https://orcid.org/0000-0003-4986-9711
Figures
Abstract
Investigations of nuclear genome size, complete mitochondrial genome (mitogenome) sequence, and morphometrics were conducted on specimens of Bulinus snails (Gastropoda: Planorbidae) collected from 14 locations across the east coast, central Kenya, and western Kenya around the Lake Victoria region (November 2013 and January 2024). Flow cytometry measurements of DNA content (C-value) revealed unexpected variation in nuclear genome size, with diploid Bulinus africanus and B. forskalii species groups showing C-values ranging from 0.76 to 1.98 pg, while tetraploid B. truncatus had a C-value of 1.82 pg. Additionally, C-values for six B. globosus specimens from different localities ranged from 1.43 to 1.98 pg. These findings suggest that bulinine snails, particularly the B. africanus species group, have undergone genome expansion, whole genome duplication (polyploidization), or both, which have not been previously recognized. Next-generation sequencing was performed to determine and annotate 14 complete mitogenome sequences. Despite the well-conserved arrangement of protein-coding genes, two versions of mtDNA genome structure, distinguished by the tRNA-D (Asp) location, were found, designated as DCF (Asp-Cys-Phe) type (in the B. forskalii group and the B. truncatus/tropicus complex) and CF (Cys-Phe) type (in the B. africanus group). Phylogenetic analyses based on complete mtDNA sequences of bulinines from Kenya, along with cytochrome c oxidase subunit I (COX1) sequences from various localities across Africa, contributed to resolving species identities and provided further support for the presence of multiple or cryptic species in the taxon B. globosus. A landmark-based morphometric analysis was ineffective in distinguishing these species. This study reveals unexpected nuclear genome size variation, provides new mitogenome sequences, and highlights the limitations of morphological analysis. It offers valuable insights into the cytogenetics, polyploidy, genomics, taxonomy, and evolution of bulinines, which serve as intermediate hosts for schistosomes responsible for human urogenital schistosomiasis and intestinal schistosomiasis in domestic and wild mammals.
Author summary
Freshwater snails of the genus Bulinus play a crucial role in supporting the larval development of schistosomes and other trematode parasites responsible for significant diseases in humans and animals in Africa; yet knowledge of their genetics and genomics remains limited. Considerable contradictions exist regarding species identification and classification. Focusing on bulinine specimens from Kenya, we employed flow cytometry, next-generation sequencing, and morphometrics for a simultaneous investigation, making the data comparable and informative. This study uncovers unexpected genome size variation, suggests the presence of unrecognized genome expansion and/or whole genome duplication (polyploidization), provides new mitogenome data, clarifies phylogenetic relationships, illuminates genome evolution, highlights the limitations of morphological analysis, and raises questions about the cytogenetics and classification of the genus Bulinus.
Citation: Zhang S-M, Adema CM, Habib MR, Lekired A, Posavi M, Laidemitt MR, et al. (2025) Complexity of schistosome vector bulinine snails in Kenya: Insights from nuclear genome size variation, complete mitochondrial genome sequence, and morphometric analysis. PLoS Negl Trop Dis 19(7): e0013305. https://doi.org/10.1371/journal.pntd.0013305
Editor: Bonnie L. Webster, Natural History Museum, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND
Received: April 15, 2025; Accepted: June 30, 2025; Published: July 14, 2025
Copyright: © 2025 Zhang 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: Fourteen mitogenome sequences were deposited in GenBank under accession numbers PV483366 to PV483379. The data will be released as soon as the paper is published.
Funding: This work was supported by National Institutes of Health (NIH) grants R01 AI170587 (to S-MZ) and R37AI101438 (to ESL). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Freshwater gastropod snails of the genus Bulinus Müller (1781) (family: Planorbidae) are the most medically important molluscan species in Africa [1]. These snails play an essential role in transmitting parasites that cause disease in humans and animals, serving as obligate intermediate hosts for the trematode parasite Schistosoma haematobium, the causative agent of urogenital schistosomiasis. Urogenital schistosomiasis accounts for over two-thirds of all schistosomiasis cases in Africa, which is home to approximately 90% of global cases [2–5]. In addition to S. haematobium, Bulinus snails also transmits other schistosomes, including S. intercalatum and S. guineensis, which cause intestinal schistosomiasis in humans, although they are less prevalent [6]. Furthermore, these snails serve as vectors for S. bovis, S. curassoni, and S. mattheei [7,8], as well as for other trematode species, particularly amphistomes [9,10], which infect livestock. Additionally, some schistosomes and their hybrids, such as S. mattheei, transmitted by bulinines, are the causative agents of zoonotic schistosomiasis [8,11,12]. Their predominance and diversification on the African continent also pose interesting biogeographical and evolutionary questions.
Accurate identification of bulinine snails is crucial for understanding schistosomiasis transmission. Historically, identification has relied primarily on morphological characteristics, supplemented by cytogenetic analysis and biochemical assays, such as protein electrophoresis. While a few species, such as B. umbilicatus, can be identified reliably using conchological features [6], there are no definitive morphological traits that clearly distinguish most Bulinus species from one another [13]. The number of bulinine species has fluctuated from more than 120 in early works cited by Wright (1971) [14] to 20 species recognized by Mandahl-Barth (1957) [15], and now to the 37 currently acknowledged, as described by Brown (1994) [6]. These 37 species are categorized into three groups and one complex: the B. africanus group (10 species), the B. forskalii group (11 species), the B. reticulatus group (2 species), and the B. truncatus/tropicus complex (14 species) [6]. For convenience, we refer to them hereafter as the africanus, forskalii, reticulatus, and truncatus/tropicus species groups in this paper.
Early cytogenetic studies suggested that the first three groups consist solely of diploids (2n = 2 x 18), while the latter includes both diploids and polyploids (4n, 6n, and 8n) [6]. Recent molecular studies focusing on species identification, population genetics, and phylogenetics have utilized partial mitochondrial DNA (mtDNA) sequences, such as cytochrome c oxidase subunit I (COX1) and nuclear ribosomal DNA (rDNA) segments, like internal transcribed spacer (ITS) [16–24]. These studies have provided valuable insights into a broad range of bulinine species across considerable geographic areas, enabled the assembly of near genus-wide phylogenies, and highlighted persistent questions regarding the presence of still-unresolved species groups and inconsistencies between morphological and molecular data. Genomic data are more informative but remain limited [25–28].
In this study, we conducted an integrative analysis of bulinine snails collected from diverse geographical localities across Kenya, including major schistosomiasis-endemic areas. By combining nuclear genome size estimation, complete mitochondrial genome sequencing, and detailed morphometric profiling, we provide novel insights into the cytogenetics, polyploidy, genomics, taxonomy, and evolution of bulinine snails in Africa.
Materials and methods
Ethics statement
The collections were approved by the National Commission for Science, Technology, and Innovation (permit numbers P/15/9609/4270 and P/21/9648), the National Environmental Management Authority (permit numbers NEMA/AGR/46/2014 and NEMA/AGR/149/2021), and the Kenya Wildlife Service (permit numbers KWS 0004754 and KWS-0045-03-21).
Snail collection
The snails for this study were collected in November 2013 and January 2024. Snails in shallow water were collected using long handheld scoops, while a metal dredger was used to collect aquatic plants from deeper waters of Lake Victoria. The snails attached to the aquatic grass were then separated and collected. In each location, the global positioning system (GPS) coordinates were recorded, and photographs of the habitat were taken. Live snails were shipped to the University of New Mexico (UNM) and maintained at the UNM Center for Evolutionary and Theoretical Immunology (CETI). The specimens were assigned temporal codes based on geographical location, but the final species determination for each specimen was based on phylogenetic analyses of mtDNA sequences (see Results section below).
Flow cytometric analysis of DNA content
DNA measurements were conducted at two different times: first in May 2022 for samples collected in November 2013 and second in April 2024 for samples collected in January 2024. For both measurements, propidium iodide-based flow cytometry was employed. The procedure and experimental design for the two measurements were the same, as described by Bu et al. (2023) [29]. In the first experiment, we used the garden plant hosta (Hosta plantaginea) (Praying Hands’ 2-2-2) as a control [29]. In the second experiment, we used the pea (Pisum sativum) (seed resource: Gene Bank Dept., CRI Prague-Ruzyne ACCENUMB: 09L010500; Pisum sativum subsp. sativum ACCENAME: Ctirad; ORIGCTY: Czechoslovakia) since the garden plant hosta was unavailable. The DNA content of the pea is 9.02 picograms (pg) (2C DNA content or diploid genome DNA) [30]. To ensure the reliability of the data, the DNA content of the genetically stable homozygous iM line Biomphalaria glabrata developed at UNM [31] was measured in both experiments (see details in the Results section).
DNA extraction, library preparation, and sequencing
The procedures for DNA extraction, quality control, library preparation, and sequencing to generate Illumina 150 bp x 2 paired-end reads and PacBio HiFi CCS reads (high-fidelity circular consensus sequencing) were outlined by Zhang et al. (2024) [32] and Bu et al. (2023) [29], respectively.
Assembly of mitogenomes
Raw reads were trimmed with Trimmomatic v0.39 to remove low-quality bases and adapter sequences [33]. Four methods were used to assemble the mitogenome from Illumina data: (1) a semi-reference-based assembly with MITOBIM 1.9.1 using an iterative baiting approach [34], (2) de novo assembly with NOVOPlasty using a seed-and-extend algorithm [35], (3) reference-guided de novo assembly with the specialized toolkit GetOrganelle [36], which employs a baiting and iterative mapping approach, and (4) de novo assembly using SPAdes 3.15.1 [37]. If an inconsistency at a particular nucleotide position was found, although very rare, the determination of the nucleotide was based on the majority consensus of the alignments, supported by verifying a functional reading frame. For mitochondrial genome assembly from HiFi reads, we employed the MitoHifi pipeline [38], which is well-suited for long reads with high coverage. HiFi reads were mapped to the reference mitogenome of B. truncatus (NC_060795.1) using Minimap2 [39]. The filtered reads were assembled with Hifiasm [40], and the resulting contigs were subjected to BLAST against the reference mitogenome. The final assembly was determined using the four datasets, with additional corrections made based on alignments and annotations.
Annotation of mitogenomes
Initial mitogenome assemblies were annotated using MITOS2 with RefSeq 63 Metazoa as a reference [41] available from Galaxy Europe [42]. The computational annotation results were used to check the completeness of the mitogenome assemblies. Missing and incomplete gene predictions (including some tRNAs) were corrected manually. Final annotation followed the criteria from Fourdrilis et al. (2018) [43] and Ghiselli et al. (2021) [44], incorporating unique aspects of mitogenome biology, including transcription as polycistronic RNA, the tRNA punctuation model (delimitation of reading frames by tRNA genes or secondary structures), and the completion of stop codons by polyadenylation of mRNA transcripts, as outlined by Zhang et al. (2022) [26]. Mitogenome maps were visualized using SnapGene software (viewer v.7.2.1) (www.snapgene.com).
Phylogenetic analyses of mitogenome and COX1 gene sequences
Complete mitochondrial DNA sequences (14,297 bp) and mitochondrial cytochrome c oxidase subunit I (COX1) gene sequences (611 bp) were aligned using MUSCLE [45] as performed in Molecular Evolutionary Genetics Analysis (MEGA X) [46]. The COX1 sequences analyzed corresponded to the standard DNA barcoding region of the gene [47]. Phylogenetic analyses included sequences from multiple Bulinus species, with Biomphalaria glabrata (iM-line) used as an outgroup. For the COX1 analysis, taxa included both reported sequences from GenBank and newly generated sequences from this study. The best-fit nucleotide substitution models were selected using the Bayesian Information Criterion (BIC) implemented in MEGA X: T92 + G + I for COX1 [48] and GTR + G + I for complete mtDNA [49]. Maximum Likelihood (ML) phylogenetic trees were constructed in MEGA X using the identified best-fit models, with branch support assessed using 1,000 bootstrap replicates [50]. Trees are presented as phylograms with branch lengths proportional to evolutionary distance.
Morphometric analysis
Shell morphometric analysis was conducted using a landmark-based approach by orienting each shell with the aperture facing the observer (Fig 1). Eleven linear shell measurements were recorded using a digital caliper (0.01 mm precision). The measurements included shell length (L), shell width (W), aperture height (A), aperture width (WA), whorl spacing (WS), diagonal shell width (WD), body whorl height above the aperture (LH), and whorl height (WH). Raw measurements were compiled into structured Excel spreadsheets, and seven morphometric ratios were derived to quantify shell proportions: shell elongation (L/W), aperture proportions (A/L), spire development ((L-A)/L), body whorl expansion (LH/L), relative aperture width (WA/WS), diagonal whorl expansion (WD/W), and sutural spacing (WH/L).
The abbreviations are provided in the Materials and Methods section.
Data preprocessing, taxonomic grouping, and descriptive statistics (mean ± SD) were performed in RStudio (version 2023.12.0 + 369) using the tidyverse package [51]. To investigate multivariate morphological patterns, Principal Component Analysis (PCA) was implemented via FactoMineR [52], with variance contributions from principal components reported. Pairwise relationships between shell variables were visualized using bivariate plots generated with ggplot2 [53] and RColorBrewer [54].
Results
Snail sampling in Kenya
In two samplings conducted 10 years apart (November 2013 and January 2024), 14 localities were sampled, as shown in Fig 2. For convenience, each locality was assigned a code corresponding to its operational taxonomic group, as detailed in Fig 2 and Table 1. Please note that not all specimens were available for all three analyses (nuclear genome size, mitogenome, and morphometrics) due to limitations in specimen availability.
The approximate localities are indicated. Detailed information for each locality (GPS) and its corresponding code is provided in Table 1. The photos in this figure were taken by the authors.
Flow cytometric analysis of nuclear DNA contents
For each code, three snails were individually measured using flow cytometry. A total of 16 coded samples were analyzed, including 14 from the genus Bulinus, representing three bulinine groups: B. africanus, B. forskalii, and B. truncatus/tropicus, along with 2 from the genus Biomphalaria (Table 1). Three specimens of Biomphalaria reported by Bu et al. (2023) [29] were included to add perspective to the analysis. All DNA contents presented in this study pertain to the C-value, which refers to the amount of DNA in a haploid cell. Table 1 presents the C-values of individual snails and their averages. Fig 3 displays the average C-values of snails from the africanus, forskalii, and truncatus/tropicus groups, as well as Biomphalaria spp. The smallest genome was 0.76 pg from B. forskalii (BuF3), while the highest DNA content, 1.98 pg, was observed in a specimen of B. globosus (BuG2), with similar amounts noted in B. truncatus (BuT19: 1.82 pg) and two other B. globosus specimens (BuG5L: 1.86 pg; BuG5: 1.88 pg). The DNA contents of a UNM-maintained homozygous iM line of Bi. glabrata [31] from this study (code: BiG24) (Table 1) and the same line previously measured and reported (BiG25) [29] were consistent (1.11 pg vs. 1.09 pg: p = 0.58). Moreover, two field-collected B. globosus samples (BuG5L and BuG5) from the same geographical locality (Asao, Kisumu), taken 10 years apart and measured at different times, yielded consistent results (1.88 pg vs. 1.86 pg: p = 0.80) (Table 1). These findings support the reliability of the data presented in this study.
Detailed information for the codes is provided in Table 1. Codes Bi25, Bi26, and Bi27 represent the iM line Bi. glabrata (10.9 pg), BB02 Bi. glabrata (1.02 pg), and Bi. pfeifferi (0.91 pg), respectively, as reported by Bu et al. (2023) [29]. All samples of B. globosus are enclosed by dotted lines.
Determination of complete mitogenome sequences and structure
Complete mitochondrial genomes (mitogenomes) from 14 bulinine specimens were sequenced, assembled, and annotated (S1 Table). The list of specimen codes and their GenBank accession numbers (in parentheses) is as follows: BuU4 (PV483366), BuU7 (PV483367), BuN6 (PV483368), BuN15 (PV483369), BuG1 (PV483370), BuG2 (PV483371), BuG5L (PV483372), BuG12 (PV483373), BuG13 (PV483374), BuF3 (PV483375), BuF16 (PV483376), BuTp7A (PV4833677), BuTp11 (PV483378), and BuTp14 (PV483379). The species and locality corresponding to the specimen codes are listed in Table 1.
Each mitogenome consists of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. The arrangement and order of the PCGs are consistent across all mitogenome sequences determined in bulinines, including those published previously [26]. A difference was observed in the location of tRNA-D (tRNA-Asp), dividing the 14 mitogenomes into two types: DCF (Asp-Cys-Phe) and CF (Cys-Phe). The distinction between the two types lies in the location of D; in the CF type, D is positioned downstream of the mitogenome between Y and W (YDWGHQL), whereas in the DCF type, DCF are grouped together. All species of the africanus group belong to the CF type, while species of the forskalii and truncatus/tropicus groups belong to the DCF type (S1 Data).
Phylogenetic analyses of mitogenome sequences
A total of 20 complete bulinine mitogenome sequences, including six from a previous report [26], were phylogenetically analyzed (Fig 4). The africanus and truncatus/tropicus species groups appear to cluster together. Two B. forskalii mitogenomes represent the first complete mitogenome sequences from the forskalii species group and cluster with the other two groups. Phylogenetic analysis of mitochondrial COX1 gene sequences supports the topology of the complete mtDNA tree (Fig 5). In the africanus group, B. globosus is more closely related to B. ugandae than to B. nasutus in both mitogenome and COX1 analyses. The phylogenetic analyses indicate that specimen BuTp7A, collected from deep water in Lake Victoria, is B. tropicus (see Discussion below). The divergence between B. truncatus and B. tropicus appears significant, although their morphology and size are very similar.
The tree is presented as a phylogram with branch lengths proportional to evolutionary distance (scale bar = 0.05 substitutions per site). Phylogenetic relationships were inferred using the GTR + G + I substitution model with 1,000 bootstrap replicates implemented in MEGA X [46]. A total of 21 complete mitogenome sequences (14,297 bp) were analyzed: 14 from the current study (BuF3, BuF16, BuTp7A, BuTp11, BuTp14, BuN6, BuN15, BuU4, BuU7, BuG1, BuG2, BuG5L, BuG12, and BuG13) and 7 published sequences, including 6 from Zhang et al. [26] and Biomphalaria glabrata (iM-line; MG431965.1) used as an outgroup. The tree demonstrates the phylogenetic relationships among major Bulinus species groups: the africanus group (including B. globosus, B. ugandae, and B. nasutus), the truncatus/tropicus complex, the forskalii group, and the reticulatus group. Branch lengths reflect the degree of evolutionary divergence between taxa.
The tree is presented as a phylogram with branch lengths proportional to evolutionary distance (scale bar = 0.05 substitutions per site). Phylogenetic relationships were inferred using the T92 + G + I substitution model with 1,000 bootstrap replicates implemented in MEGA X [46]. The analysis included 67 COX1 sequences (611 bp): 14 newly generated sequences from this study (indicated by specimen codes with GenBank accession numbers in parentheses) and 53 published sequences from GenBank representing several African Bulinus species and geographic localities. B. glabrata (iM-line; MG431965.1) was used as an outgroup.
Morphometric analysis
Morphometric analysis revealed substantial challenges in distinguishing among Bulinus species. As shown in Fig 6, visual examination of shells did not reliably differentiate the species, especially within a given species group. Further analyses of various shell morphometrics (S2 and S3 Tables) indicated that, despite statistically significant ANOVA differences across all groups (all p < 0.01; S4 Table), only the forskalii group (e.g., B. forskalii) was clearly separated from the africanus and truncatus/tropicus groups in PCA analysis (PC1: 40.1% variance; Fig 7A). The latter two groups exhibited significant overlap (Fig 7B), even after excluding B. forskalii (PC1: 56.6%, PC2: 16.8%). Within the truncatus/tropicus group, tetraploid B. truncatus and diploid B. tropicus could not be distinguished morphologically (Fig 7B and S3 Table). Similarly, within the africanus group, species such as B. globosus, B. ugandae, and B. nasutus overlapped in shell dimensions (L, W, A) and aperture characteristics (WA, WS) (S1 and S2 Figs). Additionally, B. globosus specimens from different localities showed no clear morphological differentiation (S3 Table), with overlapping distributions in bivariate plots (S1 and S2 Figs).
Details about the specimen codes, species, and their localities are provided in Table 1. The photograph was taken by S-MZ.
(A) PCA plot including all Bulinus groups, showing the first two principal components that explain 40.8% and 38.7% of the total variance, respectively. (B) PCA plot excluding B. forskalii (BuF3), with the first two principal components explaining 57.3% and 15.4% of the total variance, respectively. Shapes represent different Bulinus groups, with symbol colors corresponding to taxonomic groups (africanus, forskalii, and truncatus/tropicus).
Discussion
Unexpected variation in nuclear genome sizes
In the family Planorbidae, which includes Bulinus and Biomphalaria [6], the basic chromosome number is n = 18 [55]. Biomphalaria snails are diploid, as confirmed by cytogenetic studies, DNA content analysis, and whole genome sequencing [29,31,56–59]. Some authorities consider Bulinus snails to be in a separate family, the Buliniade, along with Indoplanorbis and a few other genera [60]. Indoplanorbis is also known to contain a haploid number of 18 chromosomes [61]. In Bulinus, chromosome numbers and instances of polyploidy were determined through chromosome counting conducted in the 1960s and 1970s [e.g., 62–65]. These studies provided invaluable information for understanding the cytogenetics of Bulinus snails but were not without limitations [64]. Due to technical constraints at that time and the challenges of collecting, transporting, and maintaining live snails, specimens collected in the field were preserved in chemical fixatives (e.g., Carnoy’s solution) and transported to the laboratory for cytogenetic analyses. These chromosome numbers were established from meiotic figures at the diakinesis stage of gonadal tissue (ovotestes). Karyotype analysis based on somatic metaphase chromosomes, which requires tissues or organs from live snails, was performed in the 1980s on only three bulinine species, B. truncatus, B. tropicus, and B. natalensis [56,66,67].
Given the challenge of obtaining somatic metaphase chromosome numbers or conducting karyotype studies, DNA content analysis is a viable option, particularly for polyploid species. Generally, within a closely related group of species, there is a correlation between chromosome number and genome size or DNA content [68,69]. Relative DNA contents among closely related species provide useful indications of ploidy levels and changes in genome sizes. The first effort to measure nuclear DNA content, focusing on three Bulinus species groups: africanus, forskalii, and truncatus/tropicus was performed using flow cytometry and presented in this study. The smallest genome size was found in B. forskalii, where all species in the forskalii group are diploid. Among the representatives of the truncatus/tropicus group we examined, in concordance with cytological studies [62–64], two general types of genome sizes were observed, with interspecific and intraspecific variation: tetraploid B. truncatus had significantly more DNA content than diploid B. tropicus. B. truncatus, including a strain from the type locality in Egypt maintained at the Biomedical Research Institute (BRI: www.afbr-bri.org) for decades, has been confirmed to have a total of 72 chromosomes by multiple cytogenetic studies [56,65,70–72], serving as a reliable reference for genetic and genomic studies in Bulinus snails.
An unexpected finding was noted in the B. africanus group, particularly for the taxon B. globosus, which is increasingly recognized as a likely species complex [22,24,26]. All species assayed in this group, including B. globosus, B. ugandae, and B. nasutus, are believed to be diploid [6]. However, the variation in DNA content (1.33 to 1.98 pg) is notably high, similar to that of the truncatus/tropicus group (1.03-1.82 pg), which contains both diploid (B. tropicus) and tetraploid (B. truncatus) species. The DNA content of B. globosus ranges from 1.43 to 1.98 pg, with the highest value (1.98 pg) slightly exceeding that of tetraploid B. truncatus (1.82 pg). The C-values of two B. globosus samples from western Kenya (BuG2: 1.98 pg; BuG5L: 1.86 pg) are higher than the C-value of B. globosus from the eastern coastal Kinango dam (BuG1: 1.43 pg) and the central regions, Kwa Katiwa dam (BuG12: 1.50 pg) and Eldoro in Taveta (BuG13: 1.36 pg). The high variability in genome size within the africanus group, particularly among different specimens of B. globosus, suggests dynamic genomic variation and adds further support to the hypothesis that B. globosus may comprise multiple or cryptic species [22,26].
B. ugandae (1.33-1.46 pg) and B. nasutus (1.41 pg) have a small range variation in genome size, but their genome sizes are larger than those of diploid species such as Biomphalaria spp. (0.91-1.11 pg) or B. forskalii (0.76 pg) and B. tropicus (1.03-1.32 pg), while being smaller than that of the tetraploid B. truncatus (1.82 pg). Notably, with the exception of Bulinus forskalii, the genome sizes of known diploid Bulinus snails are larger and more variable than those of the measured Biomphalaria species (Fig 2), despite both sharing the same basal chromosome number of n = 18, characteristic of the family Planorbidae [55]. Early-diverging South American representatives of Biomphalaria [73] are as yet unknown with respect to genome size and variability. With the evidence currently in hand, some representatives of Bulinus have subsequently undergone processes (genome expansion and/or whole genome duplication) that have resulted in much greater variability in genome size, including puzzling variations among species generally considered diploid.
Genome expansion and whole genome duplication (polyploidization)
The profound genome size variation uncovered in the current study suggests that bulinine snails, particularly the africanus group, have undergone genome expansion, whole genome duplication (polyploidization), or both processes.
Genome expansion or variation in genome size without changes in ploidy levels has been documented in many eukaryotes [74], including shrimp of the genus Synalpheus [75,76], flour beetles of the genus Tribolium [77], the fruit fly Drosophila [78], and the rotifer Brachionus plicatilis [79]. The genome size of B. globosus is comparable to that of the tetraploid B. truncatus, and significantly higher than that of recognized diploid species such as B. forskalii, B. tropicus, and Biomphalaria spp. If the B. africanus group species are indeed true diploids, they must have undergone significant genome expansion (i.e., increased chromosome sizes without changing chromosome numbers). Furthermore, given the known genome sizes of three species of Biomphalaria (Bi. glabrata, Bi. pfeifferi, and Bi. sudanica) and B. forskalii, it appears that most diploid bulinines have undergone some degree of genome expansion, while B. forskalii may have experienced slight genome depletion.
Polyploidization resulting from whole genome duplication has been found in animals, including the New Zealand snail Potamopyrgus antipodarum [80–82]. In bulinine snails, it has been documented only in the truncatus/tropicus group, where tetraploidy, hexaploidy, and octoploidy have been recorded [6]. Our genome size data imply that the africanus group may possess triploid and tetraploid forms, assuming that homologous chromosome sizes are conserved across the family Planorbidae. Triploidy has not been reported for any Bulinus species, while tetraploidy has only been found in the truncatus/tropicus group. Although early cytogenetic studies indicate that there are no polyploids in the africanus group [6], a recent population genetics study suggests that polyploidy may be present in B. globosus, a taxon from the africanus group, in Kenyan populations [24]. To clarify these intriguing questions, further investigations are needed to analyze nuclear DNA content and/or somatic chromosomes in various populations, particularly within the B. globosus species complex.
Polyploidy can influence an organism’s phenotypes, including its refractoriness to parasites. Increased allelic diversity may enable hosts to recognize a broader range of parasites [83–85]. Alternatively, the combination of subgenomes from different species may alter gene expression, also affecting the degree of parasite resistance [86–89]. Triploid P. antipodarum have greater resistance to allopatric parasites than diploids, suggesting the advantages of increased ploidy for hosts facing coevolving parasites [83]. In bulinines, the host-trematode (including schistosomes) systems are highly complex; several trematode species, including multiple schistosome parasites, as described in the Introduction section, employ bulinines as hosts. For the human parasite S. haematobium, some bulinine species or ecotypes are susceptible, while others are not [6,90]. The role of genome duplication and expansion in influencing infection outcomes presents a compelling question in evolutionary biology and schistosomiasis transmission.
The origin of polyploidy in Bulinus remains unknown. Diploid and tetraploid species from the B. truncatus/tropicus complex are widely distributed across Africa, while hexaploid and octoploid forms are confined to the highlands of Ethiopia [65]. In Kenya, diploids and tetraploids, but no hexaploids or octoploids, have been found in the highlands [1,70]. The tetraploid B. permembranacea found in the Kenyan highlands is morphologically different from B. truncatus found in other regions, suggesting at least two independent origins of tetraploidy in Africa [70]. Our findings add further complexity to the understanding of bulinine snails in Africa. Using a comparative karyotype approach, Goldman et al. (1983) hypothesized a hybrid origin for tetraploid B. truncatus [67]. Modern genomic technologies, such as subgenome analysis that have been applied in other polyploid organisms [91–94], may yield valuable insights into the origin of polyploidy in bulinines.
Conservation and variation of complete mitogenomes
Our study adds 14 new complete and annotated bulinine mitogenome sequences to public databases, bringing the total to 20 complete and annotated bulinine mitogenome sequences available to date [26]. We demonstrate that the protein-coding genes (PCGs) of all bulinine mitogenomes are well conserved. However, a difference was noted in the location of tRNA-D, which divides the Bulinus snails we sampled into two types: DCF and CF, as noted in our previous study [26]. In terms of gene organization, the sampled members of the forskalii and truncatus/tropicus groups share the same structure, referred to as the DCF type, while the africanus group exhibits the CF type. This difference results from the open reading frame (ORF) of the ND4 gene terminating with an incomplete stop codon (T--) followed by an inverted repeat (TAACAGAATTCTGTTA), forming a hairpin structure at the 3’ end of the transcript in the DCF group. The forskalii and truncatus/tropicus groups are more closely related to each other than to the africanus group from a mitogenome structure perspective.
Implications for phylogenetic analyses
The availability of twenty Bulinus and six Biomphalaria mitogenomes [26, 95, this study] provides valuable genetic markers for population genetics and phylogenetic analyses. This enables a more comprehensive investigation of mitogenome evolution in schistosomiasis-transmitting planorbid snails, which are responsible for most global schistosomiasis transmission. Our full-length mtDNA tree indicated that the B. forskalii group is more closely related to the B. truncatus/tropicus complex than to the B. africanus group (Figs 4 and 8A), aligning with the pattern revealed by mitogenome structure (see above). This relationship, despite the exclusion of the B. reticulatus group, differs from patterns revealed by COX1 and ITS (Fig 8B and 8C) [16,17,19,20,23,96–100]. Our COX1 tree also indicated that species from the B. africanus group cluster with species from the B. truncatus/ tropicus group (Figs 5 and 8B) [16,17,20,97,98,100], which is different from the finding that the B. forskalii and B. truncatus/tropicus are more closely related to each other than to the B. africanus group (Fig 8C) [19,23,99]. These variable patterns among the species groups within Bulinus suggest that establishing species concepts in Bulinus requires more evidence. Employing whole genome sequencing, rather than focusing on a few loci, may provide a more robust pattern of relationships.
References for each type of relationship (A, B, and C) are provided. The species studied from the B. reticulatus group is normally B. wright, as there are only two species (B. reticulatus and B. wright) in this group.
The phylogenetic analyses also support the complexity of the taxon B. globosus as revealed by nuclear genome size variation in this study and previous mtDNA analyses [22,24,26]. It also helps clarify ambiguous species. The specimen BuTp7A, found in the deep waters of Lake Victoria, is much smaller than B. tropicus but similar in size to B. truncatus. Morphometric analysis cannot easily differentiate the two species. In Lake Victoria, three species with type localities in the lake are present: B. ugandae (2n = 36), B. transversalis (2n = 72), and B. trigonus (2n: unknown) [6]; however, our phylogenetic analysis indicates that BuTp7A is closer to B. tropicus than to these three species. This is surprising, as B. tropicus has been commonly found elsewhere but not in deep waters. Furthermore, the DNA content of BuTp7A (1.32 pg) is higher than that of B. tropicus (BuTp14; 1.03 pg), collected from Lake Jipe, but smaller than that of tetraploid B. truncatus (1.82 pg). These findings suggest a complex relationship within the B. truncatus/tropicus group, which includes diploid, tetraploid, hexaploid, octoploid, and possibly other polyploids such as triploids.
Limitations of morphometric analysis
Among the four species groups, the forskalii group is easily identified due to its distinct long shell shape. Differentiating between B. forskalii and B. senegalensis, both from the forskalii group using morphometric analysis, is feasible, mainly due to the different whorl shapes in these species [101]. The challenge lies with the remaining africanus and truncatus/tropicus groups, which consist of 24 species and play a crucial role in schistosomiasis transmission. Similar difficulties in differentiating B. africanus group species using shell morphology alone have been reported [102]. Nevertheless, caution is warranted in morphological analysis, as misidentifications from morphological data can lead to misinformation in molecular data in public databases. It is critical to integrate molecular data for reliable species identification, particularly within taxonomically challenging groups where shell morphology exhibits high variability or overlap among species.
Bulinine species and species identification
In bulinine snails, high morphological variation, the lack of clear conchological characteristics, and the existence of ecotypes and intermediate forms between species make accurate species identification highly challenging. Early cytogenetic work relied on meiotic figures of the ovotestis and requires verification through DNA content and somatic chromosome analysis. While these snails are polyploid, it remains uncertain whether polyploidy can serve as a criterion for species distinction, given that B. permembranaceus (4n), B. hexaploidus (6n), and B. octoploidus (8n)—the polyploid complex found in Ethiopia—were primarily named based on their polyploid characteristics [6,65]. It is common for a single species to exhibit multiple ploidy levels [103]. For example, the New Zealand mud snail P. antipodarum has diploid, triploid, and tetraploid forms; yet, they all belong to one species [80–82]. The freshwater fish Misgurnus anguillicaudatus has diploid, triploid, tetraploid, pentaploid, and hexaploid forms in natural populations [104]. It seems that ploidy level may not be a reliable characteristic for species identification. At the molecular level, protein electrophoresis was previously employed to assist in species identification. For example, the primary evidence for the distinct species B. browni and B. hightoni from Kenya was established by protein electrophoretic patterns [6,105,106]. However, caution should be exercised regarding protein expression patterns, as they are controlled by gene regulation, which is highly influenced by ecosystems, environments, and developmental stages. For molecular markers such as proteins and mtDNAs, which have been commonly used, it is unclear what level of genetic differentiation can be considered species level. Furthermore, it is uncertain which molecular marker—mtDNA or nuclear marker—is best to use, given the discordance found between nuclear and mitochondrial genes (mitonuclear discordance) in many species [107–110]. Although these issues are being addressed, defining species remains a challenge, especially since they exhibit hermaphroditism. A combined study of datasets from the nuclear genome, mitochondrial genome, and morphology, as shown in this study, revealed unexpected findings and raised unanswered questions, further highlighting the complexity of bulinine snails in Africa. More comprehensive investigations from various biological perspectives, utilizing modern technologies such as genomics and AI-assisted 3D analysis, while considering specimens from type localities, are necessary to address these complex questions.
Conclusion
We measured nuclear DNA content, determined complete mitogenomes, and analyzed morphometric data of bulinine snails from three Bulinus species groups collected across various localities in Kenya. We uncovered significant variation in DNA content among bulinine specimens, suggesting genome expansion, whole genome duplication (polyploidization), or both in the B. africanus group—phenomena that were previously unrecognized, with the mechanisms still unknown. Adding 14 new complete mitogenome sequences enhances our understanding of comparative mitogenomics and provides valuable resources for studies in taxonomy, evolution, population genetics, phylogenetics, and epidemiology. Both genome size and mitogenome analyses indicate that B. globosus may comprise multiple or cryptic species (i.e., the B. globosus species complex). Our findings confirmed that morphometric data alone are inadequate for reliably identifying Bulinus species, reiterating the need for molecular approaches in bulinine identification. This study also raises significant questions about defining and identifying bulinine species as well as the relationships among species groups within the genus Bulinus. Analyzing all three types of data simultaneously highlights the complexity of bulinine snails, not only in Kenya but also across the African continent. Further studies are needed to focus on these important yet understudied species, which may enhance our understanding of bulinine biology and disease transmission.
Supporting information
S1 Table. Annotation of 14 complete mitogenomes.
https://doi.org/10.1371/journal.pntd.0013305.s001
(XLSX)
S2 Table. Raw shell morphometric measurements (mm) and derived ratios.
https://doi.org/10.1371/journal.pntd.0013305.s002
(XLSX)
S3 Table. Mean ± standard deviation (SD) of shell morphometric parameters and ratios.
https://doi.org/10.1371/journal.pntd.0013305.s003
(XLSX)
S4 Table. ANOVA results for shell morphometric variables.
https://doi.org/10.1371/journal.pntd.0013305.s004
(XLSX)
S1 Fig. Bivariate relationships of shell morphometric variables among Bulinus species.
https://doi.org/10.1371/journal.pntd.0013305.s005
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S2 Fig. Bivariate relationships of shell morphometric variables among Bulinus species after excluding B. forskalii.
https://doi.org/10.1371/journal.pntd.0013305.s006
(TIFF)
S1 Data. Map and SnapGene file of 14 bulinine mitogenomes.
https://doi.org/10.1371/journal.pntd.0013305.s007
(ZIP)
Acknowledgments
We thank the UNM Center for Advanced Research Computing (CARC), supported in part by the National Science Foundation, for providing the high-performance computing and large-scale storage resources used in this research. Bulinus truncatus, originating from Egypt, was provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201700014I.
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