# Morphological separation of adult males of Piona dispersa Sokolow, 1926 and Piona variabilis (Koch, 1836) (Acari: Hydrachnidia: Pionidae)

Marco van Wieringen, Rink Wiggers, Harry Boonstra, Henk van der Meulen

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Marco van Wieringen, Rink Wiggers, Harry Boonstra, Henk van der Meulen. Morphological separation of adult males of Piona dispersa Sokolow, 1926 and Piona variabilis (Koch, 1836) (Acari: Hydrachnidia: Pionidae). Acarologia, 2025, 65 (3), pp.756-761. 10.24349/sldh-jkc4. hal-05199140

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# Morphological separation of adult males of *Piona dispersa* Sokolow, 1926 and *Piona variabilis* (Koch, 1836) (Acari: Hydrachnidia: Pionidae)

Marco van Wieringen<sup>a</sup>, Rink Wiggers<sup>b</sup>, Harry Boonstra<sup>c</sup>, Henk van der Meulen<sup>c</sup>

## Original research

### ABSTRACT

*Piona dispersa* Sokolow, 1926, has long been considered as a form of *Piona variabilis* (Koch, 1836). However, DNA barcoding has shown that *P. dispersa* should be treated as a valid species. The females of both species can be morphologically distinguished but up till now, diagnostic features to separate males have not been found. Recent findings of this species in the Netherlands have provided an opportunity to study and compare males of both species. This paper presents clear and distinct characters to distinguish them. To validate the attribution of the males based on the observed morphological differences, tentative males of both species were linked to females through DNA barcoding.

**Keywords** water mites; morphological differences; COI

### Introduction

The water mite species *Piona dispersa* and *P. variabilis* closely resemble each other morphologically and were long considered conspecific. *Piona dispersa* was originally described by Sokolow (1926) as a variety of *P. variabilis*, based on differences in the genital field of females — acetabula lying free in the idiosoma rather than situated on sclerotized plates, as seen in the nominate form. Sokolow (1926) found no distinguishing features in males and regarded the variation in females to be intraspecific. However, phylogenetic analysis by Stålstedt *et al.* (2013) showed that *P. dispersa* is a distinct species rather than a mere phenotypic variant of *P. variabilis*. Until now, the distinguishing characteristics of males of these two species have remained unknown. Recent findings of *P. dispersa* made it possible to identify and describe the male of the species and to describe distinguishing characteristics compared to *P. variabilis*.

### Materials and methods

To tentatively attribute males to either *P. variabilis* or *P. dispersa*, only males from samples that also contained females of one of the two species were examined. The morphological features of males co-occurring with *P. variabilis* females were compared to those of males found with *P. dispersa* females. To further increase the number of available specimens for measuring the identified diagnostic features, males from locations where both species coexist were also included. Samples of *P. dispersa* and *P. variabilis* were collected from eleven and sixteen sampling locations, respectively, across the western and northern parts of the Netherlands. At

Received 23 May 2025

Accepted 16 July 2025

Published 22 July 2025

Corresponding author

Academic editor

Mąkol, Joanna

https://doi.org/10.24349/sldh-jkc4

ISSN 0044-586X (print)

ISSN 2107-7207 (electronic)

<figure><img src="image_2.png" /></figure>

van Wieringen M. *et al*.

Licensed under
Creative Commons CC-BY 4.0

OPEN ACCESS

<footnote><sup>a</sup> Van't Hoffstraat 35, 2014 RB Haarlem, the Netherlands.</footnote>

<footnote><sup>b</sup> Waardenburg Ecology, Varkensmarkt 9, 4101 CK Culemborg, the Netherlands.</footnote>

<footnote><sup>c</sup> Wetterskip Fryslân, Fryslânplein 3, 8914 AZ Leeuwarden, the Netherlands.</footnote>
six of these sites, both species co-occurred. In total, 104 male specimens of *P. dispersa* and 52 male specimens of *P. variabilis* were available for examination. All specimens were preserved in Koenike's fluid. Morphological examinations and measurements were conducted using both a stereomicroscope and a compound microscope equipped with a calibrated graticule. Three males and two females of each species are included in the collection of Naturalis Biodiversity Center in Leiden, the Netherlands.

To validate the attribution of the males based on the observed morphological differences, a DNA analysis was conducted. Genomic DNA was extracted from one male and two females of *P. dispersa* and two males and two females of *P. variabilis* using a non-destructive method. Whole specimens were incubated in lysis buffer at 56 °C for one hour. The lysis buffer consisted of 88 µL of homogenization buffer (0.4 M NaCl, 10 mM Tris, 2 mM EDTA) with the addition of 9 µL SDS (20% w/v) and 3 µL Proteinase K (20 mg/ml). After incubation, the specimens were removed, and DNA was extracted from the buffer using 1.0X magnetic beads (Rohland & Reich, 2012).

The 658-bp barcoding region of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified using the primer set designed by Folmer *et al.* (1994). PCR reactions were performed in a 25 µL volume, containing 17.8 µL Milli-Q water, 2.5 µL 10X PCR ammonium buffer, 2.5 µL dNTPs (2 mM each), 0.5 µL each of LCO1490 and HCO2198 primers (Folmer *et al.*, 1994), 0.2 µL TEMPase Hot Start DNA polymerase (5 U/µL), and 1 µL DNA template. The PCR protocol consisted of an initial denaturation at 95 °C for 15 minutes, followed by 45 cycles of 95 °C for 30 s, 46 °C for 30 s and 72 °C for 35 s, with a final extension at 72 °C for 5 minutes.

PCR products were purified by mixing 10 µL of each reaction with 7.6 µL Milli-Q water, 0.24 µL FastAP Thermosensitive Alkaline Phosphatase (1 U/µL) and 0.16 µL Exonuclease I (20 U/µL), followed by incubation at 37 °C for 30 minutes and enzyme deactivation at 80 °C for 15 minutes. Purified products were Sanger sequenced in both directions using the Eurofins Genomics LightRun service and analyzed in Geneious Prime. All sequences generated for this study have been deposited in GenBank under accession numbers PV174582–PV174588 and in BOLD, with photo's, under accession numbers PDPV001-25 to PDPV007-25. The minimum p-distance of the DNA sequence of the males to their corresponding females was estimated using BOLD tools.

The vouchers are deposited at Wetterskip Fryslân in Leeuwarden, the Netherlands.

# Results

## Morphology

At first glance, males of both species appear very similar. They both possess an elongated posterior claw of the third pair of legs (Figure 1), and their genital fields are largely similar. However, upon closer examination, several morphological differences become apparent.

Fresh specimens show a clear difference in colour between the two species, for both females and males. In *P. dispersa*, the chitin appears light blue-green or even colourless, whereas in *P. variabilis*, it is vivid blue-green (Figure 2). After preservation in Koenike's fluid, this colour fades over time.

The shape of the genital field provides a unique distinguishing characteristic between the males of the two species. In *P. dispersa*, the genital plates have a more posterior orientation than in *P. variabilis*, extending posteriorly beyond the genital cavity by a distance equivalent to 1 to 1.5 times the diameter of an acetabulum (Figures 3 and 4A). In contrast, in *P. variabilis*, this extension reaches at most half the diameter of an acetabulum (Figure 4B). This characteristic shows no overlap between the two species across all collected specimens. Furthermore, the anterior point of the genital field in *P. dispersa* is elongated and usually longer than it is wide. In *P. variabilis*, however, this point appears as a blunt triangle (Figures 3 and 4). In most specimens of *P. dispersa*, the genital plates features a notch or are deeply concave lateral to the
anterior acetabulum (Figure 3 and 4A). In *P. variabilis*, on the other hand, the genital plates are only slightly concave near the anterior acetabulum (Figure 4B).

Several other characteristics help to distinguish between the males of the two species. In particular the medial distance between the coxal plates is, on average, greater in *P. dispersa* than in *P. variabilis*, although their ranges overlap to some extend (Figure 4A and B, Table 1). Additionally, the chitinized medial margin of coxal plates III and IV is broader in *P. variabilis* than in *P. dispersa*. Furthermore, in most specimens of *P. variabilis*, the surface of coxal plates III and IV displays a relief of distinct lobed structures, whereas in *P. dispersa*, these lobes are faint or absent, resulting in a relatively smooth appearance. Cook (1974) refers to these lobed structures as muscle scars, which we believe is incorrect, as these structures are located on the outside of the coxal plates. A summary of all distinguishing features is provided in Table 1.

<figure><img src="image_3.png" /><figcaption>**Figure 1** Sixth segment of third leg with an elongated claw of male *Piona dispersa*. Photo by Christophe Brochard.</figcaption></figure>

<table><caption>**Table 1** Overview of male characteristics of *Piona dispersa* and *Piona variabilis*. N = number of specimens.</caption><thead><tr><th>Characteristic</th><th>P. dispersa ♂</th><th>P. variabilis ♂</th></tr></thead><tbody><tr><td>Genital field</td><td></td><td></td></tr><tr><td>Colour (fresh specimens)</td><td>Faint blue-green</td><td>Vivid blue-green</td></tr><tr><td>Number of acetabula</td><td>8–17 (N = 53)</td><td>7–12 (N = 25)</td></tr><tr><td>Total genital plate width</td><td>Median: 290 µm (range 250–320 µm, N = 38)</td><td>Median: 240 µm (range 200–270 µm, N = 16)</td></tr><tr><td>Genital plate extension posteriorly to genital cavity</td><td>1–1.5 times of acetabulum (N = 90)</td><td>0–0.5 times of acetabulum (N = 48)</td></tr><tr><td>Shape of genital plate near anterior acetabula</td><td>Often with a notch or deeply concave</td><td>Slightly concave</td></tr><tr><td>Anterior point of genital field</td><td>Elongated point</td><td>Blunt triangular</td></tr><tr><td>Genital cavity, posterior edge*</td><td>Rounded</td><td>More straight, with corners on the sides</td></tr><tr><td>Coxal plates</td><td></td><td></td></tr><tr><td>Medial distance at midpoint of Cx IV</td><td>Median: 22 µm (range 9–44 µm, N = 70)</td><td>Median: 8 µm (range 3–16 µm, N = 37)</td></tr><tr><td>Medial margin of Cx III+ IV</td><td>Narrow</td><td>Broad</td></tr><tr><td>Surface of Cx III+IV</td><td>Smooth, without or with faint lobed structures</td><td>Mostly with distinct lobed structures</td></tr><tr><td>Leg III, segment 5</td><td></td><td></td></tr><tr><td>Number of short setae dorsodistally</td><td>Mostly five (92%, range 3-6, N = 65)</td><td>Mostly four (80%, range 4-5, N = 25)</td></tr></tbody></table>

<footnote>* Best observed at low magnification (40x).</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 2** Fresh specimens of males in ventral view. A – *Piona dispersa*; B – *Piona variabilis*. Photos by M. van Wieringen.</figcaption></figure>

**DNA barcoding**

Based on the DNA barcoding data, our analysis revealed that the male we attributed to *P. dispersa* on morphological grounds could be associated with the female of *P. dispersa* (99.9% match). Additionally, the tentative male of *P. variabilis* could be linked to females belonging to this species (≥ 99.5% match). DNA sequences are deposited in GenBank and in BOLD Systems (Table 2).

**Discussion**

Males of *P. dispersa* and *P. variabilis* exhibit several morphological differences, although many of these features overlap between the two species. However, based on the shape of the genital

<figure><img src="image_5.png" /><figcaption>**Figure 3** Genital field of male *Piona dispersa*. (a) anterior point of the genital field, (b) notch or concavity lateral to the anterior acetabulum, (c) extension of the genital plate posterior to the genital cavity. Photo by M. van Wieringen.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 4** Ventral view of males. A – *Piona dispersa*; B – *Piona variabilis*. Photos by M. van Wieringen.</figcaption></figure>

<table><caption>**Table 2** DNA barcoded specimens, collected from two sample locations in a ditch in the inland dune area of Haarlem, the Netherlands. The table includes the coordinates of the sampling locations, the sample codes, the GenBank accession numbers, and the barcode index number codes.</caption><thead><tr><th>Coordinates</th><th>Species/sex</th><th>Collection date</th><th>Sample ID</th><th>GenBank</th><th>BOLD: BIN</th></tr></thead><tbody><tr><td rowspan="5">52.37344°N, 4.60547°E</td><td>P. variabilis ♂</td><td>07-04-2024</td><td>24-001b-1</td><td>PV174582</td><td>PDPV001-25</td></tr><tr><td>P. variabilis ♂</td><td>07-04-2024</td><td>24-001b-3</td><td>PV174583</td><td>PDPV002-25</td></tr><tr><td>P. variabilis ♀</td><td>07-04-2024</td><td>24-001b-2</td><td>PV174584</td><td>PDPV003-25</td></tr><tr><td>P. variabilis ♀</td><td>07-04-2024</td><td>24-001b-4</td><td>PV174585</td><td>PDPV004-25</td></tr><tr><td>P. dispersa ♀</td><td>20-05-2024</td><td>24-009b-3</td><td>PV174588</td><td>PDPV007-25</td></tr><tr><td rowspan="2">52.37537°N, 4.60602°E</td><td>P. dispersa ♂</td><td>27-04-2024</td><td>24-004b-3</td><td>PV174586</td><td>PDPV005-25</td></tr><tr><td>P. dispersa ♀</td><td>27-04-2024</td><td>24-004b-2</td><td>PV174587</td><td>PDPV006-25</td></tr></tbody></table>

field, the two species can be clearly distinguished. Additionally, several other morphological traits can aid in distinguishing the males of both species. For example, the differences in coloration of the chitine of fresh specimens can serve as a useful diagnostic trait. However, this feature alone is not sufficient, as coloration can be variable, as demonstrated in some *Arrenurus* species (Więcek *et al.* 2023).

The DNA analysis confirmed that the assignment of the males of both species on morphological grounds is justified.

Recent diagnostic keys have caused some confusion regarding the taxonomic classification of *P. dispersa*. In Gerecke *et al.* (2016), the male of *P. dispersa* is mistakenly paired with *Piona conglobata* (Koch, 1836), claiming that *P. dispersa* lacks an elongated claw on the third leg, a key feature that distinguishes it from *P. conglobata*. The same error is found in the Dutch diagnostic key by Smit (2018). Therefore, amendments should be made to both diagnostic keys.

*Piona dispersa* and *P. variabilis* show overlap in their habitat preferences, as evidenced by their co-occurrence at several sites in this study. It is therefore gratifying that, approximately a century after the description of the female of *P. dispersa* by Sokolow, the males of both species can now be distinguished based on morphological characteristics.
## Acknowledgements

Our gratitude goes to Christophe Brochard (Bureau Biota) for providing one of his photos. We are also thankful to Harry Smit and two anonymous reviewers for reviewing the article.

## ORCID

Marco van Wieringen https://orcid.org/0009-0001-4649-3387

Rink Wiggers https://orcid.org/0000-0002-5011-9267

Harry Boonstra https://orcid.org/0000-0002-3173-2429

Henk van der Meulen https://orcid.org/0000-0002-2253-2126

## References

* Cook D.R. 1974. Water mite genera and subgenera. Mem. Am. Entomol. Inst., 21. pp 860.

* Folmer O., Black M., Hoeh W., Lutz R., Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech., 3(5): 294-299.

* Gerecke R., Gledhill T., Pešić V., Smit H. 2016. Chelicerata, Acari III. In: R. Gerecke (Ed.). Süßwasserfauna von Mitteleuropa 7/2-3. Berlin Heidelberg: Springer-Verlag. pp. 429. https://doi.org/10.1007/978-3-8274-2689-5

* Rohland N., Reich D. 2012. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res., 22: 939-946. https://doi.org/10.1101/gr.128124.111

* Smit H. 2018. De Nederlandse watermijten (Acari: Hydrachnidia). Entomol. Tabellen, 11: 1-299.

* Sokolow I. 1926. Neue Hydracarinen aus Russisch-Karelien. Rev. Russe Entomol., 20: 165-179.

* Stålstedt J., Bergsten J., Ronquist F. 2013. "Forms" of water mites (Acari: Hydrachnidia): intraspecific variation or valid species? Ecol. Evol., 3(10): 3428-3435. https://doi.org/10.1002/ece3.704

* Więcek M., Broda Ł., Proctor H., Dabert M., Smith B.P., Dabert J. 2023. Species boundaries among extremely diverse and sexually dimorphic *Arrenurus* water mites (Acariformes: Hydrachnidiae: Arrenuridae). Syst. Appl. Acarol., 28(2): 322-342. https://doi.org/10.11158/saa.28.2.14