Investigating the Genetic Basis of Plumage Variation in the Australian Magpie, Cracticus tibicen Author Dobson, Ana Elizabeth Published 2017 Thesis Type Thesis (PhD Doctorate) School Griffith School of Environment DOI https://doi.org/10.25904/1912/275 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/365743 Griffith Research Online https://research-repository.griffith.edu.au Investigating the genetic basis of plumage variation in the Australian magpie, Cracticus tibicen Ana E. Dobson B.Sc. (Hons) Griffith School of Environment Griffith Sciences Group Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy December 2016 SUMMARY Many species exhibit some form of colour variation between groups or individuals, and studies of such colour variation, its genetic basis and functional importance can contribute unique insights into the evolutionary, ecological, demographic and genetic processes affecting the diversity of life. Through the examination of the genetics underlying colour traits, the relative importance of different selective and demographic processes in the evolution and maintenance of a trait can be inferred, enabling scientists to gain a better understanding of what drives evolutionary change in different organisms and environments. Such studies also assist in bridging the gaps in our knowledge about the interactions between genetics and ecology that are involved in phenotypic variation. Understanding the pathways linking genetic and regulatory changes to phenotypic change and evolutionary change through time contributes to our knowledge about how species develop and evolve. The Australian magpie (Cracticus tibicen), is a group-living and territorial passerine bird with a characteristic pied appearance of strongly contrasting patches of black and white plumage. The species has several different plumage forms, delineated by the colour and/or pattern of back feathers; these plumage forms hybridise where their distributions overlap. Distributions of plumage forms are highly discordant with patterns of phylogeographic structure found in this species, making a scenario of allopatric divergence followed by secondary recontact at hybrid zones highly unlikely. Similar plumage colour forms seem likely to have evolved in situ within both eastern and western Australian magpie lineages. A number of previous studies have proposed that different forms of selection may be acting on plumage colour phenotypes either side of hybrid zones to generate and maintain these polymorphic forms in the face of gene flow. In such scenarios, black backs are putatively favoured in the north of the continent, by better crypsis where vegetation is more open-canopied, and white backed forms are favoured by sexual selection for ‘brightness’ in males, in the closed-canopy style vegetation dominating the far south of the continent, in which crypsis is less important. While experiments have found evidence to support the idea black backed magpies may be favoured via crypsis north of hybrid zones, as yet a number of studies looking for evidence of sexual selection favouring white backed males have failed to find strong evidence of such an effect, and it is currently unclear why white backed forms (including varied backs) dominate the south of the continent. Previous phylogeographic analyses of the magpie suggest Pleistocene-era arid barriers to dispersal led to the genetic divergence of eastern and western groups II around 36,000 years ago, and also present some evidence for range expansion of eastern magpies following this period of aridity. Across all five of the nuclear candidate colour genes assayed for this study, tests of neutrality and selection did not identify signals consistent with selection acting on the gene(s), but did add significant support to the hypothesis of past population expansions within the recent evolutionary history of this species. It is suggested that this signature of population expansion may be linked to changes in population size and distribution of magpies during the late Pleistocene, as aridity decreased following the Last Glacial Maximum, allowing refugial populations to recolonise the interior of the continent. A long-term study site in the eastern back colour hybrid zone, in which magpies have been progressively colour banded and DNA sampled over a 25 year period, was used to investigate several aspects of the quantitative genetics of magpie back colour variation. Microsatellite parentage analyses were first used in 35 territories with reliable sighting data and in which all, or nearly all adult members had been sampled, in order to establish genetic parentage of fledglings (as this species has been shown to have high levels of extra-pair and extra-group paternity). On average, data from each territory spanned a nine year period. Observations of individuals’ back colours, scored on a five-point scale, were averaged over the lifetime of the study, and this data was used to examine both the heritability of this back colour variation and the patterns of inheritance within family groups. Both linear-mixed model and regression-based methods independently produced exceptionally similar estimates for the heritability of back colour variation in magpies, and these both indicated that this heritability is quite high: 0.92 using mixed models and 0.94 using corrected midparent/midoffspring regression. These large heritability estimates are comparable to those estimated for other melanin-based plumage traits across a wide range of bird species, and is at least partially a reflection of the need for melanic pigments to be manufactured endogenously, rather than obtained from environmental sources. This idea is reinforced by the non-significant contribution of patch quality and cohort to heritability models, indicating environmental contributions related to natal territories such as the differing quantity or quality of feeding sites between territories, and differing abundance of resources between years have little effect on the heritability of variation in this trait. Regressions of offspring back colour on parents by sexes suggest neither maternal nor paternal non-genetic effects (e.g. parent body condition) are likely to play a large role in determining offspring back colour. There was no pattern of either positive or negative III assortative mating with respect to parental back colours, which were not significantly correlated in mating pairs. Such assortative mating could have at least partially explained the maintenance of hybrid zones in the face of gene flow. Patterns of inheritance in familial groups suggest there may be some effect of dominance of black backed alleles, and these patterns together with the continuous nature of back colour phenotypes indicate that a minimum of two genes are likely to play a role in determining magpie back colour, whilst the upper limit of number of genes involved remains as yet undetermined. The high heritability estimate for back colour variation also indicates that the correlation between genotype and phenotype is very strong, and consequently future gene-mapping studies investigating this trait have a reasonable likelihood of pinning down genes of large effect that may play a role in magpie back colour. Previous studies using a candidate gene approach to identify the genetic basis of traits, such as intraspecific colour variation, have yielded a large number of genotype- phenotype associations across a wide range of animal species. The melanocortin-1 receptor (MC1R) plays an important role in the synthesis of different types of melanin, and mutations in this gene have been associated with melanin-based pigmentation variation across a wide range of vertebrate taxa, including many bird species. The majority of the MC1R gene, comprising 861 nucleotides was sequenced in 98 magpie individuals, mapped onto the chicken genome and structural features of this transmembrane receptor were identified in magpies. Although four coding region mutations in magpies led to amino acid changes, none of these were partitioned between back colour forms, subspecies or previously delineated eastern and western clades. At sites of amino acid substitutions associated with colour variation in other bird species, magpies were consistently invariable. A total of 3758 nucleotides from coding and non-coding regions of four additional candidate colour genes involved in pigmentation and melanocortin pathways were also sequenced: the Tyrosinase-related protein 1 (TYRP1), Tyrosinase (TYR), Dopachrome Tautomerase (DCT), and the Agouti-related protein (AGRP), in search of possible associations with plumage colour differences in this polymorphic species. For these four candidate genes, 30 magpie individuals, including representatives from all plumage colour forms (black back, white back and varied), both sexes, and each of eight recognised subspecies from 15 sites across the Australian continent were used to evaluate associations between genetic variation and colour variation using a series of inverse ordinal and univariate logistic regressions. IV Of the 67 variable mutations identified, 64 were not significantly associated with plumage variation or geographic or subspecies groupings, and sites of mutations important in colour variation in other species were invariable in magpies. Of the four mutations identified by regression analyses as statistically significant, only two (both located in intron