A new feather mite species of the genus Trouessartia Canestrini, 1899 (Acarina, Trouessartiidae) – an integrative description (morphology and DNA barcoding data)

Abstract A new species of the feather mite genus Trouessartia (Trouessartiidae) is described from the Large NiltavaNiltavagrandis (Blyth) (Passeriformes, Muscicapidae) in Northeast India (Meghalaya, Jaintia Hills, Shnongrim village). Trouessartianiltavae Constantinescu, sp. n. is morphologically closely related (no phylogenetic meaning) to T.bulligera Gaud, 1968 from Clytorhynchushamlini (Mayr) (Passeriformes: Monarchidae), sharing in males a unique character within the genus, by having setae e on legs IV hemispheroid, with spine-shaped apex. Males of the new species have the prodorsal shield without ornamentation, the prohysteronotal shield and lobar shield connected, and the terminal cleft parallel sided. Females have the posterior half of the hysteronotal shield ornamented with large ovate lacunae in central area and small elliptical lacunae marginally. To the morphological description of this new feather mite species we added sequence data on the mitochondrial cytochrome c oxidase subunit I gene fragment (COI). The phylogenetic relationships between Trouessartia species are briefly discussed.

The main goals of this paper are to realise the description of a new species of Trouessartia and to analyse its relationships within the genus based on molecular data. This is the first species of Trouessartia described from a host of the genus Niltava, although, as mentioned above, two presumably new species of Trouessartia have been reported by Atyeo (McClure and Ratanaworabhan 1973) from N. grandis and N. sundara, but they have never been described.
The new species of Trouessartia described herein cannot be referred to any of the seven species groups previously established in the genus (Santana 1976, Mironov and Kopij 1996, 2000, because of having a specific combination of characters.

Materials and methods
The material used in the present paper was collected near 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 selected for morphological analysis were cleared in 90% lactic acid for 24 hours and mounted on microscope slides in Hoyer's medium. Some specimens preserved in ethanol were used for genetic analysis. 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 the 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). Description of the new species of Trouessartia is given according to the standards proposed for mites of this genus and related genera (Orwig 1968, Santana 1976, and the measuring techniques of particular structures follow Mironov and González-Acuña (2013). We give the full set of measurements 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 (MGAB). The inventory numbers are given in parentheses for all type specimens.
Three paratype specimens of Trouessartia niltavae sp. n. (one male ANA838 and two females ANA839, ANA840) were used to isolate DNA using DNAeasy Tissue Kit (Qiagen). All four specimens used for molecular analyses were mounted and kept as reference vouchers for morphological examination. The specimens preserved in ethanol 96% were transferred in 180 μl ATL Buffer with 20 μl of Proteinase K and incubated overnight at 56 °C on a shaking thermoblock. After 24 h, 5μl of Proteinase K were added and incubation was continued until 72 h. For the rest of the protocol we followed the manufacturer specifications and the modification suggested by Dabert et al. (2008).
As DNA barcode we used a region near the 5` terminus of the COI gene, amplified by PCR with the degenerate primers bcdF05 (5`-TTTTCTACHAAYCATAAA-GATATTGC-3`) and bcdR04 (5`-TATAAACYTCDGGATGNCCAAAAAA-3`), according to Dabert et al. (2008). The PCR genotyping reaction was performed in a 50 μL total volume containing DNA template, 1X Green GoTaq Flexi Buffer, 2.5 mM MgCl2, each dNTP at 0.1 mM, each primer at 0.5 μM (the primers were M13 tailed) and 1.5 units of GoTaq DNA polymerase (5U/μl) (Promega, Madison, USA). The PCR products were isolated from samples containing visible bands and sent for sequencing to Macrogen (Seoul, South Korea).
Sequence chromatograms were edited and assembled with CodonCode Aligner version 3.7.1. For the phylogenetic analysis we used a dataset comprising three sequences obtained from the new species Trouessartia niltavae and 74 sequences belonging to 17 species of Trouessartia genus available in GenBank and BOLD data system and four sequences belonging to genus Calcealges (see Tabel 1), to be used as outgroup.
MEGA version 7 software (Kumar et al. 2016) was used to identify the most appropriate substitution model, which was subsequently used to generate phylogenetic trees using the Maximum Likelihood (ML) and Neighbor-Joining (NJ) methods. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) was also computed. The same software was used to compute intraspecific pairwise distances between sequences using K2P distance model (Kimura 1980).
Etymology. The specific name niltavae is derived from the generic name of the type host and is a noun in the genitive case.
Remarks. Trouessartia niltavae sp. n. is morphologically close to T. bulligera Gaud, 1968 from Clytorhynchus hamlini (Mayr) (Passeriformes: Monarchidae), shar- ing in males a unique character within the genus: setae e on tarsi IV are hemispheroid with stick-like apex. Additionally, in both sexes of these species, setae d1 are present, setae c2 and sRIII are narrowly lanceolate, with acute apex and the dorsal hysterosomal apertures (DHA) is absent. Both sexes of T. niltavae differ from those of T. bulligera by the shape of setae c2, which are spiculiform in the first species ver-   sus needle-like in the second. Males of both species have the prohysteronotal shield without ornamentation, the lamellae of opisthosomal lobes are attenuate apically and with entire margins, the translobar apodeme is present, setae g are contiguous at bases and situated on postgenital plaque. In males of T. niltavae, the prodorsal shield     is without ornamentation, the prohysteronotal shield and lobar shield have wide median connection, the terminal cleft is parallel-sided and 44-46 μm long, and terminal lobes are separated by 6-8 μm. In males of T. bulligera, the prodorsal shield has ornamentation with faint, interconnecting network of irregular lines, the prohysteronotal shield is completely separated from the lobar shield, the terminal cleft is divergent in posterior half and 75 μm in length, and terminal lobes are separated by 12 μm. In females of both species, setae h1 are lanceolate, the external copulatory tube is present, the supranal concavity is open posteriorly into terminal cleft, and the interlobar membrane occupies the anterior 1/3 of terminal cleft. In females of T. niltavae setae f2 are present, the posterior half of the hysteronotal shield is ornamented with large ovate lacunae in the central area and small elliptical lacunae marginally arranged. In females of T. bulligera setae f2 are absent, the posterior half of the hysteronotal shield has ornamentation with small elliptical lacunae in the central area and large ovate lacunae marginally arranged. DNA barcode. Representative DNA sequences: molecular voucher specimens ANA838 male (GenBank accession number MH094247), ANA839 female (Gen-Bank accession number MH094248), ANA840 female (GenBank accession number MH094249).
The best-fit base substitution model for the analyzed data was determined to be TN93+G+I. The NJ and ML trees exhibited similar topologies and branching structures (see Figures 7 and 8). In both trees our new species was grouped with Trouessartia rubecula and T. simillima, also described from the Muscicapidae family. Our analysis resolved well the analysed Trouessartia species with the exception of T. motacillae and T. jedliczkai, which were poorly resolved in both analyses. Another noticeable feature of our analysis refers to the species T. bifurcata, identified on two avian families: Sylviidae and Acrocephalidae. The two sequences of T. bifurcata introduced in our analysis exhibit a high intraspecific diversity which can be a signal of two cryptic species presently identified as T. bifurcata.