A new feather mite species of the genus Proterothrix Gaud, 1968 (Acarina, Proctophyllodidae) from the Large Niltava, Niltava grandis (Passeriformes, Muscicapidae) – an integrative description

Abstract A new species of the feather mite genus Proterothrix (Proctophyllodidae: Pterodectinae) is described from the Large Niltava Niltava grandis (Blyth) (Passeriformes, Muscicapidae) in northeast India (Meghalaya, Jaintia Hills, Shnongrim village). Proterothrix chachulae Constantinescu, sp. n. differs from all known species of the genus by having in males the aedeagus with bilobate tip. The morphological description is supplemented with molecular characterisation of a fragment f near the 5` terminus of the mitochondrial COI gene.

The genus Proterothrix Gaud, 1968 (Proctophyllodidae: Pterodectinae) includes 28 species known exclusively from the Old World (Ethiopian, Oriental and Australasian regions) (Hernandes and Valim 2014). In the Oriental region, the genus appears to be one of the most widespread proctophyllodid genera, with 13 described species (from Malaysia, Taiwan, China, Vietnam and India) and 56 potentially new species recorded by Atyeo (1973), but never described elsewhere.

Materials and methods
The material used in the present paper was collected in Shnongrim (Meghalaya, India) in January 2014. The birds were captured using mist-nets, identified and visually checked for the presence and collection of mites and released back to the wild. Mite specimens were collected manually with a needle and placed in tubes with 96% ethanol. Later, in laboratory conditions, mite specimens were cleared in 90% lactic acid for 24 hours, and mounted on microscope slides in Hoyer's medium. Drawings were made using an Olympus CX21 microscope, with a camera lucida drawing device. The bird specimens were identified according to Rasmussen and Anderton (2012) and Grimmett et al. (2011), and the taxonomy of birds follows Clements et al. (2016). The body setation of mites follows that of Griffiths et al. (1990) with the modifications by Norton (1998) concerning coxal setae, while the setation of legs follows Gaud and Atyeo (1996). The description of the new species of Proterothrix is given according to the current format used for species of pterodectine mites (Mironov and Fain 2003, Mironov 2006, Valim and Hernandes 2006) and the measuring techniques of particular structures used were described by Mironov and Proctor (2009).
The full set of measurements is given for a holotype (male) and a range of measurements for corresponding paratypes. All measurements are in micrometers (μm). The holotype and paratypes of the new species are deposited in the Acarological Collection of the "Grigore Antipa" National Museum of Natural History, Bucharest, Romania (inventory numbers are given in brackets for all type specimens).
Three paratype specimens of Proterothrix chachulae sp. n. (two males ANA747, ANA748 and one female ANA749) were used to isolate DNA using DNAeasy Tissue Kit (Qiagen). All three specimens used for molecular analyses were mounted and kept as reference vouchers for morphological examination. The specimens preserved in ethanol 96% were transferred in 180μl ATL Buffer with 20 μl of Proteinase K and incubated overnight at 56ºC on a shaking thermoblock. After 24h, 5μl of Proteinase K were added and incubation was continued until 72h. For the rest of the protocol we followed the manufacturer specifications and the modification suggested by Dabert et al. (2008).
Etymology. This species is named in honor of Oana Mirela Chachula (a biologist, the National Museum of Romanian History, Romania), for her support of our research of ectoparasites of birds from Meghalaya (India).
Remarks. Proterothrix chachulae sp. n. belongs to the wolffi species group by having almost closed coxal fields III in males and parallel-sided terminal cleft in females. Males of the new species differ from all known males of the genus by having the aedeagus with bilobate tip. Among all species of the genus, P. chachulae is closely related to P. cyornis Mironov and Tolstenkov, 2013 from Cyornis tickelliae Blyth (Passeriformes: Muscicapidae) by having the pseudorutelar lobes with acute lateral extensions in both sexes. Males of both species have the opisthosomal lobes roughly trapezoidal and short, setae h3 flattened and enlarged in basal part, a similar general proportions of aedeagus and epimerites IVa well developed, their anterior tips bearing bases of setae 4a. Proterothrix chachulae differs from that species by the following features: in males, the prodorsal shield has posterior margin with wide blunt-angular extension, the opisthosomal lobes have angular median expansion on posterior margin, a pair of small ovoid shields is located at the tips of the genital arch, seta d is bigger in diameter then e on tarsus IV; in females, the prodorsal shield bears in anterior part a few small circular lacunae, the lobar shield is without ornamentation, and the setae h3 have about 1/3 from the length of terminal appendages. In males of P. cyornis, the prodorsal shield has posterior margin slightly convex, the opisthosomal lobes have posterior margin with a small median invagination, small sclerites near the tips of genital arch are absent, seta d and e on tarsus IV are subequal in diameter; in females, the prodorsal shield bears big ovate lacunae in anterior part, the lobar shield bears few circular lacunae in anterior part, and the setae h3 have about 1/5 from the length of terminal appendages. DNA barcode. Representative DNA sequences. GenBank accession numbers for molecular voucher: ANA747 P2 male KY594726; ANA748 P1 male KY594724; ANA749 P1 female KY594725.
We sequenced a 660-pb fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene for two male and one female paratypes. In the resulted alignment we identified 8 variable sites. Two haplotypes were identified: H1 (ANA_748_P1_M and ANA_749_P1_F) and H2 (ANA_747_P2_M).
Intraspecific genetic distance between the analyzed specimens using K2P model is 0.8% (SE 0.3). All observed nucleotide substitutions were synonymous and did not change the amino acid sequence.
This reported genetic distance in the nucleotide sequence of the DNA barcode is comparable with genetic distances found for other Analgoidea species like Proctophyllodes cetti (0.87%) (Badek et al. 2008).