Mixtacandona thessalica Rossetti & Mazzini, 2025

Rossetti, Giampaolo, Sarbu, Serban M., Ștefan, Andrei, Motoc, Rozalia & Mazzini, Ilaria, 2025, Mixtacandona thessalica, a new species of ostracod (Crustacea, Ostracoda) from a sulfidic cave in central Greece, Subterranean Biology 52, pp. 111-133 : 111-133

Mixtacandona thessalica Rossetti & Mazzini sp. nov.

Figs 2 , 3 , 4 , 5 , 6 , 7 A, B

Note.

Authorship of the new species is attributed to G. R. and I. M. and should be cited as “ Rossetti and Mazzini ” in “ Rossetti et al. ” (ICZN 2000, Recommendation 51 E).

Type locality.

Sulfidic lake in Melissotrypa Cave , Elassona, Greece, 39.877778°N, 22.049167°E, 299 m a. s. l.

Type material.

Holotype: • adult ♂ (GR 972 – MZUF 691 ): soft parts dissected in glycerine in a sealed slide, valves stored dry in a micropalaeontological slide (LV damaged) . Paratypes: • one adult ♂ (GR 973 – MZUF 692 ) with soft parts dissected as the holotype, valves stored dry in a micropalaeontological slide (LV slightly damaged and fragments of RV) ; • one adult ♂ (GR 975 – MZUF 693 ) with soft parts dissected as the holotype ; • two adult ♀♀ (GR 974 – MZUF 694 and GR 985 – MZUF 695 ) with soft parts dissected as the holotype ; • two adult ♂♂ and two adult ♀♀ stored in toto in ethanol (no numbers) . All type material was collected by S. M. S. on 5 May, 2023 (GR 985 – MZUF 695 ) and 29 June, 2024 (GR 972 – MZUF 69 , GR 973 – MZUF 692 , GR 974 – MZUF 694 , GR 975 – MZUF 693 ) and stored in 96 % ethanol.

Other material examined.

About 50 specimens from the same samples of the type material, partly used for dissections and / or SEM and the remaining ones preserved in ethanol. The material is stored in the ostracod collection of the first author.

Etymology.

The specific name derives from the Latin adjective “ thessalicus ” (conjugated feminine), indicating the origin from Thessaly, the region of Greece where Melissotrypa Cave is located.

Measurements.

L of ♂♂ (n = 9): range 520–558 µm, mean ± SD 546.0 ± 12.1 µm; L of ♀♀ (n = 4): range 543–585 µm, mean ± SD 569.5 ± 18.3 µm.

Diagnosis.

Small-medium sized Mixtacandona , belonging to the laisi – chappuisi species group (see Discussion). Females slightly larger than males, but with some overlap in lengths. Cp with an elongated, lateral outline (H / L ≅ 0.45 in both sexes) and narrow in dorsal view (W <1 / 3 of L). Valve surface smooth, covered with sparse setae. LV slightly overlapping RV on all sides, more markedly in the postero-dorsal corner, especially in males. Ventral margin straight, dorsal margin gently arched, greatest height at middle length. Posterior margin rounded in females, straight in males. Aesthetasc Y on A 2 approximately as long as the first endopodal segment. A 2 with second endopodal segment subdivided in males and undivided in females, setae t 2 and t 3 transformed into bristles in males. Seta f on T 2 present. T 3 with a three-segmented endopodite (second and third endopodal segments partially fused), seta h 2 longer than last endopodal segment, seta h 3 c. as long as the endopodite.

Description.

Carapace and valves. Male Cp in lateral view with elongate, subtrapezoidal shape (Fig. 3 A, B ). Greatest H around mid-length. Ventral margin straight. RV: Dorsal margin gently arched, anteriorly slightly sloping more steeply than posteriorly. Greatest length just below mid-height. Anterior margin rounded. Posterior margin bluntly more pointed than anterior one (Fig. 3 A ). LV: general outline similar to RV but with a subtle posterior dorsal protuberance that overlaps RV, forming an obtuse angle with the dorsal margin. The characteristic posterior protuberance is more evident in males (Fig. 3 D ) than in females (Fig. 3 C ). Surface ornamentation consists of a delicate pattern similar to a vascular pattern (Fig. 3 F, G ). Simple pore canals with a lip and sensory seta (Fig. 3 G ). LV overlaps RV at both ends (Fig. 3 H, I ). Marginal pore canals short, simple and scattered. Central muscle scar arrangement as characteristic for the genus.

Soft parts. A 1 (Fig. 4 A ): first segment with two long dorsal setae and two shorter setae on the ventral margin; second segment with a short ventral seta apically; third segment with short apical setae, one ventral and one dorsal; fourth and fifth segments with a short dorsal seta and two long, unequal ventral setae greatly exceeding tip of last segment; sixth segment with a dorsal setae reaching c. 2 / 3 of the next segment and two very long ventral setae; seventh (terminal) segment bearing two very long setae and a shorter one, the latter slightly longer than aesthetasc ya. A 2 ♂ (Figs 4 B , 6 A – C ): protopodite with a long ventral seta; exopodite with one long seta and two tiny, unequal setae; endopodite four-segmented (second segment subdivided); first endopodal segment with aesthetasc Y placed at c. 1 / 3 of the ventral margin and as long as the segment, three-segmented (distal part widened), and two ventro-apical setae, one largely exceeding the last segment of endopodite and the other very tiny; second endopodal segment bearing a sub-apical aesthetasc y 1 and an apical seta ventrally, and on the internal side setae t 1-4, of which the two median transformed into bristles; third endopodal segment with sub-equal apical claws (G 1 and G 3), more dorsally another claw (G 2) slightly shorter than half the length of the previous ones, three setae (z 1-3) in sub-apical position, and a ventro-apical aesthetasc (y 2); fourth segment with two claws, one (Gm) about 70 % the length of the other (GM), and an apical aesthetasc (y 3) with its companion seta ventrally. A 2 ♀ (Figs 4 C , 6 D ): endopodite three-segmented (second segment undivided); second endopodal segment with short seta inserted at the half of the dorsal margin, unequal setae t 1–4, setae z 2 and z 3 about 2 / 5 as long as seta z 1, aesthetascs y 1 and y 2 respectively at mid and sub-apical position along the ventral margin, apical claw G 2 c. 3 / 5 the length of G 1 and G 3; third endopodal segment distally with claw Gm about 3 / 4 the length of GM, and aesthetasc y 3 with its companion seta. T 1 ♂ (Figs 4 D, E , 6 E ): endopodite palps slightly asymmetrical, left one slightly more bumped and sinuous than right one. T 2 (Figs 5 A ; 6 F ): endopodite four-segmented, second and third segments with apical short setae (f and g); fourth segment with a stout, terminal claw h 2 slightly longer than the three distal endopodal segments, flanked by setae h 1 and h 3, the latter very tiny and c. 1 / 3 of h 1. T 3 (Figs 5 B , 6 G ): protopodite with setae dp and d 2 subequal and approximately as long as the next segment, seta d 1 shorter than previous ones; endopodite three-segmented (second segment partially subdivided); first endopodal segment without setae, second segment with a tiny sub-apical seta (g); third segment with three apical setae, the first (h 1) very small, the second (h 2) a little longer than the distal endopod segment, the third (h 3) slightly shorter than the endopodite. CR (Fig. 5 C ): seta Sp inserted at about 2 / 3 of the posterior margin; seta Sa strongly reduced, claw Gp c. 3 / 4 Ga. Hemipenis (Figs 5 D , 6 H ): median part of inner margin straight, lobe a thin and pointed distally, lobe b roughly triangular with the apical part folded and thickened, lobe h thumb-shaped with chitinous distal margin. Zenker organ (Fig. 5 E ): thin and with 5 + 2 spinous whirls. Eye absent. Other appendages as typical for the genus Mixtacandona .

Differential morphological diagnosis.

Mixtacandona thessalica sp. nov. is easily distinguished from the other species of the genus having a smooth, elongated, and dorsally curved carapace by its peculiar valve outline and the marked sexual dimorphism in the posterior margin (Fig. 7 ). It can be also differentiated by the combination of chaetotaxic characters details reported above. COI sequences confirm the distinctness of this species (see below), although the available data allow comparison with a small number of congeneric species.

Molecular analysis.

Seven sequences of M. thessalica sp. nov. and four sequences of M. idrisi were obtained for COI, with a length of 663 bp. The sequence quality and the presence of indels or early stop codons were checked. Four haplotypes were present in the seven isolates of M. thessalica sp. nov.: Hap 1 ( M. thessalica sp. nov. isolate MEL 1), Hap 2 ( M. thessalica sp. nov. isolate MEL 2, M. thessalica sp. nov. isolate MEL 5, M. thessalica sp. nov. isolate MEL 6, M. thessalica sp. nov. isolate MEL 7), Hap 3 ( M. thessalica sp. nov. isolate MEL 3), and Hap 4 ( M. thessalica sp. nov. isolate MEL 4). Only one haplotype was present in the isolates of Mixtacandona idrisi : Hap 1 ( M. idrisi isolate IT 1, M. idrisi isolate IT 2, M. idrisi isolate IT 4, M. idrisi isolate IT 5). GenBank sequences were included in the analyses and the COI alignment was trimmed to the length of the shortest GenBank sequence, i. e., 515 bp. GenBank accession numbers for our sequences and the downloaded sequences are provided in the Suppl. material 1. Maximum Likelihood phylogeny and p-distances (0.178, p <0.05) indicate that the closest species to M. thessalica sp. nov. is Mixtacandona sp. n. (GenBank accession numbers MN 013108 and MN 013109 ) from the Frasassi caves in Central Italy. Both ASAP and PTP clearly distinguish M. thessalica sp. nov. as a distinct species. The occurrence of two other genera reported from Australia, Notacandona and Meridiescandona , within the clade of four Mixtacandona species is an unusual result, indicating the possibility of polyphyly in the Mixtacandona genus (Fig. 8 ). Polyphyly was also described in other morphogenera of freshwater Candonidae such as Candona , Fabaeformiscandona , and Pseudocandona ( Karanovic and Sitnikova 2017; Wysocka et al. 2019). Six sequences of M. thessalica sp. nov. were obtained for the 28 S marker and all shared the same haplotype. Five sequences of M. idrisi were also obtained and all shared the same haplotype. The sequence alignment was trimmed to the shortest GenBank sequence (279 bp) and the ML phylogeny and p-distances (0.065, p <0.05) indicate that M. thessalica sp. nov. is closest to M. idrisi . The absence of 28 S sequences in GenBank made it impossible to compare these species to other representatives of the genus Mixtacandona (Fig. 9 ).

Differential molecular diagnosis.

(COI): ‘ T’ at site 32, ‘ C’ at site 40, ‘ G’ at site 171 on the 515 bp-long alignment (corresponding to sites 116, 124 and 171, respectively, on the 663 bp full length sequence).

Distribution.

The species is only known from its type locality.

Remark.

Males and females were equally represented in the analyzed samples.

Figures

Figure 2. Mixtacandona thessalica sp. nov., ♂ (GR 975 – MZUF 693). Optical microscope image of entire body in interior of right valve after removing left valve. Scale bar: 100 μm.

Figure 3. Mixtacandona thessalica sp. nov. A ♂, Cp right lv (GR 976) B ♂, Cp left lv (GR 977) C ♀, Cp right lv, detail posterior part (GR 978) D ♂, Cp right lv, detail posterior part (GR 976) E ♂, LV iv partly damaged (0060) F ♂, detail of reticulate pattern of external valve surface (0024) G ♂, rimmed pore canal with its seta (0024) H ♀, Cp oblique dv (GR 979) I ♂, Cp oblique dv (0024). Scale bar: 200 µm (A, B, E, H, I); 150 µm (C, D); 50 µm (F); 20 µm (G).

Figure 4. Mixtacandona thessalica sp. nov. A ♂, A 1 (first segment partially twisted) (GR 972 – MZUF 691) B ♂, A 2 (GR 972 – MZUF 691) C ♀, terminal part of A 2 (GR 985 – MZUF 695) D ♂, left T 1 – palp (GR 972 – MZUF 691) E ♂, right T 1 – palp (GR 972 – MZUF 691). Scale bar: 100 μm.

Figure 5. Mixtacandona thessalica sp. nov. A ♂, T 2, distal part of endopodite (GR 972 – MZUF 691) B ♂, T 3 (GR 972 – MZUF 691) C ♂, CR (GR 972 – MZUF 691) D ♂, hemipenis (GR 972 – MZUF 691) E ♂, Zenker organ (GR 973 – MZUF 692). Scale bar: 100 μm (A – C, E); 80 μm (D).

Figure 6. Mixtacandona thessalica sp. nov. A ♂, A 2 (0061) B ♂, A 2, detail male bristles (arrows) (0061) C ♂, A 2, detail aesthetasc Y (0061) D ♀, A 2, detail exopodite (0036) E ♂, T 1 – palps (arrows) (0061) F ♀, terminal part of T 2 (0036) G ♀, branchial plate of maxillula (foreground) and T 3 (0036) H ♂, hemipenis (0061). Scale bar: 100 μm (A, B, E – H); 25 μm (C, D).

Figure 7. Comparison of outlines of species with curved dorsal valve margins assigned to the laisi – chappuisi group in the genus Mixtacandona (all figures redrawn to the same length) A Mixtacandona thessalica sp. nov., ♀, Cp left lv B Mixtacandona thessalica sp. nov., ♂, Cp left lv C Mixtacandona idrisi, ♂, Cp left lv D Mixtacandona idrisi, ♀, Cp left lv E Mixtacandona pseudocrenulata, ♂, LV iv F Mixtacandona pseudocrenulata, ♀, LV iv G Mixtacandona chappuisi, ♂, LV iv H Mixtacandona chappuisi, ♀, LV ev I Mixtacandona laisi, ♀, LV ev J Mixtacandona transleithanica, ♀, LV ev. C, D from Mazzini et al. 2017; E, F from Schäfer 1945; G, H from Klie 1943; I from Klie 1938; J from Löffler 1960.

Figure 8. Maximum Likelihood phylogenetic tree of available species in the family Candonidae using COI sequences. Species delimitation results by ASAP (dark gray) and PTP (light gray) are also shown. Bennelongia pinderi Martens et al., 2015 was used as outgroup. The tree is drawn to scale, with branch lengths measured in the number of nucleotide substitutions per site. The scalebar represents the number of substitutions per unit of branch length. Bootstrap values larger than 70 % after 1000 replicates are shown on the branches.

Figure 9. Maximum Likelihood phylogenetic tree for 28 S. Paracypria longiseta Hiruta & Kakui, 2017 was used as outgroup.