Combined description (morphology with DNA barcode data) of a new quill mite Torotrogla paenae n. sp. (Acariformes: Syringophilidae) parasitising the Kalahari scrub-robin Cercotrichas paena (Smith) (Passeriformes: Muscicapidae) in Namibia

A new quill mite species Torotrogla paenae n. sp. (Acariformes: Syringophilidae) parasitising the Kalahari scrub-robin Cercotrichas paena (Smith) (Passeriformes: Muscicapidae) in Namibia is described based on the external morphology and DNA barcode data (the mitochondrial cytochrome c oxidase subunit 1 sequences, cox1). Females of T. paenae n. sp. morphologically differ from the most similar species T. lusciniae Skoracki, 2004 by the total body length (780–830 vs 645–715 µm in T. lusciniae) and the presence of hysteronotal shields (vs absence), apunctate propodonotal and pygidial shields (vs punctate), apunctate coxal fields (vs punctate), the fan-like setae p’ and p” of legs III–IV provided with c.10 tines (vs 14–15) and the length of setae si (140–180 vs 190–210 µm) and se (160–185 vs 210–225 µm). The male of T. paenae n. sp. morphologically differs from T. lusciniae by the lateral branch of peritremes composed of 4 chambers (vs 7–8 chambers) and lengths of setae ve (45 vs 70–75 µm) and se (120 vs 165 µm).


Introduction
Quill mites (Acariformes: Syringophilidae) are tiny, permanent bird ectoparasites inhabiting quills of feathers. Syringopilids spend there all their life-cycle and also feed by piercing quill walls with dagger-like chelicerae and suck liquid components of surrounding soft tissue (Casto, 1976;Skoracki, 2011). The family is presently represented by 338 species belonging to 60 genera, which were recorded from 478 bird species from 93 families and 24 orders (Glowska et al., 2015a;Zmudzinski & Skoracki, 2017). The genus Torotrogla Kethley, 1970 is widespread globally with proven occurrence in the Palaearctic, Nearctic, Neotropical and Saharo-Arabian zoogeographical regions (Zmudzinski & Skoracki, 2017). Until now, Torotrogla has been represented by 18 species associated with several groups of passeriform hosts (35 species and 14 families) (Glowska et al., 2015a, b;Skoracki et al., 2016;Zmudzinski & Skoracki, 2017). Currently, this genus is in the field of molecular research interests, because of the phenotypic plasticity observed in T. merulae and T. rubeculi (see Glowska et al., 2013) and due to its species being hosts for endosymbiotic bacteria of the genus Wolbachia Hertig & Burt 1924 (see Glowska et al., 2015b). All this indicates that basic systematic studies on quill mites, whenever possible, should be extended with DNA data. As it has been shown, molecular tools may be successfully used for syringophilid species descriptions (Glowska et al., 2012a, b), tests of host specificity (Glowska et al., 2016), delimitation of species boundaries (Glowska et al., 2013(Glowska et al., , 2014 and detecting cryptic species (EG, unpublished data).
In this paper, a new quill mite species Torotrogla paenae n. sp. parasitising the Kalahari scrub-robin Cercotrichas paena (Smith) (Passeriformes: Muscicapidae) in Namibia is described based on morphological and DNA barcode data. Cercotrichas paena is a new host species for the family Syringophilidae and the first record of the genus Torotrogla in the Afrotropical realm.

Animal material and morphological analysis
Mite material used in the study was acquired from the collection of feathers deposited in the Smithsonian Institution, National Museum of Natural History, Department of Vertebrate Zoology, Division of Birds, Washington, DC, USA (USNM) (September 2014). Bird specimen was collected in Namibia, 2009. Drawings were made with an Olympus BH2 microscope with differential interference contrast (DIC) optics and a camera lucida. All measurements are in micrometres. The idiosomal setation follows Grandjean (1939) with modifications adapted for Prostigmata by Kethley (1990). The system of nomenclature for leg chaetotaxy follows that proposed by Grandjean (1944). The application of these chaetotaxy schemes to Syringophilidae was recently provided by Bochkov et al. (2008) with changes by Skoracki (2011). Latin and common names of the bird species follow Clements et al. (2017).
Material depositories and abbreviations: AMU, Adam Mickiewicz University, Poznań, Poland; USNM, Smithsonian Institution, National Museum of Natural History, Washington, DC, USA. The voucher slides and corresponding DNA samples are deposited in the collection of the AMU under the identification numbers as indicated below. The sequences were deposited in the GenBank database under the accession numbers MG948551 (cox1) and MG952940 (D1 region of the 28S rRNA gene).

Molecular data and analysis
Total genomic DNA was extracted from single specimens using DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) as described by Dabert et al. (2008). We used sequence data for the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the D1 region of the nuclear 28S rRNA gene. cox1 was amplified by PCR with degenerate primers Aseq01F (5 0 -GGA ACR ATA TAY TTT ATT TTT AGA-3 0 ) and Aseq03R (5 0 -GGA TCT CCW CCT CCW GAT GGA TT-3 0 ) (Glowska et al., 2014). The D1 region of the 28S rRNA gene was amplified using the primer pair 28SF0001 (5 0 -ACC CVC YNA ATT TAA GCA TAT-3 0 ) (Mironov et al., 2012) and 28SR0450 (5'-TTT GCA ACT TTC CCT CAC GG-3') (newly designed). PCR amplifications were carried out in 10 ll reaction volumes containing 5 ll of Type-it Microsatellite Kit (Qiagen), 0.5 lM of each primer, and 4 ll of DNA template using a thermocycling profile of one cycle of 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 1 min at 50°C, 1 min at 72°C, with a final step of 5 min at 72°C. After amplification PCR products were two-fold diluted with water, and 5 ll of the sample was analyzed by electrophoresis on a 1.0% agarose gel. Samples containing visible bands were purified with thermosensitive Exonuclease I and FastAP Alkaline Phosphatase (Fermentas, Thermo Scientific, Göteborg, Sweden). The amplicons were sequenced in one direction using PCR forward primers. Sequencing was performed with BigDye Terminator v3.1 on an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence chromatograms were checked for accuracy and edited using FinchTV 1.3.1 (Geospiza, Inc.) and manually aligned in GeneDoc v.2.7.000 (Nicholas & Nicholas, 1997).

DNA barcodes
The cox1 sequence data were generated from three females of Torotrogla paenae n. sp. All specimens shared the same cox1 haplotype (GenBank: MG948551). For one specimen a 305-bp fragment coding for the region D1 of 28S rRNA gene was sequenced as DNA-barcode for nuclear DNA (Gen-Bank: MG952940).