Application of extended depth of field 3 D imagery to tackle the challenges of cryptic species: a use case in the genus Betiscoides Sjoestedt, 1924 (Orthoptera, Caelifera, Lentulidae) and its taxonomic implications Author Matenaar, Daniela https://orcid.org/0009-0008-4093-930X Hessisches Landesmuseum Darmstadt (HLMD), Friedensplatz 1, 64283 Darmstadt, Germany daniela.matenaar@gmail.com text Evolutionary Systematics 2024 2024-03-25 8 1 65 90 http://dx.doi.org/10.3897/evolsyst.8.117735 journal article http://dx.doi.org/10.3897/evolsyst.8.117735 2535-0730-1-65 6B534FD5542C463687D11738CE33D9F8 F2EBA604A9625D10BE246A37D51D1264 Betiscoides nova sp. nov. Type material. The type material is deposited in the invertebrate collection of Hessisches Landesmuseum Darmstadt (HLMD). Holotype . Male, pinned. HLMD-Cael-4HT, B168, Genbank Acc. PP417675, South Africa, Western Cape province, Jonaskop, 33°58'9.75"S , 19°30'17.99"E , 1553 m above sea level, Plot 07, Restio wetland, 13 Dec 2013, leg. L. Broeder , E. Heym & D.Matenaar, Paratypes . Allotype. Female, pinned. HLMD-Cael-18AT, B166, paratypes HLMD-Cael-5-19PT, all same data as holotype. In total, five female and ten male paratypes are designated. Etymology. The specific epithet is a Latin adjective (feminine form) meaning 'extraordinary' or 'new' . It refers to the species' prominent habitus and to the fact that it is a new species characterized with the help of novel techniques. Definition. The described species is assigned to the genus Betiscoides Sjoestedt , 1924 due to its phylogenetic position and overall morphological characteristics. The species is defined by the elongate, stick-like medium to large sized body, ensiform antennae, which are triangular in cross-section; its conical head, which is in profile almost equal in length and height, large eyes, the trapezoid shape of the prosternal process with two kinds of hairs and little variation in general. The end of the abdomen has only few and short hairs. The arolium is very large. The subgenital plate is elongate and conical; the ovipositor is short with robust, recurved valves. Diagnosis. This new species differs morphologically from the described ones mostly through its robustness and higher body volume and the almost identical HL and HH. The body is less elongate than Betiscoides meridionalis. Genital valves are less acute compared to Betiscoides meridionalis . Furthermore, the new species represents a divergent evolutionary lineage (DEL 19) as stated in Matenaar et al. (2018) and is genetically distinct representing the closest relative of B. parva (p-distance: ~ 0.067). As Key (1937) stated for B. parva and B. sjostedti , also this species is far less elongate than Betiscoides meridionalis , and B. parva , B. nova and B. sjostedti seem morphologically more related to another than any of these to B. meridionalis . In general, the new species seems to be morphologically intermediate between B. parva and B. sjostedti , whereby the robustness is more similar, to B. sjostedti but regarding the general coloration of the body, the shape of the Fr, the large eyes and the angularity of the end of the abdomen, there is a resemblance with B. parva , although B. nova is smooth, whereas B. parva is hairy. Furthermore, B. nova lacks the big feet of B. parva (Figs 7 , 8 ). Figure 7. Lateral views of female allo- and paratypes of B. nova sp. nov. A. HLMD-Cael-8PT, B162; B. HLMD-Cael-16PT; C. HLMD-Cael-12PT; D. HLMD-Cael-14PT, B165; E. HLMD-Cael-18AT, B166. HLMD-Cael-18AT is the allotype. Figure 8. Lateral views of the paratypes of B. nova sp. nov. A. HLMD-Cael-7PT; B. HLMD-Cael-15PT, B167; C. HLMD-Cael-9PT; D. HLMD-Cael-6PT; E. HLMD-Cael-10PT, B163; F. HLMD-Cael-19PT; G. HLMD-Cael-11PT; H. HLMD-Cael-17PT; I. HLMD-Cael-5PT; J. HLMD-Cael-13PT. Description of the holotype. Body robust, of medium size (Figs 9A , 10A ). BL 25.81 mm, Integument finely rugose, shiny, smooth. Body of type specimen curved to the right in dorsal view, starting at the 10th tergit. Antennae 22-jointed, ~ 4.5 mm, ensiform, slightly flattened above and slightly tapering, in length longer than the head, reaching the pronotum, evenly punctured, grooves darker than the rest, fine hairs, end of each segment lined in lighter ground color. HL 3.82 mm, HDL 6.13 mm, HH 3.77 mm, head from above elongate, conical, from above app. 1.5 as long as its width at the occiput. Fv 1.6 mm, 0.78 the length of an eye, angular, the margins well raised but obtuse-angled, margins of contrasting color, forming a thin, dark brown line, narrowing from the anterior margin of the eye forwards, protruding in front of the eyes culminating at the tip of the vertex as a protruding dot. Median carinula faintly discernible on the fastigium. Head in profile very slightly concave above, face slightly incurved, angle acute (app. 57.9°). Apex of fastigium in profile slightly raised; frons oblique. Fr between the antennae in profile somewhat projecting, thin, lamelliform, lateral carinae below the antennae very narrow, shallowly sulcate, in basal part obliterated. Fc straight, distinct throughout. Eyes ovate, about twice as long as their maximum width, their surface strongly convex, both margins somewhat curved, the upper more so than the lower, lower margin slightly raised, as of callus. Ratio of eye length and fastigium length 0.60. PL 3 mm. Pronotum cylindrical, with weak median and indistinct lateral carinae; no sulcus crossing dorsum. Hind margin of metazona very slightly concave, anterior margin straight; sides of pronotum with the lower margin almost straight, sloping down- and forwards in the anterior part, posterior margin practically straight, anterior lower angle slightly more than 90°, rounded; posterior lower angle 90°, rounded. Pp trapezoid, about twice as long as broad, its margins and angles rounded, the anterior end little broader than the posterior, finely haired and rugose (anterior width: 299.27 µm , posterior width: 263.23 µm , length ~ 492 µm ), see Fig. 11A . Mesosternal interspace reduced/ closed with mesosternal lobes connected. Metasternal interspace like a pigs' nose. Anterior and middle legs strongly shortened. Carinae of anterior and middle femora with fine and sparse hairs, anterior and middle tibiae and tarsi hairy, HFL 9.47 mm, hind legs reaching beyond the end of abdomen. Hind femora robust, about five times as long as their maximum width, upper carina of femur finely toothed as of small frequent tubercles, hind tibiae with 8 outer and 10 inner spines. External apical spine of hind tibia present. Hind tarsus shorter than half of the length of the tibia. Arolium very large and of white color. Supra-anal plate elongate, 1.33 mm, acutely angular. Cercus short, conical. Supra-anal plate with the basal part about one third the length of the apical part, the longitudinal depression distinct; the apical part of the plate shaped like an equilateral triangle with the base curved and the sides straight; apical angle widely rounded; no depression on the apical angle. Subgenital plate acute and elongate (angle app. 50°), but apex rounded, comparatively sharply pointed in dorsal view, lower margin convex, upper margin straight. Outer genitalia only sparsely haired with the subgenital plate featuring a fine line of hairs along the fold. Genital apparatus about the length of 2.5 tergits. SGP: 2.33 mm, three tergits: 3.35 mm ratio: 0.6955. The total body volume is 1.33029e-07 m³ (133.029 mm3). The volume of the visible part of the eye is 1160104343.13 µm 3, the longitudinal height being 540.54 µm , and the length 2339.22 µm . The cross-sectional height is 657.29 µm and the width itself is 1372.3 µm . General coloration deep -brown. Antennae, fore and middle legs and knees are slightly paler than the brown primary color of the body. The upper part of the head, thorax and abdomen, within the callus line, is of green color. A longitudinal shallow stripe extends from the front of the head backwards along the central ridge of the body. The second and third tergit show brown markings between the callus lines; the posterior part of each tergit is of brown color, like a thin line, starting at the 5th tergit. The lateral stripe backwards from the base of the eye is beige-white. It starts at the lower hind corner of the eye (as a somewhat raised callus ridge) and stops right behind the middle leg. Eyes brown, anterior femora reddish-brown, middle femora green, tibia and tarsi dark brown, hind femora of the same green as the dorsal part, knees and upper and lower external carina of the hind femur pale brown. Hind tibia brown and very dark in the lower side, spines and spurs black-tipped. Figure 9. Holotypes of Betiscoides nova sp. nov. and Betiscoides muris sp. nov. in lateral view. A. Shows B. nova , HLMD-Cael-4HT, B168; B. Shows B. muris , HLMD-Cael-364HT, B156. Figure 10. Holotypes of Betiscoides nova sp. nov. and Betiscoides muris sp. nov. in dorsal view. A. Shows B. nova , HLMD-Cael-4HT, B168; B. Shows B. muris , HLMD-Cael-364HT, B156. Figure 11. Prosternal proceses, x 200. A. Holotype of B. nova sp. nov., HLMD-Cael-4HT, B168; B. Shows the Pp of HLMD-Cael-10PT, B163 ( B. nova ) in ventral topview. Description of the allotype. Larger than the male (Fig. 7E ). BL 30 mm, HL 5 mm, HDL 7 mm, HH 3.79 mm, Head in profile straight, angle acute (app. 57.5°). Antennae 22-jointed, 5.5 mm, ensiform, finely punctured. Fr strongly protruding, lateral carinae of frontal ridge distinct beneath the sulcus, almost until clepeus. Fastigium 0.6 the length of an eye. PL 3.7 mm, sides of pronotum with lower margin straight; anterior margin sloping forward, only very slightly incurved, posterior margin slightly incurved, sloping forward in the upper part. Anterior lower angle more than 90°, posterior lower angle 90°, both rounded. Pp trapezoid, slightly resembling the number 8, rugose and finely haired with two different kinds of hair, margins rounded, the anterior part slightly broader than the posterior (anterior width: 297.32 µm posterior width: 212.37 µm , length: ~ 562 µm ; measured in tilted angle (see Fig. 12 ). HFL 10.5 mm, hind tibiae with 10 outer and 12 inner spines, black tipped. Genital valves recurved, rounded in the apical part, hairs inside. Supra-anal plate 1.4 mm in total, subgenital plate acute. Genital valves spread. Substrate on the lower valves. Degree of protrusion acute (124°), Pr line 1 slightly longer (174 µm ) than Pr line 2 (109 µm ), Pr distance is 253,15 µm , Pr area is of 13755.38 µm 2 and protrusion vertical distance to tip is 180.92 µm . Protrusion to tip of valve distance is 311.02 µm . Upper valve degree acute of 163°, short upper valve distance: 239 µm , upper valve width 311 µm (see Fig. 13A ). General coloration brown, in dorsal view head greenish brown, pronotum and abdomen colored in mixture of orange-brown, greenish brown, end of abdomen dark brown. Ventral ridge of the body finely but densely haired, head and hind femur of yellow-green color, knee and beginning of hind tibia pale brown, apically slightly darker, arolium large and white; black claws. The volume of the visible part of the eye is 1729519566.68 µm 3. With the longitudinal height being 642.85 µm , and the length 2737.3 µm . The cross-sectional height is 646.17 µm and the width itself is 1412.78 µm . Figure 12. Image of the prosternal process of the allotype of Betiscoides nova sp. nov., HLMD-Cael-18AT, B166, x 200, captured in ventral view, with a tilt angle of 60°. Figure 13. Ovipositor valves, lateral view. A. Allotype of B. nova sp. nov. HLMD-Cael-18AT, B166; B. Allotype of B. muris sp. nov. HLMD-Cael-368AT, B155. Variation. Specimens show little differences in the shape of the head in profile, varying from described morphology of the holotype to a slightly more convex form. By contrast, the face differs concerning the distinction of the lateral carinae below the sulcus and the shape of the sulcus itself. In some specimens, the lateral carinae are distinct throughout, also beneath the sulcus. The sulcus is shaped as a mustache sometimes. Median Carina/ Central ridge of the body: Whereas the median carina is present in all specimens, the central ridge of the body is clearly distinct in males on the abdomen, starting on the third segment. This pattern is truly sex specific. Females show a distinct median carina throughout, but in three females (HLMD Cael-18AT, B166; HLMD-Cael-14PT, B165; and HLMD-Cael-16PT) the median carina culminates in a black spot at the verge of the fastigium of vertex. The prosternal process was described as laminate or lamelliform compressed by Key (1937) and Dirsh (1965) . In contrast to the other known Betiscoides species, the Pp of B. nova can be described as trapezoid with vague variability towards a square shape. In 2D some specimens seem to show a proximal concave Pp (see Fig. 11B ). Detailed 3D image analysis revealed a straight but rugose progression of the surface of the Pp (see Fig. 14 ). The curve progression showed a gradient of ~ 54 µm (HLMD-Cael-10PT, B163, see Fig. 14 ). Supra-anal plate and subgenital plate more elongate than in the holotype. Variation in spines on the hind tibia: In male, the hind tibiae had 8-9 outer and 10-11 inner spines. In females the hind tibia showed 9-10 outer spines and 11-12 inner spines. General coloration in males dark-brown. Antennae, fore and middle legs are of the same brown primary color as the body. The upper part of the head, thorax and abdomen, within the callus line, is of light brown or green color. When of green color, sometimes the hind margin of each segment of the abdomen is of light brown or orange color. Eyes brown. Hind femora pale, especially when light brown within the callus line; or green, both inside and outside, except the carina, which is sometime black-tipped, herring bone pattern distinct. Knees light brown. Hind tibiae brown and very dark in the lower side, spines and spurs black-tipped. Female: General primary coloration similar to, but slightly less dark than, that of the male. No variation toward green or light brown color within the callus line. Hind femora, both outside and inside, paler than the rest of the body, varying in one female towards yellow-green, herring bone pattern distinct, hind tibiae darker apically than basally. Detailed measurements are provided in Table 1 . Figure 14. 3D image profile analysis of the prosternal process of specimen HLMD-Cael-10PT, B163 ( B. nova sp. nov.). A. Shows the 3D image turned to profile view and the two points set to measure the distance and height progression curve of the selected part; B. Shows the depth image composition of the 3D image and the two points set to measure the distance; C. Shows the profile analysis results, including the length and height and the curve progression. Table 1. Plane and 3D measurements of the morphological characters for males and females of Betiscoides nova sp. nov. and B. muris sp. nov.

Measurements	B. nova males	B. nova females	B. muris males	B. muris females
Body length mm	22.5-27.2	29.3-30.02	21.2-24.3	26.15-29.5
Pronotum length mm	3.0-3.3	3.5-3-7	2.2-2.65	2.85-3.4
Hind femur length mm	8.7-10	9-11	6.9-8.1	8.9-10.4
Antenna segment no.	21	21	21-22	22-23
Head length mm	3.6-4.5	4.2-5	3.5-4.3	4.3-4.7
Head diagonal length mm	5.51-6.32	6.48-7.02	4.73-5.52	5.65-6.62
Head height mm	3.26-3.77	3.69-4.19	2.14-2.59	2.85-3.2
Subgenital plate length mm	2.3-3.0		2.2-3.0
Subgenital plate degree	46.3-58.3		30.7-35.8
3D measurements of the eyes
Longitudinal height µm			482.72-709.17	458.61-760.13
Cross-sectional height µm	532.59-657.29	598.04-646.17	460.35-577.58	422.86-696.96
Length µm	2254.37-2462.66	2500.83-2737.3	1943.74-2137.79	2131.68-2469.35
Width µm	1321.6-1454.8	1412.78-1524.26	1055.54-1213.7	1162.68-1344.2
Volume µm 3	1071906358.87-1812003789.7	1566466980.82-1798593679.03	587729028.76-865630822.96	894156739.30-1428871850.17
upper valve degree		146-163		134-140
upper valve distance µm		165-240		115-208
upper valve width µm		160-311		106-180
degree of protrusion		106-135		57-100
protrusion line 1 µm		171-235		118-158
protrusion line 2 µm		104-137		86-118
protrusion distance µm		236-327		174-199
protrusion vertical distance µm		161.57-180.92		126.31-154.28

The longitudinal height cannot be safely derived from the measurements since most of specimens eyes have been subject to certain degree of decay, which caused the highest part of the eyes to collapse at the highest point. Specimens HLMD-Cael- 6, 8, 9, 13, and 15PT were excluded from the cross sectional height analysis since the eyes had collapsed. Distribution, ecology and conservation status. Described from 11 males and five females from Jonaskop, Western Cape, South Africa, 2013. The species occur on top of Jonaskop in restio-dominated wetland. Abundant in patches with water covering the bare ground between the single restio plants. As the three formally recognized species of Betiscoides are considered endangered ( Hochkirch 2012a , 2012b , 2012c ), I propose this status as well for B. nova because itis only known from a single locality and its habitat is in decline.