# Counting the Invisible: New Tools to Estimate the Number of Contributors From Sequence-Based Microsatellite Genotyping of Environmental DNA Samples

Olivier Lepais, Ivan Paz-vinas

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Olivier Lepais, Ivan Paz-vinas. Counting the Invisible: New Tools to Estimate the Number of Contributors From Sequence-Based Microsatellite Genotyping of Environmental DNA Samples. Molecular Ecology Resources, 2025, 10.1111/1755-0998.70051. hal-05300732

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# Counting the Invisible: New Tools to Estimate the Number of Contributors From Sequence-Based Microsatellite Genotyping of Environmental DNA Samples

Olivier Lepais¹ | Ivan Paz-Vinas²

¹Univ. Bordeaux, INRAE, BIOGECO, Cestas, France | ²Université Claude Bernard Lyon 1, LEHNA UMR 5023, CNRS, ENTPE, Villeurbanne, France

**Correspondence:** Olivier Lepais (olivier.lepais@inrae.fr)

**Received:** 22 August 2025 | **Revised:** 10 September 2025 | **Accepted:** 18 September 2025

**Handling Editor:** Joanna Kelley

The study of intraspecific genetic variation in environmental DNA samples has recently gained momentum following its demonstration as an effective method to study population-level processes (Andres et al. 2023). Although allele frequencies can be inferred from the distribution of allele sequence coverage within a sample, the number of detected alleles can be used to estimate the number of contributors (NOC), a long-standing issue in forensc science. This development enables the estimation of the absolute abundance of a species, opening up new possibilities for population monitoring and ecological and evolutionary studies. Although promising, no dedicated tools providing a straightforward way to implement it existed. In this issue of *Molecular Ecology Resources*, Liggan et al. (2025) make a welcome contribution to the field by introducing two new R packages that facilitate the estimation of multi-locus allelic diversity and of the NOC from the sequencing of microsatellites of mixed samples obtained from environmental DNA. The AMPLICOMSAT R package determines the observed allele count (based on sequence length and sequence identity) from sequence-based microsatellite genotyping (Figure 1A). The GENOTYPEQUANT R package estimates the NOC given the number of observed alleles within mixed samples and allele frequencies of a reference population (Figure 1B). The authors conducted extensive testing of the developed method using simulation and empirical work in the laboratory and in the field, providing a convincing demonstration of its strengths and limitations, but also useful guidelines for future applications to other biological models or to address a broad range of scientific inquiries. Importantly, these advances can help support ongoing global biodiversity monitoring efforts.

The results reported by Liggan et al. (2025) are promising because the method performed well with easy-to-gather molecular data. Using 11 microsatellites and 40 individuals as references, it revealed a total of 177 alleles differing by their size or 297 microhaplotypes based on allele sequence identity (averaging 16 and 27 alleles per locus, respectively), with satisfactory accuracy with up to 20 contributors (Liggan et al. 2025). This was the case for the bull kelp gametophyte study (Figure 2), where empirical validation was conducted on eight contributors, and field sample estimates ranged from one to 12 contributors. However, more complex mixtures (> 50 contributors) as well as missing genotypes due to degraded DNA in environmental samples were more challenging, calling for caution when applying the method in these specific cases. Improving PCR efficiency or library preparation can help generate more data from difficult samples, as suggested by the authors. It is worth noting; however, that Liggan et al. (2025) were able to estimate the NOC from field samples with reasonable confidence even in the presence of high sequencing failure (50% of missing genotypes among field samples). This illustrates how the method can be successfully applied in a realistic scenario, adding up to previous case studies that also made significant advances in the field (Andres et al. 2021). In the author's study case, the additional information provided by allele sequence identity (compared to allele size) was crucial to recover the expected correlation between the NOC and the surface area sampled. This empirical result illustrates the wealth of information provided by considering all polymorphisms in the sequenced amplicon encoded as a microhaplotype. In a recent

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Molecular Ecology Resources* published by John Wiley & Sons Ltd.
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** | Microsatellite microhaplotype scoring from amplicon sequencing of individuals and environmental DNA in AMPLICOMSAT (A) and estimation of the number of contributors (haploid genotypes) in GENOTYPEQUANT (B). Note that if the output of AMPLICOMSAT can be directly used as input GENOTYPEQUANT, each package has its own purpose and can be used independently. In particular, GENOTYPEQUANT necessitates simple input data that can be generated with a wide range of upstream processing tools. This is a welcome feature that should encourage creative use of the method which may have unforeseen applications, such as for instance inferring individual ploidy in species with variable ploidy level. Courtesy of Lauran Liggan.</figcaption></figure>

application targeting 74 nuclear loci using GT-seq with bio-
informatics analysing SNPs, (Shi et al. 2025) identified 252
unique microhaplotypes among 565 Chinook salmon (mean
3.4 alleles per locus). This curated dataset provides enough
power to resolve a mixture of up to 10 individuals, with min-
imal headroom to accommodate high missing genotypes on
degraded DNA from digestive tracts. The number of observed
alleles is especially important for accurate inference. Thus,
integrating highly polymorphic variants such as microsatel-
lites is ideal to maximise the number of alleles within a short
DNA fragment. Such compact markers are also more likely to
amplify during PCR in the context of degraded environmental
DNA. It is important to note that Liggan et al. (2025) sam-
pled specific microhabitats where their studied organism was
known to be present, increasing the probability of capturing
DNA from the target species. Applying the approach to sam-
pling highly diluted DNA dispersed in the environment could
prove to be more challenging.

Simulations and the empirical validation conducted by Liggan
et al. (2025) clearly indicate room for improvement in cases
involving more complex DNA mixtures or less polymorphic
markers, including by increasing the number of markers, the
number of individuals in the reference sample or the number
of detected alleles. The latter solution can involve sequencing
longer markers, which will increase the cumulated number
of detected polymorphisms and hence the total number of ob-
served alleles, as illustrated in humans with hundreds of mac-
rohaplotypes predicted for 8 kb markers spanning multiple
microsatellites (Ge et al. 2021). As the number of observable
alleles increases, additional power is available to resolve com-
plex DNA mixtures without suffering from locus saturation
(Andres et al. 2023). The GENOTYPEQUANT R package thus
represents a welcome improvement over previous implemen-
tations, as it tolerates high numbers of alleles while offering
greater computational efficiency for handling complex mix-
tures and high polymorphism.

Another way for progress is reducing genotyping error to
detect rare variants (Andres et al. 2021), which could be

achieved through the use of the emergent digital sequencing
(Andersson et al. 2024). By tagging each original DNA mol-
ecule with a unique molecular index in early protocol steps,
digital sequencing allows determining a consensus sequence
of each original DNA strand and greatly improves the distinc-
tion between rare variants and errors introduced during PCR
and sequencing (Carlson et al. 2015). Although there are many
ways to implement digital sequencing, only a few are proba-
bly appropriate for degraded and weakly concentrated eDNA,
which calls for further specific testing and development.
Hybrid-capture sequencing, which has been successfully used
(Ai et al. 2025) to estimate the abundance of two goby species
by focusing on substitutions in enriched mitochondrial DNA,
might be more efficient than PCR-based approaches for de-
graded DNA.

Even without further technical improvements, microhaplotype
data are now readily accessible for processing in the tools de-
veloped by Liggan et al. (2025) to estimate the number of in-
dividuals releasing gametes in the environment during mating.
It opens up new possibilities to understand complex ecological
processes, such as, for instance, plant pollination through the
study of pollen transported by insects (Kämper et al. 2025) or
of wind-dispersed pollen captured by airborne passive samplers
(Lin et al. 2025). This new kind of data will provide new infor-
mation about the plant reproductive landscape. As pointed out
by the authors, their method enhances the capabilities of eDNA
to monitor further beyond presence-absence populations of
species with complex life histories, or that are microscopical,
elusive or rare enough to be monitored using traditional direct
sampling methods.

Besides, the advances made by Liggan et al. (2025) are important
to support global biodiversity monitoring efforts. Methods that
would simultaneously determine both community-level species
diversity and intraspecific diversity across multiple species re-
main panaceas for evolutionary and conservation biologists.
Although numerous challenges persist, such as accurately esti-
mating exact allele frequencies or summary statistics, like het-
erozygosities from eDNA samples, the methodological advances
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** | Picture of sporophytes and microscopic gametophytes (insert) of bull kelp. Bull kelp (*Nereocystis luetkeana*) exhibits a complex life-history, in which microscopic haploid gametophytes (insert: Microscopy image by Gabriel Montecinos), residing on the benthos, alternate with macroscopic diploid sporophytes (captured in Campbell River, British Columbia, Canada, by Bartek Radziej). The sporophyte stage forms extensive and structurally complex underwater forest ecosystems. If sporophyte abundance can be observed in the field, the population size of cryptic gametophytes remained unknown until the advent of genetic diversity analysis of environmental DNA. The tools developed by Liggan et al. (2025) make the estimation of the number of contributors from a DNA mixture easier than ever. Courtesy of Lauran Liggan, Bartek Radziej and Gabriel Montecinos.</figcaption></figure>

made by Liggan et al. (2025) and others (Andres et al. 2021) are
paving the way towards this goal.

As allelic variation is a key feature in evolutionary biology and conservation (Allendorf et al. 2024), observed allele counts obtained from species-specific microsatellites sequenced from environmental samples provide highly valuable information from a conservation standpoint. First, observed allele counts can serve as proxies for allelic richness, that is, one of the six essential biodiversity variables for monitoring genetic composition (Hoban et al. 2022). Furthermore, observed allele counts may reveal rare or private alleles in specific populations, informing about their genetic uniqueness (Kalinowski 2004). Finally, multi-specific observed allele counts can be incorporated into systematic conservation planning tools to identify priority

areas for intraspecific genetic diversity conservation (Paz-Vinas
et al. 2018).

The NOC and the derived individual densities could help ap-
proximate population census sizes (Nc), a key metric for pop-
ulation monitoring. These estimates can help compute the
headline genetic indicator A.4 of the Kunming-Montreal Global
Biodiversity Framework of the UN's Convention on Biological
Diversity (i.e., the proportion of populations within species with
an effective population size Ne above 500), given the links exist-
ing between Ne and Nc (Allendorf et al. 2024; Mastretta-Yanes
et al. 2024). As countries are increasingly deploying eDNA-
based biomonitoring programmes, integrating procedures like
those developed by Liggan et al. (2025) could help optimise these
approaches, unlocking the possibility to perform multifaceted
biodiversity monitoring—from genes to communities—in cost-
effective ways.

**Conflicts of Interest**

The authors declare no conflicts of interest.

**Data Availability Statement**

No new data produced for this article.

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