

Explanatory notes for the graphic above

Environmental level: Differences in temperature and precipitation can drive differences in food abundance and selection for local adaptation. Measuring aspects of birds' biology in different environments are needed to test whether the different lineages are adapted to their local environments.

Organismal level: Differently-geared metabolisms could be suited to different environments, and can be measured in individual birds to test whether the lineages have metabolisms that suit them to their local conditions.

Cellular function level: We can measure how the mitochondria in a bird function, and can make predictions about how this may relate to the metabolism of the bird, and also to the underlying genetic differences between individuals.

Molecular level: Recent advances in protein biochemistry of mitochondrial gene products allow us to make predictions about how genetic variation could affect proteins, in turn how those proteins are expected to affect mitochondrial function, which then leads to predictions about individual birds.

Genomic level: Advances in gene sequencing allow us look at tens of thousands of genes in an individual, to see how they differ from other individuals, and how they are organized in the genome. A particularly exciting aspect of our work is the discovery of a region of the Eastern Yellow Robin's genome that differs greatly between the two lineages, and is over-supplied with genes known to have mitochondrial functions. It may act as a 'supergene' for metabolic adaptation in the species - testing that idea is a major aspect of our program.

Organismal level (reproductive isolation and incompatibilities): If the two lineages are adapted to different environments, they may produce sub-standard offspring if they mate with each other. It is expected that they would evolve to prevent this, by recognizing each other, and mating preferentially with their own type. This would tend to reinforce the differences between the lineages.


This work would not be possible without the support of wildlife authorities including the Victorian Department of Environment, Land, Water and Planning, Parks Victoria, and the Australian Bird and Bat Banding Scheme. Monash ethics committees and ones from other institutions oversee ethical procedures and permissions. We thank collectors of all the specimens used from Australian National Wildlife Collection (ANWC) over the years and the ethics and scientific research permit agencies that granted permits for those specimens to be collected.

From 2018-2020 the project was funded by an Australian Research Council (ARC) grant DP180102359 to Sunnucks P. Beissinger SR & Stier A Can mitochondrial and nuclear co-evolution drive climate adaptation? From 2021 the project will be funded by ARC grant DP210102275 to Sunnucks P. Evolution and role of neo-sex chromosomes in mitonuclear co-evolution.

The Holsworth Wildlife Research Endowment and the Stuart Leslie Bird Research Award (BirdLife Australia) have supported Hernán Morales, Steph Falk, Lynna Kvistad, Lana Austin and Gabriel Low for their work on this project.

Some resources were derived from the ARC Linkage Grant (LP0776322) and ARC LIEF project (LE150100083). Craig White is supported by ARC Future Fellowship (FT130101493 ).

Other funding has come from various sources within Monash University, including student scholarships and a paper-writing award to Hernán.

Hernán’s scholarship was also supported by the Department of Public Education (SEP) of the Mexican Government.

Many volunteers, collaborators and colleagues have contributed their time and other resources - many are co-authors on papers.

Publications - if these are not available to you, you are welcome to email and ask for copies: paul.sunnucks(_at_)

Koch RE, Buchanan KL, Casagrande S, Crino O, Dowling DK, Hill GE, Hood WR, McKenzie M, Mariette MM, Noble DWA, Pavlova A, Seebacher F, Sunnucks P, Udino E, White CR, Salin K & Stier A (accepted) Integrating mitochondrial aerobic metabolism into ecology and evolution. Trends in Ecology & Evolution, TREE-D-20-00157R2 accepted 12/12/2020.

Gan HM, Falk S, Morales HE, Austin CM, Sunnucks P, Pavlova A. (2019) Genomic evidence of neo-sex chromosomes in the Eastern Yellow Robin Gigascience 8, 1–10 OPEN ACCESS Supplementary material Gigascience blog

Morales HE, Pavlova A, Joseph L, Sunnucks P (2015) Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance. Molecular Ecology 24, 2820–2837.

Morales HE, Pavlova A, Amos JN, Major R, Kilian A, Greening C and Sunnucks P (2018) Concordant divergence of mitogenomes and a mitonuclear gene cluster in bird lineages inhabiting different climates. Nature Ecology & Evolution 2, 1258–1267.

Morales HE, Pavlova A, Sunnucks P, Major R, Amos JN, Joseph L, Wang B, Lemmon AR, Endler JA, Delhey K. (2017a) Neutral and selective drivers of colour evolution in a widespread Australian passerine. Journal of Biogeography 44, 522–536.

Morales HE, Sunnucks P, Joseph L, Pavlova A (2017b) Perpendicular axes of differentiation generated by mitochondrial introgression Molecular Ecology doi: 10.1111/mec.14114. An earlier version is available at

Pavlova A, Amos JN, Joseph L, Loynes K, Austin JJ, Keogh JS, Stone GN, Nicholls JA and Sunnucks P (2013). Perched at the mito-nuclear crossroads: divergent mitochondrial lineages correlate with environment in the face of ongoing nuclear gene flow in an Australian bird. Evolution 67, 3412–3428.

Sunnucks P, Morales HE, Lamb AM, Pavlova A, Greening C (2017). Integrative Approaches for Studying Mitochondrial and Nuclear Genome Co-evolution in Oxidative Phosphorylation. Frontiers in Genetics 8:25. doi:10.3389/fgene.2017.00025 OPEN ACCESS