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The trematodes that cause schistosomiasis in humans require aquatic snails as intermediate hosts. Identifying the genes in snails at which allelic variation controls resistance to infection by schistosomes could lead to novel ways to break the cycle of transmission. We therefore mapped genetic variation within the BS90 population of Biomphalaria glabrata snails that controls their resistance to infection by the SmLE population of Schistosoma mansoni. A marker in the PTC2 genomic region strongly associates with variation in resistance. The S-haplotype, which confers increased susceptibility, appears to be almost completely dominant to the R-haplotype, which confers increased resistance. This result suggests a model in which the parasite must match a molecule on the host side to successfully infect. The genomic region surrounding our marker shows high structural and sequence variability between haplotypes. It is also highly enriched for genes that code for single-pass transmembrane (TM1) genes. Several of the TM1 genes present on the S-haplotype lack orthologs on the R-haplotype, which makes them intriguing candidate genes in a model of dominant susceptibility. These results add to a growing body of work that suggests TM1 genes, especially those in this exceptionally diverse genomic region, may play an important role in snail-schistosome compatibility polymorphisms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Blouin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The two main Afrotropical malaria vectors - Anopheles coluzzii and An. gambiae - are genetically distinct and reproductively isolated across West Africa. However, populations at the western extreme of their range are assigned as "intermediate" between the two species by whole genome sequence (WGS) data, and as hybrid forms by conventional molecular diagnostics. By exploiting WGS data from 1190 specimens collected across west Africa via the Anopheles gambiae 1000 Genomes network, we identified a putative taxon in the far-west (provisionally named Bissau molecular form), which did not arise by admixture but rather may have originated at the same time as the split between An. coluzzii and An. gambiae. Intriguingly, this taxon lacks insecticide resistance mechanisms commonly observed in the two main species. These findings lead to a change of perspective on malaria vector species in the far-west region with potential for epidemiological implications, and a new challenge for genetic-based mosquito control approaches., (© 2024. The Author(s).)

Schistosomiasis, afflicting >260 million people worldwide, could be controlled by preventing infection of freshwater snail vectors. Intestinal schistosomiasis, caused by Schistosoma mansoni , occurs predominantly in Sub-Saharan Africa and is vectored by Biomphalaria sudanica and related Biomphalaria species. Despite their importance in transmission, very little genomic work has been initiated in African snails, thus hindering development of novel control strategies. To identify genetic factors influencing snail resistance to schistosomes, we performed a pooled genome-wide association study (pooled-GWAS) on the offspring of B. sudanica collected from a persistent hotspot of schistosomiasis in Lake Victoria, Kenya, and exposed to sympatric S. mansoni . Results of the pooled-GWAS were used to develop an amplicon panel to validate candidate loci by genotyping individual snails. This validation revealed two previously uncharacterized, evolutionarily dynamic regions, SudRes1 and SudRes2 , that were significantly associated with resistance. SudRes1 includes receptor-like protein tyrosine phosphatases and SudRes2 includes a class of leucine-rich repeat-containing G-protein coupled receptors, both comprising diverse extracellular binding domains, suggesting roles in pathogen recognition. No loci previously tied to schistosome resistance in other snail species showed any association with compatibility suggesting that loci involved in the resistance of African vectors differ from those of neotropical vectors. Beyond these two loci, snail ancestry was strongly correlated with schistosome compatibility, indicating the importance of population structure on transmission dynamics and infection risk. These results provide the first detail of the innate immune system of the major schistosome vector, B. sudanica , informing future studies aimed at predicting and manipulating vector competence., Competing Interests: Competing Interest Statement: The authors declare no competing interests

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites and the lack of specific drugs necessitate the discovery of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of Babesia spp. ( B. bovis and B. divergens ). We identified a potent antibabesial, MMV019266, from the Malaria Box, and selected for resistance in two species of Babesia . After sequencing of multiple independently derived lines in the two species, we identified mutations in a membrane-bound metallodependent phosphatase ( phoD ). In both species, the mutations were found in the phoD-like phosphatase domain. Using reverse genetics, we validated that mutations in bdphoD confer resistance to MMV019266 in B. divergens . We have also demonstrated that BdPhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of BdPhoD alter the sensitivity to MMV019266 in the parasite. Overexpression of BdPhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting BdPhoD is a pro-susceptibility factor. Together, we have generated a robust pipeline for identification of resistance loci and identified BdPhoD as a resistance mechanism in Babesia species., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Control and elimination of schistosomiasis is an arduous task, with current strategies proving inadequate to break transmission. Exploration of genetic approaches to interrupt Schistosoma mansoni transmission, the causative agent for human intestinal schistosomiasis in sub-Saharan Africa and South America, has led to genomic research of the snail vector hosts of the genus Biomphalaria. Few complete genomic resources exist, with African Biomphalaria species being particularly underrepresented despite this being where the majority of S. mansoni infections occur. Here we generate and annotate the first genome assembly of Biomphalaria sudanica sensu lato, a species responsible for S. mansoni transmission in lake and marsh habitats of the African Rift Valley. Supported by whole-genome diversity data among five inbred lines, we describe orthologs of immune-relevant gene regions in the South American vector B. glabrata and present a bioinformatic pipeline to identify candidate novel pathogen recognition receptors (PRRs)., Results: De novo genome and transcriptome assembly of inbred B. sudanica originating from the shoreline of Lake Victoria (Kisumu, Kenya) resulted in a haploid genome size of ~ 944.2 Mb (6,728 fragments, N50 = 1.067 Mb), comprising 23,598 genes (BUSCO = 93.6% complete). The B. sudanica genome contains orthologues to all described immune genes/regions tied to protection against S. mansoni in B. glabrata, including the polymorphic transmembrane clusters (PTC1 and PTC2), RADres, and other loci. The B. sudanica PTC2 candidate immune genomic region contained many PRR-like genes across a much wider genomic region than has been shown in B. glabrata, as well as a large inversion between species. High levels of intra-species nucleotide diversity were seen in PTC2, as well as in regions linked to PTC1 and RADres orthologues. Immune related and putative PRR gene families were significantly over-represented in the sub-set of B. sudanica genes determined as hyperdiverse, including high extracellular diversity in transmembrane genes, which could be under pathogen-mediated balancing selection. However, no overall expansion in immunity related genes was seen in African compared to South American lineages., Conclusions: The B. sudanica genome and analyses presented here will facilitate future research in vector immune defense mechanisms against pathogens. This genomic/transcriptomic resource provides necessary data for the future development of molecular snail vector control/surveillance tools, facilitating schistosome transmission interruption mechanisms in Africa., (© 2024. The Author(s).)

Hookworm infection is a major public health problem in many regions of the world. Given the high levels of host morbidity and even mortality of the host caused by these infections, it is crucial to understand the genetic structure of hookworm populations. This understanding can provide insights into the ecology, transmission patterns, mechanisms of drug resistance, and the development of vaccines and immunotherapeutic strategies. Previously, we examined presumably neutral molecular markers, such as microsatellites and COI (Cytochrome C oxidase subunit 1) in Brazilian populations of Ancylostoma caninum. Here we analyze the molecular variability of a genomic fragment of the Aca-asp-2 (Ancylostoma secreted protein-2) gene from Ancylostoma caninum. This gene is a highly expressed and activated following the infection of the L3 larvae in the host. We obtained individuals of A. caninum from five different geographic locations in Brazil, sequenced and analyzed parts of the gene. The results revealed extensive polymorphism at this fragment, especially in the intronic region, indicating low selective pressure acting on these sequences. However, we also observed irregular distributions of nucleotides and polymorphisms in the coding region of this gene, resulting in the identification of 27 alleles. The data presented here contribute to expanding the understanding of population genetic studies of hookworms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Biomphalaria glabrata is a snail intermediate host for Schistosoma mansoni , a trematode responsible for human schistosomiasis. BS90 is one of the most well studied strains of B. glabrata owing to its high resistance to infection by most strains of S. mansoni . An F2 mapping study from 1999 identified two RAPD markers that associated with what appeared to be single-locus, dominant resistance by the BS90 population relative to the susceptible M-line population. One marker cannot be mapped, but the other, OPM-04 , maps to within 5 Mb of PTC2 , a region we recently showed has a very large effect on resistance within another snail population challenged by the same strain of parasite (PR1). Here we tested the hypothesis that the PTC2 region contains the causal gene/s that explain the iconic resistance of BS90 snails., Methods: We used marker-assisted backcrossing to drive the BS90 version of the PTC2 region (+/-~1 Mb on either side) into an M-line (susceptible strain) genetic background, and the M-line version into a BS90 genetic background. We challenged the offspring with PR1-strain schistosomes and tested for effects of allelic variation in the PTC2 region in a common genetic background., Results: Relative to M-line haplotypes, the BS90 haplotype actually confers enhanced susceptibility. So we reject our original hypothesis. One possible explanation for our result was that the causal gene linked to OPM-04 is near, but not in the PTC2 block that we introgressed into each line. So we used an F2 cross to independently test the effects of the PTC2 and OPM-04 regions in a randomized genetic background. We confirmed that the BS90 haplotype confers increased susceptibility, and we see a similar, although non-significant effect at OPM-04 . We discuss possible reasons why our results differed so dramatically from those of the 1999 study. We also present Pacbio assemblies of the PTC2 and flanking region in BS90 and M-line, compare with previously published PTC2 haplotypes, and discuss candidate genes that might be behind the enhanced susceptibility of the BS90 haplotype., Competing Interests: The authors declare that they have no competing interests., (© 2022 Blouin et al.)

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance., Competing Interests: The authors have declared that no competing interests exist.

Malaria has been one of the strongest selective pressures on our species. Many of the best-characterized cases of adaptive evolution in humans are in genes tied to malaria resistance. However, the complex evolutionary patterns at these genes are poorly captured by standard scans for nonneutral evolution. Here, we present three new statistical tests for selection based on population genetic patterns that are observed more than once among key malaria resistance loci. We assess these tests using forward-time evolutionary simulations and apply them to global whole-genome sequencing data from humans, and thus we show that they are effective at distinguishing selection from neutrality. Each test captures a distinct evolutionary pattern, here called Divergent Haplotypes, Repeated Shifts, and Arrested Sweeps, associated with a particular period of human prehistory. We clarify the selective signatures at known malaria-relevant genes and identify additional genes showing similar adaptive evolutionary patterns. Among our top outliers, we see a particular enrichment for genes involved in erythropoiesis and for genes previously associated with malaria resistance, consistent with a major role for malaria in shaping these patterns of genetic diversity. Polymorphisms at these genes are likely to impact resistance to malaria infection and contribute to ongoing host-parasite coevolutionary dynamics., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Anopheles gambiae complex consists of multiple morphologically indistinguishable mosquito species including the most important vectors of the malaria parasite Plasmodium falciparum in sub-Saharan Africa. Nine cryptic species have been described so far within the complex. The ecological, immunological and reproductive differences among these species will critically impact population responses to disease control strategies and environmental changes. Here, we examine whole-genome sequencing data from a longitudinal study of putative A. coluzzii in western Burkina Faso. Surprisingly, many specimens are genetically divergent from A. coluzzii and all other Anopheles species and represent a new taxon, here designated Anopheles TENGRELA (AT). Population genetic analysis suggests that the cryptic GOUNDRY subgroup, previously collected as larvae in central Burkina Faso, represents an admixed population descended from both A. coluzzii and AT. AT harbours low nucleotide diversity except for the 2La inversion polymorphism which is maintained by overdominance. It shows numerous fixed differences with A. coluzzii concentrated in several regions reflecting selective sweeps, but the two taxa are identical at standard diagnostic loci used for taxon identification, and thus, AT may often go unnoticed. We present an amplicon-based genotyping assay for identifying AT which could be usefully applied to numerous existing samples. Misidentified cryptic taxa could seriously confound ongoing studies of Anopheles ecology and evolution in western Africa, including phenotypic and genotypic surveys of insecticide resistance. Reproductive barriers between cryptic species may also complicate novel vector control efforts, for example gene drives, and hinder predictions about evolutionary dynamics of Anopheles and Plasmodium., (© 2020 John Wiley & Sons Ltd.)

Schistosomiasis is a debilitating parasitic disease infecting hundreds of millions of people. Schistosomes use aquatic snails as intermediate hosts. A promising avenue for disease control involves leveraging innate host mechanisms to reduce snail vectorial capacity. In a genome-wide association study of Biomphalaria glabrata snails, we identify genomic region PTC2 which exhibits the largest known correlation with susceptibility to parasite infection (>15 fold effect). Using new genome assemblies with substantially higher contiguity than the Biomphalaria reference genome, we show that PTC2 haplotypes are exceptionally divergent in structure and sequence. This variation includes multi-kilobase indels containing entire genes, and orthologs for which most amino acid residues are polymorphic. RNA-Seq annotation reveals that most of these genes encode single-pass transmembrane proteins, as seen in another resistance region in the same species. Such groups of hyperdiverse snail proteins may mediate host-parasite interaction at the cell surface, offering promising targets for blocking the transmission of schistosomiasis., Competing Interests: JT, SB, EP, BK, CS, BH, MB No competing interests declared, (© 2020, Tennessen et al.)

Freshwater snails are obligate intermediate hosts for numerous parasitic trematodes, most notably schistosomes. Schistosomiasis is a devastating human and veterinary illness, which is primarily controlled by limiting the transmission of these parasites from their intermediate snail hosts. Understanding how this transmission occurs, as well as the basic immunobiology of these snails may be important for controlling this disease in the future. Allelic variation in the Guadeloupe resistance complex (GRC) of Biomphalaria glabrata partially determines their susceptibility to parasitic infection, and can influence the microbiome diversity and microbial defenses in the hemolymph of these snails. In the present study, we examine the most abundant proteins present in the hemolymph of snails that are resistant or susceptible to schistosomes, as determined by their GRC genotype. Using proteomic analysis, we found that snails with different GRC genotypes have differentially abundant hemolymph proteins that are not explained by differences in transcription. There are 13 revealed hemolymph proteins that differ significantly between resistant and susceptible genotypes, nearly 40% of which are involved in immune responses. These findings build on the mounting evidence that genes in the GRC region have multiple physiological roles, and likely contribute more extensively to the general immune response than previously believed. These data also raise the intriguing possibility that the GRC region controls resistance to schistosomes, not directly, but indirectly via its effects on the snail's proteome and potentially its microbiome., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduced species often colonize regions that have vastly different ecological and environmental conditions than those found in their native range. As such, species introductions can provide a deeper understanding into the process of adaptive evolution. In the 1880s, steelhead trout (Oncorhynchus mykiss) from California were introduced into Lake Michigan (Laurentian Great Lakes, North America) where they established naturally reproducing populations. In their native range, steelhead hatch in rivers, migrate to the ocean and return to freshwater to spawn. Steelhead in Lake Michigan continue to swim up rivers to spawn, but now treat the freshwater environment of the Great Lakes as a surrogate ocean. To examine the effects of this introduction, we sequenced the genomes of 264 fish. By comparing steelhead from Lake Michigan to steelhead from their ancestral range, we determined that the introduction led to consistent reductions in genetic diversity across all 29 chromosomes. Despite this reduction in genetic diversity, three chromosomal regions were associated with rapid genetic adaptation to the novel environment. The first region contained functional changes to ceramide kinase, which likely altered metabolic and wound-healing rates in Lake Michigan steelhead. The second and third regions encoded carbonic anhydrases and a solute carrier protein, both of which are critical for osmoregulation, and demonstrate how steelhead physiologically adapted to freshwater. Furthermore, the contemporary release of diverse hatchery strains into the lake increased genetic diversity but reduced the signature of genetic adaptation. This study illustrates that species can rapidly adapt to novel environments despite genome-wide reductions in genetic diversity., (© 2018 John Wiley & Sons Ltd.)

Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes-but conservation of the master sex-determining genes-characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxonomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific "SDR cassette" contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phylogenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the "souvenir" sequence-and the resultant expansion of the hemizygous SDR over time-could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the insertion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism ("move-lock-grow") for expansion and diversification of incipient sex chromosomes., Competing Interests: The authors have declared that no competing interests exist.

The fates of genetic polymorphisms maintained by balancing selection depend on evolutionary dynamics at linked sites. While coevolution across linked, epigenetically-interacting loci has been extensively explored, such supergenes may be relatively rare. However, genes harboring adaptive variation can occur in close physical proximity while generating independent effects on fitness. Here, I present a model in which two linked loci without epistasis are both under balancing selection for unrelated reasons. Using forward-time simulations, I show that recombination rate strongly influences the retention of adaptive polymorphism, especially for intermediate selection coefficients. A locus is more likely to retain adaptive variation if it is closely linked to another locus under balancing selection, even if the two loci have no interaction. Thus, two linked polymorphisms can both be retained indefinitely even when they would both be lost to drift if unlinked. While these results may be intuitive, they have important implications for genetic architecture: clusters of mutually reinforcing genes may underlie phenotypic variation in natural populations, and such genes cannot be assumed to be functionally associated. Future studies that measure selection coefficients and recombination rates among closely linked genes will be fruitful for characterizing the extent of this phenomenon., Competing Interests: The author declares that they have no competing interests.

Freshwater snails are the intermediate hosts for numerous parasitic worms which can have negative consequences for human health and agriculture. Understanding the transmission of these diseases requires a more complete characterization of the immunobiology of snail hosts. This includes the characterization of its microbiome and genetic factors which may interact with this important commensal community. Allelic variation in the Guadeloupe resistance complex (GRC) genomic region of Guadeloupean Biomphalaria glabrata influences their susceptibility to schistosome infection and may have other roles in the snail immune response. In the present study, we examined whether a snail's GRC genotype has a role in shaping the bacterial diversity and composition present on or in whole snails. We show that the GRC haplotype, including the resistant genotype, has a significant effect on the diversity of bacterial species present in or on whole snails, including the relative abundances of Gemmatimonas aurantiaca and Micavibrio aeruginosavorus. These findings support the hypothesis that the GRC region is likely involved in pathways that can modify the microbial community of these snails and may have more immune roles in B. glabrata than originally believed. This is also one of few examples in which allelic variation at a particular locus has been shown to affect the microbiome in any species.

Premise of the Study: Recurrent formation of polyploid taxa is a common observation in many plant groups. Haploid, cytoplasmic genomes like the plastid genome can be used to overcome the problem of homeologous genes and recombination in polyploid taxa. Fragaria (Rosaceae) contains several octo- and decaploid species. We use plastome sequences to infer the plastid ancestry of these taxa with special focus on the decaploid Fragaria cascadensis., Methods: We used genome skimming of 96 polyploid Fragaria samples on a single Illumina HiSeq 3000 lane to obtain whole plastome sequences. These sequences were used for phylogenetic reconstructions and dating analyses. Ploidy of all samples was inferred with flow cytometry, and plastid inheritance was examined in a controlled cross of F. cascadensis., Key Results: The plastid genome phylogeny shows that only the octoploid F. chiloensis is monophyletic, all other polyploid taxa were supported to be para- or polyphyletic. The decaploid Fragaria cascadensis has biparental plastid inheritance and four different plastid donors. Diversification of the F. cascadensis clades occurred in the last 230,000 years. The southern part of its distribution range harbors considerably higher genetic diversity, suggestive of a potential refugium., Conclusions: Fragaria cascadensis had at least four independent origins from parents with different plastomes. In contrast, para- and polyphyletic taxa of the octoploid Fragaria species are best explained by incomplete lineage sorting and/or hybridization. Biogeographic patterns in F. cascadensis are probably a result of range shift during the last glacial maximum., (© 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

With their extraordinary diversity in sexual systems, flowering plants offer unparalleled opportunities to understand sex determination and to reveal generalities in the evolution of sex chromosomes. Comparative genetic mapping of related taxa with good phylogenetic resolution can delineate the extent of sex chromosome diversity within plant groups, and lead the way to understanding the evolutionary drivers of such diversity. The North American octoploid wild strawberries provide such an opportunity. We performed linkage mapping using targeted sequence capture for the subdioecious western Fragaria virginiana ssp. platypetala and compared the location of its sex-determining region (SDR) to those of 2 other (sub)dioecious species, the eastern subspecies, F. virginiana ssp. virginiana (whose SDR is at 0-5.5 Mb on chromosome VI of the B2 subgenome), and the sister species F. chiloensis (whose SDR is at 37 Mb on chromosome VI of the Av subgenome). Male sterility was dominant in F. virginiana ssp. platypetala and mapped to a chromosome also in homeologous group VI. Likewise, one major quantitative trait locus (QTL) for female fertility overlapped the male sterility region. However, the SDR mapped to yet another subgenome (B1), and to a different location (13 Mb), but similar to the location inferred in one population of the naturally occurring hybrid between F. chiloensis and F. virginiana (F. ×ananassa ssp. cuneifolia). Phylogenetic analysis of chromosomes across the octoploid taxa showed consistent subgenomic composition reflecting shared evolutionary history but also reinforced within-species variation in the SDR-carrying chromosome, suggesting either repeated evolution, or recent turnovers in SDR., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Disentangling the evolutionary histories of polyploids, especially those with high ploidies, can reveal fundamental processes in speciation. Despite occurring frequently during evolution, the origins of many extant polyploid plant species remain largely unknown. By integrating linkage mapping, polyploid phylogeny and sex-determining region (SDR) in a unified framework, we statistically evaluated evolutionary hypotheses concerning the origin of a recently recognized decaploid strawberry (Fragaria cascadensis). The maximum-likelihood phylogenies and topology tests across homeologous groups consistently rejected the seemingly parsimonious hypothesis of 'contemporary sympatric speciation' via hybridization between octoploid and diploid congeners. Instead, most chromosomes supported 'ancient hybrid speciation' between a maternal octoploid progenitor ancestral to extant octoploid strawberries and a paternal, extinct Fragaria iinumae-like diploid progenitor, probably in Beringia during the Pleistocene. The absence of a shared SDR between the decaploid and other Fragaria is also consistent with an older origin rather than a recent hybrid origin in situ. Our study reveals a long evolutionary history of the decaploid despite its recent discovery, and highlights the pitfalls of inferring polyploid origins from niche/range alone or combined with morphology. It can serve as an exemplary starting step towards building much-needed model systems of established polyploids that have been, and remain to be, recognized., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Genotyping-by-sequencing (GBS) was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry ( Fragaria ×  ananassa ) populations with the goal of evaluating this technique in a species with a complex octoploid genome. GBS sequence data were aligned to the F. vesca ' Fvb ' reference genome in order to call SNPs. Numbers of polymorphic SNPs per population ranged from 1,163 to 3,190. Linkage maps consisting of 30-65 linkage groups were produced from the SNP sets derived from each parent. The linkage groups covered 99% of the Fvb reference genome, with three to seven linkage groups from a given parent aligned to any particular chromosome. A phylogenetic analysis performed using the POLiMAPS pipeline revealed linkage groups that were most similar to ancestral species F. vesca for each chromosome. Linkage groups that were most similar to a second ancestral species, F. iinumae , were only resolved for Fvb 4. The quantity of missing data and heterogeneity in genome coverage inherent in GBS complicated the analysis, but POLiMAPS resolved F. ×  ananassa chromosomal regions derived from diploid ancestor F. vesca., Competing Interests: The authors declare there are no competing interests.

This corrects the article DOI: 10.1038/ncomms15451.

The aquatic planorbid snail Biomphalaria glabrata is one of the most intensively-studied mollusks due to its role in the transmission of schistosomiasis. Its 916 Mb genome has recently been sequenced and annotated, but it remains poorly assembled. Here, we used targeted capture markers to map over 10,000 B. glabrata scaffolds in a linkage cross of 94 F1 offspring, generating 24 linkage groups (LGs). We added additional scaffolds to these LGs based on linkage disequilibrium (LD) analysis of targeted capture and whole-genome sequences of 96 unrelated snails. Our final linkage map consists of 18,613 scaffolds comprising 515 Mb, representing 56% of the genome and 75% of genic and nonrepetitive regions. There are 18 large (> 10 Mb) LGs, likely representing the expected 18 haploid chromosomes, and > 50% of the genome has been assigned to LGs of at least 17 Mb. Comparisons with other gastropod genomes reveal patterns of synteny and chromosomal rearrangements. Linkage relationships of key immune-relevant genes may help clarify snail-schistosome interactions. By focusing on linkage among genic and nonrepetitive regions, we have generated a useful resource for associating snail phenotypes with causal genes, even in the absence of a complete genome assembly. A similar approach could potentially improve numerous poorly-assembled genomes in other taxa. This map will facilitate future work on this host of a serious human parasite., (Copyright © 2017 Tennessen et al.)

Considering the great efforts towards formulating a vaccine against hookworms, and the concerns about the spread of drug resistance through hookworm populations, it is justified to study the molecular diversity and population genetic structure of these nematodes. This work had the aim to develop microsatellite markers to investigate the genetic structure and the molecular diversity of Brazilian populations of Ancylostoma caninum. Seven microsatellites markers were successfully used to characterize five Brazilian populations. These findings may contribute to a better comprehension of the ecology, patterns of transmission, drug resistances and development of immunotherapeutic strategies in hookworms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.

Recombination in ancient, heteromorphic sex chromosomes is typically suppressed at the sex-determining region (SDR) and proportionally elevated in the pseudoautosomal region (PAR). However, little is known about recombination dynamics of young, homomorphic plant sex chromosomes. We examine male and female function in crosses and unrelated samples of the dioecious octoploid strawberry Fragaria chiloensis in order to map the small and recently evolved SDR controlling both traits and to examine recombination patterns on the incipient ZW chromosome. The SDR of this ZW system is located within a 280 kb window, in which the maternal recombination rate is lower than the paternal one. In contrast to the SDR, the maternal PAR recombination rate is much higher than the rates of the paternal PAR or autosomes, culminating in an elevated chromosome-wide rate. W-specific divergence is elevated within the SDR and a single polymorphism is observed in high species-wide linkage disequilibrium with sex. Selection for recombination suppression within the small SDR may be weak, but fluctuating sex ratios could favor elevated recombination in the PAR to remove deleterious mutations on the W. The recombination dynamics of this nascent sex chromosome with a modestly diverged SDR may be typical of other dioecious plants., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Whitebark pine (Pinus albicaulis) inhabits an expansive range in western North America, and it is a keystone species of subalpine environments. Whitebark is susceptible to multiple threats - climate change, white pine blister rust, mountain pine beetle, and fire exclusion - and it is suffering significant mortality range-wide, prompting the tree to be listed as 'globally endangered' by the International Union for Conservation of Nature and 'endangered' by the Canadian government. Conservation collections (in situ and ex situ) are being initiated to preserve the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. We evaluated the use of hybridization-based target capture to enrich specific genomic regions from the 27 GB genome of whitebark pine, and to evaluate genetic variation across loci, trees, and geography. Probes were designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32% of targeted positions (528,873 bp), and we were able to identify 12,390 segregating sites from 47 trees. Variations reveal strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Range showing the greatest distinctiveness and differentiation. Our results show that even under non-optimal conditions (low enrichment efficiency; inclusion of repetitive elements in baits), targeted enrichment produces high quality, codominant genotypes from large genomes. The resulting data can be readily integrated into management and gene conservation activities for whitebark pine, and have the potential to be applied to other members of 5-needle pine group (Pinus subsect. Quinquefolia) due to their limited genetic divergence.

Gynodioecy, the coexistence of females and hermaphrodites, occurs in 20% of angiosperm families and often enables transitions between hermaphroditism and dioecy. Clarifying mechanisms of sex determination in gynodioecious species can thus illuminate sexual system evolution. Genetic determination of gynodioecy, however, can be complex and is not fully characterized in any wild species. We used targeted sequence capture to genetically map a novel nuclear contributor to male sterility in a self-pollinated hermaphrodite of Fragaria vesca subsp. bracteata from the southern portion of its range. To understand its interaction with another identified locus and possibly additional loci, we performed crosses within and between two populations separated by 2000 km, phenotyped the progeny and sequenced candidate markers at both sex-determining loci. The newly mapped locus contains a high density of pentatricopeptide repeat genes, a class commonly involved in restoration of fertility caused by cytoplasmic male sterility. Examination of all crosses revealed three unlinked epistatically interacting loci that determine sexual phenotype and vary in frequency between populations. Fragaria vesca subsp. bracteata represents the first wild gynodioecious species with genomic evidence of both cytoplasmic and nuclear genes in sex determination. We propose a model for the interactions between these loci and new hypotheses for the evolution of sex determining chromosomes in the subdioecious and dioecious Fragaria., (Copyright © 2015 Ashman et al.)

Background: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails., Methodology/principal Findings: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated., Conclusions/significance: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.

Premise of the Study: Molecular phylogenies derived from all three plant genomes can provide insight into the evolutionary history of plant groups influenced by reticulation. We sought to reconstruct mitochondrial exome, chloroplast, and nuclear genome phylogenies for octoploid Fragaria and their diploid ancestors and to document patterns of incongruence between and within the cytoplasmic genomes and interpret these in the context of evolutionary origin of the octoploid strawberries., Methods: Using a genome-skimming approach, we assembled chloroplast genomes and mitochondrial exomes, and we used the POLiMAPS method to assemble nuclear sequence for octoploid species and constructed phylogenies from all three genomes. We assessed incongruence between and within cytoplasmic genomes using topology-based phylogenetic incongruence tests., Key Results: The incongruent cytoplasmic genome phylogeny with respect to the placement of octoploids suggests potential breakage in linkage disequilibrium of cytoplasmic genomes during allopolyploid origin of the octoploids. Furthermore, a single mitochondrial chimeric gene with a putative role in cytoplasmic male sterility yields a phylogeny that is inconsistent with the rest of the mitochondrial genome but consistent with the chloroplast phylogeny, suggesting intracellular gene transfer between heteroplasmic mitochondria, possibly driven by selection to overcome the effects of mito-nuclear incompatibility in octoploid origins., Conclusions: This work expands on the current understanding of evolutionary history of the octoploid ancestors of cultivated strawberry. It demonstrates phylogenetic incongruence between cytoplasmic genomes in octoploids with respect to diploid ancestors, indicating breakage in linkage disequilibrium of cytoplasmic genomes. We discuss potential organism-level processes that may have contributed to the observed incongruence in Fragaria., (© 2015 Botanical Society of America, Inc.)

Schistosomiasis, a neglected global pandemic, may be curtailed by blocking transmission of the parasite via its intermediate hosts, aquatic snails. Elucidating the genetic basis of snail-schistosome interaction is a key to this strategy. Here we map a natural parasite-resistance polymorphism from a Caribbean population of the snail Biomphalaria glabrata. In independent experimental evolution lines, RAD genotyping shows that the same genomic region responds to selection for resistance to the parasite Schistosoma mansoni. A dominant allele in this region conveys an 8-fold decrease in the odds of infection. Fine-mapping and RNA-Seq characterization reveal a <1Mb region, the Guadeloupe Resistance Complex (GRC), with 15 coding genes. Seven genes are single-pass transmembrane proteins with putative immunological roles, most of which show strikingly high nonsynonymous divergence (5-10%) among alleles. High linkage disequilibrium among three intermediate-frequency (>25%) haplotypes across the GRC, a significantly non-neutral pattern, suggests that balancing selection maintains diversity at the GRC. Thus, the GRC resembles immune gene complexes seen in other taxa and is likely involved in parasite recognition. The GRC is a potential target for controlling transmission of schistosomiasis, including via genetic manipulation of snails.

Whole-genome duplications are radical evolutionary events that have driven speciation and adaptation in many taxa. Higher-order polyploids have complex histories often including interspecific hybridization and dynamic genomic changes. This chromosomal reshuffling is poorly understood for most polyploid species, despite their evolutionary and agricultural importance, due to the challenge of distinguishing homologous sequences from each other. Here, we use dense linkage maps generated with targeted sequence capture to improve the diploid strawberry (Fragaria vesca) reference genome and to disentangle the subgenomes of the wild octoploid progenitors of cultivated strawberry, Fragaria virginiana and Fragaria chiloensis. Our novel approach, POLiMAPS (Phylogenetics Of Linkage-Map-Anchored Polyploid Subgenomes), leverages sequence reads to associate informative interhomeolog phylogenetic markers with linkage groups and reference genome positions. In contrast to a widely accepted model, we find that one of the four subgenomes originates with the diploid cytoplasm donor F. vesca, one with the diploid Fragaria iinumae, and two with an unknown ancestor close to F. iinumae. Extensive unidirectional introgression has converted F. iinumae-like subgenomes to be more F. vesca-like, but never the reverse, due either to homoploid hybridization in the F. iinumae-like diploid ancestors or else strong selection spreading F. vesca-like sequence among subgenomes through homeologous exchange. In addition, divergence between homeologous chromosomes has been substantially augmented by interchromosomal rearrangements. Our phylogenetic approach reveals novel aspects of the complicated web of genetic exchanges that occur during polyploid evolution and suggests a path forward for unraveling other agriculturally and ecologically important polyploid genomes., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Gynodioecy is a sexual system wherein females coexist with hermaphrodites. It is of interest not only because male-sterile plants are advantageous in plant breeding but also because it can be a crucial step in the evolutionary transition to entirely separate sexes (dioecy) from a hermaphroditic ancestor. The gynodioecious diploid wild strawberry, Fragaria vesca ssp. bracteata (Rosaceae), is a member of a clade with both dioecious and cultivated species, making it an ideal model in which to study the genetics of male sterility. To create a genetic map of F. v. ssp. bracteata, we identified informative polymorphisms from genomic sequencing (3-5x coverage) of two outbred plants from the same population. Using targeted enrichment, we sequenced 200 bp surrounding each of 6575 polymorphisms in 48 F1 offspring, yielding genotypes at 98% of targeted sites with mean coverage >100x, plus more than 600-kb high-coverage nontargeted sequence. With the resulting linkage map of 7802 stringently filtered markers (5417 targeted), we assessed recombination rates and genomic incongruities. Consistent with past work in strawberries, male sterility is dominant, segregates 1:1, and maps to a single location in the female. Further mapping an additional 55 offspring places male sterility in a gene-dense, 338-kb region of chromosome 4. The region is not syntenic with the sex-determining regions in the closely related octoploids, F. chiloensis and F. virginiana, suggesting either independent origins or translocation. The 57 genes in this region do not include protein families known to control male sterility and thus suggest alternate mechanisms for the suppression of male function.

Motivation: The goal of any parentage analysis is to identify as many parent-offspring relationships as possible, while minimizing incorrect assignments. Existing methods can achieve these ends, but they require additional information in the form of demographic data, thousands of markers and/or estimates of genotyping error rates. For many non-model systems, it is simply not practical, cost-effective or logistically feasible to obtain this information. Here, we develop a Bayesian parentage method that only requires the sampled genotypes to account for genotyping error, missing data and false matches., Results: Extensive testing with microsatellite and SNP datasets reveals that our Bayesian parentage method reliably controls for the number of false assignments, irrespective of the genotyping error rate. When the number of loci is limiting, our approach maximizes the number of correct assignments by accounting for the frequencies of shared alleles. Comparisons with exclusion and likelihood-based methods on an empirical salmon dataset revealed that our Bayesian method had the highest ratio of correct to incorrect assignments.

As a first step toward understanding how rare variants contribute to risk for complex diseases, we sequenced 15,585 human protein-coding genes to an average median depth of 111× in 2440 individuals of European (n = 1351) and African (n = 1088) ancestry. We identified over 500,000 single-nucleotide variants (SNVs), the majority of which were rare (86% with a minor allele frequency less than 0.5%), previously unknown (82%), and population-specific (82%). On average, 2.3% of the 13,595 SNVs each person carried were predicted to affect protein function of ~313 genes per genome, and ~95.7% of SNVs predicted to be functionally important were rare. This excess of rare functional variants is due to the combined effects of explosive, recent accelerated population growth and weak purifying selection. Furthermore, we show that large sample sizes will be required to associate rare variants with complex traits.

Exome sequencing is poised to yield substantial insights into human genetic variation and evolutionary history, but there are significant challenges to overcome before this becomes a reality.

Few genetic differences between human populations conform to the classic model of positive selection, in which a newly arisen mutation rapidly approaches fixation in one lineage, suggesting that adaptation more commonly occurs via moderate changes in standing variation at many loci. Detecting and characterizing this type of complex selection requires integrating individually ambiguous signatures across genomically and geographically extensive data. Here, we develop a novel approach to test the hypothesis that selection has favored modest divergence at particular loci multiple times in independent human populations. We find an excess of SNPs showing non-neutral parallel divergence, enriched for genic and nonsynonymous polymorphisms in genes encompassing diverse and often disease related functions. Repeated parallel evolution in the same direction suggests common selective pressures in disparate habitats. We test our method with extensive coalescent simulations and show that it is robust to a wide range of demographic events. Our results demonstrate phylogenetically orthogonal patterns of local adaptation caused by subtle shifts at many widespread polymorphisms that likely underlie substantial phenotypic diversity., Competing Interests: The authors have declared that no competing interests exist.

Exome sequences, which comprise all protein-coding regions, are promising data sets for studies of natural selection because they offer unbiased genome-wide estimates of polymorphism while focusing on the portions of the genome that are most likely to be functionally important. We examine genomic patterns of polymorphism within 10 diploid autosomal exomes of European and African descent. Using coalescent simulations, we show how polymorphism, site frequency spectra, and intercontinental divergence in these samples would be influenced by different modes of positive selection. We examine putatively selected loci from four previous genome-wide scans of SNP genotypes and demonstrate that these regions indeed show unusual population genetic patterns in the exome data. Using a series of conservative criteria based on exome polymorphism, we are able to fine-scale map signatures of selection, in many cases pinpointing a single candidate SNP. We also identify and evaluate novel candidate selection genes that show unusual patterns of polymorphism. We sequence a portion of one novel candidate locus, IVL, in 74 individuals from multiple continents and examine global genetic diversity. Thus, we confirm, narrow, and supplement existing catalogs of putative targets of selection, and show that exome data sets, which are likely to soon become common, will be powerful tools for identifying adaptive genetic variation.

Ranatuerins are antimicrobial peptides of the innate immune system found in ranid frogs. We previously presented evidence that a positive selective sweep had fixed a single allele at the Ranatuerin2 locus in the northern leopard frog (Rana pipiens). In this paper, we further investigate the evolutionary history of ranatuerins as follows. First, we sequenced Ranatuerin2 in additional individuals of R. pipiens and related frog species and compared diversity and divergence at these sequences with that at four putatively neutrally evolving loci. Second, we asked whether the evolutionary patterns observed at Ranatuerin2 were typical for ranatuerin loci by sequencing our samples at a paralogous locus, Ranatuerin2b, and performing the same neutrality tests. Ranatuerin2b also showed strong and significant evidence of at least one selective sweep. Third, we used the neutral loci to independently resolve conflicting hypotheses about phylogenetic relationships among our study species. Both the neutral loci and the ranatuerin loci supported an older phylogeny inferred from allozyme data and strongly rejected a more recent phylogeny inferred from mitochondrial DNA. Finally, in order to test whether the sweep was driven by the evolution of substantially new peptide function, we used the phylogeny to reconstruct the hypothetical Ranatuerin2 peptide that existed before the sweep. We synthesized this peptide and tested its activity and that of the extant peptide against six bacterial pathogens of frogs. We observed antibacterial activity but found no significant functional differences between the two peptides.

The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes.

Balancing selection is common on many defense genes, but it has rarely been reported for immune effector proteins such as antimicrobial peptides (AMPs). We describe genetic diversity at a brevinin-1 AMP locus in three species of leopard frogs (Rana pipiens, Rana blairi, and Rana palustris). Several highly divergent allelic lineages are segregating at this locus. That this unusual pattern results from balancing selection is demonstrated by multiple lines of evidence, including a ratio of nonsynonymous/synonymous polymorphism significantly higher than 1, the ZnS test, incongruence between the number of segregating sites and haplotype diversity, and significant Tajima's D values. Our data are more consistent with a model of fluctuating selection in which alleles change frequencies over time than with a model of stable balancing selection such as overdominance. Evidence for fluctuating selection includes skewed allele frequencies, low levels of synonymous variation, nonneutral values of Tajima's D within allelic lineages, an inverse relationship between the frequency of an allelic lineage and its degree of polymorphism, and divergent allele frequencies among populations. AMP loci could be important sites of adaptive genetic diversity, with consequences for host-pathogen coevolution and the ability of species to resist disease epidemics.

Motivation: Functional divergence among proteins is often assumed to be strongly influenced by natural selection, as inferred from the ratio of non-synonymous nucleotide divergence (d(N)) to synonymous nucleotide divergence (d(S)). That is, the more a mutation changes protein function, the more likely it is to be either selected against or selectively favored, and because the d(N)/d(S) ratio is a measure of natural selection, this ratio can be used to predict the degree of functional divergence (d(F)). However, these hypotheses have rarely been experimentally tested., Results: I present a novel method to address this issue, and demonstrate that divergence in bacteria-killing activity among animal antimicrobial peptides is positively correlated with the log of the d(N)/d(S) ratio. The primary cause of this pattern appears to be that positively selected substitutions change protein function more than neutral substitutions do. Thus, the d(N)/d(S) ratio is an accurate estimator of adaptive functional divergence., Contact: tennessj@science.oregonstate.edu, Supplementary Information: Supplementary data, including GenBank Accession numbers, are available at Bioinformatics online.

The mitochondrial cytochrome oxidase I gene was partially sequenced for 164 Ancylostoma caninum individuals, originating from five different localities in Brazil, with the aim of describing the genetic diversity and genetic structure of Brazilian hookworm populations. Allelic and nucleotide diversity were moderate (overall h=0.88 and pi=0.016) and were similar among cities. There was moderate genetic differentiation among the populations sampled (approximately Phi(ST)=0.12) and a weak but nonsignificant correlation between geographical and genetic distance. This genetic structure was similar to that observed among populations of the human hookworm, Necator americanus, but distinct from that typically found in trichostrongylid nematode parasites of livestock. Thus, a pattern of different genetic structures among different groups of nematodes is emerging. We also observed a few individuals that had a highly divergent mtDNA sequence (almost 7% sequence divergence from the other sequences). These results in combination with data from other studies suggest that A. caninum populations worldwide consist of a mix of previously differentiated populations, or perhaps even cryptic species. This study contributes to the knowledge of genetic structure and diversity of hookworms, which in turn will be useful in developing methods for their control.