New fossil stilt-legged mites of Neophyllobius Berlese, 1886 (Acariformes, Camerobiidae) from Eocene Baltic amber

Abstract. The fossil record of the family Camerobiidae has been represented by only one species, Neophyllobius succineus Bolland and Magowski, 1990, described from Eocene Baltic amber. These prostigmatan mites are distinguishable by their distinctly long and slender stilt-like legs, and they are associated with aboveground vegetation where they hunt for other small invertebrates. This paper enhances the knowledge of fossil stilt-legged mites. Two new fossil species, N. electrus new species and N. glaesus new species, are described from samples of Baltic amber, and remarks on their morphology and taphonomy are provided. The discovery is complemented with a discussion on morphological singularities (the shape of the prodorsum, the location of setae h1 and h2 in living specimens, and lengths of genual setae), an anomaly of hypertrophied seta (found in the N. glaesus holotype), and some biogeographical issues.


Introduction
Mites of the family Camerobiidae (Acariformes, Prostigmata, Raphignathoidea) are recognizable by an almost round (especially when it comes to specimens mounted on microscope slides), dorsoventrally flattened idiosoma that is suspended on long, slender, stilt-like legs, mostly with long setae, which yield a characteristic appearance (Bolland, 1986. Camerobiids are free-living predators of small invertebrates (e.g., plant-associated mites and crawlers of scale insects [Hemiptera, Coccoidea]). Camerobiids hunt their prey on aboveground vegetation (including tree bark) but have also been found in the litter (Gerson et al., 2003;). In the literature, more than 160 extant species have been described in seven genera: Acamerobia Fan and Walter, 2011;Bisetulobius du Toit, Theron, and Ueckermann, 1998;Camerobia Southcott, 1957;Decaphyllobius Bolland, 1986;Neophyllobius Berlese, 1886;Tillandsobius Bolland, 1986;and Tycherobius Bolland, 1986. Most species are monotypic, and the reason for this is the single, rare occurrence of these mites, which probably do not occur in larger aggregations as adults (Bolland, 1986(Bolland, , 2001. The historical aspect of the developing systematics hypotheses of the family and its constituent genera was summarized by Fan and Walter (2011).
Baltic amber is one of the richest sources of animal remains, which are exceptionally well preserved in the form of threedimensional inclusions. Despite the long history of research on fossil organisms (Koch and Berendt, 1854 published the first study that included mites), new information is continually provided on the structure of the so-called Eocene Amber Forests (Weitschat and Wichard, 2002;Seyfullah et al., 2018). In addition, because of a diverse paleoacarofauna including the presence of minute and weakly sclerotized mites, this Lagerstätte is essential for further paleoacarological studies (Sidorchuk, 2018).
The fossil record of the superfamily Raphignathoidea is scarce and consists of only two described species: Mediolata eocenia Kuznetsov, Khaustov, and Perkovsky, 2010 (Stigmaeidae) from Rovno amber and Neophyllobius succineus Bolland and Magowski, 1990 from Baltic amber (Dunlop et al., 2019). None of the studied inclusions was subjected to appropriate grinding techniques (which were developed later) helpful for studying microarthropods (Sidorchuk, 2013;Sidorchuk and Vorontsov, 2018), and quality pictures of the specimens were not taken (there is only one black-and-white photograph in Kuznetsov et al., 2010).
This work presents descriptions of two new fossil species, Neophyllobius electrus n. sp. and N. glaesus n. sp., which were found in samples of Baltic amber (Fig. 1). These decriptions are accompanied by detailed images, line drawing interpretations, and reconstructions of their habitus. Thus, the paper expands the knowledge about the Camerobiidae mites inhabiting the extinct Eocene ecosystem where resin originated.
Amber preparation.-Amber samples were cut using a handheld cutting tool (Proxxon Micromot 60/E), and then obtained pieces were polished according to methods of preparation described by Sidorchuk (2013), using the tools introduced by Sidorchuk andVorontsov (2016, 2018). Because of cracks along a natural amber fissure and the small size of the preparation, some fragments were embedded in Buehler EpoThin 2 epoxy resin between two round glass coverslips. One sample that had not been embedded in the resin was placed in a test tube filled with aqueous thymol solution. All preparations are labeled following the information given in this paper.
Observations and imaging.-Observations were done with a light compound microscope Nikon Eclipse Ni-U equipped with differential interference contrast (DIC) optics, 10× plan apochromatic dry, and 40× and 60× apochromatic waterimmersion lens objectives. Image stacks were obtained with a Nikon DS-Ri2 microscope camera using Nikon NIS-Elements D imaging software (Nikon Corporation). All images were corrected for light, tone, noise, and sharpness using Adobe Photoshop Lightroom (Adobe Systems). Layered images were obtained by processing the focal planes with Helicon Focus Pro (Helicon Soft Ltd) rendering method A; minor retouch of the final image has been conducted to make some morphological structures visible. Drawings are interpretations of studied inclusions and were made with Adobe Photoshop (Adobe Systems) with the aid of a graphic tablet Wacom Intuos Pro on the basis of obtained pictures. Reconstructions of the species' habitus are based on the studied material, data from the literature, and pictures of living specimens. Original images are available through Figshare data-set collections (see details provided in the Material sections of individual descriptions of species).
Measurements.-All measurements are made and given herein in micrometers (μm), and they are rounded to the nearest integer. Measurements have been made with the aid of Nikon NIS-Elements D (Nikon Corporation) calibrated for used objectives. Due to the nature of preservation, measurements of organs and body parts that are oriented in three-dimensional space should be considered as minimum estimates.
Repositories and institutional abbreviations.-Type specimens examined in this study are deposited in the following institutions: Senckenberg Museum of Natural History Görlitz (SMNG), Görlitz, Germany, and Geological-Paleontological Institute and Museum of the University of Hamburg (GPIH), now CeNak-Centrum für Naturkunde, Hamburg, Germany.
Terminology.-Terminology used here follows that developed in works by F. Grandjean (reviewed by Travé and Vachon, 1975; general terminology combined by van der Hammen, 1980). Its application to Camerobiidae follows Kethley (1990) and Fan and Walter (2011). The left and right sides of the body and its structures correspond to those of the dorsal view. Symbols and abbreviations used in the text are explained in the legends of the corresponding figures. Leg setae were designated with diligence on the basis of three-dimensional specimens. However, the nature of the appendages (long, slender, stilt-like) may cause difficulty in the correct determination of lateral and ventral setae; hence, the chaetotaxy of setae l and v on tibiae should be considered a simplification. Application of Grandjean's system for leg phanerotaxy has been already applied in the recent literature (e.g., Fan and Walter, 2011;Paredes-Leon et al., 2016;Khaustov and Abramov, 2017).
Journal of Paleontology 94(4):696-715 700 smooth genital setae g (seta visible on the right side, only the base of seta visible on the left side). Three bases of pseudanal setae ps1-3 present on the right, but only two bases discernible on the left. Oval breaks in the striation situated in the proximity of anogenital region visible (probably areas of simple cupules ih).
Etymology.-The specific epithet electrus is an adjective derived from the Latin noun electrum in the nominative, which is translated into amber. Zmudzinski-New fossil Neophyllobius spp. from Baltic amber Remarks.-N. electrus n. sp. is morphologically similar to the extant species N. meyerae Bolland, 1991 by the presence of only one midventral seta on each tarsus I-III. However, it differs from this species by having an additional third seta on femora III, probably l ′′ (N. meyerae has only two setae); lack of setae pdx (present in N. meyerae); a slightly different idiosomal pattern, especially in terms of vi-ve distance; and significant differences in length between individual pairs within medial and lateral rows of idiosomal setae (there are only slight differences in N. meyerae). There are also some similarities between the new species and the Recent camerobiid N. panici . These two species have only one midventral seta vs on each tarsus IV, four setae on femur I, three setae on femur II, and significant differences in length ratio between the median setae. N. electrus n. sp. differs from this species by the presence of only one midventral seta on each tarsus I-III (two setae present in N. panici), lack of setae pdx (present in N. panici), having additional third setae on femora III, probably l ′′ (N. panici has only two setae), shorter setae d on genua (in N. panici seta d is at least three times longer than the genu), and idiosomal setae barbed (in N. panici these setae are nodular). By the presence of three setae on femora III, two setae on femora IV, and the longest setae d1 among the idiosomal setae, N. electrus n. sp. is also similar to the other extant species N. trisetosus De Leon, 1958. The new species differs by the presence of only one midventral seta on each tarsus I-IV (N. trisetosus has two midventral setae on each tarsus I-IV) and by the absence of setae pdx (which are present in N. trisetosus).
N. electrus n. sp. is morphologically similar to the fossil species N. glaesus n. sp. described herein. Both species have relatively short dorsal setae d of genua I-IV and the same chaetotaxy formulae of femora 4-3-3-2 and tibiae 9-8-8-7. N. electrus differs from N. glaesus by the presence of fourteen pairs of idiosomal setae (setae pdx present in N. glaesus), ve-sci distance 50-52 μm (29-36 μm in N. succineus), gnathosoma partially covered by the prodorsum (in N. glaesus prodorsum completely covers it), the presence of only one midventral seta vs on each tarsus (N. glaesus has two setae vs on each tarsus), and shorter dorsal setae d on tibiae than lateral l and ventral v setae (N. glaesus has these setae similar in length). A comparison of all three fossil Neophyllobius species from Baltic amber is summarized in Table 1.
The original sample of amber had a natural fissure filled with air that passed through the inclusion. Hence, to preserve and polish the specimen, the sample was separated into two preparations (see Materials). Nevertheless, the mite imprint in the resin is comparable in quality to modern specimens mounted on microscope slides, even thin striae on the legs and the idiosoma, and clavate tarsal solenidia are clearly visible. Residues of the cuticle, internal organs, and probably food remnants form orange-brown artifacts that hinder the visibility of certain structures such as the anogenital area. Left leg I is incomplete (whole tarsus and a half of tibia missing); those parts were probably lost before embedding in the resin. Interestingly, the apotelae of the legs are poorly preserved, so claws as relatively strongly sclerotized rigid structures should be better remained.
Neophyllobius glaesus new species Figures 1.1, 7-13; Table 1 Holotype.-Adult female ( Fig. 1.1) in two polished pieces of amber. One piece contains an almost complete specimen, and it is placed in a tube with a thymol aqueous solution (GPIH 4995a). Another contains the incomplete left tarsus II ( Fig. 12.3) and is embedded in epoxy resin between two round glass coverslips (GPIH 4995b). Type material is deposited in GPIH under collection number GPIH 4995. See Zmudzinski (2020b) for original series of pictures of the specimen.
Journal of Paleontology 94(4):696-715 Etymology.-The specific epithet glaesus is an adjective derived from the Latin noun glaesum in the nominative, which is translated into amber.
Remarks.-N. glaesus n. sp. is morphologically very similar to the Recent species N. hypoleanae . Both species have relatively short dorsal setae d of genua I-III; setae c1 are shorter and d1 longer than interval to setae next behind them; setae bv ′′ (most proximal) of femora I are distinctly shorter than setae d (most distal); epimeral setae 1a-c are different in length; number of setae on femora I-IV are 4-3-3-2, respectively; each tarsus of legs I-IV has two midventral setae vs; dorsal setae d of palpfemur are only slightly longer than lateral setae l ′′ . However, N. glaesus has short dorsal setae d on genua IV (N. hypoleanae has these setae at least two times longer than the length of the genu); distances between setae ve and sci are distinctly greater (in N. hypoleanae bases of ve are situated just in front of sci); setae e1 45 μm long and f1 41 μm are distinctly shorter (these setae in N. hypoleanae are 60 μm long); and setae f1 do not reach level of bases of setae h1 (in N. hypoleanae setae f1 reach behind bases of setae h1).
N. glaesus n. sp. is morphologically similar to the fossil species N. succineus Bolland and Magowski, 1990. Both species have a relatively short dorsal setae d of genua I-IV; fifteen pairs of idiosomal setae (setae pdx present) without significant differences in their lengths; two midventral setae vs on each tarsus; and the dorsal setae d of tibiae similar in length to the lateral l and ventral v setae. The new species differs from N. succineus by the presence of three setae on femora III (N. succineus has only two of them); chaetotaxy formulae of tibiae I-IV is 9-8-8-7 (8-7-7-6 in N. succineus); distinctly shorter idiosomal setae e1 42-44 μm and h1 (28-30 μm) (75 and 64 μm, respectively, in N. succineus); distance ve-sci 29-36 μm (12 μm in N. succineus); and gnathosoma wholly covered by the prodorsum (in N. succineus prodorsum covers it partially). A comparison of all three fossil Neophyllobius species from Baltic amber is summarized in Table 1.
Unlike the inclusion of N. electrus, almost the entire individual was preserved inside the preparation. The cuticle imprint is translucent, especially on the legs and in the middle of the idiosoma. Residues of the cuticle, internal organs, and probably food remains form brown artifacts within the inclusion. Their highest concentration occurs on the edges of the idiosoma forming a dense border. This makes it difficult to interpret some structures, especially on the ventral opisthosoma. The piece containing the tarsus and part of the tibia of left leg II broke off and was embedded in epoxy resin to preserve such minute amber fragment. Left tarsus I had already been cut or polished in the sample.

Discussion
Common remarks for studied inclusions.-The most recent and valid diagnosis of Neophyllobius has been proposed by Fan and Walter (2006). There is little doubt that the fossil specimens belong to this genus. Although the preservation does not reveal an exact number of peritremal loops, there is at least one in each inclusion because some peritremal structures are visible microscopically. Palptrochanters are nude, palpfemora have two setae on each, and each palpgenu has one seta. Idiosoma bears 14 or 15 pairs of setae. Genital valves have one pair of setae g. Anal valves have probably three pairs of pseudanal setae ps1-3. Solenidion ω is present on each basal half of tarsi I and II. Phanerotaxies of legs are also typical, as in Recent representatives of Neophyllobius.
There is noticeable sexual dimorphism in the family Camerobiidae (already present in nymphs, see Bolland, 2001). As in other raphignathoid families, males have a sclerotized aedeagus, and the genital opening is situated terminally or dorsoterminally. In addition, there are two solenidia w on each tibia I-II, and tarsal solenidia ω are enlarged and/or elongated. The lack of these features excludes the possibility that the specimens Zmudzinski-New fossil Neophyllobius spp. from Baltic amber 709 are males. There are also other sex differences such as the length and position of some leg setae, but they are objectively comparable only if there are two sexes available to study. Camerobiid protonymphs are easily distinguished by the absence of setae on the epimera of legs IV and the nude trochanters IV (seta v ′ not developed yet). These setae are present on the studied specimens.  stated for the first time that there are two nymphal stases within the family. However, deutonymphs are more difficult to distinguish from adults without reference material of all stases. Deutonymphs have generally longer setae d on the genua. Specimens of each new fossil species have relatively short genual setae, were found solely (larvae and nymphs are more commonly found in small aggregations), and probably have well-developed genital valves and complete chaetotaxy. Hence, the specimens are considered in this work as adult females, which seems to be the most probable determination of stase and sex. It is important for the reader to be aware of the difficulty of interpreting the fossil material, especially if it is present only in the form of sole individuals of rare mite groups.
Asymmetric hypertrophy of seta h2 in N. glaesus.-Mostly neglected, François Grandjean's works on deviations and their possible evolutionary significance (summarized and extended in Grandjean, 1971Grandjean, , 1972Grandjean, , 1973 echo even with individual discoveries, as is the case with the inclusion of N. glaesus n. sp. According to Grandjean, deviations can be divided into vertitions, which are important in evolutionary terms and reflect the changes that may occur in future generations permanently, and anomalies, which are more rare, random changes that are not of evolutionary significance (Grandjean, 1971). Deviations are most often asymmetrical and include the absence of some idionymous organ (any holotrichous seta is an example of such), its doubling, change of size, or different location. Other scholars have also studied morphological abnormalities, mostly in terms of teratological singularities but also in the context of regulatory genes (e.g., Southcott, 1997;Weigmann, 2010;Bingül et al., 2017). Abnormalities occurring among populations of Tycherobius stramenticola Bolland, 1986 andT. polonicus Bolland, 1986, mostly in the form of asymmetrical absence of setae, were  (Koç and Akyol, 2007). The inclusion of N. glaesus n. sp. has a hypertrophied (i.e., enlarged, thickened) left seta h2 (Fig. 12.4). The right one is similar to other idiosomal setae. There are no species of Neophyllobius with setae h2 distinctly thicker than other idiosomal setae, and this kind of asymmetry has not been reported for Camerobiidae; hence, the observed deviation is more likely to be an anomaly rather than a vertition. It would be interesting to study the microanatomical structure of such an abnormality, whether enlargement and thickening are associated with more intense cuticle deposition, cell proliferation, or all of these factors and whether the abnormality is affected by any individual mutation or is an error in ontogenetic development. Note also that hypertrophic setae occur in both acariform (e.g., Fernandez et al., 2014) and parasitiform mites (e.g., Mašán and Fenďa, 2014) and mostly have some functions in sexual behavior. Hypertrophic setae may have originated at first from rare, random deviations (enlarging, thickening), which having a genetic background, were subject to sexual selection.
Zmudzinski-New fossil Neophyllobius spp. from Baltic amber without highly sclerotized cuticular structures, lose valuable information about the exact shape of their bodies. The flexible cuticle, which is additionally often softened before final preparation and flattened under two microscope slides (for the essential preparation techniques, see Evans, 1992;, gives only an approximate shape of the body. In nature, it is subject to other forces and factors shaping the habitus, for example, through muscle attachments and its compositional structure (Alberti and Coons, 1999). A solid preparation made by the forces of nature, as inclusions in Journal of Paleontology 94(4):696-715 amber can be called, preserves the almost intact appearance of a once-living animal. Most of the studies on Camerobiidae were carried out on the basis of just such flattened individuals mounted on microscopic slides (some recent examples: Bolland, 2001;Akyol, 2013;Zeity and Gowda, 2013;Khanjani et al., 2014). This process generally causes the gnathosoma to be unnaturally situated slightly on the ventral part of the body but usually at the same level as the idiosoma. Scanning electron microscopic techniques were used for the first time by Bolland (1986) and then by Fan and Walter (2006) to image protonymphal Decaphyllobius gersoni Bolland, 1986 and adult Tycherobius stipula Walter and Fan, 2006 among Camerobiidae. Images from their papers clearly show that the prodorsum forms a well-distinguishable part of the anterior idiosoma in the shape of a trapezoid, partially protecting the gnathosoma from above. Khaustov and Abramov (2017) also used a scanning electron microscope, but the entire individual was not imaged. The studied inclusion of N. glaesus n. sp. has a similarly shaped prodorsum at the edges of which are placed vertical setae directed forward, and it almost entirely covers the gnathosoma. The specimen of N. electrus n. sp. has a round prodorsal idiosoma, but it distinctly forms a fold over the gnathosoma. Although no comprehensive studies have been conducted on the behavior of camerobiids, it can be hypothesized that the prodorsum may serve a protective function for the gnathosoma, and its shape may be taxonomically important. This feature dates back to at least the middle Eocene, from which Baltic amber probably originates (Weitschat and Wichard, 2002). Bolland and Magowski (1990) indicated that the terminal situation of setae h1 in N. succineus is rather exceptional because, in extant species, these setae are situated rather on the dorsal side of the idiosoma. In N. electrus the same case can be found-setae h1 are placed marginally terminating the idiosoma. However, in N. glaesus, setae h1 are located clearly on the dorsal surface of the posterior idiosoma at some distance from the end of the body. So it seems that when interpreting this feature, the microscopic preparations of Recent specimens do not distort the true image of the setae situation. It is not excluded that the terminal location of the h1 setae may constitute a plesiomorphic character state in the genus Neophyllobius, or at least this state could have been more common in the Eocene species.
Brittleness of legs in Camerobiidae. -Bolland and Magowski (1990) stated that legs of camerobiids are difficult to manipulate and are prone to breaking off; hence, the complete specimen of N. succineus is a quite curious finding. The studied inclusions have an almost complete set of legs. In N. glaesus the tarsus of left leg I had been polished before the sample reached the author. In N. electrus, a tarsus and a half of the tibia of left leg I are lacking, and this loss most likely happened before the mite was trapped in leaking resin. Through these observations, the following questions arise. Since the legs of camerobiids are long and prone to breaking, is there a mechanism to regenerate lost parts by adding tissues after molting? Is there a mechanism for regeneration in adults, and if not, how does limb loss affect survival and thus the fitness of individuals? It seems that the ability of limb regeneration among Prostigmata is the weakest and with the highest mortality rate compared with other higher groups of mites (Rockett and Woodring, 1972) and takes place during molting. New, more-comprehensive studies of the regeneration capacity carried out on different representatives of prostigmatan mites may bring novel discoveries.
Lengths of genual setae d.-A curious feature among the representatives of the genus Neophyllobius that has never been discussed is a pattern of lengths of dorsal genual setae d. These setae may be short, with length equal to or slightly longer (two or three times) than that of the genu, or they may be very long, with lengths exceeding the combined length of the genu and the following tibia. Intermediate forms are probably present. However, we require more detailed studies and measurements of this character issuing precise criteria (e.g., setae reaching to half of the tibiae are common among species). N. bequartiodendri , N. texanus McGregor, 1950, and N. trisetosus De Leon, 1958 are examples of species with short setae d on each genu I-IV. N. euonymi Bolland and Ripka, 2000, N. curtipilis, De Leon, 1958, and N. saxatilis Halbert, 1923 have setae d short on genua I-III but distinctly long on genua IV. N. farrieri De Leon, 1958, N. fissus De Leon, 1967, and N. niloticus Bolland, 1991 all have setae d on genua I-IV very long. N. armenica N. sycomorus Zaher andGomaa, 1979 have the setae d long on genua I, III, IV but short on genua II. In some other genera of Camerobiidae-Bisetulobius, Camerobia, Decaphyllobius, and Tillandsobiusthese setae are always short, that is, the same length as the genu or slightly longer (Bolland, 1986). However, in Tycherobius, there may also be a different pattern of setae length, and Acamerobia has some setae d distinctly longer Walter 2006, 2011). Both N. electrus n. sp. and N. glaesus n. sp. have short dorsal setae of each genu I-IV. It is noticeable that the same pattern is present in N. succineus; hence, this feature is common for all known fossil Camerobiidae. It may be a plesiomorphic state of that characteristic, but a sample error is also likely, so the next findings may or may not falsify this hypothesis. If these setae have begun to lengthen in the Cenozoic era, determining the primary cause and function of the final product could be an interesting task. Dorsal setae d are simple setae, not morphologically distinct from other leg setae, so a more probable function of them is sensory or/and protective rather than chemosensory. Minute setae κ of genua I-II, which are probably present in all species but indiscernible in fossils, may be chemostimuli-responsive.
Neophyllobius, the most numerous and the oldest genus.-Mites of the genus Neophyllobius have been found on all continents except Antarctica . Their worldwide distribution can be due to their exceptional dispersal abilities or their long evolutionary history (camerobiids are slow-moving mites, and no dispersal forms or behaviors have been observed). The three known inclusions of Camerobiidae are evidence that the characteristics of the genus Neophyllobius were well developed in the Eocene, and the degree of morphological difference between the fossil species Zmudzinski-New fossil Neophyllobius spp. from Baltic amber 713