Oreochromis niloticus) is among the most farmed finfish worldwide, distributed across different environmental conditions. Its wide distribution has mainly been facilitated by several breeding programs and widespread dissemination of genetically improved strains. In the first Nile tilapia study exploiting a whole‐genome pooled sequencing (Poolseq) approach, we identified the genetic structure and signatures of selection in diverse, farmed Nile tilapia populations, with a particular focus on the GIFT strain, developed in the 1980s, and currently managed by WorldFish (GIFTw). We also investigated important farmed strains from The Philippines and Africa. Using both SNP array data and Poolseq SNPs, we characterized the population structure of these samples. We observed the greatest separation between the Asian and African populations and greater admixture in the Asian populations than in the African ones. We also established that the SNP array data were able to successfully resolve relationships between these diverse Nile tilapia populations. The Poolseq data identified genomic regions with high levels of differentiation (FST) between GIFTw and the other populations. Gene ontology terms associated with mesoderm development were significantly enriched in the genes located in these regions. A region on chromosome Oni06 was genetically differentiated in pairwise comparisons between GIFTw and all other populations. This region contains genes associated with muscle‐related traits and overlaps with a previously published QTL for fillet yield, suggesting that these traits may have been direct targets for selection on GIFT. A nearby region was also identified using XP‐EHH to detect genomic differentiation using the SNP array data. Genomic regions with high or extended homozygosity within each population were also identified. This study provides putative genomic landmarks associated with the recent domestication process in several Nile tilapia populations, which could help to inform their genetic management and improvement.Nile tilapia ( For example, a fast‐growing strain called With the rapid development of genomic tools in farmed animal species, many studies over recent years have identified genomic regions showing evidence of selection, primarily focused on terrestrial livestock. These selection “signatures” have been associated with domestication and breed development, as well as production traits and environmental adaptation , (ii) a Z‐transformed heterozygosity score (ZHp) and (iii) two haplotype‐based homozygosity analyses: iHS and XP‐EHH. We then explored the biological functions of candidate genes discovered in the outlier regions and assessed their potential in the domestication process of these Nile tilapia populations.Genetic studies in Nile tilapia are aided by the rapid development of genomic tools for this species  from seven populations were analyzed in this study, focused on major strains from Asia and Africa, which dominate worldwide production. The first sample comprised 100 fish from generation 15 of the GIFT strain from the breeding nucleus managed by WorldFish  and located in the Aquaculture Extension Center in Jitra, Malaysia. These individuals were previously sampled for a SNP discovery study to develop a SNP array , two GIFT‐derived populations: (ii) Genetically Enhanced Tilapia Excellent  that has been selected for growth for more than 10 years. The geographical location and genetic background of the studied populations are shown in Figure Each pool of the six remaining populations were composed of 50 individuals. These included four populations from The Philippines with different genetic background: (i) a GIFT population originally from the GIFT breeding program developed in the late 1980s . Raw sequencing data from the DNA pools were quality filtered and adapters removed using the fastp v0.20.0 software  were merged into a single dataset, hereafter called GIFTd. Variants were called jointly across the six population pools with the software Freebayes v1.0.2 . In addition, fish with a genotyping call rate below 95% were also filtered, leading to the exclusion of one fish from BEST. Also, one pair of samples from each of the Abbassa and GIFTw populations were excluded as they shared an IBD > 99%. Thus, the total data set comprised 100 fish and 16,938 SNPs that were shared across populations.In order to assess the utility of the open access high‐density Thermofisher Axiom 65 K SNP array ) were calculated for each population using the ‐‐het function in Plink v1.90  constructed based on the allele frequencies across populations, using the core model from Baypass v2.2  was performed. This was done independently for the Poolseq and the SNP array datasets. For the former, population structure was assessed through a scaled covariance matrix  values ranging from 1 to 15 were assessed, with 10 replicates per K value. The likelihoods and average ΔK for each K value were estimated through the command‐based program STRUCTURE HARVESTER , using the computeprob and inferalpha functions in Structure. We observed the log‐likelihood fluctuating stochastically around a relatively constant value, indicating that the model converged for all K values during the burn‐in period.Additionally, population structure was assessed based on this latter dataset, through a hierarchical Bayesian analysis implemented in the STRUCTURE software  and used these to calculate the pH statistic in 625‐SNP windows with 312‐SNP overlap, for consistency with the Poolfstat analyses. For each window we calculated∑nMAJand∑nMIN are the sums of nMAJ and nMIN across the window. We then Z‐transformed the Hp values across all windows, that is, ZHp=Hp−μHpσHp, where μHp and σHp are the mean and standard deviation, respectively, of the Hp distribution across all windows. We defined outlier windows as those comprising the bottom 0.1% of the distribution of ZHp (i.e. lowest heterozygosity), and again defined the central position for each of these windows as the “top position.”We used the n et al. , 2012 ton et al.  for Pool2.7meanunXP−EHH and SDunXP−EHH represent the average and standard deviation of the unstandardized (un) unXP‐EHH(s) = lniESpop1siESpop2s, and iES represents the integral over the EHH curve.We applied two further approaches to capture genomic regions under recent selection, based on the SNP array data. By using the R package rehh v.3.2.2  was ≥4.For both analyses, we defined a marker as an outlier if the –log or from the flanking markers of the region (where a region comprised multiple markers). The selection of 50 kb was based on the known distribution of gene lengths in the O. niloticus genome, shown to be <50 kb for over 90% of genes  and the middle of the outlier window  was required to be no greater than 50 kbp.We also looked for regions in common between the Poolseq and haplotype‐based SNP array analyses, specifically between 33.1GIFTw biological replicates and each of the two technical replicates for Abbassa, BEST, FAST, GETExcel, GIFTp, and Kenya , the average coverage within populations ranged from 92 to 104 M reads. A high positive correlation of the allele frequencies for the filtered bi‐allelic SNPs (0.95–0.97) was estimated between the technical replicates within each population, allowing us to merge the datasets and improve the total depth coverage without compromising genotyping accuracy  and Asian (FAST and GIFT) populations as well as the FAST strain from the GIFT strains, while the second principal component further separates strains within Africa and Asia. The GIFT populations have similar PC1 values, while the position of the GIFTp population on the PC2 axis is intermediate between GIFTd and GIFTw.To characterize the genetic structure among the Nile tilapia populations, we used the 1.7 M SNPs that segregated within each of the assessed populations and also surpassed the MAF >0.4 quality control threshold. The first two principal components explained nearly 66% of the genetic structure, with 38.12% and 27.79% for the first and second components, respectively Figure . The firK was maximized for K = 2, suggesting that two clusters best described the data. The first separation (K = 2) was seen between the dominant cluster in Kenya  and the other populations, followed by separation of both African populations and FAST from the GIFT and GIFT‐derived populations (K = 4). Greater values of K  revealed further structure within the populations. For K = 5, GETExcel showed the greatest level of admixture while Abbassa, GIFTw and FAST showed the least, with the highest ancestry fraction to the majority cluster  seen for these populations. GIFTp showed greater admixture than GIFTw, with most GIFTw individuals showing ancestry fractions >85% to the majority (green) cluster. This cluster was also represented in GIFTp individuals, but at a lower level. The PCA and Structure results indicated that three individuals from the Kenya population cluster separately from the rest of that population. These individuals show a very distinct ancestry pattern in the Structure analysis and thus it does not appear that they were mislabeled or swapped with other samples.By using the 17 K SNPs identified by the SNP array, a similar genetic structure to that obtained with Poolseq data was observed Figure . As expeKenya, all populations showed an slight excess of heterozygosity as compared with expected levels, indicated by the inbreeding coefficient (F) ranging from −0.046 to −0.008 . In accordance with the PCA results, highest genetic differentiation was observed between the Asian non‐GIFT population (FAST) and the African populations .All genome‐wide pairwise ns Table , with FFST patterns across the genome revealed peaks in differentiation between pairs of Nile tilapia populations, including comparisons between GIFTw and the other strains , the s Figure –S4. Genoed Table . Consists Figure , with ouy Figure .FST regions were found in common across multiple comparisons with the GIFTw strain  found as an outlier in all comparisons except those involving FAST and another ~0.5 Mbp region  and Oni11 (36.53–36.71 Mbp) were found only for comparisons between GIFTw and both GIFTp and GIFTd. Additional regions showing differentiation between GIFTw and other strains were identified on Oni17 and Oni19.Several outlier n Figure ; Table 3GIFTw and the other strains, we also noted a region on Oni12 (21.33–21.69 Mbp) showing differentiation between FAST and other strains .Although our primary focus was on differentiation between s Figure –S4. TherGIFTw and the other populations  identified outlier SNPs in a 1 Mb region on Oni12 (17.16–18.04 Mbp), with the most extreme statistics seen in the comparisons with GIFTd and GIFTp. A similar pattern was observed on Oni06, although the outlier markers were located within a larger region (18.57–34.46 Mbp), which was considerably reduced (to 18.57–26.34 Mbp) when the comparison with Abbassa was excluded. It is also worth noting that when GIFTw was compared with FAST, a single outlier marker was found on Oni10 (16.19 Mbp), which was not seen for any of the other comparisons.The cross‐population statistic XP‐EHH analysis of the SNP array data highlighted several genomic regions showing differentiation between s Figure . For insGIFTp with other populations. For instance, a 1.5 Mb window was observed on Oni19 (19.91–21.48 Mbp) in comparisons with Abbassa, Kenya and GIFTd  between the XP‐EHH (SNP array) and FST (Poolseq) analyses. One was on Oni06, when comparing with GIFTd, where the outlier markers were located at 24.42 and 24.39 Mbp for FST and XP‐EHH, respectively. The other was on Oni12, when comparing with Abbassa, where the outlier markers were located at 17.39 and 17.35 Mbp for FST and XP‐EHH, respectively. No common regions were found among the remaining comparisons.When considering the 3.4Hp values across windows ranged from 0.21 (SD = 0.08) for Kenya to 0.28 (SD = 0.06) for GIFTd. The genomic regions that have been under positive artificial or natural selection are likely to show reduced heterozygosity. Thus, regions of excess homozygosity across the genome were identified based on the ZHp metric . A number of these regions overlapped with outlier regions identified for the other Asian populations , Oni15 (Kenya and GIFTd), Oni16 (Abbassa and GIFTp), and Oni22 .The genome‐wide average ns Table . In addiGIFTw, there was no overlap between the high differentiation regions and those showing reduced heterozygosity (low ZHp). For the other populations, there were several overlaps. These include regions on Oni04 (~35 Mbp) for GIFTd; Oni15 (~28 Mbp) for GIFTd and Kenya; Oni16  for Abbassa and GIFTp; Oni18 (~3 Mbp) for Kenya and Oni22 (~34–38 Mbp) for GIFTp, Abbassa and Kenya.For Kenya population, for which 17 chromosomes showed at least one outlier region. This high number might be explained due to the presence of individuals that clustered away from the rest of the population, as seen in the Structure and PCA analyses. This pattern of numerous outlier regions was not observed for the remaining populations. For instance, most of the outlier regions were located on Oni02, Oni07, and Oni20 for both the Abbassa and FAST populations. Regarding the GIFTd population, a total of 11 chromosomes had regions showing signals of selection, whereas for the other GIFT strains, fewer but more distinct peaks were found. These were located on Oni10 and Oni19 for GIFTp and on Oni03 and Oni07 for GIFTw. None of these regions were classified as common with those identified through ZHp for the Poolseq data.Several selection signatures were identified through the iHS analysis of the SNP array data Figure . The lar3.5GIFTw, we identified loci based on the Poolseq analyses, that is, those underlying the regions of high differentiation between GIFTw and the other populations (FST) and those with high levels of homozygosity (ZHp) in GIFTw; due to limitations of the Nile tilapia reference genome, some could not be associated with annotated genes. Regarding the FST analysis, a total of 67 loci (43 located on Oni06) were found in regions showing differentiation between GIFTw and at least two populations  data to investigate relationships between commercial Nile tilapia populations and used the Poolseq data to identify regions of the genome showing evidence of selection, based on population differentiation and low diversity measures. The latter analyses had the aim of characterizing the impact of the complex selection process to which the GIFT strain has been subjected.4.1FST and Structure analyses showed similar patterns: the greatest divergence was between the African and Asian Nile tilapia populations. In line with previous analyses of low‐density genomic data  but the Asian populations were generally more admixed than the two African ones (Abbassa and Kenya). This complex pattern in the Asian populations is consistent with results from the study of Hamilton and colleagues  cluster at K = 5, showing substantially greater representation than the other clusters , suggesting the possibility of multiple targets of selection. Nearby markers were also detected by XP‐EHH (using SNP array data).The strongest signatures of genomic differentiation seen in the Poolseq analysis of GIFTw and all other strains, providing particularly strong evidence for selection. Of the 16 genes in this region, we note that NFIX has been associated with primary slow muscle fiber formation in zebrafish , showing differentiation with GIFTd, FAST and Kenya strains, also includes genes of interest. Of particular note, LUC7L2 has been identified as differentially expressed in comparisons of early and late muscle fiber growth in zebrafish , was also previously detected as being highly differentiated in the above‐mentioned Cadiz et al.  showed differentiation between the FAST population and other populations, suggesting the possibility of selection related to the development of the FAST strain. One of the three annotated genes in this region, CCSER1, has been identified as associated with growth and feed efficiency in several studies of terrestrial livestock , close to the highly differentiated region in GIFTd mentioned above , and the other on Oni16 (32.98–33.08). The former region includes genes of note, including UTP18, which has been associated with compensatory muscle growth and flesh quality recovery after spawning in rainbow trout  very stringent procedures for defining common signals and differences in marker density (high density Poolseq versus low‐density SNP array), which may have limited the precise overlap of identified regions, and (ii) differences in the type of signatures and selection pressures that can be captured by different methods  shows less evidence of admixture than the strain from the Philippines (GIFTp) from which it was derived, suggesting founder effects or that specific elements of its genetic ancestry have been promoted due to strong selection. This study also revealed a number of signatures of diversifying selection in the GIFT genome, with enrichment for genes related to mesoderm development. These genomic signatures are particularly evident on Oni06, which has been previously identified as containing selection signatures and QTL for growth traits and also contains several genes known to be associated with muscle‐related traits. Finally, this study demonstrates the utility of Poolseq, a cost‐effective approach to whole‐genome sequencing, for uncovering signatures of selection.This study is the first pooled whole‐genome sequencing analysis of farmed Nile tilapia strains. The characterization of Asian and African strains demonstrated their complex ancestries, particularly for the Asian populations. We also showed that the WorldFish GIFT strain (The authors declare that they have no competing interests.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Figure S7Click here for additional data file.Figure S8Click here for additional data file.Figure S9Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.


Hexaglandula corynosoma is a specialist species that uses a single bird species as a definitive host. Southwellina hispida is a generalist species that uses a broad spectrum of definitive hosts to complete its life cycle. In the current research, sequences of cytochrome c oxidase subunit 1 (cox1) from mitochondrial DNA were generated from 44 specimens of H. corynosoma and 76 of S. hispida distributed sympatrically in 6 biogeographic provinces of Mexico with the objective of characterizing and comparing the population genetic structure of 2 acanthocephalan species with opposing life strategies. The phylogeographic studies indicated that the populations of both species lacked a phylogeographic structure and exhibited high haplotype diversity, low nucleotide diversity and low Fst values among the biogeographic provinces; in combination with negative values on the neutrality test, this suggests that the populations of acanthocephalans are expanding. Paratenic hosts are key for the transmission from intermediate to definitive hosts in the generalist species. However, the inclusion of paratenic hosts does not play a principal role in the population genetic structure of S. hispida within its distribution along the coasts of Mexico.Polymorphidae is a monophyletic group of acanthocephalans distributed worldwide. Within this family,  The acanthocephalans were identified by conventional morphological criteria following Petrochenko . In addiH. corynosoma and 56 as S. hispida were placed individually in tubes and digested overnight at 56°C in a solution containing 10 mm Tris–HCl (pH 7.6), 20 mm NaCl, 100 mm Na2-EDTA (pH 8.0), 1% Sarkosyl and 0.1 mg mL−1 proteinase K. Following digestion, DNA was extracted from the supernatant using the DNAzol reagent  according to the manufacturer's instructions. The cox1 of the mitochondrial DNA was amplified using the forward primer 5′-AGTTCTAATCATAA(R)GATAT(Y)GG-3′ and reverse primer 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′  consisted of 10 μL of each primer, 2.5 μL of 10× buffer, 2 mm MgCl2 and 1 U of Taq DNA polymerase . PCR cycling parameters for the molecular marker consisted of denaturation at 94°C for 1 min, 35 cycles of 94°C for 1 min, 40°C for 1 min and 72°C for 1 min, followed by a post-amplification incubation at 72°C for 10 min. Sequencing reactions were performed using ABI Big Dye  terminator sequencing chemistry, and reaction products were separated and detected using an ABI 3730 capillary DNA sequencer. Contigs were assembled and base-calling differences resolved using Codoncode Aligner version 9.0.1  and submitted to the GenBank dataset  downloaded from GenBank , downloaded from GenBank . Intrapopulation variation was summarized using standard statistics: number of haplotypes (H), number of segregating sites (S), haplotype diversity (Hd), nucleotide diversity (Pi) and average number of nucleotide differences (K), were all calculated using the program DnaSP v.5.10  in coastal Mexico show morphological characteristics that match those assigned to H. corynosoma by Nickol et al. , and 5 others were shared haplotypes . The most frequent haplotype  corresponded to specimens from 4 biogeographic provinces . Most of the identified haplotypes were separated from one another by 1, 2, 3 or up to 5 substitutions (see Hd = 0.970) and nucleotide diversity was low (pi = 0.00555) among the populations from the 4 biogeographic provinces sampled  of Mexico. Neutrality tests  were negative for all regions . Despite the large geographic distances, the Fst values were low, ranging from −0.007 to 0.058 , and only 1 was shared  between 2 biogeographic provinces (Veracruzan and the Yucatán Peninsula). Most of the identified haplotypes were separated by several substitutions (see Hd = 0.993) was very high and nucleotide diversity was low (pi = 0.01166) among the populations from the 3 biogeographic provinces sampled . Neutrality tests  were negative for all regions . Despite the large geographic distances, the Fst values were low, ranging from −0.019 to 0.059  by Travassos in H. corynosoma has been recorded in Puerto Rico, the USA and Mexico in the same definitive host species , spotted shag Phalacrocorax punctatus (Sparrman) and Otago little blue penguin, Eudyptula novaehollandiae (Forster) from New Zealand, which ranged from 0.00 to 0.32%  Aznar et al.  in central Mexico, which ranged from 0.0 to 3.0%  Travassos, 1926, which ranged from 0.00 to 1.6% , Stewart Island shag , spotted shags (P. punctatus Sparrman), yellow-eyed penguins (Megadyptes antipodes Hombron and Jacquinot), New Zealand brill (Colistium guntheri Hutton) and New Zealand sole (Peltorhamphus novaezeelandiae Gunther), which ranged from 0.00 to 2.8%  recovered from the California sea lion , South American sea lions (Otaria flavescens Shaw), South American fur seals , Magellanic penguins (Spheniscus magellanicus Forster) and cystacanths recovered from marine fishes in Argentina, which ranged from 1.0 to 1.7%  and H. corynosoma  did not show a phylogeographic structure; therefore, the haplotypes could not be grouped into their own geographic clusters. In fact, the specimens of S. hispida were not correlated with their definitive host family  resemble the specialist model. To date, only a few phylogeographic studies of generalist acanthocephalans have been conducted using cox1 as a molecular marker. For example, Profilicollis altmani , which has a broad distribution across North and South America and parasitizes multiple species of intermediate hosts and diverse species of marine birds that act as definitive hosts , showed a lack of population genetic structure with high haplotype diversity and low nucleotide diversity, suggesting that P. altmani have experienced a period of rapid population growth in the past  and oystercatchers (Haematopus spp.) and as cystacanths in the shore crab Hemigrapsus crenulatus (Milne-Edwards) distributed along the east coast of New Zealand's South Island  showed similar population genetics patterns. This pattern was not consistent with the specialist–generalist variation hypothesis (SGVH), which predicts that: (i) populations of specialists may be less connected and more subdivided into smaller populations than generalists; (ii) specialists are expected to have lower effective population sizes than generalists and to be composed of populations with less gene flow and (iii) generalists will show high effective population sizes, high genetic flow and highly structured populations  than those of ardeid hosts , suggesting that these piscivorous birds may act as suboptimal definitive hosts.The 2 species of acanthocephalans analysed here are considered to be typical components of the helminth fauna of piscivorous birds in the Americas. Of the 19 piscivorous birds recorded as definitive hosts, 11 belong to the family Ardeidae, which represent 58% of the host diversity, suggesting that the ardeids could be the ancestral hosts of S. hispida and H. corynosoma showed similar patterns. However, these species use different strategies to complete their life cycles. The main ecological difference between S. hispida and H. corynosoma is the inclusion of paratenic hosts in S. hispida  (García-Prieto et al., S. hispida within its distribution along the coasts of Mexico.The population genetic structures of


Lodoicea maldivica, a dioecious palm that produces the largest seed of any plant. Previous research has suggested that restricted seed and pollen dispersal in populations resulted in strong spatial genetic structure. We used microsatellites to genotype young plants and their potential parents at four sites across the species’ entire natural range. We determined the most likely parents of each young plant based on the spatial separation of each parent pair, their genetic relatedness, and the level of correlated paternity. We identified both parents  for 139 of 493 young plants. Mean distance between parental pairs was 26.8 m. Correlated paternity was low (0.168), indicating that mother trees were often pollinated by several fathers. Parental pairs were more closely related than expected by chance, suggesting outbreeding depression. Our results highlight the apparent strong mate choice for close kin in parent pairs of surviving offspring. We discuss the alternative biological processes that could lead to this, including the potential for break-up of favourable allelic combinations necessary for the development of the palm’s very large seed. Management implications include germinating seeds where they naturally fall, using a diverse range of male plants as pollen donors for hand pollination, and protecting the native community of gecko pollinators.We studied spatial patterns of kinship in the offspring of the endangered  Species vary, however, in their vulnerability to inbreeding depression; plants with mixed mating systems, for example, are usually less affected than outbreeding species4. Also, species that occur naturally in small or isolated populations may be less susceptible to negative effects of inbreeding, presumably because deleterious alleles have been purged from their populations over many generations5. The effectiveness of purging remains unclear, however, and inbred populations may accumulate genetic load gradually, with negative consequences for their long-term fitness6.The genetic relatedness of mating pairs has long been understood as a critical factor influencing offspring vigour. It is often found that mating between closely related individuals leads to reduced fitness or inbreeding depression2. In some cases, however, outbreeding results in lower fitness, especially when the two individuals come from different populations. Possible genetic reasons for such outbreeding depression include adaptive differentiation among populations, fixed chromosomal differences between populations, and the existence of coadapted gene complexes9. Whereas the first two factors are most likely to appear in crosses between widely separated or ecologically distinct populations or sub-species (e.g.10), the break-up of coadapted gene complexes may also occur at a small spatial scale within populations11. For example, several experimental studies have shown that crossing distance within a site can significantly affect the fitness of progeny, sometimes with inbreeding depression at short distances and outbreeding depression at large distances. When the herb Delphinium nelsonii was hand-pollinated across different distances within a population11, progeny from the intermediate crossing distances (3 and 10 m) grew approximately twice as large as the more inbred (1 m) and outbred (30 m) progeny. Similarly, both inbreeding and outbreeding depression were detected in an experimental study of the Australian shrub Grevillea mucronulata, with all measures of fitness  being higher for pollen transfers across intermediate compared to maximum and minimum distances12.Compared to inbreeding, matings between less related individuals usually exhibit greater fitness5. Introducing distant genotypes into a threatened population is not without risk, however, since it could lead to outbreeding depression14. A particular problem in screening for outbreeding depression, especially in long-lived, slow-growing species, is that any fitness reductions may be delayed until the F2 or later generations, due to the time taken for deleterious interactions to become exposed2.The effects of inbreeding depression have become a major concern for plant conservation, as the distributions of many species become increasingly fragmented. This concern has prompted numerous efforts to restore ecological connectivity in the landscape and also to promote outbreeding by increasing the genetic diversity of small populationsLodoicea maldivica (hereafter Lodoicea). This dioecious palm is best known for its huge seed—by far the largest in the plant kingdom—which can weigh as much as 18 kg. The species forms dense, almost monospecific stands on two small islands, Praslin and Curieuse, in the Indian Ocean archipelago of Seychelles. In the absence of any mechanism other than gravity for dispersing fruits, the huge seedlings of Lodoicea typically grow in clusters around the mother plant. The very limited seed dispersal is reflected in the genetic make-up of populations, which exhibit high levels of inbreeding and a strongly developed fine-scale spatial genetic structure (FSGS)17.We studied possible inbreeding and outbreeding depression in populations of the endangered coco de mer palm Lodoicea life history, three possibilities relating to its breeding system seem worth investigating: (1) inbreeding depression occurs due to very short seed dispersal distances and a strongly developed FSGS; (2) outbreeding depression occurs, perhaps linked to the break-up of coadapted gene complexes involved in the development of the huge seed; (3) both inbreeding and outbreeding occur, as reported in some other plant species12. To distinguish between these possibilities, we used microsatellites to characterise the genotypes of young Lodoicea and all possible father and mother plants growing in their vicinity. These data were then used to determine the most likely parents of each offspring plant, allowing us to calculate their spatial separation (pollination distance) and genetic relatedness (kinship coefficient). Finally, we compared the distribution of kinship coefficients for parental pairs with the expected distribution if mating was random. In this comparison, fewer closely related parental pairs than expected by random mating would indicate inbreeding depression (or avoidance), while fewer than expected distantly related parental pairs would indicate outbreeding depression. These analyses were performed separately for male and female offspring to investigate whether there were any sex-specific differences in inbreeding or genetic diversity, as reported for some dioecious species , Fond Peper (FP) and Fond Ferdinand (FF)—and one was on the island of Curieuse (CU).The coco de mer ica Fig.  is an en2 and contained between 34 and 193 plants of varying ages. Within each cluster, we geo-referenced (Garmin 60CSx) the locations of all potential mother trees and mapped the relative positions of younger plants using a tape measure and compass. Distances between the boundaries of adjacent clusters ranged from 13 to 360 m. We also recorded the GPS locations of all possible father trees within a radius of 80–120 m of each cluster. Leaf tissue was collected from all georeferenced plants in accordance with relevant institutional, national and international guidelines and legislation. Permits to conduct the research and export samples were granted by the Seychelles Bureau of Standards and the Ministry of Environment, Energy and Climate Change, respectively.In each sub-population, we selected and mapped four ‘clusters’ of young plants, referred to here as offspring, that appeared to be the product of natural regeneration and showed minimal signs of human disturbance Fig. . The 16 Lodoicea at ecogenics GmbH , using Roche 454 sequencing. For details of the polymorphic microsatellite loci and PCR conditions see19. Fragment length was analysed using the internal size marker LIZ 500 HD in an ABI3730 capillary sequencer (Applied Biosystems), and scored with GeneMarker 2.6.020 . Twelve nuclear microsatellite markers were designed for 22. Females and males from within the cluster were included as candidate parents. This analysis simulated 10,000 offspring, with the minimum number of typed loci set at 9, the proportion of mistyped loci set at 1%, and with 96.1% of the loci typed. The proportions of candidate mothers and fathers sampled were set at 0.8 and 0.5, respectively. The strict confidence level of 95% for the trio assignment (mother-father-offspring) was used. Combined exclusion probability for the first and second parents were 0.9961 and 0.9999, respectively. We assumed that the mother and father of each assigned juvenile were the nearest assigned female and male plants, respectively23.We used the delta maximum-likelihood exclusion analysis in CERVUS 3.024, using SPAGeDi25) of male–female pairs using the genotype data for adult plants. We then compared the kinship of parental pairs  with those of a reference group comprising all possible pairings of the assigned mothers (what we consider as random mating). For this latter group, we included all male trees growing within a radius of 105 m of each mother tree, this being the maximum distance over which all living male trees were sampled.To investigate possible outbreeding or inbreeding, we calculated kinship coefficients . From the growth rate data in that study, we estimated that our offspring plants ranged between 7 years for the youngest seedlings (trunkless plants with one or two leaves) to around 60 years for the oldest immature plants , with an average of about 44 years. Adding 7 years for the time it takes for a seed to mature, we obtained an estimate of 51 years as the average interval between pollination and time of sampling. The same study also estimated the average reproductive lifespan of Lodoicea  as about 100 years for male trees. On the basis of these estimates, we conclude that roughly half of all father trees would have died by the time of our study.Since young Using pooled data for the four sites, we calculated separate linear regressions of kinship against distance (square-root transformed) for non-parental and parental pairs. We then performed an analysis of covariance (ANCOVA) to test how kinship was affected by pair type, whilst controlling for distance and site. This analysis showed that kinship declined significantly with distance between male/female pairs. We therefore used the residuals of the linear regression of kinship against square-root transformed distance for all pairs to compare kinship of parental and non-parental pairs (t-test). This analysis was performed separately for each site and using the pooled data.We investigated possible biases in our data arising from the fact that we could only assign fathers to half of all offspring (e.g. if dead fathers were less related to the mother than those we sampled). For this, we performed one-way analysis of variance to investigate possible variation in kinship among age cohorts of offspring  and also correlated kinship of offspring against height of the fathers (as a proxy for age).26.All analyses were performed using R version 4.2.2Lodoicea sampled in four sub-populations on Praslin and Curieuse. We were then able to assign probable fathers to 139 (52%) of these offspring. Thus, for 28% of the offspring sampled, both a probable father and mother could be assigned, with this percentage varying among the 16 clusters. Maternal assignment rates were lower than we had expected, given that we deliberately sampled clusters of offspring growing close to a probable mother tree. This was probably because some clusters included the offspring of female trees that had already died. The overall assignment rate for fathers corresponded closely with our estimate that about half of father trees were dead. Consistent with the progressive mortality of older fathers, paternal assignment rates declined with increasing age of offspring from 67% for seedlings to 25% for immature trees.Using the genotype data, we determined the probable mothers of 269 (55%) of the 493 young 27) for all families with at least two offspring was 0.168, indicating a high level of multiple paternity.The 139 offspring in our sample were produced through matings between 43 female trees and 85 male trees, representing 103 parental pairs. The numbers of offspring from individual females varied widely; while over half of females were associated with one or two offspring, there were a few larger clusters of up to 15 offspring from one female Fig. . CorrelaThe mean distance between mothers and fathers, calculated from the distances between the 139 parental pairs, was 26.8 m, with no significant difference in these distances for male and female offspring . Two thirds of parent pairs occurred over distances of less than 30 m and the longest distance recorded was 103 m  using (a) all possible pairs of male and female trees within each sub-population  negative relationship, indicating that the kinship of male/female pairs declined with distance . Analysis of covariance revealed no difference in the slopes of the two regressions (P > 0.05), but a highly significant difference in their intercepts (P < 0.001). There was also a significant interaction (P < 0.05) between pair type and site (see Supplementary Tables Linear regressions of kinship against distance (sqrt-transformed) were calculated for 2382 non-parental pairs and 103 parental pairs. For non-parental pairs, there was a significant  and highly significant inbreeding coefficients in these natural populations (FIS17). The results presented here about the species’ breeding system help in interpreting the genetic structure of Lodoicea populations, including some potentially paradoxical findings in the two previous studies.A molecular genetic study of remnant Lodoicea populations can be explained by predominantly short-distance dispersal of both pollen and seeds. In a previous study, we found that mean seed dispersal was 8.7 m, with most offspring growing in dense clusters close to the mother trees17. Only on steep slopes did seeds occasionally slide longer distances over the forest floor and establish up to 100 m from the mother. Here, we show that mean pollen dispersal  is also mainly over short distances (mean 26.8 m), though sometimes occurs over distances up to 103 m. A few fathers may have grown beyond the maximum sampling distance (105 m), but—to judge from paternal assignment rates and mainly short pollen dispersal distances , during which the surrounding pollination landscape would probably have changed considerably. Fourth, unlike other species of Borasseae, most Lodoicea fruits produce only a single seed. It has been suggested that a reduction of ovule number to one per fruit is an evolutionary trend that reduces sibling rivalry31, which would be especially significant for a tree with very limited seed dispersal18. Finally, it is possible that matings occasionally occur over much longer distances than we could detect. Male Lodoicea are on average longer lived than females, sometimes growing to a height of over 30 m, while females rarely reach more than 20 m. Despite possible outbreeding depression, even very rare pollinations over long distances from long-lived emergent trees might be sufficient to maintain a high degree of genetic uniformity across the species’ range.Several factors may explain the apparent paradox of high inbreeding and FSGS coupled with high genetic diversity and weak between-population differentiation. The first is dioecy, which ensures that inbreeding is never as intense as in some monoecious species. On the other hand, spatial genetic structure in populations of dioecious species also tends to be much more strongly developed than in monoecious and hermaphrodite species, as has also been shown within the genus Kinship analyses showed that the likelihood of a male–female pair producing offspring was influenced by the pair’s genetic relatedness. After controlling for distance and site (Supplementary Table 5). Furthermore, as a dioecious species, Lodoicea is protected from the extreme inbreeding that can occur in selfing populations. Modelling studies indicate that lethal mutations are more efficiently purged through sib-mating than through selfing32. In the oil palm Elaeis guineensis, for example, selfing led to a marked inbreeding depression, while sib-crossing not only had a less depressive effect, but was sometimes even superior to outcrossing33. This is not to suggest that Lodoicea is immune to the effects of inbreeding, however; high levels of even biparental inbreeding could be associated with a gradual increase in genetic load, with negative consequences in the long term6.The apparent absence of inbreeding depression could be due to the purging of deleterious alleles, as has been suggested for other species in which inbreeding is normal . A study of pollen dispersal and genetic diversity in the dioecious rainforest tree Fontainea picrosperma in Queensland, Australia36 found mean pollen dispersal distances similar to those for Lodoicea, with 63% of seeds sired by male trees located within 30 m of the mother. Levels of inbreeding were negligible, however, suggesting that long-distance gene flow was sufficient to maintain genetic diversity at a stable level across the species’ range. The most comparable study to ours concerns seed and pollen dispersal and spatial genetic structure in the large-seeded dioecious palm Phytelephas aequatorialis37. In their study site in Ecuador, the authors detected a strongly developed FSGS over distances up to 35 m and inferred mean dispersal distances of 35 m and 68 m for seeds and pollen, respectively. They concluded that the FSGS was maintained mainly by limited seed dispersal, while more extensive pollen dispersal was sufficient to counteract inbreeding.There have been few other studies of dioecious species with restricted pollen dispersal with which our data can be compared, and some of these are ecologically very different  were more closely related than expected from pollen dispersal distances40, which the authors suggested could be due to genetic influences upon flowering phenology, with closely related individuals showing more synchronous flowering, or to the behaviour of pollinators. These possibilities seem unlikely in the case of Lodoicea. Not only is there no evidence for the kind of site heterogeneity, for example in soil conditions or pollinator community, that might produce such an effect, but the outbreeding probability was similar across all distances . This suggests that there could be a dosage effect: when a large number of pollen grains are transferred to the stigma of a distant tree through hand pollination, the chance that one of them will be of a compatible genotype is greater than when only a few grains are transferred by a natural pollinator that requires closed forest to ensure high pollination success44. However, we cannot disregard potentially heritable differences also in flower attractants, which we could not determine with our study.Support for this idea comes from the frequent occurrence of non-viable abnormal fruits, which develop for a year or more but fail to produce any endosperm. In a survey byLodoicea exhibit a strongly developed fine-grained genetic structure, and the possible ecological or genetic consequences of disrupting this structure are unknown. In the past, planting schemes aimed at restoring populations have sometimes transferred seeds over long distances. Given the indications that outbreeding depression could operate, seeds should be allowed to grow as closely as possible to where they fall or are collected. For the same reason, hand pollination should usually be performed using a broad range of pollen donors to allow maternal selection of the best allelic combinations. It is worth adding that hand pollinations provide a valuable opportunity to learn more about the breeding system  and it would be useful to record such pollinations, and the subsequent seed production.Our results have implications for management. Local populations of Lodoicea is presumably maintained by pollinators that transfer pollen over mainly short distances. Various pollination mechanisms have been proposed, including wind, flies, slugs and trigonid bees46, but a recent study suggests that the most important pollinators in natural forest are endemic geckos . It seems likely that the movement patterns of geckos within closed forest help maintain the high level of multiple paternity that we recorded. If so, the absence of geckos could explain the low seed set and high proportion of abnormal fruits often to be observed in isolated plants44. Maintaining a healthy population of endemic geckos should therefore be a high management priority. In addition, we suggest that any planting schemes or habitat restoration should use seeds collected in the same area over time.The genetic structure of Lodoicea.In conclusion, we suggest that conservationists should take a more nuanced view of inbreeding and its significance for threatened populations. While it poses a severe threat for many plant species, especially those that formerly occurred in large, unfragmented populations, it may be less critical for species that are prone to limited gene flow, as demonstrated here for Supplementary Information 1.Supplementary Information 2.


Mytilus unguiculatus in seven coastal areas of China. The results showed that M. unguiculatus has high genetic diversity with genetic structure differences observed between the Qingdao population and the other six populations. The genetic structure of M. unguiculatu is relatively weak. These findings provide a molecular biological basis for the rational development and protection of wild germplasm resources of Mytilus unguiculatus in China and can serve as a data reference for the formulation of reasonable breeding programs.Microsatellite markers are widely used in genetic breeding and population genetic structure analysis. In this study, microsatellite markers are used to analyze the genetic diversity and population genetic structure of Mytilus unguiculatus plays an important role in mussel aquaculture in China due to its characteristic and nutritive value. In this study, 10 microsatellite loci are used to study the genetic diversity and genetic structure of seven location populations of M. unguiculatus in coastal areas of China. The results of amplification and genotyping show that the observed heterozygosity (Ho) and the expected heterozygosity (He) are 0.61~0.71 and 0.72~0.83, respectively. M. unguiculatus has high genetic diversity. The inbreeding index (FIS) of M. unguiculatus is significantly positive (FIS: 0.14~0.19), indicating that inbreeding might exist within populations. The genetic structure of M. unguiculatus is weak within populations from the East China Sea All results showed that genetic differences existed between the Qingdao population from the Yellow Sea and other populations from the East China Sea. It does not detect a population bottleneck event or expansion event in the populations. The results from this study can be used to provide important insights in genetic management units and sustainable utilization of M. unguiculatus resources and provide a better understand of genetic structure of marine bivalve with similar planktonic larval stage in the China Sea.The hard-shelled mussel  Mytilus unguiculatus  is an offshore warm–temperate benthic shellfish. The shell of M. unguiculatus is tip and thin, and the shell surface is often brown. In East Asia, it is widely distributed in the littoral of the Japan Sea, Bohai Sea, Yellow Sea, East China Sea, and South China Sea [M. unguiculatus is widely consumed by people [M. unguiculatus is one of the important aquaculture shellfish species in China. With the increasing demand of people, M. unguiculatus have become one of the key fishing targets. However, the generation replacement rate of M. unguiculatus is slow, and large-scale and high-intensity harvesting is bound to reduce mussel resources [M. unguiculatus.hina Sea ,2. In Chhina Sea ,4. Due ty people . M. unguesources ,7. ThereMicrosatellite DNA  is a molecular marker that is widely distributed in the genome of eucaryon. It consists of a repetitive DNA sequence with a certain number of base pairs (1 to 6 bp) ,9. MicroM. unguiculatus are mainly studied using mitochondrial molecular markers [M. unguiculatus. The objectives of this study were to investigate the genetic diversity and population genetic structure of M. unguiculatus using microsatellite markers and compare the results with those obtained using mitochondrial molecular markers. This study aims to provide information on the geographical distribution patterns and evolutionary processes of this species, as well as to fill knowledge gaps in related research fields. Additionally, the new data obtained will aid fishery resource managers in assessing present resources and developing conservation strategies.Currently, the population genetics of  markers ,21,22. TM. unguiculatus crossed from the Yellow Sea to East China Sea  (The seven sampling locations of  Xiamen) . In tota Xiamen) . SpecimeAll primers used in this study were selected from Fu et al. . Ten paiFinally, capillary electrophoresis genotyping was performed using the BiopticQsep100 DNA Analyzer in Shanghai Generay Biotech Co. Ltd.  under the following conditions: 1 µL PCR product, 1 µL GS500LIZ (P/N 4322682) size standard (Applied Biosystems), and together with 9 µL HiDi-formamide (Applied Biosystems).Na), observed (Ho), and expected (He) heterozygosity were evaluated using GenAlEx v6.501 [Ar) by setting the minimum sample size to 46 and the genetics inbreeding index (FIS). The MICRO-SATELLITE ANALYSER (MSA) v4.05 was used to estimate the genetics differentiation index (FST) [HWE) and the p value. Arlequin v3.5 [After genotyping, the number of alleles (x v6.501 . The FSTx v6.501  was usedex (FST) . The proex (FST)  was useduin v3.5  was perfuin v3.5  to estimuin v3.5 ,32 perfouin v3.5 , Stepwisuin v3.5  and Two-uin v3.5  with 10%uin v3.5 . The Wiluin v3.5 . Finallyuin v3.5  was perfNJ phylogenetic tree among seven populations based on the Nei’s standard genetics distance [The program Populations v1.2  was useddistance . The sofdistance  for evaldistance  was usedNa) ranged from 5.5 (TS) to 7.1 (YH). The observed and expected heterozygosity (Ho and He) varied from 0.61 (QD) to 0.71 (YH), and 0.72 (QD) to 0.83 (YH). The inbreeding index (FIS) ranged from 0.14 (YH) to 0.19 . All loci did not significantly deviate from HWE . Results of AMOVA showed that genetic differentiation among groups was over 4.1% of total variation, with 1.3% attributed to variation among populations within groups and the remaining 94.6% attributed to within populations  were not significant. The gene flow (Nm) ranged from 3.80 to 833.08, indicating frequent gene exchange between populations at different sampling locations  to infinity on QD (CI = 372.6 − infinity). In the bottleneck test, based on three different models, it was not significant that the heterozygote exceeded any other populations. The allele distribution conforms to the L-shaped distribution without bottleneck effect events. It indicates that no genetic bottleneck has recently occurred in the populations of M. unguiculatus along the coastline of the China Sea , moderate differentiation (0.05 < FST < 0.15), large differentiation (0.15 < FST < 0.25), and very large genetic differentiation (FST > 0.25). Therefore, the pairwise FST (0.003~0.065) was quite small for the populations studied. The pairwise FST values, including Qingdao population, were higher than others. The phylogenetic tree , and local circulation, upwelling, and eddy currents  [M. unguiculatus in China, and its mass breeding season is in winter every year. During this period, a large number of mussel larvae will be dispersed in the sea. M. unguiculatus with a long planktonic period (about 35d) [M. unguiculatus is relatively mature, and aquaculture practitioners typically obtain seedlings from the main producing areas and transport them to breeding sites. The aquaculture method involves using attached substrates for seedling culture in natural sea areas. This open breeding method leads to a large number of cultured sample larvae being dispersed in the sea during the breeding season and becoming mixed with wild mussel larvae. As a result, a weakening of the genetic structure of M. unguiculatus can occur due to the large number of cultured sample larvae mixing with wild mussel larvae and spreading with ocean currents.The results of this study indicated that e water) ,59,60,61e water) . Marine e water) ,64,65. Tout 35d)  drift foM. unguicultas did not show significant traces of bottleneck effects, indicating that crossbreeding between populations is likely the main factor contributing to the current level of genetic variation. Inbreeding among populations could result in the loss of genetic information, making it undetectable through molecular methods. Although an artificial seedling technique has been developed for farming M. unguiculatus, most of the seedling parents still originate from the natural producing area in the East China Sea. The increase in breeding demand has led to the embezzlement of natural habitats, resulting in a decline in natural samples and a reduction in genetic variation. At present, the breeding industry of M. unguicultas lacks natural population protection management, which can lead to the dispersal of a large number of cultured larvae into the sea during the breeding season. These larvae can mix with natural mussel larvae and weaken the genetic structure of M. unguiculatus in the wild population. Therefore, to conserve the germplasm resources of M. unguiculatus, we recommend reducing the genetic exchange between cultured and natural populations and minimizing the contamination of genetic information in natural populations.The investigated seven populations of M. unguiculatus is critical in revealing its situation in the natural environment. In this study, seven M. unguiculatus populations in coastal areas of China were analyzed by microsatellite markers. The results showed that M. unguiculatus had high genetic diversity. Qingdao and other populations had genetic differentiation. The overall genetic structure was weak, and the presence and size of any bottleneck effect on genetic variation were difficult to assess. The genetic data generated in this study could provide insights into genetic management units and the utilization of M. unguiculatus resources.Population genetics research on


Human activities have left indelible traces in the gene pool of livestock as well as recently domesticated wild animal species which are manifested by changes in their genome structure, often accompanied by the significant loss of biodiversity, especially in the case of small local populations. Despite the benefits of deer farming, intensive artificial selection associated with domestication can lead to several negative effects, including founder effects, inbreeding depression, or rapid decline in effective population size, which are also often present in traditional livestock. This study investigates the effect of human-mediated selection on the diversity of seven farmed red deer populations compared to two wild populations through the quantification of the level of genomic heterozygosity, inbreeding, admixture, and gene flow. These results will help to improve traditional breeding schemes and assist in a more sustainable utilisation of available animal genetic resources.eH = 0.19) and the lowest genomic inbreeding (HOMF = 0.04) found in Slovak wild population confirmed our hypothesis that artificial selection accompanied by bottlenecks has led to the increase in overall genomic homozygosity. The Bayesian approach and DAPC consistently identified three separate genetic groups. As expected, the farmed populations were clustered together, while the Slovak and Spanish populations formed two separate clusters. Identified traces of genetic admixture in the gene pool of farmed populations reflected a strong contemporary migration rate between them. This study suggests that even if the history of deer farming has been shorter than traditional livestock species, it may leave significant traces in the genome structure.In this study, we analysed the effect of human-mediated selection on the gene pool of wild and farmed red deer populations based on genotyping-by-sequencing data. The farmed red deer sample covered populations spread across seven countries and two continents . The Slovak and Spain wild red deer populations (the latter one in a large game estate) were used as control outgroups. The gene flow intensity, relationship and admixture among populations were tested by the Bayesian approach and discriminant analysis of principal components (DAPC). The highest gene diversity ( Cervus elaphus) has been historically affected either by environmental changes or especially by anthropogenic activity [C. e. hispanicus) based on nuclear DNA microsatellites as opposed to mitochondrial DNA [The genetic diversity of European red deer populations  ,11. TranIn addition to the translocation of individuals considered of high-genetic quality, human-mediated artificial selection is another long-standing practice of red deer population management, both in the wild and on farms. In wild populations, the traditional hunting practice, largely for meat, is based on selective harvesting (negative selection) of non-fit animals predominantly. The exception was selective shooting for age class of trophy culmination in males/stags by trophy hunters. Over time, artificial selection has shifted more to trophy value and less to meat. However, classic positive genetic selection of males and estimated female breeding value for trophy value was only conducted in farms (as it is impossible to conduct in the wild). More recently, in game estates and deer parks, selected animals have also been transported and released into wild populations contained within fenced areas. In addition to the selection of national populations of deer, it is important to bear in mind the strong influence of New Zealand and British populations of deer, which, being the first to be selected for trophy in the last quarter of 20th century (see below), have been the source of genetic improvement for trophy  influencing crosses with national populations in farms of many European countries .In wild populations, similar to translocation and introgression, artificial selection significantly affects the genetic variability and fitness of local autochthonous populations ,22. In tWhile translocation and selective hunting have affected the genetic diversity of European deer populations for centuries, increasing land fragmentation  accompanied by isolation of populations is now common . A partiCervus elephus sp.), fallow deer (Dama dama) and European mouflon (Ovis musimon) [In the second half of the last century, farming of wild ungulates, especially cervids, became popular overseas, but also in western Europe. Although deer farming dates back possibly 2000 years, it was rather common in the middle age, at least in Britain , the fimusimon) . Spain hmusimon) ).The first world exporter of deer venison, deer subproducts, and the number of farms and animals in a single country is New Zealand, with about one million farmed deer . TherefoIn sustainable agriculture, deer farming is considered a modern phenomenon that benefits both the country and its inhabitants. The use of marginal land to produce high-quality venison production can be regarded as the main advantage of deer farming. The development of deer farming in most European countries like Slovakia was slower than in New Zealand. Deer has radically different historical reasons for farming or harvesting in western and Asian cultures: for trophies (and secondary product its venison) in western culture, whereas in China and the rest of Asia has been used for 2000 years in their traditional medicine, mainly growing antler . Only New Zealand, Russia and former Soviet countries like Kazakhstan, and to a lesser extent, Argentina, farm deer mainly for velvet or in addition to venison . As an eTherefore, human activities are expected to have left indelible traces in the genomes of farmed as well as wild deer. In this study, we aimed to analyse the gene pool of two wild and seven farmed red deer populations to test the effect of human-mediated selection on their genome structure and quantify potential gene flow between them using genotyping-by-sequencing (GBS) data.Cervus elaphus elaphus) and Spanish red deer (formerly Cervus elaphus hispanicus)  . When wee et al.  and Dodde et al. . The nuce et al. .-UFastqToTagCountPlugin -c 1 -e PstI; -UMergeTaxaTagCountPlugin -c 3; -UTagCountToTagPairPlugin -e 0.03; -UTagPairToTBTPlugin; -UMapInfoToHapMapPlugin -mnMAF 0.01 -mxMAF 0.5. We identified initially 69,379 SNPs with an average depth of reads across SNPs and samples of 3.144 and call rate of 0.655. Genotype information obtained was then transformed using VCFtools v3.0 [SNP calling for GBS data was conducted by Tassel v3.0.174  based onols v3.0  to the iEven though red deer populations under study belong to the same species, breeding goals and selection criteria could differ depending on the country of origin. In this study, the effect of human activity on the gene pool of wild as well as farmed red deer populations was derived from the level of observed and expected heterozygosity and genomic inbreeding. The applied approach assumed that processes such as translocation, introgression and selective breeding significantly changed the intrapopulation level of overall heterozygosity.oH) and expected heterozygosity (eH) and genomic inbreeding (HOMF), PLINK v1.9 [HOMF was derived from the genome-wide homozygous excess according to the formula:O is observed homozygous genotype counts for each individual, E is expected homozygous genotype counts under the Hardy–Weinberg equilibrium (HWE) for each individual and N is the total number of genotypes.For the calculation of observed FST=He StAMPP .X and Y, where ix and iy are the frequencies of the i-th allele in populations X and Y, the probability of matching two randomly selected genes is X and Y. The probability of matching two randomly selected genes, one from each population, is then I is the normalised gene identity (or genetic identity) between the X and Y populations. xJ, y,J and xyJ are the arithmetic mean of xj, y,j and xyj over all loci in the genome, including monomorphic ones. Nei’s genetic distances within and between populations were calculated using the R package StAMPP [Nei’s genetic distance theory assumes that if two populations with low genetic distances are similar, they have most likely common ancestors. When considering two populations, lated as :(3)D=−lne StAMPP .Genetic variance distributed within and between analysed populations was quantified by discriminant analysis of principal components using the R package Adegenet v2.1.3 . This muThe degree of differentiation within and between populations was then tested through unsupervised cluster analysis, also called superparamagnetic clustering (SPC) . In the The degree of genetic admixture between populations was quantified based on the Bayesian approach implemented in Structure v2.3.6 . The anaeH values were higher than the observed heterozygosity. When the expected heterozygosity values are higher than observed, the populations are assumed to deviate from the Hardy–Weinberg equilibrium and, in that case, they can be significantly affected by factors such as inbreeding, migration, selection, or genetic drift. French and Slovak wild populations showed the lowest differences between observed and expected heterozygosity, which suggests that the bottleneck effect  had a minor effect on the population gene pool. On the contrary, the highest difference between observed and expected heterozygosity was found in the Hungarian and Spanish populations.Generally, the average observed and expected heterozygosity was relatively low in all analysed populations of red deer . ObserveThe MAF level observed across animals and SNPs within each analysed population corresponds to the obtained level of heterozygosity . The SpaGenomic inbreeding derived from the observed and expected homozygous genotype counts ranged from 0.035 (Slovak wild population) to 0.359 (Spanish populations) . SimilarSTF fixation index (STF index (0.077 ± 0.033) suggested a certain degree of genetic connectedness among the analysed red deer populations, which subsequently confirmed the Nei’s genetic distances (in average 0.025 ± 0.006).In the first step, two commonly accepted parameters were used to quantify genetic relationships between populations, Nei’s genetic distances and on index . The aveSTF index was observed between the Spanish population and the French (STF = 0.141), Slovak (STF = 0.134) or New Zealand populations (STF = 0.138). A similar genetic distance was observed between the wild Slovak population and either the New Zealand (STF = 0.125) and the French population (STF = 0.112). On the contrary, very low genetic distances were observed between the German, Latvian, and Polish farmed populations (STF in the range of 0.020–0.027). Relatively low genetic distance was also found between the Slovak farmed and the Hungarian farmed population (STF = 0.015).As expected, because of the origin of populations, the highest genetic distance derived from the AD in the range of 0.027–0.039). Similar to the STF index, the second population showing a high genetic distance from other populations in the analysis was the Slovak wild populations, which revealed a relatively high degree of differentiation, mainly in the case of farmed populations from France and New Zealand (AD = 0.035 resp. AD = 0.036). Matrices of Nei’s genetic distances confirmed that Slovak farmed and Hungarian populations are genetically closely connected with AD value of 0.013. A similar level of genetic distances was also found between populations from Germany, Poland, and Latvia (AD in the range of 0.013–0.014).In the case of Nei’s genetic distances, the Spanish population also showed the highest degree of genetic differentiation from others  used to discover SNPs in the genome of analysed red deer populations belongs to the group of genotyping methods that utilise next-generation sequencing (NGS) technologies. GBS is a simplified reduced-representation sequencing approach similar to RAD sequencing that is popular in animal and plant genetics as a low-cost alternative to whole genome sequencing and SNP genotyping microarrays ,49,50,51The removal of markers that do not conform to Hardy–Weinberg equilibrium expectations is commonly used as part of quality control in population-genetic studies. However, it was not applied in this study. The hypothesis we tested assumes that due to human-mediated selection as well as other factors, a change in allele frequencies can be observed in the genome of the tested farmed red deer populations compared to wild and thus they do not necessarily exhibit Hardy–Weinberg equilibrium. Wild animal species often exhibit population substructure or genetic differentiation due to factors like geographic barriers, limited gene flow, or local adaptation and varying levels of inbreeding due to small population sizes or social structures. These factors can violate the assumptions of Hardy–Weinberg equilibrium, resulting in departures from equilibrium even in the absence of genotyping errors. It was showed that departure from HWE with excess of heterozygosity is suggestive for genotyping errors and, on the other side, loss of heterozygosity pointed to natural forces such as population substructure . PearmanOne of the factors that may have contributed to the reduction of heterozygosity in analysed populations was the bottleneck effect (in farmed populations founder effect) because farmed populations generally arise from a limited number of founders. In addition, most breeders buy animals from the best or high-quality farms that bought from the best, thus further increasing the bottleneck effect. However, the bottleneck effect also affects wild populations, mainly due to anthropogenic factors. During the 20th century, the diversity of all wild red deer populations was affected by the 1st and 2nd World Wars, which led to a drastic decline in the population size. Although some populations were later restored with what remained from genetic diversity, their gene pool was affected by intensifying agricultural activity and urbanisation. The lowest observed heterozygosity was found in the deer population from Spain. This is most likely the results of two facts: 1) the human pressure for use of land and need of hunting to improve the diet left at the beginning of 20th century only three populations in Spain : centralAn important factor that indicates the effect of selection, migration, or genetic drift on the population genetic structure is the difference between the observed and expected heterozygosity, i.e., the deviation from the Hardy–Weinberg equilibrium. From analysed populations, significant deviations were found for the Hungarian and Spanish populations. Especially in the case of the Spanish population, it is likely to be the result of the bottlenecks mentioned above, or else, its geographical isolation from other local deer populations under study. In the Hungarian population, heterozygosity may have been affected mainly by the intensive selection of animals for trophy quality .It was shown that the usability of genetic markers in population genetic studies depends on their polymorphic nature, i.e., level of minor allele frequency . The oveHOMF = 0.035), in this study derived from the observed and expected homozygous genotype counts. On the other hand, the observed excess of homozygous genotypes proportion in the Spanish population gene pool is probably the result of the historical bottlenecks mentioned above or its geographical isolation and the unbalanced effect of founders; thus, obtained HOMF value represents mainly the historical load of inbreeding. The founder effect was also fully reflected in the gene pool of farmed red deer populations, with an average HOMF from 0.04 to 0.30. Similar levels of HOMF have also been found in other livestock and companion animals showing at the same time a high level of intra-population homozygosity, e.g., local dog breeds [As expected, based on the level of observed and expected heterozygosity, the Slovak wild population showed the lowest level of genomic inbreeding .The results of the DAPC showed a high degree of genetic connectedness between analysed farmed red deer populations. Increasing profits often leads deer breeders to buy breeding animals from abroad, thus exchanging genetic material between countries. Genetic differences between countries create a precondition for accelerating genetic progress, e.g., through the heterosis effect . BecauseSlovak wild and Spanish populations showed only a low level of genetic admixture, which confirms their phenotypic and genotypic divergence from other populations under study. Based on the DAPC results, we assumed that the group of farmed populations would be divided into four genetic clusters: first, composed of German populations, second, composed of the French population, third, consisting of Slovak farmed, Hungarian, and New Zealand populations and the fourth consisted of populations from Poland and Latvia. However, the observed admixture proportion only partially confirmed this assumption. Compared to previous results, traces of genetic variants coming from the New Zealand population were observed in the gene pool of all evaluated farmed populations. The highest proportion of these variants was found in the gene pool of Slovak farmed, German, Polish, and Latvian populations. The observed level of gene flow between populations was consistent with the admixture proportion; thus, identified traces of genetic admixture in the gene pool of farmed populations reflected a strong contemporary migration rate among them.Previous studies were mostly oriented to the analysis of red deer populations spread across a particular country ,78,79, bThis study suggests that although the history of deer farming is relatively short compared to other traditional livestock species, it may leave significant traces in their gene pool. All of the approaches used indicated that intensive gene flow within farmed red deer populations has led to significant changes in allele frequencies compared to the wild populations. Due to higher levels of genetic diversity and homogeneity between sampling areas in farms, the genetic changes caused by human-mediated selection can be evolved much faster than in wild populations, even being subjected to the negative selective force of hunting management. It is also the first transnational evaluation of genetic diversity in red deer that has used a sequenced-based discovery and genotyping approach comprising a large number of DNA variants, which is not subject to ascertainment bias effects common in alternative approaches.


Perca flavescens), a species with an ~40‐day pelagic larval duration (PLD), collected from 20 sites circumscribing Lake Michigan. We also developed a novel, integrative approach that couples detailed biophysical models with eco‐genetic agent‐based models to generate “predictive” values of genetic differentiation. By comparing predictive and empirical values of genetic differentiation, we estimated the relative contributions for known drivers of population connectivity . For the main basin populations , we found that high gene flow led to low overall levels of genetic differentiation among populations (FST = 0.003). By far the best predictors of genetic differentiation were connectivity matrices that were derived from periods of time when there were strong and highly dispersive currents. Thus, these highly dispersive currents are driving the patterns of population connectivity in the main basin. We also found that populations from the northern and southern main basin are slightly divergent from one another, while those from Green Bay and the main basin are highly divergent (FST = 0.11). By integrating biophysical and eco‐genetic models with genome‐wide data, we illustrate that the drivers of population connectivity can be identified in high gene flow systems.How to identify the drivers of population connectivity remains a fundamental question in ecology and evolution. Answering this question can be challenging in aquatic environments where dynamic lake and ocean currents coupled with high levels of dispersal and gene flow can decrease the utility of modern population genetic tools. To address this challenge, we used RAD‐Seq to genotype 959 yellow perch ( Here, we use this integrative approach across a 500 km latitudinal gradient to examine patterns of population connectivity in an ecologically and economically important fish species found throughout the Laurentian Great Lakes (hereafter Great Lakes).There are several challenges with identifying which currents serve as the primary drivers of population connectivity. First, high‐resolution oceanographic data and coupled biophysical models are required from numerous time points. Thus, high‐quality oceanographic data are requisite. Second, genetic differentiation is often used as a proxy for population connectivity, yet contemporary evolutionary processes  and past evolutionary legacies  can confound genetic estimates of population connectivity Waples, . Third, Perca flavescens), an ecologically and economically important fish with an approximately 40‐day pelagic larval duration . In fact, most yellow perch populations throughout the Great Lakes are dominated by a single mtDNA haplotype  distributed throughout the yellow perch genome , have high site fidelity, particularly with respect to spawning grounds  tissue digestion with Proteinase K incubated at 56°C, DNA was extracted in plates using standard kit protocols and eluted from the silica membrane with 200uL Tris Low‐EDTA buffer. Extractions were quantified using a Quant‐it™ PicoGreen® dsDNA Assay (Invitrogen), and DNA was normalized to a quantity of 200 ng or approximately 20 ng/μL.SbfI followed by ligation with indexed adaptors. Barcoded libraries were pooled into master libraries of 96 individuals and fragmented to ~300–500 bp with 12 30s cycles in a Q500 sonicator (Qsonica). Fragmented DNA was bound to Dynabeads™ M‐280 Streptavidin magnetic beads (Invitrogen) and washed with buffer to remove nontarget fragments. Following purification with AMPure XP beads (Beckman Coulter), master libraries were passed in series through the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® at the End Prep step for (1) end repair and ligation of master library barcodes, (2) a 250‐bp insert size selection, and (3) a 12‐cycle PCR enrichment. Successful size selection and enrichment were confirmed with visualization of products on a 2% agarose E‐Gel (Invitrogen). Products underwent a final AMPure XP purification clean‐up followed by quantification with a Qubit® 2.0 Fluorometer. A total of 10 master libraries, each containing 96 individually barcoded samples, were sent to Novogene  for PE150 sequencing on one lane of the Illumina Novaseq S4 platform.Libraries for restriction site‐associated DNA (RAD) sequencing were prepared following the BestRAD protocol  and expected heterozygosity (HE) were calculated in R 4.0.2  was calculated using the R package hierfstat v0.5.7  were calculated in hierfstat v0.5.7  because the orders of magnitude higher e et al.  to incore et al.  and yelle et al. . Three oe et al.  for a toe et al. . This pre et al. .i local populations. In practice, the multinomial distribution was implemented prior to reproduction so that we knew precisely how many offspring to create in each local population, which increased computational efficiency; however, the actual dispersal of individuals occurred after reproduction.Because many aquatic organisms are characterized by high variance in reproductive success . Thus, we obtained values of the exact same estimator calculated with two entirely independent approaches. Because the same estimator was used, a perfect fit between the predictive values and the empirical values would fall directly on a 1:1 line . Thus, to estimate the accuracy and precision of the predictive values we used simple linear regression and calculated the slope and correlation  between the empirical and predictive values, where a perfect fit would have a slope and R2 = 1 , larval retention , and goodness of fit. Lastly, the percent of variation explained by each parameter was estimated as the correlation between a specific predictor and the goodness of fit between predictive and empirical FST. The eco‐genetic model and all downstream analyses were written in R version 4.0.2  were genotyped at 9302 loci Table . The eston Table . An averFST values ranged from −0.001 to 0.148 ) and ∆K suggested two optimal clusters (K = 2)  Figure . These r) Figure , we foun) Figure . Results) Figure . By cont) Figure . The obs Figures  and S9.  Figures  and S10. Figures  and S10.3.2FST values and the genotype‐derived, empirical FST values, we found that the best predictors of empirical FST occurred when there was high population connectivity . The single best‐fitting connectivity matrix also characterized a time period with high connectivity . When examining the relationship between connectivity and the mean current strength, we found a positive relationship  between the current‐derived, predictive y Figure . Conversn Figure . Stated p Figure , suggestFST  were, on average, worse at predicting empirical estimates of genetic differentiation.When we examined the joint effect of release year and week, we found that the specific week of release alone had the highest predictive ability of any single parameter Figure . In geneFST (and not just the slope and correlation) did not assist with model evaluation  explained the majority of differences in predictive ability Figure . Vertican Figure . Lastly,s Figure .4For an aquatic species with a pelagic larval stage, we demonstrated that genetic differentiation across a 500 km latitudinal gradient is best predicted by strong and highly dispersive currents. Our results suggest that abiotic and biotic factors that increase dispersal distances  will favor population connectivity and contribute to observed population structure. More generally, the observation that genetic differentiation in large aquatic systems may best be determined by strong and highly dispersive currents has broad implications. First, many examinations of population connectivity rely solely on data from biophysical models and often average or integrate current data over lengthy periods of time , which further highlights the importance of managing Green Bay and main basin yellow perch as distinct stocks. This confirmation of previous work by Miller (FST = 0.018). Thus, for management purposes it may be appropriate to consider separate management strategies for perch from different regions of Green Bay . We also documented subtle population structure within the main basin itself (Figure FST equal to 0.003. Of note, yellow perch populations from Traverse Bay were much more similar to main basin yellow perch populations than Green Bay yellow perch were to main basin populations. This result suggests that the local, and perhaps fine‐scale, currents within Traverse Bay result in more connectivity with the main basin of Lake Michigan than is currently appreciated. From a population genetic perspective, it is interesting that Green Bay yellow perch show characteristics in common with fishes that lack a pelagic larval stage (higher FST; isolation‐by‐distance; Figure FST; no pattern of isolation‐by‐distance Figure At a regional level, we found that Green Bay was highly genetically differentiated from the main basin (mean y Miller  is most y Miller , but othy Miller  tagged 6y Miller  and measy Miller . If thisy Miller . Anotherf Figure  where noIn conclusion, we found that populations of an ecologically and commercially important fish with a 40‐day pelagic larval stage are connected by highly dispersive currents. Identifying the characteristics of currents that drive population connectivity can allow for better conservation and management decisions such as identifying the appropriate size and spacing of protected areas (Baetscher et al., The authors declare no conflicts of interest.Data S1.Click here for additional data file.


Pustulosa pustulosa is a freshwater species with a relatively wide latitudinal distribution that extends from southern Ontario, Canada, to Texas, USA. Considerable morphological and geographic variation in the genus Pustulosa (formerly Cyclonaias) has led to uncertainty over species boundaries, and recent studies have suggested revisions to species-level classifications by synonymizing C. aurea, C. houstonensis, C. mortoni, and C. refulgens with C. pustulosa (currently P. pustulosa). Owing to its wide range and shallow phylogenetic differentiation, we analyzed individuals of P. pustulosa using mitochondrial DNA sequence data under a population genetics framework. We included 496 individuals, which were comprised of 166 samples collected during this study and 330 additional sequences retrieved from GenBank. Pairwise ΦST measures based on ND1 data suggested there may be up to five major geographic groups present within P. pustulosa. Genetic differentiation between regions within Texas was higher compared to populations from the Mississippi and Great Lakes populations, which may reflect differences in historical connectivity. Mitochondrial sequence data also revealed varying demographic histories for each major group suggesting each geographic region has also experienced differential population dynamics in the past. Future surveys should consider exploring variation within species after phylogeographic delimitation has been performed. In this study, we begin to address this need for freshwater mussels via the P. pustulosa system.Unionid mussels are among the most imperiled group of organisms in North America, and  Unionid mussels (order: Unionida) belong to one of the most imperiled group of organisms in North America with two-thirds of the 298 currently recognized species being either threatened, vulnerable, endangered, or presumed/possibly extinct . ApproxiQuadrula  species to the genus Cyclonaias (Pustulosa (Pustulosa (Cyclonaias) pustulosa, has a wide latitudinal distribution ranging from southern Ontario, Canada to Texas, USA. Considerable phenotypic plasticity and intrinsic shell variation could be partly responsible for taxonomic confusion within and among species of Quadrula, Cyclonaias, and Pustulosa  as C. pustulosa (currently P. pustulosa) based on analysis of genetic and morphological data  has been performed within the St. Croix River  and three basins in Ontario, Canada  from 2015 to 2018 . We coll2, ~1–10 ng of genomic DNA, and nuclease-free H2O to volume. The second PCR contained half volumes of all reagents and 0.25 to 0.5 μL of PCR product, depending on the relative concentration of amplicons assessed via band intensity after gel electrophoresis. The thermal profile for both reactions followed an initial denaturation at 95 °C for 5 min; then 34 cycles of 95 °C for 1 min., 55 °C for 1 min., and 72 °C for 1 min. 50 s; and a final elongation at 72 °C for 7 min.To compensate for variable DNA yields from non-lethal swabs and to increase sample size, we performed a nested PCR protocol using primers targeting the mitochondrial NADH dehydrogenase 1 gene (ND1). We focused on this marker to leverage the available reference data for other populations in GenBank . The iniOQ656460–OQ656625). Previously published sequences showing uncorrected sequence similarity greater than 96% regardless of named taxon were also incorporated into the downstream analyses; however, we excluded any sequences with ambiguities . We cycle sequenced both DNA strands independently using BigDye v3.1 , which we purified using Sephadex G-50 Superfine  gel filtration medium. DNA sequencing was performed on an Applied Biosystems 3500 Genetic Analyzer. We used Geneious 9.1.8  to edit and assemble bidirectional chromatograms and to build and trim alignments, and we accessioned all newly generated sequence data into GenBank  and adjusted the resulting P-values via Bonferroni correction. To evaluate haplotype sharing among populations, we visualized haplotype diversity and abundance in a median-joining network using POPART  were either grouped according to the ranges of previously described species now comprising sensu latoP. pustulosa  or grouped by unionid biotic provinces. Specifically, six provinces  were previously delineated using a combination of ecological and genetic data .We compared two utations . Populatdata see . We sougTo infer the demographic history of each group using mitochondrial haplotype data, we estimated values for Tajima’s D and performed mismatch distribution analyses under a model of spatial expansion in Arlequin. We constructed a plot using the observed number of site differences between sequence pairs and the expected distribution under a constant population size model.geodist (geodist) function (dist.dna (ape)  function (kde2d (MASS)  . To testfunction  and set d (MASS)  to overlsensu latoP. pustulosa  (P. pustulosa group showed high haplotype diversity (H = 229) across 163 polymorphic sites (S).We generated ND1 sequence data for individuals collected in Texas, USA and Ontario, Canada and incorporated publicly available data from other studies . Altogetnsu lato . A 528 bST among river drainages showed partitioning of ND1 variation according to geography (ST 0 to 0.11) than populations within Texas (ΦST 0 to 0.85). The among group variation was higher (47.7%) when rivers were grouped by historical nominal species groups (Scenario A), and lower when rivers were grouped by unionid biotic provinces   .Based on the population-level genetic partitioning results, individuals from drainages within the Mississippi basin and the Great Lakes were placed into Group I; individuals from the Pascagoula/Pearl populations were placed into Group II; individuals from the Sabine, Neches, Trinity, and San Jacinto rivers were placed into Group III; individuals from the Brazos and Colorado were placed into Group IV; individuals from the Guadalupe, San Antonio, and Nueces rivers were placed into Group V; . PlottedD were significantly negative for all groups , suggesting a selective sweep or demographic expansion for each group. Group V had the least negative value , while Groups I , III , and IV  had more negative values  pattern of IBD when considering pairwise raw genetic distances and Haversine geographic distances. However, the greatest density of pairwise comparisons were centered at approximately 400 Km and 0.02 genetic distance (P = 0.894) and IV  showed no significant pattern of isolation by distance, while Groups III  and V  showed weak but significant pairwise relationships between geographic and genetic distances. A moderate and significant IBD pattern was detected among Texas P. pustulosa populations that were included in Groups III, IV, and V   but significant (distance . Therefo= 0.001) .Cyclonaias are synonyms of C. pustulosa (currently P. pustulosa) owing to the lack of monophyly and minimal genetic distances  showed that geographic groups of sensu latoP. pustulosa  exhibit population differentiation  genetically nested into regional groups, which were generally congruent to the distribution of previously described species within sensu latoP. pustulosa  (C. pustulosa (Group I), C. mortoni (Group III), C. houstonensis (Group IV), and C. aurea (Group V)  .P. pustulosa populations from rivers draining into the Mississippi and the Great Lakes (Lake Erie basins) experienced high gene flow historically, which is evidenced by low population differentiation among river drainages , but Groups III and V showed weak but significant patterns of IBD. However, when analyzed together, populations within Groups III, IV, and III showed a moderate IBD pattern that demonstrates the contrasting population dynamics of P. pustulosa within Texas when compared to the rest of the range  exhibited a more ragged distribution indicating stable historical populations, while Groups I (Mississippi–Great Lakes), III , and IV (Brazos–Colorado) showed primarily unimodal distributions suggesting historical bottlenecks and subsequent demographic expansions, likely at different times given the differences in mode . These r sharing  may have sharing . Howeversensu latoPustulosa pustulosa  may comprise a single species based on phylogenetic delimitation methods  were found for populations from the Pascagoula/Pearl rivers and future investigations should increase population-level sampling in Louisiana and Mississippi and other undersampled areas . Increased sampling may reveal additional geographically partitioned groups. For example, haplotypes from Group V form two separate clusters in the haplotype network , Imperiled (S2), or Critically Imperiled (S1) . Population genetics studies on other unionid mussel species have also suggested considering genetically distinct populations in management strategies , evolutionary trajectories and likely represent distinct ESUs. Therefore, assisted migration between regional groups should be avoided to prevent potentially disrupting adaptive gene complexes  encompassing a similar sampling regime for aquatic organisms that included rivers from Texas, the Mississippi, and Great Lakes basins in a single study. Most studies provide valuable phylogeographic treatments of related aquatic species or species delimitation within closely related groups , which included samples from Texas, the mid and lower Mississippi basin, and Florida, used both microsatellites and mitochondrial control region haplotypes to infer the population genetics of this species  than among populations within the Mississippi basin , and higher differentiation between Texas populations and those within the Mississippi basin (average ΦST = 0.28)  see . While cST) among P. pustulosa populations when compared to rivers within the Mississippi drainage and rivers associated with the Great Lakes. Such high differentiation among river basins in Texas has been shown by previous ecological and genetic analyses of mussels and fish, which mussels need for large-scale dispersal during their parasitic larval stage on host fish. For example, Spotted Bass showed large genetic divergence between river basins (e.g., Texas Rivers were characterized by more population subdivision (higher estimates of pairwise Φr basins , and musr basins . Yet, mons e.g.,  are stil10.7717/peerj.15974/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15974/supp-2Supplemental Information 2Click here for additional data file.