van Wieringen, Marco ¹ ; Wiggers, Rink ² ; Boonstra, Harry ³ and van der Meulen, Henk ⁴

¹✉ Van't Hoffstraat 35, 2014 RB Haarlem, the Netherlands.
²Waardenburg Ecology, Varkensmarkt 9, 4101 CK Culemborg, the Netherlands.
³Wetterskip Fryslân, Fryslânplein 3, 8914 AZ Leeuwarden, the Netherlands.
⁴Wetterskip Fryslân, Fryslânplein 3, 8914 AZ Leeuwarden, the Netherlands.

2025 - Volume: 65 Issue: 3 pages: 756-761

Original research

Keywords

Abstract

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 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.

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 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.

References

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