Two New Species of the Mite Genus Stereotydeus Berlese, 1901 (Prostigmata: Penthalodidae) from Victoria Land, and a Key for Identiﬁcation of Antarctic and Sub-Antarctic Species

: Two new mite species belonging to the genus Stereotydeus Berlese, 1901 were discovered from locations along the coast of Victoria Land, continental Antarctica. Previous records of this genus in the area under study only reported the presence of S. belli and S. mollis . Although those studies included no morphological analyses, it has since been assumed that only these species were present within the area. Specimens of S. ineffabilis sp. nov. and S. nunatakis sp. nov. were obtained, sometimes in sympatry, from four different localities in Central and South Victoria Land and are here described and illustrated using optical and scanning electron microscopy (SEM) techniques. Features useful for identiﬁcation of the two new Stereotydeus species include the size of the specimens, the length of the apical segment of pedipalps, the presence/absence of division of the femora, the position of solenidia, the shape and disposition of the rhagidiform organs on the tarsi, the shape of the apical setae of the tarsi, the numbers of aggenital setae and the position of the anal opening. A key to 14 of the 15 currently described Antarctic and sub-Antarctic Stereotydeus species is provided.


Introduction
Free-living mites are amongst the most abundant and widespread group of Antarctic arthropods [1,2], with the best-represented groups being Prostigmata, Oribatida and Mesostigmata. Within the continental and maritime Antarctic regions, 40 different species have been recorded considering only the two first orders, with 12 genera from six families for Prostigmata and eight genera from seven families for Oribatida [3]. Within the Prostigmata, one of the most represented families is Penthalodidae, which includes the cosmopolitan genus Stereotydeus Berlese, 1860 [4]. To date, a total of 13 Stereotydeus species have been recorded in Antarctica: six from continental Antarctica, one from maritime Antarctica, and seven from the sub-Antarctic (Table A1). Three members of the genus (S. belli (Trouessart, 1902), S. delicatus Strandtmann, 1967 and S. punctatus Strandtmann, 1967) are known from Northern Victoria Land and two (S. mollis Womersley and Strandtmann, 1963 and S. shoupi Strandtmann, 1967) from Southern Victoria Land and the immediate vicinity of the central Transantarctic Mountains (Table A1). Since the original morphological studies conducted on Antarctic Stereotydeus species (e.g., [2,[5][6][7]), little research has been carried out on the genus in recent years, with studies investigating their physiology, ecology [8][9][10] and molecular diversity [11][12][13]. With the combination of great morphological and genetic variability emerging from these studies and the incomplete and fragmented information about their distribution in coastal regions of Victoria Land, we conducted a morphological taxonomy study of two newly discovered Stereotydeus species from the region. A more detailed population genetic study inclusive of several Stereotydeus species (Brunetti et al., unpublished data) will provide a wider picture of the distribution in Victoria Land of taxa of the genus. These are of interest both in terms of taxonomic knowledge of the genus and in underpinning the development of future conservation plans in the Antarctic.

Samples Collection and Preparation
Stereotydeus specimens were collected from four different locations along the Victoria Land coast (Table 1) Table 1) were placed on a slide with few drops of lactic acid (20%) at room temperature for three weeks, allowing the samples to clear. Individuals were then observed under a Leica DM RBE microscope. For preparing illustrations, a camera lucida attached to a Leica DM LB microscope was used. The specimens used were then transferred to slides in Hoyer's mounting medium for permanent preservation. Table 1. Sampling location details including respective area codes and coordinates, the date of collection, the altitude of the sampling site and numbers of the specimens prepared for optical microscopy (n.) and scanning electron microscopy (SEM). Some individuals were also prepared for scanning electron microscope (SEM) observation (Table 1). To better clean the samples, an additional washing step with absolute ethanol was performed. Then, the ethanol was removed by evaporation in a Balzer CPD 010 reaching the CO 2 critical point. Individuals were then placed on an aluminum stub and coated with gold-palladium in a Balzer MED 010, before observation using a Philips XL20 electron microscope.

Morphological Characters under Study
Characters used to identify the genus Stereotydeus include the presence of a trilobed epirostrum with epivertex at the base of the middle lobe and genitalia in a nearly circular camerostome covered by two valves separated from the body by a distinct suture [7]. Characters used to separate different species include the division in two segments of the femora, the degree of development of the epirostrum and dorsal sculpturing. In addition, the following features were useful in distinguishing the species described here from those already known: body length, the position of solenidia, length of the apical (4th) segment of pedipalps and length of the movable digits of the chelicerae, presence of rhagidial organs on the tibiae, the symmetry or asymmetry of the rhagidiform organs on the tarsi and the shape of the apical setae on top of the tarsi, numbers of the aggenital setae and the position (distal/ventral) of the anal opening on the hysterosoma. Etymology: From Latin, "ineffabilis" meaning inexpressible, ineffable because of its fragile nature.

Systematics
Description: Soft-bodied mite with a barely visible sculptured pattern on the prodorsum and sclerotization almost absent. The body length of the Holotype is 408.44 µm; the average length of adult specimens studied is 414 µm, with values ranging from 369 to 460 µm (±25 µm). The size is smaller than S. mollis [2,5]. The shape of the body is similar to the other species of the genus. The propodosoma is divided from the hysterosoma by a distinct sejugal furrow. The morphological features are illustrated in  Dorsal side: The epirostrum is trilobed but with the two lateral lobes weakly developed and with a slightly striated epivertex with two ciliated setae at the base of the middle lobe (Figures 1a and 5c). The eyes are convex and lightly striated. Three pairs of slit pores are present on each side of the dorsum (Figures 1a and 5a); as for other species of the genus, the hysterosoma carries 8 pairs of plumose setae (Figures 1a and 5a).
Ventral side: As in other species of the genus, the genitalia are situated in a circular camerostome protected by two valves which, under the optical microscope, laterally are hardly separated from the body wall (as in S. mollis) (Figures 1b and 5b). Seven pairs of internal genital setae are present (Figures 1b and 5b). Each genital valve has 6 setae, of which the fourth is more lateral. The aggenital setae are present in 5 pairs, but often the specimens observed showed asymmetry in the number with a total of 9 setae (Figures 1b and 5b). The anal opening in ventral and distal position is smaller than the genital pore, and it is covered by two valves and surrounded by 3 pairs of plumose setae.
Gnathosoma: Rostrum triangular with 2 pairs of nude apical setae. Chelicerae plump, finely pubescent; movable digit about the same length as the fixed digit; two setae at the base of the movable digit and shorter than the digit itself ( Figure 1c). Pedipalps finely pubescent with the terminal segment slightly slender and same length as the sub-terminal segment (Figure 1d) with basal dorso-lateral rhagidiform organ and 7 apical setae. the latter rhagidial organ on tarsi I and II (Figures 2-4). Chaetotaxy of the legs very variable: trochanters with 1 seta, femora I, II, III and IV with 17 in INE and 16 (or 15; one is weaker and less developed, sometimes missing) in PRI, 15,11 (INE) and 9 (PRI) and 8 setae, respectively; genua I, II, III and IV with 8, 6 (INE) or 7 (PRI), 5 and 5 setae, respectively; all tibiae with 6 setae each; tarsi I, II, III and IV with 18 (sometimes 17 in INE), 13,9 (INE) or 12 (PRI) and 13, respectively; leaf-shaped, brush-like setae between the claws on top of the empodia (Table 2; Figures 3 and 4).        Legs: The legs are slender and shorter than the body: the second pair is shorter than the other pairs but almost comparable in length with the third while the first pair is slightly shorter than the fourth (Figures 3 and 4). Coxal setal formula: 3, 1, 4, 3. All femora are undivided. Solenidia: basal and dorsal on genua I, II and III; at 2/3 (mid-basal) of the tibia's length in all legs of the specimens. Tibiae I and II with a small apical rhagidial organ, as in S. mollis (Figure 5b,e). Tarsi I and II with three different rhagidiform organs: the two apicals are smaller than the basal one, longer and obliquely placed in a confluent (tarsi I) or continuous (tarsi II) field (Figures 2 and 5d). In the samples from Prior Island, the three rhagidiform organs on tarsi I and II are often in line with the basal one, only slightly oblique in confluent fields (Figure 5b). A small nude round seta is present at the base of the latter rhagidial organ on tarsi I and II (Figures 2-4). Chaetotaxy of the legs very variable: trochanters with 1 seta, femora I, II, III and IV with 17 in INE and 16 (or 15; one is weaker and less developed, sometimes missing) in PRI, 15, 11 (INE) and 9 (PRI) and 8 setae, respectively; genua I, II, III and IV with 8, 6 (INE) or 7 (PRI), 5 and 5 setae, respectively; all tibiae with 6 setae each; tarsi I, II, III and IV with 18 (sometimes 17 in INE), 13, 9 (INE) or 12 (PRI) and 13, respectively; leaf-shaped, brush-like setae between the claws on top of the empodia (

Inexpressible Island
Dorsal I 0 0 10- 11  5  3  8  II  0  0  9  4  3  5  III  0  0  6  2  3  4  IV  0  0  4  3  3  5   Ventral   I  3  1  5  3  3  10  II  1  1  6  3  3  8  III  4  1  3  3  3  8  IV  3  1  4  2  3  8   Prior Island   Dorsal   I  0  0  10  5  3  7-8  II  0  0  9  3  3  5  III  0  0  7  2  3  4  IV  0  0  4  3  3  5   Ventral   I  3  1  7  3  3  10  II  1  1  6  3  3  8  III  4  1  4  3  3  5  IV  3  1  4  2  3  8 Remarks: Comparing S. ineffabilis adults with S. mollis as described in [2,5,7,14], the characters that help to positively identify and distinguish S. ineffabilis were: (i) the smaller size of the adults; (ii) the number of the aggenital setae (10 or 9 in S. ineffabilis, 8 in S. mollis); (iii) asymmetry in the tarsal rhagidial organs in S. ineffabilis specimens; (iv) the terminal (4th) segment of the pedipalps being as long as the sub-terminal (3rd) and bearing 7 setae in S. ineffabilis (while in S. mollis the 4th segment has a cluster of 8 setae and is longer than the 3rd segment); (v) the equal length of the movable and fixed digits of the chelicerae in S. ineffabilis (while in S. mollis the 4th segment is longer than the 3rd) and (vi) the position of the solenidia on the tibiae and the genua. Two specimens (one male from Campo Icaro and one from Inexpressible Island) showed a wrinkle (like a hint of division) at the apical quarter of the femora (in I and II, the wrinkle is barely visible while in III and IV it is clearer) and ventral but almost terminal position of the anal opening. Another specimen from INE prepared for SEM showed wrinkles at the terminal part of the femora. The indication of femora divisions observed under the optical microscope technique may be due to the occurrence of the folding of the cuticle as shown with the SEM technique.
Stereotydeus nunatakis sp. nov. Brunetti Material Examined for the Description: Starr Nunatak, 1 + 6 slides (2 + 2 ♀, 0 + 2 ♂, 0 + 2 nymphs) and Prior Island, 2 slides with single individuals (2 ♀). All specimens collected by A. Carapelli and deposited in the Collection of the Department of Life Sciences at the University of Siena.
Etymology: named after the ice-free ridge at the holotype locality, Starr Nunatak, continental Antarctica.
Description: Soft-bodied mite well sclerotized with a clearly visible sculptured pattern on the dorsum. The body length of the holotype is 566.89 µm; the average length of the adult specimens studied is 563 µm, with values ranging from 537 to 582 µm (±13.57). The adults of the species are comparable in size with S. belli [5]. The shape of the body is similar to the other species of the genus and the furrow dividing the propodosoma from the hysterosoma is evident both ventrally and dorsally. The morphological features are illustrated in Figures 6-8.
Taxonomy 2021, 1, FOR PEER REVIEW 10 Description: Soft-bodied mite well sclerotized with a clearly visible sculptured pattern on the dorsum. The body length of the holotype is 566.89 µm; the average length of the adult specimens studied is 563 µm, with values ranging from 537 to 582 µm (±13.57). The adults of the species are comparable in size with S. belli [5]. The shape of the body is similar to the other species of the genus and the furrow dividing the propodosoma from the hysterosoma is evident both ventrally and dorsally. The morphological features are illustrated in Figures 6-8.    Dorsal side: The epirostrum is strongly trilobed and lightly striated and the epivertex is almost smooth with two ciliated setae at the base of the middle lobe (Figures 6a and 8d). Propodosomal area is strongly sclerotized, more than the rest of the dorsum showing an evident reticulated pattern that lightly fades in the proximity of the sejugal furrow (Figures 6a and 8d). The eyes are convex and striated. Three pairs of slit pores are present on each side of the dorsum (Figure 6a): the first pair is horizontal (parallel to the sejugal furrow), and the two other pairs are oblique ( Figure 6b); as for other species of the genus, the hysterosoma carries 8 pairs of plumose setae (as shown in the Figures 6a and 8a) almost all of the same length.
Ventral side: As in the other species of the genus, the genitalia are situated in a circular camerostome protected by two valves which laterally are well distinct from the body wall (as in S. belli). Seven pairs of internal genital setae (Figure 6a). Each genital valve holds 6 setae, of which the fourth is more lateral. The aggenital setae are present in 5 pairs, but specimens were also observed with asymmetry in the number (4/5 and 5/6) (Figures 6b and 8b). The anal opening in ventral and distal position is smaller than the genital pore, and it is covered by two valves and surrounded by 3 pairs of plumose setae.
Gnathosoma: Rostrum triangular with 2 pairs of apical nude setae. Chelicerae are plump and finely pubescent, and the movable digit is longer than the fixed digit ( Figure 6c). Two nude setae, one as long as the movable digit at the base of the fixed digit while the second is about half of the length of the movable digit, and it is positioned lower than the latter. Pedipalps finely pubescent with the terminal segment same length as the sub-terminal segment with 7 apical setae and basal dorso-lateral rhagidiform organ (Figures 6c and 8c).
Legs: The legs are slender and shorter than the body: the second pair is shorter, and the fourth pair which is longer than the others, while the first and third pairs are almost similar in length (Figure 7). Coxal setal formula: 3, 1, 4, 3. Trochanters all bearing 1 seta. All femora are divided. Solenidia: mid-basal (2/3) on tibiae I, II and IV and genua I and II, medial on tibia and genu III (but sometimes also mid-basal). Small apical rhagidial organ is present also on tibiae I and II. Tarsi I and II with three symmetrical rhagidiform organs similar in length laying in a common field ( Figure 8e); a small nude round seta is present at the base of the proximal rhagidial organ on both tarsi I and II. Chaetotaxy of the legs: femora: 12/5, 10/5, 7/4, 6/2; genua: 11, 6, 5, 5; all tibiae with 6 setae each; tarsi: 21, 14, 12, 15; leg setae plumose except for 1-2 pairs at the tips of all tarsi clavate and brush-like ( Figure 8f); bulb-shaped, brush-like setae between the claws on top of the empodia (Table 3; Figure 7). Table 3. Chaetotaxy of S. nunatakis legs. Dorsal and ventral refer to surfaces of the leg segments; chaetotaxy in the femur shown as basal/apical segment; solenidia are excluded from the count, whereas trichobothria are included (see Figure 7 for the details and position leg chaetotaxy). c. coxa, tr. trochanter, f. femur, g. genu, ti. tibia and ta. tarsus.

Leg
c. tr. f. g. ti. ta. I  0  0  9/3  4  3  9  II  0  0  6/3  3  3  6  III  0  0  5/3  2  3  4  IV  0  0  2/2  3  3  7   Ventral   I  3  1  3/2  7  3  12  II  1  1  4/2  3  3  8  III  4  1  2/1  3  3  8  IV  3  1  4/-2  3  8 Remarks: Comparing S. nunatakis adults with other Stereotydeus species, the species appears more closely related to S. punctatus as described in Strandtmann [7] and to S. belli as described in Womersley and Strandtmann, and Strandtmann [5,7], sharing division of all the femora at the apical quarter, the prominent dorsal sculpturing and the apical (4th) segment of the pedipalps being as long as the 3rd, while, for the size of the adults, the number and position of the rhagidial organs on tarsi I and II of S. nunatakis are comparable only with S. belli as described in Womersley and Strandtmann [5]. Conversely, the characters that help to positively identify and distinguish adults of S. nunatakis from both S. belli and S. punctatus were: (i) the number of aggenital setae (10 with possible asymmetry of 9 or 11 in S. nunatakis, while 8 in S. punctatus and 22 in S. belli); (ii) the terminal (almost apical) position of the anal opening in S. nunatakis (while sub-terminal in S. belli and dorsal in S. punctatus); (iii) the position of the solenidia is mid-basal on tibiae and genua I, II and tibia IV and medially on tibia and genu III in S. nunatakis (while in S. punctatus all are basal on tibiae I-IV and genua I-III, and in S. belli are apical on tibiae and genua I, II and tibia III and medial on genua III and tibia IV); (iv) the bulb-shaped and brush-like setae terminally on the tarsi in S. nunatakis (while in S. punctatus and S. belli these setae are slender and plumose, not different from all the others present on the legs); (v) the 4th segment of the pedipalps bearing 7 plumose setae in S. nunatakis (while 8 in S. belli) and, finally, (vi) the chaetotaxy of the legs.

Key to the Antarctic and Sub-Antarctic Stereotydeus Species
This key includes 14 of the 15 Stereotydeus species described to date from continental and maritime Antarctica and the sub-Antarctic islands. Stereotydeus intermedius Trouessart, 1907 is excluded from the key due to the lack of information and data about this species [7,[15][16][17] (see Tables A1-A7 in Appendix A). Additional information about morphological characteristics useful for identification is provided in Appendix A (Tables A1-A7).
This key was constructed based on the original morphological descriptions of the known species. Besides S. ineffabilis and S. nunatakis, specimens of S. belli, S. punctatus and S. delicatus from Victoria Land were also available for morphological examination. A separate molecular phylogenetic analysis of these five species is currently being carried out (Brunetti et al., unpublished data).

Discussion
The two newly described species are placed in the genus Stereotydeus following the key of the free-living mites of Antarctica provided by Strandtmann [7] and in particular because of the presence of a trilobed epirostrum with epivertex at the base of the middle lobe, the genitalia placed in a distinct and nearly circular camerostome and covered by two valves and the dorsum with a more or less defined reticulated pattern [6].
It was possible to distinguish Stereotydeus ineffabilis sp. nov. from the other species belonging to the genus because of (i) the small size of the adults (ca. 100 µm smaller if compared to, e.g., S. mollis; see Table A2); (ii) the fourth segment of the pedipalps being as long as the third (longer in the other Stereotydeus species from North Victoria Land (NVL), shorter in S. shoupi from South Victoria Land (SVL); Figure 1d, Table A2) and bearing seven setae (while e.g., S. belli and S. mollis have a cluster of eight setae; Figure 1d, Table A2); (iii) the equal length of the movable and fixed digits of the chelicerae (movable digit longer than the fixed one in S. belli, S. mollis and S. villosus [5]; see Figure 1c); (iv) the presence on the hysterosoma of two faintly visible longitudinal grooves (absent in e.g., S. mollis and S. shoupi; Figure 1a, Table A3) and of three dorso-lateral slit pores (absent in S. mollis and S. shoupi; Figures 1a and 5a Table A5); (ix) the position of the solenidia on the tibiae and the genua (Figures 3 and 4, Table A6) and (x) the asymmetry and different length of the tarsal rhagidial organs (Figures 2-4, Table A7).
For Stereotydeus nunatakis sp. nov., it was possible to distinguish it from the other species of the genus because of (i) the large size of the specimens (compared to S. mollis and S. shoupi recorded in SVL; Table A2) Table A6).

Conclusions
Several decades after the first descriptions of Stereotydeus species from Antarctica, the current study describes two new species, S. ineffabilis sp. nov. and S. nunatakis sp. nov. With the exclusion of S. intermedius Trouessart 1907, the key presented here and the information in Appendix A provide robust and reliable tools for the rapid identification of Antarctic and sub-Antarctic representatives of Stereotydeus. Together with the new species described herein, future molecular genetic studies of three additional species from Victoria Land (S. belli, S. punctatus and S. delicatus) will shed light on the phylogenetic relationships within the genus and increase knowledge of the distribution of Stereotydeus species in continental Antarctica. Funding: This study was funded by the Italian Program of Research in Antarctica (PNRA16_00234) to A.C. Partial support was also provided by the University of Siena. P.C. is supported by NERC core funding to the British Antarctic Survey's 'Biodiversity, Ecosystems and Adaptation' Team. The paper also contributes to the SCAR 'State of the Antarctic Ecosystem' (AntEco) international program.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The list of the data presented in this study is available on https: //steu.shinyapps.io/MNA-generale/.

Acknowledgments:
We would like to thank Alessandro Gradi and Massimo Migliorini for their technical assistance. We wish to thank also the two anonymous reviewers for their helpful comments.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Appendix A
To provide a clear overview of some morphological characteristics useful for the identification of all the Antarctic and sub-Antarctic Stereotydeus species known to date, different synoptic tables are provided below. All information regarding morphology has been extrapolated from the original descriptions of the authors, with additional personal observations by C.B. (cited in the tables).          Figure 4) Undivided S. shoupi [7], Figure 3 Ventral (almost sub-terminal o.d. Figure 3a) Slender, shorter than the body, abundant setae than other species of the genus Well distinct (o.d. Figure 3a) Undivided S. meyeri [7], Figure 5 Sub