Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues.

Formalin fixation of natural history specimens and histopathological material has historically been viewed as an impediment to successful genomic analysis. However, the development of extraction methods specifically tailored to contend with heavily crosslinked archival tissues, re-contextualises millions of previously overlooked specimens as viable molecular assets. Here, we present an easy-to-follow protocol for screening archival wet specimens for molecular viability and subsequent genomic DNA extraction suitable for sequencing. The protocol begins with non-destructive assessment of specimen degradation and preservation media conditions to allow both museum curators and researchers to select specimens most likely to yield an acceptable proportion (20-60%) of mappable endogenous DNA during short-read DNA sequencing. The extraction protocol uses hot alkaline lysis in buffer (0.1M NaOH, 1% SDS, pH 13) to simultaneously lyse and de-crosslink the tissue. To maximise DNA recovery, phenol:chloroform extraction is coupled with a small-fragment optimised SPRI bead clean up. Applied to well-preserved archival tissues, the protocol can yield 1-2 µg DNA per 50 mg of tissue with mean fragment sizes typically ranging from 50-150 bp, which is suitable to recover genomic DNA sufficient to reconstruct complete mitochondrial genomes and achieve up to 25X nuclear genome coverage. We provide guidance for read mapping to a reference genome and discuss the limitations of relying on small fragments for SNP genotyping and de novo genome assembly. This protocol opens the door to broader-scale genetic and phylogenetic analysis of historical specimens, contributing to a deeper understanding of evolutionary trends and adaptation in response to changing environments.

Gene expression of male pathway genes sox9 and amh during early sex differentiation in a reptile departs from the classical amniote model.

BACKGROUND: Sex determination is the process whereby the bipotential embryonic gonads become committed to differentiate into testes or ovaries. In genetic sex determination (GSD), the sex determining trigger is encoded by a gene on the sex chromosomes, which activates a network of downstream genes; in mammals these include SOX9, AMH and DMRT1 in the male pathway, and FOXL2 in the female pathway. Although mammalian and avian GSD systems have been well studied, few data are available for reptilian GSD systems. RESULTS: We conducted an unbiased transcriptome-wide analysis of gonad development throughout differentiation in central bearded dragon (Pogona vitticeps) embryos with GSD. We found that sex differentiation of transcriptomic profiles occurs at a very early stage, before the gonad consolidates as a body distinct from the gonad-kidney complex. The male pathway genes dmrt1 and amh and the female pathway gene foxl2 play a key role in early sex differentiation in P. vitticeps, but the central player of the mammalian male trajectory, sox9, is not differentially expressed in P. vitticeps at the bipotential stage. The most striking difference from GSD systems of other amniotes is the high expression of the male pathway genes amh and sox9 in female gonads during development. We propose that a default male trajectory progresses if not repressed by a W-linked dominant gene that tips the balance of gene expression towards the female trajectory. Further, weighted gene expression correlation network analysis revealed novel candidates for male and female sex differentiation. CONCLUSION: Our data reveal that interpretation of putative mechanisms of GSD in reptiles cannot solely depend on lessons drawn from mammals.

Developmental dynamics of sex reprogramming by high incubation temperatures in a dragon lizard.

BACKGROUND: In some vertebrate species, gene-environment interactions can determine sex, driving bipotential gonads to differentiate into either ovaries or testes. In the central bearded dragon (Pogona vitticeps), the genetic influence of sex chromosomes (ZZ/ZW) can be overridden by high incubation temperatures, causing ZZ male to female sex reversal. Previous research showed ovotestes, a rare gonadal phenotype with traits of both sexes, develop during sex reversal, leading to the hypothesis that sex reversal relies on high temperature feminisation to outcompete the male genetic cue. To test this, we conducted temperature switching experiments at key developmental stages, and analysed the effect on gonadal phenotypes using histology and transcriptomics. RESULTS: We found sexual fate is more strongly influenced by the ZZ genotype than temperature. Any exposure to low temperatures (28 °C) caused testes differentiation, whereas sex reversal required longer exposure to high temperatures. We revealed ovotestes exist along a spectrum of femaleness to male-ness at the transcriptional level. We found inter-individual variation in gene expression changes following temperature switches, suggesting both genetic sensitivity to, and the timing and duration of the temperature cue influences sex reversal. CONCLUSIONS: These findings bring new insights to the mechanisms underlying sex reversal, improving our understanding of thermosensitive sex systems in vertebrates.

Truncated jarid2 and kdm6b transcripts are associated with temperature-induced sex reversal during development in a dragon lizard.

Sex determination and differentiation in reptiles are complex. In the model species, Pogona vitticeps, high incubation temperature can cause male to female sex reversal. To elucidate the epigenetic mechanisms of thermolabile sex, we used an unbiased genome-wide assessment of intron retention during sex reversal. The previously implicated chromatin modifiers (jarid2 and kdm6b) were two of three genes to display sex reversal-specific intron retention. In these species, embryonic intron retention resulting in C-terminally truncated jarid2 and kdm6b isoforms consistently occurs at low temperatures. High-temperature sex reversal is uniquely characterized by a high prevalence of N-terminally truncated isoforms of jarid2 and kdm6b, which are not present at low temperatures, or in two other reptiles with temperature-dependent sex determination. This work verifies that chromatin-modifying genes are involved in highly conserved temperature responses and can also be transcribed into isoforms with new sex-determining roles.

Thicker eggshells are not predicted by host egg ejection behaviour in four species of Australian cuckoo.

Defences of hosts against brood parasitic cuckoos include detection and ejection of cuckoo eggs from the nest. Ejection behaviour often involves puncturing the cuckoo egg, which is predicted to drive the evolution of thicker eggshells in cuckoos that parasitise such hosts. Here we test this prediction in four Australian cuckoo species and their hosts, using Hall-effect magnetic-inference to directly estimate eggshell thickness in parasitised clutches. In Australia, hosts that build cup-shaped nests are generally adept at ejecting cuckoo eggs, whereas hosts that build dome-shaped nests mostly accept foreign eggs. We analysed two datasets: a small sample of hosts with known egg ejection rates and a broader sample of hosts where egg ejection behaviour was inferred based on nest type (dome or cup). Contrary to predictions, cuckoos that exploit dome-nesting hosts (acceptor hosts) had significantly thicker eggshells relative to their hosts than cuckoos that exploit cup-nesting hosts (ejector hosts). No difference in eggshell thicknesses was observed in the smaller sample of hosts with known egg ejection rates, probably due to lack of power. Overall cuckoo eggshell thickness did not deviate from the expected avian relationship between eggshell thickness and egg length estimated from 74 bird species. Our results do not support the hypothesis that thicker eggshells have evolved in response to host ejection behaviour in Australian cuckoos, but are consistent with the hypothesis that thicker eggshells have evolved to reduce the risk of breakage when eggs are dropped into dome nests.

Lineage diversity within a widespread endemic Australian skink to better inform conservation in response to regional-scale disturbance.

Much attention is paid in conservation planning to the concept of a species, to ensure comparability across studies and regions when classifying taxa against criteria of endangerment and setting priorities for action. However, various jurisdictions now allow taxonomic ranks below the level of species and nontaxonomic intraspecific divisions to be factored into conservation planning-subspecies, key populations, evolutionarily significant units, or designatable units. Understanding patterns of genetic diversity and its distribution across the landscape is a key component in the identification of species boundaries and determination of substantial geographic structure within species. A total of 12,532 reliable polymorphic SNP loci were generated from 63 populations (286 individuals) covering the distribution of the Australian eastern three-lined skink, Bassiana duperreyi, to assess genetic population structure in the form of diagnosable lineages and their distribution across the landscape, with particular reference to the recent catastrophic bushfires of eastern Australia. Five well-supported diagnosable operational taxonomic units (OTUs) existed within B. duperreyi. Low levels of divergence of B. duperreyi between mainland Australia and Tasmania (no fixed allelic differences) support the notion of episodic exchange of alleles across Bass Strait (ca 60 m, 25 Kya) during periods of low sea level during the Upper Pleistocene rather than the much longer period of isolation (1.7 My) indicated by earlier studies using mitochondrial sequence variation. Our study provides foundational work for the detailed taxonomic re-evaluation of this species complex and the need for biodiversity assessment to include an examination of cryptic species and/or cryptic diversity below the level of species. Such information on lineage diversity within species and its distribution in the context of disturbance at a regional scale can be factored into conservation planning regardless of whether a decision is made to formally diagnose new species taxonomically and nomenclaturally.

Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation.

Pogona vitticeps has female heterogamety (ZZ/ZW), but the master sex-determining gene is unknown, as it is for all reptiles. We show that nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1), a gene that is essential in mammalian sex determination, has alleles on the Z and W chromosomes (Z-nr5a1 and W-nr5a1), which are both expressed and can recombine. Three transcript isoforms of Z-nr5a1 were detected in gonads of adult ZZ males, two of which encode a functional protein. However, ZW females produced 16 isoforms, most of which contained premature stop codons. The array of transcripts produced by the W-borne allele (W-nr5a1) is likely to produce truncated polypeptides that contain a structurally normal DNA-binding domain and could act as a competitive inhibitor to the full-length intact protein. We hypothesize that an altered configuration of the W chromosome affects the conformation of the primary transcript generating inhibitory W-borne isoforms that suppress testis determination. Under this hypothesis, the genetic sex determination (GSD) system of P. vitticeps is a W-borne dominant female-determining gene that may be controlled epigenetically.

Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal†.

The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.

Unlocking inaccessible historical genomes preserved in formalin.

Museum specimens represent an unparalleled record of historical genomic data. However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.

Effects of natural nest temperatures on sex reversal and sex ratios in an Australian alpine skink.

Altered climate regimes have the capacity to affect the physiology, development, ecology and behaviour of organisms dramatically, with consequential changes in individual fitness and so the ability of populations to persist under climatic change. More directly, extreme temperatures can directly skew the population sex ratio in some species, with substantial demographic consequences that influence the rate of population decline and recovery rates. In contrast, this is particularly true for species whose sex is determined entirely by temperature (TSD). The recent discovery of sex reversal in species with genotypic sex determination (GSD) due to extreme environmental temperatures in the wild broadens the range of species vulnerable to changing environmental temperatures through an influence on primary sex ratio. Here we document the levels of sex reversal in nests of the Australian alpine three-lined skink (Bassiana duperreyi), a species with sex chromosomes and sex reversal at temperatures below 20 °C and variation in rates of sex reversal with elevation. The frequency of sex reversal in nests of B. duperreyi ranged from 28.6% at the highest, coolest locations to zero at the lowest, warmest locations. Sex reversal in this alpine skink makes it a sensitive indicator of climate change, both in terms of changes in average temperatures and in terms of climatic variability.

Temperature-Induced Sex Reversal in Reptiles: Prevalence, Discovery, and Evolutionary Implications.

Sex reversal is the process by which an individual develops a phenotypic sex that is discordant with its chromosomal or genotypic sex. It occurs in many lineages of ectothermic vertebrates, such as fish, amphibians, and at least one agamid and one scincid reptile species. Sex reversal is usually triggered by an environmental cue that alters the genetically determined process of sexual differentiation, but it can also be caused by exposure to exogenous chemicals, hormones, or pollutants. Despite the occurrence of both temperature-dependent sex determination (TSD) and genetic sex determination (GSD) broadly among reptiles, only 2 species of squamates have thus far been demonstrated to possess sex reversal in nature (GSD with overriding thermal influence). The lack of species with unambiguously identified sex reversal is not necessarily a reflection of a low incidence of this trait among reptiles. Indeed, sex reversal may be relatively common in reptiles, but little is known of its prevalence, the mechanisms by which it occurs, or the consequences of sex reversal for species in the wild under a changing climate. In this review, we present a roadmap to the discovery of sex reversal in reptiles, outlining the various techniques that allow new occurrences of sex reversal to be identified, the molecular mechanisms that may be involved in sex reversal and how to identify them, and approaches for assessing the impacts of sex reversal in wild populations. We discuss the evolutionary implications of sex reversal and use the central bearded dragon (Pogona vitticeps) and the eastern three-lined skink (Bassiana duperreyi) as examples of how species with opposing patterns of sex reversal may be impacted differently by our rapidly changing climate. Ultimately, this review serves to highlight the importance of understanding sex reversal both in the laboratory and in wild populations and proposes practical solutions to foster future research.

Two transcriptionally distinct pathways drive female development in a reptile with both genetic and temperature dependent sex determination.

How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox; 'CaRe' status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.

High elevation increases the risk of Y chromosome loss in Alpine skink populations with sex reversal.

The view that has genotypic sex determination and environmental sex determination as mutually exclusive states in fishes and reptiles has been contradicted by the discovery that chromosomal sex and environmental influences can co-exist within the same species, hinting at a continuum of intermediate states. Systems where genes and the environment interact to determine sex present the opportunity for sex reversal to occur, where the phenotypic sex is the opposite of that predicted by their sex chromosome complement. The skink Bassiana duperreyi has XX/XY sex chromosomes with sex reversal of the XX genotype to a male phenotype, in laboratory experiments, and in field nests, in response to exposure to cold incubation temperatures. Here we studied the frequency of sex reversal in adult populations of B. duperreyi in response to climatic variation, using elevation as a surrogate for environmental temperatures. We demonstrate sex reversal in the wild for the first time in adults of a reptile species with XX/XY sex determination. The highest frequency of sex reversal occurred at the highest coolest elevation location, Mt Ginini (18.46%) and decreased in frequency to zero with decreasing elevation. We model the impact of this under Fisher's frequency-dependent selection to show that, at the highest elevations, populations risk the loss of the Y chromosome and a transition to temperature-dependent sex determination. This study contributes to our understanding of the risks of extinction from climate change in species subject to sex reversal by temperature, and will provide focus for future research to test on-the-ground management strategies to mitigate the effects of climate in local populations.

Genetic barcoding of museum eggshell improves data integrity of avian biological collections.

Natural history collections are often plagued by missing or inaccurate metadata for collection items, particularly for specimens that are difficult to verify or rare. Avian eggshell in particular can be challenging to identify due to extensive morphological ambiguity among taxa. Species identifications can be improved using DNA extracted from museum eggshell; however, the suitability of current methods for use on small museum eggshell specimens has not been rigorously tested, hindering uptake. In this study, we compare three sampling methodologies to genetically identify 45 data-poor eggshell specimens, including a putatively extinct bird's egg. Using an optimised drilling technique to retrieve eggshell powder, we demonstrate that sufficient DNA for molecular identification can be obtained from even the tiniest eggshells without significant alteration to the specimen's appearance or integrity. This method proved superior to swabbing the external surface or sampling the interior; however, we also show that these methods can be viable alternatives. We then applied our drilling method to confirm that a purported clutch of Paradise Parrot eggs collected 40 years after the species' accepted extinction date were falsely identified, laying to rest a 53-year-old ornithological controversy. Thus, even the smallest museum eggshells can offer new insights into old questions.

Corticosterone does not have a role in temperature sex reversal in the central bearded dragon (Pogona vitticeps).

Environmental sex determination (ESD) is common among ectothermic vertebrates. The stress axis and production of stress hormones (corticosteroids) regulates ESD in fish, but evidence of a similar influence in reptiles is sparse and conflicting. The central bearded dragon (Pogona vitticeps) has a system of sex determination involving the interplay between sex chromosomes (ZZ/ZW female heterogamety) and the thermal environment. High egg incubation temperatures induce sex reversal of the ZZ genotype, feminizing chromosomally male individuals. Here we show that corticosterone elevation is not associated with sex reversal in the central bearded dragon, either during embryonic development or adulthood. We also demonstrate experimentally that sex determination is not affected by corticosterone injection into the yolk. This strongly suggests that stress axis upregulation by high temperature during incubation does not cause sex reversal in P. vitticeps. Our work is in general agreement with other research in reptiles, which suggests that the stress axis does not mediate sex in reptiles with ESD. Alternative biological systems may be responsible for capturing environmental conditions during reptile development, such as cellular calcium and redox regulation or the action of temperature-sensitive splicing factors.

Ovotestes suggest cryptic genetic influence in a reptile model for temperature-dependent sex determination.

Sex determination and differentiation in reptiles is complex. Temperature-dependent sex determination (TSD), genetic sex determination (GSD) and the interaction of both environmental and genetic cues (sex reversal) can drive the development of sexual phenotypes. The jacky dragon (Amphibolurus muricatus) is an attractive model species for the study of gene-environment interactions because it displays a form of Type II TSD, where female-biased sex ratios are observed at extreme incubation temperatures and approximately 50 : 50 sex ratios occur at intermediate temperatures. This response to temperature has been proposed to occur due to underlying sex determining loci, the influence of which is overridden at extreme temperatures. Thus, sex reversal at extreme temperatures is predicted to produce the female-biased sex ratios observed in A. muricatus. The occurrence of ovotestes during development is a cellular marker of temperature sex reversal in a closely related species Pogona vitticeps. Here, we present the first developmental data for A. muricatus, and show that ovotestes occur at frequencies consistent with a mode of sex determination that is intermediate between GSD and TSD. This is the first evidence suggestive of underlying unidentified sex determining loci in a species that has long been used as a model for TSD.

Reproductive phenotype predicts adult bite-force performance in sex-reversed dragons (Pogona vitticeps).

Sex-related differences in morphology and behavior are well documented, but the relative contributions of genes and environment to these traits are less well understood. Species that undergo sex reversal, such as the central bearded dragon (Pogona vitticeps), offer an opportunity to better understand sexually dimorphic traits because sexual phenotypes can exist on different chromosomal backgrounds. Reproductively female dragons with a discordant sex chromosome complement (sex reversed), at least as juveniles, exhibit traits in common with males (e.g., longer tails and greater boldness). However, the impact of sex reversal on sexually dimorphic traits in adult dragons is unknown. Here, we investigate the effect of sex reversal on bite-force performance, which may be important in resource acquisition (e.g., mates and/or food). We measured body size, head size, and bite force of the three sexual phenotypes in a colony of captive animals. Among adults, we found that males (ZZm) bite more forcefully than either chromosomally concordant females (ZWf) or sex-reversed females (ZZf), and this difference is associated with having relatively larger head dimensions. Therefore, adult sex-reversed females, despite apparently exhibiting male traits as juveniles, do not develop the larger head and enhanced bite force of adult male bearded dragons. This pattern is further illustrated in the full sample by a lack of positive allometry of bite force in sex-reversed females that is observed in males. The results reveal a close association between reproductive phenotype and bite force performance, regardless of sex chromosome complement.

Cellular calcium and redox regulation: the mediator of vertebrate environmental sex determination?

Many reptiles and some fish determine offspring sex by environmental cues such as incubation temperature. The mechanism by which environmental signals are captured and transduced into specific sexual phenotypes has remained unexplained for over 50 years. Indeed, environmental sex determination (ESD) has been viewed as an intractable problem because sex determination is influenced by a myriad of genes that may be subject to environmental influence. Recent demonstrations of ancient, conserved epigenetic processes in the regulatory response to environmental cues suggest that the mechanisms of ESD have a previously unsuspected level of commonality, but the proximal sensor of temperature that ultimately gives rise to one sexual phenotype or the other remains unidentified. Here, we propose that in ESD species, environmental cues are sensed by the cell through highly conserved ancestral elements of calcium and redox (CaRe) status, then transduced to activate ubiquitous signal transduction pathways, or influence epigenetic processes, ultimately to drive the differential expression of sex genes. The early evolutionary origins of CaRe regulation, and its essential role in eukaryotic cell function, gives CaRe a propensity to be independently recruited for diverse roles as a 'cellular sensor' of environmental conditions. Our synthesis provides the first cohesive mechanistic model connecting environmental signals and sex determination pathways in vertebrates, providing direction and a framework for developing targeted experimentation.

Museum Epigenomics: Charting the Future by Unlocking the Past.

Epigenomic state preserved in museum specimens could be leveraged to provide unique insights into gene regulation trends associated with accelerating environmental change during the Anthropocene. We address the challenges facing museum epigenomics and propose a collaborative framework for researchers and curators to explore this new field.