# An exceptionally well-preserved Eocene fossil mite, Histiogaster altilis sp. n. (Acari: Astigmata), from tree sap: Evidence of morphological and ecological niche conservatism, with a review of fossil Astigmata

Vasiliy B Kolesnikov, Dmitry D Vorontsov, Evgeny E Perkovsky, Pavel B Klimov

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Vasiliy B Kolesnikov, Dmitry D Vorontsov, Evgeny E Perkovsky, Pavel B Klimov. An exceptionally well-preserved Eocene fossil mite, Histiogaster altilis sp. n. (Acari: Astigmata), from tree sap: Evidence of morphological and ecological niche conservatism, with a review of fossil Astigmata. Acarologia, 2025, 65 (1), pp.213 - 241. 10.24349/c35e-8bmj. hal-05006796

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# An exceptionally well-preserved Eocene fossil mite, *Histiogaster altilis* sp. n. (Acari: Astigmata), from tree sap: Evidence of morphological and ecological niche conservatism, with a review of fossil Astigmata

Vasiliy B. Kolesnikov IDa,b, Dmitry D. Vorontsov Id,c, Evgeny E. PerkovskyId,d, Pavel B. Klimov IDe

## Original research

### ABSTRACT

We describe *Histiogaster altilis* sp. nov. (Acari: Astigmata: Acaridae), a newly identified species of mite from Eocene Rovno amber (34–37 Ma), based on both male and female specimens. The exceptional preservation of the fossils, coupled with advanced amber preparation and imaging techniques—including high-resolution microscopy, differential interference contrast (DIC) with transmitted light, and confocal laser scanning microscopy (CLSM) equipped with a super-resolution detector—enabled detailed examination of nearly all diagnostic features. These data allowed us to perform a comprehensive comparison between this fossil species and extant representatives of the genus *Histiogaster*. Based on morphological similarities with extant species of known biology, and the presence of syninclusions such as fossilized phloem sap emulsions and fungal or bacterial hyphae, we hypothesize that this fossil mite likely fed on fermented tree sap and associated microorganisms. Our findings indicate that species within the genus *Histiogaster* have undergone minimal morphological changes since the Eocene, while continuing to occupy their ancestral ecological niche. We also review fossil Astigmata and provide an annotated checklist of 14 fossil astigmatid records, and exclude 8 records, including the genus *Palaeotyroglyphus* Dubinin, 1962, from Astigmata.

**Keywords** astigmatid mites; Rovno amber; morphology; taxonomy; paleontology

**Zoobank** http://zoobank.org/B3A38FFE-645E-4E3C-BC3D-171AE3B5C384

### Introduction

Mites belonging to the hyporder Astigmata (= Astigmatina) include more 6000 (Schatz *et al.* 2011) free-living and parasitic species living in terrestrial, freshwater and synanthropic habitats (OConnor, 2009). However, in contrast to oribatid mites, fossil records of Astigmata are scarce (Dunlop *et al.* 2011). Currently, there are 24 records, but only 14 can be confidently attributed

Received 12 October 2024

Accepted 13 March 2025

Published 26 March 2025

Corresponding author

Pavel B. Klimov ID:
pklimov@purdue.edu

Academic editor
Pfingstl, Tobias

https://doi.org/10.24349/c35e-8bmj

ISSN 0044-586X (print)
ISSN 2107-7207 (electronic)

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

Kolesnikov V. B. et al.

Licensed under
Creative Commons CC-BY 4.0

OPEN ACCESS

<footnote>a Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, 152742 Borok, Yaroslavl Province, Russia.</footnote>

<footnote>b Institute of Environmental and Agricultural Biology (X-BIO), Tyumen State University, 625003 Tyumen, Russia.</footnote>

<footnote>c Koltzov Institute of developmental biology, RAS, Russia.</footnote>

<footnote>d Natural History Museum of Denmark, 2100 Copenhagen, Denmark.</footnote>

<footnote>e Department of Biological Sciences, Purdue University, Lilly Hall of Life Sciences, West Lafayette, IN 47907, USA.</footnote>
to Astigmata (Table 1). Of these, eight species are known from heteromorphic deutonymphs (a specialized dispersal stage), while only five are represented by adults and other feeding stages (Table 1). Phoretic stages of astigmatid mites have a much higher likelihood of preservation in amber because their insect carriers are likely to be trapped in amber and fossilized. Once entrapped in resin, heteromorphic deutonymphs may detach from their arthropod host or be preserved while still attached to the host, providing insight into their ancient host associations (Kolesnikov *et al.*, 2023). In contrast, adult and other feeding stages are less likely to fossilize due to their limited mobility and habitat specificity. Fossilization is most likely to occur when these mites inhabit environments in close proximity to resin-producing trees, such as subcortical habitats where resin can easily entrap them. The fossil species *Glaesacarus rhombeus* (Koch et Berendt, 1854) (Sidorchuk and Klimov, 2011) is an example of such a mite species, as its feeding stages are particularly abundant in Eocene amber. A modestly sized piece of Baltic amber can contain dozens of *G. rhombeus* specimens. These mites were reported as less common in Rovno amber (Perkovsky *et al.*, 2007), but they are the most important mites in Rovno amber as well. While we have detailed knowledge of the external morphology of *G. rhombeus*, its ecology and biology remain speculative due to the lack of close extant relatives that could provide ecological insights.

Our survey of Rovno amber mites yielded a single amber piece containing several excep-
tionally well-preserved males and females of a mite belonging to the modern genus *Histiogaster*
Berlese, 1883, family Acaridae. This genus includes 24 modern species living in subcortical
habitats, including galleries of bark beetles (Woodring, 1966), tree sap flows (Türk and Türk,
1957; Bugrov, 1997), decaying wood and polypore fungi, and other fermented materials (Zach-
vatkin, 1941; Woodring, 1966; Klimov and Tolstikov, 2011; Pfammatter *et al.*, 2016; Klimov
*et al.*, 2022). Among most other acarid genera, males of *Histiogaster* are distinct by having
characteristic four-lobed, fan-like projections on the posterior opisthosoma, which serve for
attachment to females during mating and pre-copulatory guarding (Klimov *et al.* 2022). The
only genera that have a similar structure are *Paulacarellus* (unrelated to *Histiogaster*) inhabiting
sea wrack in the marine supralittoral zone (Fain, 1977; Klimov, 2000) and *Horstiella* (related
to *Histiogaster*, our data) associated with bees of the genus *Epicharis* from the Neotropical
region (Ochoa and OConnor, 2000).

In this study, we use advanced imaging (high-resolution microscopy, DIC in combination
with transmitted light and CLSM with a super-resolution detector) and amber preparation
techniques to examine the fine morphological details of this newly discovered fossil mite,
*Histiogaster altilis* sp. n. We compared its morphological traits with those of modern
*Histiogaster* species with known ecologies to infer the paleohabitat of the fossil. Additional
evidence from the amber piece, such as its physical characteristics (suggestive of phloem sap
presence) and syninclusions (e.g., bacterial or fungal hyphae that could indicate fermented
substances), further aided our paleoecological reconstruction. *Histiogaster altilis* sp. n.
provides valuable evidence of the long-term evolutionary stability of this genus and its
specialized ecological niche since the Eocene.

**Methods**

Amber processing

The amber pieces were trimmed and polished following the protocols outlined by Sidorchuk and Vorontsov (2018). To optimize high-resolution immersion optics and allow observation of specimens from multiple angles, the amber was polished to leave a space of ≤100 µm between the surface and the inclusion. The processed amber pieces were then stored in water.

Imaging

For microscopic observation, processed pieces of amber were mounted on a coverslip in a
saturated fructose solution, then the coverslip was inverted and studied under a microscope
<table><caption>Table 1. Fossil records of mites identified as Astigmata</caption><thead><tr><th>Identification</th><th>Developmental stage</th><th>Phoretic host</th><th>Age</th><th>Reference</th><th>Notes</th></tr></thead><tbody><tr><td>Levantoglyphus sidorchukae<br>Klimov, Vorontsov, Azar, Sidorchuk, Braig, Khaustov, Tolstikov, 2021<br>(Levantoglyphidae)</td><td>phoretic deutonymphs</td><td>found without a host</td><td>Early Cretaceous (Lebanese amber, 125-129 Ma)</td><td>Klimov et al. 2021</td><td></td></tr><tr><td>Plesioglyphus lebanotermi Sendi, Klimov, Kolesnikov, Káčerová, Bonino, Azar, Robin, 2025<br>(Schizoglyphidae)</td><td>phoretic deutonymphs</td><td>Lebanotermes veltzae<br>(Blattodea)</td><td>Early Cretaceous (Lebanese amber, 125-129 Ma)</td><td>Azar 2007; Sendi et al., 2025</td><td></td></tr><tr><td>Congovidia glesoconomorphi<br>Kolesnikov et Klimov, 2023<br>(Hemisarcoptidae)</td><td>phoretic deutonymphs</td><td>Glesoconomorphus<br>ekaterinae (Coleoptera:<br>Myceridae)</td><td>Late Eocene (Rovno amber, 34-37<br>Ma)</td><td>Telnov et al. 2021 (as<br>"Winterschmidtiidae"); Kolesnikov<br>et al. 2023</td><td></td></tr><tr><td>Glaesacarus rhombeus (Koch &<br>Berendt, 1854) (Glaesacaridae)</td><td>all feeding stages and eggs<br>(except phoretic<br>deutonymphs)</td><td>free-living</td><td>Late Eocene (Baltic amber 34-37<br>Ma, Rovno amber, 34-37 Ma)</td><td>Koch and Berendt 1854; Sidorchuk<br>and Klimov 2011; Perkovsky et al.<br>2007</td><td></td></tr><tr><td>Histiostoma ovalis species group<br>(Histiostomatidae)</td><td>phoretic deutonymph</td><td>Phloeosinus sp.<br>(Coleoptera:Scolytidae)</td><td>Late Eocene (Baltic amber, 34-37<br>Ma)</td><td>Wirth & Garonna 2015</td><td></td></tr><tr><td>Histiogaster altilis sp.n. (Acaridae)</td><td>adult</td><td>free-living</td><td>Late Eocene (Rovno amber, 34-37<br>Ma)</td><td>Vorontsov et al. 2023 (as<br>Histiogaster sp); Pepato et al.<br>2022 (as Histiogaster sp.</td><td>Described in this study</td></tr><tr><td>Acaridae or Histiostomatidae</td><td>phoretic deutonymph</td><td>Dasuniana emicans<br>(Araneae: Dysderidae)</td><td>Eocene (Baltic amber, 34-37 Ma)</td><td>Dunlop 2012</td><td></td></tr><tr><td>Acaridae</td><td>phoretic deutonymph</td><td>Collohmannia<br>weiterschani (Acari:<br>Oribatida:<br>Collohmanniidae)</td><td>Eocene (Baltic amber, 34-37 Ma)</td><td>Kolesnikov et al. 2024</td><td></td></tr><tr><td>Astigmata, probably Acaridae</td><td>phoretic deutonymph</td><td>found without a host</td><td>Eocene (Baltic amber 34-37 Ma)</td><td>Halbwachs et al. 2021 (as<br>unidentified palynomorph, Plate 8,<br>Fig. 12)</td><td></td></tr><tr><td>Amphicalvolia huri Türk, 1963<br>(Winterschmidtiidae)</td><td>phoretic deutonymphs</td><td>found without a host</td><td>Miocene (Mexican Chiapas amber,<br>15-20 Ma)</td><td>Türk 1963</td><td>Belongs to either Winterschmidti or<br>Parawinterschmidti (Klimov et al. 2021,<br>OConnor 2022)</td></tr><tr><td>Atopomelidae (OConnor, pers.<br>comm.)</td><td>feeding stages</td><td>Mammal hair</td><td>Early Miocene (Dominican amber,<br>16-18 Ma)</td><td>Poinar 1988 (as Listrophoroidea)</td><td></td></tr><tr><td>Astigmata</td><td>phoretic deutonymphs</td><td>ambrosia beetle<br>(Coleoptera:<br>Platypodinae)</td><td>Early Miocene (Dominican amber,<br>16-18 Ma)</td><td>Santiago-Blay et al. 2012</td><td></td></tr><tr><td>Astigmata (genus unrecognized)<br>(specimen 128-g)</td><td>unknown, probably a feeding<br>stage</td><td>unknown</td><td>Quaternary (Mizunami copal)</td><td>Aoki 1974 (taxon names written in<br>Japanese)</td><td></td></tr><tr><td>Astigmata (genus unrecognized)</td><td>nymph</td><td>free-living</td><td>Dolomite from the base of the<br>Saline Series at the junction of<br>Jankush Nulla and Warchha<br>Gorge, Warchha, Punjab, Pakistan.<br>Early Permian (Ghazi and<br>Mountnev, 2011).</td><td>George 1952 (as "Parasitidae")</td><td>Assigned to Palaeotyroglyphus by Dubinin<br>(1962) as "Gamasus fossilis Mani, 1945", which<br>belongs to Mesostigmata*</td></tr><tr><td colspan="6"><strong>Not Astigmata</strong></td></tr><tr><td>Ectoparasitic feather mites</td><td>eggs</td><td>Dinosaur feathers</td><td>Early Cretaceous (Nova Olinda,<br>Crato Formation, late Aptian)</td><td>Martill and Davis 1998</td><td>Not Astigmata, possible misidentification for<br>Cheyletoidea (Bochkov, 2009).</td></tr><tr><td>Palaeotyroglyphus fossilis (Mani,<br>1945)</td><td>nymph</td><td>free-living</td><td>Worli Hill, Mumbai, India.<br>Maastrichtian to Selandian,<br>66-61.7 Ma (Mantilla et al. 2022)</td><td>Dubinin, 1962 (as "larva, from the<br>Deccan Intertrappean sample,<br>Punjab, Neogene (?)"; type species:<br>"Gamasus fossilis Mani, 1945"</td><td>Dubinin (1962) designated Gamasus fossilis<br>Mani, 1945 (Mesostigmata) as the type species<br>for the genus Palaeotyroglyphus Dubinin, 1962.<br>However, he misidentified this species, mistaking<br>it for "Parasitidae" described by George (1952),<br>which actually belongs to Astigmata. According<br>to ICZN Article 70.1.1, when designating a type<br>species from a previously established nominal<br>species, it is assumed that the species has been<br>correctly identified. Therefore, despite the<br>misidentification, Palaeotyroglyphus should be<br>classified within Mesostigmata*<br>nomina dubia (Dunlop et al. 2011); could be a<br>Bdellidae based on the original description: "the<br>body is ovoid, tapering at the front, with the head<br>projecting like a tip"<br>Not Astigmata, misidentification for<br>Endeostigmata (probably Nanorchestidae)</td></tr><tr><td>Acarus resinosus Presl, 1822</td><td>unknown</td><td>unknown</td><td>Eocene (Baltic amber, 34-37 Ma)</td><td>Presl, 1822</td><td></td></tr><tr><td>"Sarcoptes kutchensis" Agnihotri,<br>Singh, Subramanian et Acharya,<br>2023</td><td>adult</td><td>free-living</td><td>Middle Eocene (Kutch amber)</td><td>Agnihotri et al 2024</td><td></td></tr><tr><td>Specimens unrecognized as mites</td><td>free-living</td><td>free-living</td><td>Warchha 14; Salt and Marl KS<br>from Khewra, respectively, from<br>Salt Range, Punjab, Pakistan.<br>Early Permian (Ghazi and<br>Mountnev, 2011).</td><td>Mani 1946 (as "Mite (leg)";<br>"gamasid mite (protonymph)")</td><td>Attributed to Palaeotyroglyphus fossilis by<br>Dubinin (1962), but both specimens are<br>unrecognizable as mites</td></tr><tr><td>"Tyroglyphites miocenicus"<br>Pampaloni, 1902</td><td>adult</td><td>free-living</td><td>Neogene (Sicily, Italy)</td><td>Pampaloni 1902</td><td>Not Astigmata, misidentification for<br>Lohmanniidae</td></tr><tr><td>"Acarus indicus" Kumar, Ja Jha,<br>Bhattacharya & Pande, 2011</td><td>nymph</td><td>free-living</td><td>Originally reported from Permian<br>sediments in the Chamba Valley,<br>Himachal Pradesh, India, it likely<br>represents contamination by a<br>modern mite</td><td>Kumar et al. 2011</td><td>Not Astigmata, misidentification for juvenile<br>Nothridae (Norton, pers. comm.)</td></tr><tr><td>"Acarus siro (Linnaeus, 1758)"</td><td>nymph</td><td>free-living</td><td>Originally reported from Permian<br>sediments in the Chamba Valley,<br>Himachal Pradesh, India, it likely<br>represents contamination by a<br>modern mite</td><td>Kumar et al. 2011</td><td>Not Astigmata, misidentification for juvenile<br>Nothridae (Norton, pers. comm.)</td></tr></tbody></table>

<footnote>*The genus was originally proposed as follows (Dubinin, 1962): "Palaeotyroglyphus W. Dubinin, nom. nov. (= Gamasus Mani, 1945, 1946). Genotype—Gamasus fossilis Mani, 1945; Larva (and not protonymph as written by Mani, 1945 and George, 1952) (Fig. 1290)". However, Gamasus fossilis Mani, 1945, clearly belongs to Mesostigmata based on the presence of peritremes, ambulacra with two claws, and the shape and setation of the legs. In contrast, George (1952) described and illustrated an astigmatid mite but identified it as a "gamasid mite". This mite appears in Fig. 1290 of Dubinin (1962). It is therefore likely that Dubinin (1962) considered George's mite to be identical to Mani's mite without actually examining Mani's description.</footnote>
(see Vorontsov and Voronezhskaya 2022 for details). Images were taken in transmitted and reflected light using a Nikon E-800 compound microscope equipped with an Olympus OM-D E-M10-II digital camera. Image z-stacks (images taken consequently from multiple focal planes) were processed in Adobe Lightroom for color correction, noise reduction, and sharpness enhancement. Focus stacking, both automatic and manual, was performed in Helicon Focus. Confocal scanning was done using a Zeiss LSM-880 CLSM equipped with AiryScan detector. A 488 nm Argon laser was used for excitation, while detection range was set to 500–700 nm. To view and manipulate the confocal image stacks, we used the following software packages: Zeiss ZEN, FIJI (Schindelin *et al.* 2012), FastStone Image Viewer, and Helicon Focus. The original image stacks and three-dimensional reconstructions are available through Figshare (https://doi.org/10.6084/m9.f figshare.28632650; https://doi.org/10.6084/m9.f figshare.22101911).

## Terminology and measurements

The idiosomal chaetotaxy terminology follows Griffiths *et al.* (1990), while the terminology for the palp and leg chaetotaxy is based on Grandjean (1939) and Griffiths (1970). The terminology of coxisternal setae follows Norton (1998). The terminology of the spermatheca follows that of Klimov and OConnor (2003). The terminology for the ovipore and related structures follows Evans (1992), as applied to oribatids. For consistency with Oribatida, the structure commonly referred to as the 'vagina' in Astigmata is termed the ovipositor. The following abbreviations are used: A—aedeagus; GO—Grandjean's organ; gla—opisthonotal gland openings; O—plicated zone of the ovipositor; S—genital papillae; Sp—spermatheca; V—ovipositor. All measurements are provided in micrometres (μm).

## Results

### Systematic paleontology

**Class Arachnida Cuvier, 1812**

**Order Sarcoptiformes Reuter, 1909**

**Suborder Oribatida Duges, 1834**

**Family Acaridae Latreille, 1802**

**Genus Histiogaster Berlese, 1883**

Type species. *Tyroglyphus carpio* Kramer, 1881, by original designation.

**Histiogaster altilis** sp. n.

Zoobank: E453FB5A-EA48-4904-8CB0-D52EA6510ABF
(Figs 1–18)

### Material

Rovno amber originated on the Volhynian Uplift (Chemyreva *et al.* 2024). We studied two pieces of Eocene Rovno amber mined at Pugach quarry (Klesov, Rovno Oblast) from the amber collection of the Schmalhausen Institute of Zoology, Kiev (SIZK), with the following collection numbers: SIZK K-15219 and SIZK K-15220. These two amber pieces were cut from a single original piece of amber (catalog number 3-694, weight 10.3 g, dimensions 40x34x15 mm). Amber was dark-yellow (Fig.1A–C), with many areas being nearly opaque due to numerous micro-inclusions (Fig.1D), which we interpreted as fossilized emulsion of phloem sap (Fig.1DFHI). However, at a microscopic level, the amber was transparent. One of the amber pieces (K-15219) contained filamentous fungal hyphae (Fig.1EG). A total of 11 adult mite specimens were found (8 females and 3 males). These mites were easily recognizable
as a species of *Histiogaster* due to the presence of four fan-like projections on the posterior opisthosoma in males. Brief descriptions of these specimens are provided below.

**SIZK K-15220-B:** holotype, a well-preserved female (Fig. 2–5). Almost all structures, including the spermatheca, are visible.

**SIZK K-15219-B:** paratype, a well-preserved female, compressed laterally (Fig.6–7). The legs and gnathosoma are better visible in the lateral aspects.

**SIZK K-15220-A1:** paratype, a moderately preserved female (Fig. 8), the gnathosoma is bent ventrally and is visible in the frontal view. Specimens A1, A2 and A3 were situated close to each other. Specimen A1 was separated for study, while specimens A2 and A3 were not separated (Fig.14A).

**SIZK K-15220-A2, K-15220-A3, K-15220-C:** moderately-well preserved females (Fig.9). In most specimens, legs and gnathosoma are visible, but the idiosomal cuticle is deformed and opaque, which does not allow to link setae with their alveoli or to find short setae.

**SIZK K-15219-A:** a well-preserved male specimen (Fig.10–14), with visible legs, dorsal and ventral structures, gnathosoma, and genitalia. The amber piece is polished into a six-sided shape to provide access to dorsal, ventral, lateral, frontal, and caudal views. The gnathosoma is bent ventrally and visible from the frontal aspect.

**SIZK K-15219-C:** a well-preserved male (Fig.15, 17–18) from a partially degraded layer of amber.

**SIZK K-15219-D:** a partially preserved female.

**SIZK K-15219-E:** a moderately-well preserved male (Fig.16), anal suckers are visible in the ventral aspect.

**SIZK K-15220-D:** a partially preserved female (Fig.9 F).

## Type deposition

The holotype (SIZK K-15220-B), 2 paratypes (SIZK K-15219-B, K-15220-A1), non-type specimens of *H. altilis* (SIZK K-15219-A, K-15219-C, K-15219-D, K-15219-E, K-15220-A2, K-15220-A3, K-15220-C, K-15220-D) and syninclusions are deposited in the amber collection of the Schmalhausen Institute of Zoology NAS of Ukraine, Kiev (SIZK).

## Etymology

The specific name (*altilis* Lat. adj., fattened) refers to the large, corpulent body of the fossil mite.

## Diagnosis

Body length 280–520, width 280–360. Grandjean’s organ arched, with forked tip. Supracoxal setae *scx* short, spiniform, twice as long as its width at base. Female with 3 pseudanal *ps*<sub>1-3</sub> and 3 adanal setae *ad*<sub>1-3</sub>; setae *ad*<sub>1</sub>, *ps*<sub>1</sub>, *ad*<sub>2</sub> and *ps*<sub>2</sub> situated in one diagonal row. Canal of spermatheca relatively wide (wider than bases of *ps* and *ad*), curved and not looped, shorter than anal slit. Internal canal of spermatheca not visible. Plicated zone of ovipositor well developed, its length and width at least 1/3 length and width of the non-plicated zone of the ovipositor. Coxal apodemes free, distance between posterior coxal apodemes II and apodemes III shorter than length of coxal apodemes III. In male, pseudanal setae *ps*<sub>3</sub> filiform (weakly spiniform), situated at anterior margin of anal suckers; setae *ps*<sub>2</sub> short, spiniform, wider than *ps*<sub>3</sub>. In male, dorsal part of opisthosomal shield shorter than wide; pattern of dorsal opisthosomal shield absent. In male, opisthosomal fan-like lobes overlap at bases. Male genital apparatus situated between coxal fields IV. Lateral edge of genital capsule forms a very broad arc, not bent medially. Aedeagus long (more 2 times longer than anal setae *ps*<sub>3</sub>), not protruding beyond anterior margin of genital capsule.

## Description

### Female
<figure><img src="image_3.png" /><figcaption>**Figure 1**. Pieces of amber after preliminary trimming: A, B –parts of the pieces SIZK K-15219 and K-15220, respectively. Both pieces contain clouds of micro-inclusions, interpreted here as emulsion of phloem sap; C,E,G –plexus of spiral filamentous hyphae, shown at different magnifications (part of the amber piece SIZK K-15219); D, F, H, I –microinclusions filling the amber piece, at different magnifications. Note the mite specimen (K-15219-E), imaged before preparation, in F.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** *Histiogaster altilis* sp. n., adult female, holotype, specimen K-15220-B, dorso-lateral view, micrographs: A –focus-stacked light micrograph, showing the imprint in amber (outer contour) and, inside it, fossilized remains of a mite; B –maximum intensity projection of confocal stack. Note the alveoli of setae e1 brightly fluorescent; C –gnathosoma, super-resolution (Airyscan) confocal stack, maximum intensity projection; inset shows supracoxal seta (scx) separately stacked in Helicon Focus; D, E –bifurcate Grandjean's organ was best resolved in transmitted light (100x, DIC) and in the CLSM scan (63x, Airyscan).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Figure 3**. *Histiogaster altilis* sp. n., adult female, holotype, specimen K-15220-B ventral view, micrographs: A –habitus image; B –gnathosoma, DIC micrograph; C –same, confocal image; D –tarsus I; E –legs III and IV; F –anal region; G –genital region. All except B, maximum intensity projections of confocal stacks.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

**Figure 4.** *Histiogaster altilis* sp. n., adult female, holotype, specimen K-15220-B: A –dorsal aspect; B –ventral aspect; C –genital structures; D –anal region; arrow indicates inseminatory canal. Internal structures shown by dotted red line. Green outline shows the contour of the mite exoskeleton inside the amber imprint.
<figure><img src="image_7.png" /><figcaption>**Figure 5** *Histiogaster altilis* sp. n., adult female, holotype, specimen K-15220-B: A, B –legs I; C, D –legs II; E, F –legs III; G, H –legs IV; I –gnathosoma with palpi, ventral aspect. Green outline shows the contour of the mite exoskeleton inside the amber imprint.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 6.** *Histiogaster altilis* sp. n., adult female, specimen K-15219-B, paratype, lateral views, micrographs: A –maximum intensity projection of confocal stack. B –DIC light micrograph. C – chelicera, enlarged.</figcaption></figure>

*Gnathosoma*. Subcapitulum elongated, seta *h* present (Figs 3B,C, 5I). Chelicerae elongated; movable finger with 3 teeth observed, fixed finger with 3 teeth observed (Fig. 6C, 7A, 9G); seta *cha* short, needle-like (Fig. 8D). Palps (Figs 8D, 3B, C, 5I) with setae *a*, *cm* and *sup*, *elcp* and solenidia *ul''*, *ul'*, ω. Labrum serrate distally (Fig.8D, arrow); pectinate processes at the labrum base not observed.
<figure><img src="image_9.png" /><figcaption>**Figure 7.** *Histiogaster altilis* sp. n., adult female, specimen K-15219-B, paratype: A –lateral view; B, C –right and left legs I, respectively; D, E –legs II; F, G –legs III; H, I –legs IV. Green outline shows the contour of the mite exoskeleton inside the amber imprint.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Figure 8** *Histiogaster altilis* sp. n., adult female, specimen K-15220-A1, paratype, ventral and frontal views: A –ventral view, maximum intensity projection of confocal stack. Gnathosomal structures and most of the legs are projected ventrally; B –frontal view, showing gnathosoma as if in traditional dorsal projection, focus-stacked transmitted light image; C –gnathosoma and legs 1, focus-stacked confocal scans. D – gnathosoma, super-resolution (Airyscan) confocal stack, maximum intensity projection. Note that, despite the comparatively low fluorescence of the mite structures in A and the significant level of fluorescence in the amber, high-resolution scans (C, D) reveal fine morphological details.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Figure 9**. *Histiogaster altilis* sp. n., females: A – specimens K-15220-A2 and A3 in the block of amber; B – specimen K-15220-C; C, E – specimen K-15220-A2 and A3, respectively; D – specimen K-15220-C; F, specimen K-15220-D; G – specimen K-15220-C, gnathosoma and legs I, Airyscan super-resolution image. A-C, E, F, focus-stacked light micrographs. D, G, maximum intensity projections.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Figure 10.** *Histiogaster altilis* sp. n., adult male, specimen K-15219-A, dorso-lateral view, micrographs: A, B –DIC light micrographs showing the dorsal aspect of the mite and the bifurcate Grandjean's organ; C –maximum intensity projection of the confocal stack. Note the bright fluorescence of setae *e*₁ clearly shown against the body; D, E, F –unidentified microscopic structures attached to (or originating from) the posterior part of the body; D, E –dorsal view; F –posterior view. One of the possible interpretations could be that these are fractures in amber.</figcaption></figure>

*Idiosoma*. Prodorsal shield (Figs 2C, 4A) long, evenly punctate, lateral margins almost straight. Setae *vi* filiform, distance between *vi*-*vi* 4–5 (n=4), setae *ve* absent. Setae *se* long, distance between *se*-*se* 78–80. Setae *si* absent. Supracoxal setae *scx* thick, twice as long as width at its base (Fig. 2C). Grandjean's organ (GO) arched, bifurcated (Figs 2D, E). Setae *c*p long, filiform, setae *c*₃ short, filiform. Setae *e*₁, *e*₂, *h*₁, *h*₂ and *h*₃ filiform, subequal. Setae *e*₁ not reaching posterior end of body. Setae *c*₁, *c*₂, *d*₁, and *d*₂ absent. Opisthonotal gland openings (*gla*) slightly posterior to setae *e*₁ (Fig. 4A). Coxal setae *1a*, *3a*, *3b*, *4a* and genital setae *g* subequal (Figs 3G, 4B, C). Setae *4a* posterior to setae *g*. Sternum simple (Fig. 4B). Distance between anterior apodemes II–III shorter than length of apodemes III. Anterior apodemes III and IV, free, not fused medially (coxal fields III open) (Figs 3A, 4B). Anal opening (Figs 3F, 4D) with 3 pairs of short pseudanal setae (*ps*₁, *ps*₂, *ps*₃) and three pairs of adanal setae (*ad*₁, *ad*₂, *ad*₃). Setae *ad*₁, *ps*₁, *ad*₂ and *ps*₂ situated in a diagonal row. Setae *ps*₃ and *ad*₃ situated
<figure><img src="image_13.png" /><figcaption>**Figure 11** . *Histiogaster altilis* sp. n., adult male, specimen K-15219-A, ventro-lateral view, confocal images: A –whole mite; B –bifurcate Grandjean's organ; C –parts of gnathosoma and palpi; D, E –genital structures. All images are maximum intensity projections except E, which is a manually focus-stacked image from the part of the stack shown in D.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure 12.** *Histiogaster altilis* sp. n., adult male, specimen K-15219-A: A –lateral view, combined transmitted and reflected light; B –enlarged leg IV; C –gnathosoma, palpi and legs I, frontal view; D –enlarged gnathosoma; B, C, D –maximum projections of confocal stacks; B, D –Airyscan super-resolution mode.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 13**. *Histiogaster altilis* sp. n., adult male, specimen K-15219-A: A – dorso-lateral view; B – ventro-lateral view; C – gnathosoma; D – genital structures. Green outline shows the contour of the mite exoskeleton inside the amber imprint.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 14.** *Histiogaster altilis* sp. n., adult male, specimen K-15219-A: A –part of gnathosoma in lateral view and right legs I and II; B –right leg III; C –right leg IV; D –left legs I and II; E –left leg III. Green outline shows the contour of the mite exoskeleton inside the amber imprint.</figcaption></figure>

near anterior end of anal opening; *ps*₃ closer to the middle of the anal opening, *ad*₃ closer to anterior end of anal opening. Distance between *ps*₁-*ps*₁ and *ad*₁-*ad*₁ less than distance between *h*₃-*h*₃; distances *ad*₂-*ad*₂ and *h*₃-*h*₃ subequal; distance *ps*₂-*ps*₂ longer than distance *h*₃-*h*₃. Inseminatory canal of spermatheca (Figs 3F, 4D) relatively wide, 2–3 times wider than bases of setae *ps* and *ad*; curved and not looped; length shorter than anal slit, directly enters to spermatheca. Internal canal of spermatheca absent. Efferent ducts and their transverse base not observed. Spermatheca (Sp) deformed, poorly visible. Ovipore (V) situated between epimers III and IV, wide (Figs 3A, 4B). Plicated zone (O) of the ovipositor (Figs 3G, 4C) well developed, its length and width at least 1/3 length and width of the non-plicated zone of the ovipositor. Genital papillae (S) larger than diameter of base of genital setae (Fig. 4C).

**Legs.** Legs shorter than body, all segments free; empodial claws short, no longer that ambulacra (Figs 3A,D,E, 4A, B, 5A–H, 6, 7, 9). Leg setation: trochanters 1-1-1-0, femora 1-1-0-1, genua 2(2)-2(1)-1(1)-0, tibia 2(1)-2(1)-1(1)-1(1), tarsus 12(3+ε)-12(1)-10-10.
<figure><img src="image_17.png" /><figcaption>**Figure 15**. *Histiogaster altilis* sp. n., adult male, specimen K-15219-C, micrographs: A –dorsal view, maximum intensity projections of confocal stack. Note the high level of amber fluorescence around the mite; B –ventral view, confocal stack, background subtraction, inverted maximum intensity projection; C –genitalia, lateral view, confocal image; D –projection of the opisthosomal shield with a 4-lobed, fan-like structure, characteristic of *Histiogaster* males, ventral view, DIC light micrograph; E –genitalia, ventral view, DIC light micrograph; F –genitalia, ventral view, confocal image, maximum intensity projection; G –anal suckers, confocal image, maximum intensity projection.</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Figure 16**. *Histiogaster altilis* sp. n., adult male, specimen K-15219-E, micrographs: A –ventral view, maximum intensity projection of confocal stack; B –anal suckers, confocal Airyscan super-resolution mode, maximum intensity projection; C, D –the same, partial stacking showing the ps<sub>3</sub> setae.</figcaption></figure>

I and II with filiform setae (*pR* I-II, *sR* III). Femoral setae *vF* I-II and *wF* IV filiform. Genual setae *cG* I-II, *mG* I-II and *nG* III weakly spiniform. Tibial setae *gT* I-II, *hT* I-II and *kT* III-IV weakly spiniform. Tarsus I-II without foliate setae; setae *ra*, *d*, *f*, *la* filiform, long, *e* and *ba* spiniform, short; *wa* weakly spiniform, short; *p*, *u*, *s*, *q*, *v* very short, spiniform. Distance between *wa*-s much larger than diameter of bases of *s*. Tarsus III with filiform setae *d*, *r*, *f*; weakly spiniform *w*; spiniform *e*; and very short, spiniform *p*, *u*, *s*, *q*, *v*. Tarsus IV with filiform setae *d*, *f*; weakly spiniform *w* and *r*; strongly spiniform *e*; very short, spiniform *p*, *u*, *s*, *q*, *v*.
<figure><img src="image_19.png" /><figcaption>Figure 17. *Histiogaster altilis* sp. n., adult male, specimen K-15219-C: A –dorsal aspect; B –ventral aspect; C –caudal aspect.</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure 18.** *Histiogaster altilis* sp. n., adult male, specimen K-15219-C: A –gnathosoma, palpi and legs I, ventral aspect; B, C –legs II; D, E –legs III; F, G –legs IV.</figcaption></figure>

Solenidion ω₁ on tarsi I–II with widened end, club-shaped, situated closer to base of tarsus; ω₂ of tarsus I short, with rounded tip, situated somewhat more basal and posterior or directly adjacent to ω₁; ω₃ on tarsus I bacilliform, subequal to ω₁, situated apically on tarsus; tibial solenidia φ I–IV long, tapering; σ' of genua I long, 3 times shorter than φ I; σ" of genua I bacilliform, three times shorter than σ'; solenidia σ of genua II–III with pointed ends. Famulus ε of tarsus I short, pointed, adjacent to solenidion ω₁ and seta *ba*.

*Measurements* (n=4). Idiosomal length 280, 340, 400, 520, width 280–330. Gnathosoma: subcapitulum length 60, width 27–30, seta *h* 15–16, chelicerae 50–57, seta *cha* 3–4, palp seta *a* 4–5, *cm* 11 and *sup* 9, solenidion ω 2–3. Prodorsal shield length 50–72, width 42–44.
Idiosomal setae: *vi* 30–45, *se* 70, *scx* 3, *c*<sub>p</sub> 52–95, *c*<sub>3</sub> 10–14, *e*<sub>1</sub> 20–25, *e*<sub>2</sub> 30–40, *h*<sub>1</sub> 80–90, *h*<sub>2</sub> 50–70, *h*<sub>3</sub> 55–72, *la*, *3a*, *3b*, *4a* and *g* 9–12, *ps*<sub>1</sub>, *ps*<sub>2</sub>, *ps*<sub>3</sub>, *ad*<sub>1</sub>, *ad*<sub>2</sub> and *ad*<sub>3</sub> 5–7. Grandjean's organ length 16–18. Canal of spermatheca 22; length of spermatheca 50, width 30. Ovipore 65. Anal opening 84. Leg I: length 100–140, trochanter 20, femur 30–35, genu 16–20, tibia 18–22, tarsus (including pretarsus) 35–50, pretarsus (excluding claw) 8–10, claw 11–15; ω<sub>1</sub> I 11–15, ω<sub>2</sub> I 4–5, ω<sub>3</sub> I 16–20, famulus ε 3, *ba* I 4–6, *wa* I 5, *e* I 6–7, *d* I 37–40, *ra* I 30–33, *f* I 25, *la* I 10, *s* I, *p* I, *q* I, *u* I and *v* I 3–5, φ I 56–80, *gT* I 6, *hT* I 5–6, σ'' I 8–10, σ' I 23–25, *cG* I 6, *mG* I 10–12, *vF* I 35–50, *pR* I 20–23. Leg II: length 92–110, trochanter 11–15, femur 20–23, genu 15–20, tibia 14–20, tarsus (including pretarsus) 42–50, pretarsus (excluding claw) 10, claw 10; ω II 10–11, *ba* II 5, *wa* II 6–7, *e* II 5–6, *d* II 31–34, *ra* II 30–32, *f* II 22–30, *la* II 9–10, *s* II, *p* II, *q* II, *u* II and *v* II 3–5, φ II 50–55, *gT* II 7, *hT* II 6–7, σ II 10–13, *cG* II 6–7, *mG* II 6–7, *vF* II 22–32, *pR* II 9–11. Leg III: length 110–120, trochanter 20, femur 20, genu 20, tibia 18–23, tarsus (including pretarsus) 41–60, pretarsus (excluding claw) 10, claw 10–13; *w* III 7–8, *e* III 8, *d* III 40–85, *r* III 30–50, *f* III 25–35, *s* III, *p* III, *q* III, *u* III and *v* III 3–6, φ III 46–55, *kT* III 10, σ III 10, *nG* III 10, *sR* III 12. Leg IV: length 100–122, trochanter 20–25, femur 20, genu 18–20, tibia 18–22, tarsus (including pretarsus) 40–65, pretarsus (excluding claw) 10–11, claw 10; *w* IV 11–15, *e* IV 8–10, *d* IV 50–120, *r* IV 18–20, *f* IV 60–80, *s* IV, *p* IV, *q* IV, *u* IV and *v* IV 3–6, φ IV 20–30, *kT* IV 9–14, *wF* IV 15.

Male

*Gnathosoma*. As in female (Figs 11C, 12C, D, 13C, 18A).

*Idiosoma*. Prodorsal shield (Figs 13A) long, evenly punctate, lateral margins almost straight. Setae *vi* filiform, distance between *vi-vi* 4 (n=2); setae *ve* absent. Setae *se* long, distance between *se-se* 90. Setae *si* absent. Supracoxal setae *scx* not observed. Grandjean's organ bifurcated (Figs 10B, 11B, 12C). Setae *c*<sub>p</sub> long, filiform, setae *c*<sub>3</sub> short, filiform. Setae *e*<sub>1</sub>, *e*<sub>2</sub>, *h*<sub>1</sub>, *h*<sub>2</sub> and *h*<sub>3</sub> filiform, subequal. Setae *e*<sub>1</sub> not reaching end of body. Setae *c*<sub>1</sub>, *c*<sub>2</sub>, *d*<sub>1</sub>, *d*<sub>2</sub> absent. Opisthonotal gland (*gla*) (Fig. 17A) openings slightly posterior to setae *e*<sub>1</sub>. Dorsal part of opisthosomal shield shorter than wide; pattern of dorsal opisthosomal shield absent (Figs 17A,C). Four fan-like projections overlap at their bases (Figs 10A, 11A, 13A,B, 15A,B,D, 17). Coxal setae *la*, *3a*, *3b*, *4a* and genital setae *g* subequal (Figs 13B, 15E, 17B). Setae *4a* posterior to setae *g* (Fig. 11E). Sternum simple (Figs 11A, 13B). Distance between anterior coxal apodemes II–III shorter than length of anterior apodemes III. Apodemes III and IV not observed. Genital opening situated between apodemes IV (Figs 11A, 13B, 15B, 17B). Lateral edges of genital capsule form a very broad arc, not bent medially (Figs 11D,E, 13D, 15E,F). Aedeagus (A) (Figs 11D,E, 13D) long, 2 times longer than length of setae *ps*<sub>3</sub>, not protruding beyond anterior edge of genital capsule. Anal sucker rounded in outline (Figs 15G, 16, 17B,C). Setae *ps*<sub>3</sub> weakly spiniform, situated at anterior edge of anal suckers (Figs 15G, 16C,D, 17B,C). Setae *ps*<sub>2</sub> short, spiniform, wider than *ps*<sub>3</sub> (Fig. 15G). Setae *ps*<sub>1</sub> absent, *ih* not observed.

*Legs*. Legs I–III as in females (Figs 12A–C, 14, 18). Tarsus IV with filiform setae *f*; spiniform *w*; weakly spiniform *r*; very short, spiniform *p*, *u*, *s*, *q*, *v*; setae *d* and *e* transformed to suckers. Solenidion φ of tibiae IV short, bacilliform.

*Measurements* (n=2). Idiosomal length 460–510, width 350–360. Gnathosoma: subcapitulum length 65, width 30, seta *h* 15, chelicerae 50–64, seta *cha* 5, palp seta *a* 5–6, *cm* 15, *sup* 8, solenidion ω 3–4. Prodorsal shield length 60, width 50. Idiosomal setae: *vi* 42, *se* 110–113, *c*<sub>p</sub> 70–100, *c*<sub>3</sub> 10, *e*<sub>1</sub>, *e*<sub>2</sub> 54, *h*<sub>1</sub> 70–100, *h*<sub>2</sub> 63–76, *la*, *3a*, *3b*, *4a* and *g* 9–10, *ps*<sub>2</sub> 4, *ps*<sub>3</sub> 8, genital capsule 40. Aedeagus 22. Anal opening 50–66. Diameter of anal suckers 25–28. Leg I: length 120–150, trochanter 20, femur 38, genu 25, tibia 24, tarsus (including pretarsus) 48, pretarsus (including claw) 10, claw 13; ω<sub>1</sub> I 13–15, ω<sub>2</sub> I 6, ω<sub>3</sub> I 15, famulus ε 3–5, *ba* I 7–8, *wa* I 6, *e* I 7, *d* I 30–35, *ra* I 25–45, *f* I 23, *la* I 11, *s* I, *p* I, *q* I, *u* I and *v* I 3–6, φ I 70–88, *gT* I 7, *hT* I 10, σ'' I 10, σ' I 26–28, *cG* I 9, *mG* I 8–10, *vF* I 30–48, *pR* I 8. Leg II: length 122, trochanter 15, femur 37, genu 17–26, tibia 17–22, tarsus (including pretarsus) 50, pretarsus (including claw) 10, claw 12; ω II 13, *ba* II 8, *wa* II 7, *e* II 6, *d* II 25–35, *ra* II 30–40, *f* II 30, *la* II 7, *s* II, *p* II, *q* II, *u* II and *v* II 3–6, φ II 50–55, *gT* II 7, *hT* II 9–10, σ II 10–11, *cG* II 6, *mG* II 11–12, *vF* II 30–40, *pR* II 7. Leg III: length 130, trochanter 22, femur 30, genu 26–30, tibia 26, tarsus (including
pretarsus) 40, pretarsus (excluding claw) 10, claw 11–13; *w* III 9, *e* III 6–7, *d* III 60, *r* III 20, *f* III 40, *s* III, *p* III, *q* III, *u* III and *v* III 4–7, *φ* III 45–50, *kT* III 9–10, *σ* III 13, *nG* III 6–7, *sR* III 10. Leg IV: length 140, trochanter 25, femur 35, genu 25–28, tibia 25–30, tarsus (including pretarsus) 40, pretarsus (excluding claw) 9–10, claw 12; *w* IV 11, *e* IV width 7–9, *d* IV width 8–9, *r* IV 6, *f* IV 35–50, *s* IV, *p* IV, *q* IV, *u* IV and *v* IV 4–7, *φ* IV 8, *kT* IV 11, *wF* IV 10.

**Heteromorphic deutonymph** unknown.

**Remarks**

Given that all specimens originated from a single large piece of amber, we can assume that they are conspecific and probably belong to a single population. The remarkable degree of preservation of some of these specimens has allowed us to accurately compare the fossil species with modern species of the genus *Histiogaster*.

Currently, species of the genus *Histiogaster* are divided into two groups: the *carpio*-species group and the *arborsignis*-species group. In the *carpio* group, the body is wide, apodemes III and IV are free, the distance between posterior apodemes II and III is shorter than apodeme III length, the female ovipore is situated between apodemes III and IV, the plicate zone of the ovipositor is normally developed (at least 1/2 the width of ovipositor), genital papillae wider, and the opisthonotal shield is without tree-like pattern in males. In the *arborsignis*-species group, the body is elongated, apodemes III and IV are connected, the distance between posterior apodemes II and apodemes III is longer than the length of apodeme III, the female ovipore is situated between apodemes IV, plicated zone of the ovipositor is greatly reduced (less than 1/2 the width of ovipositor) in females, genital papillae are small, and in males the opisthonotal shield has a tree-like pattern (Klimov *et al.* 2022). *Histiogaster altilis* sp. n. belongs to the *carpio*-species group.

*Histiogaster altilis* sp. n. is most similar to *H. anops* Griffiths, 1963 as both species have a wide and short canal of spermatheca, wider than the bases of setae *ps* and *ad* (in other species this canal is thin, not wider than the bases of setae *ps* and *ad*) (Griffiths 1963). The new species differs significantly from *H. anops* in the arrangement of three pairs of pseudanal setae (*ps*) and three pairs of adanal setae (*ad*): in *H. altilis* sp. n. setae *ad*₁, *ps*₁, *ad*₂ and *ps*₂ are situated on the same diagonal row relative to the anal opening (*vs*. these setae are not arranged in the same row, setae *ps*₁ are distinctly posterior to *h*₃ and *ad*₂ are distinctly anterior to *ps*₂ in *H. anops*).

Males of *H. altilis* sp. n. and *H. anops* have needle-shaped setae *ps*₃ (Fig.15G), but in *H. altilis* sp. n. setae *ps*₃ are clearly narrower than *ps*₂ (*vs*. seta *ps*₃ slightly wider than *ps*₂ in *H. anops*). *Histiogaster altilis* sp. n. males have overlapping fan-like opisthosomal projections (Fig.11A, 15D), while in *H. anops* these projections are separated. Tarsal setae *f* I–II and *ra* I–II in *H. altilis* sp. n. (Fig.12C, Fig.3D) are filiform, while in *H. anops* these setae are widened.

The similarities between *H. altilis* sp. n. and *H. anops* suggest that this condition of the spermatheca (short and thick canal and simple saccular spermatheca) is plesiomorphic. The absence of eyes in the heteromorphic deutonymphs of *H. anops* also seems plesiomorphic, but this cannot be confirmed until heteromorphic deutonymphs of *H. altilis* sp. n. are found.

Twisted (or corkscrew-shaped) setae have been observed in *H. altilis* sp. n. (Figs 3 E, F) and in other fossil mites preserved in amber (Khaustov *et al.*, 2021). Since setae of this shape have never been observed in modern specimens of the same genera or families, we believe that initially straight setae become twisted during the process of fossilization.

**Discussion**

**Habitat of *Histiogaster altilis* sp. n.**

Fossil mites of *H. altilis* sp. n. likely lived close to the site where resin was released, as indicated by the presence of several males and females that probably belong to the same population. Additionally, the numerous microinclusions around the mites, which we interpret as phloem sap emulsion, along with abundant filamentous hyphae, suggest proximity to a
rich and likely organic substrate. It should be noted that these hyphae are likely to belong to resinicolous fungi related to those found both in Cretaceous ambers and in extant coniferous resins (Speranza et al., 2015; Peñalver et al., 2025), which, in our opinion, supports that the Eocene resin was enriched with a sugary tree sap. Resinicolous fungi were also reported from Rovno amber (Sukhomlyn et al., 2021).

Moreover, *H. altilis* sp. n. is morphologically similar to *H. carpio* (Kramer, 1882) and *H. iberica* Kadzhaya, 1959, which are modern inhabitants of tree sap flows. However, the fossil mite also shares similarities with *H. anops*, which inhabits the larval tunnels of two species of pine weevils (*Hylobius pales* (Herbst, 1797) and *Pachylobius picivorus* (Germar, 1824)) in the southern United States (Davis and Hunter, 1963). Considering all available evidence, we propose that *H. altilis* sp. n. inhabited fermented tree sap, like many modern *Histiogaster* species (Türk and Türk, 1957; Bugrov, 1997; Klimov et al., 2022). However, we cannot entirely exclude the possibility of an association with beetle galleries given the data at hand.

It is interesting to compare the newly described *H. altilis* sp. n. with another fossil astigmatan, *Glaesacarus rhombeus*, which is common in Baltic and Rovno amber (Perkovsky et al. 2007). The probable microhabitats of *G. rhombeus* may include insect burrows in wood, sap flow, or water-filled tree holes (Sidorchuk and Klimov, 2011), although subcortical spaces are the most likely habitat. One view suggests that this species could forage on hardened resin surfaces and possibly feed on carrion (Grünemaier, 2017). Surprisingly, despite the abundance of this species in Eocene amber, modern congeners have not yet been found. In contrast, mites of the genus *Histiogaster* have been able to maintain a conserved phenotype and ecology, presumably feeding on tree sap flows since the Eocene.

## Advanced imaging

Here, we approached the limit of optical resolution in amber by using modern confocal microscopy (Vorontsov and Voronezhskaya, 2022; Vorontsov et al., 2023). This was needed to observe minute taxonomically important features, like the shape of the Grandjean's organ (Fig.2E) or the male's anal suckers (Fig.16B-D). To reach high resolution of imaging, we (1) fine-polished the amber pieces, leaving only a very thin layer of amber over the inclusion and (2) used high-resolution DIC microscopy and CLSM with a super-resolution detector. We found that successful application of CLSM highly depends on the level of fossil autofluorescence, which is usually better in deeper, less deteriorated layers of amber. Deteriorated surface layers of amber become fluorescent and mask the fluorescence from the fossil (Vorontsov et al., 2023). Among the specimens that we studied here, K-15219-C and K-15220-A1 were found closer to the surface of the original pieces, while K-15219-A and -B were extracted from deeper layers (compare background fluorescence in Fig.10C, Fig.11A and Fig.6A to Fig.8A). However, even partially deteriorated amber did not prevent high-resolution imaging (Fig. 8CD). As a result, with our advanced imaging technique, we scored almost all anatomical characters. In contrast, the most complete fossils typically preserve less one third of the anatomical characters used to infer evolutionary relationships among living species (Edgecombe, 2010).

## Acknowledgments

D.D.V. thanks Evert Lindquist (Agriculture and Agri-Food Canada) and Roy Norton (State University of New York, USA) who provided support for his research in high-resolution imaging of fossil Acari. We thank Dr. Kimiko Okabe (Forestry and Forest Products Research Institute, Tsukuba, Japan) for translating Aoki's 1974 paper. We are grateful to Anatoly P. Vlaskin (SIZK) for cutting and polishing the amber pieces and to Elena Voronezhskaya (IDB RAS) for help with confocal microscopy. CLSM images were taken using the equipment of the Core Centrum of the Institute of Developmental Biology RAS. This contribution is a continuation of research started by the late Ekaterina Sidorchuk.
Funding

The work of DDV was conducted under the IDB RAS research program №0088-2024-0011.
VBK was supported by the Ministry of Science and Higher Education of the Russian Federation
within the framework of the Federal Scientific and Technical Program for the Development
of Genetic Technologies for 2019-2027 (agreement №075-15-2021-1345, unique identifier
RF—193021X0012).

ORCID

Vasiliy B. Kolesnikov https://orcid.org/0000-0001-6177-7858

Dmitry D. Vorontsov https://orcid.org/0000-0002-1701-1748

Evgeny E. Perkovsky https://orcid.org/0000-0002-7959-4379

Pavel B. Klimov https://orcid.org/0000-0002-9966-969X

References

Agnihotri P., Singh H., Subramanian K. A., Acharya S. 2024. Scanning electron microscopy of *Sarcoptes kutchensis*, a new species of a Middle Eocene sarcoptid mite in amber from the Umarsar Lignite Mine of Kutch, Western India. Historical Biology, 36(12): 2847-2853. https://doi.org/10.1080/08912963.2023.2281579

Aoki, J. 1974. On the fossil mites in Mizunami amber from Gifu Prefecture, Central Japan. Bulletin of the Mizunami Fossil Museum, 1: 397-399 [in Japanese]

Azar D. 2007. Preservation and accumulation of biological inclusions in Lebanese amber and their significance. Comptes Rendus Palevol, 6: 151-156. https://doi.org/10.1016/j.crpv.2006.10.004

Berlese A. 1883. Sopra due nuovi generi di Acari italiani. Lettura fatta alla R. Accademia di Padova, 33: 45-52.

Bochkov A. V. 2009. A review of mites of the parvorder Eleutherengona (Acariformes: Prostigmata) - permanent parasites of mammals. Acarina Supplement No 1: 3-149.

Bugrov S.A. 1997. Free-living astigmatic mites (Acariformes, Astimata) of the fauna of the Moscow region. Zoologicheskiy Zhurnal, 76(2): 147-156. [In Russian]

Chemyreva V. G., Legalov A. A., Perkovsky E. E. 2024. A new genus of Ambositrinae (Hymenoptera, Diapriidae) from Rovno amber and remarks on the Eocene distribution of the subfamily. Ecologica Montenegrina, 79: 104-112. https://doi.org/10.37828/em.2024.79.9

Davis R., Hunter P. F. 1963. Biological studies of a *Histiogaster* mite (Acarina: Acaridae) associated with pine reproduction weevils. Annals of the Entomological Society of America, 56: 682-687.
https://doi.org/10.1093/aesa/56.5.682

Dubinin D. V. 1962. Class Acaromorpha. Ticks, mites or gnathosomous chelicerates, *In*: Rohdendorf, B.B. (Ed.) Fundamentals of Paleontology. Volume 9. Arthropoda, Tracheata, Chelicerata. [Osnovy paleontoligii. A manual for paleontologists and geologists of the USSR, translated by IPST Staff]. Smithsonian Institution Libraries and National Science Foundation, Washington, D.C., pp. 681-722.

Dunlop J. A., Penney D., Jekel D. 2011. A summary list of fossil spiders and their relatives. In: Platnick N. (Ed.), The world spider catalog, version 12.0. American Museum of Natural History, New York, 258 pp.

Dunlop J. A., Wirth S., Penney D., McNeil A., Bradley R. S., Withers P. J., Preziosi R. F. 2012. A minute fossil phoretic mite recovered by phase-contrast X-ray computed tomography. Biology Letters, 8: 457-460. https://doi.org/10.1098/rsbl.2011.0923

Edgecombe G. D. 2010. Palaeomorphology: fossils and the inference of cladistic relationships. Acta Zoologica, 91(1): 72-80. https://doi.org/10.1111/j.1463-6395.2009.00426.x

Evans G. O. 1992. Principles of Acarology. CABI Publishing Wallingfrod, Oxon OX10 8DE, UK, 563 p.

Fain A. 1977. Nouvelles observations sur les acariens recoltes par le Dr. J. Trave aux lies Saint-Paul et Nouvelle-Amsterdam (Astigmates). Acarologia, 18(3): 553-564.

George V. P. 1952. On some arthropod microfossils from India. Agra University Journal of Research, 1: 83-108

Ghazi S., Mountney N. P. 2011. Petrography and provenance of the Early Permian Fluvial Warchha Sandstone, Salt Range, Pakistan. Sedimentary Geology, 233(1-4): 88-110. https://doi.org/10.1016/j.sedgeo.2010.10.013

Grandjean F. 1939. La chaetotaxie des pattes chez les Acaridae. Bulletin de la Société Zoologique de France, 64: 50-60.

Griffiths D. A. 1963. A new species of *Histiogaster* belonging to the family Acaridae Ewing and Nesbitt 1942 (Acarina). Acarologia, 5: 61-70

Griffiths D. A. 1970. A further systematic study of the genus *Acarus* L., 1758 (Acaridae, Acarina), with a key to species. Bulletin of the British Museum (Natural History). Zoology series, 19: 85-118.

Griffiths D. A., Atyeo W. T., Norton R. A., Lynch C. A. 1990. The idiosomal chaetotaxy of astigmatid mites. Journal of Zoology, 220: 1-32. https://doi.org/10.1111/j.1469-7998.1990.tb04291.x
Grünemaier M. 2017. Not just hyphae - the amber mite *Glaesacarus rhombeus* as a forager on hardened resin surfaces and a potential scavenger on trapped insects. Palaeodiversity, 10: 129-134. https://doi.org/10.18476/pale.v10.a9

Halbwachs H., Bässler C., Worobiec E. 2021. Palynomorphs in Baltic, Bitterfeld and Ukrainian ambers: a comparison. Palynology, 45(3): 441-457. https://doi.org/10.1080/01916122.2020.1863274

Kadzhaya G. Sh. 1959. A new species of the genus *Histiogaster* in Gruzia (Acarina, Tyroglyphoidea). Soob. Akad. Nauk Gruz. S. S. R. 23:75-78.

Khaustov A. A., Vorontsov D. D., Perkovsky E. E., Lindquist E. E. 2021. Review of fossil heterostig-
matic mites (Acari: Heterostigmata) from late Eocene Rovno Amber. I. Families Tarsocheyli-
dae, Dolichocybidae and Acarophenacidae. Systematic and Applied Acarology, 26(1): 33-61.
https://doi.org/10.11158/saa.26.1.3

Klimov P.B. (2000) *Paulacarellus faini* sp. n., a new species of acarid mite (Acariformes: Acaridae) from the Russian Far East. *Acarologia*, 41: 249-253.

Klimov P. B., Kolesnikov V. B., Khaustov A. A., Ermilov S. G., Tolstikov A. V. 2022. The mite genus *Histiogaster* (Acari: Acaridae) in the Eastern Palaearctic, with a description of two new species and report of pre-copulatory guarding. *Acarina*, 30(2): 121-147. https://doi.org/10.21684/0132-8077-2022-30-2-121-147

Klimov B. B., OConnor B. M. 2003. Phylogeny, historical ecology and systematics of some mushroom-associated mites of the genus *Sancassania* (Acari: Acaridae), with new generic synonyms. Invertebrate Systematics, 17(4): 469-514. https://doi.org/10.1071/IS02050

Klimov P. B., Tolstikov A. V. 2011. Acaroid mites of Northern and Eastern Asia (Acari: Acaroidea). Acarina, 19(2): 252-264.

Klimov P. B., Vorontsov D. D., Azar D., Sidorchuk E. A., Braig H. R., Khaustov A. A., Tolstikov A. V. 2021. A transitional fossil mite (Astigmata: Levantoglyphidae fam. n.) from the early Cretaceous suggests gradual evolution of phoresy-related metamorphosis. Scientific Reports 11(1): e15113. https://doi.org/10.1038/s41598-021-94367-2

Koch C. L., Berendt G. C. 1854. Die im Bernstein befindlichen Crustaceen, Myriopoden, Arachniden und Apteren der Vorwelt. P. 124 in G. C. Berendt (ed.) Die in Bernstein befindlichen organischen Reste der Vorwelt, gesammelt, in Verbindung mit Mehreren bearbeitet und herausgegeben. Commission der Nicolaischen Buchhandlung, Berlin.

Kolesnikov V. B., Vorontsov D. D., Perkovsky E. E., Vasilenko D. V., Klimov P. B. 2023. Confocal autofluorescence microscopy revealed the fine morphology of the amber preserved mite *Congovidia glesoconomorphi* sp. nov. (Acari: Hemisarcoptidae) phoretic on a mycterid beetle. *Palaeoentomology* 006(6): 665-678. https://doi.org/10.11646/palaeoentomology.6.6.8

Kolesnikov V. B., Vorontsov D. D., Sidorchuk E. A. 2024. Seven new species from Eocene Baltic amber reveal surprising diversity and suggest possible speciation scenarios in the recentual family Collohmanniidae (Acari: Oribatida). Zootaxa, 5553(1): 1-78. https://doi.org/10.11646/zootaxa.5553.1.1

Kramer P. 1881. Ein *Dermaleichus*-artiger *Tyroglyphus*. Zoologischer Anzeiger, 4: 619.

Kumar P., Jha N., Battachaharya D. D., Pande A. C. 2011. Acarid mites from Early Permian sediments of the Chamba Valley, Himachal Pradesh, India. Journal of the Palaeontological Society of India, 56: 171-179. https://doi.org/10.1177/0971102320110206

Mani M. S. 1945. Arthropod fossil from India. Indian Journal of Entomology, 6: 61-64.

Mani M. S. 1946. On some arthropod fossil from India. Proceedings of the National Academy of Sciences, India. Section B, 16(2-4): 43-56.

Mantilla G. P. W., Renne P. R., Samant B., Mohabey D. M., Dhobale A., Tholt A. J., Tobin T. S., Widdowson M. Anantharaman S., Dassarma D. C., Mantilla J. A. W. 2022. New mammals from the Naskal intertrappean site and the age of India's earliest eutherians. Palaeogeography Palaeoclimatology Palaeoecology, 591: 110857. https://doi.org/10.1016/j.palaeo.2022.110857

Martill D. M., Davis P. G. 1998. Did dinosaurs come up to scratch? Nature, 396: 528-529. https://doi.org/10.1038/25027

Norton R. A. 1998. Morphological evidence for the evolutionary origin of Astigmata (Acari: Acariformes). Experimental and Applied Acarology, 22: 559-594. https://doi.org/10.1023/A:1006135509248

Ochoa R., OConnor B. M. 2000. Revision of the genus *Horstiella* (Acari: Acaridae): mites associated with Neotropical *Epicharis* bees (Hymenoptera: Apidae). Annals of the Entomological Society of America, 93: 713-737. https://doi.org/10.1603/0013-8746(2000)093%5B0713:ROTGHA%5D2.0.CO;2

OConnor B. M. 2009. Cohort Astigmatina. In: Krantz, G.W., Walter, D. E. (Eds), *A manual of acarology*. Texas Tech University Press, pp. 565-657.

OConnor B. M. 2022. Two new genera of winterschmidtiine mites (Acari: Astigmata: Winterschmidtiidae) associated with beetles in the family Bostrichidae (Coleoptera: Polyphaga: Bostrichoidea). Systematic and Applied Acarology, 27 (2): 209-219. https://doi.org/10.11158/saa.27.2.4

Pampaloni L. 1902. I resti organici nel disodile di Melilli in Sicilia. Palaeontographia Italica, 8:121-130

Pastorelli G., Shashoua Y., Richter J. 2013. Surface yellowing and fragmentation as warning signs of depolymerisation in Baltic amber. Polymer Degradation and Stability, 98(11): 2317-2322. https://doi.org/10.1016/j.polymdegradstab.2013.08.009

Peñalver E., Speranza M., Delclòs X. 2025. Fungal cortices in kauri resin from New Zealand as taphonomic and ecological analogues of Cretaceous amber cortices: an actualistic approach. Spanish Journal of Palaeontology. https://doi.org/10.7203/sjp.29855

Pepato A. R., Costa S. G. dos S., Harvey M. S., Klimov P. B. 2022. One-way ticket to the blue: A large-scale, dated phylogeny revealed asymmetric land-to-water transitions in acariform mites (Acari: Acariformes). Molecular Phylogenetics and Evolution, 177: 107626. https://doi.org/10.1016/j.ympev.2022.107626
Perkovsky E. E., Rasnitsyn A. P., Vlaskin A. P., Taraschuk M. V. 2007. A comparative analysis of the Baltic and Rovno amber arthropod faunas: representative samples. African Invertebrates, 48(1): 229-245.

Pfammatter J. A., Coyle D. R., Gandhi K. J. K., Hernandez N., Hofstetter R. W., Moser J. C. Raffa K. F. 2016. Structure of phoretic mite assemblages across subcortical beetle species at a regional scale. Environmental Entomology, 45(1): 1-13. https://doi.org/10.1093/ee/nvv150

Poinar G.O. 1988. Hair in Dominican amber: Evidence for tertiary land mammals in the Antilles. Experientia, 44: 88-89. https://doi.org/10.1007/BF01960261

Presl J. S. 1822. Additamenta ad faunam protogaeam, sistens descriptiones aliquot animalium in succino inclusorum. In Presl, J. S. & Presl, C. B. (eds). Deliciae Pragenses Historiam Naturalem Spectantes. Tome I. Calvae, Pragae, viii + 244 pp. https://doi.org/10.5962/bhl title.46467

Santiago-Blay J. A., Jolivet P. Verma K. K. 2012. A natural history of conspecific aggregations in terrestrial arthropods, with emphasis on cycloalexy in leaf beetles (Coleoptera: Chrysomelidae). Terrestrial Arthropod Reviews, 5: 289-355. https://doi.org/10.1163/18749836-05031054

Schatz H., Behan-Pelletier V. M., OConnor B. M., Norton R. A. 2011. Suborder Oribatida van der Hammen, 1968. In: Zhang, Z.-Q. (Ed.) Animal biodiversity: An outline of higher-level classification and survey of taxonomic richness. Zootaxa, 3148: 141-148. https://doi.org/10.11646/zootaxa.3148.1.26

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.-Y., White D. J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A. 2012. Fiji: An Open-Source Platform for Biological-Image Analysis. Nature Methods, 9: 676-682. https://doi.org/10.1038/nmeth.2019

Sendi H., Klimov P. B., Kolesnikov V. B., Káčerová J., Bonino E., Azar D., Robin N. 2025. The oldest continuous association between astigmatid mites and termites preserved in Cretaceous amber reveals the evolutionary significance of phoresy. BMC Ecology and Evolution, 25: 16. https://doi.org/10.1186/s12862-025-02351-5

Seyfullah L. J., Beimforde C., Dal Corso J., Perrichot V., Rikkinen J., Schmidt A. R. 2018. Production and preservation of resins - past and present. Biological Reviews, 93(3): 1684-1714. https://doi.org/10.1111/brv.12414

Sidorchuk E. A., Klimov P. B. 2011. Redescription of the mite *Glaesacarus rhombeus* (Koch & Berendt, 1854) from Baltic amber (Upper Eocene): evidence for female-controlled mating. Journal of Systematic Palaeontology, 9(2): 183-196. https://doi.org/10.1080/14772019.2011.566585

Sidorchuk E. A., Vorontsov D. D. 2018. Preparation of small-sized 3D amber samples: state of the technique. Palaeoentomology, 1(1): 80-90. https://doi.org/10.11646/palaeoentomology.1.1.10

Speranza M., Ascaso C., Delclòs X., Peñalver E. 2015. Cretaceous mycelia preserving fungal polysaccharides: taphonomic and paleoecological potential of microorganisms preserved in fossil resins. Geologica Acta, 13(4): 363-385. https://doi.org/10.1344/GeologicaActa2015.13.4.8

Sukhomlyn M. M., Heluta V. P., Perkovsky E. E., Ignatov M. S., Vasilenko D. V. 2021. First record of fungus of the family Mycocaliciaceae in Rovno amber (Ukraine). Paleontological Journal, 55(6): 684-690. https://doi.org/10.1134/S0031030121060125

Telnov D., Perkovsky E. E., Vasilenko D. V., Yamamoto S. 2021. The first fossil Coleoptera record from the Volyn Region, Ukraine, with description of a new *Glesoconomorphus* (Coleoptera, Mycteridae) in syninclusion with Winterschmidtiidae (Acari) and a key to species. Zookeys, 1068: 189-201. https://doi.org/10.3897/zookeys.1068.75391

Türk E. 1963. A new tyroglyphid deutonymph in amber from Chiapas, Mexico. University of California Publications in Entomology, 31: 49-51.

Türk E. and Türk F. 1957. Systematik und Ökologie der Tyroglyphiden Mitteleuropas. In: H. J. Stammer (Ed.). Beiträge zur Systematik und Ökologie mitteleuropäischer Acarina. Band 1. Tyroglyphidae und Tarsonemini. Akademische Verlagsgesellschaft Geest & Portig K.-G., Leipzig, pp. 3-231.

Volgin V. I. 1966. A new genus and new species of mites of the family Acaridae (Acarina, Acariformes). Trudy Akad. Nauk SSSR, 37: 286-296.

Vorontsov D. D., Voronezhskaya E. E. 2022. Pushing the limits of optical resolution in the study of the tiniest fossil arthropods. Historical Biology, 34(12): 2415-2423. https://doi.org/10.1080/08912963.2021.2017920

Vorontsov D. D., Kolesnikov V. B., Voronezhskaya E. E., Perkovsky E. E., Berto M. M., Mowery J., Ochoa R., Klimov P. B. 2023. Beyond the limits of light: an application of super-resolution confocal microscopy (sCLSM) to investigate Eocene amber microfossils. Life, 13(4): 865. https://doi.org/10.3390/life13040865

Wirth S. F., Garonna A. P. 2015. *Histiostoma ovalis* (Histiostomatidae, Acari) associated with Ips sexdentatus (Scolytinae, Curculionidae, Coleoptera): Ecology and mite redescription on the basis of formerly unknown adults and nymphs. International Journal of Acarology, 41: 415-428. http://dx.doi.org/10.1080/01647954.2015.1050062 https://doi.org/10.1080/01647954.2015.1050062

Woodring J. P. 1966. North American Tyroglyphidae (Acari): The Genus *Histiogaster*, with Descriptions of four New Species. Proc. Louis. Ac. Sc., 29: 113-136.

Zachvatkin A. A. 1941. Tiroglifoidnye kleshchi (Tyroglyphoidea) [=Tyroglyphoid Mites (Tyroglyphoidea)]. In: Zernov, S. A. (Ed.), Akademiya Nauk SSSR, Moscow-Leningrad, Fauna SSSR, Paukoobraznuye, 475 pp. (in Russian)