De novo genome assemblies of two cryptodiran turtles with ZZ/ZW and XX/XY sex chromosomes provide insights into patterns of genome reshuffling and uncover novel 3D genome folding in amniotes.

Understanding the evolution of chromatin conformation among species is fundamental to elucidate the architecture and plasticity of genomes. Nonrandom interactions of linearly distant loci regulate gene function in species-specific patterns, affecting genome function, evolution, and, ultimately, speciation. Yet, data from nonmodel organisms are scarce. To capture the macroevolutionary diversity of vertebrate chromatin conformation, here we generate de novo genome assemblies for two cryptodiran (hidden-neck) turtles via Illumina sequencing, chromosome conformation capture, and RNA-seq: Apalone spinifera (ZZ/ZW, 2n = 66) and Staurotypus triporcatus (XX/XY, 2n = 54). We detected differences in the three-dimensional (3D) chromatin structure in turtles compared to other amniotes beyond the fusion/fission events detected in the linear genomes. Namely, whole-genome comparisons revealed distinct trends of chromosome rearrangements in turtles: (1) a low rate of genome reshuffling in Apalone (Trionychidae) whose karyotype is highly conserved when compared to chicken (likely ancestral for turtles), and (2) a moderate rate of fusions/fissions in Staurotypus (Kinosternidae) and Trachemys scripta (Emydidae). Furthermore, we identified a chromosome folding pattern that enables "centromere-telomere interactions" previously undetected in turtles. The combined turtle pattern of "centromere-telomere interactions" (discovered here) plus "centromere clustering" (previously reported in sauropsids) is novel for amniotes and it counters previous hypotheses about amniote 3D chromatin structure. We hypothesize that the divergent pattern found in turtles originated from an amniote ancestral state defined by a nuclear configuration with extensive associations among microchromosomes that were preserved upon the reshuffling of the linear genome.

The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Transplant Bellwether: Endothelial Cells in Antibody-Mediated Rejection.

Ab-mediated rejection of organ transplants remains a stubborn, frequent problem affecting patient quality of life, graft function, and grant survival, and for which few efficacious therapies currently exist. Although the field has gained considerable knowledge over the last two decades on how anti-HLA Abs cause acute tissue injury and promote inflammation, there has been a gap in linking these effects with the chronic inflammation, vascular remodeling, and persistent alloimmunity that leads to deterioration of graft function over the long term. This review will discuss new data emerging over the last 5 y that provide clues into how ongoing Ab-endothelial cell interactions may shape vascular fate and propagate alloimmunity in organ transplants.

The transcriptional repressor B cell lymphoma 6 regulates CXCR3 chemokine and human leukocyte antigen II expression in endothelial cells.

Interferon gamma (IFN-γ) induces an endothelial proimmunogenic phenotype through the JAK/STAT1 pathway, which can shape the activation of alloreactive leukocytes in transplant rejection. In immune cells, the DNA-binding protein B cell lymphoma 6 (BCL6) controls the transcription of inflammatory genes. This study tested if BCL6 modulates IFN-γ-induced gene expression in endothelial cells. In vitro, BCL6 was IFN-γ-inducible in primary human endothelium, along with CXCR3 chemokines and human leukocyte antigen (HLA). BCL6, HLA II, and CXCL9 were also increased in human cardiac transplants during acute rejection. Knockdown of BCL6 augmented, whereas overexpression and BTB domain inhibitors (BCL6-BTBi) suppressed, HLA II and CXCR3 chemokine expression but not HLA I. Further, BCL6 had a greater effect on HLA-DR and DP but was less involved in regulating HLA-DQ expression. The effect correlated with BCL6 binding motifs in or near affected genes. The BCL6 DNA recognition sequence was highly similar to that of STAT1, and BTBi reduced STAT1's transcriptional activity in vitro. Our results show for the first time that BCL6 selectively controls IFN-γ-induced endothelial gene expression, advancing our understanding of the endogenous mechanisms regulating donor immunogenicity.

Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection.

Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFß-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

JAKinibs prevent persistent, IFNγ-autonomous endothelial cell inflammation and immunogenicity.

The adhesion and subsequent activation of T cells is a critical step in local inflammatory responses, particularly of alloreactive leukocytes in rejection of transplanted donor tissue. Interferon (IFN)γ is an adaptive cytokine that promotes endothelial cell (EC) expression of pro-adhesive factors and costimulatory molecules. We recently reported that IFNγ-induced endothelial cell antigen-presenting capacity was protracted after cytokine withdrawal. This study sought to determine what intracellular signaling mediates this chronic endothelial activation by IFNγ. The durability of interferon signaling in human aortic endothelial activation was tested. Pro-adhesive and costimulatory gene expression, phenotype, secretome, and Janus kinase (JAK)/STAT phosphorylation in human primary endothelial cells were measured under chronic and transient IFNγ stimulation, with various JAK inhibitors. IFNγ reporter cells were tested for STAT1 transcriptional activity with JAK inhibition and suppressors of cytokine signaling (SOCS) overexpression, under continuous and priming conditions. The consequences of even short exposure to IFNγ were long-lasting and broad, with sustained elevation of adhesion molecules and chemokines up to 48 h later. JAK/STAT and interferon response factor expression were likewise durable, dependent on new transcription but autonomous of continuous IFNγ. Persistent STAT new transcription and JAK signaling in the endothelium was required to maintain a pro-adhesive and proimmunogenic phenotype after IFNγ withdrawal since both could be prevented by cycloheximide but only by JAKinibs with potency against JAK2. Finally, the suppressor of cytokine signaling SOCS1 failed to emerge in primed endothelial cells, which likely accounted for prolonged inflammatory gene expression. The results reveal a sustained JAK-dependent perturbation of endothelial function and suggest that JAKinibs may have therapeutic benefits in dampening vascular inflammation and allogeneic leukocyte activation.NEW & NOTEWORTHY The central question investigated in this study is why vascular endothelium remains inflamed and what underlying signaling is responsible. The new results show that the resolution of endothelial-controlled inflammation may be impaired or delayed because Janus kinase (JAK)/STAT activation is maintained autonomous of interferon (IFN)γ presence, and the late phase negative regulator suppressors of cytokine signaling (SOCS)1 fails to be induced.

Sensitization in transplantation: Assessment of Risk 2022 Working Group Meeting Report.

The Sensitization in Transplantation: Assessment of Risk workgroup is a collaborative effort of the American Society of Transplantation and the American Society of Histocompatibility and Immunogenetics that aims at providing recommendations for clinical testing, highlights gaps in current knowledge, and proposes areas for further research to enhance histocompatibility testing in support of solid organ transplantation. This report provides updates on topics discussed by the previous Sensitization in Transplantation: Assessment of Risk working groups and introduces 2 areas of exploration: non-human leukocyte antigen antibodies and utilization of human leukocyte antigen antibody testing measurement to evaluate the efficacy of antibody-removal therapies.

Molecular Signature of Antibody-Mediated Chronic Vasculopathy in Heart Allografts in a Novel Mouse Model.

Cardiac allograft vasculopathy (CAV) limits the long-term success of heart transplants. Generation of donor-specific antibodies (DSAs) is associated with increased incidence of CAV clinically, but mechanisms underlying development of this pathology remain poorly understood. Major histocompatibility complex-mismatched A/J cardiac allografts in B6.CCR5-/- recipients have been reported to undergo acute rejection with little T-cell infiltration, but intense deposition of C4d in large vessels and capillaries of the graft accompanied by high titers of DSA. This model was modified to investigate mechanisms of antibody-mediated CAV by transplanting A/J hearts to B6.CCR5-/- CD8-/- mice that were treated with low doses of anti-CD4 monoclonal antibody to decrease T-cell-mediated graft injury and promote antibody-mediated injury. Although the mild inhibition of CD4 T cells extended allograft survival, the grafts developed CAV with intense C4d deposition and macrophage infiltration by 14 days after transplantation. Development of CAV correlated with recipient DSA titers. Transcriptomic analysis of microdissected allograft arteries identified the Notch ligand Dll4 as the most elevated transcript in CAV, associated with high versus low titers of DSA. More importantly, these analyses revealed a differential expression of transcripts in the CAV lesions compared with the matched apical tissue that lacks large arteries. In conclusion, these findings report a novel model of antibody-mediated CAV with the potential to facilitate further understanding of the molecular mechanisms promoting development of CAV.

Evolution and dosage compensation of nucleolar organizing regions (NORs) mediated by mobile elements in turtles with female (ZZ/ZW) but not with male (XX/XY) heterogamety.

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.

Effects of semi-constant temperature on embryonic and hatchling phenotypes of six-tubercled Amazon River turtles, Podocnemis sextuberculata.

PURPOSE: We evaluated how constant incubation temperatures affect life-history traits pre-hatching and post-hatching of the six-tubercled Amazon River turtle, Podocnemis sextuberculata. METHODS: We incubated eggs from natural nests at ten semi-constant temperatures between 22.26 ± 1.01 °C and 37.37 ± 0.38°C (2013) and at six temperatures between 25.75 ± 0.22 °C and 36.17 ± 0.15°C (2016). In 2013, we raised hatchling for 90 days to evaluate effects of temperature on early hatchling growth. We evaluated maternal effects in 2016. RESULTS: P. sextuberculata displays temperature-dependent sex determination and produces males at colder and females at warmer temperatures (TSD Ia). The estimated pivotal temperature was 33.73 ± 0.15 °C and the transitional range of temperatures (TRT) 1.16 ± 0.59 °C. Semi-constant temperatures below 26 °C and above 38 °C were lethal. Intermediate temperatures (32.25 °C and 31.5 °C, respectively) were optimal for hatching success and produced larger hatchlings that grew slower early in life compared to colder or warmer conditions, which produced smaller hatchlings. Warmer incubation temperatures within the optimal range (28°C-37 °C) accelerated embryonic development. In contrast, comparisons of 30, 60 and 90 days-old suggests that warmer incubation temperatures reduced growth and mass gain rates post-hatching, such that incubation temperature effects on body size at emergence disappeared by 3 months of age. CONCLUSIONS: Six-tubercled Amazon River turtles showed the highest pivotal temperature reported for any turtle. The relatively narrow TRT may limit the evolutionary potential of this vulnerable turtle in the face of global warming. Future incubation experiments at a finer scale (33°C-36 °C) are warranted to refine the sex-ratio reaction norm. Field studies that monitor natural nests are imperative to evaluate conservation measures and the effect of female-biased illegal hunting and climate change. By providing data about the thermal biology of an understudied lineage of non-model species, our study helps fill gaps in our understanding of the evolution of vertebrate sex determination and its potential adaptive value.

Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.

Sensitization in transplantation: Assessment of risk (STAR) 2019 Working Group Meeting Report.

The purpose of the STAR 2019 Working Group was to build on findings from the initial STAR report to further clarify the expectations, limitations, perceptions, and utility of alloimmune assays that are currently in use or in development for risk assessment in the setting of organ transplantation. The goal was to determine the precision and clinical feasibility/utility of such assays in evaluating both memory and primary alloimmune risks. The process included a critical review of biologically driven, state-of-the-art, clinical diagnostics literature by experts in the field and an open public forum in a face-to-face meeting to promote broader engagement of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics membership. This report summarizes the literature review and the workshop discussions. Specifically, it highlights (1) available assays to evaluate the attributes of HLA antibodies and their utility both as clinical diagnostics and as research tools to evaluate the effector mechanisms driving rejection; (2) potential assays to assess the presence of alloimmune T and B cell memory; and (3) progress in the development of HLA molecular mismatch computational scores as a potential prognostic biomarker for primary alloimmunity and its application in research trial design.

HLA Class II-Triggered Signaling Cascades Cause Endothelial Cell Proliferation and Migration: Relevance to Antibody-Mediated Transplant Rejection.

Transplant recipients developing donor-specific HLA class II (HLA-II) Abs are at higher risk for Ab-mediated rejection (AMR) and transplant vasculopathy. To understand how HLA-II Abs cause AMR and transplant vasculopathy, we determined the signaling events triggered in vascular endothelial cells (EC) following Ab ligation of HLA-II molecules. HLA-II expression in EC was induced by adenoviral vector expression of CIITA or by pretreatment with TNF-α/IFN-γ. Ab ligation of class II stimulated EC proliferation and migration. Class II Ab also induced activation of key signaling nodes Src, focal adhesion kinase, PI3K, and ERK that regulated downstream targets of the mammalian target of rapamycin (mTOR) pathway Akt, p70 ribosomal S6 kinase, and S6 ribosomal protein. Pharmacological inhibitors and small interfering RNA showed the protein kinases Src, focal adhesion kinase, PI3K/Akt, and MEK/ERK regulate class II Ab-stimulated cell proliferation and migration. Treatment with rapalogs for 2 h did not affect HLA-II Ab-induced phosphorylation of ERK; instead, mTOR complex (mTORC)1 targets were dependent on activation of ERK. Importantly, suppression of mTORC2 for 24 h with rapamycin or everolimus or treatment with mTOR active-site inhibitors enhanced HLA-II Ab-stimulated phosphorylation of ERK. Furthermore, knockdown of Rictor with small interfering RNA caused overactivation of ERK while abolishing phosphorylation of Akt Ser473 induced by class II Ab. These data are different from HLA class I Ab-induced activation of ERK, which is mTORC2-dependent. Our results identify a complex signaling network triggered by HLA-II Ab in EC and indicate that combined ERK and mTORC2 inhibitors may be required to achieve optimal efficacy in controlling HLA-II Ab-mediated AMR.

Discovery of Putative XX/XY Male Heterogamety in Emydura subglobosa Turtles Exposes a Novel Trajectory of Sex Chromosome Evolution in Emydura.

The discovery of sex chromosome systems in non-model organisms has elicited growing recognition that sex chromosomes evolved via diverse paths that are not fully elucidated. Lineages with labile sex determination, such as turtles, hold critical cues, yet data are skewed toward hide-neck turtles (suborder Cryptodira) and scant for side-neck turtles (suborder Pleurodira). Here, we used classic and molecular cytogenetics to investigate Emydura subglobosa (ESU), an unstudied side-neck turtle with genotypic sex determination from the family Chelidae, where extensive morphological divergence exists among XX/XY systems. Our data represent the first cytogenetic description for ESU. Similarities were found between ESU and E. macquarii (EMA), such as identical chromosome number (2n = 50), a single and dimorphic nucleolus organizer region (NOR) localized in a microchromosome pair (ESU14) of both sexes (detected via FISH of 18S rDNA). Only the larger NOR is active (detected by silver staining). As in EMA, comparative genome hybridization revealed putative macro XX/XY chromosomes in ESU (the 4th largest pair). Our comparative analyses and revaluation of previous data strongly support the hypothesis that Emydura's XX/XY system evolved via fusion of an ancestral micro-Y (retained by Chelodina longicollis) onto a macro-autosome. This evolutionary trajectory differs from the purported independent evolution of XX/XY from separate ancestral autosomes in Chelodina and Emydura that was previously reported. Our data permit dating this Y-autosome fusion to at least the split of Emydura around 45 Mya and add critical information about the evolution of the remarkable diversity of sex-determining mechanisms in turtles, reptiles, and vertebrates.

Chromosomal Rearrangements during Turtle Evolution Altered the Synteny of Genes Involved in Vertebrate Sex Determination.

Sex-determining mechanisms (SDMs) set an individual's sexual fate by its genotype (genotypic sex determination, GSD) or environmental factors like temperature (temperature- dependent sex determination, TSD), as in turtles where the GSD "trigger" remains unknown. SDMs co-evolve with turtle chromosome number, perhaps because fusions/fissions alter the relative position/regulation of sexual development genes. Here, we map 10 such genes via FISH onto metaphase chromosomes in 6 TSD and 6 GSD turtles for the first time. Results uncovered intrachromosomal rearrangements involving 3 genes across SDMs (Dax1, Fhl2, and Fgf9) and a chromosomal fusion linking 2 genes (Sf1 and Rspo1) in 1 chromosome in a TSD turtle (Pelomedusa subrufa) that locate to 2 chromosomes in all others. Notably, Sf1 and its repressor Foxl2 map to Apalone spinifera's ZW chromosomes but to a macro- (Foxl2) and a microautosome (Sf1) in other turtles potentially inducing SDM evolution. However, our phylogenetically informed analysis refutes Foxl2 (but not Sf1) as Apalone's master sex-determining gene. The absence of common TSD-specific or GSD-specific rearrangements underscores the independent evolutionary trajectories of turtle SDMs. Further comparative analyses using more genes from the sexual development network are warranted to inform genome evolution and its contribution to enigmatic turnovers of vertebrate sex determination.