Hidden Species Emerge as Vienna Museum Revisits Its Tiny Pauropod Collection

Pauropods, minute myriapods measuring between 0.3 and 2 millimetres, are blind arthropods with weakly chitinised cuticles and biramous antennae, a feature unique among their relatives. Although roughly 835 species have been described worldwide, their taxonomy rests on intricate morphological characters, and their soft bodies make them notoriously difficult to collect, preserve and study. Now a comprehensive re-examination of the Pauropoda collection at the Natural History Museum Vienna (NHMW) has shown just how much hidden diversity can be recovered from specimens that have sat in ethanol for more than a century.

The collection, one of the most underrepresented groups within the museum’s Myriapoda holdings, previously comprised 34 lots of specimens preserved in 75 percent ethanol and 21 permanent slides mounted in media such as Canada balsam, Hoyer’s medium and Swan medium, together corresponding to 17 species. Seven of the ethanol lots and 12 of the slides were marked as types. As part of the restudy, 84 new permanent slides were prepared by mounting specimens in Polyvinyl-Lactophenol, which the authors report enabled the taxonomic study of these minute species by providing detailed information on their morphological diagnostic characters.

The revision paid off quickly. Re-identification of the historical material revealed overlooked taxa, including species and a subspecies new to the collection such as Allopauropus danicus, Stylopauropus beauchampi, Donzelotauropus limitaneus and Stylopauropus pedunculatus brevicornis, and allowed the nomenclature of others to be updated. Among the most revealing cases were the ethanol lots stored under the name Pauropus huxleyi Lubbock, 1868. When these specimens were mounted on new slides and examined, they turned out to belong to a range of other taxa, including Pauropus furcifer Silvestri, 1902, Stylopauropus pedunculatus (Lubbock, 1867), Allopauropus danicus (Hansen, 1902), an undetermined Allopauropus of the gracilis species group, an undetermined Decapauropus, and a Stylopauropus. Similarly, specimens originally assigned to Stylopauropus pedunculatus revealed Stylopauropus beauchampi Remy, 1947, the subspecies Stylopauropus pedunculatus brevicornis Remy, 1935, which had not previously been listed for Austria, and Donzelotauropus limitaneus (Remy, 1962).

The centrepiece of the study concerns two morphologically very similar species whose holotypes are held in Vienna. Acopauropus hastatus Attems, 1895 was collected and described more than 130 years ago, while Acopauropus attemsi Hasenhütl, 1986 was described much later, based on freshly collected samples from Siegelsdorf in Carinthia and on specimens discovered among Attems’ type material of A. hastatus that had never been considered in the original description. The two species differ subtly in the pattern of median tergal “warts” on tergites I to IV, characters regarded as reliable for distinguishing members of the family Eurypauropodidae. Both species were collected from different localities in southern Austria, and in two instances they were found in syntopy, indicating an overlap in their distribution ranges.

To test whether these subtle morphological differences truly reflect separate species, the researchers applied an integrative approach combining morphology with DNA barcoding. Thanks to the ABOL project, the Austrian Barcode of Life, representatives of Pauropoda were sequenced using a non-destructive DNA extraction method. In this protocol, whole genomic DNA is extracted from complete specimens following an adapted kit protocol with an increased incubation time of six hours; after digestion, the remaining exoskeleton is stored in 96 percent ethanol and mounted on permanent slides in Polyvinyl-Lactophenol, so that the same individual can serve both genetic and taxonomic studies. This is a decisive advantage for tiny, fragile organisms whose cuticle would normally be destroyed by conventional extraction.

Thirteen COI 5′ fragments were generated from freshly collected specimens of the Eurypauropodidae, including new samples of A. attemsi and A. hastatus collected in Styria, and deposited in the BOLD dataset DS-EPAUAT alongside previously published sequences. The resulting Maximum Likelihood tree, based on 1,000 bootstrap replicates, largely shows species-specific clusters matching the morphological identifications. The two focal species are clearly separated: A. attemsi and A. hastatus differ by a genetic distance of 23.9 percent under the Kimura 2-parameter model, a divergence far exceeding what would be expected within a single species and supporting the morphology-based hypothesis of two distinct taxa. The single representative of A. otteri lies between them, with a comparable genetic distance of 16 percent to A. hastatus.

The data also revealed striking patterns within species. The intraspecific variation between the two sequenced specimens of A. attemsi reaches 15 percent, and the authors note that this might correlate with a geographic separation of 140 kilometres along the southern Alpine border, a region where soil organisms show vast variation and diversification, although more specimens are needed to confirm this hypothesis. The main clade of A. hastatus varies by only 1 percent, representing two populations around Graz in Styria. Yet one specimen, morphologically identified as A. hastatus from Hartelsgraben in the Gesäuse National Park, appears as an outlier with 24 percent distance to the main clade. The specimen was re-sequenced and produced the same result, and since populations of the related A. ornatus from corresponding sampling areas show no comparable genetic variation, the authors argue this cannot easily be explained by geography alone, calling for further investigation of this taxon and more sampling. A. ornatus itself shows an intraspecific variation of only 0.6 percent, while two undetermined Eurypauropodidae specimens from France share identical sequences and stand 35 percent apart from their nearest neighbour, Trachypauropus graecensis.

The catalogue work also documented the losses that inevitably accumulate over decades. The jar originally holding the syntypes of Eurypauropus cycliger Latzel, 1884, from which Latzel had described five specimens, was found empty after successive loans, and the specimens are considered permanently lost. A second ethanol lot of Trachypauropus latzeli (Cook, 1896) sent on loan was likewise found empty. Two Canada balsam slides of A. hastatus from Weizklamm and Rehbergsattel bei Lunz appear to have been destroyed or lost during examination, according to older correspondence between the former curator Jürgen Gruber and the researcher Klaus Hasenhütl. By checking original publications, acquisition notes, loan forms and correspondence, the team was able to reconstruct the history of the collection and sort, label and photograph all surviving specimens.

The study places the Vienna holdings in a wider context. The Austrian fauna of Pauropoda encompasses 42 species classified in nine genera and three families, with the highest diversity recorded in Styria and Carinthia, and recent sampling has further stretched the known distribution ranges of A. attemsi and A. hastatus, confirming their co-occurrence in syntopy. Globally, molecular knowledge of the group remains sparse: only 110 public records of Pauropoda, covering 14 species, were available in BOLD as of 17 August 2026, and information on relationships among the main subgroups and families is still missing. The taxonomy of the group traditionally relies almost entirely on cuticular characters, particularly structures of the pygidium and anal plate, antennal morphology and chaetotaxy, with tergal protuberances playing a role in some subgroups.

For the authors, the message extends well beyond one museum drawer. Well-preserved natural history collections, they argue, are a perpetual source of undescribed species waiting on the shelves to be discovered, and even studied taxa may have been misidentified or mixed with others. With non-destructive extraction methods now enabling DNA barcodes and morphological vouchers to be obtained from the very same individual, obscure and previously inaccessible taxa such as pauropods are becoming far more tractable, and the authors expect this picture to change as such approaches become more widely used.

Subject of Research: Revision and DNA barcoding of the Pauropoda collection at the Natural History Museum Vienna

Article Title: The Pauropoda ( Arthropoda , Myriapoda ) collection in the Natural History Museum Vienna, with notes on Acopauropus attemsi Hasenhütl, 1986

Article References: Akkari, N., Macek, O., Hasenhütl, K., & Szucsich, N. (2026). The Pauropoda (Arthropoda, Myriapoda) collection in the Natural History Museum Vienna, with notes on Acopauropus attemsi Hasenhütl, 1986. Annals of the Natural History Museum Vienna, 127, 197–210. https://doi.org/10.3897/anhmw.212757

Image Credits: AI Generated

DOI: 10.3897/anhmw.212757

Keywords: Pauropoda, Myriapoda, Natural History Museum Vienna, DNA barcoding, COI, non-destructive DNA extraction, Acopauropus attemsi, Acopauropus hastatus, taxonomy, natural history collections, Austria, type specimens