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Research Article
Mixtacandona thessalica, a new species of ostracod (Crustacea, Ostracoda) from a sulfidic cave in central Greece
expand article infoGiampaolo Rossetti‡, Serban M. Sarbu§|, Andrei Ștefan§, Rozalia Motoc, Ilaria Mazzini#
‡ University of Parma, Parma, Italy
§ “Emil Racoviţă” Institute of Speleology, Cluj Napoca, Romania
| California State University, Chico, United States of America
¶ “Grigore Antipa” National Museum of Natural History, Bucharest, Romania
# CNR - IGAG, Area della Ricerca di Roma 1, Rome, Italy

Corresponding author: Ilaria Mazzini ( ilaria.mazzini@igag.cnr.it )

Academic editor: Fabio Stoch
© 2025 Giampaolo Rossetti, Serban M. Sarbu, Andrei Ștefan, Rozalia Motoc, Ilaria Mazzini.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Rossetti G, Sarbu SM, Ștefan A, Motoc R, Mazzini I (2025) Mixtacandona thessalica, a new species of ostracod (Crustacea, Ostracoda) from a sulfidic cave in central Greece. Subterranean Biology 52: 111-133. https://doi.org/10.3897/subtbiol.52.142113
ZooBank: urn:lsid:zoobank.org:pub:B8B54667-384D-4498-BE7F-BBA4439AEAFD
Open Access

Abstract

The genus Mixtacandona Klie, 1938 (Crustacea, Ostracoda, Candonidae) includes 21 living non-marine species, all subterranean, with Palearctic distribution. Here we report on Mixtacandona thessalica sp. nov., collected in a sulfidic cave in central Greece. It can be considered an extremophile species because of its ability to thrive in an environment with high concentrations of sulfide and reduced chemical compounds. Mixtacandona thessalica sp. nov. belongs to the species group laisi–chappuisi, one of the five groups in which species of the genus are conventionally placed. A detailed morphological description of the valves and the soft parts of the new species is offered. In addition, COI and 28S genetic markers were sequenced. Mixtacandona thessalica sp. nov. is easily distinguishable from the other species in the genus by its peculiar carapace outline and the marked sexual dimorphism of the posterior margin of valves, as well as by details of chaetotactic characters. The discovery of Mixtacandona thessalica sp. nov. increases the number of non-marine ostracod species known from Greece, which is still rather scarce compared to other Mediterranean countries due to the scarcity of studies. Ten hypogeal species of living ostracods have already been reported from Greece, six of which are considered endemic, and among them three belong to the genus Mixtacandona. It is stressed that a comprehensive review of this genus by combining a thorough morphological approach and molecular techniques, is most needed to assess its phylogenetic relationships within the family Candonidae.

Keywords

Candonidae, COI and 28S sequences, Melissotrypa Cave, morphology, subterranean ostracods, sulfidic environment, taxonomy

Introduction

Ostracods are a class of bivalve crustaceans that have undergone an extraordinary evolutionary radiation and are characterized by an almost continuous fossil record (Horne 2003). Ostracods appeared in marine environments during the late Cambrian (Siveter et al. 2024), and the first invasions of continental waters date back to the Carboniferous (Rodriguez-Lazaro and Ruiz-Muñoz 2012; Iglikowska 2014). Ostracods have been used as model organisms in numerous evolutionary, ecological and biogeographical studies (Martens and Horne 2000).

The extant non-marine ostracods all belong to the order Podocopida G.O. Sars, 1866, in which more than 2300 species in about 270 genera have been described (Meisch et al. 2019). Non-marine ostracods have colonized a wide variety of aquatic and semi-terrestrial ecosystems, including subterranean habitats such as hyporheic zones, alluvial aquifers and caves. A considerable share of the subterranean ostracod species described so far belong to the subfamily Candoninae Kaufmann, 1900, in which three groups of species can be distinguished based on the degree of colonization of the subterranean realm (Danielopol 1980): in the first there are no exclusively subterranean species (stygoxenes, e.g. the genus Candona Baird, 1845), the second includes both epigean and subterranean species (e.g. species in the genera Fabaeformiscandona Krstić, 1972 and Pseudocandona Kaufmann, 1900), while the third consists of subterranean dwellers only (stygobites, e.g. the genera Phreatocandona Danielopol, 1973 and Mixtacandona Klie, 1938). Accordingly, the degree of morphological and physiological adaptations to subterranean environments also varies, e.g. reduction in body size, anophthalmia, chemo– and mechanoreceptor development, predominantly or exclusively sexual reproduction, and reduction in the number of eggs produced (Moldovan 2018).

Most species of subterranean ostracods are considered endemic, but this estimation is undoubtedly affected by the still limited number of studies that have analyzed these invertebrate faunas.

Freshwater caves (i.e. those that are not marine or anchialine) frequently host ostracods. Martínez (2023) reported that studies conducted in 1577 freshwater caves across the world produced 389 records of non-marine ostracods, totaling 141 species of which 59 were exclusively found in caves. Among this category of subterranean ecosystems, chemosynthesis-based sulfidic caves are particularly interesting due to the peculiar chemical characteristics of their waters and, despite the extremely stressful environmental conditions, the possibility of hosting unexpectedly rich faunas (Engel 2007; Hillebrand-Voiculescu 2018; Pop et al. 2023; De Waele et al. 2024). Regarding ostracods, records from these ecosystems are extremely limited. Mixtacandona sp. and Pseudolimnocythere sp., two new putative species, were found in sulfidic waters of the Frasassi cave system, Italy (Peterson et al. 2013; Sarbu et al. 2022). Pseudocandona movilaensis Iepure, Wysocka & Namiotko, 2023 was described from a sulfidic cave in Romania (Iepure et al. 2023). Popa et al. (2019) reported the presence of ostracods belonging to the genus Mixtacandona from Melissotrypa cave in central Greece, pointing out close morphological similarities with M. idrisi Mazzini & Rossetti, 2017, a species described from a cave near Palermo in Sicily (Mazzini et al. 2017) and possibly occurring as a fossil in north-eastern Spain (Iriarte et al. 2023).

In this paper, a complete morphological description of the valves and soft parts of the Mixtacandona ostracods from Melissotrypa Cave is provided, allowing us to describe it as a new species. In addition, the COI and 28S DNA markers were sequenced for comparison with the molecular data available for other congeneric species and to assess the phylogenetic relationships of Mixtacandona within the family Candonidae Kaufmann, 1900.

Materials and methods

Study area

Melissotrypa Cave (39°52'40"N, 22°02'57"E) is a hypogenic cave located close to Elassona, in central Greece, at an altitude of 299 m (Vaxevanopoulos 2006). The cave contains a 5 m deep sulfidic lake, and two non-sulfidic lakes in the southern and eastern maze areas (Fig. 1). Temperatures of 16° to 19 °C have been measured in the air and in the lakes, respectively. The rich food base for its abundant and diverse subterranean invertebrate community (Popa et al. 2019) is generated by chemosynthesis in situ by sulfur-oxidizing microorganisms that use hydrogen sulfide from the cave water and dioxygen from the cave atmosphere. Numerous specimens of ostracods belonging to the genus Mixtacandona Klie, 1938 have been observed in the Sulfidic Lake and in the Gallery Lake. Four additional endemic stygobitic species have been described from Melissotrypa Cave: two gastropods (Iglica hellenica Falniowski & Sarbu, 2015 and Daphniolla magdalenae Falniowski & Sarbu, 2015), one isopod (Turcolana lepturoides Prevorčnik, Konec & Sket, 2016), and one amphipod (Niphargus gammariformis Borko, Collette, Brad, Zakšek, Flot, Vaxevanpoulos, Sarbu & Fišer, 2019); two oligochaetes (Haplotaxis sp. nov. and Delaya sp. nov.), one planarian (Dendrocoelum sp. nov.), and one amphipod (Bogidiella sp. nov.) are still waiting to be described (Popa et al. 2019).

Figure 1. 

Plan of Melissotrypa Cave (modified after Vaxevanopoulos 2006). The location of the cave in the Greek Mainland is marked with a star.

Ostracod sampling and morphological analysis

Ostracods were collected during two surveys (5 May, 2023 and 29 June, 2024) using a 180 µm hand net and were preserved in 70% ethanol. Sorting and dissections of ostracods were done under a stereomicroscope (Zeiss 47 50 22). Carapaces were measured under a light microscope equipped with a calibrated micrometer ocular. In most cases, intact valves could not be preserved due to their extreme fragility. Whenever possible, partially damaged valves or valve fragments were stored dry in micropalaeontological slides. Digital images of carapaces and valves were acquired using scanning electron microscopy (ZEISS EVO MA10 – SEM). The dissected soft parts were mounted in glycerine on microscope glass slides and sealed using nail polish. Line drawings of soft parts were made with the help of a camera lucida-equipped microscope. The chaetotaxy of the soft parts follows Meisch (2000). All the figured specimens are adults.

The specimens examined in this study labeled with the abbreviation GR followed by a number are stored in the first author’s ostracod collection at the University of Parma, Italy. Specimens used for SEM of soft parts are labeled with a four-digit number. Type material is deposited in the crustacean collection of La Specola Museum of Natural History, Zoology Section a Florence, Italy; the number after the acronym MZUF indicates the collection number of the deposited specimens.

Molecular analyses

The specimens used in the molecular analyses were stored in 96% ethanol and kept at -20 °C until further processing (n = 7 specimens of M. thessalica sp. nov., and n = 5 specimens of M. idrisi). DNA extraction was carried out using the QIAmp® DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer specifications. A fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the ZplankF1 (5’-TCTASWAATCATAARGATATTGG-3’) and ZplankR1 (5’-TTCAGGRTGRCCRAARAATCA-3’) primer pair (Prosser at al. 2013), and a fragment of the nuclear 28S rDNA was amplified using the 28SA (5’-GACCCGTCTTGAAGCACG-3’) and 28SB (5’-TCGGAAGGAACCAGCTAC-3’) primer pair (Whiting 2002). PCRs were performed in 40 μl volumes containing 1X AccuStart II ToughMix (Quantabio, Beverly MA, USA), 1X Loading Dye, 0.1 μM each primer and DNA template. Thermal cycling for COI amplification consisted of an initial denaturation at 94 °C for 3 minutes followed by 35 cycles of denaturation at 94 °C for 30 seconds, primer annealing at 51 °C for 30 seconds and elongation at 72 °C for 50 seconds. The cycling conditions for 28S were the same, except for the primer annealing temperature, which was 55 °C. Amplification was confirmed on 1.5% (w/v) agarose gels stained with ethidium bromide, and sequencing was performed at a sequencing facility (Macrogen, Amsterdam, The Netherlands).

The resulting chromatograms were visually inspected and edited using Chromas v.2.6.6 (Technelysium Ltd., South Brisbane, Australia) and assembled in CodonCode Aligner v.3.7.1 (CodonCode Corporation, MA, USA). The manually curated sequences were checked against GenBank (Sayers et al. 2022) using BLASTn (Altschul et al. 1990) and the most similar sequences from the Candonidae family were downloaded and included in the analysis. Sequence alignment for the two sets (COI and 28S) were done on the MAFFT web server (https://mafft.cbrc.jp/alignment/server/index.html, accessed on 21 October 2024) using default parameters, phylogenetic reconstruction was performed by Maximum Likelihood on the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/, accessed on 21 October 2024), and the phylogenetic trees were visualized in FigTree v.1.4 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 23 March 2023). The substitution saturation for COI was checked in DAMBE v.7.3.32 (Xia 2017) using the method described in Xia et al. (2003). Species delimitation analysis was done using the distance-based method ASAP (Assemble Species by Automatic Partitioning; Puillandre et al. 2021) and the tree-based method PTP (Poisson Tree Processes; Zhang et al. 2013). Basic sequence statistics and haplotype identity analysis were performed in DnaSP v.6 (Rozas et al. 2017) and p-distances between species were calculated in MEGA v.7 (Kumar et al. 2016). MOLD v.1.4 was used to identify diagnostic nucleotide combinations (DNCs) in the COI sequence alignment, which can be used to provide formal diagnoses of taxa (Fedosov et al. 2022).

Abbreviations used in the text and figures

Valves. Cp: carapace; dv: dorsal view; ev: external view; H: height; iv: internal view; L: length; LV: left valve; lv: lateral view; RV: right valve; W: width.

Soft parts. A1: antennule; A2: antenna; T1: first thoracopod (maxilliped); T2: second thoracopod (walking leg); T3: third thoracopod (cleaning leg); CR: caudal ramus; exo: exopodite on A2; ya: aesthetasc on A1; t1-4 and z1–3: setae or male bristles on A2; Y and y1-3: aesthetascs on A2; G1-3, GM, Gm: claws on A2; f, g and h1-3: setae and claws on T2 and T3; d1-2 and dp: setae on T3; Sa and Sp: anterior and posterior setae on CR; Ga and Gp: anterior and posterior claws on CR; a, b and h: outer, inner and medial lobes of hemipenis.

Results

Taxonomic account

Class Ostracoda Latreille, 1802

Subclass Podocopa G.O. Sars, 1866

Order Podocopida G.O. Sars, 1866

Suborder Cypridocopina Baird, 1845

Superfamily Cypridoidea Baird, 1845

Family Candonidae Kaufmann, 1900

Subfamily Candoninae Kaufmann, 1900

Tribe Candonini Kaufmann, 1900

Mixtacandona Klie, 1938

Diagnosis of the genus

(after Mazzini et al. 2017). carapace 0.48–0.80 mm long, W < 1/3 L. Calcified inner lamella of valves narrow, marginal pore canals rare. Valve shape in lateral view trapezoidal, triangular or elongated, surface smooth or slightly ornamented. A2 of male with or without male bristles, aesthetasc Y conspicuously long (≥ 60% of first endopodal segment). T1–respiratory plate with three filaments. Male T1–palps (clasping organs) only slightly asymmetrical. T3 four– or five-segmented; protopodite with three setae (d1, d2, and dp), seta f often present, distal segment with one long (h3) and two very short setae (h1 and h2). CR with short Sp. Hemipenis with a finger-shaped outer lobe, M-process absent. Eye pigment absent. Exclusively stygobitic species.

Mixtacandona thessalica Rossetti & Mazzini, sp. nov.

https://zoobank.org/7343C8DE-9834-4C6C-8878-D8539C5AAEB5
Figs 2, 3, 4, 5, 6, 7A, 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 51E).

Type locality.

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

Type material.

Holotype : • adult ♂ (GR972–MZUF691): soft parts dissected in glycerine in a sealed slide, valves stored dry in a micropalaeontological slide (LV damaged). Paratypes: • one adult ♂ (GR973–MZUF692) with soft parts dissected as the holotype, valves stored dry in a micropalaeontological slide (LV slightly damaged and fragments of RV); • one adult ♂ (GR975–MZUF693) with soft parts dissected as the holotype; • two adult ♀♀ (GR974–MZUF694 and GR985–MZUF695) 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 (GR985–MZUF695) and 29 June, 2024 (GR972–MZUF69, GR973–MZUF692, GR974–MZUF694, GR975–MZUF693) 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 A2 approximately as long as the first endopodal segment. A2 with second endopodal segment subdivided in males and undivided in females, setae t2 and t3 transformed into bristles in males. Seta f on T2 present. T3 with a three-segmented endopodite (second and third endopodal segments partially fused), seta h2 longer than last endopodal segment, seta h3 c. as long as the endopodite.

Figure 2. 

Mixtacandona thessalica sp. nov., ♂ (GR975–MZUF693). Optical microscope image of entire body in interior of right valve after removing left valve. Scale bar: 100 μm.

Description.

Carapace and valves. Male Cp in lateral view with elongate, subtrapezoidal shape (Fig. 3A, 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. 3A). 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. 3D) than in females (Fig. 3C). Surface ornamentation consists of a delicate pattern similar to a vascular pattern (Fig. 3F, G). Simple pore canals with a lip and sensory seta (Fig. 3G). 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.

Figure 3. 

Mixtacandona thessalica sp. nov. A ♂, Cp right lv (GR976) B ♂, Cp left lv (GR977) C ♀, Cp right lv, detail posterior part (GR978) D ♂, Cp right lv, detail posterior part (GR976) 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 (GR979) I ♂, Cp oblique dv (0024). Scale bar: 200 µm (A, B, E, H, I); 150 µm (C, D); 50 µm (F); 20 µm (G).

Soft parts. A1 (Fig. 4A): 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. A2 ♂ (Figs 4B, 6A–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 y1 and an apical seta ventrally, and on the internal side setae t1-4, of which the two median transformed into bristles; third endopodal segment with sub-equal apical claws (G1 and G3), more dorsally another claw (G2) slightly shorter than half the length of the previous ones, three setae (z1-3) in sub-apical position, and a ventro-apical aesthetasc (y2); fourth segment with two claws, one (Gm) about 70% the length of the other (GM), and an apical aesthetasc (y3) with its companion seta ventrally. A2 ♀ (Figs 4C, 6D): endopodite three-segmented (second segment undivided); second endopodal segment with short seta inserted at the half of the dorsal margin, unequal setae t1–4, setae z2 and z3 about 2/5 as long as seta z1, aesthetascs y1 and y2 respectively at mid and sub-apical position along the ventral margin, apical claw G2 c. 3/5 the length of G1 and G3; third endopodal segment distally with claw Gm about 3/4 the length of GM, and aesthetasc y3 with its companion seta. T1 ♂ (Figs 4D, E, 6E): endopodite palps slightly asymmetrical, left one slightly more bumped and sinuous than right one. T2 (Figs 5A; 6F): endopodite four-segmented, second and third segments with apical short setae (f and g); fourth segment with a stout, terminal claw h2 slightly longer than the three distal endopodal segments, flanked by setae h1 and h3, the latter very tiny and c. 1/3 of h1. T3 (Figs 5B, 6G): protopodite with setae dp and d2 subequal and approximately as long as the next segment, seta d1 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 (h1) very small, the second (h2) a little longer than the distal endopod segment, the third (h3) slightly shorter than the endopodite. CR (Fig. 5C): seta Sp inserted at about 2/3 of the posterior margin; seta Sa strongly reduced, claw Gp c. 3/4 Ga. Hemipenis (Figs 5D, 6H): 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. 5E): thin and with 5+2 spinous whirls. Eye absent. Other appendages as typical for the genus Mixtacandona.

Figure 4. 

Mixtacandona thessalica sp. nov. A ♂, A1 (first segment partially twisted) (GR972–MZUF691) B ♂, A2 (GR972–MZUF691) C ♀, terminal part of A2 (GR985–MZUF695) D ♂, left T1–palp (GR972–MZUF691) E ♂, right T1–palp (GR972–MZUF691). Scale bar: 100 μm.

Figure 5. 

Mixtacandona thessalica sp. nov. A ♂, T2, distal part of endopodite (GR972–MZUF691) B ♂, T3 (GR972–MZUF691) C ♂, CR (GR972–MZUF691) D ♂, hemipenis (GR972–MZUF691) E ♂, Zenker organ (GR973–MZUF692). Scale bar: 100 μm (A–C, E); 80 μm (D).

Figure 6. 

Mixtacandona thessalica sp. nov. A ♂, A2 (0061) B ♂, A2, detail male bristles (arrows) (0061) C ♂, A2, detail aesthetasc Y (0061) D ♀, A2, detail exopodite (0036) E ♂, T1–palps (arrows) (0061) F ♀, terminal part of T2 (0036) G ♀, branchial plate of maxillula (foreground) and T3 (0036) H ♂, hemipenis (0061). Scale bar: 100 μm (A, B, E–H); 25 μm (C, D).

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.

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.

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.: Hap1 (M. thessalica sp. nov. isolate MEL1), Hap2 (M. thessalica sp. nov. isolate MEL2, M. thessalica sp. nov. isolate MEL5, M. thessalica sp. nov. isolate MEL6, M. thessalica sp. nov. isolate MEL7), Hap3 (M. thessalica sp. nov. isolate MEL3), and Hap4 (M. thessalica sp. nov. isolate MEL4). Only one haplotype was present in the isolates of Mixtacandona idrisi: Hap1 (M. idrisi isolate IT1, M. idrisi isolate IT2, M. idrisi isolate IT4, M. idrisi isolate IT5). 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 MN013108 and MN013109) 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 28S 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 28S sequences in GenBank made it impossible to compare these species to other representatives of the genus Mixtacandona (Fig. 9).

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 28S. Paracypria longiseta Hiruta & Kakui, 2017 was used as outgroup.

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.

Discussion

All the 22 extant recognised species of the genus Mixtacandona including M. thessalica sp. nov. have a western-Palearctic distribution (Meisch et al. 2019). Actually, the number of species is expected to be considerably higher, considering the difficulties involved in sampling subterranean aquatic ecosystems and the taxonomic expertise required to describe species that are often very small and whose identification relies on minute morphological features. As reported by Wysocka et al. (2019), the subfamily Candoninae is one of the most taxonomically difficult lineages of non-marine Ostracoda. In the literature, there are numerous reports of living and fossil Mixtacandona, some even of putative new species, but with largely insufficient or completely missing descriptions.

Mixtacandona thessalica sp. nov. can be considered an extremophile species, based on the chemical characteristics of the water in Melissotrypa Cave, with high concentrations of sulfide and reduced compounds. It can be assumed that its presence in this habitat is the result of adaptive processes that occurred over long timescales. Information on the ecology of this species is still lacking. It is possible that, as observed for Mixtacandona sp. in the Frasassi cave system, this species occupies oxygenated water layers close to the sulfidic chemocline, where sulfur-oxidizing bacteria abound and constitute an almost unlimited trophic supply (Peterson et al. 2013). The sex ratio in the population of M. thessalica sp. nov. was close to 1:1. In many stygobitic crustacean species, a balanced sex ratio or even a predominance of males may result in a reproductive advantage in environments characterized by substantial thermal stability, where females display asynchronous mating behavior (Tabilio Di Camillo et al. 2023).

The discovery of Mixtacandona thessalica sp. nov. allows us to expand the list of species known from the ostracod fauna of Greece, which, compared to other Mediterranean countries, is still rather limited due to the scarcity of studies (Danielopol 1981; Karanovic 2003; Marrone et al. 2019a, 2019b). There are 10 hypogeal species of living ostracods reported from Greece so far, of which six are currently considered endemic, and among them three belong to the genus Mixtacandona (Table 1). The latter are all included in the laisi–chappuisi species group, namely the one with rectangular or trapezoidal outline in lateral view, small to middle sized and with smooth valve surface among the five groups established by Danielopol (1978) for this genus. The different groups can be separated based on size, shape and ornamentation of the carapace (for a discussion on their validity for taxonomic or zoogeographic purposes, see Mazzini et al. 2017). It is therefore plausible to speculate that this geographical area potentially played a crucial role as a speciation hotspot for this group of Mixtacandona species, representatives of which have existed in Europe since at least the Miocene (Danielopol 1972, 1980; Mazzini et al. 2017). It can also be seen from Table 1 that the only known cave ostracod is Mixtacandona thessalica sp. nov. This further confirms how research on subterranean biodiversity is still limited in Greece, a country that has a complex geological landscape in which the Hellenic Speleological Society has recorded > 10000 caves (Trimmis 2015).

Table 1.
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List of extant subterranean species of ostracods known for Greece (Danielopol 1981; Karanovic 2003). The asterisk indicates species endemic to Greece. A putative new species of Mixtacandona (reported as Trapezicandona sp. nov.?) from a cave in Greece (Valavani et al. 2024) is not listed here because not yet fully illustrated and formally described.

Species Family Habitat Locality
*Candonopsis thienemani Schäfer, 1945 Candonidae well Armenio
*Candonopsis trichota Schäfer, 1945 Candonidae well Armenio
Kliella hyaloderma Schäfer, 1945 Kliellidae well Armenio
Kovalevskiella bulgarica (Danielopol, 1970) Limnocytheridae well Igoumenitsa
*Kovalevskiella dani Karanovic, 2003 Limnocytheridae well Lesbos
Kovalevskiella rudjakovi (Danielopol, 1969) Limnocytheridae well Lesbos
*Mixtacandona peliaca (Schäfer, 1945) Candonidae well Armenio
*Mixtacandona pseudocrenulata (Schäfer, 1945) Candonidae well Larissa
“ “ well Euboea
*Mixtacandona thessalica sp. nov. Candonidae cave Elassona
*Nannokliella dictyoconcha Schäfer, 1945 Kliellidae well Armenio
*§Pseudolimnocythere hartmanni Danielopol, 1979 Loxoconchidae well Euboea

§ Aiello et al. (2020) reported P. hartmanni as a fossil from the Gulf of Salerno, Italy. If confirmed, this record would invalidate the endemic status for this species (see Rossetti et al. 2022).

As the carapace is the first point of contact with the external environment, many ostracod species have evolved a highly plastic carapace, whereas the soft parts remain much more conservative (Karanovic et al. 2019). For example, it has been suggested that triangular and trapezoidal shapes are the result of homoplastic adaptations in species dwelling in subterranean environments (Iepure et al. 2023). In the case of Mixtacandona, which consists exclusively of stygobitic species, very different carapace morphologies are observed, but without a strict relation to habitat type, e.g. alluvial aquifers or caves. At least one species, M. botosaneanui Danielopol, 1973, has been found in both environments (Danielopol 1982; Danielopol and Hartmann 1986).

The taxonomy of the Candoninae remains unclear and generic relationships ambiguous due to a number of homoplasies (Wysocka et al. 2019). This also applies to Mixtacandona. In species for which detailed descriptions of the soft parts are available, a marked variability is found in the chaetotaxy. Such differences in appendage morphology are probably sufficient for the establishment of new genera. A comprehensive review of the genus, including the re-examination of all available types, is therefore advisable.

To solve complex questions of taxonomy, phylogeny, but also biogeographical models related to the genus Mixtacandona and other ostracod taxa, it is deemed necessary to combine a purely morphological approach with molecular techniques. Such an approach seems appropriate to resolve the phylogenetic inconsistencies in the Candoninae. Wysocka et al. (2019) mention the decoupling between morphological species assignments and genetic data between other non-marine ostracods as well. So, probably, more sequenced markers, with varying degrees of variability and more sequenced ostracod species could shed more light and help disentangle these complex phylogenies. But, for now, the almost complete absence of publicly available sequences for species of Mixtacandona makes it impossible to assess their phylogenetic relationships and more molecular studies are clearly needed. This is more evident in the case of the 28S marker, which is completely absent from sequence databases for Mixtacandona, but the short overlap (279 bp) in the sequence alignment between the sequences from this study was still enough to separate M. thessalica sp. nov. and M. idrisi. The genera Candona and Fabaeformiscandona appear polyphyletic on 28S as well (Fig. 9), but we could not assess the potential 28S polyphyly of Mixtacandona, since there are no available sequences to compare them to. Molecular methods have made it possible to document the occurrence of a considerable number of cryptic species of non-marine ostracods, which are indistinguishable from each other through even extremely thorough morphological analysis (Kilikowska et al. 2024). Several approaches using DNA sequencing data have been proved to have a great potential in improving our knowledge on freshwater meiofaunal biodiversity, especially in habitats difficult to sample, such as caves (Schenk and Fontaneto 2020). At the same time, detailed information on the morphology, intraspecific variability and sexual dimorphism of ostracod valves is essential to provide diagnostic characters that can be used in palaeontological research.

Acknowledgments

We thank the members of the speleological teams from Greece and Romania who helped us reach the subterranean sites and assisted with the field work. Luisa Dainelli (University of Pisa) kindly helped with the shipment of specimens used in this study. Dr Matteo Paciucci (CNR-IGAG) is thanked for patiently taking the SEM photos of the specimens. This research was partially funded by Biodiversa+, the European Biodiversity Partnership under the 2021–2022 BiodivProtect joint call for research proposals, co-funded by the European Commission (GA N°101052342) and with the funding organizations Ministry of Universities and Research (Italy), Agencia Estatal de Investigación—Fundación Biodiversidad (Spain), Fundo Regional para a Ciência e Tecnologia (Portugal), Suomen Akatemia—Ministry of the Environment (Finland), Belgian Science Policy (Belgium), Agence Nationale de la Recherche (France), Deutsche Forschungsgemeinschaft e.V.—BMBF-VDI/VDE INNOVATION + TECHNIK GMBH (Germany), Schweizerischer Nationalfonds zur Forderung der Wissenschaftlichen Forschung (Switzerland), Fonds zur Förderung der Wissenschaftlichen Forschung (Germany), Ministry of Education, Science and Sport (Slovenia), and the Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania). Two anonymous reviewers are acknowledged for their careful and constructive comments, which greatly improved the quality of this manuscript.

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Supplementary material

Supplementary material 1 

Ostracod samples

Andrei Ștefan

Data type: xlsx

Explanation note: List of GenBank accession numbers for our sequences and the downloaded sequences.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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