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X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes

['John Mattick', 'Institute For Genome Science', 'University Of Maryland', 'Baltimore', 'Maryland', 'United States Of America', 'Silvia Libro', 'New England Biolabs', 'Ipswich', 'Massachusetts']

Date: 2021-12

Abstract The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (π X /π A = 0.2), which is lower than the expected (π X /π A = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Author summary Almost a billion people receive >7.7 billion doses of treatment aimed at eliminating lymphatic filariasis, which is caused by three filarial nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Drug resistance and adaptation are both associated with pathogen success as well as higher levels of genetic diversity. In an examination of genetic diversity in Brugia malayi and Brugia pahangi, we observed a lack of genetic diversity over a third of the genome that is found on chromosome X. These species have neo-X chromosomes where a chromosome X fused with an autosome. Using publicly-available published data, the other filarial nematodes of greatest human significance are also found to have a similar lack of genetic diversity on their neo-X chromosomes. The two filarial nematodes with publicly-available data that lack neo-X chromosomes did not have this lack of genetic diversity. This lack of sequence diversity in B. malayi, W. bancrofti, and O. volvulus could have profound effects on all traits encoded on chromosome X.

Citation: Mattick J, Libro S, Bromley R, Chaicumpa W, Chung M, Cook D, et al. (2021) X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes. PLoS Negl Trop Dis 15(10): e0009838. https://doi.org/10.1371/journal.pntd.0009838 Editor: Peter U. Fischer, Washington University School of Medicine, UNITED STATES Received: December 18, 2020; Accepted: September 24, 2021; Published: October 27, 2021 Copyright: © 2021 Mattick 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. Data Availability: All sequencing data is deposited in the SRA and can be found under the following run numbers, which are also detailed in S1 and S2 Tables: TRS B. malayi males: SRR3111504, SRR3111510, SRR3111514, SRR3111517. FR3 B. malayi males: SRR3111544, SRR3111568, SRR3111579, SRR3111581. The Liverpool School of Tropical Medicine B. malayi males: SRR3111629, SRR3111630, SRR3111634, SRR3111636, SRR3111640. Washington University B. malayi males: SRR3111318, SRR3111319, SRR3111488, SRR3111493, SRR3111498, SRR5190290, SRR5190289, SRR5190290, SRR5190289. Lucknow, India B. malayi males: SRR3111731, SRR3111738, SRR3111864, SRR3112012. Thailand B. malayi males: SRR12884294, SRR12884293, SRR12884292, SRR12884291. FR3 B. pahangi males: SRR7229557, SRR7244205, SRR13482041, SRR13482040, SRR13482039. FR3 B. pahangi females: SRR10997235, SRR10997259, SRR10997264, SRR10997265, SRR10997290, SRR10997293, SRR10997301, SRR10997315, SRR10997319, SRR10997320, SRR10997325. Kuala Lumpur B. pahangi males: SRR7226912, SRR7227476, SRR7227477, SRR7227478, SRR7227479. In addition, we used the following files from the SRA for the analysis presented in this paper. W. bancrofti samples: SRR8188284, SRR8188279, SRR8188269, SRR8188264, SRR8188300, SRR8188272, SRR8188273, SRR8188271. O. volvulus samples: SRR2924837, SRR2924836, SRR2924835, SRR2924834, SRR2924832, SRR2924830, SRR2924828, SRR2924826, SRR2924824, SRR2924823, SRR2924812, SRR2924811, SRR2924784, SRR2924783, SRR2924782, SRR2924781, SRR2924780, SRR2924779, SRR2924778, SRR2924733, SRR2924722, SRR2924721, SRR2924720, SRR2924719, SRR2924532, SRR2924467, SRR2924442, SRR2924439, SRR2924434, SRR2924383, SRR2924326, SRR2924211. L. loa samples: SRR3136724, SRR3136977, SRR3136722, SRR3136723, SRR3136973, SRR3136975, SRR3136979, SRR3140170, SRR3140171, SRR3136725, SRR3136972, SRR3136976. C. elegans samples: SRR9322180, SRR9322887, SRR9322632, SRR9322850, SRR9322406, SRR9322439, SRR9322420, SRR9322366, SRR9322508, SRR9322360, SRR9322172, SRR9322278, SRR9321994, SRR9322517, SRR9322739, SRR9322241, SRR9322893, SRR9322681, SRR9322222, SRR9322809, SRR9322720, SRR9322512, SRR9322507, SRR9322002, SRR9322671, SRR9322510, SRR9322657, SRR9322769. D. immitis samples: SRR10533236, SRR10533238, SRR10533239, SRR10533240, SRR10533237, SRR13154013, SRR13154014, SRR13154015, SRR13154016, SRR13154017. D. melanogaster samples: SRR189389, SRR306629, SRR306612, SRR306616, SRR306614, SRR306609, SRR306621, SRR306619, SRR306624, SRR306611, SRR306618, SRR189102, SRR218317, SRR189040, SRR189101, SRR189105. All code used for data analysis and generation of figures used in this project can be found at https://github.com/jeanmattique/BrugiaPopulationGenomics. Funding: This project has been funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under grant U19AI110820 (MM, JMF, JDH), as well as by the grants for in-lab maintenance of parasite (B. malayi, specifically) by the Council of Scientific and Industrial Research (CSIR- INDIA) viz projects Superainstitutional (SIP0026, CSIR 2006-11) and SplenDID (BSC 0104, CSIR 2012-17). This research work was also partially supported by Faculty of Medicine (Grant No. PAR-2563-07268) and Office of Research Administration, Chiang Mai University, Thailand, to AS (Atiporn Saeung). WASHU B. malayi life cycle maintenance was supported by the Barnes-Jewish Hospital Foundation. Adult B. malayi and B. pahangi males were obtained from the FR3 through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH. The funders have no role in this study design, data collection and analysis, or in the manuscript preparation. Competing interests: The authors have declared that no competing interests exist.

Introduction Brugia malayi, Wuchereria bancrofti, and Brugia timori are filarial nematodes (roundworms) that are responsible for lymphatic filariasis in humans with almost a billion people receiving >7.7 billion doses of treatment through lymphatic filariasis elimination efforts [1]. All filarial nematodes undergo a complex reproductive cycle that includes multiple larval stages within an arthropod vector followed by more larval stages, sexual development, and reproduction in vertebrate hosts [2]. Of the three filarial species responsible for human lymphatic filariasis, only a subset of B. malayi strains can be maintained in small animals in the laboratory, a prerequisite for rigorous laboratory-based studies. These laboratory populations are critical to our understanding of filarial biology, and are commonly used for anti-filarial drug trials [3]. Brugia pahangi can also be maintained in a laboratory life cycle, infects cats and dogs, and is occasionally zoonotic. B. pahangi and B. malayi use mosquito insect vectors and can co-infect dogs and cats [4]. Male B. malayi and female B. pahangi can produce viable offspring following mating in laboratory conditions [5,6], but the extent to which this happens successfully in nature is unknown. In addition to lymphatic filariasis, filarial nematodes are responsible for other diseases of medical and veterinary important, including human onchocerciasis [7] caused by the filarial nematode Onchocerca volvulus, human loiasis [8] caused by Loa loa, and dog and cat heartworm caused by Dirofilaria immitis [9]. Onchocerca volvulus [10], Brugia malayi [11–13], and Brugia pahangi [14] all have nearly complete genomes with chromosome-level assemblies of autosomes and chromosome X, while chromosome Y has yet to be resolved in any filarial nematode. Draft genomes are available for many other filarial nematodes [15], including W. bancrofti [16], L. loa [17], and D. immitis [18]. The genomes of all filarial nematodes are represented by six Nigon elements [12,19,20] that reflect conserved chromosomal segments that likely reflect the ancestral chromosome state in many nematodes, similar to Muller elements in Drosophila species [21]. In the case of filarial nematodes, the composition of these elements was primarily determined through homology to the completed genomes of O. volvulus, Caenorhabditis elegans, and/or B. malayi [12,19,20]. An important resource for filarial nematode research is the Filariasis Research Reagent Resource Center, better known as FR3, which maintains both B. malayi and B. pahangi worms across the life cycle in both Mongolian gerbils (jirds; Meriones unguiculatus) and cats [3]. At FR3, B. malayi and B. pahangi are passaged in cats via a mosquito vector. First, blood containing microfilariae is drawn from multiple cats, and pooled together. Then, this pooled blood is fed to mosquitos to allow microfilariae to develop to infective third-stage larvae (L3) which are extracted from mosquitos and introduced into an uninfected cat. Not all mosquitos survive infection with microfilariae, and not all infective L3 worms that are introduced into cats mature into viable adults. Infective L3s are also used to inoculate Mongolian gerbils that are used as a source of much of the material that is distributed by FR3. There are several steps where bottlenecks could occur, and different labs that maintain the life cycle have their own methods to prevent bottlenecks. Genetic diversity can be influenced by bottlenecks, polyandry, population size, sex-biased population size, sex-biased or sex-exclusive inheritance, the rate of recombination, the mutation rate, and selection [22,23]. Bottlenecks occur when there is a rapid reduction in the population size such that allele frequencies shift dramatically [24] and have been studied in other parasite species [25–27]. These bottlenecks can significantly reduce genomic variation, but the presence of alleles that confer survival advantages can also generate selective sweeps that produce similar reductions in genomic variation [28]. Sex chromosomes add additional complexity to genetic diversity. For instance, in heteromorphic sex chromosomes like those in X-Y sex determination systems (which includes some filarial nematodes), the X chromosome has reduced genetic diversity by virtue of reduced effective population size. In a population with random mating (e.g. one without polyandry), this results in ~0.75 variance on chromosome X and ~0.25 variance on chromosome Y relative to the autosomes, but in species with multiple mating, this variance can be reduced even further [29]. Though multiple centers across the globe maintain B. malayi in laboratories, many of these laboratory populations are derived from the same initial population. Several cats were experimentally infected in the early 1960s with a sub-periodic zoophilic B. malayi strain that is reported to be derived from a human patient from Malaysia [30] and distributed to numerous places by Prof. Dr. C. P. Ramachandran [31,32]. Recipients included the Central Drug Research Institute, Lucknow, India, and the University of California Los Angeles (UCLA), among others. Most modern B. malayi laboratory lines are descended from this latter line at UCLA [3], including populations maintained and distributed by TRS labs and the NIAID-funded Filariasis Research Reagent Resource Center (FR3). FR3 and TRS supply one another worms when either laboratory has issues with their populations. In addition, investigators acquire worms from FR3 and/or TRS to establish their own culture collections and replenish with worms as needed, including the laboratories of Prof. Mark Taylor and Dr. Joseph Turner in the Liverpool School of Tropical Medicine and Dr. Gary Weil and Dr. Ramakrishna Rao at Washington University in St. Louis. A further B. malayi line was established independently from an infected woman in Narathiwat Province, southern Thailand, and has been maintained at The Faculty of Tropical Medicine, Mahidol University, Bangkok, then Chiang Mai University, Thailand, for ~40 years with no mixing with the other laboratory lineages [33]. The B. pahangi lineage at FR3 is thought to have been established in the 1970s [34] from a green leaf monkey. Because B. pahangi and B. malayi share very similar life cycles, the procedure for laboratory maintenance for both species at FR3 is similar. Using samples of B. malayi and B. pahangi from multiple laboratory centers as well as natural samples of B. pahangi that were acquired from wild cats [35], we sought to investigate the genomic diversity within these Brugia populations. Given the potential for frequent bottlenecks both in nature and the laboratory, there is the repeated and significant risk of a founder effect that we sought to examine. To this end, we have employed public data from other filarial nematodes, including W. bancrofti, L. loa, O. volvulus and D. immitis in order to place this population diversity in the context of the broader filarial nematode family.

Materials and methods B. malayi library preparation and sequencing Adult male worms were provided from the following B. malayi centers: Washington University in St. Louis, MO, USA; Liverpool School of Tropical Medicine, UK; TRS Laboratories, Athens, GA, USA; FR3, Athens, GA, USA; Central Drug Research Institute, Lucknow, India; and Chiang Mai University, Chiang Mai, Thailand (S1 Text). Adult male worms were sequenced, since females are typically gravid precluding obtaining their individual genome. While virgin females would be a viable alternative, the difficulties in isolating them would have precluded us from obtaining many of the samples used here. Frozen single adult males recovered from the host gerbil were homogenized separately in 50 μl Buffer G2 from the genomic DNA buffer set (Qiagen) supplemented with RNase A (Qiagen) to 200 μg/mL. Homogenization was performed in a 1.5 mL microcentrifuge tube using a disposable micro pestle (Kimble-Chase). The homogenate was removed to a fresh tube and then the pestle and original tube were washed with an extra 0.95 mL of G2 buffer with RNase which was then added to the sample. The homogenized sample was then processed according to the protocol for tissue samples described in the genomic DNA handbook (Qiagen) and using genomic-tip 20/G gravity flow columns (Qiagen) except 80 U proteinase K (New England Biolabs) were used. Elution buffer QF was prewarmed to 50°C to increase DNA recovery. The DNA was precipitated by centrifugation as recommended, but in the presence of 20 μg glycogen (Invitrogen). Genomic DNA was sheared to ~380 bp with an ultrasonicator (Covaris) and used to construct indexed PE Illumina libraries using the NEBNext Ultra DNA kit (New England Biolabs). All samples were sequenced on the Illumina HiSeq 2500 with a read length of 100 bp, except for W_male_2 and W_male_6, which were sequenced on the Illumina HiSeq 4000 with a read length of 150 bp. While the data was generated specifically for this study, the data from a subset of samples were used in a previously published study to aid in identification of sex chromosomes and as such these methods are previously described for those samples [12]. B. pahangi library preparation and sequencing Adult B. pahangi male worms were provided from the following locations: FR3 laboratories, at both University of Georgia, Athens, GA, USA; University of Wisconsin, OshKosh, WI, USA (S1 Text) and University of Malaya, Kuala Lumpur, Malaysia [35]. Adult females were obtained from FR3 laboratories and pooled for the purposes of this analysis. Pooled adult female samples were prepared as described in Mattick et al [14]. Endemic isolates from Malaysia were prepared in an identical fashion to the Brugia malayi samples described above. Frozen single adult males obtained from FR3 and recovered from the same host gerbil were separately homogenized under liquid nitrogen in 1.5 mL microcentrifuge tubes. The samples were processed according to the Qiagen DNeasy blood and tissue insect protocol using 180 μl buffer ATL and 20 μL proteinase K. The samples were processed according to the manufacturer’s recommendations and eluted in 200 μL of buffer AE. After DNA isolation, the pooled adult female sample and the B. pahangi male FR3_UWO_Bp1AM_09 sample were sequenced on the Illumina HiSeq2500 from KAPA Hyper libraries with 150 bp paired-end reads. For all other B. pahangi samples, genomic DNA was sheared to ~380 bp with an ultrasonicator (Covaris) and prepared into an indexed, paired-end Illumina library using the NEBNext Ultra DNA kit. These samples were sequenced on the Illumina HiSeq 4000 with 150 bp paired end reads. Sample variant calling and processing for all individual nematode species Each individual B. pahangi, B. malayi, O. volvulus, D. immitis, C. elegans and Drosophila melanogaster sample was mapped against its respective genome (GCA_000002995.5, GCA_012070555.1, GCA_000002985.3, GCA_001077395.1, GCA_000499405.2, GCA_000001215.4) [14,36–40] using BWA MEM [41] with the following settings: -M -a. The resulting BAM files were all sorted and de-duplicated using the Picard tools SortSam and MarkDuplicates, respectively [42] using default parameters for both. Single Nucleotide Variants (SNVs) were jointly called for each sample using Genomic Variant Call Format (GVCF) files generated using the Genome Analysis Tool kit (GATK) [43] with the HaplotypeCaller with the--read-filter MappingQualityReadFilter setting. The resulting GVCF files were merged and jointly called for SNVs using the GATK GenomicsDBImport and GenotypeGVCFs functions, then filtered using a manual filter with the following settings:--filter-name "QD"--filter-expression "QD < 5.0"--filter-name "QUAL"--filter-expression "QUAL < 30.0"--filter-name "DP"--filter-expression "DP < 14.0"--filter-name "MQ"--filter-expression "MQ < 30.0"--filter-name "MQRankSum"--filter-expression "MQRankSum < -12.5"--filter-name "ReadPosRankSum"--filter-expression "ReadPosRankSum < -8.0"--filter-name "FS"--filter-expression "FS > 60.0". For male samples from species where chromosome structure was known (B. malayi, B. pahangi), the autosomes were called with a ploidy of 2, while the X chromosome was called at a ploidy of 1. For female samples from species where chromosome structure was known (O. volvulus), the autosomes and X chromosome were called with a ploidy of 2. Filtration in samples called with a ploidy of 1 were filtered with--filter-name "DP"--filter-expression "DP < 7.0" to reflect the reduced sequencing depth on those sequences. Putative known pseudoautosomal regions from B. malayi, B. pahangi, and O. volvulus were excluded from variant analysis. Sample variant calling and processing for multi-individual samples Each multi-individual W. bancrofti sample was mapped against its respective genome (GCA_000002995.5, GCA_012070555.1) [14,37] using BWA MEM [44] with the following settings: -M -a. The resulting BAM files were all sorted and de-duplicated using the Picard tools SortSam and MarkDuplicates respectively [42] using default parameters for both. SNVs were called using the Freebayes software, specifically the freebayes-parallel feature using default parameters. SNV density and Pi analysis SNV density can allow for the identification of regions of the genome that are under- or over-represented in variants relative to the entire genomic sequence. SNV density across each of the chromosomes was calculated over 10-kbp sliding non-overlapping windows, considered as 20,000 possible variant sites with homozygous variants counting for 2 site changes and heterozygous variants counting as 1 site change. Pi was calculated using VCFtools over 10 kbp non-overlapping windows for all samples with a genomic coverage > 80% (S1 Table) for samples with a ploidy of 2. Because VCFtools requires diploid sites, the R package PopGenome [45] was used with default parameters to calculate Pi for B. malayi, B. pahangi and O. volvulus X chromosomes. Plots of SNV density and Pi were generated using the ggplots2 package in R [46], with the 10-kbp regions as the X-axis and Pi as the Y-axis. A density plot for Pi for each species was generated using the geom_density function of ggplots with default settings on the 10-kbp values of Pi across each chromosome. SNV density and Pi were assigned to Nigon elements, which were determined as previously described [12]. Briefly, contigs were mapped against B. malayi, O. volvulus and C. elegans using the NUCmer tool from the MUMmer package v.3.23 [47], and contigs were assigned to a specific Nigon element based on the largest match against each specific Nigon element. Principal component analysis was conducted on all autosomal variants in Brugia malayi and Brugia pahangi individuals using PLINK v.1.9 [48] with the--pca parameter. The resulting primary two principal components for each species were plotted using the geom_point function of ggplots with default settings in R. Phylogenetic relationships Phylogenetic relationships for chromosome X and the autosomes were developed by first obtaining current genomes for B. timori, W. bancrofti and O. volvulus from WormBase [49]. Conserved nematode genes from these genomes, in addition to B. malayi and B. pahangi, were predicted using BUSCO v.4.06 package and its nematoda_odb10 database [50]. To ensure orthology, the genomes that were not in chromosome form (i.e. B. timori and W. bancrofti) were aligned against B. malayi using the NUCmer tool from the MUMmer package v.3.23 [47]. Contigs were binned to a chromosome based on maximum match length, and genes were assigned to chromosome X or the autosomes based on their contig matches. Genes present in all 5 species were aligned using TranslatorX [51] and filtered to include only those that were <15% dissimilar (>85% similarity) at the amino acid level and had at most a difference of 10% in gene length amongst all 5 orthologues. This left a total of 38 genes on chromosome X, and 228 genes on the autosomes. Trees were generated for these sequences using IQ-TREE with default parameters [52], and plotted using iTOL [53]. Mitochondrial sequences (NC_004298.1, CM022469.1, NC_016186.1, AP017686.1) for each species were obtained from GenBank, and aligned at the nucleotide level using MAFFT v.7.427 [54]. The mitochondrial tree was generated and plotted in an identical manner to the autosome and chromosome X trees. Ethics statement All animals in the US were handled in accordance with guidelines defined by the Animal Welfare Act (A3381-01), Association for Assessment and Accreditation of Laboratory Care International (AAAALAC), PHS Policy for the Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. Animal work for FR3 was approved under the University of Georgia Athens Institutional Animal Care and Use protocol A2010 12–005 and A2013 11–009 or the University of Wisconsin OshKosh under IACUC protocol number 0026-000246-R2-01-12-17. All animal research at TRS was approved under Institutional Animal Care and Use Protocol 13–03 or 14–03. All animal work at WUSM was approved under WUSM Institutional Animal Care and Use Protocol 20120025. The study in Lucknow India bears IAEC approval number 129/08/Para/IAEC/renew (84/09) dated April 27, 2009. All experiments on animals at Liverpool School of Tropical Medicine were approved by the ethical committees of Liverpool School of Tropical Medicine and the University of Liverpool and were conducted according to Home Office Legislation, the revised Animals (Scientific Procedures) Act of 1986 (project license numbers 3002974, P86866FD9). Approval for using gerbils for sample work in Malaysia was granted by the University of Malaya Animal Care and Use Committee (Ref. No. PAR/29/06/2012/RM [R]). The protocol for samples obtained from Thailand was approved by the Institutional Animal Care and Use Committee (Protocol Number 15/2562) of the Faculty of Medicine, Chiang Mai University, Chiang Mai province, Thailand.

Discussion B. malayi and B. pahangi filarial nematodes populations have genetic diversity that is consistent with the known separation over time of these populations (Fig 3). The greatest difference is seen between endemic nematodes and laboratory populations in the case of B. pahangi, or between independently derived laboratory populations in the case of B. malayi. To a lesser extent there are differences between nematodes that were derived from the same human sample but have been maintained separately for decades reflected in the differences between Lucknow and the FR3 samples. Lack of access to clinical samples precluded their inclusion in this study. While the passage of laboratory populations through non-native hosts could impact the genetic diversity, introducing new bottlenecks and selective pressures, the lack of diversity on neo-X chromosomes was found in at least two populations for each of four species with known neo-X fusions (B. malayi, B. pahangi, W. bancrofti, and O. volvulus) and was absent from the two filarial nematodes that lack such fusions (L. loa and D. immitis). Further population level data and the completion of filarial nematode genomes will likely shed further light on the factors influencing genetic diversity in filarial nematodes as well as parasitic nematodes more broadly. A significant difference in genetic diversity was observed between autosomes and chromosome X. Genetic diversity can be influenced by bottlenecks, polyandry, rate of recombination, mutation rate, selection, and effective population size [22,23]. The loss of genetic diversity on chromosome X is not limited to just laboratory populations (and the bottlenecks associated with laboratory propagation) since natural populations of W. bancrofti and B. pahangi have the same loss of diversity. Although polyandry and population shrinkage may also contribute to loss of diversity in filarial nematodes, it is quite likely to be similar for all of the examined filarial nematodes given their life history. The rate of recombination is expected to be suppressed in sex chromosomes relative to autosomes [61], which is supported by the significant reduction in intrachromosomal inversions observed in the Brugia chromosome X relative to its autosomes [12]. In addition, chromosome Y has an abundance of repeats and transposable elements that prevented its assembly [12], and these repetitive elements are predicted to play a critical role in the further suppression of recombination [62]. In mammals and birds, the higher mutation rate in males over females leads to differences in the mutation rate between autosomes and sex chromosomes [63], while in at least one plant [64] the autosome and sex chromosome mutations are approximately equal. Differences in mutation rate on the sex chromosomes in mammals are associated with more rounds of replication in male gametes, which is likely also the case in filarial nematodes. However, we expect male gametogenesis to be similar between all examined filarial nematodes, such that the differences we observe are not likely attributed to the mutation rate. Genetic diversity can also be influenced by sex-biased effective population size, sex-biased inheritance, and sex-exclusive inheritance [22,23]. While we cannot rule out the effects of sex-biased inheritance or sex-exclusive inheritance, we suggest that they would likely be the same across all examined filarial nematodes. Across nematodes and even filarial nematodes, there is a diversity of sex chromosomes, with XO sex determination being common, but XY being present, and even some nematodes having three sexes [65]. Among the filarial nematodes examined, L. loa and D. immitis are thought to be XO [66], with Brugia spp. and Onchocerca spp. being XY [66] resulting from different neo-X fusions [12]. In the absence of selection and no sex bias in reproduction, the expected population size for an organism with heteromorphic XY chromosomes, like Brugia and Onchocerca filarial nematodes, the autosome:(chromosome X):(chromosome Y) allelic frequency is 4:3:1. As a consequence, a reduction of nucleotide diversity is expected on heteromorphic sex chromosomes, with π X /π A ~ 0.75 [10,22]. Similarly, nematodes with XO sex determination would have an expected autosome:(chromosome X):(chromosome Y) allelic frequency of 4:3:0 with π X /π A ~ 0.75. However, we observe π X /π A ~ 0.2 for both Brugia species. Upon examination of other filarial nematodes, a reduction in π X /π A similar to that in Brugia spp. was observed for W. bancrofti and O. volvulus, all four of which have neo-X chromosomes that emerged after fusion of chromosome X with an autosome. In the case of filarial worms, different neo-X chromosomes were formed at least twice by the fusion of two Nigon elements [12,19,20]. The common Nigon element in these fusion events appears to be Nigon-D, which is likely the ancestral sex chromosome of filarial nematodes [12,19,20]. The chromosomal fusion event in the ONC3 clade, containing Onchocerca spp., joined Nigon-D and Nigon-E, while the chromosomal fusion in the ONC5 clade, containing Brugia spp. and Wuchereria sp., joined Nigon-D and Nigon-X (Fig 5). Both times that there is a loss in diversity on chromosome X in this study, there is a concomitant neo-X fusion. And conversely, where there is not a neo-X fusion, there is not the loss of diversity (i.e. L. loa and D. immitis). As such this lack of genetic diversity on chromosome X seems consistent with the formation of the neo-X chromosomes prior to several speciation events, like that of Brugia spp. and W. bancrofti (Fig 5). Chromosomal fusion events are known to reduce genomic diversity in species as the effective population size of the sex chromosome is reduced and novel genes and dosage mechanisms must be generated to compensate for the fusion [67,68]. For example, in Sylvoidea bird species, a loss of diversity on chromosome Z (the equivalent of chromosome X in ZW systems) is attributed to a neo-sex chromosome fusion [69]. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Phylogenetic relationships related to sex chromosome Nigon content. The phylogenetic relationship of filarial nematodes is shown as adapted from Lefoulon et al. [75]. Nigon element assignments for the sex chromosomes are shown when known or inferred previously [12]. The loss of diversity on the sex chromosome co-occurs with the instances of chromosomal fusions between Nigon-IV and Nigon-X in ONC5 and between Nigon-IV and Nigon-V in ONC3, but does not appear to be present in species that do not contain the chromosomal fusion in either the ONC3 or ONC5 clades. https://doi.org/10.1371/journal.pntd.0009838.g005 Chromosomal fusions may not be the only source of diversity loss on chromosome X. For example, Haemonchus contortus, a parasitic nematode, does not show evidence of a recent chromosomal fusion. Yet the H. contortus π X /π A is 0.36 [70], which is also lower than neutral expectation of π X /π A ≃ 0.75. This decrease in H. contortus was attributed to host sex biases due to reproductive fitness being over-dispersed between males and females from polyandry and high fecundity [70]. However, filarial nematodes only seem to have this lack of genetic diversity on neo-X chromosomes despite likely polyandry and high fecundity across many or most filarial nematodes. In nematodes, there has also been a transition in the sex chromosomes. Nigon-D is likely the ancestral chromosome for all Rhabditida nematodes, with a conversion of Nigon-X to chromosome X in Rhabditina nematodes, which includes C. elegans [12]. This transition does not appear to be associated with a difference in genetic diversity for chromosome X upon comparisons of C. elegans and the filarial nematodes without neo-X fusions, like D. immitis and L. loa. (Fig 2). It is possible that altering the sex determining Nigon element is not enough to cause diversity loss, and that it is specifically associated with chromosomal fusion. Alternatively, it is possible that enough time has elapsed to eliminate the signature associated with that transition at least with the resolution with which it was examined here. The same processes that subject chromosome X to decreased genetic diversity and Muller’s ratchet also affect chromosome Y to a much larger degree [63,71]. In filarial nematodes, we do not have an assembled chromosome Y, and are limited to male-specific contigs attributed to chromosome Y. But the high repetitiveness of the sequences [12] suggests that filarial nematode Y chromosomes are undergoing a degeneration consistent with neo-Y formation. Although chromosomal fusions appear to be associated with diversity loss in filarial worms, it is not yet clear if this will be found universally in other parasitic nematodes. This lack of chromosome X genetic diversity is important since most medically important filarial nematodes have neo-X fusions with a third of all genetic material being on chromosome X, representing a substantial loss of sequence diversity. Genetic material on chromosome X also undergoes recombination at a lower rate than the rest of the genome [61]. Thus the sex chromosome is more susceptible to Muller’s Ratchet [72], which is a process whereby deleterious mutations accumulate in the absence of recombination. This loss of diversity on such a large portion of the genome could have significant consequences. In other parasites, drug resistance and adaptability are associated with a higher level of genetic diversity, and its absence can prevent an organism from developing strategies of coping with adverse events [73].

Conclusions Populations were examined that were derived from two independent isolates of B. malayi and B. pahangi. For B. malayi this includes several populations derived from a human from Malaysia and a population from an infected woman in Thailand. For B. pahangi this includes the populations derived from a green leaf monkey from Malaysia and from naturally infected Malaysian cats. We observe a profound lack of sequence diversity on chromosome X in all independent populations of B. malayi and B. pahangi that is consistent with reduced chromosome X diversity in other sequenced filarial nematodes with neo-X chromosomes. Given the importance that sequence diversity has with respect to adaptability and the size of chromosome X, which is a third of the genome, this lack of sequence diversity in a third of the genome in medically important filarial nematodes is likely to have a large effect on the evolutionary trajectory of these species.

Acknowledgments Adult B. malayi and B. pahangi males were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH, which procures material from the NIH/NIAID Filarial Research Reagent Resource Center (FR3) with morphological voucher specimens stored at the Harold W. Manter Museum at University of Nebraska, accession numbers P2021-2032. The NIAID, USA, and Central Drug Research Institute, India, played no part in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0009838

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