Two new species of the family Nippobodidae (Acari, Oribatida), including a description of the leg-folding process

Abstract Nippobodespanemorfissp. n. and Leobodestrypasissp. n. are described by means of optical and Scanning Electron Microscopy (SEM) and compared to other congeners. The leg-folding process is described and illustrated. Nippobodespanemorfissp. n. is characterised by interlocking, double hook-shaped, posterior prodorsal condyle and anterior zone humeral apophysis; posterior prodorsal depression present. Tutorium a large lamina defining a pocket-shaped structure; bothridial opening ovoid, situated at the bottom of a U-shaped structure; deep, rounded-ovoid anterior notogastral depression present; ten pairs of notogastral setae; c setae looped, dentate, sharply tipped. Marginal setae h3, p3 on large promontories, followed by deep V-shaped incision; notogaster completely surrounded by circumgastric depression; lateral genital zone with locking structure constituted by longitudinal cuticular elevation, with promontories and a parallel furrow involved in the leg-folding process; genital plate smaller than anal plate. Leobodestrypasissp. n. is characterised by: the presence of posterior prodorsal depression and anterior notogastral depression; bridge-shaped anterior prodorsal condyles; heart-shaped frontal prodorsal orifice; ten pairs of notogastral setae; posterior prodorsal condyle and humeral condyle interlocked, forming double hook-like structure; circumgastric furrow surrounding entire notogaster; setae lp, h2, h1 situated on shallow medial furrow; notogastral setae lm, lp, h1, h2 medially aligned; p1, p2, p3, h3 marginally situated. Legs I-IV, tutorium, pedotectum I, and pedotectum II involved in leg folding which is inferred to be a protection mechanism.


Materials and methods
Specimens studied by means of light microscopy followed the techniques described by Grandjean 1949 andKrantz andWalter 2009. Specimens studied under SEM, followed the techniques of Fernandez 1990a, 1990b;Alberti et al. 1991Alberti et al. , 1997Alberti et al. , 2007Fernandez et al. 1991. Equipment used was the same as for previous studies (see Fernandez et al. 2016).
Optical drawings should be considered semi-schematic with regard to cuticular microsculpture and setal shape. The shape of these specimens made it difficult to orientate the material and obtain the same position consecutively. Studies with SEM provided high levels of precision and detailed Figures; another very important aspect was the positioning system, permitting orientation of material with a much higher level of precision, as well as being able to return to an initial position.
Body measurements taken: total length (from tip of rostrum to posterior edge of notogaster); width (widest part of notogaster). Setal measurements taken on three specimens under SEM. Leg chaetotaxy studies used optical microscopy (standard, polarised, and phase contrast) and SEM.
Setal formulae of legs include the number of solenidia (in parentheses); tarsal setal formulae include the famulus (ε). All measurements are given in micrometres (μm).
Rostrum round, with a conspicuous groove parallel to margin ( Figure 23 indicated by ¿) large hump visible in front of groove (Figures 14).

trajectory indicated by l).
Posterior notogastral view (Figure 7). Deep ovoid p.p.d as well as n.a.d clearly visible; setae c situated on paraxial zone of h.ap.
Trajectory of s.c indicated by l; externally to s.c, flat surface of notogaster extending from s.c to notogastral margin; scalloped zone (behind setae h 3 , p 3 ), some distance from s.c, not interrupting its trajectory.
Remarks. Future ontogenetic studies are necessary in order to confirm nomination of notogastral setae. As only the adult stase was available for study, we used standard, previously used notation (see Morphological terminology). We were unable to locate information on the palp in previous studies. Setae l' of genu II were indicated by Chen and Wang (2007) as bifurcate; however, in our studies only one instance of bifurcate setae l' was observed. Another particularity is the presence of (it) on tarsus IV. The femoral groove was observed, though not indicated in any previous study.
Lamella (Lam) forming conspicuous curved ribbon (Figure 56, 59); running more or less parallel to Tu margin; setae le situated on promontories on apical zone. Pd I: prominent lamina, directing forward, slightly tilted down. Pd II a small lamina, rounded apex; on basal zone a small hump directing outwards ( Figure 62). The area immediately above b.ng is flat, smooth, surrounding the entire notogaster ( Figure 62, trajectory indicated by 6); this flat surface, forms a prominent la.le, parallel to h.ap ( Figure 62); la.le anterior zone, ribbon shaped, (Figure 59, 62, 63); b.ng slightly convex ( Figure 62).

Discussion
Aoki, when establishing the new genus Nippobodes in 1959, initially included it in the family Carabodidae. Almost sixty years later, we propose that the family Nippobodidae presents a series of characters linking these families, as knowledge of the families Nippobodidae and Carabodidae has grown significantly in the intervening years. We consider here only some elements that indicate important similarities: 1) prodorsal posterior depression and notogastral anterior depression situated either side of d.sj; 2) the projection of h.ap overlapping the posterior area of prodorsum; 3) the structures involved in leg folding such as tutorium, pedotectum I, genu (functioning as a hinge), femoral groove in femur III, shapes of femurs. These three elements are insufficient for a comparison, but highlight some aspects indicating a possible relationship between the families. More detailed analysis is required, but hampered by the lack of immature specimens of Nippobodidae and for the greater part of Carabodidae.
Unfortunately descriptions of genera in the family Nippobodidae are often superficial, and in many instances the frontal and posterior views were neglected although  ,dp,v,l" d,l" l',v (ft) they could potentially provide important information. Leg chaetotaxy is problematic and we endeavour to obtain new material in order to study legs in a larger number of specimens. Much of our study material was collected many years ago, and does not permit detailed study, resulting in leg chaetotaxy necessarily being considered provisional. It has been difficult to find a species related to Nippobodes panemorfis sp. n. due to its particular characteristics. Nippobodes flagellifer Chen & Wang, 2007 displays the most characters in common, as both species present similar disposition of: setae ro on tubercle near lateral margin of prodorsum; setae le inserted on tubercle on anterior on lamella; sensillus curved, sickle-shaped, swollen medially; posterior prodorsal condyles interlocking with notogastral humeral apophyse (but dissimilar in shape). Notogastral surface smooth; ten pairs of notogastral setae.
The taxonomy of Leobodes trypasis sp. n. is complex. The species is difficult to compare to other congeners due to their dissimilarity, and the often simplified original descriptions impede adequate comparison. However, there are similarities to L. anulatus Aoki, 1965, such as the presence of a heart-shaped prodorsal orifice, but occurring in a dorsal and not frontal position as in L. trypasis. (Figures 77-88) Fernandez et al. (2013a) have studied the folding of legs as a part of the protection mechanism in various genera of the family Carabodidae. We were fortunate to have the opportunity to examine this process in vivo on adults of Carabodes sp. under light microscopy and document the different steps. Additionally, material was available for SEM-studies, facilitating comparison with other SEM images. For this paper we were unable to conduct in vivo studies of the leg-folding process, but based on a series of observed morphological characteristics and a large number of SEM observations, we do not doubt the presence of similar functions and processes as observed in genera of the family Carabodidae.

Leg-folding process
Some morphological characteristics, however, suggest some variation in aspects of this mechanism.
To understand this process one needs, first of all, to embark on detailed studies of leg structures in Nippobodes (Figures 48-53) and Leobodes (Figures 73-76), as well as other body structures related to leg positioning. Due to the high number of images obtained from more than fifty specimens, selected from a total of more than three hundred animals, only the most representative images of this mechanism have been included for the sake of clarity.

Structures involved
The legs The following is generally observed: tibia-tarsal articulation by means of a small section of synarthrodial membrane, allowing limited movement. Tibia and tarsus are long  and narrow, facilitating positioning either in a pocket-shaped structure delimited by the tutorium (See below), or behind pedotectum I, femur III, and IV (Figures 77-88). Particular characteristics present in superior part of femur I, allows for partial concealment under the lateral prodorsal zone, in front of the bothridial zone ( Figure 82). Femur II presents a slightly curved, smooth posterior surface (Figure 83), coapting with the posterior area of femur III. These structures and surfaces of femurs III and IV permit perfect coaptation, to allow tibia and tarsus to slip in behind and be concealed by them (Figures 82, 87).
Leg III plays a vital role. The femoral groove on femur III is a rather deep, triangular to ovoid groove, with a small seta near the depression. The groove and seta permit an-choring of femur IV into the groove. Femora III and IV each presents a ventral carina, permitting the tibia and tarsus to be concealed under them (Figures 75,76,82,84).
The tiny genu plays a fundamental role as hinge, and generally presents a reduced number of setae (73)(74)(75)(76). The particular shape of the anterior zone of the femur improves the genu-hinge function and assists in the tibia-tarsus in the required position.

Tutorium
The tutorium plays a very particular role, forming a pocket-shaped structure with the lateral prodorsal zone, permitting concealment of tibia-tarsus I. The pocket shaped structure ends in a sharp point, which protects the leg, and houses the claw (Figures 56,57). Pedotectum I conceals the tibia and tarsus of leg II.

Lateral zone of body
The lateral area of body is adapted to receive the legs, with depressions and smooth areas to facilitate their positioning, along with tutorium, pedotectum I, and between legs II, III, and IV.
The "locking structure" Ventrally, behind leg IV, a locking structure is observed. It consists of a longitudinal furrow (on Figures 42, 62, 80, 88, trajectory indicated by l), ending in a bean-shaped structure (on Figures 42, 80, 87, 88 indicated by v), with a lateral promontory (Figures 42,88, dicated by S). Tibia-tarsus IV is inserted into the longitudinal furrow ( Figure 80) and the claw positioned in the depression of the bean-shaped structure (Figures 80, 87, 88). The femur resembles a lid closing a box, preventing the tibia-tarsus from moving and anchoring the entire leg in one position (Figure 81, 82, 88).

The process
Phase 1 (Figure 77): Initial position prior to leg folding, arrows indicating the directions in which legs will move. Phase 2 ( Figure 78): Leg I: femur moves backwards and approaches bothridial zone; this movement is facilitated by the genu functioning as a hinge. By rotating, it permits the tibia and tarsus to approach the margin of tutorium. Then, the tibia and tarsus are positioned ready to initiate installation into the tutorium pocket. Leg II: the femur moves upward and backward, approaching the posterior zone of femur III; the tibia and tarsus approach the margin of pedotectum I and move downwards, to conceal those two segments behind pedotectum I. Leg III: rotates towards the posterior and femur III moves closer to femur II. The tibia and tarsus slide in under ventral trochanteric-femoral carinas for concealment. Leg IV: the femur is directed backwards in order to locate the femoral groove and settle into it. The tibia and tarsus move back to settle into the longitudinal depression indicated by l ( Figure 80).
Phase 3 ( Figure 79): Leg I. Femur I approaches the final position on the bothridial zone; the tarsus and tibia are almost completely concealed behind the tutorium and embedded in the pocket tutorial depression. Leg II. Femur is coapted to the posterior zone of femur III. The tibia and tarsus are almost completely hidden behind pedotectum I. Legs III and IV are very close to each other; femur IV is almost entirely within the femoral groove of femur III. Tibia and tarsus IV are installed in the longitudinal depression of the locking structure and tibia and tarsus III slide in and are concealed under femoral and trochanteral carinae; the claw is visible between trochanters III and IV.
Phase 4, the final position ( Figure 81). Leg I: apical dorsal area of femur positioned under the anterior part of bothridium (indicated by 3 ). The genu rotates inwards and its dorsal part, as well as the tibia and the tarsus are concealed deep in the tutorial pocket depression. Leg II. The genu turns inwards to position the tibia and tarsus in the optimal position; the tibia is slightly curved. With slight rotation of the genu, the tibia is similar in shape to pedotectum I; the tibio-tarsus articulation gives important rigidity. The tibia and tarsus descend and are perfectly concealed behind pedotectum I. Leg III. The posterior zone of femur II glides underneath the anterior part of femur III. Tibia and tarsus III slide in under the carina of trochanter-femur III for concealment, and the tarsal claw is visible between trochanters III and IV. Leg IV: the posterior part of femur IV is placed inside the femoral groove of femur III. Femur IV is inclined upwards to enable the tibia and tarsus to glide in underneath the femur, and tibia and tarsus IV are placed into the longitudinal furrow of the locking structure. To conclude the mechanism, femur IV acts as a lid, blocking this segment and concealing the leg segments in the longitudinal depression, with the claw placed in a horizontal position resting on the bean-shaped structure of the locking structure ( Figure 80).
During the final stages of the coaptation process, the relationship between the legs and body depressions can be described as follows: tibia and tarsus IV are located in the longitudinal furrow (of the locking structure), concealed by the femur; the posterior part of femur IV anchors in the groove of femur III; the apical distal expansion of femora III and IV partially conceal the genu; apical zone of tarsus III is situated between trochanter III and trochanter IV (Figures 79, 81). The f.g allows the posterior part of femur IV to fold into femur III.

Supplementary SEM images
In Figure 82 the final position has almost been reached, with an indication of structures involved, and arrows indicating the final movement to conclude the process. Figure 83 indicates the step where femur II is directed to the anterior part of femur III. The surfaces of the femurs about to come into contact can clearly be seen to fit together perfectly, and other structures such as the setae are located in such a way that they do not impede this process. On the posterior zone of Femur III, the femoral groove shows the seta v, which will assist in anchoring femur IV inside the depression. Figure  84 shows the displacement of femur IV towards the femoral groove (femur III). The carina of the trochanter and femur is clearly visible, which will permit the tarsus and tibia of leg III and leg IV to be partially concealed. Figure 85 posterior view, indicates the final position of legs I, II. The arrows indicate the position of tibia and tarsus I and II, concealed behind the pedotectum I and tutorium. Figure 86 Shows the lateral view of final position of legs I and II. Figure 87, posterior view. Tarsus III and tarsus IV are clearly visible, as well as the claw of tarsus IV and the bean-shaped structure of the locking structure supporting claw IV. Figure 88 shows theset of fundamental elements of legs III and IV, as well as cuticular surfaces necessary for the leg-folding process.

Similarities and differences in leg folding between Carabodidae and Nippobodidae
The system is very similar in the two families, and importantly, the following are common to both: all legs are involved in the process; the presence of the femoral groove on femur III; a tiny genu, which plays the role of a hinge; the involvement of pedotecum I and tutorium to conceal legs I and II.
Differences: 1) In Nippobodidae leg I is concealed in a pocket structure formed by the attachment of the tutorium to the lateral wall of the prodorsum. This connection to the prodorsal body wall resulting in the formation of the pocket structure is very different to Carabodidae (see Fernandez et al. 2013a). 2) The complexity of the locking structure in Nippobodidae, specifically the longitudinal depression where tibia and tarsus IV are inserted, femur IV which functions as a lid, and the bean-shaped structure where the claw rests, are dissimilar to what is observed in Carabodidae.