Introduction
In recent years, there has been a growing concern regarding the conservation of parasites that do not harm humans or do not impede conservation attempts (e.g. Dougherty et al., Reference Dougherty, Carlson, Bueno, Burgio, Cizauskas, Clements, Seidel and Harris2016; Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020). This is because parasites are integral parts of the ecosystems from several points of view, e.g. species diversity, biomass, evolution of host immunity and importance in food webs (Dougherty et al., Reference Dougherty, Carlson, Bueno, Burgio, Cizauskas, Clements, Seidel and Harris2016). Moreover, they are affected by anthropogenic environmental changes and can go extinct together with their hosts (Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020). However, tools for parasite conservation have rarely been tested (Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020). Particularly, molecular tools (e.g. DNA barcoding and reconstruction of demographic histories through molecular data) have not been used extensively in non-medically relevant parasites and they can be useful for estimating general population parameters [e.g. genetic diversity and trends of effective population size (Ne); Criscione et al., Reference Criscione, Poulin and Blouin2005; Selbach et al., Reference Selbach, Jorge, Dowle, Bennett, Chai, Doherty, Eriksson, Filion, Hay, Herbison, Lindner, Park, Presswell, Ruehle, Sobrinho, Wainwright and Poulin2019; Strobel et al., Reference Strobel, Hays, Moody, Blum and Heins2019; Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020]. Genetic diversity may be linked to local adaption or life history, and its estimation may help to understand ecological traits or even infer potential population bottlenecks (van Schaik et al., Reference van Schaik, Dekeukeleire and Kerth2015; Tobias et al., Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017; Radačovská et al., Reference Radačovská, Čisovská Bazsalovicsová, Šoltys, Štefka, Minárik, Gustinelli, Chugunova and Králová-Hromadová2022). Ne is known to greatly influence the genetic diversity and viability of populations (Criscione et al., Reference Criscione, Poulin and Blouin2005; Pérez-Pereira et al., Reference Pérez-Pereira, Wang, Quesada and Caballero2022), but estimating it in parasites may come with some caveats depending on the studied system (Criscione et al., Reference Criscione, Poulin and Blouin2005; Strobel et al., Reference Strobel, Hays, Moody, Blum and Heins2019). Nevertheless, estimates of Ne can be helpful for considering the taxa's conservation status (Pérez-Pereira et al., Reference Pérez-Pereira, Wang, Quesada and Caballero2022). Unfortunately, the current lack of genetic resources makes it difficult for researchers to enact timely conservation plans, putting some species at risk of extinction (Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020).
Methods for estimating Ne trends from genetic data of non-model organisms are being developed (e.g. Liu and Fu, Reference Liu and Fu2020). This has been possible thanks to protocols that allow researchers to generate genome-wide data without reference sequences (Peterson et al., Reference Peterson, Weber, Kay, Fisher and Hoekstra2012; Nunziata and Weisrock, Reference Nunziata and Weisrock2018). For example, restriction-site associated DNA sequencing (RADseq) approaches have been shown to work well with non-model organisms and became popular in population and conservation genetic studies due to their relatively low cost, versatility and ability to generate data for up to tens of thousands of loci (Peterson et al., Reference Peterson, Weber, Kay, Fisher and Hoekstra2012; Nunziata and Weisrock, Reference Nunziata and Weisrock2018). Furthermore, the produced data can be used with bioinformatic tools for estimating population trends at least 20 generations before a decline (Nunziata and Weisrock, Reference Nunziata and Weisrock2018) and genetic structure (e.g. Dalapicolla et al., Reference Dalapicolla, do Prado, Percequillo and Knowles2021). This is interesting from a molecular conservation parasitology perspective, given that non-medically relevant parasites often lack reference data (Selbach et al., Reference Selbach, Jorge, Dowle, Bennett, Chai, Doherty, Eriksson, Filion, Hay, Herbison, Lindner, Park, Presswell, Ruehle, Sobrinho, Wainwright and Poulin2019). Alternatively, even single-locus methods for Ne estimation are available, allowing researchers to use previously released data (e.g. sequences from GenBank) for demographic inferences, although having more loci leads to better parameter estimation (Ho and Shapiro, Reference Ho and Shapiro2011).
In this study, we used multiple protocols and methods for generating molecular data to study population genetic structure (i.e. potential genetic differentiation based on host or geography: e.g. van Schaik et al., Reference van Schaik, Dekeukeleire and Kerth2015 and Dalapicolla et al., Reference Dalapicolla, do Prado, Percequillo and Knowles2021) and the changes of Ne over time in freshwater horsehair worms, also called ‘gordiids’ (phylum Nematomorpha, class Gordioida; Sup. Info) in Taiwan. Such worms are regarded as one of the least studied animal group, especially from a molecular perspective (Bolek et al., Reference Bolek, Schmidt-Rhaesa, De Villalobos, Hanelt, Thorp and Rogers2015; Tobias et al., Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017) and they have interesting ecological and conservation characteristics (Sato et al., Reference Sato, Watanabe, Fukushima and Tokuchi2014; Bolek et al., Reference Bolek, Schmidt-Rhaesa, De Villalobos, Hanelt, Thorp and Rogers2015; Sup. Info). The methods used in this study can be applied to other parasitic groups for conservation and biodiversity-oriented studies. Specifically, a double-digest restriction-site-associated DNA sequencing (ddRADseq) dataset including all 3 known horsehair species in Taiwan (Sup. Info) was generated. Additionally, DNA sequence data from the mitochondrial cytochrome c oxidase subunit I (COXI) region were generated and used for evaluating population and genetic status in combination with previously released data (Chiu et al., Reference Chiu, Huang, Wu and Shiao2011, Reference Chiu, Huang, Wu and Shiao2016, Reference Chiu, Huang, Wu and Shiao2017, Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020). This mitochondrial marker was used due to the lack of any other previously released molecular data on Taiwanese horsehair worms. Although the use of mitochondrial data for genetic diversity studies can be controversial, COXI is regarded as a good proxy for evaluating the conservation status of species population for the first time (Petit-Marty et al., Reference Petit-Marty, Vázquez-Luis and Hendriks2021 and references therein). Additionally, such marker was also utilized in a previous study about hairworms' population genetic structure (Tobias et al., Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017) and its availability in public repositories is expected to increase, given its use in barcoding (Selbach et al., Reference Selbach, Jorge, Dowle, Bennett, Chai, Doherty, Eriksson, Filion, Hay, Herbison, Lindner, Park, Presswell, Ruehle, Sobrinho, Wainwright and Poulin2019). With both datasets, population genetic structure and demographic history were estimated and compared. More focus was given to Chordodes formosanus, since they are relatively easier to sample and are more common (Chiu, Reference Chiu2017). A country-wide ddRADseq dataset was generated for the said species, allowing us to estimate Ne and population genetic structure at Taiwan main island-level. Additionally, the Taiwanese and Japanese populations' trends were compared using the COXI dataset.
Materials and methods
Sampling
Gordiids were sampled over a span of 14 years, from 2007 to 2021 (Fig. 1: Sup. Table 1). Chordodes formosanus, the most common species in Taiwan (Chiu, Reference Chiu2017), was sampled country-wide, while specimens of Acutogordius taiwanensis and Gordius chiashanus were collected from fewer localities (Fig. 1; Sup. Table 1). Specimens were collected either by hand in bodies of water (i.e. streams and man-made ponds), by sampling the hosts and then immersing them until the adult worm came out, or by collecting the dead gordiids on the ground, which is the most common method for finding them in Taiwan (Chiu, Reference Chiu2017). The samples were fixed in 95% alcohol and stored at −20°C. Species recognition was based on DNA barcoding, given that the majority of the samples were too ruined for morphological identification. A total of 38 C. formosanus specimens, 10 A. taiwanensis and 3 G. chiashanus were collected (see SRA BioProject PRJNA914055); however, DNA extraction and sequencing was not successful on all of them (Sup. Table 1), probably due to the low quality of some of the specimens.
Sampling localities for Chordodes formosanus (black), Acutogordius taiwanensis (purple) and Gordius chiashanus (red), with a map showing elevation and river network in Taiwan. Localities from previous studies have been included. The purple dot with black borders shows an area of sympatry between C. formosanus and A. taiwanensis. The legend on the right shows elevation in metres.
DNA extraction, COXI amplification and ddRADseq protocol
For DNA extraction, we used a modified version of the Qiagen DNeasy Animal Tissue Protocol (Qiagen, Hilden, Germany). We modified the original protocol by using double-distilled water (ddH2O) instead of elution buffer for dilution, and we eluted 60 μL of DNA, given the low concentration of DNA. Additionally, we left the tissue to incubate overnight at 60°C.
The amplification of the COXI sequences through polymerase chain reaction (PCR) was done using the universal primer set LCO1490 and HC02198 (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994) and was performed in a total volume of 25 μL using PuReTaq Ready-To-Go PCR Beads (Cytiva, formerly GE Healthcare, Marlborough, United States). The PCR was initiated at 95°C for 5 min, followed by 40 cycles at 95°C for 1 min, 50°C for 1 min and 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR products were purified following the NautiaZ Gel/PCR DNA Purification Mini Kit protocol (Nautia Gene, Taipei, Taiwan). Sanger Sequencing was done by the DNA Sequencing Core Facility of the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
An edited version of the Peterson et al. (Reference Peterson, Weber, Kay, Fisher and Hoekstra2012) protocol was used for generating the ddRADseq library. We modified the original method by using between 100 and 140 ng of genomic DNA per sample, 4 μL of ddH2O for bead clean-up and 60 μL of ddH2O for elution before measuring DNA concentration with Qubit. Single-end sequencing of 100-nucleotide base pair (bp) lengths were carried out using an Illumina HiSeq2500 system.
COXI analyses
The COXI forward and reversed sequences from each individual were visualized with MEGA X (ver. 10.1.8; Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018). The alignments were done with MAFFT 7.471 (Katoh and Standley, Reference Katoh and Standley2013) using the L-INS-i algorithm. The sequences were trimmed manually and consensus sequences were saved from the alignments.
COXI sequences at least 400 bp long for each taxon from previous studies (Chiu et al. Reference Chiu, Huang, Wu and Shiao2011, Reference Chiu, Huang, Wu and Shiao2016, Reference Chiu, Huang, Wu and Shiao2017, Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020; Sup. Table 2) were downloaded from GenBank (Clark et al., Reference Clark, Karsch-Mizrachi, Lipman, Ostell and Sayers2016: last assessed 4th November 2022), aligned with the newly generated sequences using MAFFT 7.471 with the L-INS-i algorithm and manually trimmed while being visualized with MEGA X. In the case of A. taiwanensis, an additional sequence of a hairworm from Myanmar (MF983649) was included for population genetic structure analyses; said sequence has been shown to fall inside the known COXI variability of A. taiwanensis in previous studies (Chiu et al., Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020) and therefore it should represent a sequence from the said species. In total, 433 bp were recovered for 85 specimens of C. formosanus (36 newly generated), 414 bp for 31 A. taiwanensis (3 newly generated) and 382 bp for 26 individual of G. chiashanus (1 newly generated). The newly generated sequences are available in GenBank (OQ121045-OQ121047 for A. taiwanensis, OQ121048-OQ121083 for C. formosanus, OQ121084 for G. chiashanus).
Possible population genetic structure, hypothetical haplotypes (i.e. unsampled sequences) and genetic diversity indices [nucleotide diversity (pi) and Tajima's D: Nei and Li, Reference Nei and Li1979; Tajima, Reference Tajima1989] were inferred by a haplotype network in PopArt (Leigh and Bryant, Reference Leigh and Bryant2015), generated with the TCS 1.21 algorithm (Clement et al., Reference Clement, Posada and Crandall2000). Estimation of Ne trends was calculated by running Coalescent Bayesian Skyline plot analyses on BEAST2 (version 2.6.3: Bouckaert et al., Reference Bouckaert, Vaughan, Barido-Sottani, Duchêne, Fourment, Gavryushkina, Heled, Jones, Kühnert, De Maio, Matschiner, Mendes, Müller, Ogilvie, du Plessis, Popinga, Rambaut, Rasmussen, Siveroni, Suchard, Wu, Xie, Zhang, Stadler and Drummond2019). Such analyses allow the estimation of trends from mitochondrial data, even if only one locus is available, and recover well with Pleistocene trends (Ho and Shapiro, Reference Ho and Shapiro2011). Furthermore, previous studies were able to recover demographic trends with less than 300 bp of mitochondrial sequences (Ho and Shapiro, Reference Ho and Shapiro2011 and references therein); therefore, the alignments should be long enough for allowing trends' estimations. The most likely nucleotide substitution model for each species was inferred using ModelTest, implemented in raxmlGUI (Edler et al., Reference Edler, Klein, Antonelli and Silvestro2021) according to Akaike information criterion (AIC; Akaike, Reference Akaike, Parzen, Tanabe and Kitigawa1998). A Relaxed Clock Log model, with a clock rate of roughly 0.0013 per million years, was set. We estimated this clock rate by aligning full COXI sequences from the only available Chordodes and Gordius mitogenomes from GenBank (MG257764 and MG257767), dividing the number of variable sites by the total number of sites and then dividing again by 2 before dividing by 110, which represents the estimated age in million years of the oldest known gordiid fossil (Poinar and Buckley, Reference Poinar and Buckley2006). Note that estimates for the appearance of crown group Nematomorpha range from around the mid-Cambrian to the mid-Cretaceous (Howard et al., Reference Howard, Giacomelli, Lozano-Fernandez, Edgecombe, Fleming, Kristensen, Ma, Olesen, Sørensen, Thomsen, Wills, Donoghue and Pisani2022), but that these estimates include the possible divergence time of the Nectonema lineage from gordiids too. Therefore, we prefer to refer to the fossil datum, since it is the earliest possible divergence point for the Chordodidae/Gordiidae lineage. Additionally, the C. formosanus Japanese and Taiwanese samples were split for estimations, while the Myanmar sequence was removed for A. taiwanensis.
ddRADseq analyses
Two different pipelines for processing the ddRAD data were used for C. formosanus: ipyrad (version 0.9.84: Eaton and Overcast, Reference Eaton and Overcast2020) and Stacks (version 2.62: Rochette et al., Reference Rochette, Rivera-Colón and Catchen2019). For the ipyrad pipeline, we performed de-novo assembly with a Phred Qscore offset of 43, 90% clust threshold, 0.5 maximum of heterozygotes in consensus, and minimum sample locus at 80% of all the samples, while keeping other settings at their default values. Samples with less than 300 loci were discarded for further analyses. For Stacks, the protocol listed in Rivera-Colón and Catchen (Reference Rivera-Colón, Catchen, Verde and Giordano2022) was followed and all the sites (variant and invariant) were outputted in the Variant Call Format (VCF) file. In total, 27 individuals were retained for C. formosanus from both pipelines. In the case of A. taiwanensis and G. chiashanus, only 2 individuals per species had at least 200,000 reads after trimming and therefore we only used Stacks for their assemblies, with the ‘r’ flag set at 1 instead of 0.8.
To check the genetic population genetic structure of C. formosanus, tools from ipyrad and R were used with both the ipyrad and Stacks output. The Stacks VCF file was filtered to remove the loci without SNPs and the SNPs with more than 20% missing data, using an edited version of previously released R scripts (Dalapicolla et al., Reference Dalapicolla, do Prado, Percequillo and Knowles2021). Additionally, the filtered VCF file was converted to a HDF5 database using the converter inside the ipyrad-analysis toolkit (Eaton and Overcast, Reference Eaton and Overcast2020) to make it compatible with the tools implemented inside ipyrad. After this, k-means principal component analyses (PCA) were run 100 times in the ipyrad-analysis tools suite. Moreover, the snapclust clustering method implemented into the ‘adegenet’ R package (Beugin et al., Reference Beugin, Gayet, Pontier, Devillard and Jombart2018) was used. The best number of clusters (k) was calculated by computing the AIC and the Bayesian information criterion (BIC; Schwarz, Reference Schwarz1978), while picking the value for which both criteria were lower. The snapclust analyses were run with both the ipyrad full output STRUCTURE file and a STRUCTURE file generated from the filtered Stacks VCF thanks to PGDSpider (Lischer and Excoffier, Reference Lischer and Excoffier2012). Furthermore, for analysing possible admixture between geographically separated C. formosanus individuals, RADpainter and fineRADstructure (Malinsky et al., Reference Malinsky, Trucchi, Lawson and Falush2018) were used and the results were plotted with R. The RADpainter file from the Stacks output was used for such software, while the ‘hapsFromVCF’ command generated an input file with the ipyrad-outputted VCF.
For population demographic analyses of all the taxa, a site-frequency spectra (SFS) file was generated using the easySFS script (https://github.com/isaacovercast/easySFS) by converting the Stacks VCF file with the invariant sites, choosing the number of individuals that maximize the number of segregating sites. Note that easySFS takes into consideration the fact that missing data are present, and therefore we used the full Stacks VCF output for SFS conversion. After that, Stairway Plot 2 (version 2.1.1: Liu and Fu, Reference Liu and Fu2020) was used per species following the software's manual. Given the current knowledge on Taiwanese horsehair worms (Chiu, Reference Chiu2017; Chiu et al., Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020), generation time was set at 1 per year. As for the mutation rate, given the lack of whole-genome data for horsehair worms, a spontaneous mutation of rate of 2 × 10−9 per site per generation, which is one of the nematode Pristionchus pacificus (Weller et al., Reference Weller, Rödelsperger, Eberhardt, Molnar and Sommer2014), was set. We argue that, even if nematodes and hairworms have been split for hundreds of millions of years (Howard et al., Reference Howard, Giacomelli, Lozano-Fernandez, Edgecombe, Fleming, Kristensen, Ma, Olesen, Sørensen, Thomsen, Wills, Donoghue and Pisani2022), mutation rates in invertebrates tend to be around the same order of magnitude (10−9; e.g. Konrad et al., Reference Konrad, Brady, Bergthorsson and Katju2019) and therefore should not influence the results in a significant way.
Results
Coxi analyses
In total, there were 54 different mitochondrial haplotypes in C. formosanus, 9 of which were hypothetical and 28 of which were made up by a single individual (Fig. 2A). Acutogordius taiwanensis presented 9 haplotypes, none of which were hypothetical and 5 of which were made up by a single individual; however, 5 haplotypes were separated from the most common one by just 1 mutation (Fig. 2B). 24 haplotypes, with 5 hypotheticals, were counted for G. chiashanus, and 15 of these were made up by a single individual (Fig. 2C).
Haplotype networks based on COXI fragments. (A) Chordodes formosanus; (B) Acutogordius taiwanensis; (C) Gordius chiashanus.
Pi was 297585 for C. formosanus, around 0.001 for A. taiwanensis and 67063.1 for G. chiashanus. C. formosanus had 54 segregating sites and 30 parsimony-informative ones, while A. taiwanensis had 11 and 4 and G. chiashanus had 30 and 23, respectively. Tajima's D for C. formosanus was around 3.889 × 107, while it was around −2.692 for A. taiwanensis and around 1.209 × 107 for G. chiashanus; this statistic was significant (P = 0) for both C. formosanus and G. chiashanus, but not for A. taiwanensis (P ≈ 1) (Sup. Table 3).
For the Coalescent Bayesian Skyline plot analyses, the number of variable sites was 49 for the Taiwanese samples of C. formosanus, while it was 12 for the Japanese ones. After removing the Myanmar sequence, 7 variable sites were retained for A. taiwanensis. We did not change the alignment file for G. chiashanus, and therefore 30 variable sites remained (Sup. Data). The best substitution model for the Taiwanese samples of C. formosanus was TrN + G4, while TIM1 was the best for the Japanese ones. HKY + I and TPM3uf + I were the best models for A. taiwanensis and G. chiashanus, respectively (Sup. Table 4). The Bayesian Skyline Plot results show how both the populations of C. formosanus are having a small decline, while A. taiwanensis's population tends to be stable (Fig. 3). G. chiashanus's population size seemed to be increasing until recently (Fig. 3).
Bayesian Sky Plots based on COXI fragments per each species/population.
ddRADseq analyses
The ipyrad trimming step led to an average read number of 2195578.39 per C. formosanus sample (including samples that were discarded because of the low number of reads or low number of retained loci). The ipyrad final output for C. formosanus had a SNP matrix size of 2248, with 17.82% missing sites, while the total sequence matrix size was 52233, with 17.59% missing sites. The lowest number of loci in assembly per an individual was 331, while the highest one was 548 and the average was around 473.889 loci per samples (Sup. Data).
For the Stacks output, an average read number of 1672668.836 per sample was retained (including samples that were discarded because of low number of reads). The optimal settings for C. formosanus were obtained with both the number of allowed mismatches (M) and the number of allowed mismatches between individual loci and the catalogue of loci (n) set to 6. For A. taiwanensis and G. chiashanus, the best results were obtained with M and n set to 12 and 9, respectively. The number of R80 loci was 1876 for C. formosanus, 1388 for A. taiwanensis and 2484 for G. chiashanus. After removing the SNPs with 20% of the missing data, 4243 SNPs were retained for PCA and snapclust analyses for C. formosanus (Sup. Data).
Both PCA and snapclust analyses, conducted using both the output VCFs (ipyrad and filtered Stacks), failed to trace any structure whatsoever in C. formosanus, with both the AIC and BIC computed by snapclust agreeing that k = 1 was the optimal number of clusters for both datasets (Fig. 4; Sup. Figs 1 and 2). The same pattern was observed using fineRADstructure with both input files, given that no cluster formed based on shared coancestry (Fig. 5; Sup. Fig. 3).
(A) PCA results for Stacks ddRAD-seq data for Chordodes formosanus. (B) AIC and BIC values from snapclust analyses for C. formosanus based on the same data as the PCA. The PCA and snapclust results based on ipyrad are available as Supplementary Figs 1 and 2.
Co-ancestry matrix for Chordodes formosanus based on Stacks ddRAD-seq data. The one from ipyrad data is available as Supplementary Fig. 3.
The Stairway Plot output for C. formosanus shows several population drops during the Pleistocene and an estimated Ne of around 1000 individuals in more recent times (Fig. 6; Sup. Fig. 4). However, only 2 drops were detected for both A. taiwanensis and G. chiashanus, and their estimated Ne in recent times were larger (around 300,000 for the former and 150,000 for the latter; Sup. Figs 5 and 6).
Stairway plot for Chordodes formosanus. The original version and the ones for Acutogordius taiwanensis and Gordius chiashanus are available as Supplementary Figs 4–6.
Discussion
COXI and ddRADseq were used for evaluating population genetic structure and trends of Ne in the considered gordiids. The trends for the most sampled species, C. formosanus, were similar in the Pleistocene according to both datasets, although the Stairway Plot based on ddRAD was able to detect more changes in the last 100,000 years. For the other 2 species, the scale of the recorded events with COXI differed between each other and C. formosanus, while the ddRAD datasets were too small for detecting genetic structure and recent trends. In all the 3 species, no differentiation based on geography was found according to COXI. The results associated with each species and datasets are discussed below.
COXI diversity analyses
Both C. formosanus and G. chiashanus exhibit high levels of intraspecific diversity and do not exhibit a geographical genetic structure based on mitochondrial data. High mitochondrial diversity without population genetic structure in Nematomorpha species has been shown in taxa from New Zealand (Tobias et al., Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017), hinting at a high dispersal ability or a multigenerational dispersal process. In the case of the previous study, the authors were surprised to discover a lack of genetic structure in Euchordodes nigromaculatus, because the species was known to parasitize wētās (family: Anostostomatidae), which are definitive hosts that do not disperse well and exhibit a strong population genetic structure. Additionally, the known paratenic hosts (i.e. used for transport before the final infection and not for development: Criscione et al., Reference Criscione, Poulin and Blouin2005; Bolek et al., Reference Bolek, Schmidt-Rhaesa, De Villalobos, Hanelt, Thorp and Rogers2015) should not be able to disperse greatly in the mountain range inhabited by E. nigromaculatus (Tobias et al., Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017). However, new studies have revealed that this New Zealand species can also infect cockroach Celatoblatta quinquemaculata, a common insect in the mountains of central Otago (Doherty et al., Reference Doherty, Filion and Poulin2022), which is the area considered by Tobias et al. (Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017) for the study. This suggests that this cockroach shows more dispersal ability than the wētās. Therefore, it could be the case that dispersal is facilitated by definitive hosts. Note that dispersal by definitive hosts does not exclude the possibility of a multigenerational process, as highlighted by Tobias et al. (Reference Tobias, Yadav, Schmidt-Rhaesa and Poulin2017). Given the scarce knowledge of paratenic hosts used by Taiwanese horsehair worms, we should not exclude them as possible vectors also.
In the case of G. chiashanus, only one definitive host (a millipede species from the genus Spirobolus) and only one potential paratenic host [the mayfly Ephemera orientalis (Chiu et al., Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020), which is known to be present in different East Asian countries (Lee et al., Reference Lee, Hwang and Bae2008)] are known. The mayfly does not seem to be commonly infected (Chiu et al., Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020), which raises questions about both the paratenic and definitive host ranges of G. chiashanus: such ranges may include other species that are able to disperse in middle elevation areas, given the high diversity shown by the haplotype network. Another hairworm species with terrestrial adult ecology, Gordius terrestris, prefers earthworms as natural paratenic hosts (Anaya et al., Reference Anaya, Hanelt and Bolek2021) and G. chiashanus might do the same. All the possible explanations for dispersal listed here (by paratenic hosts, by definitive hosts, by multigenerational process) do not exclude each other, and it is likely that hairworm dispersal is a complex process that may include all the options listed above.
While A. taiwanensis showed less intraspecific genetic diversity than the other 2 considered taxa, it must be noted that almost all the samples for this species came from a single region of Taiwan, Yilan County (Chiu et al., Reference Chiu, Huang, Wu and Shiao2017; this study). Meanwhile, the COXI sequences of C. formosanus were generated from individuals from multiple localities in Taiwan and Japan (Chiu et al. Reference Chiu, Huang, Wu and Shiao2011, Reference Chiu, Huang, Wu and Shiao2016, Reference Chiu, Huang, Wu and Shiao2017; this study), and the ones for G. chiashanus came from multiple middle elevation mountain localities in central-south Taiwan (Chiu et al., Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020; this study). These differences in sampling areas can be explained by the varying difficulties encountered when sampling the adult horsehair worms (Sup. Info). Sampling is usually regarded as a significant issue while studying Nematomorpha, due to the lack of standardized collecting methods and the short lifespan of the adults (Bolek et al., Reference Bolek, Schmidt-Rhaesa, De Villalobos, Hanelt, Thorp and Rogers2015). Furthermore, biological differences among species may make some taxa easier to collect compared to others (Sup. Info). As a result of sampling difficulties, it is likely that the genetic diversity of A. taiwanensis is extremely underestimated, as already suggested by Chiu (Reference Chiu2017) and Chiu et al. (Reference Chiu, Huang, Wu, Lin, Chen and Shiao2020), and the species may have gone unnoticed in many areas. Alternatively, A. taiwanensis may be the most vulnerable Taiwanese hairworm species to human actions (Chiu et al., Reference Chiu, Huang, Wu and Shiao2016); previous studies highlighted the possibility of hairworm extirpation caused by anthropic activities (Sato et al., Reference Sato, Watanabe, Fukushima and Tokuchi2014). Therefore, A. taiwanensis may have gone extinct in some areas in Taiwan due to human disturbance.
ddRADseq diversity analyses for Chordodes formosanus in Taiwan
Even with genome-wide data, C. formosanus did not show any sign of population genetic structure according to geographical origins (Fig. 4). Together with its known distribution and COXI haplotype, the results suggest a panmictic population with great dispersal ability. However, gordiids disperse poorly by themselves when they are at the larval stage (Bolek et al., Reference Bolek, Schmidt-Rhaesa, De Villalobos, Hanelt, Thorp and Rogers2015; Chiu et al., Reference Chiu, Huang, Wu and Shiao2016; Chiu, Reference Chiu2017) and the river network in Taiwan prevents non-flying organisms associated with freshwater from dispersing, given its fragmentation (Shih et al., Reference Shih, Hung, Schubart, Chen and Chang2006). That would block potential dispersal by river flow at a country-wide level. Furthermore, the presence of this species in Lyudao (Chiu et al., Reference Chiu, Huang, Wu and Shiao2011), a volcanic island that has never been connected to the main island of Taiwan (Yang et al., Reference Yang, Lee, Chen, Cheng, Knittel, Punongbayan and Rasdas1996), implies sea crossing. Previous modelling efforts noticed a very high similarity between the mantis hosts' range and the range of C. formosanus, hinting at dispersal by definitive hosts, although the dispersal by paratenic ones cannot be excluded (De Vivo and Huang, Reference De Vivo and Huang2022; Sup. Info). Therefore, dispersal by active dispersing flying insect hosts (paratenic and/or final ones) with or without a multigenerational process can be a possible explanation for this lack of geographic structure shown in our results.
Demographic history inferences
The demographic analyses using the COXI datasets were able to trace historical demography through 2 million years for C. formosanus (with the Taiwanese population going a little bit deeper in time). The same approach revealed Ne trends up to roughly 6 million years ago for G. chiashanus and 250,000 years of demographic history for A. taiwanensis. This difference in scale may have been caused by the lower variability of the A. taiwanensis sequences (7 variable sites in total), which in turn was caused by a limited geographic sampling. In general, it is known that mitochondrial sequence data usually tend to recover demographic histories in the Pleistocene (Ho and Shapiro, Reference Ho and Shapiro2011); however, such data are less informative with recent history compared to RADseq (Nunziata and Weisrock, Reference Nunziata and Weisrock2018). The COXI-based Bayesian Skyline Plots revealed recent drops in Ne for both Taiwanese and Japanese populations of C. formosanus over the last 250,000 years. Several drops in the same period were found by Stairway Plot using the ddRADseq data. Additionally, both approaches found a demographic increase a little bit before 2 million years ago for the Taiwanese population. That said, Stairway Plot analyses with ddRADseq data were able to detect more events in the last 100,000 years for the Taiwanese population of C. formosanus, compared to the Coalescent Bayesian Sky Plot based on COXI sequences, probably due to the differences in number of loci between the 2 datasets (e.g. Cristofari et al., Reference Cristofari, Liu, Bonadonna, Cherel, Pistorius, Le Maho, Raybaud, Stenseth, Le Bohec and Trucchi2018) and the different mutation rate in mitochondrial genes (Ho and Shapiro, Reference Ho and Shapiro2011).
The demographic histories reconstructed using genome-wide SNP data and the Stairway Plot were very large for A. taiwanensis and G. chiashanus, but at the same time, the software failed to recover any possible trends in recent years. This was caused by the very small sample size, which were 2 diploid individuals per species. Specifically, the number of historical events that can be estimated using Stairway Plot, and any programs that take site frequency spectrum as inputs and calculate composite likelihoods, is constrained to the number of site frequency categories. As a result, for a folded site frequency spectrum, as implemented here, the use of 2 individuals will only result in 2 site frequency categories, and thus only 2 historical demographic episodes can be estimated (Liu and Fu, Reference Liu and Fu2020). The 2 species also have extremely large Ne estimated based on the Stairway Plot results. It is known that sample size can impact the estimated demographic parameters using RADseq data (Nunziata and Weisrock, Reference Nunziata and Weisrock2018) and therefore we attribute the high Ne estimates to the sampling size, which makes the inferences unreliable.
For C. formosanus, however, more events were recorded from the Stairway Plot analyses. That said, it is surprising that this species, which is regarded as the most common in Taiwan, is showing signs of decline – although the current Ne should still be large enough to sustain the population (Pérez-Pereira et al., Reference Pérez-Pereira, Wang, Quesada and Caballero2022). The recent demographic decrease might be the result of extirpation events, possibly caused by changes in land use in Taiwan, hypothesized after niche modelling in De Vivo and Huang (Reference De Vivo and Huang2022). It is known that land-use changes can influence helminths, although the effects seem to depend on both host's and parasite's ecology (e.g. Chakraborty et al., Reference Chakraborty, Reddy, Tiwari and Umapathy2019; Portela et al., Reference Portela, dos Santos and dos Anjos2020).
Future directions
In this study, we evaluated the use of different genetic and analytical tools for estimating demographic history and population genetic structure, which informed us about potential declines and dispersal traits. From a conservation perspective, the negative trend shown by C. formosanus would deserve more attention in the future, given its confirmation by both Coalescent Skyline and Stairway Plots. A. taiwanensis should also be monitored, due to its known smaller geographical range compared to the other 2 evaluated species and potential sensitivity to anthropic changes (Sup. Info). For G. chiashanus, additional samples should be collected for testing the inferred decline shown by COXI Coalescent Skyline Plots with genome-wide data too and see if recent (last 100,000 years) declines happened. In our datasets, sampling bias for some taxa was present and can influence the results. For possible future studies on the conservation genetics of parasites, we give 3 suggestions that can help with evaluating a parasite's conservation status:
(i) consider the target species' ecologies, given that they can highly influence their population genetic structure (van Schaik et al., Reference van Schaik, Dekeukeleire and Kerth2015; Radačovská et al., Reference Radačovská, Čisovská Bazsalovicsová, Šoltys, Štefka, Minárik, Gustinelli, Chugunova and Králová-Hromadová2022), Ne estimates (Criscione et al., Reference Criscione, Poulin and Blouin2005; Strobel et al., Reference Strobel, Hays, Moody, Blum and Heins2019) as well as the sampling strategy, given that some parasites have life cycles that can influence when and how to collect them (e.g. van Schaik et al., Reference van Schaik, Dekeukeleire and Kerth2015);
(ii) try to get the geographically broadest and biggest sample size possible; for example, in this study a small sample size in the ddRADseq dataset did not allow us to estimate enough recent trends for 2 taxa, while the COXI dataset for A. taiwanensis was too biased to one area, which did not allow us to properly estimate the diversity of the species;
(iii) define the molecular and bioinformatic tools used; a direct estimate of Ne for parasites can be tricky to calculate (e.g. Strobel et al., Reference Strobel, Hays, Moody, Blum and Heins2019; Carlson et al., Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020). Given this, we suggest focusing on trends instead of raw numbers. Additionally, previous studies showed possible limits of single-locus analyses (Ho and Shapiro, Reference Ho and Shapiro2011). Therefore, we suggest using as many loci as possible (see Peterson et al., Reference Peterson, Weber, Kay, Fisher and Hoekstra2012 and Nunziata and Weisrock, Reference Nunziata and Weisrock2018 for potential protocols). However, single-locus data are often the kinds of data that are the most available for parasites (Selbach et al., Reference Selbach, Jorge, Dowle, Bennett, Chai, Doherty, Eriksson, Filion, Hay, Herbison, Lindner, Park, Presswell, Ruehle, Sobrinho, Wainwright and Poulin2019). Therefore, if budget and resources are limited, Sanger sequencing can be pursued (but see Radačovská et al., Reference Radačovská, Čisovská Bazsalovicsová, Šoltys, Štefka, Minárik, Gustinelli, Chugunova and Králová-Hromadová2022 for caveats with mitochondrial sequences in polyploid species). The use of depositories such as GenBank can be useful for retrieving previously released sequences and therefore increase both sample size and loci used while utilizing software such as BEAST2 that can use such data. That said, it is crucial to check for a possible population genetic structure before demographic trend analyses, since it influences the results (Ho and Shapiro, Reference Ho and Shapiro2011).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182023000641
Acknowledgements
We would like to thank all the people who helped us collect samples all around Taiwan. We would also like to thank Ming-Chung Chiu for helping us with designing the sampling strategy and discussing the paper and the biology of the involved species, Hsiang-Yun Lin for sampling assistance, Justin Pelofsky for revising the grammar of this article, Xiaoming Liu for helping us with Stairway Plot 2's settings, the DNA Sequencing Core Facility of the Institute of Biomedical Sciences, Academia Sinica for Sanger sequencing, the NGS High Throughput Genomics Core at Biodiversity Research Center, Academia Sinica, for providing Illumina sequencing and the anonymous reviewers who gave us suggestions on the manuscripts.
Authors’ contributions
MDV extensively sampled the specimens around the country, performed the bioinformatic analyses and wrote the original draft. MDV and WYC performed molecular work. MDV and JPH conceived and designed the study. All the authors designed the methodology, corrected the original draft and approved the manuscript's content.
Financial support
MDV was supported by a scholarship from Taiwan International Graduate Program (TIGP) and from TIGP Research Performance Fellowship 2022. JPH was supported by a grant from Ministry of Science and Technology, Taiwan (MOST 108-2621-B-001-001-MY3) and internal research support from Academia Sinica.
Competing interests
None.
Ethical standards
Not applicable.
Data availability
The COXI sequences produced in this study are available in GenBank (accession numbers: OQ121045- OQ121084). The ddRAD Stacks-trimmed reads are available in Sequence Read Archive (BioProject number: PRJNA914055). Supplementary Data (i.e. logs and outputs) for this article can be found in Zenodo (https://zenodo.org/record/7659651, doi: 10.5281/zenodo.7659651). A preprint of this article, based on the manuscript's first version, is available on BioRxiv (https://www.biorxiv.org/content/10.1101/2023.02.21.529467v1, doi:10.1101/2023.02.21.529467).
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