﻿Two new species of feather mites (Acariformes, Astigmata) from the black-tailed godwit, Limosalimosa (Charadriiformes, Scolopacidae), in Korea

﻿Abstract Two new species of feather mites are described from two individuals of the black-tailed godwit, Limosalimosa (Linnaeus, 1758), in Korea: Alloptes (Conuralloptes) neolimosaesp. nov. (Analgoidea, Alloptidae) and Phyllochaetalimosaesp. nov. (Pterolichoidea, Syringobiidae). Males of A. (C.) neolimosaesp. nov. are distinguished from A. (C.) limosae in having the hysteronotal shield with a straight anterior margin, setae h2 enlarged and slightly flattened in the basal half, and the terminal lamella monotonously transparent without sclerotized patches; females differ in having legs IV with ambulacral discs extending to or slightly beyond the level of setae f2. The discovery of P.limosaesp. nov. represents the first record of the feather mite genus Phyllochaeta on godwits of the genus Limosa Brisson, 1760 (Scolopacidae, Limosinae). Males of P.limosaesp. nov. are distinguished from P.secunda in having the terminal cleft semi-ovoid with a length-to-width ratio of 1.7, and the terminal membranes with 15 or 16 finger-shaped denticles; females differ in having the hysteronotal shield bearing faint longitudinal striations in the posterior third and lacking lacunae, and setae c1 situated posterior to the level of setae c2. Additionally, we obtained partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene from A. (C.) neolimosaesp. nov. and estimated genetic distances from 10 other Alloptes species based on comparisons of COI sequences.


Introduction
Feather mites comprise two superfamilies (Analgoidea and Pterolichoidea) of astigmatan mites within the order Sarcoptiformes and are permanent parasites or commensal ectosymbionts that colonize particular microhabitats in the plumage and on the skin of birds (Gaud and Atyeo 1996;Dabert and Mironov 1999;Proctor 2003). Species and supraspecific taxa of feather mites generally exhibit a high level of host-specificity that is apparently caused by specialization to particular microhabitats and dispersal primarily by direct physical contact between host individuals Proctor and Owens 2000;Dabert 2005).
The black-tailed godwit, Limosa limosa (Linnaeus, 1758) is a wader that is widely distributed in the Palearctic realm but has a disjunctive breeding range (Engelmoer and Roselaar 1998;Gill et al. 2007). This bird is subdivided into four subspecies [L. l bohaii Zhu, Piersma, Verkuil & Conklin, 2020, L. l. islandica Brehm, 1931, L. l. limosa (Linnaeus, 1758, and L. l. melanuroides Gould, 1846] based on morphological and genetic characteristics, and is also recognized by classification authority such as the International Ornithological Congress (IOC) (Gill et al. 2021;Zhu et al. 2021). In Korea, this bird species occurs as a passage migrant generally observed during autumn and spring migrations (Lee et al. 2014).
In this paper, we describe two new species of the genera Alloptes Canestrini, 1879 and Phyllochaeta Dubinin, 1951, which were found on two individuals of L. limosae in Korea. Additionally, we present DNA barcodes for the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences from the newly described Alloptes species and estimate genetic distances with other Alloptes species based on comparison of COI sequences.

Material sampling
Carcasses of two black-tailed godwits  were provided by the Chungnam Wild Animal Rescue Center (CNWARC). These birds were initially rescued in Asan and Seosan-si (si = City), Chungcheongbuk-do (do = Province) but later died during the course of treatment. Mite samples were collected from wing feathers of the two godwits under a dissecting microscope using a preparation needle and thereafter preserved in 95% ethanol. These were subsequently cleared in 10% lactic acid at room temperature for one day and then mounted on microscope slides using PVA mounting medium (BioQuip, Rancho Dominguez, California, USA). Mite specimens were observed under a light microscope (DM2500; Leica, Wetzlar, Germany). Figures were drawn and photographed with a drawing tube and microscopic digital camera (7D; Canon, Tokyo, Japan) attached to a light microscope, respectively, and were assembled and edited using Adobe Illustrator and Photoshop CS5 (Adobe Systems Incorporated, San Jose, California, USA).
Descriptions of two new species are presented herein following the standard formats adopted for the families Alloptidae and Syringobiidae (Dabert 2003;Mironov and Palma 2006;Hernandes et al. 2017;Han et al. 2021). Terminology, idiosomal, and leg chaetotaxy follow Gaud and Atyeo (1996), with minor corrections for the coxal setae proposed by Norton (1998). All measurements are in micrometers (μm). All examined specimens are deposited at the National Institute of Biological Resources (NIBR), Korea. The classification and scientific names of birds follow Gill et al. (2021).

DNA sequencing and molecular analysis
Genomic DNA of the new Alloptes species was extracted from the whole body of two isolated individuals found on CNWARC no. CN12-402, and a single leg per individual from two individuals discovered on CNWARC no. CN17-265, using a Tissue DNA Purification Kit (Cosmogenetech Inc., Seoul, Korea) according to the manufacturer's instructions ( Table 1). The exoskeletons remaining after DNA extraction were mounted on microscope slides using methods described above for species identification.
A COI barcode fragment was amplified using KOD-Plus (Toyobo, Osaka, Japan) in conjunction with two universal primers (bcd05F [5´-TTTTCTACHAAYCATAAA-GATATTGC-3´] and bcd04R [5´-TATAAACYTCDGGATGNCCAAAAAA-3´]) under the following conditions: an initial denaturation for 2 min at 94 °C; 40 cycles at 98 °C for 15 s, 50 °C for 30 s, and 68 °C for 60 s; and a final extension at 68 °C for 5 min (Dabert et al. 2008). The amplified products were sequenced using an ABI3100 automated sequencer (Perkin Elmer, Foster City, California, USA). Sequence assembly, alignment, and trimming were performed using Geneious v. 8.1.9 software (Biomatters, Auckland, New Zealand) (Kearse et al. 2012). However, despite performing similar procedures for the newly described Phyllochaeta species, we were unable to obtain the corresponding COI sequences.
COI sequences obtained for the new Alloptes species were aligned with those of 10 other Alloptes species registered in the National Center for Biotechnology Information (NCBI) database using Geneious v. 8.1.9 (Table 1). Pairwise distances between sequences were computed using a Kimura two-parameter (K2P) substitution model with Mega X v. 10.1.7 software (Kumar et al. 2018).
Remarks. The specimens of A. (C.) limosae used here to illustrate morphological differences are those examined by Han and Min (2019a).
In contrast to the original description of A. (C.) limosae by Dubinin (1951: fig. 65) and the illustration by Vasyukova and Mironov (1991: fig. 72), the drawing of that species by Gaud (1972: fig. 26a) clearly shows that the males are characterized by an opisthosoma having distinctly S-shaped lateral margins and enlarged setae h2. This tends to indicate that the specimens examined by Gaud are probably those of the same species we describe herein, A. (C.) neolimosae sp. nov.
The occurrence of two closely related species of the genus Alloptes on Limosa limosa could most probably be explained by their origin from the common ancestor in different parts of the geographic range of this host. The black-tailed godwit has a very wide nesting range in Eurasia, from Iceland to Chukotka peninsular, which is split into several isolated populations in eastern part of Asia (Gill et al. 2021;Zhu et al. 2021). Since we found both mites, A. (C.) limosae and A. (C.) neolimosae sp. nov., on the same individual of Limosa limosa, it is possible to speculate that bird populations where these species originated are presently mixed or rejoined.

Species (Genbank accession no.) COI distances (%)
Setae cG of genua I and II spiniform. Tarsi III and IV with small apicoventral spines between bases of setae s and r. Legs IV with ambulacral discs almost extending to level opisthosomal lobe apices. Setae d and e of tarsi IV spine-like, situated on distal end of tarsus, solenidion φ of tibia IV extending to proximal margin of ambulacral disc (Fig. 7A-D).
Female (  lateral sclerites adjacent to hysteronotal shield, posterior parts of these sclerites gradually turned onto ventral side of hysterosoma. Lateral dorsal setae c2 short, filiform, 50-52 in length. Setae d2 and e2 represented by macrosetae, 280-290 and 305-320 long, respectively; setae f2 large spiculiform, 73-76 long. Distance between dorsal setae: c2:d2 130-137, d2:e2 102-105, e2:h3 80-85, h2:h2 76-80, h3:h3 54-59. Epimerites I and II with narrow sclerotized areas, posterior end of sternum with triangular sclerotized plate. Epigynum small, roughly semicircular, 16-18 long, 27-28 wide. Setae g situated posterior to level of setae 3a and genital papillae. Setae ad short, 7-12 in length. Setae ps2 and ps3 filiform, 80-82, and 65-72 long, respectively. Structure and setation of legs I and II as in males. Solenidion φ of leg I approximately as long as this leg. Setae cG of genua I and II blade-shaped. Tarsi III and IV without ventral blunt-angular extension. Solenidion φ of tibia IV slightly shorter than corresponding tarsus (Fig. 7E, F).  Differential diagnosis. The genus Phyllochaeta currently comprises 15 species known to infest birds of the genera Actitis, Arenaria, Calidris, Charadrius, Limnodromus, Prosobonia, and Rostratula (Charadriiformes, Scolopacidae) (Dabert 2003). The newly described species Phyllochaeta limosae sp. nov., discovered here for the first time from Limosa limosa, and is most similar to Phyllochaeta secunda Dabert, 2003 described from the short-billed dowitcher, Limnodromus griseus (Gmelin, 1798) (Scolopacidae), with respect to the following features: in males of both species, ornamentation of the hysteronotal shield is absent, the opisthosomal lobes are shovel-shaped with distinctly enlarged distal parts, the terminal membranes have numerous finger-like denticles along the medioterminal margin, ventral apophyses of legs I and II are absent, and genual setae cGI and cGII are spine-like; in females, dorsal setae e1 are situated closer to the level of hysteronotal gland openings gl than to setae e2. Phyllochaeta limosae sp. nov. differs from P. secunda in the following characteristics: in males, the terminal cleft is semi-ovoid with a length-to-width ratio of 1.7, the terminal membranes have 15 or 16 finger-shaped denticles, the anterolateral extensions of the prodorsal shield are pointed, the hysteronotal shield is uniformly sclerotized, the tarsi of leg III and IV have bluntangular ventral extensions, and setae 4a and 4b are approximately situated at the same transverse level; in females, the hysteronotal shield bears faint longitudinal striations in the posterior third and lacks any lacunae, and setae c1 are situated posterior to the level of setae c2. In males of P. secunda, the terminal cleft is distinctly wider (length/width ratio about 1.0), terminal membranes have 9 or 10 finger-shaped denticles, anterolateral extensions of the prodorsal shield are widely rounded, the hysteronotal shield has a pair of longitudinal heavily sclerotized bands near the terminal cleft, ventral apophyses of tarsi III, IV are hooked, and setae 4a are situated posterior to the level of setae 4b; in females, the hysteronotal shield is monotonously punctate and has a pair of ovate lacunae at the level of setae e2, and setae c1 and c2 are approximately at the same transverse level.
Remark. The origin of Phyllochaeta limosae sp. nov. on Limosa limosa is enigmatic and disputable. Godwits (Limosinae, Limosa) and curlews (Numeniinae, Numenius) forming most basal lineages within Scolopacidae were previously known to bear only syringobiids of the genus Limosilichus Vasyukova & Mironov, 1986. This genus is apparently restricted to godwits and curlews, and most its species are monoxenous Mironov 1990, 1991;Dabert 2003). While the genus Phyllochaeta currently including 15 species is widely distributed on waders of the tribes Arenariini, Tringini, and Scolopacini (Scolopacinae). Two species, P. secunda and P. gracilis Vasyukova & Mironov, 1986, which are close to P. limosae sp. nov., are specific to dowitchers Limnodromus griseus and L. scolopaceus (Say, 1823), respectively. Therefore, it is most reasonable to hypothesize that the ancestor of P. limosae sp. nov. had been transferred to the black-tailed godwit from some dowitchers.
Etymology. The specific name is taken from the generic name of the type host and is a noun in apposition.