Additional file 2: TableS1. Summary of 346 apple accessions and resequencing data used in the CNV analysis. Table S2. Summary of CNV events in different accessions. Table S3. Extent of apple genome features impacted by non-overlapping CNVs. Table S4. List of all CNVRs and CN estimated by CNVnator in the apple genomes. Table S5. The genes completely or partially inside (50% overlap) of the identified CNVRs in the apple genome. Table S6. GO Biological Process enrichment for all CNVR genes. Table S7. Population-differentiated CNV-Genes between cultivars and wild relatives. (Here, wild relatives only consist of M.sieversii and M.sylvestris).

Salmonids are an important cultural and ecological resource exhibiting near worldwide distribution between their native and introduced range. Previous research has generated linkage maps and genomic resources for several species as well as genome assemblies for two species. We first leveraged improvements in mapping and genotyping methods to create a dense linkage map for Chinook salmon Oncorhynchus tshawytscha by assembling family data from different sources. We successfully mapped 14,620 SNP loci including 2,336 paralogs in subtelomeric regions. This improved map was then used as a foundation to integrate genomic resources for gene annotation and population genomic analyses. We anchored a total of 286 scaffolds from the Atlantic salmon genome to the linkage map to provide a framework for the placement 11,728 Chinook salmon ESTs. Previously identified thermotolerance QTL were found to co-localize with several candidate genes including HSP70, a gene known to be involved in thermal respo...

HLA frequencies show widespread variation across human populations. Demographic factors as well as selection are thought to have shaped HLA variation across continents. In this study, a worldwide comparison of HLA class I and class II diversity was carried out. Multidimensional scaling techniques were applied to 50 HLA-A and HLA-B (class I) as well as 13 HLA-DRB1 (class II) first-field frequencies in 200 populations from all continents. Our results confirm a strong effect of geography on the distribution of HLA class I allele groups, with principal coordinates analysis closely resembling geographical location of populations, especially those of Africa-Eurasia. Conversely, class II frequencies stratify populations along a continuum of differentiation less clearly correlated to actual geographic location. Double clustering analysis revealed finer intra-continental sub-clusters (e.g., Northern and Western Europe vs. South East Europe, North Africa and Southwest Asia; South and East Africa vs. West Africa), and HLA allele group patterns characteristic of these clusters. Ancient (Austronesian expansion) and more recent (Romani people in Europe) migrations, as well as extreme differentiation (Taiwan indigenous peoples, Native Americans), and interregional gene flow (Sámi, Egyptians) are also reflected by the results. Barrier analysis comparing DST and geographic location identified genetic discontinuities caused by natural barriers or human behavior explaining inter and intra-continental HLA borders for class I and class II. Overall, a progressive reduction in HLA diversity from African to Oceanian and Native American populations is noted. This analysis of HLA frequencies in a unique set of worldwide populations confirms previous findings on the remarkable similarity of class I frequencies to geography, but also shows a more complex development for class II, with implications for both human evolutionary studies and biomedical research.

Additional file 12 : Table S5. LD patterns in Africans and non-Africans for highly differentiated SNPs listed in Table 1.

SequenceDataRaw sequence data and pileupsSNP matrixSNP matrix produced from the .vcf genotype calling file by VCFTOOLSout.012allSNPSNP data (5985 SNPs in total) discovered from both herring populations in vcf 4.1 formatfixed_loci_indexFixed loci (1567 in total) on distinct alleles in the two sample locations. Each element in this file is an index of a SNP in "allSNP.vcf".herring_detailsOffice 2010 excel format table of details concerning the BLAST analysis for the study "High degree of cryptic population differentiation in the Baltic Sea herring Clupea harengus". The table columns include "RAD_id", "Reference", "E-value", "Similarity" and "Sample sequence in FASTA". The calculated values are collected with Blast2GO program. For additional descriptions, see README.txt.

High-quality carnivore genomes from roadkill samples enable species delimitation in aardwolf and bat-eared fox

Rémi Allio¹*, Marie-Ka Tilak¹, Céline Scornavacca¹, Nico L. Avenant², Erwan Corre³, Benoit Nabholz1, and Frédéric Delsuc¹*

Affiliations

¹Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, EPHE, Université de Montpellier, France remi.allio@umontpellier.fr marie-ka.tilak@umontpellier.fr celine.scornavacca@umontpellier.fr benoit.nabholz@umontpellier.fr frederic.delsuc@umontpellier.fr

²National Museum and Centre for Environmental Management, University of the Free State, Bloemfontein, South Africa navenant@nasmus.co.za

³CNRS, Sorbonne Université, FR2424, ABiMS, Station Biologique de Roscoff, 29680 Roscoff, France corre@sb-roscoff.fr

*Correspondence: remi.allio@umontpellier.fr, frederic.delsuc@umontpellier.fr

Running head

Genomics from roadkill samples

Abstract

In a context of ongoing biodiversity erosion, obtaining genomic resources from wildlife is becoming essential for conservation. The thousands of yearly mammalian roadkill could potentially provide a useful source material for genomic surveys. To illustrate the potential of this underexploited resource, we used roadkill samples to sequence reference genomes and study the genomic diversity of the bat-eared fox (Otocyon megalotis) and the aardwolf (Proteles cristata) for which subspecies have been defined based on similar disjunct distributions in Eastern and Southern Africa. By developing an optimized DNA extraction protocol, we successfully obtained long reads using the Oxford Nanopore Technologies (ONT) MinION device. For the first time in mammals, we obtained two reference genomes with high contiguity and gene completeness by combining ONT long reads with Illumina short reads using hybrid assembly. Based on re-sequencing data from few other roakill samples, the comparison of the genetic differentiation between our two pairs of subspecies to that of pairs of well-defined species across Carnivora showed that the two subspecies of aardwolf might warrant species status (P. cristataand P. septentrionalis), whereas the two subspecies of bat-eared fox might not. Moreover, using these data, we conducted demographic analyses that revealed similar trajectories between Eastern and Southern populations of both species, suggesting that their population sizes have been shaped by similar environmental fluctuations. Finally, we obtained a well resolved genome-scale phylogeny for Carnivora with evidence for incomplete lineage sorting among the three main arctoid lineages. Overall, our cost-effective strategy opens the way for large-scale population genomic studies and phylogenomics of mammalian wildlife using roadkill.

Figures & Tables

Figure 1. Disjunct distributions of the aardwolf (Proteles cristata) and the bat-eared fox (Otocyon megalotis) in Eastern and Southern Africa. Within each species, two subspecies have been recognized based on their distributions and morphological differences (Clark, 2005; Koehler and Richardson, 1990).

Figure 2. Representation of the mitochondrial genetic diversity within Carnivora with a) the mitogenomic phylogeny inferred from 142 complete Carnivora mitogenomes including those of the two populations of aardwolf (Proteles cristata) and bat-eared fox (Otocyon megalotis) and b) intraspecific (orange) and the interspecific (red) genetic diversities observed for the two mitochondrial markers COX1 and CYTB.

Figure 3. Comparison of 503 mammalian genome assemblies from 12 taxonomic groups using bean plots of the a) number of scaffolds, and b) scaffold N50 values ranked by median values. Thick black lines show the medians, dashed black lines represent individual data points, and polygons represent the estimated density of the data. Note the log scale of the Y axes. The bat-eared fox (Otocyon megalotis) and aardwolf (Proteles cristata) assemblies produced in this study using SOAPdenovo and MaSuRCA are indicated by asterisks. Bean plots were computed using BoxPlotR (Spitzer et al., 2014).

Figure 4. BUSCO completeness assessment of 67 Carnivora genome assemblies visualized as bar charts representing percentages of complete single-copy (light blue), complete duplicated (dark blue), fragmented (yellow), and missing (red) genes ordered by increasing percentage of total complete genes. The bat-eared fox (Otocyon megalotis) and aardwolf (Proteles cristata) assemblies produced in this study using MaSuRCA and SOAPdenovo are indicated by asterisks.

Figure 5. Genetic differentiation indices obtained from the comparison of intraspecific (orange) and interspecific (red) polymorphisms in four pairs of well-defined Carnivora species and for the subspecies of aardwolf (Proteles cristata) and bat-eared fox (Otocyon megalotis) (grey).

Figure 6. PSMC estimates of the change in effective population size over time for the Eastern (orange) and Southern (blue and purple) populations of a) bat-eared fox and ) aardwolf. mu = mutation rate of 10^-8mutations per site per generation and g = generation time of 2 years. Vertical red lines indicate 20kyrs and 40kyrs.

Figure 7. Phylogenomic tree reconstructed from the nucleotide supermatrix composed of 14,307 single-copy orthologous genes for 52 species of Carnivora plus one outgroup (Manis javanica). The family names in the legend are ordered as in the phylogeny.

Table 1. Summary of sequencing and assembly statistics of the genomes generated in this study.

Additional files

Figure S1: Plot of the quality of Nanopore long reads base-called with either the fastor the high accuracyoption of Guppy v3.1.5. The quality of the base-calling step has a large impact on the final quality of the assemblies by reducing the number of contigs and increasing the N50 value.

Figure S2: Definition of the genetic differentiation index (GDI) based on the F-statistic (FST). The main difference between these two indexes is the use of heterozygous allele states for GDI rather than real polymorphism for the FST. Green = π_within, Orange = π_between, Blue = Population A, Red = Population A+B.

Figure S3: Graphical representation of the results of contamination analyses performed with BlobTools for a) the aardwolf (Proteles cristata) and b) the bat-eared fox (Otocyon megalotis).

Table S1: Pairwise patristic distances estimated for the 142 species based on the phylogenetic tree inferred with the 15 mitochondrial loci (2 rRNAs and 13 protein-coding genes).

Table S2: Results of Bayesian dating for the two nodes leading to the Proteles cristataspp. and theOtocyon megalotisspp.. Divergence time estimates based on UGAM and LN models are reported with associated 95% credibility intervals for each MCMC chain.

Table S3: Sample details and assembly statistics (Number of contigs/scaffolds and associated N50 values) for the 503 mammalian assemblies retrieved from NCBI (https://www.ncbi.nlm.nih.gov/assembly) on August 13th, 2019 with filters: “Exclude derived from surveillance project”, “Exclude anomalous”, “Exclude partial”, and using only the RefSeq assembly for Homo sapiens.

Table S4: Genome completeness assessment of MaSuRCA and SOAPdenovo assemblies obtained for Proteles cristataand Otocyon megalotistogether with the 63 carnivore assemblies available at NCBI and DNAZoo (https://www.dnazoo.org/assemblies) on August 13th, 2019 using Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 with the Mammalia OrthoDB 9 BUSCO gene set.

Table S5: Annotation summary and supermatrix composition statistics of the 53 species used to infer the genome-scale Carnivora phylogeny.

Table S6: Sample details, Illumina sequencing, and assembly statistics of the 10 newly assembled carnivoran mitochondrial genomes.

Table S7: Node calibrations used for the Bayesian dating inferences based on mitogenomic data.

Table S8: Results of contamination analyses performed with BlobTools for the aardwolf (Proteles cristata).

Table S9: Results of contamination analyses performed with BlobTools for the bat-eared fox (Otocyon megalotis).

Table S10: Summary information for the Carnivora genomes available either on Genbank, DNAZoo (https://www.dnazoo.org) and the OrthoMaM database as of February 11th, 2020. The “OMM” column indicates if the genome was available on OMM (yes) or not (no). The “Annotation” column indicates whether the genome was already annotated (yes) or not (no).

Zenodo supplementary files

General_pipeline.sh contains the command lines used for the study

1- Mitogenomics

Barcoding gap analyses: Barcoding gap analysis.zip

FST test results. This table includes results for all loci included in each FST analysis. Loci are reported with positions from human genome build hg19 and are aligned with corresponding loci (hg18) from Trynka et al. 2011. (XLSX)

The ecological theory of adaptive radiation predicts that the evolution of phenotypic diversity within species is generated by divergent natural selection arising from different environments and competition between species. Genetic connectivity among populations is likely also to have an important role in both the origin and maintenance of adaptive genetic diversity. Our goal was to evaluate the potential roles of genetic connectivity and natural selection in the maintenance of adaptive phenotypic differences among morphs of Arctic charr, Salvelinus alpinus, in Iceland. At a large spatial scale, we tested the predictive power of geographic structure and phenotypic variation for patterns of neutral genetic variation among populations throughout Iceland. At a smaller scale, we evaluated the genetic differentiation between two morphs in Lake Thingvallavatn relative to historically explicit, coalescent-based null models of the evolutionary history of these lineages. At the large spatial scale, populations are highly differentiated, but weakly structured, both geographically and with respect to patterns of phenotypic variation. At the intralacustrine scale, we observe modest genetic differentiation between two morphs, but this level of differentiation is nonetheless consistent with strong reproductive isolation throughout the Holocene. Rather than a result of the homogenizing effect of gene flow in a system at migration-drift equilibrium, the modest level of genetic differentiation could equally be a result of slow neutral divergence by drift in large populations. We conclude that contemporary and recent patterns of restricted gene flow have been highly conducive to the evolution and maintenance of adaptive genetic variation in Icelandic Arctic charr.

This dataset contains genetic and landscape data of 32 Primula veris populations in Muhu island in Estonia. The study populations are on two 2x2 km study landscapes. Genetic samples were collected in 2014. Landscape data was extracted from maps dated 2015. Data is divided into node- and link-based data. Node-based data contains genetic diversity data of the P. veris populations. Link-based data contains genetic differentiation between population pairs and landscape data in buffers surrounding a straight line between population pairs. Methods To generate the genetic information, the leaves of Primula veris were collected from study populations and DNA was extracted from the leaves. Extracted DNA was prepared for library using ddRAD (Peterson, Weber, Kay, Fisher, & Hoekstra, 2012) method and sequenced. Genetic data was filtered geoinformatically (see Träger et al. 2021) and population-based genetic diversity indices (unbiased expected and observed heterozygosity, uHe and Ho, respectively) were calculated using GENALEX version 6.503 (Peakall & Smouse, 2005, 2012) and mean nucleotide diversity (π) was calculated using vcftools v0.1.12b (Danecek et al., 2011) within a window of 125 bp over all loci for each population. Inbreeding coefficients (FIS) and genetic differentiation (FST) were calculated using the package `genepop´ (Rousset, 2008) in R version 3.4.2 (R Core Team, 2017). Pairwise mean assignment probability (MAP) was calculated with the package AssignPop (Chen et al., 2018). For calculating MAP, we used assignment tests. We performed assignment tests for which we filtered out loci with low variance (threshold at 0.95) and used Monte-Carlo cross-validation. All loci (100%) were used as training data. The classification method for prediction was linear discriminant analysis. The resulting pairwise probabilities (membership accuracies across all individuals) were directional (e.g. 1 to 2, 2 to 1). We added these pairs together and divided them by two, resulting in one value per population pair (MAP; following van Strien et al., 2014). Study populations were sampled at the scale of 2 2x2 km study landscapes (Koguva, Lepiku) and a 250 m buffer around the 2x2 km landscapes was added, resulting in two 2.5x2.5 km squares. We calculated the proportional amount of landscape elements surrounding the straight line between population pairs in a buffer with a width of 100 m. We only calculated this within one landscape. We transformed the landscape data from vector data to 10x10 m raster data for resistance surface analysis. References:

Chen, K.-Y., Marschall, E. A., Sovic, M. G., Fries, A. C., Gibbs, H. L., & Ludsin, S. A. (2018). assignPOP: An r package for population assignment using genetic, non-genetic, or integrated data in a machine-learning framework. Methods in Ecology and Evolution, 9(2), 439–446. https://doi.org/10.1111/2041-210X.12897 Danecek, P., Auton, A., Abecasis, G., Albers, C. A., Banks, E., DePristo, M. A., Handsaker, R. E., Lunter, G., Marth, G. T., Sherry, S. T., McVean, G., & Durbin, R. (2011). The variant call format and VCFtools. Bioinformatics, 27(15), 2156–2158. https://doi.org/10.1093/bioinformatics/btr330 Peakall, R., & Smouse, P. E. (2005). genalex 6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes, 6(1), 288–295. https://doi.org/10.1111/j.1471-8286.2005.01155.x Peakall, R., & Smouse, P. E. (2012). GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics, 28(19), 2537–2539. https://doi.org/10.1093/bioinformatics/bts460 Rousset, F. (2008). genepop’007: A complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources, 8(1), 103–106. https://doi.org/10.1111/j.1471-8286.2007.01931.x R Core Team. (2017). R: A language and environment for statistical computing. RFoundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. Reinula, I., Träger, S., Järvine, H-T., Kuningas, V-M., Kaldra, M., Aavik, T. (2024). Beware of the impact of land use legacy on genetic connectivity: A case study of the long-lived perennial Primula veris. Biological Conservation, xx. Träger, S., Rellstab, C., Reinula, I., Zemp, N., Helm, A., Holderegger, R., Aavik, T. (2021).Genetic diversity at putatively adaptive but not neutral loci in Primula veris responds to recent habitat change in semi-natural grasslands bioRxiv 2021.05.12.442254; doi: https://doi.org/10.1101/2021.05.12.442254 van Strien, M. J., Keller, D., Holderegger, R., Ghazoul, J., Kienast, F., & Bolliger, J. (2014). Landscape genetics as a tool for conservation planning: Predicting the effects of landscape change on gene flow. Ecological Applications, 24(2), 327–339. https://doi.org/10.1890/13-0442.1

THE ORNATE SPINY LOBSTER P. ORNATUS IN THE SOUTH-EAST ASIAN ARCHIPELAGOOrnatus_Sampling_Figure_01_add_ScaleBar_addSeas copy.tifTable S1Spatial distribution of control region haplotypes among Panulirus ornatus from six localities in the South-East Asian archipelago.Table S2Null allele frequencies in original dataset and after correction calculated by using FreeNA 3.0. Values above 10% are in bold.Table S3Summary table of analysis of molecular variance (AMOVA) describing the partitioning of genetic variation for six Panulirus ornatus populations in original dataset and after correction based on 10 microsatellite loci.Table S4Genetic differentiation between Panulirus ornatus from collection locations using pairwise FST for microsatellite loci in original dataset (lower value) and after correction (upper value). No significant value was found after correction using FDR.Table S5Migration rate (and standard errors) in each population that are migrants derived from other populations per gene...

Spreadsheet: The first sheet of the spreadsheet contains notes on (1) the formatting of the entries and (2) how we coded missing values.

Table 1: Microsatellites loci targeted to determine levels of genetic differentiation and population structure of Bull Trout in the Athabasca River basin.

Table 2: Expected heterozygosity, allelic richness and total private alleles at each sampling site.

Targeted gene flow (TGF) could bolster the adaptive potential of isolated populations threatened by climate change, but could also lead to outbreeding depression. Here, we explore these possibilities by creating mixed- and within-population crosses in a terrestrial-breeding frog species threatened by a drying climate. We reared embryos on wet and dry soils and quantified fitness-related traits upon hatching. TGF produced mixed outcomes in hybrids which depended on crossing direction (origin of gametes from each sex). North-south crosses led to low embryonic survival if eggs were of a southern origin, and high malformation rates when eggs were from a northern population. Conversely, east-west crosses led to one instance of hybrid vigour, evident by increased fitness and desiccation tolerance of hybrid offspring relative to offspring produced from within-population crosses. These contrasting results highlight the need to experimentally evaluate the outcomes of TGF for focal species across generations prior to implementing management actions.,We explored the potential of targeted gene flow (TGF) to mitigate declines in population-level fitness using the crawling frog, Pseudophryne guentheri, a species in which populations are threatened by habitat loss and declining winter rainfall. We evaluated TGF within a laboratory setting by creating pure and reciprocal crosses among four geographically distant populations. Two populations occurred in low-rainfall regions at the northern edge of the species’ range, and two other populations were from higher-rainfall regions close to the centre the species range. We then assessed phenotypic traits in the resulting offspring, comparing individuals reared on wet soils (-10 kPa, a benign treatment) to those reared on drier soils (-400 kPa) that significantly reduce survival and hatchling fitness. Detailed methods from manuscript: We collected adult P. guentheri from four geographically separated breeding sites, situated at two latitudes, in May and June 2017 (Table 1). Sites spanned a ~460 mm annual rainfall gradient, with site A receiving the most rain per year and site D receiving the least (Table 1, Fig. 1). Pseudophryne guentheri collected from breeding populations at each site show variation in desiccation tolerance, with adults and embryos from site A being the most sensitive to dry conditions37. Population genetic analysis39 has demonstrated high levels of inbreeding in all populations (Table 1), and genetic differentiation among P. guentheri populations is high (overall FST = 0.186), which suggests low levels of contemporary dispersal. P. guentheri from sites A and B form distinct genetic clusters39, indicating low historical gene flow despite their close proximity (100 km), whereas P. guentheri from sites C and D show admixture, but are genetically distinct from populations A and B39. In total, 15-16 calling males from each population were collected by hand and in pit-fall traps. Gravid females were more difficult to collect due to their cryptic behaviours, and so sampling was restricted to 5-13 females from each of three sites (A, B and C; Table 1). All frogs were temporarily housed in small (4.4 L) plastic terraria containing moist sphagnum moss, and transported to the University of Western Australia within two days of collection. There, frogs were fed a diet of pinhead crickets and kept in a controlled-temperature room at 16 °C with an 11/13 h light/dark photoperiod to mimic winter conditions. Breeding design and in vitro fertilisations. Egg clutches of each female were divided equally into four groups, and fertilised with sperm from males originating from each of the four populations, resulting in one pure and three hybrid crosses. To control for potential parental compatibility (i.e. specific pairwise male-by-female) effects on offspring fitness38,69, a sperm mixture, containing sperm from five random males from the appropriate population, was used to fertilise the eggs of each female in each population88. Sperm was obtained from testes macerates after euthanizing males via ventral immersion in <0.03% benzocaine solution, followed by double pithing. Sperm was stored on ice in 25-458 μL (adjusted according to the weight of the testes) standard amphibian ringer (SAR; 113mM NaCl, 2mM KCl, 1.35 mM CaCl2). This buffer allows storage of sperm for extended periods (days–weeks) without substantial declines in motility89,90. Sperm concentrations were measured using an improved Neubauer haemocytometer (Hirschmann Laborgeräte, Eberstadt, Germany) and sperm suspensions were diluted with 1:1 SAR to 100 sperm per μL. Upon arrival at the laboratory, females were gently squeezed to determine whether ovulation had occurred. Approximately 35% of females had ovulated naturally while in transit and their eggs were gently stripped. For the remaining females, ovulation was induced via two subcutaneous injections of the hormone LHRHa over the course of two days37,87. Approximately 10 hours after the second injection, eggs were gently stripped from each female. In all instances, freshly stripped eggs were moistened with SAR and distributed equally among four small petri dishes. A standardised number of sperm from five random males collected at each site was pipetted onto one edge of each petri dish, mixed gently with the pipette tip, and then activated with a pre-calculated volume of 1:4 SAR solution38. This resulted in eggs from all females in the experiment being fertilised from males collected from sites A, B, C and D. Each dish was then manually agitated for 20 seconds to promote fertilisation. After 15 minutes, eggs were temporarily submerged in water, backlit and photographed using a digital imaging camera (Leica DFC320) attached to a light microscope (Leica MZ7.5) at 6.3 Χ magnification. These images were used to measure the ovum diameter of 50 randomly-selected eggs from each female, using ImageJ software91. Fertilisation success was initially scored one hour after mixing eggs and sperm by counting eggs that had rotated (Gosner79 stage 1). However, eggs from populations A and B took substantially longer to show signs of fertilisation when mixed with sperm from populations C and D. We therefore scored fertilisation success a second time, six hours after sperm and eggs were mixed. Incubation treatments. Fertilised eggs from each cross were reared on sandy loam soil at two water potentials (ψ): a wet soil (ψ = -10 kPa) and dry soil (ψ = -400 kPa). The soil was previously collected from a separate P. guentheri breeding site, and the soil water potentials represented a range found in natural nest sites (N. J. Mitchell, unpubl. data). Embryo incubation and soil preparation were performed as described in Rudin-Bitterli et al.92. Briefly, soil was oven-dried at 80°C for 24 hours, distributed into small containers and rewetted with an appropriate mass of deionised water using a water-retention curve previously determined for the soil sample (N. J. Mitchell, unpubl. data). The water content of the soil (g/g/ of oven dry soil) was approximately 50% in the wet treatment, and 21% in the dry treatment, and containers were sealed with a lid after wetting. Fertilised eggs from each cross were selected at random and distributed onto soils within 7 – 9 hours of fertilisation. Small plastic rings (nylon plumbing olives, 12 mm in diameter) were labelled and placed around eggs to identify individual crosses. Sealed containers were then placed in incubators set at 16 ± 0.5 °C, and embryos were monitored every two days. Any dead eggs were removed and discarded. Response variables. Putative fitness from within- and between-population crosses, reared in dry and wet rearing environments, was assessed at hatching. At 33 days after fertilisation (when embryos were approximately at Gosner79 Stage 26), hatching was induced by placing embryos individually in small test tubes containing 2 ml of deionised water38. Embryos were then monitored at least every 30 min until hatching, defined as when an individual completely escaped their egg capsule. Embryonic survival was recorded for each family as the percentage of fertilised eggs that hatched. Swimming performance was recorded 6 - 12 hours after hatching on a subset of hatchlings (N = 633 across all within and between-population crosses). For this purpose, individual hatchlings were placed in a petri dish (diameter = 150 mm) containing water 10 mm deep. After an initial acclimation period of 1 min, the tail of each hatchling was nudged with a glass cannula to elicit a burst swimming response. A video camera (Canon PowerShot G16, recording at 60 fps) installed 300 mm above the petri dish was used to film three burst swimming responses for each hatchling, and their movement was later tracked and analysed using EthoVision v8.5 software93. EthoVision enabled the quantification of the following swimming parameters: maximum velocity (cm s-1), mean velocity (cm s-1) and total distance moved (cm). We also recorded mean meander (deg cm-1), a measure of the straightness of the swimming response, as dry rearing environments can lead to asymmetrically shaped hatchlings37,38 that swim in a more circular motion. A hatchling was considered to be moving when it exceeded 0.45 cm s-1. As each video recording contained three burst swimming responses with periods of no movement in between them, EthoVision only analysed frames in which a hatchling moved faster than 0.45 cm s-1 (consequently merging the three swimming responses for each hatchling). Immediately following the swimming performance trials, hatchlings were euthanized in <0.03% benzocaine and preserved in 10% neutral buffered formalin. Wet masses of preserved hatchlings were recorded to the nearest 0.001 g after blotting on tissue. Hatchlings were then photographed in lateral view (while submerged in water to minimize refraction) using a digital imaging camera (Leica DFC320) attached to a light microscope

Reduced representation (RRL) sequencing approaches (e.g., RADSeq, genotyping by sequencing) require decisions about how much to invest in genome coverage and sequencing depth, as well as choices of values for adjustable bioinformatics parameters. To empirically explore the importance of these “simple” methodological decisions, we generated two independent sequencing libraries for the same 142 individual lake whitefish (Coregonus clupeaformis) using a nextRAD RRL approach: (1) a larger number of loci at low sequencing depth based on a 9mer (library A); and (2) fewer loci at higher sequencing depth based on a 10mer (library B). The fish were selected from populations with different levels of expected genetic subdivision. Each library was analyzed using the STACKS pipeline followed by three types of population structure assessment (FST, DAPC and ADMIXTURE) with iterative increases in the stringency of sequencing depth and missing data requirements, as well as more specific a priori population maps. Library B was always able to resolve strong population differentiation in all three types of assessment regardless of the selected parameters, largely due to retention of more loci in analyses. In contrast, library A produced more variable results; increasing the minimum sequencing depth threshold (-m) resulted in a reduced number of retained loci, and therefore lost resolution at high -m values for FST and ADMIXTURE, but not DAPC. When detecting fine population differentiation, the population map influenced the number of loci and missing data, which generated artefacts in all downstream analyses tested. Similarly, when examining fine scale population subdivision, library B was robust to changing parameters but library A lost resolution depending on the parameter set. We used library B to examine actual subdivision in our study populations. All three types of analysis found complete subdivision among populations in Lake Huron, ON and Dore Lake, SK, Canada using 10,640 SNP loci. Weak population subdivision was detected in Lake Huron with fish from sites in the north-west, Search Bay, North Point and Hammond Bay, showing slight differentiation. Overall, we show that apparently simple decisions about library construction and bioinformatics parameters can have important impacts on the interpretation of population subdivision. Although potentially more costly on a per-locus basis, early investment in striking a balance between the number of loci and sequencing effort is well worth the reduced genomic coverage for population genetics studies. More conservative stringency settings on STACKS parameters lead to a final dataset that was more consistent and robust when examining both weak and strong population differentiation. Overall, we recommend that researchers approach “simple” methodological decisions with caution, especially when working on non-model species for the first time.

The Pairwise Population Differentiation dataset consists of 106 columns, described in Table 2 of the original publication, comprised of data from 199 studies on 193 species and 14,703 pairwise population comparisons, with the latter equaling the number of rows. The first 36 columns summarize taxonomic, population, marker type, and pairwise population comparison data, while the remaining 70 columns contain data on animal and plant life history (Dataset 3 and Dataset 4, respectively). CSV format.

Between-population distance (dXY) and differentiation (FST) for pairwise population contrasts.

Categories and statistics for 84 SNPs. SNPs that did not meet the criterion for BLAST e- values are left blank. For substitution type, Syn is a putatively synonymous substitution and NS is a putatively nonsynonymous substitution. – designates when an allele is fixed. SNPs that have a He (heterozygosity) over 0.35 generally dropped out all genotyping scores when the call stringency was increased. * is p

Pufferfish from the genus Takifugu are vital commercial resources in East Asia. Within the genus, the taxonomic status of two commercially important species, T. rubripes and T. chinensis, remains ambiguous, especially given their morphological variability. Recent observations of suspected hybrids between T. rubripes and T. chinensis on Jeju Island, South Korea, displaying intermediate phenotypes, have further confused their classification. In this study, we analyzed 73 pufferfish, including wild-caught T. rubripes, T. chinensis, suspected hybrids, and farm-bred T. rubripes, using 16 microsatellite loci to explore their population structure and evolutionary relationships. The Bayesian clustering and principal coordinate analysis showed minimal genetic differentiation among the wild populations, regardless of phenotype. This finding suggests that T. rubripes and T. chinensis might represent a single species with considerable morphological diversity. In contrast, farm-bred T. rubripes exhibited significant genetic differentiation from wild populations, likely due to domestication-induced genetic drift. These results challenge the existing taxonomic distinctions between T. rubripes and T. chinensis and highlight the profound impact of aquaculture on the genetics of captive populations. This study underscores the necessity for ongoing research into the taxonomy and population genetics of the T. rubripes-chinensis complex to guide conservation and management strategies and stresses the importance of genetic monitoring in pufferfish aquaculture to counteract inbreeding and genetic drift.

Geographic origins, sample sizes, haplotypes and their frequencies of the 35

     Meconopsis integrifolia

     populations studied.
    (DOC)

Distributions of

     Exyra semicrocea

     haplotypes by sampling localities/populations across the southeastern United States Coastal Plain.
    (DOC)

For abbreviations of population names see Table 1.Pairwise population differentiation (G”ST) of S. granulata along the Dijle and Demer river systems, corrected for unknown dosage of alleles.