A new Paraleius species (Acari, Oribatida, Scheloribatidae) associated with bark beetles (Curculionidae, Scolytinae) in Canada

Abstract Bark beetles (Scolytinae) are hosts to a broad diversity of mites (Acari), including several genera of Oribatida (Sarcoptiformes). Of these, Paraleius (Scheloribatidae) species are the most frequently collected oribatid mites associated with bark beetles. A new species was discovered while surveying the acarofauna of bark beetles in Eastern Canada and is described as Paraleius leahae sp. n. (Oribatida, Scheloribatidae). This species was collected from two host beetle species, Hylastes porculus Erickson and Dendroctonus valens LeConte, in Ontario, New Brunswick and Nova Scotia. The genus Paraleius is rediagnosed, Metaleius is considered a synonym of Paraleius, and the proposed synonymy of Paraleius with Siculobata is rejected. The three known species are Paraleius leontonycha (Berlese), P. leahae sp. n., and P. strenzkei (Travé), comb. n. The barcode region of cytochrome oxidase subunit I (COI) was amplified from P. leahae sp. n.


Introduction
A broad assemblage of wood-burrowing beetles (Cerambycidae, Buprestidae, Scolytinae), and associated mites, nematodes, and fungi reside under the bark of dead, dying or living trees. Bark beetles (Curculionidae, Scolytinae) are a diverse group of wood-borers that feed and mate in the cambium or xylem of numerous tree species worldwide (Wood 1982). Mites are among one of the most diverse and common associates of scolytines, and in temperate forests some bark beetle species are associated with 15-20 mite species (Lindquist 1969).
Oribatid mites (Acari, Oribatida) dwell primarily in soil or forest litter, though many are found in arboreal habitats and a few occur in aquatic systems (Norton and Behan-Pelletier 2009). Several genera of oribatid mites are also found under tree bark in scolytine galleries, or dispersing phoretically on bark beetles (Moser and Roton 1971). Phoresy is relatively uncommon in oribatid mites, and typically phoretic species are not host specific and the association with their host appears to be passive (Norton 1980). Paraleius leontonycha (Berlese, 1910) (Scheloribatidae) is the most frequently encountered oribatid species on bark beetles, although it does not occur in high abundance or prevalence (Knee et al. 2013). Paraleius leontonycha is a broad host generalist with a Holarctic distribution, collected from 17 scolytine species and found in the galleries of 10 other bark beetle species (Knee et al. 2013, Ahadiyat andAkrami 2015). A recent survey of the mesostigmatic and oribatid mite fauna of bark beetles in eastern Ontario (Knee et al. 2013) uncovered a new species of the monotypic genus Paraleius Travé, 1960 associated with two scolytine species, Hylastes porculus and Dendroctonus valens. Herein, I propose and describe Paraleius leahae sp. n., including the barcode region of COI. I also provide a revised generic diagnosis for Paraleius, and resolve some taxonomic issues surrounding Paraleius and closely related genera.

Sampling and identifications
Bark beetle specimens collected with Lindgren funnel traps in eastern Ontario by Knee et al. (2013), and Eastern Canada by the Canadian Food Inspection Agency (CFIA) staff as part of the Invasive Alien Species Monitoring program, were examined for associated mites. Scolytines were identified to species using a dissecting microscope and keys from Bright (1976). The presence, abundance, and attachment location of oribatid mites was recorded. All mites were collected and preserved in 95% ethanol for later identification and/or molecular analysis. Specimens used for illustrations were mounted in Hoyer's medium on temporary cavity slides. Permanent slide-mounted mites were cleared in 85% lactic acid, mounted in polyvinyl alcohol medium (6371A, BioQuip Products, Rancho Dominguez, California, United States of America), and cured on a slide warmer at 40ºC for 3-4 days.
Oribatid collections at the Canadian National Collection of Insects, Arachnids, and Nematodes (CNC), State University of New York College of Environmental Science and Forestry (SUNY-ESF), and John Moser's collection at the United States Department of Agriculture (USDA) in Pineville, Louisiana were examined for Paraleius specimens.
Slide-mounted specimens were examined using a Leica DM2500 compound microscope and Leica ICC550 HD camera, with differential interference contrast illumination (DIC). Initial drawings of mites were made with pencil on paper using a camera lucida. These were later merged in Adobe Photoshop CS5 and redrawn in Adobe Illustrator CS5 using an Intuos 3 Graphics Tablet from WACOM Co., Ltd. (Saitama, Japan).
Morphological terminology used in this study follows that developed by F. Grandjean (see Travé andVachon 1975 for references andNorton andBehan-Pelletier 2009 for overview). Notogastral setation follows the unideficient nomenclature detailed by R.A. Norton in Balogh and Balogh (1988). The following conventions for measurements are used: prodorsal setae, measured on permanent slide-mounted specimens (ro, rostral seta; le, lamellar seta; in, interlamellar seta; ex, exobothridial seta; bo, bothridial seta); total length, measured dorsally from tip of rostrum to posterior margin of the notogaster on specimens in cavity slides; total width, measured at widest part of the notogaster on specimens in cavity slides. Total length and width were measured only for the few mites that were stored in ethanol and not mounted on permanent slides, all other measurements were performed on five to seven slide mounted mites. Leg setation is presented as the number of setae per segment (including the famulus on tarsus I), with solenidial counts in parentheses, in the following order: trochanter-femur-genutibia-tarsus. All measurements are in micrometres (µm); lengths presented with mean followed by the range in parenthesis. Type specimens are deposited in the Canadian National Collection of Insects, Arachnids, and Nematodes, at Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.

Molecular methods
Genomic DNA was extracted from whole specimens for 24 hours using a DNeasy Tissue kit (Qiagen, Inc., Santa Clara, California, United States of America). Following extraction, mites were removed from the extraction buffer, vouchers were-slide mounted, and genomic DNA was purified following the DNeasy Tissue kit protocol. PCR amplifications were performed in a total volume of 25 µl, with 14.7 µl ddH2O, 2.5 µl 10× ExTaq buffer, 0.65 µl 25 mM MgCl2, 1.0 µl of each 10 µM primer, 2.0 µl 10 mM dNTPs, 0.15 µl ExTaq DNA polymerase, and 3 µl genomic DNA template. Primer pairs PHF1 (5'-CWACAAAYCAYAAAGATATTGG-3') and PHR1 (5'-TAHACYTCHGGRTGVCCRAAAAAYCA-3') were used to amplify a 641 bp fragment of the 5'-end of COI. PCR amplification was performed on an Eppendorf ep Gradient S Mastercycler (Eppendorf AG, Hamburg, Germany), using the following protocol: initial denaturation cycle at 94 °C for 3 min, followed by 45 cycles of 94 °C for 45 s, primer annealing at 40 °C for 45 s, 72 °C for 1 min, and a final extension at 72 °C for 5 min. Amplified products and negative controls were visualized on 1% agarose electrophoresis gels, and purified using pre-cast E-Gel CloneWell 0.8% SYBR Safe agarose gels (Invitrogen, Carlsbad, California, United States of America).
Sequencing reactions followed the protocol of Knee et al. (2012), and sequencing was performed at the Agriculture and Agri-Food Canada, Ottawa Research and Development Centre, Core Sequencing Facility (Ottawa, Ontario, Canada). Sequence chromatograms were edited and contiguous sequences were assembled using Sequencher v5.3 (Gene Codes Corp., Ann Arbor, Michigan, United States of America). Sequence for Paraleius leahae sp. n. has been submitted to GenBank (KY402259).
Remarks. Travé (1960) described Paraleius as closely resembling Hemileius Berlese, 1916 with the distinction of the following characters: rostrum extended medially, forming narrow point; bothridial seta capitate; sublamella absent; seta ex medium sized and barbed; Ah expressed as saccule; heterodactyl claws with pronounced central claw; solenidia of tibiae III and IV microcephalic. Travé's diagnosis lacked a few additional characters which have been included in the revised diagnosis above: notogaster anterior margin convex, bothridium inserted close to the lamella, numerous spicules on bothridium. To accommodate the new species herein described the description for three character states from Travé's original diagnosis were modified: bothridial seta shape, pretarsal dactyly, and solenidia of tibiae III and IV microcephalic or not.
While Weigmann (1969) treated Paraleius, Metaleius and Siculobata as distinct genera, he later (2006) considered Paraleius and Metaleius to be junior synonyms of Siculobata based on a shared lamellar complex. However, this complex is not identical: Siculobata has a rudimentary sublamella, while Paraleius and Metaleius lack a sublamella. The synonymization of these genera also overlooks several other distinct character states shared by Paraleius and Metaleius that Siculobata does not possess. These include: rostrum with narrow medial point, anterior margin of notogaster convex, seta ex medium sized and barbed, Ah expressed as saccule, and bothridial seta inserted dorsolaterally close to lamella. Fredes and Martinez (2013) did not follow Weigmann's (2006) proposed synonymy and provided a diagnosis for Siculobata sensu stricto that excludes Paraleius and Metaleius. Based on their concepts and on the aforementioned shared character states, I also reject the synonymization of Paraleius with Siculobata, but synonymize Metaleius and Paraleius. Each of the latter genera has been monotypic to this point, so the revised diagnosis for Paraleius is based on Paraleius leontonycha, Paraleius leahae sp. n., and Paraleius strenzkei (Travé, 1960), comb. n.
In his checklist of the world oribatid mite fauna, Subías (2004) placed Wallworkiella Hammer, 1979 as a subgenus of Paraleius, with the single species Paraleius (Wallworkiella) nasalis (Hammer, 1979). No explanation or justification was provided by Subías. In an unpublished online update (Subías 2016), possibly following Weigmann's classification, he instead placed Wallworkiella as a subgenus of Siculobata. However, Wallworkiella differs from Paraleius by having five pairs of notogastral sacculi, homo-tridactylous tarsi, and inflated tarsal pulvilli. Additionally, Wallworkiella does not belong to Siculobata based upon the concept of Fredes and Martinez (2013). Clearly, the generic and species level relationships of Scheloribatidae require further research and revisions, but the demotion of Wallworkiella to subgeneric rank under either Paraleius or Siculobata is unsupported. Integument. Body cuticle red-brown. Notogastral surface and venter appear smooth, but with fine granulate structure at higher magnification (100x). Small microtubercles on epimeral surface (Fig. 4). Small microtubercles medially on subcapitulum between h setae.

Leg Trochanter Femur
Genetics. There are no other sequences of Paraleius or Metaleius on GenBank; however, GenBank blast searches of the COI sequence (KY402259) of P. leahae sp. n. generally matches that of other poronotic brachypyline oribatid mites. Further analysis was not performed.
According to Grandjean (1959) microcephalic solenidia are found only in arboricolous or saxicolous species. Paraleius leontonycha, P. leahae sp. n. and P. strenzkei are arboricolous species, the former has microcephalic solenidia and the latter two species lack this feature. The tips of solenidia on tibiae III and IV are delicate and prone to breakage, so it is possible that they are microcephalic in P. leahae; however, I examined more than 100 specimens without finding microcephalic tips.

Distribution and biology
Paraleius leontonycha and P. leahae are quite similar morphologically, and it is possible that the latter has been misidentified as the former in the past. These two species are also ecologically similar in being corticolous and phoretic on bark beetles. The feeding biology of P. leahae and P. leontonycha is poorly understood, but fungal hyphae have been observed in the gut of slide mounted specimens of both species.
Paraleius leontonycha is the most commonly collected and widely distributed oribatid phoretic on bark beetles, however this species occurs infrequently and in low abundance (Norton 1980, Knee et al. 2013. Paraleius leontonycha has a Holarctic distribution; whereas, P. leahae has only been collected in Eastern Canada (Ontario, New Brunswick and Nova Scotia). Paraleius sp. and P. leontonycha collections at the CNC, SUNY-ESF, and the USDA were examined for P. leahae specimens. These collections contained material from across Canada (AB, BC, NB, NFLD, ON, QC), parts of the United States of America (AK, AZ, CA, LA, TX, UT, WI), parts of Europe (Croatia, Germany, Spain, Sweden, Switzerland), Mexico, Honduras, and Japan. All of the material examined from these collections represented P. leontonycha; no misidentified P. leahae were uncovered.
Typically the association between oribatid mites and their scolytine hosts is considered to be passive and with low host specificity (Norton 1980). Paraleius leontonycha is a host generalist, collected from 17 species of bark beetles (Knee et al. 2013, Ahadiyat andAkrami 2015). In contrast, P. leahae is a host specialist, collected from only two bark beetle species, Hylastes porculus and Dendroctonus valens. These two host species are not closely related species, but they are ecologically similar, as both species live in the stumps and roots of dead or dying conifers (Wood 1982). Multiple bark beetle species often occupy the same tree concurrently and occasionally their galleries cross, thus providing mites with an opportunity to transfer host species (Moser et al. 1971). Paraleius leahae shows a marked preference for only these two bark beetle species despite opportunities to switch host species. Hylastes porculus and D. valens are hosts to many species of mites; 16 other species of mites were collected from each of these host species in eastern Ontario including P. leontonycha (Knee et al. 2013). Paraleius leahae was the most abundant species collected out of the 33 species of mites collected from bark beetles in eastern Ontario using general lures (α-pinene and 95% ethanol) and Lindgren funnel traps (Knee et al. 2013). Paraleius leahae challenges the assumptions that bark beetle associated oribatid mites are uncommon and are not host specific.  (Travé, 1960)