Characterisation and cross-amplification of sex-specific genetic markers in Australasian Egerniinae lizards and their implications for understanding the evolution of sex determination and social complexity

Abstract

Sex is a pervasive factor that underpins functional phenotypic variation across a range of traits. Although sex can usually be distinguished morphologically, in some species this is not possible. The development of genetic markers for sex identification is, thus, key if we are to incorporate sex into an understanding of ecological or evolutionary process. Here we develop genetic markers for the identification of sex within an iconic Australian lizard group, the Egernia group, which is notable for its complex social behaviour. We used restriction-site associated DNA sequencing to characterise sex-specific genetic sequences for a key member of the group, Liopholis whitii, and designed primers for four of these putative sex-specific sequences. These primers amplified across some, but not all, species of the group. Our results provided several important insights. They suggest conservatism of a XX/XY sex determination system within the group as well as sex-specific genomic regions that appear independent of the conserved genomic regions identified in other skink species. More broadly, the development of sex markers for the Egernia group opens up a range of potential research questions related to the role that sex plays in the mediation of social behaviour and, through this, the emergence and stability of social life.

Using a handful of transcriptomes to detect sex-linked markers in a lizard with homomorphic sex chromosomes

“…The amplification of several of our sexing markers across four other Carinascincus species suggest that the sex determining system of C. ocellatus is shared across the genus. However, the fact that all markers failed in two more distantly related skinks casts doubt on the conservation of sex determination across all Scincidae, as previously suggested based on work in the Egernia subfamiliy and in the ground skink Scincella melanostica 25,50 . Testing the PCR markers developed here and by others 25,44,51 across a larger panel species would benefit our understanding of scincid lizard sex chromosome evolution.…”

“…However, the fact that all markers failed in two more distantly related skinks casts doubt on the conservation of sex determination across all Scincidae, as previously suggested based on work in the Egernia subfamiliy and in the ground skink Scincella melanostica 25,50 . Testing the PCR markers developed here and by others 25,44,51 across a larger panel species would benefit our understanding of scincid lizard sex chromosome evolution. More broadly, our study highlights the benefits of increasing the number of available sex-linked genetic markers and molecular sexing tests in taxonomic groups with poorly studied sex determination.…”

“…This in turn enables the identification of sex-linked regions based on the clustering of candidate sex-linked markers derived from only a small number of individuals. In contrast, the alignment of RRS reads (usually done with BLAST) tends to have very poor success when no close reference genome is available (1–10% 20,25,26,42,43 ), such that there is a stronger reliance on large sample sizes to increase confidence on whether candidate markers are sex-linked. This was the case for sex-linkage inference of C. ocellatus DArT-seq markers, as only 2% of the approximately 300 sex-linked markers aligned to BLAST’s nucleotide collection 38,44 .…”

“…The most common approach for species with homomorphic sex chromosomes relies on reduced-representation sequencing (RRS) of multiple phenotypically sexed individuals, and the identification of sex-limited loci or variants. This approach is more affordable than whole genome sequencing, and has proved successful across a wide-range of organisms 14,[17][18][19][20][21][22][23][24][25][26][27][28][29][30] . However, it has some drawbacks.…”

Using a Handful of Transcriptomes to Detect Sex-Linked Markers and Develop Molecular Sexing Assays in a Species with Homomorphic Sex Chromosomes

“…Over 70% of transcripts assembled here aligned to the wall lizard genome, despite an estimated divergence time of 174 Myr ( Kumar et al 2017 ). In contrast, alignment of raw RRS reads (usually done with Blast) tends to have poor success when no close reference genome is available (1% to 10%; Brelsford et al 2017 ; Nielsen et al 2019 , 2020 ; Keating et al 2022 ; Bouffet-Halle et al 2022 ). In C. ocellatus , a previous RRS study identified ∼300 sex-linked markers of which only 2% aligned to Blast's nucleotide collection ( Hill et al 2018 ; Cornejo-Páramo et al 2020 ).…”