Sheltered life beneath elytra: three new species of Eutarsopolipus (Acari, Heterostigmatina, Podapolipidae) parasitizing Australian ground beetles

In this study, we conducted a summer sampling of carabid beetles in eastern Australia to identify their associated parasitic mites. Here, we describe three new species of the genus Eutarsopolipus from under the elytra (forewings) of three native carabid species (Coleoptera: Carabidae): Eutarsopolipus paryavae n. sp. (pterostichi group) from Geoscaptus laevissimus Chaudoir; Eutarsopolipus pulcher n. sp. (leytei group) from Gnathaphanus pulcher (Dejean); and Eutarsopolipus chlaenii n. sp. (myzus group) from Chlaenius flaviguttatus Macleay. We further provide an identification key of the world species of pterostichi and leytei species groups as well as closely related species of the myzus group possessing similar characters including short cheliceral stylets. The significant diversity of Eutarsopolipus recovered here suggests that the current knowledge about Australian podapolipid mites (specially Eutarsopolipus) is still in its infancy and deserves further study.


Introduction
Beetles are among the most successful animals on the planet, accounting for about 25% of described species [10,46]. Their success is partly attributed to their modified, sclerotized forewings, known as elytra, that protect their body against physical damage, desiccation, predation and thermal stress, enabling them to occupy a wide range of ecological niches [33,48]. The subelytral space serves as a suitable microhabitat for a broad range of organisms such as mites, pseudoscorpions and nematodes that occupy this niche temporarily or permanently [6,36,37]. Some mites have evolved to be permanent ectoparasites in the subelytral spaces of beetles, imbibing beetle haemolymph using piercing stylets [2,7]. This parasitic association sometimes occurs in one part of a mite's life cycle. For example, in Parasitengona (Acariformes: Prostigmata), larvae are parasites of many insects and are sometimes found under the elytra of terrestrial and aquatic beetles, while the nymphs and adults are free-living predators of immature stages of small arthropods [51,52]. However, some taxa represent evolutionary transitions from phoresy towards parasitism, as in a few canestrinioid mites (Astigmata) in which deutonymphs remain phoretic on the thoracic venter of some carabid beetles, whereas the other stages (feeding stages) are subelytral parasites of the same hosts [15,49]. Some other groups are real parasites with *Corresponding author: a.katlav@westernsydney.edu.au a Dr. Hamidreza Hajiqanbar died on October 18, 2021, at the age of 48, when this paper was in press. This article is dedicated to the memory of Dr. Hamidreza Hajiqanbar, who cherished research as his life joy and made a major contribution to the world's insect-associated mites.
The cohort Heterostigmatina (Acariformes: Prostigmata) is a large group of morphologically diverse mites, among which numerous species are associated with arthropods [49]. Some species are subelytral symbionts of various beetles with their associations varying from facultative or obligate phoresy to parasitoidism or parasitism [25,28,30,31]. Several species are potential biocontrol agents against pest beetles. For example, the species of the families Pyemotidae and Acarophenacidae are known as insect ectoparasitoids, with the former mostly attacking juvenile stages of bark beetles and stored-product beetles and the latter egg ectoparasitoids of various beetle families [8,25,29,50].
All members of the family Podapolipidae are specialized obligate external (and rarely internal) parasites of various insects [18], among which at least 20 genera are subelytral ectoparasites of different beetle families, mainly Carabidae, Chrysomelidae, Coccinellidae, and Scarabaeidae [21,23,38,45]. These mites are sexually transmitted, i.e. the motile stages of the mite (larvae or adult females) move from one host individual to another during copulation [17]. Parasitisation with these mites can negatively affect host fitness. For example, in some ladybirds, individuals parasitised with Coccipolipus suffer lower fecundity and egg viability [17] and sometimes reduced longevity [40]. Beyond this, these mites can modify host sexual and behavioural traits to boost their transmission success among individual hosts [1]. For example, in the milk weed leaf beetle, males parasitized by Chrysomelobia tend to more frequently contact other males, and are more successful in mating competition compared to unparasitised males; and this facilitates the mite's higher transmission rate [1].
Four genera of Podapolipidae are exclusively associated with carabid beetles: Dorsipes (22 species), Eutarsopolipus (99 species), Ovacarus (3 species) and Regenpolipus (5 species) [11,13,19,26,27,44]. Apart from Ovacarus, which is an endoparasite of the reproductive tracts of some carabids, the rest are subelytral ectoparasites [11]. Species of Eutarsopolipus are versatile in morphology and are currently grouped into ten species groups [42]. Most of the species are specific to a single host species. However, a few parasitize more than one host species [41] or more rarely more than one genus [26], yet the possibility of them being cryptic species remains untested. More interestingly, in some cases more than one species can parasitize one host species [42] and sometimes they are specialized to different microhabitats such as the elytral cavity, on hindwings or on the dorsal abdomen of their host [39].
Australia is anticipated to harbour rich Eutarsopolipus fauna given its large diversity of carabid beetles [5]. This is inferred from small sampling efforts that have recently been conducted in some regions, and yet that discovered a considerable number of new species [31,[41][42][43][44]. Here, we describe three new species of Eutarsopolipus belonging to three different species groups (leytei, myzus, pterostichi) from three native Australian carabid beetles, raising the total number of Australian Eutarsopolipus to 30 species. All these species were recovered following a minimal sampling effort at one site, again corroborating the hypothesis that Australia is home to diverse podapolipid fauna awaiting discovery.

Materials and methods
Carabid host beetles were collected at night on the ground, near an outdoor LED solar light lamp in Richmond, New South Wales, in February 2020. The subelytral area of the beetles (preserved in 75-80% ethanol) was subsequently examined for mite infestation. Mite specimens were cleared in a mixture of Nesbitt's fluid and a small amount of glycerine slide mounted in Hoyer's medium. Mite morphology was studied using a light microscope (Olympus BX51) equipped with phase contrast illumination. Mites from Queensland specimens of the carabid host Gnathaphanus pulcher were removed from dried beetles as described in Seeman [42] and examined using a Nikon 80i microscope equipped with differential interference contrast. All measurements are given in micrometres for holotypes and the range of measurements for five selected paratypes (in parentheses), if available. Distances between setae were measured from the base of one seta to the other; setae with their acetabulum remnant only were categorised as vestigial setae and those with their setae not extending past the acetabulum as microsetae (m). Terminology and setal notation were adapted from Lindquist [32]. The species group assignment follows that of Seeman [42]. Host beetles were all identified with the help of Geoff Monteith. Species group: pterostichi -Key characters of the group based on adult female: stigmata and tracheae absent; genua II-III without setae [42].     Deposition of type material: The holotype, one adult female, 2 male and 2 larval female paratypes are deposited at ANIC (ANIC 52-003953-58). 1 adult female, 2 males and 1 larval female paratypes are deposited at QM (QMS 117000-04). The remaining paratypes (TMU SP-20200211, 1-3), 10 non-type males and the host beetle specimen are deposited at AC-DE-TMU.

Abbreviations
Etymology: The new species is named after the first author's mother, Shams Paryav, the collector of the host beetle samples, in gratitude of her immense engagement in material collections.

Differential diagnosis
Within the pterostichi species group, the new species is most similar to E. fischeri Husband, 1998 and E. teteri Husband & Husband, 2009 in having ambulacra II and III with a pair of claws each and ambulacra I with one claw and femur I with two setae. However, it differs from both species in having cheliceral stylets longer than 60 (vs. shorter than 40 in both species), setae h 1 9-12 (absent in E. teteri and microsetae in E. fischeri) and seta k on tibia I absent (seta k on tibia I present in both species). The setal counts alone mask further differences. In E. paryavae and E. fisheri, the setae on femur I are the tiny setae d and l 0 , but in E. teteri seta l 0 is absent and v 00 is present. Another important difference is the absence of a solenidion on tarsus II, which is present in E. teteri and probably present in E. fischeri (present in male and larva, absent or obscured in females). All the important characters among these three species are compared for all life stages in Table 1 and a key to the world species of the pterostichi group of Eutarsopolipus (based on adult females) is presented in Figure 4.
Species group: leytei -Key characters of the group based on adult females: stigmata and tracheae present; ambulacral claws II-III present; genu II-III with setae [42]. Etymology: The new species name "pulcher" is adopted after the species name of the carabid host beetle G. pulcher meaning "beautiful" in Latin that is associated with the beautiful metallic colouration patterns of elytra in this beetle. Furthermore, this epithet has a proper relevance to the beautiful trifurcate setae u 0 on tarsi II-III in adult females of the new mite species.

Differential diagnosis
This new species is unique in Eutarsopolipus by having trifurcate setae u 0 on tarsi II-III. However, among species with simple claws on legs I (unlike E. biuncatus Seeman, 2021 and E. janus Seeman, 2021 with bifurcate claws on legs I), it is most similar to E. leytei Husband & Raros, 1989 with femur I seta lʹ very short, not reaching genual base in adult females; but it is readily distinguishable from this species by longer setae v 1 11-14 (m-5 in E. leytei) and shorter cheliceral stylets being at most 51 in E. pulcher n. sp. vs. 68 in E. leytei.
The new species further differs from E. dastychi with setae v 1 longer than ch and setae c 1 , c 2 , d and f shorter than 8 in adult females (vs. setae v 1 shorter than ch and setae c 1 , c 2 , d and f longer than 15 in adult females of E. dastychi). The male of E. pulcher n. sp. resembles that of E. orpheus with all ventral and dorsal setae (except sc 2 ) being microsetae, but it differs from E. orpheus with setae ch longer than 8 (ch microsetae in male of E. orpheus). The larval female of E. pulcher n. sp. is similar to E. orpheus with h 1 shorter than 70 and h 2 shorter than 2, but it is readily distinguishable from E. pulcher n. sp. by shorter setae sc 1 , sc 2 , c 1 , c 2 , d, 3a and 3b ( Table 2). All the important characters among the species of leytei group are compared for all life stages (excluding E. leytei with unknown male) in Table 2 and keys to the world species (based on adult females) are presented in Figure 9.
Etymology: The species epithet "chlaenii" refers to the generic name of the carabid host beetle Chlaenius flaviguttatus.
Authorship: Note that the authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of International Code of Zoological Nomenclature [24].

Differential diagnosis
The new species belongs to a subgroup of the myzus species group that shares a combination of the following characters in adult females: ambulacrum I claw well-developed, idiosoma without lateral bulges or posteriorly without wrinkled lobes, shield C not divided, femur I seta l 0 developed (not microseta), and cheliceral stylets less than 35 lm long [13]   in E. oconnori). All the important characters among these five species of the myzus species group are compared for all life stages (excluding E. chlaenii n. sp. with unknown male) in Table 3. Among adult females of the myzus species group with a strong claw on ambulacrum I, lateral bulges or posterior wrinkled lobes and entire shield C, six species have short cheliceral stylets (less than 35 lm long). The key to this subgroup is presented in Figure 12.

Discussion
Among all Eutarsopolipus, leytei is apparently the most primitive group that represents the putative plesiomorphies of a well-developed tracheal system as well as retention of genual I-III setae (2-1-1) and all femoral I setae (3 setae). Conversely, the pterostichi group with a missing tracheal system and genual I-III setae (0-0-0), reduction of femoral I setation (2 setae) and sometimes reduction/absence of ambulacral claws [as in E. echinatus, 43] may be relatively more derivative than the other Australian Eutarsopolipus [31,43]. However, the myzus group, possessing a combination of plesiomorphies (well-developed tracheal system and ambulacral claws) and some apomorphies [reduction of femoral I setation (2 setae) and absence of genual I-III setae (0-0-0)], may hold an intermediate position. It is surprising that in our study such considerable species diversity was detected in a single location following a minimal sampling effort preformed across fewer than three weeks. This may substantiate the previously held notion that Australia exhibits diverse Eutarsopolipus fauna with a wide gradient of morphological variations [42]. Despite a few sporadic studies on Australian Eutarsopolipus, six out of the ten known species groups that exist across the world (including ochoai, megacheli and secundus) have so far been recorded from Australia ( [31,42,44], present study). However, the rich diversity of Australian carabid beetles may posit the idea that the current knowledge about their associated Eutarsopolipus mites is still in its infancy; therefore, more extensive faunistic studies in different regions could potentially lead to the discovery of enormous diversity in Eutarsopolipus.
With the description of E. chlaenii, this study reports the myzus group for the first time in Australia, thereby extending its distribution to Oceania, and beyond the previously recorded Holarctic, Afrotropical and Oriental realms [12,22]. About half of the species of this group (13/25) are parasites of carabids of the genus Chlaenius Bonelli [12,22]. Furthermore, the finding of E. pulcher n. sp. from G. pulcher is the second record of the leytei group from a native carabid of the genus Gnathaphanus Macleay, 1825 (tribe Harpalini). Recently, a study in the same location found another species, E. orpheus from under the elytra of Gnathaphanus melbournensis (Castelnau, 1867), probably suggesting more specific association of the leytei group with carabids of Gnathaphanus. This carabid genus is apparently native to the Australasian and Oriental regions and represents more than 15 species in Australia [4] with G. pulcher and G. melbournensis being highly abundant in eastern Australia [3]. It is interesting, however, that the only Palearctic representative of the leytei group, E. dastychi, was found from Calathus of the carabid tribe Sphodrini [20] which is phylogenetically diverged from the carabid tribe Harpalini. This kind of counterintuitive host range is even more profound among the myzus and pterostichi groups, both of which are associated with carabids of the two distantly related subfamilies, Harpalinae and Scaritinae [26,42], suggesting that several episodes of host switching may have contributed to the evolution of their host associations.
Carabid beetles are generalist predators that feed on a variety of small invertebrates including important agricultural pests and thus serve as important biocontrol agents [34]. However, their ecological interactions are often hard to predict [9]. It is unknown how the parasitic role of Eutarsopolipus mites can shape the ecology and evolution of carabids, yet incorporation of such information may contribute to models predicting interaction networks of carabids for future biocontrol programs.