Insulin/IGF signaling (IIS) regulates developmental and metabolic plasticity. Conditional regulation of insulin-like peptide expression and secretion promotes different phenotypes in different environments. However, IIS can also be regulated by other, less-understood mechanisms. For example, stability of the only known insulin/IGF receptor in C. elegans, DAF-2/INSR, is regulated by CHIP-dependent ubiquitination. Disruption of chn-1/CHIP reduces longevity in C. elegans by increasing DAF-2/INSR abundance and IIS activity in adults. Likewise, mutation of a ubiquitination site causes daf-2(gk390525) to display gain-of-function phenotypes in adults. However, we show that this allele displays loss-of-function phenotypes in larvae, and that its effect on IIS activity transitions from negative to positive during development. In contrast, the allele acts like a gain-of-function in larvae cultured at high temperature, inhibiting temperature-dependent dauer formation. Disruption of chn-1/CHIP causes an increase in IIS activity in starved L1 larvae, unlike daf-2(gk390525). CHN-1/CHIP ubiquitinates DAF-2/INSR at multiple sites. These results suggest that the sites that are functionally relevant to negative regulation of IIS vary in larvae and adults, at different temperatures, and in nutrient-dependent fashion, revealing additional layers of IIS regulation.

Calochortus tolmiei Hook. & Arn., a bulbous monocot with cat's ear flowers in the angiosperm family Liliaceae, is a perennial herb native to northern California, Oregon, and Washington. Calochortus exhibits substantial morphological and karyotype diversity with multiple floral forms and a haploid chromosome number varying from six to 10. Here, we present the first high-quality reference assembly in Liliaceae for C. tolmiei, with a scaffolded assembly of 2.9 Gbp with a N50 of 296 Mbp. Notably, 92% of the assembled genome is scaffolded into 10 pseudomolecules, matching the documented chromosome count of C. tolmiei. The genome contains 31,049 protein-coding genes, with 86.2% being functionally annotated. The closest reference-quality genome assembly to C. tolmiei is from Chionographis japonica (Willd.) Maxim. (Melianthiaceae), which diverged approximately 83 MYA, providing a valuable genomic resource in the Liliales, an order which lacks genomic resources.

Sex-determining region Y box 2 (Sox2) is a critical transcription factor for embryogenesis and neural stem and progenitor cell (NSPC) maintenance. While distal enhancers control Sox2 in embryonic stem cells (ESCs), enhancers closer to the gene are implicated in Sox2 transcriptional regulation in neural development. We hypothesize that a downstream enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), regulates Sox2 transcription in neural stem cells and we investigate this in NSPCs derived from mouse ESCs. Using functional genomics and CRISPR-Cas9 mediated deletion analyses we investigate the role of SRR2-18 in Sox2 regulation during neural differentiation. Transcriptome analyses demonstrate that loss of even one copy of SRR2-18 disrupts the region-specific identity of NSPCs, reducing the expression of genes associated with more anterior regions of the embryonic nervous system. Homozygous deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which causes differentiation into posterior neural fates, specifically the caudal neural tube. This study highlights the importance of precise Sox2 regulation by SRR2-18 in neural differentiation.

Conducting genomic selection in plant breeding programs can substantially speed up the development of new varieties. Genomic selection provides more reliable insights when it is based on dense marker data, in which the rare variants can be particularly informative. Despite the availability of new technologies, the cost of large-scale genotyping remains a major limitation to the implementation of genomic selection. We suggest to combine pooled genotyping with population-based imputation as a cost-effective computational strategy for genotyping SNPs. Pooling saves genotyping tests and has proven to accurately capture the rare variants that are usually missed by imputation. In this study, we investigate adding iterative coupling to a joint model of pooling and imputation that we have previously proposed. In each iteration, the imputed genotype probabilities serve as feedback input for adjusting the per-sample prior genotype probabilities, before running a new imputation based on these adjusted data. This flexible setup indirectly imposes consistency between the imputed genotypes and the pooled observations. We demonstrate that repeated cycles of feedback can take advantage of the strengths in both pooling and imputation when an appropriate set of reference haplotypes is available for imputation. The iterations improve greatly upon the initial genotype predictions, achieving very high genotype accuracy for both low and high frequency variants. We enhance the average concordance from 94.5% to 98.4% at limited computational cost and without requiring any additional genotype testing.

Chemical signaling can play a crucial role in predator-prey dynamics. Here, we present evidence that ink from the common cuttlefish (Sepia officinalis) targets olfactory receptor proteins in shark, potentially acting as a predator deterrence. We apply in silico 3D docking analysis to investigate the binding affinity of various odorant molecules to shark olfactory receptors of two shark species: cloudy catshark (Scyliorhinus torazame) and white shark (Carcharodon carcharias). Pavoninin-4 (a known shark-repellent compound), displayed selectivity in binding to receptors in the white shark. In contrast, the primary component of cuttlefish ink, melanin, displayed the highest binding affinities to all shark olfactory receptor proteins in both species. Taurine, another important ink component, exhibited standard to strong bindings for both species. Trans-4,5-epoxy-(E)-2-decenal ("blood-decenal"), an odorant associated with the smell of blood displayed strong binding affinities to all shark olfactory receptors, similar to that of melanin. These findings provide new insights into the molecular interplay between cephalopod inking behavior and their shark predators, with cuttlefish ink likely exploiting the narrow band of the shark olfactory repertoire.

The demographic history of a population, and the distribution of fitness effects (DFE) of newly arising mutations in functional genomic regions, are fundamental factors dictating both genetic variation and evolutionary trajectories. Although both demographic and DFE inference has been performed extensively in humans, these approaches have generally either been limited to simple demographic models involving a single population, or, where a complex population history has been inferred, without accounting for the potentially confounding effects of selection at linked sites. Taking advantage of the coding-sparse nature of the genome, we propose a 2-step approach in which coalescent simulations are first used to infer a complex multi-population demographic model, utilizing large non-functional regions that are likely free from the effects of background selection. We then use forward-in-time simulations to perform DFE inference in functional regions, conditional on the complex demography inferred and utilizing expected background selection effects in the estimation procedure. Throughout, recombination and mutation rate maps were used to account for the underlying empirical rate heterogeneity across the human genome. Importantly, within this framework it is possible to utilize and fit multiple aspects of the data, and this inference scheme represents a generalized approach for such large-scale inference in species with coding-sparse genomes.

Orphan crops serve as essential resources for both nutrition and income in local communities and offer potential solutions to the challenges of food security and climate vulnerability. Tef [Eragrostis tef (Zucc.)], a small-grained allotetraploid, C4 cereal mainly cultivated in Ethiopia, stands out for its adaptability to marginal conditions and high nutritional value, which holds both local and global promise. Despite its significance, tef is considered an orphan crop due to limited genetic improvement efforts, reliance on subsistence farming, and its nutritional, economic, and cultural importance. Although pre-Semitic inhabitants of Ethiopia have cultivated tef for millennia (4000-1000 BCE), the genetic and environmental drivers of local adaptation remain poorly understood. To address this, we resequenced a diverse collection of traditional tef varieties to investigate their genetic structure and identify genomic regions under environmental selection using redundancy analysis, complemented by differentiation-based methods. We identified 145 loci associated with abiotic environmental factors, with minimal geographic influence observed in the genetic structure of the sample population. Overall, this work contributes to the broader understanding of local adaptation and its genetic basis in tef, providing insights that support efforts to develop elite germplasms with improved environmental resilience.

As part of an ongoing effort to generate comprehensive resources for the experimental analysis of fourth chromosome genes in Drosophila melanogaster, the Fourth Chromosome Resource Project has used CRISPR mutagenesis with single guide RNAs to isolate mutations in 62 of the 80 fourth chromosome, protein-coding genes. These mutations were induced on a fourth chromosome bearing a basal FRT insertion to facilitate experimental approaches involving FLP recombinase-induced mitotic recombination. To permit straightforward comparisons among mutant stocks, most of the mutations were generated on isogenic fourth chromosomes, which were then crossed into a common genetic background. Of the 119 mutations, 84 are frameshift mutations likely to be null alleles, 29 are small, in-frame deletions and 6 have yet to be characterized molecularly. The mutations were tested for recessive lethal, female sterile and visible phenotypes. Stable stocks for most of the mutations have been submitted to repositories in the United States and Japan for public distribution.

The conserved MAP3K DLKs are widely known for their functions in synapse formation, axonal regeneration and degeneration, and neuronal survival, notably under traumatic injury and chronic disease conditions. In contrast, their roles in other neuronal compartments are much less explored. Through an unbiased forward genetic screening in C. elegans for altered patterns of GFP-tagged DLK-1 expressed from the endogenous locus, we have recently uncovered a mechanism by which the abundance of DLK-1 is tightly regulated by intraflagellar transport in ciliated sensory neurons. Here, we report additional mutants identified from the genetic screen. Most mutants exhibit increased accumulation of GFP::DLK-1 in sensory endings, and the levels of misaccumulated GFP::DLK-1 are exacerbated by loss of function in cebp-1, the b-Zip transcription factor acting downstream of DLK-1. We identify several new mutations in genes encoding proteins functioning in intraflagellar transport and cilia assembly, in components of BBSome, MAPK-15 and DYF-5 kinases. We report a novel mutation in the chaperone HSP90 that causes misaccumulation of GFP::DLK-1 and up-regulation of CEBP-1 selectively in ciliated sensory neurons. We also find that the guanylate cyclase ODR-1 constrains GFP::DLK-1 abundance throughout cilia and dendrites of AWC neurons. Moreover, in odr-1 mutants, AWC cilia display distorted morphology, which is ameliorated by loss of function in dlk-1 or cebp-1. These data expand the landscape of DLK-1 signaling in ciliated sensory neurons and underscore a high degree of cell- and neurite- specific regulation.

The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR-Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.

Haplotyped-resolved phased assemblies aim to capture the full allelic diversity in heterozygous and polyploid species to enable accurate genetic analyses. However, building non-collapsed references still presents a challenge. Here, we used long-range interaction Hi-C reads (high-throughput chromatin conformation capture) and HiFi PacBio reads to assemble the genome of the apomictic cultivar Basilisks from Urochloa decumbens (2n = 4x = 36), an outcrossed tetraploid Paniceae grass widely cropped to feed livestock in the tropics. We identified and removed Hi-C reads between homologous unitigs to facilitate their scaffolding and employed methods for the manual curation of rearrangements and misassemblies. Our final phased assembly included the four haplotypes in 36 chromosomes. We found that 18 chromosomes originated from diploid U. brizantha and the other 18 from either U. ruziziensis or diploid U. decumbens. We also identified a chromosomal translocation between chromosomes 5 and 32, as well as evidence of pairing exclusively within subgenomes, except for a homoeologous exchange in chromosome 21. Our results demonstrate that haplotype-aware assemblies accurately capture the allelic diversity in heterozygous species, making them the preferred option over collapsed-haplotype assemblies.

Atahualpa is a rural village located in coastal Ecuador, a region that has been inhabited by people as early as 10,000 years ago. The traditional diet of their indigenous inhabitants is rich in oily fish and they have, therefore, served as a model for investigating the beneficial effects of such a diet. However, the genetic background of this population has not been studied. In this study, we sequenced the genomes of Atahualpa residents to look for variants under natural selection, which could mediate the effects of oily fish intake. DNA was extracted from 50 blood samples from randomly selected individuals recruited in the Atahualpa Project Cohort. After applying various filters, we calculated genome-wide genotype likelihoods from 33 samples, and combined data from those samples with data from other populations to investigate how the Atahualpa population is genetically related to these populations. Using selection scans, we identified signals of natural selection that may explain the above-mentioned dietary effects. The genetic ancestry in Atahualpa residents is 94.1% of indigenous American origin, but is substantially diverged from other indigenous populations in neighboring countries. Significant signatures of natural selection were found in the Atahualpa population, including a broad selection signal around the SUFU gene, which is a repressor of Hedgehog pathway signaling and associated with lipid metabolism, and another signal in the upstream region of LRP1B which encodes low-density lipoprotein (LDL) receptor related protein 1B. Our selection study reveals genes under selection in the Atahualpa population, which could mediate the beneficial effects of oily fish intake in this population.

Retinopathy with Vitamin E Deficiency (RVED) is a familial disease in the English Cocker Spaniel (ECS) dog breed. Ophthalmic abnormalities observed in RVED-affected ECS include lipofuscin granule deposition within the tapetal fundus and subsequent retinal degeneration resulting in visual deficits. Affected dogs may also exhibit neurological signs that include ataxia and hindlimb proprioceptive deficit. In all cases, circulating plasma concentrations of α-tocopherol are low. This study sought to investigate the genetic basis of RVED in the ECS breed. We undertook a genome-wide association study comprising 30 ECS with normal fundic examinations aged 6 years or older (controls) and 20 diagnosed with RVED (cases) and identified a statistically associated signal on chromosome 29 (Praw = 1.909×10-17). Whole genome sequencing (WGS) of two cases identified a 102bp deletion in exon 1 of the Alpha Tocopherol Transfer Protein gene (TTPA), truncating the protein by 34 amino acids. The c.23_124del variant segregated with RVED in a total of 30 cases and 43 controls. Variants in TTPA are causal for Ataxia with Vitamin E Deficiency (AVED) in humans which is a phenotypically similar disease to RVED. The identification of the canine variant is extremely significant as the availability of a DNA test will allow for identification of presymptomatic dogs and early therapeutic intervention which may prevent development of retinopathy and improve neurological signs. Breeders can also use the DNA test to efficiently eradicate the disease from this breed.

In recent years, black beans (Phaseolus vulgaris L.) have gained popularity in the U.S., with improved seed yield and canning quality being critical traits for new cultivars. Achieving genetic gains in these traits is often challenging due to negative trait associations and the need for specialized equipment and trained sensory panels for evaluation. This study investigates the integration of genomics and phenomics to enhance selection accuracy for these complex traits. We evaluated the prediction accuracy of single and multi-trait genomic prediction (GP) models, incorporating near-infrared spectroscopy (NIRS) data and markers identified through genome-wide association studies (GWAS). The models demonstrated moderate prediction accuracies for yield and canning appearance, and high accuracies for color retention. No significant differences were found between single-trait and multi-trait models within the same breeding cycle. However, across breeding cycles, multi-trait models outperformed single-trait models by up to 45% and 63% for canning appearance and seed yield, respectively. Interestingly, incorporating significant SNP markers identified by GWAS and NIRS data into the models tended to decrease prediction accuracy both within and between breeding cycles. As genotypes from the new breeding cycle were included, the models' prediction accuracy generally increased. Our findings underscore the potential of multi-trait models to enhance the prediction of complex traits such as seed yield and canning quality in dry beans and highlight the importance of continually updating the training dataset for effective GP implementation in dry bean breeding.

Recombination is advantageous over the long-term, as it allows efficient selection and purging deleterious mutations. Nevertheless, recombination suppression has repeatedly evolved in sex and mating-type chromosomes. The evolutionary causes for recombination suppression and the proximal mechanisms preventing crossing overs are poorly understood. Several hypotheses have recently been suggested based on theoretical models, and in particular that divergence could accumulate neutrally around a sex-determining region and reduce recombination rates, a self-reinforcing process that could foster progressive extension of recombination suppression. We used the ascomycete fungus Podospora anserina for investigating these questions: a 0.8 Mbp region around its mating-type locus is non-recombining, despite being collinear between the two mating types. This fungus is mostly selfing, resulting in highly homozygous individuals, except in the non-recombining region around the mating-type locus that displays differentiation between mating types. Here, we test the hypothesis that sequence divergence alone is responsible for recombination cessation. We replaced the mat- idiomorph by the sequence of the mat+ idiomorph, to obtain a strain that is sexually compatible with the mat- reference strain and isogenic to this strain in the MAT-proximal region. Crosses showed that recombination was still suppressed in the MAT-proximal region in the mutant strains, indicating that other proximal mechanisms than inversions or mere sequence divergence are responsible for recombination suppression in this fungus. This finding suggests that selective mechanisms likely acted for suppressing recombination, or the spread of epigenetic marks, as the neutral model based on mere nucleotide divergence does not seem to hold in P. anserina.

Occurrence of degenerative interactions is thought to serve as a mechanism underlying hybrid unfitness in most animal systems. However, the molecular mechanisms underpinning the genetic interaction and how they contribute to overall hybrid incompatibilities are limited to only a handful of examples. A vertebrate model organism, Xiphophorus, is used to study hybrid dysfunction, and it has been shown from this model that diseases, such as melanoma, can occur in certain interspecies hybrids. Melanoma development is due to hybrid inheritance of an oncogene, xmrk, and loss of a co-evolved tumor modifier. It was recently found that adgre5, a G protein-coupled receptor involved in cell adhesion, is a tumor regulator gene in naturally hybridizing Xiphophorus species Xiphophorus birchmanni (X. birchmanni) and Xiphophorus malinche (X. malinche). We hypothesized that 1 of the 2 parental alleles of adgre5 is involved in regulation of cell growth, migration, and melanomagenesis. Accordingly, we assessed the function of adgre5 alleles from each parental species of the melanoma-bearing hybrids using in vitro cell growth and migration assays. In addition, we expressed each adgre5 allele with the xmrk oncogene in transgenic medaka. We found that cells transfected with the X. birchmanni adgre5 exhibited decreased growth and migration compared to those with the X. malinche allele. Moreover, X. birchmanni allele of adgre5 completely inhibited melanoma development in xmrk-transgenic medaka, while X. malinche adgre5 expression did not exhibit melanoma suppressive activity in medaka. These findings provide evidence that adgre5 is a natural melanoma suppressor and provide new insight in melanoma etiology.

Aulacorthum solani is a worldwide agricultural pest aphid capable of feeding on a wide range of host plants. This insect is a vector of plant viruses and causes injury to crops including stunted growth from the loss of phloem. We found that the publicly available genome for A. solani is contaminated with another aphid species, and we produced a new genome using a barcoded isogenic laboratory line. We generated Oxford Nanopore and Illumina reads to assemble a draft genome, and we sequenced RNA to aid in the annotation of our assembly. Our A. solani genome is 671 Mbp containing 7,020 contigs with an N50 length of 196 kb with a BUSCO completeness of 98.6%. Out of the 24,981 genes predicted by E-GAPx, 22,804 were annotated with putative functions based on homology to other aphid species. This genome will provide a useful resource for the community of researchers studying aphids from agricultural and genomic perspectives.

Evidence suggests that increases in ploidy have occurred frequently in the evolutionary history of organisms and can serve adaptive functions to specialized somatic cells in multicellular organisms. However, the sudden multiplication of all chromosome content may present physiological challenges to the cells in which it occurs. Experimental studies have associated increases in ploidy with reduced cell survival and proliferation. To understand the physiological challenges that suddenly increased chromosome content imposes on cells, we used S. cerevisiae to ask how much chromosomal DNA cells may contain and what determines this limit. We generated polyploid cells using 2 distinct methods causing cells to undergo endoreplication and identified the maximum ploidy of these cells, 32-64C. We found that physical determinants that alleviate or exacerbate cell surface stress increase and decrease the limit to ploidy, respectively. We also used these cells to investigate gene expression changes associated with increased ploidy and identified the repression of genes involved in ergosterol biosynthesis. We propose that ploidy is inherently limited by the impacts of growth in size, which accompany whole-genome duplication, to cell surface integrity.

Cnidarians (sea anemones, corals, hydroids, and jellyfish) are a key outgroup for comparisons with bilaterial animals to trace the evolution of genomic complexity and diversity within the animal kingdom, as they separated from most other animals 100s of millions of years ago. Cnidarians have extensive diversity, yet the paucity of genomic resources limits our ability to compare genomic variation between cnidarian clades and species. Here we report the genome for Edwardsia elegans, a sea anemone in the most specious genus of the family Edwardsiidae, a phylogenetically important family of sea anemones that contains the model anemone Nematostella vectensis. The E. elegans genome is 396 Mb in length and predicted to encode approximately 49,000 proteins. We annotated large conservation of macrosynteny between E. elegans and other Edwardsiidae anemones as well as conservation of both microRNAs and ultra conserved noncoding elements previously reported in other cnidarians species. We also highlight microsyntenic variation of clustered developmental genes and ancient gene clusters that vary between species of sea anemones, despite previous research showing conservation between cnidarians and bilaterians. Overall, our analysis of the E. elegans genome highlights the importance of using multiple species to represent a taxonomic group for genomic comparisons, where genomic variation can be missed for large and diverse clades.

Small Leucine-Rich Proteoglycans (SLRPs) are a major family of vertebrate proteoglycans. In bony vertebrates, SLRPs have a variety of functions from structural to signaling and are found in extracellular matrices, notably in skeletal tissues. However, there is little or no data on the diversity, function and expression patterns of SLRPs in cartilaginous fishes, which hinders our understanding of how these genes evolved with the diversification of vertebrates, in particular regarding the early events of whole genome duplications that shaped gnathostome and cyclostome genomes. We used a selection of chromosome-level assemblies of cartilaginous fish and other vertebrate genomes for phylogeny and synteny reconstructions, allowing better resolution and understanding of the evolution of this gene family in vertebrates. Novel SLRP members were uncovered together with specific loss events in different lineages. Our reconstructions support that the canonical SLRPs have originated from different series of tandem duplications that preceded the extant vertebrate last common ancestor, one of them even preceding the extant chordate last common ancestor. They then further expanded with additional tandem and whole-genome duplications during the diversification of extant vertebrates. Finally, we characterized the expression of several SLRP members in the small-spotted catshark Scyliorhinus canicula and from this, inferred conserved and derived SLRP expression in several skeletal and connective tissues in jawed vertebrates.

Genetic studies of Plasmodium parasites increasingly feature relatedness estimates. However, various aspects of malaria parasite relatedness estimation are not fully understood. For example, relatedness estimates based on whole-genome-sequence (WGS) data often exceed those based on sparser data types. Systematic bias in relatedness estimation is well documented in the literature geared towards diploid organisms, but largely unknown within the malaria community. We characterise systematic bias in malaria parasite relatedness estimation using three complementary approaches: theoretically, under a non-ancestral statistical model of pairwise relatedness; numerically, under a simulation model of ancestry; and empirically, using data on parasites sampled from Guyana and Colombia. We show that allele frequency estimates encode, locus-by-locus, relatedness averaged over the set of sampled parasites used to compute them. Plugging sample allele frequencies into models of pairwise relatedness can lead to systematic underestimation. However, systematic underestimation can be viewed as population-relatedness calibration, i.e., a way of generating measures of relative relatedness. Systematic underestimation is unavoidable when relatedness is estimated assuming independence between genetic markers. It is mitigated when relatedness is estimated using WGS data under a hidden Markov model (HMM) that exploits linkage between proximal markers. The extent of mitigation is unknowable when a HMM is fit to sparser data, but downstream analyses that use high relatedness thresholds are relatively robust regardless. In summary, practitioners can either resolve to use relative relatedness estimated under independence, or try to estimate absolute relatedness under a HMM. We propose various tools to help practitioners evaluate their situation on a case-by-case basis.