The structure of the TH/INS locus and the parental allele expressed are not conserved between mammals.

Parent-of-origin-specific expression of imprinted genes is critical for successful mammalian growth and development. Insulin, coded by the INS gene, is an important growth factor expressed from the paternal allele in the yolk sac placenta of therian mammals. The tyrosine hydroxylase gene TH encodes an enzyme involved in dopamine synthesis. TH and INS are closely associated in most vertebrates, but the mouse orthologues, Th and Ins2, are separated by repeated DNA. In mice, Th is expressed from the maternal allele, but the parental origin of expression is not known for any other mammal so it is unclear whether the maternal expression observed in the mouse represents an evolutionary divergence or an ancestral condition. We compared the length of the DNA segment between TH and INS across species and show that separation of these genes occurred in the rodent lineage with an accumulation of repeated DNA. We found that the region containing TH and INS in the tammar wallaby produces at least five distinct RNA transcripts: TH, TH-INS1, TH-INS2, lncINS and INS. Using allele-specific expression analysis, we show that the TH/INS locus is expressed from the paternal allele in pre- and postnatal tammar wallaby tissues. Determining the imprinting pattern of TH/INS in other mammals might clarify if paternal expression is the ancestral condition which has been flipped to maternal expression in rodents by the accumulation of repeat sequences.

Atrazine disorganises laminin formation and reduces cell numbers in the tammar testis during early differentiation.

In brief: Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation. Abstract: The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20-40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17ß-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17ß-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40-50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

Body fat and circulating leptin levels in the captive short-beaked echidna (Tachyglossus aculeatus).

It is possible that the reproductive strategy of the short-beaked echidna is related to seasonal changes in fat deposition and energy availability, regulated by seasonal changes in endocrine function. We predicted that circulating leptin levels would be directly proportional to adiposity during most of the year, but that a change in this relationship would occur during the pre-breeding season to allow increased fat deposition. To test this hypothesis, we made use of a captive colony of echidnas to describe and quantify changes in fat distribution and the adipostatic hormone leptin. First we assessed seasonal changes in circulating leptin levels, body mass and adiposity for three male and three female adult echidnas maintained on a standard diet. Second, we explored the relationship between circulating leptin levels and increased caloric intake for an additional five adult female echidnas that were provided with supplemented nutrition. Third we visualised fat distribution in male and female adult echidnas using magnetic resonance imaging (MRI) before and after the breeding season, to determine where fat is deposited in this species. For echidnas maintained on the standard diet, there were no seasonal changes in body mass, body fat or plasma leptin levels. However, female echidnas provided with supplemented nutrition had significantly elevated plasma leptin levels during the breeding season, compared to the pre-and post- breeding periods. MRI showed substantial subcutaneous fat depots extending dorso-laterally from the base of the skull to the base of the tail, in both sexes. Pre-breeding season, both sexes had considerable fat deposition in the pelvic/rump region, whilst the female echidna accumulated most fat in the abdominal region. This study shows that male and female echidnas accumulate body fat in the pelvic/rump and the abdominal regions, respectively and that circulating leptin may promote fattening in female echidnas during the breeding season by means of leptin resistance. However, further research is required to evaluate the precise relationship between seasonal changes in leptin and adiposity.

Genome of the endangered eastern quoll (Dasyurus viverrinus) reveals signatures of historical decline and pelage color evolution.

The eastern quoll (Dasyurus viverrinus) is an endangered marsupial native to Australia. Since the extirpation of its mainland populations in the 20th century, wild eastern quolls have been restricted to two islands at the southern end of their historical range. Eastern quolls are the subject of captive breeding programs and attempts have been made to re-establish a population in mainland Australia. However, few resources currently exist to guide the genetic management of this species. Here, we generated a reference genome for the eastern quoll with gene annotations supported by multi-tissue transcriptomes. Our assembly is among the most complete marsupial genomes currently available. Using this assembly, we infer the species' demographic history, identifying potential evidence of a long-term decline beginning in the late Pleistocene. Finally, we identify a deletion at the ASIP locus that likely underpins pelage color differences between the eastern quoll and the closely related Tasmanian devil (Sarcophilus harrisii).

Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted.

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.

An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus).

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristic curves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

The alternate pathway of androgen metabolism and window of sensitivity.

Since the discovery in 1968 that dihydrotestosterone (DHT) is a major mediator of androgen action, a convincing body of evidence has accumulated to indicate that the major pathway of DHT formation is the 5α-reduction of circulating testosterone in androgen target tissues. However, we now know that DHT can also be formed in peripheral tissues by the oxidation of 5α-androstane-3α,17ß-diol (adiol). This pathway is responsible for the formation of the male phenotype. We discuss the serendipitous discovery in the tammar wallaby of an alternate pathway by which adiol is formed in the testes, secreted into plasma and converted in peripheral tissues to DHT. This alternate pathway is responsible for virilisation of the urogenital system in this species and is present in the testes at the onset of male puberty of all mammals studied so far. This is the first clear-cut function for steroid 5α-reductase 1 in males. Unexpectedly, the discovery of this pathway in this Australian marsupial has had a major impact in understanding the pathophysiology of aberrant virilisation in female newborns. Overactivity of the alternate pathway appears to explain virilisation in congenital adrenal hyperplasia CAH, in X-linked 46,XY disorders of sex development. It also appears to be important in polycystic ovarian syndrome (PCOS) since PCOS ovaries have enhanced the expression of genes and proteins of the alternate pathway. It is now clear that normal male development in marsupials, rodents and humans requires the action of both the classic and the alternate (backdoor) pathways.

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials.

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, chromosomal ends were conspicuously polarized in a bouquet configuration and synapsis progressed exclusively from the telomeres towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophase I in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, the bouquet polarization was incomplete and ephemeral, γH2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

Conserved H3K27me3-associated chromatin remodelling allows STRA8 but not MEIOSIN expression in mammalian germ cells.

In brief: Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other. Abstract: In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

The swan genome and transcriptome, it is not all black and white.

BACKGROUND: The Australian black swan (Cygnus atratus) is an iconic species with contrasting plumage to that of the closely related northern hemisphere white swans. The relative geographic isolation of the black swan may have resulted in a limited immune repertoire and increased susceptibility to infectious diseases, notably infectious diseases from which Australia has been largely shielded. Unlike mallard ducks and the mute swan (Cygnus olor), the black swan is extremely sensitive to highly pathogenic avian influenza. Understanding this susceptibility has been impaired by the absence of any available swan genome and transcriptome information. RESULTS: Here, we generate the first chromosome-length black and mute swan genomes annotated with transcriptome data, all using long-read based pipelines generated for vertebrate species. We use these genomes and transcriptomes to show that unlike other wild waterfowl, black swans lack an expanded immune gene repertoire, lack a key viral pattern-recognition receptor in endothelial cells and mount a poorly controlled inflammatory response to highly pathogenic avian influenza. We also implicate genetic differences in SLC45A2 gene in the iconic plumage of the black swan. CONCLUSION: Together, these data suggest that the immune system of the black swan is such that should any avian viral infection become established in its native habitat, the black swan would be in a significant peril.

Getting out of a mammalian egg: the egg tooth and caruncle of the echidna.

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, development of the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence. Unlike therian tooth germs that develop by placode invagination, the echidna egg tooth developed by evagination, similar to the first teeth in some reptiles and fish. The egg tooth ankylosed to the premaxilla, rather than forming a tooth root with ligamentous attachment found in other mammals, with loss of the egg tooth associated with high levels of activity odontoclasts and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium on top of the nose expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with typical reptilian teeth, suggesting that this tooth has been conserved from a common ancestor of mammals and reptiles.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus).

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0-18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.

Principles of 3D chromosome folding and evolutionary genome reshuffling in mammals.

Studying the similarities and differences in genomic interactions between species provides fertile grounds for determining the evolutionary dynamics underpinning genome function and speciation. Here, we describe the principles of 3D genome folding in vertebrates and show how lineage-specific patterns of genome reshuffling can result in different chromatin configurations. We (1) identified different patterns of chromosome folding in across vertebrate species (centromere clustering versus chromosomal territories); (2) reconstructed ancestral marsupial and afrotherian genomes analyzing whole-genome sequences of species representative of the major therian phylogroups; (3) detected lineage-specific chromosome rearrangements; and (4) identified the dynamics of the structural properties of genome reshuffling through therian evolution. We present evidence of chromatin configurational changes that result from ancestral inversions and fusions/fissions. We catalog the close interplay between chromatin higher-order organization and therian genome evolution and introduce an interpretative hypothesis that explains how chromatin folding influences evolutionary patterns of genome reshuffling.

Comparing the potential for maternal-fetal signalling in oviparous and viviparous lizards.

The evolution of a placenta requires several steps including changing the timing of reproductive events, facilitating nutrient exchange, and the capacity for maternal-fetal communication. To understand the evolution of maternal-fetal communication, we used ligand-receptor gene expression as a proxy for the potential for cross-talk in a live-bearing lizard (Pseudemoia entrecasteauxii) and homologous tissues in a related egg-laying lizard (Lampropholis guichenoti). Approximately 70% of expressed ligand/receptor genes were shared by both species. Gene ontology (GO) analysis showed that there was no GO-enrichment in the fetal membranes of the egg-laying species, but live-bearing fetal tissues were significantly enriched for 50 GO-terms. Differences in enrichment suggest that the evolution of viviparity involved reinforcing specific signalling pathways, perhaps to support fetal control of placentation. One identified change was in transforming growth factor beta signalling. Using immunohistochemistry, we show the production of the signalling molecule inhibin beta B (INHBB) occurs in viviparous fetal membranes but was absent in closely related egg-laying tissues, suggesting that the evolution of viviparity may have involved changes to signalling via this pathway. We argue that maternal-fetal signalling evolved through co-opting expressed signalling molecules and recruiting new signalling molecules to support the complex developmental changes required to support a fetus in utero. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Reproductive behaviour before and after oestrus and oviposition in the captive short-beaked echidna (Tachyglossus aculeatus).

CONTEXT: Most of our current knowledge regarding echidna reproductive behaviour is based on qualitative measurements; therefore, it is unclear if specific behavioural cues could be utilised in their captive reproductive management. AIMS: This study aimed to identify quantitative changes in general and reproductive behaviour of echidna breeding pairs and pregnant females that might facilitate the detection of oestrus and impending oviposition and provide a summary of reproductive behaviour observed in a captive colony over a three-year observation period. METHODS: Three echidna breeding pairs and two trios were monitored daily for seven reproductive and eight general behaviours during the 2020 breeding season. After confirmed copulation, females were monitored for four egg-laying and eight general behaviours until egg incubation. General observations of reproductive behaviours during the 2018-2020 breeding seasons were recorded as part of routine husbandry. KEY RESULTS: For breeding pairs, there was a significant rate of change over time before and after copulation for the behaviours 'urogenital sniffing', 'rolling' and 'copulation attempt'. For pregnant females, time engaged in 'pacing' significantly increased while 'time eating' and the 'quantity of food eaten' significantly decreased on the day of oviposition. We were not able to identify oestrus from specific behaviours, but our observations suggest that the female echidna's period of receptivity is less than 24h. CONCLUSIONS: The frequency that males express 'urogenital sniffing', 'rolling' and 'copulation attempt' toward the female can be used to alert zookeepers that copulation has likely occurred. Increased pacing, reduced feeding time and quantity of food eaten can aid zookeepers to identify impending oviposition. IMPLICATIONS: This study demonstrates that there are quantifiable changes in specific echidna behaviours that can be incorporated into zoo husbandry practices to improve the reproductive management of this species.

Placental imprinting of SLC22A3 in the IGF2R imprinted domain is conserved in therian mammals.

BACKGROUND: The eutherian IGF2R imprinted domain is regulated by an antisense long non-coding RNA, Airn, which is expressed from a differentially methylated region (DMR) in mice. Airn silences two neighbouring genes, Solute carrier family 22 member 2 (Slc22a2) and Slc22a3, to establish the Igf2r imprinted domain in the mouse placenta. Marsupials also have an antisense non-coding RNA, ALID, expressed from a DMR, although the exact function of ALID is currently unknown. The eutherian IGF2R DMR is located in intron 2, while the marsupial IGF2R DMR is located in intron 12, but it is not yet known whether the adjacent genes SLC22A2 and/or SLC22A3 are also imprinted in the marsupial lineage. In this study, the imprinting status of marsupial SLC22A2 and SLC22A3 in the IGF2R imprinted domain in the chorio-vitelline placenta was examined in a marsupial, the tammar wallaby. RESULTS: In the tammar placenta, SLC22A3 but not SLC22A2 was imprinted. Tammar SLC22A3 imprinting was evident in placental tissues but not in the other tissues examined in this study. A putative promoter of SLC22A3 lacked DNA methylation, suggesting that this gene is not directly silenced by a DMR on its promoter as seen in the mouse. Based on immunofluorescence, we confirmed that the tammar SLC22A3 is localised in the endodermal cell layer of the tammar placenta where nutrient trafficking occurs. CONCLUSIONS: Since SLC22A3 is imprinted in the tammar placenta, we conclude that this placental imprinting of SLC22A3 has been positively selected after the marsupial and eutherian split because of the differences in the DMR location. Since SLC22A3 is known to act as a transporter molecule for nutrient transfer in the eutherian placenta, we suggest it was strongly selected to control the balance between supply and demand of nutrients in marsupial as it does in eutherian placentas.

Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus).

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.

Evolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals.

Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals.

Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.