DOI: 10.5061/dryad.ffbg79d4z
title: Data from: Sampling strategy matters: eDNA-based assessment and extrapolation of myxozoan diversity in a model stream system

description: Determining true species diversity is particularly challenging for
 parasitic organisms. Environmental DNA (eDNA) metabarcoding has
 revolutionized diversity studies by increasing both efficiency and
 precision. This approach is especially relevant for parasitic species,
 like myxozoans that spend part of their life cycle outside their hosts,
 often during hostswitching stages. However, the adequacy of sampling depth
 and methodology in capturing total diversity often remains uncertain. To
 address this, we used a small stream as a model system and collected 60
 small-volume aquatic sediment samples. Using an eDNA metabarcoding
 approach with myxozoan-specific primers, including newly designed primers
 for Malacosporea and the Paramyxidium group, we identified 30 myxozoan
 OTUs. These were predominantly from the Myxobolus clade but represented a
 broad spectrum of freshwater myxozoans, including Malacosporea.
 Rarefaction and extrapolation analyses estimated the potential maximum
 myxozoan diversity at 40 OTUs, 25% higher than the observed diversity.
 Although approximately a quarter of OTUs were probably missed, our
 analysis suggested that additional sampling would not be efficient, as
 most newly detected OTUs would overlap with those already identified or
 belong to very rare species. We demonstrated that eDNA-based methods
 reliably detect myxozoan diversity in small sediment volumes and that
 statistical approaches can effectively estimate true diversity and assess
 the need for further sampling at a given locality.
description: # Data from: Sampling strategy matters: eDNA-based assessment and
 extrapolation of myxozoan diversity in a model stream system Dataset DOI:
 [10.5061/dryad.ffbg79d4z](10.5061/dryad.ffbg79d4z) ## Description of the
 data and file structure In this repository, we provide the data and
 necessary information for our paper titled “Sampling strategy matters:
 eDNA-based assessment and extrapolation of myxozoan diversity in a model
 stream system”. We include resulting myxozoan OTUs, OTU table and R-code
 we used to produce all analyses in this paper. ### Files and variables
 #### File: R-code_Lisnerova_et_al_OIKOS_2.zip **Description:**  This R
 script reproduces the statistical analyses and figures presented in the
 manuscript: Lisnerová, M. et al. (2025) published in Oikos. ##### Files -
 `R-code_Lisnerova_et_al_OIKOS.R` – main script with all analyses and
 figure generation and details - `community_presence.csv: `The table shows
 the presence/absence matrix of species across samples. Each row
 corresponds to a species, while each column (H1–H60) represents a sample.
 A value of “1” indicates that the species was detected in the given
 sample, whereas “0” indicates absence. - `species_list.csv`: The table
 lists the species detected in each sample. Rows correspond to individual
 samples (H1–H60), while columns (C1–C14) indicate the species recorded in
 each sample. The column “Order” shows the numerical order of samples.
 Empty cells indicate that no additional species were detected for that
 sample. #### File: Lisnerova_et__al_myxozoan_OTUs_sequences.fasta
 **Description:**  Resulting sequences of OTUs assigned to myxozoans used
 in the present study. #### File: Lisnerova_et_al_Myxozoan_OTUs_table.xlsx
 **Description:** Resulting OTU table used for analyses with read accounts
 for individual OTUs per individual samples. Green text indicates positive
 samples, i.e., those with more than 20 reads, while red text with 0
 indicates samples with fewer than 20 reads. #### File:
 Full_phylogenetic_tree.pdf **Description:** Output ML full tree with
 bootstraps ## Code/software Bioinformatic analyses: We merged forward and
 reverse reads using FastqJoin (Aronesty 2011), control the quality of
 reads using FastX Toolkit (Gordon and Hannon 2010), demultiplex the data
 with specific Python script with subsequent barcode trimming, and
 operational taxonomic units (OTUs) were clustered to generate sets of
 unique sequences at an OTU radius of 3% and the OTU table was constructed
 using USEARCH (Edgar 2010). Only OTUs containing more than 20 reads were
 included in the analysis to avoid false-positive detections.  Statistical
 analyses: Statistical analyses were calculated using R software (R
 Development Core Team 2021). To investigate the relationship between the
 number of obtained OTUs and sample size, the number of OTUs was modelled
 as a function of number of samples collected. Out of the total pool of
 samples (60), always one sample was randomly drawn (with replacement) from
 the sample pool and number of newly found OTUs were recorded. This was
 repeated 60 times to match the actual number of collected samples
 resulting with increasing (cumulative) number of OTUs as a response to
 increasing sample number. This was repeated 1,000 times to get mean value
 of number of OTUs per corresponding sample size. The means were plotted
 with 90% prediction intervals, which gives the probability range for
 alternative observations.   To estimate (using extrapolation) a potential
 maximum number of OTUs of myxozoans and completeness of the samples (the
 relative proportion of the total number of species estimated for full
 community belonging to field-sampled OTUs) in the stream based on their
 incidence data, we used the Hill numbers (Hill 1973) implemented in the
 *R* package *iNEXT* (Hsieh et al. 2016). This approach quantifies
 biodiversity by combining information on the number of species (or OTUs)
 and their relative frequencies – in our case, based on incidence
 (presence/absence across samples) rather than abundance. It allows us to
 estimate diversity not only for the observed sample size (interpolation),
 but also to predict expected diversity for bigger sampling effort, i.e.
 higher sampling completeness (extrapolation). We have used diversity order
 *q*=0 corresponding to OTUs richness and Chao2 estimator for incidence
 data. It is important to note that this estimation can be sensitive to
 very rare OTUs occurrences, however this uncertainty is reflected in the
 provided confidence intervals. The potential diversity and completeness of
 the samples was simulated for 300 samples, where the model became
 asymptotic. ## Access information Other publicly accessible locations of
 the data: * n/a Data was derived from the following sources: * n/a