Oziella sibirica, Chetverikov, Philipp E., Beaulieu, Frédéric, Cvrković, Tatjana, Vidović, Biljana & Petanović, Radmila U., 2012

Chetverikov, Philipp E., Beaulieu, Frédéric, Cvrković, Tatjana, Vidović, Biljana & Petanović, Radmila U., 2012, Oziella sibirica (Acari: Eriophyoidea: Phytoptidae), a new eriophyoid mite species described using confocal microscopy, COI barcoding and 3 D surface reconstruction, Zootaxa 3560, pp. 41-60 : 45-57

Oziella sibirica sp. nov. Chetverikov

Description of external morphology ( Figs. 1–6 ; Table 2 ). PROTOGYNE FEMALE (n=8). Idiosoma vermiform, whitish, 352 (321–374) long, 59 (53–65) wide. Prodorsal shield ( Figs. 1 , 2 , 3 C & 4B) subtriangular, with anterior margin weakly rounded and slightly indented medially; ornamentation consisting of median, two admedian and five submedian lines ( Fig. 1 B). Median line straight, visible in posterior two-thirds of shield, usually entire or can be divided into 2–3 fragments. Two admedian lines straight in anterior third of shield, slightly sinuous in posterior two-thirds, together forming a bottle-like figure, usually with a gap in anterior one-third of prodorsal shield. Submedian-1 lines short, visible in anterior one-third of prodsorsal shield. Additional short line can be present between admedian and submedian-1. Submedian-2 can be straight and short ( Fig. 2 A & 2B) or as long as shield and then straight in anterior half but slightly sinuous in posterior half ( Fig. 2 C & 2D). Submedian-3 lines running from anterior shield margin, medially to ve tubercles, ending well in front of sc tubercles; those lines consisting of 2–3 fragments or may be entire, with a small curve posteriorly ( Fig. 2 B & 2C). In some individuals, submedian-3 appear to be bifurcate, with median branch merging with posterior portion of submedian-2 ( Fig. 2 B & 2C); a small sub-circular (almost eye-like), apparently smooth area visible (only under CLSM) in some specimens inbetween submedian-2 and -3 and anterad to sc tubercles ( Fig. 2 A, 2B & 2D). Two short lines, submedian-4 and -5, behind each ve tubercle, flanked laterally by an eye-like structure, appearing more-or-less flat (under phase contrast) or as an oval protuberance (under CLSM), about 6 long and 4 wide. Surface of prodorsal shield anterior to ve with small granulations. Prodorsal shield 32 (32–36) long, 36 (36–46) wide; ve 10 (8–10) long, directed forward and slightly laterally; tubercles 21 (20–26) apart; sc 1 (0.5–1.5) long, directed posteromedially, tubercles 17 (16–20) apart. Distance between tubercles of ve and sc 18 (18–19). Gnathosoma 25 (24–27) long. Dorsal pedipalp genual setae d 10 (7–10) long. Leg I 30 (28–31) long, tibia 5 (5–7), l' 2 (2–2), tibial solenidion φ 7 (6–7); tarsus 5 (5–6), u' 2 (1–2) long, ft' 2 (1–3) long, ft'' 9 (8–11) long, ω 8 (7–9) long, without knob; empodium 5/4-rayed, 7 (7–7) long; l'' 14 (13–16) long, femur with a small ventrolateral spine, bv 2 (1–3) long. Leg II 26 (25–27) long, tibia 5 (5–6), l' absent, tarsus 5 (5–6) long, u' 2 (1–2) long, ft' 2 (2–3) long, ft'' 8 (8–10) long, ω 9 (8–10) long, without knob; empodium 5/4-rayed, 6 (6–7) long; l'' 15 (12–17) long, bv 2 (1–2) long. Coxae with numerous oval microtubercles and thin, indistinct lines. Subcapitular plate round, flat. Setae 1b 14 (10–15) long, 16 (16–19) apart; 1a 16 (11–17) long, 12 (12–14) apart; 2a 36 (31–38) long, 28 (26–31) apart. Prosternal apodeme weakly pronounced, three-forked. Epigynium 11 (10–11) long, 23 (22–24) wide, with indistinct four long and four short lines, absent in some individuals; 3a 8 (7–8) long. Opisthosoma with 77 (73–80) ventral and 80 (75–82) dorsal microtuberculate annuli; 4 (3–5) annuli present before epigynium. Setal lengths: c1 20 (18–24), c2 19 (16–19), d 7 (6–8), e 5 (5–7), f 26 (22–28), h1 4 (3–4), h2 64 (50–72); 7 (7–8) annuli between rear shield margin and c1 tubercles, 6 (5–7) annuli from rear prodorsal shield margin to c2; 15 (14–16) annuli between c2 and d, 20 (20–22) annuli between d and e, 27 (24–28) annuli between e and f, and 9 (8–9) annuli between f and h1.

MALE (n=5). Similar in shape and colour to female; about 0.9 times the size of female. Prodorsal shield ornamentation and chaetotaxy similar to that of female. Eugenital setae not visible neither with conventional optical microscope nor in CLSM ( Fig. 4 D). All measurements of males given in Table 2 .

LARVA (n=9) ( Fig. 5 ). Idiosoma wormlike, whitish, 206 (182–225) long, 43 (38–46) wide. Prodorsal shield ornamentation similar to that of female except median and admedian lines often broken and with gaps, and eyelike structures indistinct in some individuals. Leg I 19 (17–23), empodium 4/3-rayed, femur with very weak ventrolateral spine. Coxae and coxisterna with numerous microtubercles; 12 (10–14) annuli present before setae 3a. Opisthosoma with 76 (70–86) dorsal annuli and 61 (57–66) ventral annuli. Other measurements given in Table 2 .

NYMPH (n=8) ( Fig. 6 ). Idiosoma wormlike, whitish, 287 (273–318) long, 53 (49–60) wide. Prodorsal shield ornamentation similar to that of the female except median line and eye-like structures indistinct in some individuals. Leg I 23 (22–25), femur with small ventrolateral spine, empodia 4/3- or 4/4-rayed. Coxae and coxisterna with numerous microtubercles; 11 (11–12) annuli present before setae 3a. Opisthosoma with 73 (70–77) dorsal annuli and 72 (68–74) ventral annuli. Other measurements given in Table 2 .

Host plant. Carex macroura Meinsh. (Cyperaceae) . Mites were found living inside the leaf sheaths, causing no apparent damage.

Type material. Female holotype (slide #167-11), 4 female paratypes, 4 nymph and 4 larvae (all on the same slide) deposited in Saint-Petersburg State University, Department of Invertebrate Zoology (Universitetskaya naberejnaya, 7/9, Saint-Petersburg, 199034, Russia). Other paratypes (9 females, 7 males, 21 nymphs and 11 larvae on slides #168-11, #169-11 and #170-11) deposited in the Acarological Collection of the Zoological Institute of Russian Academy of Sciences ( ZIN RAS). All specimens collected from RUSSIA: Republic of Buryatia, Barguzinsky District, Maximiha village; from Carex macroura , in a firdominated ( Abies sibirica ) forest, on the bank of Lake Baikal (53°26'64''N, 108°73'55''E), 24 June 2011, coll. P. E. Chetverikov.

Distribution. Oziella sibirica sp. nov. is at present recorded only from the Republic of Buryatia, Barguzinsky District, Russia. However, Carex macroura , the host plant of O. sibirica sp. nov., is a widely distributed East European-Asian forest species, which also occurs in the Ural, Preuralye and South Siberia regions of Russia, East Kazakhstan (Saur mountains), North-West China (Dzungaria) and Mongolia ( Malyshev 1990 p. 123; Egorova 1999 p. 290; Rotreklová et al. 2011, p. 45), so future surveys could reveal a wider distribution for this new mite species.

Etymology. The specific epithet, sibirica , is a feminine singular adjective derived from the name of Eastern region of Russia, the Sibir (Siberia), where the new species was collected.

...... continued on the next page Epigynium/epiandrium length11 ± 0.5 10–11 4 ± 0.5 3–4 – – – – Epigynium/epiandrium width 23 ± 0.6 22–24 23 ± 0.7 22–24 – – – – Differential diagnosis. The new species is very similar to Oziella yuccae Keifer 1954 and Oziella rufensis Manson 1970 but can be easily distinguished from them by its extremely short setae sc, the number of empodial rays, its longer body and at least twice shorter opisthosomal setae c1 and d ( Table 3 ). Although the descriptions of O. yuccae , O. rufensis and O. sibirica may suggest that these species could also be distinguished by the prodorsal shield ornamentation and the position of c1 setae, we doubt that these characters are reliable for species diagnosis because they vary within species. Moreover, illustrations in the description of O. rufensis are currently inadequate.

The three aforementioned species were so far each collected from distinct monocot plants belonging to three plant families ( Agavaceae , Cyperaceae and Juncaceae ) on three different continents ( Table 3 ).

Species of Oziella

Character

O. sibirica sp. nov. O. yuccae O. rufensis Remarks on the position of setae 3a in immature instars. In contrast to adult mites which have 3–5 annuli before the epigynium or epiandrium, the immatures of Oziella sibirica sp. nov. possess an average 11–12 annuli before setae 3a ( Figs. 5 & 6 ). Similar ratios of 2–3 times more annuli in immatures versus adults were noted in the phytoptid genera Phytopus Dujardin 1851 , Setoptus Keifer 1944 , Boczekella Farkas 1965 and Trisetacus Keifer 1952 (P.E. Chetverikov, unpublished data 2008–2012). In adult eriophyoids, setae 3a are always intimately flanking the external genitalia. The genital shield (more or less) closely appressed to the coxisternal plates is thought to be one of the apomorphies of Eriophyoidea ( Lindquist 1996b, p. 303). In this context, the relatively remote position of setae 3a in immature instars of phytoptids could represent a more plesiomorphic character state in contrast to the immatures of representatives of the families Eriophyidae Nalepa, 1898 and Diptilomiopidae Keifer 1944 , which as we know usually have setae 3a positioned quite more anteriorly. In Novophytoptus , setae 3a are positioned even further posteriorly. For instance, larvae and nymphs of N. rostratae Roivainen 1947 have 12–15 and 19–22 annuli before setae 3a, respectively (P.E. Chetverikov, unpublished data 2005–2007 and 2011–2012). Furthermore, this posterior position of 3a is retained in adults: 10–12 annuli separate the epigynium from coxae II in N. rostratae , and 12–16 in other species ( Lindquist 1996a p. 20; Chetverikov & Sukhareva 2007). This could indicate a more ancient origin of the genus Novophytoptus relatively to other phytoptid genera, if this condition is plesiomorphic. However, this posterior position of setae 3a and the epigynium could represent a secondarily derived condition associated with the transformation of the body of Novophytoptus in adapting to living inside air cavities and sheaths of herbaceous monocotyledonous plants of the families Poaceae , Cyperaceae and Juncaceae ( Chetverikov & Sukhareva 2007; Chetverikov 2010; E. E. Lindquist pers. comm. 13 December 2011).

Variability of empodium morphology. Recently, some of us have shown that the study of variability of empodia may help separate cryptic species or distinguish morphologicaly similar protogyne and deutogyne females belonging to the same species (Chetverikov et al. 2009; Chetverikov 2011). Therefore, we recommend that descriptions of new taxa include detailed description of the morphology of the empodia, based on light microscopy (with phase or differential interference contrast) but ideally also on scanning electron or confocal laser scanning microscopy and its variability in both adult and immature stages. Immatures of O. sibirica sp. nov. possess two empodium variants, with larvae usually having 4/3-rayed empodia (rarely 4/4-rayed empodia), and nymphs 4/3- about as frequently as 4/4-rayed empodia ( Figs. 5 F, 6G & 6H; Table 4 ). In addition, a single nymph was found with an abnormal empodium on right leg II with two anterior rays elongate and flattened ( Fig. 6 I). All studied male and female adults have 5/4-rayed empodia, except a single female having one 4/4- and three 5/4- rayed empodia. Thus, O. sibirica sp. nov. exhibit a gradual increase in the number of empodial rays during ontogenesis from 4/3-rayed empodia in the larval stage to 5/4-rayed empodia in the adult stage, and with a significant proportion of the nymphs with intermediate, 4/4-rayed empodia.

Possession of eye-like structures ( Figs. 1 , 2 , 3 C, 4B, 5B & 6C). CLSM imaging carried out at different layers indicated that these structures are dense, ovoid globes that protrude 1–2 mkm outside the body and 3–4 mkm deep inside. The eye-like structures of O. sibirica sp. nov. are similar in shape and position (laterad ve setae) to those observed in Novophytoptus stipae Keifer (1962) (Phytoptidae) . In Phytoptus oculatus Smith (1977) and in the few eriophyoids known to have lens-like structures (see Flechtmann et al. 1995 and taxa listed therein), these appear to be slightly more posterior. Unpublished observations (P. Chetverikov, December 2011) based on CLSM indicate that members of additional phytoptid and eriophyid genera possess similar eye-like structures. Such a threedimensional structure suggests that it is almost certainly a true light-sensitive organ (R. Ochoa & E. Lindquist pers. comm. May 2012 & June 2012, respectively), and its presence in many eriophyoid taxa, including phytoptids, suggests that it was also present in ancestral eriophyoid stock, as suggested by Smith (1977).

Description of the internal genitalia. The genital apparatus of eriophyoid mites is difficult to study because it is small and internal. According to a transmission electron microscopy (TEM) study of eriophyoid mite anatomy by Nuzzaci and Alberti (1996, pp. 138–142), the female reproductive system includes soft organs (ovary, oviduct and muscles), and more or less sclerotized, chitinous structures: (1) paired spermathecae, connected to a (2) genital chamber via (3) spermathecal ducts or tubes (each provided with a special valve); the chamber is walled anterodorsally by (4) a genital apodeme, and covered ventrally by an (5) epigynium (genital coverflap). Elements 1–4 are internal whereas the epigynium is essentially external or superficial. The soft parts of the reproductive system are visible by TEM only, whereas the sclerotized elements are sometimes visible and more easily studied under light microscopy. Among light microscopy techniques, the most powerful for studying internal genitalic structures of eriophyoids was recently shown to be confocal laser scanning microscopy ( Chetverikov 2012b). The reproductive systems of eriophyoid mites remain poorly studied and no standards exist for describing them. Here, using O. sibirica sp. nov. as an incipient model, we propose a relatively simple protocol for describing the most distinct parts of the internal genitalia of eriophyoid mites based on CLSM image analysis ( Figs. 7 , 8 & 9 ). The method will probably need to be adjusted to accommodate the variation in shape and structures across taxa, and also for progress in the interpretation and homologization of structural components. For example, some structures have not yet been discussed in the literature (although coarsely illustrated in species descriptions) and their variation in form and function are essentially unknown. A more or less sclerotized, longitudinal structure is present medially, meeting with the genital apodeme anteriorly and the paired spermathecal tubes posteriorly. The function of this structure is yet to be determined. Here, it is provisionally called ‘longitudinal bridge’. Posteriorly, this structure appears to be relatively thicker and more rigid, whereas the anterior part seems to be composed of a more pliable, membranous tissue ( Figs. 7 , 8 & 9 ; see also Chetverikov 2012b). Another structure, tentatively identified as a distal segment of the spermathecal tube, was also found in the new species, O. sibirica sp. nov. (herein called ‘prespermathecal swelling’; Figs. 7 , 8 B, 9E–H). Moreover, the architecture of the genital apodeme varies broadly and additional measurements (lengths, angles) could be taken for describing this adequately in other species.

Because of several artifacts and the variability of specimen-clearing during the slide-mounting process, a large proportion (e.g. over two-thirds) of the slide-mounted specimens did not show accurately or in entirety, the structures of the internal genitalia (e.g. spermathecae, apodemes). Therefore, studying the internal genitalia of eriophyoids under CLSM requires the examination of many specimens before appropriate images can be obtained and structures measured and understood (see also Chetverikov 2012b).

Description of the internal genitalia of Oziella sibirica sp. nov. Spermathecae ellipsoid ( Fig. 8 A), oriented posterolaterally (40º–50º angle with longitudinal body axis), without any bend or bulge, 12.1 long, 4.7 wide (see Table 5 for standard deviations & ranges). Proximal part of spermathecal tube ( Fig. 8 C) short (3.3) with margins hard to distinguish. A spherical prespermathecal swelling (representing presumably the distal segment of spermathecal tube) ( Fig. 8 B). 3.6 long, 3.4 wide, forming an angle of 90º–120º with spermatheca. Longitudinal ‘ bridge’ ( Fig. 8 D) 17.0 long, posteriorly thicker for approximately 2/5 of its length, and more membranous and pliable anteriorly. Genital apodeme a transverse plate curved lateroposterad, with each of the left and right halves 14.9 long (if measured as a straight line, excluding fold), forming together a rounded (or pointed, Fig. 7 A & 7D) angle of 80º–100º medially (as if each half forms two straight lines); varying from a subtriangular shape, with almost straight halves ( Fig. 7 A–7C), to a flattened bell shape ( Fig. 7 D). Transverse apodeme distally folded on itself: first anteriorly, towards itself, and then posteriorly and tightly; each of these folds 2.2 long (note that the last fold distally connects to the anterior end of the rim of the genital aperture, and is sometimes visible, as in Figs. 7 B & 8).

Structure Mean±SD Min–max COI sequence. The barcode region (sensu Hebert et al. 2004) of mitochondrial cytochrome oxidase subunit I gene (COI) in Oziella sibrica sp. nov. was successfully amplified and sequenced. This sequence is 657 bp long and available from GenBank under accession number NCBI JQ342669 . Base pair frequencies show that the region is AT-rich (A: 0.205, C: 0.137, G: 0.164, T: 0.494).

Figures

FIGURE 2. Variation of the prodorsal shield design among four females of Oziella sibirica sp. nov. (black & white inverted CLSM images). Admedian lines colourised in red (A & D), additional lines in green (B) and red (C; the same female as in Fig. 2 A & 2 B). Scale bar = 30 mkm.

FIGURE 3. Oziella sibirica sp. nov., female. A. Ventral view of the mite, B. Coxigenital area, C. Female prodorsal shield; D. Female internal genitalia, E. Empodium; F. Epiandrium, G. Leg I (arrow indicates a spine), H. Leg II. Scale bar: A = 130; B & C = 45; D = 35; E = 15; F = 45; G & H = 35.

FIGURE 4. Microphotographs under light phase contrast microscopy (A & B) and CLSM (C & D) of Oziella sibirica sp. nov. A. Coxigenital region; B. Female prodorsal shield (arrows indicate the eye-like structures); C. Position and view of internal genitalia inside female; D. Epiandrium on male venter. Scale bar A, B, C, D = 20 mkm.

FIGURE 5. Oziella sibirica sp. nov., larva. A. Ventral view; B. Prodorsal shield and anterior part of opisthosoma; C. Coxisternal area and anteroventral region of opisthosoma; D. Right leg I, dorsal view; E. Right leg II, dorsal view; F. Typical 4 / 3 - rayed empodium. Scale bar: A = 100; B & C = 45; D & E = 35; F = 10.

FIGURE 6. Oziella sibirica sp. nov., nymph. A. Dorsal view of the mite; B. Ventral view; C. Prodorsal shield; D. Coxigenital area; E. Right leg I (arrow indicates a spine); F. Right leg II; G. Typical 4 / 3 - rayed empodium; H. Typical 4 / 4 - rayed empodium; I. Abnormal empodium. Scale bar: A & B = 140; C & D = 50; E & F = 45; G, H & I = 12.

FIGURE 7. Variation found in the internal genitalia of Oziella sibirica sp. nov. specimens.

FIGURE 8. CLSM image of the internal genitalia of Oziella sibirica sp. nov. A. Spermatheca; B. Pre-spermathecal swelling (distal segment of spermathecal tube); C. Proximal segment of spermathecal tube; D. Longitudinal bridge; E. Transverse apodeme; F. Laterodistal fold of transversal apodeme.

FIGURE 9. Morphometrics of the internal genitalia of Oziella sibirica sp. nov. Length (A – B) and width (C – D) of spermatheca; length (E – F) and width (G – H) of prespermathecal swelling (distal segment of spermathecal tube); length (I – J) of proximal segment of spermathecal tube; length (J – K) of longitudinal bridge; half-length (K – L) of genital apodeme; maximal distance between left and right parts of transversal apodeme (L – M).

FIGURE 1. 3 D model of Oziella sibirica sp. nov. prodorsal shield (the same female as Fig. 2 C). A. Gray scale prodorsal shield, B. Colourised prodorsal shield (notifications of lines follows that of Amrine et al. 1994 & Amrine et al. 2003; admedian lines colourised in red, additional line between admedian and submedian- 2 line colourised in black). Scale bar A & B = 30 mkm. Note: setae ve and c 1 are short on images A & B because only proximal parts of the setae of eriophyoid mites can be observed on CLSM images using blue laser, 405 nm (Chetverikov 2012 b).

Tables

TABLE 2. Morphological measurements of Oziella sibirica sp. nov.

CHARACTER	FEMALE (n=8) MALE (n=5) Mean±SD Min–max Mean±SD Min–max	LARVA (n=9) NYMPH (n=8) Mean±SD Min–max Mean±SD Min–max
Body length	353 ± 15.6321–374 310 ± 8.7 301–322	206 ± 14.1 182–225 287 ± 13.7 273–318
Body width	59 ± 3.5 53–65 56 ± 1.9 53–58	43 ± 2.5 38–46 53 ± 5.0 49–60
Prodorsal shield length	34 ± 1.6 32–36 31 ± 1.0 30–32	25 ± 1.4 22–27 30 ± 1.5 28–32
Prodorsal shield width	42 ± 3.7 36–46 43 ± 1.9 40–44	35 ± 1.9 31–37 41 ± 2.4 37–44
Gnathosoma length	25 ± 1.1 24–27 26 ± 0.8 25–27	19 ± 0.9 18–20 21 ± 1.0 20–23
v (apical s.)	8 ± 1.1 7–10 7 ± 1.3 6–9	1 ± 0.4 0.5–2 4 ± 0.8 3–5
ve (dorsal s. I)	9 ± 0.7 8–10 9 ± 0.9 8–10	5 ± 0.7 4–6 6 ± 0.6 5–7
ve–ve distance	23 ± 2.2 20–26 24 ± 0.4 23–24	19 ± 0.8 18–20 21 ± 1.1 20–23
sc (dorsal s. II)	1 ± 0.3 0.5–1.5 1 ± 0.0 1–1	1 ± 0.2 0.5–1 1 ± 0.2 0.5–1
sc–sc distance	18 ± 1.4 16–20 18 ± 0.5 17–18	14 ± 0.3 14–15 16 ± 1.1 15–18
ve–sc distance	19 ± 0.5 18–19 19 ± 1.0 18–20	15 ± 0.7 14–16 17 ± 0.7 16–18

TABLE 3. Morphological differences between Oziella sibirica sp. nov., Oziella yuccae Keifer and Oziella rufensis Manson.

Body length	353 (321–374)	290–340	234–303
No. empodial rays	5/4, asymmetrical	7/8, asymmetrical	6, symmetrical?
ve	8 (7–10)	12	13–19
sc	1 (0.5–1.5)	5	11–19
c1	21 (18–24)	40	71–107
d	7 (6–8)	15	18–23
Host plant	Asian sedge, Carex macroura Meinsh. [ Cyperaceae ]; widely distributed in East Europe	Soapweed yucca , Yucca glauca Nutt. [ Agavaceae ]; North American endemic	Red woodrush, Luzula rufa var. rufa Edgar [ Juncaceae ]; New Zealand endemic
References	This paper; Egorova 1999	Keifer 1954; Hess & Robbins 2003	Manson 1970; Moore & Edgar 1970

TABLE 4. Proportion of mites having each variant of empodia (4 / 3, 4 / 4 and 5 / 4 - rayed) on legs I & II of active stages of Oziella sibirica sp. nov.

Stage	N 4/3	Both 4/3 and 4/4	4/4	Both 4/4 and 5/4	5/4
Larva	9 8 (89%)	1 (11%)	–	–	–
Nymph	8 3 (37.5%)	3 (37.5%)	2 (25%)	–	–
Female	8 –	–	–	1 (12.5%)	7 (87.5%)
Male	5 –	–	–	–	5 (100%)