Heterocypris exodonta sp. nov. (Ostracoda, Cyprididae), morphological and molecular description of a high altitude asexual microcrustacean from the Nam Co region, Southern Tibetan Plateau

207 Heterocypris exodonta sp. nov. (Ostracoda, Cyprididae), morphological and molecular description of a high altitude asexual microcrustacean from the Nam Co region, Southern Tibetan Plateau Mauricio Bonilla-Flores1, Ivana Karanovic2, Paula Echeverría-Galindo3, Peter Frenzel4, Liseth Pérez5, Nicole Börner6, Katharina Dulias1, Junbo Wang7, Antje Schwalb1 1 Institute of Geosystems and Bioindication, Technische Universität Braunschweig, 38106 Braunschweig, Germany 2 Department of Life Science, Research Institute for Convergence of Basic Science, Hanyang University, 04763 Seoul, Republic of Korea 3 Institute of Organic Biogeochemistry in Geo-Systems (OBG), RWTH Aachen University, 52056 Aachen, Germany 4 Institute of Geosciences, University of Jena, 07749 Jena, Germany 5 Institute of Geosciences, Kiel University, 24118 Kiel, Germany 6 Max-Planck-Institut für Biogeochemie, 07745 Jena, Germany 7 State Key Laboratory of Tibetan Plateau Earth System, Environment and Resources (TPESER), Institute of Tibetan Plateau Research, Chinese Academy of Sciences, Beijing 100101, China Corresponding author: Mauricio Bonilla-Flores ([email protected]) Copyright: © Mauricio Bonilla-Flores et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract A new ostracod species, Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov. is described from a high-altitude temporary pond near Nam Co, Southern Tibetan Plateau. Detailed morphological analyses of valves, soft body, and the partial mitochondrial COI (cytochrome c oxidase subunit I) sequence distinguish this species from its close congeners, Heterocypris incongruens and Heterocypris salina. Key taxonomic features include the morphology of the female genital lobe and the structure of the internal opening of normal pores, particularly variations in the female genital aperture at the intersection of the seminal duct and the oviduct. Pore openings in H. exodonta Bonilla-Flores & Karanovic, sp. nov. and H. incongruens exhibit a turbine shape, while in H. salina they display a simple aperture. Phylogenetic analysis based on the COI dataset supports the new species description and suggests potential synonymy and cryptic species within the H. salina and H. incongruens complexes. Ecologically, these species mainly inhabit temporary water bodies with high oxygen levels and likely follow an r-type ecological strategy, being opportunistic in colonizing new habitats, reproducing via parthenogenesis, and undergoing rapid population growth. This study enhances the understanding of Southern Tibetan Plateau ostracod biodiversity, providing crucial morphological and molecular data for species identification, particularly for parthenogenetic species lacking standard male morphological characters. Additionally, this research offers methods to prevent misidentifications and misinterpretations in (paleo-)ecological studies, contributing significantly to the broader knowledge of taxonomy and ecology of ostracods from the Southern Tibetan Plateau. Due to the high morphological plasticity of Heterocypris incongruens, our findings highlight the need for caution when identifying similar species, as external resemblance may conceal genetic divergence. Key words: Asexual reproduction, freshwater ecosystems, high altitude, ostracods, taxonomy, temporary ponds Academic editor: Simone Nunes Brandão Received: 24 October 2024 Accepted: 1 October 2025 Published: 19 December 2025 ZooBank: https://zoobank.org/ FB9BAF5C-C20E-4481-9794EF60D1A0AA8C Citation: Bonilla-Flores M, Karanovic I, Echeverría-Galindo P, Frenzel P, Pérez L, Börner N, Dulias K, Wang J, Schwalb A (2025) Heterocypris exodonta sp. nov. (Ostracoda, Cyprididae), morphological and molecular description of a high altitude asexual microcrustacean from the Nam Co region, Southern Tibetan Plateau. ZooKeys 1264: 207–248. https://doi. org/10.3897/zookeys.1264.140174 ZooKeys 1264: 207–248 (2025) DOI: 10.3897/zookeys.1264.140174 208 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Introduction During the past two decades, the documented diversity of identified ostracods from the Southern Tibetan Plateau (STP) has increased from 30 to 89 species (Wrozyna et al. 2009a, b; Yu et al. 2009; Mischke et al. 2010; Mischke 2012; Peng et al. 2021). However, ongoing discoveries highlight the need to address taxonomic inconsistencies within this extensive and largely unexplored region (Mischke 2012; Peng et al. 2021). Genera such as Heterocypris Claus, 1892, Ilyocypris Brady & Norman, 1889, and the family Candonidae Kaufmann, 1900, present taxonomic challenges, as morphological features are similar and descriptions are mostly available for valves only (Mischke 2012). This has led to potential inaccuracies in estimating biodiversity, as certain cryptic species require a review of soft part morphology and DNA sequences for species differentiation (Echeverría-Galindo et al. 2021). Nam Co, situated at 4730 m a.s.l. in the south-central part of the STP, is surrounded by temporary ponds that sustain aquatic species adapted to extreme conditions (Blaustein and Schwartz 2001; Jacobsen and Dangles 2017). The genus Heterocypris, mainly composed of desiccation-tolerant species, commonly inhabits these temporary habitats (Meisch 2000; Schwalb et al. 2002; Aguilar-Alberola and Mesquita-Joanes 2011; Mischke 2012; Zhai and Zhao 2014; Akita et al. 2016). Despite its high diversity, with 70 known species (Meisch et al. 2024), the genus diagnosis still poses problems in proper species classification. For instance, the right valve has a row of denticles on its anterior and posterior edges, and it is overlapped by the opposing left valve (Purper and Würdig-Maciel 1974; Martens et al. 2019). The same characteristics are present in Cyprinotus species, members of the same subfamily, except for the presence of a hump in Cyprinotus (Martens et al. 2019; Savatenalinton 2020). The challenge also lies in species delineation due to the remarkably similar soft body morphology (Meisch 2000), highlighting the importance of combining valve characteristics and exploring additional features to differentiate between species. The presence of cryptic species, morphologically indistinguishable but genetically divergent, further complicates taxonomic differentiation (Bode et al. 2010), impacting ecological and paleoenvironmental assessments (Lord et al. 2012). Resolving taxonomic issues in Heterocypris, therefore, requires an integrative taxonomic approach (Padial et al. 2010), combining morphological and molecular methods. Parthenogenetically reproducing ostracods pose additional problems in species delineation. Characters such as the morphology of the female genital opening (often labeled as a lobe) are rarely used for species identification, but they can be useful taxonomic characters (Smith and Kamiya 2007; Bonilla-Flores et al. 2024. These lobes, in external view, are located in the posterior region of the body, near the base of the uropodal ramus. The anterior part has a rigid oval shape, while the posterior part displays a tubular junction (intersection) that internally connects to the oviducts, and also to the seminal ducts (Smith and Kamiya 2007). The appearance of this intersection is variable in the Cyprididae family, such as Amphicypris argentinensis (Fontana & Ballent, 2005), Dolerocypria mukaishimensis Okubo, 1980 (Karanovic & Lee, 2012), Chrissia dongqianhuensis Kong, Karanovic & Yu, 2014 and Stenocypris major (Baird, 1859), and Cypris pretusi Mesquita-Joanes, Aguilar-Alberola, Palero & Rueda, 2020. Three Heterocypris species have been documented from the STP and the Qinghai province: H. incongruens (Ramdohr, 1808), H. salina (Brady, 1868), and 209 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet H. vandouwei (Brehm, 1923). While the first two were recorded from both areas, the latter was only found in Qinghai province (Peng et al. 2021). Our study describes a new species of Heterocypris discovered in a temporary pond near the Nam Co Research Station (NAMORS) in September 2019, thus contributing to the diversity of ostracods on the STP. The aims of this study are to describe a new species of Heterocypris; to identify reliable taxonomic characters that distinguish it from the close congeners, H. incongruens and H. salina; and to use the mitochondrial COI gene to reconstruct its phylogenetic relationship with selected Heterocypris and Cyprinotus species. The study focused on sites in the south-central Tibetan Plateau (STP), particularly around the lakes Nam Co, Taro Co, Xuru Co, and Luo Ma (Fig. 1). Many of the temporary ponds surveyed are located at elevations higher than 4500 m a.s.l.; however, additional sites at lower altitudes from Mexico and Germany were also included (see Table 1). The Tibetan lakes are generally endorheic and strongly influenced by solar radiation, the Indian Summer Monsoon, and the Westerlies (Zhu et al. 2008). Winters are dry, and precipitation occurs mainly from May to September (Anslan et al. 2020). The local surrounding of the lake exhibits an average annual air temperature of 0 °C (Wang et al. 2020) and an estimated mean annual precipitation of 406 mm (Anslan et al. 2020). Furthermore, Gou et al. (2017) noted ice cover from early January through April in the lake’s vicinity and in Nam Co itself. Materials and methods Sampling and material handling Ostracods were collected from surface sediments of a temporary pond near Nam Co, using a spatula and a hand net with a mesh size of 125 µm (Echeverría-Galindo et al. 2021; Vences et al. 2024). The material used to describe the new species corresponds to specimens previously referred to as Heterocypris aff. salina by Vences et al. (2024) and as Heterocypris cf. salina by Echeverría-Galindo et al. (2021). In both studies, these names were used provisionally due to the lack of a formal species description. In total, we examined material from eight sites (mainly in ponds), including samples from Nam Co collected in 2019, and more recent specimens from Mexico City (2021) and Braunschweig, Germany (2021) (see Table 1). Additionally, we included females of H. exodonta sp. nov., H. incongruens, and H. salina collected from other regions of the Southern Tibetan Plateau. These specimens originate from unpublished studies conducted in the vicinity of Luo Ma, Taro Co, and Xuru Co. Locations and environmental data of the samples are given in Table 1. To verify taxonomy, we decided to compare the new species with morphologically similar species previously recorded from the STP. A total of ten adult females of each species were dissected using a stereo microscope (Leica MZ75); the right and left valves of each specimen were photographed to record their color. Female genital lobes were photographed with a KeyenceTM microscope (VHX-E100; objective ×100–×500). The valves were then placed onto micropaleontological slides, and subsequently photographed using scanning electron microscopy (SEM, ZEISSTM EVO Ls 25) at the Institute for Chemical and Thermal Process Engineering, Faculty of Mechanical 210 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Engineering, TU Braunschweig. The material from the STP will be deposited at the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, China. The material of H. incongruens from Mexico will be deposited at the National Collection of Crustaceans, Institute of Biology, National Autonomous University of Mexico. The material of H. salina collected in Braunschweig will be deposited at the Zoological Museum, Hamburg, Germany (ZMH). Terminology and chaetotaxy of the limbs follow Broodbakker and Danielopol (1982), and Meisch (2000), except for the most posterior appendage (uropodal ramus) which follows Meisch (2007). Chaetotaxy of the antenna follows Horne (2005), and systematics refers to Meisch et al. (2024). Molecular analysis and phylogenetic methods Genetic material extracted from the following four species was used: Cyprinotus cassidula Smith & Chang, 2020 (as a reference outgroup), Heterocypris exodonta sp. nov., H. incongruens, and H. salina (Table 2). Lysis buffer was prepared following established protocols for nematodes (Williams et al. 1992). All PCR reactions were conducted in a 25 μl volume, comprising 5 μl of DNA template, 2.5 μl of 10 × ExTaq Buffer, 0.25 μl of TaKaRa ExTaq (5 units/μl), 2 μl of dNTP Mixture (2.5 mM each), 1 μl of each primer, and 13.25 μl of distilled water. Partial sequences of mitochondrial COI were amplified using the following PCR protocol: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, at 46 °C for 30 s, at 72 °C for 1 min, and a final extension at 72 °C for 10 min. This gene was amplified with universal Folmer primers (Folmer et al. 1994). The presence of DNA was confirmed using 1% agarose gel electrophoresis. After a treatment with LaboPass PCR Purification Kit (Cosmo Genetech), Table 1. Environmental data from sampling sites of Heterocypris species (He = Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov., Hi = Heterocypris incongruens (Ramdohr, 1808), Hs = Heterocypris salina (Brady, 1868)). WD: water depth, EC: electrical conductivity, WT: water temperature, DO: dissolved oxygen, Alk: alkalinity, ND: no data. (GPS coordinates referenced to the WGS84 geodetic system). Species Sample reference number Locality Date Latitude, Longitude Altitude m a.s.l. Habitat WD (m) EC [µS/cm] WT [°C] pH DO [mg/l] alk [mmol/l] He NC-0919 Nam Co, STP 13.09.2019 30°47.40'N, 90°57.60'E 4728 Pond 0.2 ND ND ND ND ND He, Hi, Hs TIP11-84 Taro Co, STP 21.09.2011 31°11.11'N, 84°19.81'E 4577 Pond ND 1342 10.1 9.2 3.5 10.7 Hi HI-M19 San Nicolas Tetelco, Mexico City 24.03.2021 19°12.41'N, 98°58.24'W 2271 Flowerpot 0.1 ND ND ND ND ND Hi TIP08-4 Luo Ma town, STP 08.09.2008 31°16.49'N, 91°48.48'E 4587 Small lake 0.1 227 12.9 8.8 5.2 0.2 Hi TIP11-99 Taro Co, STP 23.09.2011 31°19.42'N, 84°19.40'E 4578 Pond near inflow 0.2 236 16.6 9.4 2.9 1.4 Hs HS-G19 Braunschweig, Germany 26.03.2021 52°16.23'N, 10°31.95'E 73 Flowerpot 0.2 ND ND ND ND ND Hs TIP11-86 Taro Co, STP 21.09.2011 31°11.21'N, 84°18.21'E 4588 Pond 0.1 1017 16.2 9.5 5.8 7.1 Hs TIP12-H55 Pond near Xuru Co, STP 23.06.2012 30°10.17'N, 86°26.37'E 4728 Pond 0.1 1639 22.5 8.6 10.4 15.5 211 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet the PCR products were sequenced using an ABI automated capillary sequencer (Macrogen, Seoul, South Korea), employing the same sets of primers. Finch TV 1.4.0 (http://www.geospiza.com/Products/finchtv.shtml) was used to assess the quality of signal and to identify sites with potential low resolution, which were corrected by comparing forward and reverse strands. BLAST algorithm (Altschul et al. 1990) was used to check the identity of obtained sequences. All sequences were deposited in GenBank, and the accession numbers are listed as terminals on the phylogenetic trees. Table 2. Sampling sites of Heterocypris and Cyprinotus species (He = Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov., Hi = Heterocypris incongruens (Ramdohr, 1808), Hs = Heterocypris salina (Brady, 1868), Cc = Cyprinotus cassidula (Smith & Chang, 2020) were used for molecular analysis. n: number of specimens, ND: no data. Species Sample reference number Locality Date Latitude, Longitude Altitude (m a.s.l.) Habitat Water dept (m) nSex Cc CKF Seonjudo park, South Korea 08.2022 37°54.00'N, 126°54.00'E 165 ND ND 5Female Cc CKF Seonjudo park, South Korea 08.2022 37°54.00'N, 126°54.00'E 8ND ND 2Male He NC-0919 Nam Co, STP 13.09.2019 30°47.40'N, 90°57.60'E 4728 Pond 0.2 10 Female Hi HI-M19 San Nicolas Tetelco, Mexico City 24.03.2021 19°12.41'N, 98°58.24'W 2271 Flowerpot 0.1 12 Female Hs HS-G19 Braunschweig, Germany 26.03.2021 52°16.23'N, 10°31.95'E 73 Flowerpot 0.2 12 Female Figure 1. A. Map showing the Tibetan Plateau and B. Sampling localities of Heterocypris species within the study area (source: Demis 2023). Abbreviations: He = Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov., Hi = Heterocypris incongruens (Ramdohr, 1808), Hs = Heterocypris salina (Brady, 1868). 212 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet The obtained sequences, with those retrieved from GenBank, were aligned using MEGA11 (Tamura et al. 2021). The evolutionary model was tested using the IQ-TREE Web Server (Trifinopoulos et al. 2016), applying Akaike information criterion (Akaike 1974). Bayesian Inference, implemented in BEAST v. 2.5 (Bouckaert et al. 2014), was used to estimate phylogenetic relationships. The analysis ran for 10 million generations, sampling every 1000 generations. Tracer (Rambaut et al. 2018) was used to visualize the results of BEAST analyses, and FigTree v. 1.4.3 (available from http://github.com/rambaut/figtree) was used for tree visualization. Results Taxonomic account Class Ostracoda Latreille, 1802 Subclass Podocopa Sars, 1866 Order Podocopida Sars, 1866 Suborder Cypridocopina Baird, 1845 Superfamily Cypridoidea Baird, 1845 Family Cyprididae Baird, 1845 Subfamily Cyprinotinae Bronstein, 1947 Genus Heterocypris Claus, 1892 Brief diagnosis. (after Meisch 2000 and Karanovic 2012): valves asymmetrical, the left valve (LV) longer and higher, overlapping the right valve (RV). In the dorsal area, LV lacks a hump, or gibbosity, and the RV has margins bearing denticles (Victor and Fernando 1980; Martens et al. 2019). Antenna with natatory setae extending beyond the tips of terminal claws, maxillular palp two-segmented, with the third endite bearing two-segmented and serrated teeth. Walking leg (T2) with a small seta d1; cleaning leg (T3) with a seta at the tip of the segment transformed into a pincer organ; uropodal ramus well developed. Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov. https://zoobank.org/C2AC540D-7614-4CEE-A80E-F53CCA9794B6 Figs 2–5 Heterocypris aff. salina: Vences et al. 2024: 1–9. Heterocypris cf. salina: Echeverría-Galindo et al. 2021: 982–996. Type locality. Temporary pond near Nam Co, Southern Tibetan Plateau. Material examined. Holotype: China • 1 female, Nam Co, Southern Tibetan Plateau, appendages mounted on a glass slide, and the valves stored in a micropaleontological slide. Size RV: length = 1214 µm, height = 652 µm; LV: length = 1220 µm, height = 690 µm. Echeverría-Galindo et al. (2021) leg. Paratypes: China • 7 females, in addition, eleven RV and nine LV separated; same location as holotype. Size RV: length = 994–1322 µm, height = 544–732 µm; LV: length = 1001–1337 µm, height = 555–734 µm, each specimen’s soft body mounted on a glass slide; valves stored in micropaleontological slides. 213 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Additional material. China • 1 female collected from Taro Co, STP, site TIP1184 (Table 1). Size RV: length = 1113 µm, height = 610 µm; LV: length = 1135 µm, height = 624 µm; P. Frenzel leg. Figure 2. Valve details of Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov. from Nam Co (ID: NC-0919-E01) and Taro Co (ID: TIP11-84-E01), Southern Tibetan Plateau. Nam Co specimens: A1. RV and A2. LV lateral internal view of adult specimens. A3. Posterior internal view of RV; A4. Close-up of posterior external view of RV, with denticles in the margin. A5, A6. Close-ups of normal pores (turbine shape) without bristles and lip, in the central-internal region of RV. Taro Co specimens: B1. RV and B2. LV in lateral external view. B3. Anterior external view with denticles on RV; B4. Posterior internal view with denticles on RV. B5. Pore (turbine shape without bristle) in internal view, and B6. Simple pore in the central-internal region of RV. 214 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Diagnosis. (adult females, Figs 2, 3) Valves elongated in lateral view. LV overlaps RV on all free margins. It is distinguished by a flattened dorsal margin and a pronounced row of blunt denticles on the anterior and posterior margins of the RV. The inner lamella is broad, and the inner list is well-developed, particularly in the LV. On the internal side, normal pores with a turbine shape but without a bristle in the center. The exopod seta in A2 is relatively shorter than the length of the second endopodal segment. Upper lip has a broad lip with a patch of pseudochaetae present laterally, immediately above the mouth opening. The second endopodal segment in T2 is proportionally short and the claw h2 is long. General shape of lobes is elongated, medium width; curvature of lobes is smooth, continuous; shape of apex is truncate with retrograde beak. Dimensions. Female, RV: length = 994–1322 µm, height = 544–732 µm; LV: length = 1001–1337 µm, height = 555–734 µm. Description of holotype. Valves: Surface of both valves with numerous normal rimmed (type A2; Danielopol et al. 2018) pores, each carrying one seta, as well as striations, and foveolae (Fig. 3A7–A8, B7–B8). In lateral view, lacks a hump on the dorsal side. RV with broad inner lamella and anterior and posterior margins with a well-differentiated row of denticles blunt, with inner list along anterior margin. LV with broad inner lamella in internal view (Fig. 2A2, B2). Normal pores on inner side with turbine-shaped opening and without a bristle in the center (Fig. 2A5, A6, B5, B6). Color yellowish (Fig. 3). Description of soft parts: Antennule (Fig. 4A). Seven-segmented: segment I with three long posterodistal setose setae, and one short, smooth seta anteromedially. Segment II with one smooth distal seta anteriorly, and a Rome organ posteriorly. Segment III with one short, smooth distal seta on each side. Segment IV with four distal setae: two long setae located anteriorly, and two shorter, smooth setae spanning to segment VII. Segments V and VI with same chaetotaxy as segment IV, however, the ventral setae are longer. Additionally, segment VI bears a short alpha seta not present in the preceding segments. Segment VII with three setae distally (two long, one short; long ones setose, short one smooth), plus aesthetasc ya, which is 67 µm long, 2× longer than the terminal segment with 30 µm. Antenna (Fig. 4B). Five-segmented: coxa with three short setae; protopod or basis robust, with a smooth ventral distal seta. First endopodal segment with aesthetasc Y with distal dots; with five long swimming setae, and one small additional seta at the anterodistal side. Additionally, this segment includes one long setose seta posterodistally. Second endopodal segment with four t-setae (t1–t4), and aesthetascs y1, and y2, all situated posteromedially to posterodistally. Distally, this segment bears three long, thin z-setae and claws G1, G2, and G3. Claws G1 and G3 are approximately equal in length, while G2 is slightly shorter. Terminal segment with an aesthetasc y3 and claws GM and Gm; the former being more robust and slightly longer than the latter. Exopodal seta measures 205 µm in length, longer than the first (162 µm) and second (98 µm) endopodal segments. The second endopodal segment is 0.48× the length of the exopodal seta. Upper lip and rake-like organ (Fig. 5A, B). Measurements on the upper lip were taken as follows: l—length (263 µm), h—height (61 µm), hp—position of the maximum height (186 µm), in accordance with Karan-Žnidaršič and Petrov (2014). A large patch of dense pseudochaetae was present laterally, immediately above the mouth opening. Rake-like organ with nine teeth on the right and ten on the left. 215 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 3. Microscopic and SEM images of the valves of Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov. from Nam Co (ID: NC-0919-E01) and Taro Co (ID: TIP11-84-E01), Southern Tibetan Plateau. Nam Co: A1. LV and A2. RV in lateral external view (whole carapace). A3. LV and A4. RV in lateral internal view. A5. LV and A6. RV in external view. A7, A8. Normal rimmed pores, striation, and foveolae in the external surface of RV. Taro Co: B1. LV and B2. RV lateral external view (whole carapace). B3. LV and B4. RV in lateral internal view. B5. LV and B6. RV in lateral external view. B7, B8. Normal rimmed pore, striations, and foveolae on the external surface of RV. 222 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Upper lip (Fig. 9A) has a distinctive internal reticulation. Measurements on the upper lip were taken as follows: length (290 µm), height (77 µm), position of the maximum height (208 µm), in accordance with Karan-Žnidaršič and Petrov (2014). A patch of medially positioned setae is in the middle region, approximately within the first third of the length from the mouth opening. Rake-like organ with nine teeth on the right and ten on the left. Mandibular coxa (Fig. 8C). With seven teeth and one plumose subdistal seta. Mandibular palp (Fig. 8D). Four-segmented: first segment bears a respiratory plate with six rays of similar length, plus three long distal setae, and an α seta. Second segment with a β seta, with four additional setae, including three long distal ones. Third segment features a γ seta on the apical part, and this same segment has nine setae in the distal region. Fourth segment features five setose setae, each measuring twice the length of the segment. Maxillula (Fig. 8E). Palp two-segmented: the first one bears a long, smooth seta with four setose setae, and the second segment is quadrangular in shape with five smooth long setae. The third endite holds two serrated two serrated Zahnborsten, plus five distal and a subdistal setae. Second endite with eight setae, and first endite with nine distal setae and two setae with rays in the basal region. T1 (Fig. 8F). With two setae a, one b, and one d at the endopod. Exopod with six hirsute rays; protopod with 13 setose distal setae. T2 (Fig. 9B). Five-segmented: the first one with a short seta d1. The second segment bears a long distal seta (e). The third segment has a setose distal seta (f). Segment four possesses a distal long seta (g) with a shorter seta. Segment five with short setae h1 and h3 and a claw-like seta h2 with spinules. The length of the second endopodal segments measures 108 µm, representing 59% of the length of the claw, which measures 183 µm. T3 (Fig. 9C). Four-segmented: first segment bears a short, smooth d1-seta. In the distal region, there is a long setose seta d2, and a seta dp as long as the third segment. Second segment features a setose distal seta (e). Third segment displays a medial f-seta, a reduced h1 seta in the distal part (fused 4th segment), a segmented h2 seta, and a long h3 setose seta. Uropodal ramus (Fig. 9D). Elongated structure with two distal claws—Ga (174 µm), exceeding Gp (122 µm) in length—and one shorter setose Sp (94 µm). A Sa seta is present, shorter than Sp, and two caudal ramus attachments are observed. Female genital lobes (Fig. 9E) have an intersection with a triangular shape. General shape of lobes is elongated and relatively broad; curvature of lobes is more open and straighter; shape of apex is pointed, without truncation. Reproduction. Commonly asexual populations (Meisch 2000), with records of males from a man-made pond, Kahramanmaraş, Turkey (Yavuzatmaca and Külköylüoğlu 2019), and several sexual records in Europe (Horne and Martens 1999) Distribution. Recorded from numerous places, it is cosmopolitan (Meisch 2000). Habitat. The species predominantly inhabits temporary ponds with vegetation cover (Meisch 2000; Mischke 2012). In accordance with our limnological data (Table 1), this species was found in freshwater to brackish, alkaline to oxygenated waters (average 3.8 mg/l). Ganning (1971) found this species up to a salinity of 16 psu at the Baltic Sea coast. 223 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 8. Heterocypris incongruens (Ramdohr, 1808), female, Mexico City (ID: HI-M19-I01). A. Antennule; B. Antenna; C. Mandibular coxa; D. Mandibular palp; E. T1; F. Maxillula. 224 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 9. Heterocypris incongruens (Ramdohr, 1808), female from Mexico City, Mexico (ID: HI-M19-I01). A. Upper lip and rake-like organs; B. T2; C. T3; D. Uropodal ramus with attachment and E. Female genital lobes. 225 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Heterocypris salina (Brady, 1868) Figs 10–13 Cypris salina Brady, 1868: 368–369, pl. 26, figs 8–13. Cyprinotus fretensis Brady & Robertson, 1870: figs 48, 49 (Meisch and Broodbakker 1993). Cyprinotus salina (Brady, 1868): Müller 1900: 76, pl. 16, figs 1, 2, 10, 12. Heterocypris salina (Brady, 1868), nov. comb.: Klie 1932: 588. Cyprinotus salinus (Brady, 1868): Wagner 1957: 30–31, pl. 9, figs 1–6. Cyprinotus salinus (Brady, 1868): Jordan et al. 1962: 76, 77, pl. I, figs 6, 7; pl. V, figs 57–61; pl. VIII, figs 80, 82, 83. Cyprinotus salinus (Brady, 1868): Devoto 1965: 332, fig. 26. Cyprinotus salinus (Brady, 1868): Diebel and Pietrzeniuk 1975: 1213, pl. VII, figs 5, 6. Cyprinotus salinus (Brady, 1868): Diebel and Pietrzeniuk 1978: 90, pl. 24, figs 5, 6. Heterocypris salina salina (Brady, 1868): Freels 1980: 28, pl. 3, figs 1–6. Heterocypris salina (Brady, 1868): Meisch 2000: 354–357, fig. 148. Material examined. Germany • 10 dissected females, (size RV length = 1012– 1107 µm, height = 629–652 µm; LV length = 1098–1133 µm, height = 671– 694 µm). Botanical Garden, TU Braunschweig; M. Bonilla-Flores leg. China • 2 females from Taro Co, STP (TIP11-84) (size RV length = 1068–1061 µm, height = 660–667 µm; LV length = 1079–1092 µm, height = 741–741 µm); • 2 females from Taro Co (TIP11-86) (RV length = 1011–1146 µm, height = 633–732 µm; LV length = 1024–1151 µm, height = 616–693 µm); • 1 female from Xuru Co, STP (TIP12-H55) (size RV length = 960 µm, height = 569 µm; LV length = 983 µm, height = 618 µm); P. Frenzel leg. Diagnosis. (Adult female, Figs 10, 11) (adapted from Meisch 2000 and Kubanç et al. 2007): valves compressed and triangular in the dorsal region. RV smaller than LV, LV overlapping RV. RV has row denticles on anterior and posterior margins in external view, with broad inner lamella and inner list (Fig. 10A1, A3, A4, B1, B3, B4). LV with a broad inner lamella, anteriorly with inner list, in the posteromedial region with a slight fold (Fig. 10A2–B2). Both valves display a brown coloration pattern, characterized by a pair of lighter stripes (Fig. 11B1, B2). Normal pores on the internal valve surface with simple apertures. The upper lip presents dense pseudochaetae laterally, just above the mouth opening. Female genital lobes with an oval shape, with no discernible projection at the intersection, but the shape of the apex is truncated, and an interlacing projection at the intersection forms a ring. Dimensions. Adult females, RV ranges: length = 970–1194 µm, height = 577–755 µm; LV ranges: length = 989–1189 µm, height = 603–724 µm. Valves. Laterally, dorsal side arched. RV has a broad inner lamella with small denticles along the anterior and posterior margins. LV displays a broad inner lamella accompanied by an inner list. Normal pores on the internal valve surface with simple apertures (Fig. 10A5, A6, B5, B6). Similarly, multiple normal rimmed pores are present on the external valve surface, with a single bristle (Fig. 11A7, A8, B7, B8). Some striations were observed for organisms from Germany (Fig. 11B7). Description of soft parts. (Figs 12, 13): Antennule (Fig. 12A). Seven-segmented: segment I with two long setose setae on the posterodistal side and one short seta on the anterodorsal region. Segment II with a short seta on the anterior side. Segment III carries two distal setae, with the anterodorsal seta 226 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 10. SEM images of valve details of Heterocypris salina (Brady, 1868) from Taro Co, Southern Tibetan Plateau (ID: TIP11-84-S01), and Braunschweig, Germany (ID: HS-G19-S01). Taro Co: A1. RV and A2. LV in internal view of adult specimens. A3. Denticles on the anterior margin, and A4. The posterior margin of the RV, internal view. A5, A6. Normal pores with simple openings located in the central inner region of the RV. Specimens from Braunschweig: B1. RV and B2. LV adult specimens, internal views. B3, B4. Denticles in the posterior region of the RV. B5, B6. Normal pores with simple opening located in the central inner area of the RV. 227 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 11. Microscopic image and SEM images of the valves of Heterocypris salina (Brady, 1868) from Taro Co, Southern Tibetan Plateau (ID: TIP11-84-S01), and Braunschweig, Germany (ID: HS-G19-S01). Taro Co: A1. LV and A2. RV, external view. LV (A3) and RV (A4), internal view. LV (A5) and RV (A6) in external view. A7, A8. Foveolae and normal rimmed pores with bristles on the external surface of RV. Specimens from Braunschweig: B1. LV and B2. RV, external view. B3. LV and B4. RV in internal view. B5. LV and B6. RV, external view. B7, B8. Show striations and normal rimmed pores with bristles on the external surface of RV. 228 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet reaching segment VII. Segment IV bears two long anterodorsal setae and two short posterodistal setae. Segment V features four long setae in the distal region. Segment VI with an α-setae in the distal part, with four long setae. Segment VII presents three long setae distally and one aesthetasc ya with 65 µm length, roughly twice as long as the terminal segment (31 µm). Antenna (Fig. 12B). Five-segmented: coxa has two short hairy setae and one smooth short seta. Basis with a ventrodistal long seta. Exopod long seta as long as the first endopodal segment, which bears a segmented aesthetasc Y, a stout ventroapically seta, one short and five long natatory setae in the apical dorsal part. The second endopodal segment has four t-setae and two aesthetascs (y1 in the posteromedial part and y2 in the posterodistal part). This segment also bears two medial setae, and distally three long, slender setae (z) and three claws (G1, G2, and G3). Claws G1 and G3 are approximately equally long, while G2 is shorter. The terminal segment presents two claws (GM and Gm), and the aesthetasc y3. Exopod long seta (140 µm length) longer than the first (119 µm) and second (96 µm) endopodal segment, respectively; the exopod long seta is approximately 1.46× the length of the second endopodal segment. Upper lip (Fig. 13A). Measurements on the upper lip were taken as follows: l—length (245 µm), h—height (64 µm), hp—position of the maximum height (168 µm), in accordance with Karan-Žnidaršič and Petrov (2014). Dense pseudochaetae were present laterally, just above the mouth opening. Rake-like organs are formed by eight teeth on the right, and nine on the left. Mandibular coxa (Fig. 12C) with seven teeth, the largest one with small setae on its ventral part. Teeth three, four, and seven lack setae between them. Mandibular palp (Fig. 12D). Four-segmented: the first segment bears two setose setae and one smooth seta, and an additional α-seta. The second segment has four setose setae, one long smooth seta, and one short setose β-seta on the inner part, and three smooth setae on the outer part. The third segment features eleven setae; four long smooth setae, with one thick γ-seta. In the most distal region, there are three short, setose setae and three smooth setae. The last segment has three setose setae in the distal region and one smooth seta. Maxillula (Fig. 12E). Maxillular palp two-segmented, the first one bears a long, smooth seta with four long and smooth distal setae, the second segment of the palp is subrectangular with four smooth setae. First endite with eight setae in distal part, and two setae in the basal region. Seven setae in second endite. The third endite has two serrated Zahnborsten, plus five smooth setae. T1 (Fig. 12F). With two setae a, one b, and one d. Exopod with six hirsute rays, protopod with 13 setose distal setae. Endopod with three distal setae, h1 and h3 longer than h2. T2 (Fig. 13B). Five-segmented: segment I with a short seta d1. Second segment bears a long anterodistal e-seta. Third segment with a distal setose f-seta. Segment four with a distally located g-seta with a shorter seta. Fifth segment with three h-setae; the claw-shaped seta h2 with dentition. The length of the second endopodal segment measures 95 µm, representing 61% of the length of the claw which measures 156 µm. T3 (Fig. 13C). Four-segmented: first segment bears a short, smooth seta d1. In the distal region, with a long d2-seta, and a setose dp-seta. Second segment with a setose distal e-seta. Third segment in the distal part (fused 4th segment) has a small h1-seta, seta h2 is striated, and seta h3 long. 229 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 12. Female Heterocypris salina (Brady, 1868) from Braunschweig, Germany (ID: HS-G19-S01). A. Antennule; B. Antenna; C. Mandibular coxa; D. Mandibular palp; E. T1; F. Maxillula. 230 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Figure 13. Female Heterocypris salina (Brady, 1868) from Braunschweig, Germany (ID: HS-G19-S01). A. Upper lip and rake-like organs; B. T2; C. T3 with detail of the tip; D. Caudal ramus with attachment, and E. Female genital lobes. 231 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Uropodal ramus (Fig. 13D) with an elongated shape and two distal claws – Ga (137 µm), exceeding Gp (97 µm) in length – plus one shorter setose Sp (80 µm). Additionally, with a smooth Sa seta, shorter than Sp, and two caudal ramus attachments. Female genital lobes (Fig. 13E). Oval and elongated shape, curvature of lobes; shape of apex is truncated, without a beak, and an interlacing projection at the intersection forms a ring. Reproduction. Only asexual populations were found in Germany and STP. Males were recorded from Crete, Greece (Petkowski et al. 2000). Distribution. Widespread distribution (Meisch 2000). Habitat. Heterocypris salina is an eurytopic species, tolerant to high conductivity, typical of shallow and temporary ponds (Meisch 2000). Furthermore, according to our limnological data (Table 1) and previous publications, the species is found in fresh, brackish, and saline waters (Ganning 1971), and alkaline and oxygenated waters (average 6.5 mg/l). Upper salinity limits are given as 8.6‰ (Vesper 1975) and even up to 20‰ (Griffiths and Holmes 2000). It is also tolerant to organic pollution (Mezquita et al. 1999). Molecular analysis The COI alignment included 30 sequences, with 14 newly obtained (Code OR91) and the remainder downloaded from GenBank (Fig. 14). The alignment spanned 715 base pairs, but sequence lengths varied from 317 to 683 base pairs. Within group p-distances (Table 3) ranged from 1% (in Heterocypris exodonta sp. nov. and Heterocypris sp.) to 9% (in H. salina from Egypt and Germany). The largest between-group distances (Table 3) were observed between H. exodonta sp. nov. and Cyprinotus cingalensis Brady (1889), equaling 25%, and the smallest were between H. salina and H. spadix Munakata, Tanaka & Kakui, 2021, equaling 10%. The JC69 (Jukes and Cantor 1969) model was selected as the most appropriate model for DNA evolution. In the resulting phylogenetic tree (Fig. 14), H. exodonta sp. nov. clustered with H. cf. salina sequences from the STP (Echeverría-Galindo et al. 2021; Vences et al. 2024), as they derive from the same material. However, to avoid further confusion, the name was changed to H. exodonta sp. nov. The Table 3. Pairwise genetic distances (p-distances) based on cytochrome c oxidase subunit I (COI) sequences among species of Heterocypris Claus, 1892 and Cyprinotus Brady, 1886, calculated using 1000 bootstrap replications. Values represent average distances between groups; diagonal entries (in bold or along the row) indicate within-group variation. Sequences of Heterocypris exodonta Bonilla-Flores & Karanovic, sp. nov. are compared with closely related species and outgroups. Species H. sp. H. incongruens H. salina H. spadix C. sp. C. cingalensis C. cassidula H. exodonta sp. nov. 0.01 H. sp. 0.23 0.01 H. incongruens 0.22 0.19 0.06 H. salina 0.24 0.20 0.20 0.09 H. spadix 0.24 0.21 0.20 0.10/ C. sp. 0.23 0.20 0.20 0.21 0.22/ C. cingalensis 0.25 0.21 0.21 0.19 0.21 0.20/ C. cassidula 0.24 0.21 0.21 0.19 0.21 0.23 0 238 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet this structure is not clearly discernible. We propose the use of the female genital lobes to differentiate species with similar morphology, although further analysis and comparisons among different species are necessary to validate their potential as a taxonomic character and their utility in future descriptive studies. Molecular differences In recent years, the combination of traditional taxonomy, based on morphological characteristics, and DNA barcoding using the CO1 gene for identifying ostracod species has become extremely useful (Nigro et al. 2016). Molecular methods are particularly useful when species share similarities in limb characters and valve morphology (Macario-González et al. 2018). Despite this, there is limited research on molecular analyses in non-marine ostracods in the STP. Echeverría-Galindo et al. (2021) identified eight species based on morphology and molecular data. However, for the species identified as Heterocypris cf. salina, the abbreviation “cf.” and “aff.” was used to indicate uncertainty in its identification due to its resemblance to H. salina. However, our study, based on morphological and genetic data, suggests that the sequences originally attributed to Heterocypris cf. salina belong to H. exodonta sp. nov., and consequently, the name will be updated in GenBank. Most importantly, the new species forms a distinct branch on the phylogenetic tree and displays high p-distances from all available COI sequences. Results of our phylogenetic analysis and p-distance calculations suggest that Heterocypris salina and H. incongruens both represent species complexes (Fig. 14). Our findings complement a recently published study by Kilikowska et al. (2024), which examined 13 populations of H. salina from across Europe using 28S rRNA and COI. The authors found that the complex consists of four genetic species, but combining their data with ours is beyond the scope of this paper. While the p-distances observed between Egyptian (Ali et al. 2018) and German populations of H. salina are sufficiently high to support their independent species status, the distances between the German H. salina and H. spadix (described from Japan) are much smaller, which may suggest their synonymy. Unfortunately, Kilikowska et al. (2024) did not incorporate any morphological studies, as is the case with many recent publications on genetic ostracod species (e.g., Bode et al. 2010; Schön et al. 2012, 2017). Cryptic species, if left undescribed, can hinder accurate biodiversity assessments (Platnick 2013), with serious consequences for conservation efforts. Another important result of our analysis is that neither Heterocypris nor Cyprinotus appears to be monophyletic. This has been suggested previously (Kong et al. 2014; Yoo et al. 2017), and further studies are needed to resolve this issue. Ecology The three species studied are representatives of shallow and temporary waters, with a capacity for rapid reproduction and colonization of new habitats (Meisch 2000; Mischke 2012). Heterocypris incongruens is commonly found in temporary ponds in both the southern and northern parts of the Tibetan Plateau and other parts of the world (e.g., Meisch 2000; Schwalb et al. 2002; Mischke 2012; Akita et al. 2016), where the habitats in the ponds are composed of fine mud and organic matter, providing food (e.g., algae) (Purper and Würdig-Maciel 239 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet 1974). Subsequently, the phenology of the ostracods responds when these ponds periodically dry, and with the presence of rainwater, populations grow again (Rossi et al. 2011). Rossi et al. (2011) consider H. incongruens as an r-strategy species, meaning it is an opportunistic species, colonizing temporary ponds, with a short generation time, reproduction mainly by parthenogenesis, rapid population growth, and even cannibalistic behavior. The latter could influence population density regulation. These ecological strategies result in genetic variability, with a high level of clonal diversity (Rossi and Menozzi 2012). Heterocypris exodonta sp. nov. is found in temporary ponds on the Southern Tibetan Plateau, and it can be considered a freshwater ostracod, also with r-type ecological strategies, similar to H. incongruens. During this study, we also observed cannibalistic behavior of adults towards recently molted juveniles. This behavior has multiple hypotheses, including lack of food, competition, and population regulation; however, laboratory studies and observations are required to corroborate these hypotheses (Rossi et al. 2011). Finally, Heterocypris salina can also be found in shallow water bodies (Petkowski et al. 2000), and is considered thermo-euryplastic. It is an indicator of high salinity, with development occurring in summer (Janz et al. 2001). In sediments from Elk Lake, Minnesota, dating to the mid-Holocene, this species was recognized as euryhaline and used as a paleo-indicator of a shallow saline lake (Smith et al. 2002). This is corroborated by the work of Meisch and Broodbakker (1993) on the Canary Islands, where they determined that H. salina inhabits water bodies with conductivity ranges between 360–6130 µS/cm. Geographic distribution Heterocypris exodonta sp. nov. has a more restricted geographical distribution, so far, it is known only from the STP. In contrast, H. incongruens and H. salina have broader distribution ranges spanning Asia, Europe, Africa, and the northern and southern regions of the Americas. However, it is important to note that the cosmopolitan distribution of the latter two species must be confirmed and further examined through molecular analysis to investigate potential cryptic species, as it is confirmed by our molecular study. According to our phylogeny, H. salina from Germany and Egypt, as well as H. incongruens from Mexico, India, and possibly Turkey, may belong to different species. Our phylogenetic results suggest that Heterocypris salina from Germany and Egypt, as well as H. incongruens from Mexico, India, and possibly Turkey, may represent distinct species rather than a single cosmopolitan taxon. This pattern aligns with recent findings by Kilikowska et al. (2024), who identified multiple cryptic genetic lineages within H. salina, highlighting a higher-than-expected species diversity in morphologically similar populations. These results reinforce the importance of integrative approaches, combining molecular data with morphology and biogeography, to resolve species boundaries in ostracods. Conclusions The recent discovery and subsequent morphological characterization of Hetero cypris exodonta sp. nov. from the Southern Tibetan Plateau offers a novel taxonomic perspective within the Heterocypris genus. The focus on 240 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet female genital lobes, particularly the varying shapes of the intersection region of the seminal duct and the oviduct, serves as a crucial diagnostic feature, especially in asexual species and populations where male morphological features are lacking. This study provides the first detailed description of the internal structure of pore openings in species of the genus Heterocypris. The inner morphology was found to differ between species: H. exodonta sp. nov. and H. incongruens exhibit turbine-shaped internal pores, whereas H. salina shows a simple, unstructured aperture. These differences, reported here for the first time within the genus, may serve as valuable diagnostic characters in species delimitation and future systematic studies. Our molecular analyses, specifically targeting the Cytochrome c Oxidase Subunit 1 gene (CO1), has allowed a comprehensive differentiation of H. exodonta sp. nov. from closely related species, H. incongruens and H. salina. These findings not only reinforce the descriptive aspects of traditional taxonomy but also provide valuable insights into the genetic differences, additionally to observed morphological variations. This meticulous morphological delineation is important in preventing misidentification of H. exodonta sp. nov., thus ensuring a clear distinction from other species. By integrating molecular, morphological, and ecological insights, this study considerably advances our understanding of ostracod biodiversity and evolution. Heterocypris exodonta sp. nov. currently has limited records on the STP. Increased sampling efforts in future studies may expand its distribution. In contrast, the widespread distribution of both H. incongruens and H. salina spans diverse regions, including Asia, Europe, and both northern and southern areas of the Americas, but probably represents species complexes. Acknowledgements We thank all the colleagues from Nam Co Observation and Research Station for Multisphere (NAMORS) of the Institute of Tibetan Plateau Research, Chinese Academy of Sciences. We are grateful for the support by Nancy Mercado Salas, crustacean collection of the Zoological Museum of Hamburg, for allowing the use of the camera lucida microscope. Simone Schulze from the Faculty of Mechanical Engineering, Institute for Chemical and Thermal Process Engineering, TU Braunschweig, took the SEM pictures of ostracods. We thank Sten Anslan and Wengang Kang for their support during field work. Michael Kraft, director of the botanical garden, TU Braunschweig, and Thorsten Marschall, in charge of the greenhouses are acknowledged for allowing us to collect ostracods. Thanks to Janine Melzig and Nora Kraatz for their assistance in sorting, counting, and measuring the ostracod valves. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. 241 ZooKeys 1264: 207–248 (2025), DOI: 10.3897/zookeys.1264.140174 Mauricio Bonilla-Flores et al.: Heterocypris exodonta sp. nov., a high-altitude microcrustacean from Tibet Use of AI No use of AI was reported. Funding This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the project “Geo-ecosystems in transition on the Tibetan Plateau” (TransTiP; 317513741/ GRK2309) and the project from Science and Technology Department of Tibet (XZ202101ZD0006G). Author contributions Mauricio Bonilla-Flores: Conceptualization, Formal analysis, Investigation, Data curation, Writing – original draft. Ivana Karanovic: Resources, DNA amplification, phylogenetic analysis, Writing – reviewing and editing. Paula Echeverría-Galindo: Conceptualization, Methodology, Investigation, Resources, Writing – review and editing. Peter Frenzel: Methodology, Supervision, Visualization, Writing – review and editing. Liseth Pérez: Conceptualization, Supervision, Visualization, Methodology, Writing – review and editing. Nicole Börner: Resources, Writing – review and editing. Katharina Dulias: Writing – review and editing. Junbo Wang: Supervision, Methodology, Writing – review and editing. Antje Schwalb: Conceptualization, Funding acquisition, Resources, Writing – review and editing. Author ORCIDs Mauricio Bonilla-Flores https://orcid.org/0000-0002-5593-6522 Ivana Karanovic https://orcid.org/0000-0002-9002-9952 Paula Echeverría-Galindo https://orcid.org/0000-0003-0210-4607 Peter Frenzel https://orcid.org/0000-0002-3821-4632 Liseth Pérez https://orcid.org/0000-0002-5256-3070 Nicole Börner https://orcid.org/0000-0001-8833-6665 Katharina Dulias https://orcid.org/0000-0002-3938-6663 Junbo Wang https://orcid.org/0000-0003-2335-519X Antje Schwalb https://orcid.org/0000-0002-4628-1958 Data availability All of the data that support the findings of this study are available in the main text. References Aguilar-Alberola JA, Mesquita-Joanes F (2011) Population dynamics and tolerance to desiccation in a crustacean ostracod adapted to life in small ephemeral water bodies. 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