Two new species of the genus Analges (Astigmata: Analgoidea) are described from two species of accentors (Passeriformes: Prunellidae): Analges himalayanus sp. nov. from the Altai Accentor P. himalayana (Blyth), and A. slovakiensis sp. nov. from the Alpine Accentor Prunella collaris (Scopoli). Both species are closely related to A. bidentatus Giebel, 1871 described from the Dunnock Prunella modularis (Linnaeus). We extended the standard morphological descriptions of feather mites by species delimitation analyses carried out on DNA barcode sequences.
Introduction
Feather mites of the genus Analges Nitzsch, 1818 (Analgoidea: Analgidae) live on the down feathers or downy parts of contour feathers of passerine birds. All representatives of this genus are rather uniformly shaped with strongly developed hook-like lateral apophyses on femora I and II used for clasping hair-like barbs, strongly hypertrophied legs III in males, and poorly sclerotized hysteronotum usually lacking of any shields in females (Gaud & Atyeo 1996).
Analges is the largest genus of the family with over 50 described species, mostly from European birds (Gaud 1974; Mironov 1985, 1996; Sohn 1995; Mironov & Kopij 1996; Su et al. 2013; Pedroso & Hernandes 2018). Traditional morphological systematics of this genus is complicated by the strong and continuous polymorphism of males, mainly expressed in the body size and structure of hypertrophied legs III, on the one hand, and extreme morphological uniformity of females, on the other hand. Reliable identification of many known species can be made only with heteromorphic males (Mironov & Kopij 1996).
In the present paper we describe two new species of Analges from accentors (Passeriformes: Prunellidae), the Alpine Accentor Prunella collaris (Scopoli) and the Altai Accentor P. himalayana (Blyth). We compared these new species with the most phenotypically similar and probably closely related Analges bidentatus Giebel 1871, which inhabits the third species of accentors, the Dunnock Prunella modularis (Linnaeus). For the first time in the descriptions of Analges species, we augment the standard morphological analysis with statistical delimitation methods based on DNA barcode markers. We propose this procedure as a new formal standard for feather mite descriptions in the cases when DNA barcode sequences are available.
Material and methods
Mites from Prunella collaris were collected by M.J. in Slovakia in 2015 while the material from P. himalayana was collected in Kyrgyzstan in 2012. The type material is deposited at the Department of Animal Morphology, Adam Mickiewicz University, Poznan, Poland (AMU). For comparative material we used Analges bidentatus from P. modularis, represented by slide-mounted specimens (ZISP 1393, Russia, Kaliningrad province, Curonian Spit, Biological station Rybachy, 55°05′18″N, 20°44′03″E, 23 May 1980, S.V. Mironov) and alcohol-preserved specimens (SVM-12-0420-2, Rybachy, 20 April 2012, S.V. Mironov, and AMU01766, Germany, near Osnabrück, 1991, J. Dabert). We had also a DNA isolate from A. bidentatus collected by J.D. from P. modularis in Osnabrück, Germany, in 1991 (AMU 01765).
Morphological descriptions of the new species follow the recent standards applied for Analgesand related genera (Mironov 1985; Mironov & Kopij 1996). The idiosomal and leg chaetotaxy follow Gaud and Atyeo (1996); nomenclature for coxal setation was used with corrections proposed by Norton (1998). Drawings were made with a Leica DM5500 B microscope with DIC illumination and equipped with a camera lucida. All measurements are in micrometres and were assessed using images taken by a Leica DFC450 digital camera and calculated by LAS v.4.6.1 software (2105, Leica Microsystems CMS GmbH). Measuring standards for particular structures are as follows: (i) length of idiosoma is measured from the anterior margin of the propodosoma to the posterior margin of the opisthosoma, excluding the terminal lamella in males; width of idiosoma is measured at the level of the humeral setae cp; (ii) hysterosoma length is measured from the level of the posterior margins of the scapular shields to the posterior margin of the opisthosoma, excluding the terminal lamella in males; (iii) distance between idiosomal setae of the same pair is the distance between their bases; distance between different pairs of setae is the distance between the their bases of either side of the body (the mean of two measures is taken for each specimen).
The standard morphological descriptions of new species are supplemented with barcode data of the mitochondrial cytochrome c oxidase subunit I gene 661 bp fragment (COI) and 808 bp of the nuclear D2 region of 28S rDNA. The sequencing protocol follows Mironov et al. (2012). Statistical species delimitation by barcode data was carried out on COI alignment by four methods: (1) comparison of pairwise Kimura 2-parameter (K2P) distances was computed in MEGA 7.0 (Kumar et al. 2016); intergroup and intragroup distances and 10 fold species screening threshold were estimated according to Hebert et al. (2004), (2) gap analysis between putative species was carried by the ABGD method (Puillandre et al. 2011) with default settings, (3) gene genealogies were estimated in TCS 1.21 (Clement et al. 2000) using statistical parsimony networks (SP) (Templeton et al. 1992). The 95% connection limit for species boundary was applied for assessing putative species (Hart & Sunday 2007), (4) the posterior probability (PP) of species distinctiveness was estimated by Bayesian method based on the multispecies coalescent model as implemented in STACEY package for BEAST 2 (Jones 2017).
Morphological descriptions
Family Analgidae Trouessart et Mégnin, 1884
Subfamily Analginae Trouessart et Mégnin, 1884
Genus Analges Nitsch, 1918
Analges himalayanus sp. nov. (Figs. 1–3, 5A, D, 7C)
Description. HETEROMORPH MALE (Figs. 1, 3A, B, E, 5A). Idiosoma, length × width, 398 in holotype (349–398 in two paratypes) × 250 (206–231), hysterosoma length 314 (263–311). Prodorsal shield longitudinally trapezoidal with a pair of median ridges, length excluding posterior processes 83 (75–83), width at posterior margin 84 (71–84). Posterior suprategumental processes of prodorsal shield long triangular with acute tips, length 15 (15–16) (Fig. 1B). Postero-medial part of scapular shields with triangular suprategumental extensions. Hysteronotal shield: gradually expanded anteriorly with maximum width at anterior angles; anterior margin deeply concave; concavity about ¼ of shield length; maximum length 251 (216–256), maximum width 184 (136–168); surface uniformly dotted. Posterior margin of opisthosoma rounded, in some paratypes a small rectangular lamella present. Supranal concavity small, slit-like, 28 (23–35) long. Setae vi relatively long, nearly as long as prodorsal shield. Scapular macrosetae se and setae si situated on postero- lateral extensions of prodorsal shield; setae se separated by 70 (60–71) and extending beyond level of cupules im, setae si extending beyond level of setae d1. Setae c2 situated on anterior margins of narrow and long humeral shields and approximately as long as idiosoma. Short setae d1 set close to anterior margin of hysteronotal shield. Bases of setae d2 and e2 adjoining and set on anterior angles of the hysteronotal shield; setae d2 approximately equal to body length, setae e2 about 1.5 times longer than body. Setae f2 longer than the distance between their bases. Distances between dorsal setae and pores: c2:e2 65 (61–63), d1:d1 78 (62–77), e1:e1 76 (62–77), e1:e2 146 (123–139), e1:f292 (79–86), h2:h2 43 (35–37), e1:gl 25 (27–29), gl:im 28 (25–27).
Epimerites I fused as a Y, sternum long with bifurcate end, area between free parts of epimerites sclerotized (Fig. 1A). Posterior ends of epimerites II expanded, truncate. Coxal fields III with a pair of small humeral spurs set laterally at bases of trochanters III. Epiandrum bow-like; length × width 26 (28–38) × 47 (41–44). Length of aedeagus 24 (17–20)1*. Adanal shield shaped as a longitudinal plate, with slightly narrowed anterior part and concave anterior margin, posterior branches extending to the midway level between adanal suckers and setae ps2. Maximum length of adanal shield 99 (96–100), maximum width 61 (57–59), posterior cleft of this shield 49 (35–50) in length. Adanal suckers 14 (12–14) in diameter. Cupules ih set at level of setae ps2. Setae 4a situated at level of setae g. Setae 4b extending beyond bases of setae ps3; setae 3a extending posteriorly far beyond tarsi III. Distances between ventral setae: 4b:g 43 (43–50), g:ps3 106 (98–105), ps3:ps2 46 (42–43).
Femur I with large spatuliform lateral apophysis, femur II with small triangular lateral apophysis (Figs. 3A, B). Femur III enlarged, about twice as wide in apical part than in basal one; inner margin with three distal spines (Fig. 5A); medial spine long and straight, directed mesally, two smaller triangular spines (dorsal and ventral ones) set at its base; dorsal spine acute, ventral spine with rounded tip. Genu III two times wider than long. Tibia about 1.5 times longer than wide at base. Tarsus III with vestigial ambulacral stalk and ambulacrum; inner margin without projection. Setae cG on genua I and II similar in shape, thickened basally with distal long filiform part (Figs. 3A, B). Setae sR on femur III and f on tarsus III approximately as long as the whole leg III. Setae e and d of tarsus IV button-like, seta r fine, shorter than the segment (Fig. 3E). Legs IV with tarsi extending beyond level of body terminus.
HOMEOMORPH MALE (one paratype, Figs. 5D, 7C). Only features different to heteromorph male are presented. Idiosoma, length × width, 352 × 195, hysterosoma length 263. Length of prodorsal shield excluding posterior processes 79, width at posterior margin 69. Maximum length of the hysteronotal shield 222, maximum width 135. Posterior margin of opisthosoma regularly rounded with small rounded terminal lamella. Supranal concavity 30 long. Scapular macrosetae seseparated by 59 and reaching the body terminus, setae si not reaching the level of setae d1. Setae d2and e2 both approximately equal to body length. Distances between dorsal setae and pores: c2:e2 55, d1:d1 61, e1:e1 62, e1:e2 119, e1:f2 81, h2:h2 39, e1:gl 22, gl:im 19.
Epiandrum, length × width, 25 × 42. Length of aedeagus 16. Adanal shield similar in shape to one of heteromorph male (Fig. 7C). Maximum length of adanal shield 86, maximum width 53, posterior cleft of this shield 44 in length. Adanal suckers 12 in diameter. Setae 3a reaching at most the tips of tarsi III. Distances between ventral setae: 4b:g 40, g:ps3 91, ps3:ps2 43.
All podomeres of the leg III of similar width (except small tarsus) and lacking apophyses. (Fig. 5D). Genu III as wide as long.
FEMALE (3 paratypes, Figs. 2, 3 C, D). Idiosoma, length × width, 418–528 × 164–229, length of hysterosoma 317–427. Prodorsal shield shaped as in male, with exception of long postero-lateral angles extending well beyond level of setae se (Fig. 2B), length excluding posterior processes 90–92, width at posterior margin 93–103, length of posterior suprategumental processes 18–20. Scapular shields shaped as in male. Opisthosoma bluntly rounded. Hysteronotal shield absent. Scapular setae se and setae c2 extending beyond level of setae d2; setae si filiform, length not exceeding distance between their bases. Setae d2 reaching the bases of setae e2. Setae e2 not longer than distance between them. Setae f2 short filiform. Distance between dorsal setae se:se 74–77, c2:c2 145–159, c2:d2 90–103, d2:d2 121–134; d2:e2 116–131, e2:e2 120–136, e2:h2 129–138, h2:h2 79–96.
Epimerites I free. Epigynum bow-shaped, 19–22 long, 45–47 wide, setae 4b situated on tips of epigynum (Fig. 2A). Apodemes of oviporus as inverted V. Copulatory opening set ventrally, immediately posterior to the anal slit. Setae g set near ends of oviporal apodemes, genital papillae mesal to g. Setae 1a short, not reaching the epigynum. Setae 4b reaching the bases of setae 4a; setae 4a longer than half the distance 4a:ps3. Setae 3a reaching the bases of setae g, set slightly posterior to level of setae 4b. Distances between ventral setae: 4b:g 42–44, 4b:4b 39–43, 3a:3a 120–138, g:g100–116, 4a:4a 40–44, g:4a 46–52.
Legs I, II shaped as in male. Legs IV not reaching level of setae ps3. Tarsi III and IV with small ventral extension bearing seta s III and w IV, respectively (Figs. 3C, D).
Type material. Heteromorph male holotype, 2 heteromorph male, 1 homeomorph male, and 3 female paratypes (AMU 01763) from Prunella himalayana, adult, KYRGYZSTAN: Chuy Region, Panfilov District, Ala-Bel Pass, 42°14′47″N, 73°03′08″E, 2 July 2012, coll. M. Janiga.
Type deposition. Holotype and paratypes in the Department of Animal Morphology, AMU, Poznan, Poland.
DNA barcode. GenBank accession numbers: MH593806–MH593810 (COI). Sequencing of the nuclear DNA failed.
Etymology. The specific epithet is derived from the specific name of the type host.
Differential diagnosis. Analges himalayanus sp. nov. is close to A. bidentatus in having well-developed lateral apophysis in femur II in both sexes (Figs. 2A, 3B, 6A). In heteromorph males of these species, the median spine of the femur III is distinctly longer than two remaining spines, and the base of femur III is as wide as tibia III (Figs. 5A, C). The new species differs from A. bidentatusby the following features: in heteromorph males, the hysteronotal shield is uniformly dotted (Fig. 1B), the adanal shield is longer than wide (Fig. 1A), femur of leg III (excluding spines) is nearly as long as wide and genu is about 2–2.5 times wider than long and (Fig. 5A). In heteromorph males of A. bidentatus, the hysteronotal shield has wavy longitudinal striae in antero-medial part (Fig. 8), the adanal shield is as long as wide (Fig. 7A), the femur of leg III is distinctly longer than wide and genu is slightly wider than long (Fig. 5C). In homeomorph males of A. himalayanus sp. nov., the adanal shield is distinctly narrower in the anterior than in the posterior part; in A. bidentatus, the anterior part of the adanal shield is laterally expanded and much wider than the posterior part (Figs. 7C, D).
FIGURE 2.
Analges himalayanus sp. nov., female. A—ventral view, B—dorsal view, co—copulatory opening.
FIGURE 3.
Analges himalayanus sp. nov., legs. A—leg I, heteromorph male, B—leg II, heteromorph male, C—tibia and tarsus IV, female, D—leg III, female, E—tarsus IV, heteromorph male. A, B—ventral view, C–E—dorsal view.
Analges slovakiensis sp. nov. (Figs. 4, 5B, 6B-G, 7B)
Description. HETEROMORPH MALE (Figs. 4, 5B, 6B–G). Idiosoma, length × width, 425 in holotype (405–440 in four paratypes) × 267 (247–316), hysterosoma length 321 (308–342). Prodorsal shield longitudinally trapezoidal with a pair of median ridges, length excluding posterior processes 91 (89–92), width at posterior margin 94 (88–98). Posterior suprategumental processes of prodorsal shield long triangular with rounded tips, length 17 (16–17) (Fig. 4B). Postero-mesal part of scapular shields with triangular suprategumental extensions. Hysteronotal shield: gradually expanded anteriorly with maximum width at level of setae d1; anterior margin of shield deeply concave; concavity about ¼ of the shield length; maximum length 269 (251–275), maximum width 186 (179–197). Hysteronotum uniformly dotted. Posterior margin of opisthosoma rounded, with small rectangular lamella. Supranal concavity small, keyhole-like, 32 (31–38) long. Setae virelatively long, nearly as long as prodorsal shield. Scapular macrosetae se and setae si situated on lateral extensions of prodorsal shield; setae se separated by 79 (73–81) and reaching level of cupules im, setae si extending beyond level of setae d1. Setae c2 situated on anterior margins of narrow and long humeral shields and approximately as long as idiosoma. Setae d1 short, set close to anterior margin of hysteronotal shield. Bases of setae d2 and e2 adjoining and set on anterior corners of the hysteronotal shield; setae d2 shorter than body length, setae e2 about 1.5 of body length. Setae f2longer than distance between their bases. Distances between dorsal setae and pores: c2:e2 71 (70–91), d1:d1 78 (62–77), e1:e1 76 (62–77), e1:e2 165 (150–162), e1:f2 88 (85–91), h2:h2 44 (40–48), e1:gl 32 (31–32), gl:im 19 (16–22).
Epimerites I fused as a Y; sternum long, with bifurcate posterior end, area between free parts of epimerites sclerotized (Fig. 4A). Posterior ends of epimerites II expanded, slightly concave. Coxal fields III with a pair of small humeral spurs set laterally near trochanters III. Epiandrum bow-like, length × width 32 (28–29) × 46 (47–51). Length of aedeagus 28 (22–28)2*. Adanal shield roughly shaped as inverted longitudinal trapezium with anterior part distinctly wider than posterior one; anterior margin of this shield rounded and convex, posterior branches extending to the halfway point between adanal suckers and setae ps2; maximum length of 119 (110–122), maximum width 83 (94–97), posterior cleft 58 (49–51) in length. Adanal suckers 15 (14–15) in diameter. Cupules ih set posterior to the level of setae ps2. Setae 4a situated at level of setae g or slightly posterior. Setae 4bextending beyond bases of setae ps3; setae 3a not extending beyond tarsi III. Distances between ventral setae: 4b:g 52 (44–64), g:ps3 104 (100–112), ps3:ps2 50 (48–55).
Femur I with large spatuliform lateral apophysis (Fig. 4A), femur II without lateral apophysis or apophysis vestigial (Figs. 6D–G). Femur III enlarged, about 1.5 times wider in apical part than in basal one; inner margin with three triangular spines of similar size (Fig. 5B); medial spine directed adaxially, and dorsal and ventral spines at its base directed perpendicularly to the axis of medial spine. Genu III about two times wider than long. Tibia III 1.6–1.7 times longer than wide at base. Tarsus IV with vestigial ambulacral stalk and ambulacrum; inner margin without projection. Setae cG on genua I and II thickened basally, with distal long filiform part, seta cGII with minute tongue-like membrane at base of filiform part (Fig. 6D). Setae sR on femur III approximately as long as the whole leg III, setae f on tarsus III shorter, not longer than 2/3 the length of sRIII. Setae e and d of tarsus IV button-like, setae r subequal in length to this segment. Legs IV with basal parts of tarsi extending to level of body terminus.
HOMEOMORPH MALE (one paratype, Fig. 7B). Only features different to heteromorph male are presented. Idiosoma length × width, 347 × 188, hysterosoma length 267. Length of prodorsal shield excluding posterior processes 76, width at posterior margin 67. Maximum length of the hysteronotal shield 212, maximum width 114. Posterior margin of opisthosoma regularly rounded with small rounded terminal lamella. Supranal concavity 33 long. Scapular macrosetae se separated by 56 and reaching the body terminus, setae si not reaching the level of setae d1. Setae d2 and e2both approximately equal to body length. Distances between dorsal setae and pores: c2:e2 66, d1:d150, e1:e1 65, e1:e2 108, e1:f2 83, h2:h2 41, e1:gl 23, gl:im 17.
Epiandrum, length × width, 25 × 41. Length of aedeagus 17. Adanal shield different in shape to that of heteromorph male, regularly trapezoidal, wider at base than in anterior part (Fig. 7B). Maximum length of adanal shield 83, maximum width 56, posterior cleft of this shield 32 in length. Adanal suckers 10 in diameter. Setae 3a extending posteriorly far beyond the tips of tarsi III. Distances between ventral setae: 4b:g 39, g:ps3 97, ps3:ps2 40.
All podomeres of leg III of similar width (except small tarsus) and lacking apophyses. Genu III as wide as long.
FEMALE (3 paratypes, Figs. 6B, C). Idiosoma, length × width, 420–518 × 192–218, length of hysterosoma 318–416. Prodorsal shield shaped as in male, with exception of postero-lateral corners that extending as triangular extensions well beyond the bases of setae se, length excluding posterior processes 94–98, width at posterior margin 93–113, length of posterior suprategumental processes 18–19. Scapular shields shaped as in male. Opisthosoma bluntly rounded. Hysteronotal shield absent. Scapular setae se and setae c2 extending posteriorly beyond the level of setae d2, setae siminute. Setae d2 reaching the bases of setae e2. Setae e2 as long as distance between them or longer. Setae f2 minute. Distance between dorsal setae se:se 77–81, c2:c2 158–173, c2:d2 80–98, d2:d2 120–136; d2:e2 100–115, e2:e2 118–142, e2:h2 102–145, h2:h2 81–87.
Epimerites I free. Epigynum bow-shaped with slightly enlarged tips, 18–22 long, 41–48 wide, setae 4b situated on tips of epigynum. Apodemes of oviporus as inverted V. Copulatory opening set ventrally immediately posterior to the anal slit. Setae g set near ends of oviporal apodemes, genital papillae mesal to g. Setae 1a short, not reaching the epigynum. Setae 4b reaching bases of setae 4a; setae 4a longer than half the distance between 4a:ps3. Setae 3a set posterior to level of setae 4b. Distances between ventral setae: 4b:g 44–53, 4b:4b 36–41, 3a:3a 99–138, g:g 79–107, 4a:4a 36v37, g:4a 42–64.
Legs I, II shaped as in male. Lateral apophyses on femur II always present, generally better developed than in males (Figs. 6B, C). Legs IV, depending on individual, may reach or not reaching the level of setae ps3. Tarsi III and IV with small ventral extension bearing seta s III and w IV, respectively.
Type material. Heteromorph male holotype, 4 heteromorph male, 1 homeomorph male, 3 female paratypes (AMU 01764) from Prunella collaris nestling, SLOVAKIA, Low Tatra mountains, Chopok, 8 July 2015, coll. M. Janiga.
Type deposition. Holotype and paratypes in the Department of Animal Morphology, AMU, Poznan, Poland.
DNA barcode. GenBank accession numbers: MH593800–MH593805 (COI), MH593798 (D2)
Etymology. The specific epithet is derived from Slovakia, the country of the type material origin.
Differential diagnosis. Analges slovakiensis sp. nov. is close to males of A. himalayanus sp. nov. in having a uniformly dotted hysteronotal shield (Figs. 1B, 4B). In heteromorph males, genu III is at least two times wider than long (Figs. 5A, B) and the adanal shield is distinctly longer than wide (Figs. 1A, 4A). In homeomorph males of both species the adanal shield is distinctly narrower in the anterior part than in the posterior (Figs. 7B, C). These features clearly distinguish these two species from A. bidentatus, in which the hysteronotal shield is striated antero-medially (Fig. 8) and the adanal shield is expanded laterally in anterior part (Figs. 7A, D) in both homeo- and heteromorph males; heteromorph males have genu III at most 1.5 times wider than long (Fig. 5C). Analges slovakiensis differs from A. himalayanus by the following characters: in heteromorph males, all spines of femur III are of similar size (Fig. 5B) and the adanal shield is much wider in the anterior than in the posterior part (Fig. 4A). In heteromorph males of A. himalayanus, the median spine of femur III is distinctly longer than two remaining spines (Fig. 5A) and the adanal shield has almost parallel lateral margins and distinctly narrowed anterior part (Fig. 1A). In homeomorph males of A. slovakiensis sp. nov., the adanal shield is shaped as a narrow and long trapezoid being slightly wider at the base than in the anterior part, and the length of the posterior cleft of this shield is ca one–third the total shield length (Fig. 7B); in A. himalayanus, this shield is narrowed in the anterior half only and the posterior cleft is over the half of the total shield length (Fig. 7C).
FIGURE 5.
Legs III of Analges males, dorsal view. A—A. himalayanus sp. nov., heteromorph, B—A. slovakiensis sp. nov., heteromorph, C—A. bidentatus Giebel, 1871, heteromorph, D—A. himalayanus sp. nov., homeomorph.
FIGURE 6.
Femur and genu II of Analges males, ventral view. A—A. bidentatus, male, B–G—A. slovakiensis sp. nov., B,C—females, D–G—males.
FIGURE 7.
Adanal shield of Analges males. A—Analges bidentatus, heteromorph, B—A. slovakiensis sp. nov., homeomorph, C—A. himalayanus sp. nov., homeomorph, D— A. bidentatus, homeomorph.
FIGURE 9.
Analges species delimitation results for COI. A—Automatic Barcode Gap Discovery, distribution of pairwise differences (above) and ranked pairwise differences (below). The analysis supports the specific status of three Analges populations and also points on more close relationship between two new species than either of them with A. bidentatus. B – Statistical parsimony network broken at 95% connection limit. The three subnetworks correspond to three Analges species. Empty circles—hypothetical haplotypes, numbers—mutated positions in COI sequence. Abbreviations for host species in mite haplotypes: Prmo (red)— Prunella modularis, Prco (blue)—P. collaris, Prhi (green)—P. himalayana.
Molecular species delimitation
All species delimitation methods applying COI barcode sequences univocally supported species-level status of each of the Analges populations inhabiting particular prunellid hosts: A. bidentatus Giebel, 1871 from Prunella modularis, A. himalayanus sp. nov. from P. himalayana, and A. slovakiensis sp. nov. from P. collaris. The intraspecific K2P distances for all sequences were 0.0-0.7%, while the distances between groups were much higher: A. himalayanus vs A. slovakiensis—8.8–9.8%, A. bidentatus vs A. himalayanus—11.9–12.3%, and A. bidentatus vs A. slovakiensis—12.1–13.0% (Supplementary data Tab. S1). Thus in all cases the minimal value for interspecific distance was greater than 10× maximal observed intraspecific distance. However the intraspecific distances may be underestimated due to small number of independent populations of the same species. The ABGD gap analysis reconstructed three groups of sequences corresponding to three Analges species and discovered a distinct gap between intraspecific and interspecific distances (Fig. 9A). The statistical parsimony network (TCS) was broken into three pieces corresponding to three analysed species (Fig. 9B). Finally, the coalescent method implemented in STACEY package showed significant posterior probability PP=0.64 for three species as true clustering; the best alternative clustering was ten times less probable (PP=0.05) (Supplementary data Tab. S2).
Species delimitation based on nuclear region D2 was possible only for Analges slovakiensis and A. bidentatus; we failed to obtain these data for A. himalayanus. Pairwise distance between sequences of A. slovakiensis (MH593798) and A. bidentatus (MH593799) was 0.027 (SD 0.006) that support the specific level of divergence between both compared species.
Discussion
Classical phenotypic species definition in the genus Analges is a complicated task because of great intraspecific morphological variability expressed in continuous polymorphism of males, and extremely uniform appearance of females of different species. This problem is amply evident in both new species described here. The observed intraspecific variation concerns morphometric characters (e.g. body dimensions or setal distances in both sexes) as well as discrete features (e.g. shape of femoral apophyses on legs II and presence/absence of a terminal lamella in males). Females of three analysed species are phenotypically indistinguishable, not only in discrete features but also in morphometric characters. However, it is possible that further analyses of more representative number of specimens may reveal some statistically supported differences in morphometric data (e.g. setal lengths or length proportions).
Despite their phenotypic similarity, closely related Analges species from prunellids can be distinguished by a few discrete characters in males, such as the shape of hypertrophied legs III in heteromorphs, the shape of adanal shield and the sculpture of hysteronotal shield in heteromorphs and homeomorphs. But the strongest evidence for species definition in this case turned out the molecular data. COI DNA barcode sequences, analysed by different methods of species delimitation, univocally indicate three species that are strongly genetically separated. We suggest adding this method to morphological analysis as a standard (or quite desirable) procedure in future species descriptions in all cases when suitable DNA material is available.
According to molecular analyses, Analges himalayanus seems to be a sister-species of A. slovakiensis, while A. bidentatus is more distant from these pair of species. This relationship is concordant with the phylogenetic relationships of the birds (Liu et al. 2017), which suggests cospeciation between these Analges species and their prunellid hosts.
Acknowledgements
We are thankful to colleagues and students of the Žilina University—Ľudmila Kubašková, Anna Mičková, Martin Lukáň and Imrich Kašlík—for their help in the laboratory preparation of mites. Special thanks for Mirka Dabert, Molecular Biology Techniques Laboratory, Faculty of Biology, AMU for her effort in extracting DNA barcodes for the present study.
References
Appendices
Appendix
TABLE S1.
Estimation of K2P distances between Analges COI sequences from prunellid hosts. Prmo—from Prunella modularis, Prco—from P. collaris, Prhi—from P. himalayana. Red—Analges slovakiensis sp. nov. vs A. himalayanus sp. n., yellow—A. bidentatus vs A. slovakiensis sp. nov, blue—A. bidentatus vs A. himalayanus sp. nov, white—intraspecific distances in A. slovakiensis sp. nov (left), A. himalayanus sp. nov (center), and A. bidentatus (right).
TABLE S2.
Posterior probability (PP) of alternative clustering of Analges COI sequences from prunellid hosts. Only clusterings of frequency five or above (of 2001) are shown. Abbreviations for corresponding host species: Prmo—Prunella modularis, Prco—P. collaris, Prhi—P. himalayana.
