Integrative Taxonomy of Cyclocyprididae Kaufmann, 1900 (Ostracoda: Podocopa) with Description of a New Genus and Species

© 2023 Academia Sinica, Taiwan Open Access Integrative Taxonomy of Cyclocyprididae Kaufmann, 1900 (Ostracoda: Podocopa) with Description of a New Genus and Species Maria Bisquert-Ribes1,* , Juan Rueda1, Ferran Palero1, Sukonthip Savatenalinton2, and Francesc Mesquita-Joanes1 1Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Catedràtic José Beltrán Martinez, 2, Paterna, 46980, Spain. *Correspondence: E-mail: [email protected] (Bisquert-Ribes) E-mail: [email protected] (Rueda); [email protected] (Palero); [email protected] (Mesquita-Joanes) 2Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham 44150, Thailand. E-mail: [email protected] (Savatenalinton) Received 11 September 2022 / Accepted 6 June 2023 / Published 26 July 2023 Communicated by Benny K.K. Chan The two widespread ostracod genera Cypria Zenker, 1854 and Physocypria Vávra, 1897 are traditionally distinguished based on the presence or absence of tubercles on the right valve margin. However, recent research based on soft body parts has uncovered new cryptic genera within Cypria and Physocypria. Following this line of research, a new Cyclocyprididae genus and species, Vizcainocypria viator gen. nov. sp. nov., is here described from individuals collected in rice fields and wetlands of the Iberian Peninsula. Vizcainocypria is compared with Cypria, Physocypria, Dentocypria Savatenalinton, 2017, Keysercypria Karanovic, 2011, Brasilocypria Almeida et al., 2023, and Claudecypria Almeida et al., 2023 based on morphological evidence. Besides the presence or absence of tubercles on the right valve, these genera can be distinguished according to their mandibular palp, second thoracopod, caudal ramus, and male hemipenis. Molecular analyses using mitochondrial (COX1), and nuclear (28S rDNA) genes provide further support for the differentiation of Cypria, Dentocypria, Physocypria and Vizcainocypria gen. nov. The present study highlights the importance of using an integrative taxonomy approach, combining shell and soft-body parts morphology and molecular data, to characterize the rich diversity of freshwater ostracods. Key words: DNA barcoding, Identification key, Physocypria, Vizcainocypria, Wetland Citation: Bisquert-Ribes M, Rueda J, Palero F, Savatenalinton S, Mesquita-Joanes F. 2023. Integrative taxonomy of Cyclocyprididae Kaufmann, 1900 (Ostracoda: Podocopa) with description of a new genus and species. Zool Stud 62:40. doi:10.6620/ZS.2023.62-40. BACKGROUND The most widely accepted classification of Ostracoda is probably the one proposed by Hartmann and Puri (1974). They considered that the family Candonidae Kaufmann, 1900 comprised three subfamilies, namely Candoninae Kaufmann, 1900, Cyclocypridinae Kaufmann, 1900, and Paracypridinae Sars, 1923. This classification has been followed by most ostracodologists working on living taxa (e.g., Martens 1992; Martens et al. 1998; Wouters 1999; Meisch 2000; Maddocks 2005), but paleontologists have followed alternative classifications. While Moore (1961) proposed ranking the three subfamilies as families despite lacking information on soft body parts, Liebau (2005) followed the classification proposed by Hartmann and Puri (1974) for both fossil and living taxa. Among researchers working on living ostracods in the Nearctic, one of the most influential works were those of Delorme (1970a b), which used the family rank for Candonidae and Cyclocyprididae, although he later used the subfamily rank for Cyclocypridinae, first including them in the family Cyprididae (Delorme 2001) and finally in the Candonidae (Smith and Delorme 2010). Karanovic (2011) and several other authors (Külköylüoğlu et al. 2017; Savatenalinton 2017; Külköylüoğlu 2018; Meisch Zoological Studies 62:40 (2023) doi:10.6620/ZS.2023.62-40 1 © 2023 Academia Sinica, Taiwan et al. 2019; Pieri et al. 2020; Almeida et al. 2023) followed Hartmann and Puri (1974) and considered the Cyclocypridinae as a subfamily within the Candonidae. Nevertheless, Hiruta et al. (2016) showed Candonidae to be paraphyletic, using molecular data, and proposed raising the three candonid subfamilies (i.e., Candoninae, Paracypridinae and Cyclocypridinae) to family rank within the Cypridoidea superfamily. Karanovic and Cho (2017) also provided support for this change in ostracod classification with new molecular evidence. Although the subfamily rank is still maintained for the three candonid taxa in the most recent list of freshwater ostracods of the world (Meisch et al. 2019), we will use the family rank for the Cyclocyprididae from now on in this manuscript. In a comprehensive review of the family, Karanovic (2011) noted that most authors assigned almost every Cypria-like species with tubercles on the valve margin to the genus Physocypria, and she proposed a new classification using soft parts. She recognized six genera within the Cyclocyprididae (as Cyclocypridinae in her publication): Allocypria Rome, 1962, Cyclocypris Brady and Norman, 1889, Cypria, Keysercypria, Kempfcyclocypris Karanovic, 2011 and Physocypria (Karanovic 2011 2012). Savatenalinton (2017) established a new genus, Dentocypria, to include four new species from Thailand with tubercles on the right valve margin and a tooth on the internal antero-ventral part of the left valve, which differ from Physocypria in its soft parts (Savatenalinton 2017). Later on, Külköylüoğlu (2018) erected the monotypic genus Namiotkocypria Külköylüoğlu, 2018 to include a new species from North American groundwaters (i.e., Namiotkocypria haysensis Külköylüoğlu, 2018). While Karanovic (2011) classification prioritized soft parts over valve characters, Meisch et al. (2019) supported a more traditional approximation on the basis that shell traits are observable in both living and fossil ostracods. Thus, according to Meisch et al. (2019) the family Cyclocyprididae (Cyclocypridinae in their list) comprised eight genera (Allocypria, Cyclocypris, Cypria, Dentocypria, Kempfcyclocypris, Mecynocypria Rome, 1962, Namiotkocypria and Physocypria), splitting Keysercypria species among Physocypria and Cypria, and rejecting the synonymy of Mecynocypria and Physocypria established by Karanovic (2011). Recently, two new genera, Brasilocypria and Claudecypria, have been raised for species with tubercles on the right valve margin (belonging to Physocypria s.l.) collected from Brazilian floodplains (Almeida et al. 2023). Furthermore, Almeida et al. (2023) reestablished the validity of Keysercypria, although in a more restricted form. Considering all these new contributions, the family Cyclocyprididae is now composed of eleven genera. Recent surveys from rice fields and wetlands in the southern and eastern Iberian Peninsula revealed the presence of a new Cyclocyprididae species differing from all previously known taxa, and for which the genus Vizcainocypria gen. nov. is here erected. The classification of controversial genera (e.g., Cypria, Dentocypria, Keysercypria, and Physocypria) is discussed and clarified with new diagnoses, and a genus-level identification key is provided, based both on the morphology of valves and soft parts, and validating the use of hemipenis morphology for genuslevel classification within the Cyclocyprididae. In addition, a new diagnosis for the Cyclocyprididae and Mecynocypria, and brief comments on Allocypria, Brasilocypria and Claudecypria are provided. Preliminary phylogenetic analyses were also carried out based on mitochondrial (COX1) and nuclear (28S) sequences to check for support (or its lack thereof) of morphological classifications. Finally, taxonomic characters used for distinguishing between the close genera Brasilocypria, Claudecypria, Cypria, Dentocypria, Keysercypria, Physocypria, and Vizcainocypria gen. nov. are discussed. MATERIALS AND METHODS Specimen collection, dissection, and scientific drawings Sampling was mostly carried out as part of multiple surveys in the Albufera Natural Park (eastern Iberian Peninsula) between 2013 and 2021. Ostracods were collected with a 250-µm hand net and immediately preserved in 96% ethanol. In the laboratory, individual specimens were isolated using Pasteur pipettes under a Leica M205 C stereoscope. Ostracod dissections followed Namiotko et al. (2011). Soft parts were placed in a glass slide with HydroMatrix® for permanent preparations, and valves were stored dry in micropaleontological slides. Digital drawings were completed using a graphic tablet and Adobe Illustrator 2020 (https://www.adobe.com/products/illustrator. html), in combination with photographs taken with a Nikon Eclipse E-800 microscope. Scanning electron microscopy (SEM) images were obtained with a Hitachi S-4800 microscope at the Central Service for Experimental Research (SCSIE) of the University of Valencia. Images of whole individuals were obtained with a Nikon D3400 attached to a Leica M205 C stereoscope. Limb chaetotaxy descriptions follow Broodbakker and Danielopol (1982) as revised by Martens (1987) and Meisch (2000). page 2 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan The following abbreviations are used in the text and figure captions: A1, antennula; A2, antenna; AT, Afrotropical; AU, Australasian; CpD, carapace dorsal view; CpF, carapace frontal view; CpL, carapace lateral view; CR, caudal ramus; EA, East Asia; H, height; IP, Iberian Peninsula; L, length; LV, left valve; LVi, left valve interior view; Md, mandible; Mxl, maxillula; NA, Nearctic; NT, Neotropical; OL, Oriental; PA, Palearctic; RV, right valve; RVi, right valve interior view; SEA, South East Asia; T1, T2, T3, first, second and third thoracopods; US, United States; W, width. DNA analyses Ethanol-fixed ostracods were individually transferred to 1.5 mL eppendorfs using a thin brush. Single specimens were digested at 55°C overnight using 180 µL T1 Buffer and 20 µL proteinase K, and DNA was extracted with the Nucleospin DNA extraction kit (Macherey-Nagel™) following the manufacturer’s instructions. The cytochrome oxidase subunit I mitochondrial gene (COX1) was amplified using ArF1: 5'-GCNCCWGAYATRGCNTTYCCNCG-3' (Gibson et al. 2014) and Fol-degen-rev: 5'-TANACYTCNGG RTGNCCRAARAAYCA-3' (Yu et al. 2012) primers, already tested with success on decapod crustaceans (Genis-Armero et al. 2022). The large ribosomal subunit (28S) nuclear gene region was amplified using a newly designed pair of primers: 5'-CCCGTCTTGAAACACG GACCAAGGAG-3' and 5'-GTTCGATTAGTCTTTCG CCCCTATAC-3'. Amplifications were carried out using ~10 ng of genomic DNA in a reaction containing 1 U of Taq polymerase (Amersham), 1x buffer (Amersham), 0.2 mM of each primer and 0.12 mM dNTPs. The polymerase chain reaction (PCR) thermal profile included an initial denaturation step at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 30 s and a final extension at 72°C for 20 min. Sequences were obtained using the Big-Dye ReadyReaction kit ver. 3.1 (Applied Biosystems) on an ABI Prism 3770 automated sequencer at the MACROGEN sequencing facilities. Chromatograms for each DNA sequence were checked with BioEdit v7.2.5 (Hall 1999) and sequence alignments were conducted with Muscle v3.6 (Edgar 2004). Model selection was carried out for each sequence alignment using the Bayesian Information Criterion (BIC) as implemented in ModelTest-NG v0.1.7 (Darriba et al. 2020). Maximum likelihood phylogenetic reconstruction was then completed with the corresponding DNA substitution model for each gene with ultrafast bootstrap (1000 replicates) as implemented in IQ-TREE v2.0 (Minh et al. 2020). Finally, to allow for comparison with previous COX1 genetic distance estimates obtained within and between ostracod species and genera by Nigro et al. (2016), Kimura 2-Parameter (K2P) genetic distances and their standard errors were estimated from our COX1 dataset using MEGA X (Kumar et al. 2018). RESULTS SYSTEMATICS Class Ostracoda Latreille, 1802 Subclass Podocopa Sars, 1866 Order Podocopida Sars, 1866 Suborder Cypridocopina Baird, 1845 Superfamily Cypridoidea Baird, 1845 Family Cyclocyprididae Kaufmann, 1900 Diagnosis (after Cyclocypridinae sensu Meisch (2000) and Karanovic (2012)): Carapace short, less than 1 mm in length, relatively stout in lateral view (except Allocypria, with elongated valves), moderately compressed to ovate in dorsal view. Eyes fused with a single eye cup. A1 usually 7-segmented and Rome organ present. Male sexual bristles on A2 (transformed t2 and t3 setae) present or absent, swimming-setae usually well-developed, sometimes reduced and rarely absent. Endopod of female T1 developed, transformed into 2-segmented prehensile palps in males. T3 (cleaning leg) always 4-segmented, last segment bearing two short (h1 and h2) and one long (h3) setae. Zenker organ with seven spine whorls (five along the central tube, and one at each end of the organ) and proximal part (entrance) spherically enlarged (not funnel shaped). CR not reduced and sp-seta insertion around medial part of CR. Differential diagnosis: The family Cyclocyprididae can be distinguished from other Cypridoidea families through its Zenker organ (not funnel-shaped proximally and with seven spine whorls) and other characters of the soft parts and valves. In the case of the Zenker organ of Cyprididae Baird, 1845, it has at least 8 spine whorls and their T3 has a pincer organ in most species (absent in Cyclocyprididae). The Zenker organ of Ilyocyprididae Kaufmann, 1900 has 15–20 spine whorls, and members of this family have a subrectangular carapace (ovate or subovate in Cyclocyprididae). Members of the Notodromadidae Kaufmann, 1900 present a unique Zenker organ, funnel-shaped in both extremes and with spines not arranged in separate whorls. Moreover, members of this family also present a divided eye (not divided in Cyclocyprididae) and external eye tubercles (not present in Cyclocyprididae). Candonidae s.str. (previously subfamily Candoninae) lack the Rome organ of A1 and the swimming-setae of A2, which are page 3 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan usually present and well-developed in Cyclocyprididae. Moreover, these two families can be differentiated by the prehensile palps of T1 in males; 2-segmented in Cyclocyprididae and 1-segmented in Candonidae. Paracyprididae differs from Cyclocyprididae by the presence of d1 and d2 setae on T2 (d2, and sometimes also d1, absent in Cyclocyprididae) and the marine ecological affinities of most of its representatives (freshwater in the Cyclocyprididae). However, more studies on Paracyprididae are needed to be able to give a more reliable differential diagnosis at the family level. Cypria Zenker, 1854 Cypris (Cypria) Zenker, 1854: 77. Cypria s.l. Brady and Norman, 1889: 68. Cypria s.str. Vávra, 1891: 62. Candocypria Furtos, 1933: 458, Pl. 8, Figs. 13–17; Pl. 14, Figs. 22–23. Bentocypria Kovalenko, 1987: 99. Keysercypria (partim) Karanovic, 2011: 23, Fig. 17. Type species: Cypria exsculpta (Fischer, 1855) Brady and Norman, 1889. Diagnosis: Carapace in lateral view short, ovate or subovate; in dorsal view, laterally compressed. RV margin without tubercles, LV overlapping RV, sometimes with an internal antero-ventral tooth. A1 7-segmented, natatory setae of A2 well-developed. Penultimate segment of male A2 divided, t2 and t3 seta transformed into sexual bristles. Terminal segment of Md palp three or four times longer than wide. Terminal segment of Mxl palp squared. Third endite of the Mxl with bristles. Prehensile palps asymmetrical. Basal d1seta of T2 absent. Terminal segment of T3 subquadrate, g-seta very short. CR well-developed, with a short spseta (no longer than half the length of Gp), claws welldeveloped. Hemipenis with two lobes, a-lobe (outer) usually longer than b-lobe (inner), Zenker organ with seven spine whorls. Other species: The list of Cypria species and their synonyms can be found in Meisch et al. (2019). Distribution: PA, NA, NT, AT, AU and OL. Remarks: Karanovic (2011) provided the last diagnosis for Cypria. She opted to prioritize the soft part morphology over the valve characters to differentiate between the two classical genera, Cypria and Physocypria. She allocated some Physocypria species with tubercles on the RV margin to the genus Cypria. Meisch et al. (2019) followed a more conservative approach and returned to the classical differentiation between Physocypria (with tubercles) and Cypria (without tubercles). In the present work, Cypria and Physocypria are rearranged using both soft parts morphology and valve information (see below for details). Karanovic (2011) placed C. pellucida and C. obtusa in Keysercypria based on the hemipenis morphology and the chaetotaxy of T2 and T3. However, none of these two species have tubercles on the RV, while all other species in Keysercypria do (see below), for that reason we have maintained these two species in Cypria. Savatenalinton (2017) already noted that some species of Cypria s.l. (i.e., C. bicolor and C. javana) could belong to a different genus due to their variability in some appendages (e.g., long sp-seta on CR) and the morphology of the male copulatory organ. However, many Cypria species descriptions are incomplete, so we decided to maintain these dubious species in Cypria and strongly recommend a revision of the genus. In fact, the hemipenis morphology is very variable within Cypria, probably due to the number of species accumulated over the years. Surprisingly, the hemipenis of the type species, Cypria exsculpta, is very different to its congeners. While the hemipenis lobes of C. exsculpta are both broad, with an a-lobe longer, with pointed tip, and a b-lobe with rounded tip, most Cypria species have elongated lobes with both tips either pointed or rounded. The morphology of the hemipenis of Cypria exsculpta is reminiscent of the hemipenis of Cyclocypris, the type genus of the family, which may indicate that C. exsculpta hemipenis represents the ancestral state within the genus. Dentocypria Savatenalinton, 2017 Type species: Dentocypria mesquitai Savatenalinton, 2017. Diagnosis: As in Savatenalinton (2017). Other species: Species described by Savatenalinton (2017), D. crenulata (Sars, 1903) comb. nov., D. dumonti Martens, 1982 comb. nov. Distribution: AT, NT, PA and OL. Remarks: New combinations are established here for D. crenulata comb. nov. and D. dumonti comb. nov., reallocating these species from Physocypria to Dentocypria, because both taxa have tubercles on the RV margin, a very elongated terminal segment of the Md palp, absence of d1-seta on T2 basal segment, and a long sp-seta on the CR. All those traits do not fit with the description of Physocypria bullata Vávra, 1897 (see Karanovic 2011), the type species of the genus. In addition, D. dumonti comb. nov. has a subtriangular protrusion on the right prehensile palp of the male, a character unique to Dentocypria. Moreover, the morphology of the hemipenis lobes is closer to Dentocypria than to Physocyria (see below). The transfer of these two species from Physocypria page 4 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan s.l. to the genus Dentocypria has zoogeographical implications. The genus Dentocypria was considered endemic to Thailand (Savatenalinton 2017), but with the new combinations this genus is now present in other parts of Asia and in Africa; Dentocypria crenulata was originally described from Sumatra (Indonesia), and D. dumonti was described from Somalia (Africa). Other species assigned to Physocypria may also belong to Dentocypria, but descriptions are often incomplete and generic position cannot be ascertained. The length of the Md palp terminal segment is unknown in P. furfuracea (Brady, 1886), P. larensis Hartmann, 1964 and P. minuta Victor and Michael, 1975, but they do not have d1-seta on T2 (a distinguishing character for Physocypria), and the sp-seta of the CR is long. Their distribution (OL and PA) is also congruent with Dentocypria distribution; however, more information is needed to confirm their generic position. Museum material of these species should be checked paying special attention to hemipenis and prehensile palp morphology. Keysercypria Karanovic, 2011 Physocypria (partim) Meisch et al., 2019: 87. Type species: Keysercypria affinis (Klie, 1933) Karanovic 2011. Diagnosis (modified after Karanovic (2011)): Carapace in lateral view usually ovate. RV margin tuberculated anteriorly and posteriorly, LV overlapping RV anteriorly. Surface of carapace smooth, sometimes with long-setae. A1 7-segmented, five long natatory setae of A2 reaching beyond tip of terminal claw, outermost seta completely reduced. Penultimate segment of male A2 divided, t2 and t3 setae transformed into sexual bristles. Terminal segment of Md palp extremely elongated (> 5x longer than wide). Terminal segment of Mxl palp squared. Third endite of the Mxl with bristles. Male T1 with asymmetrical prehensile palps, first segment of right prehensile palp usually with a robust finger-like protrusion. Basal d1-seta of T2 absent. Terminal segment of T3 subquadrate, g-seta very short, d2-seta on T3 absent. CR well-developed, with long sp-seta (longer than half of Gp length). Hemipenis with b-lobe short, pointed distally and shorter than a-lobe; a-lobe elongated, finger-like, thin in its middle length, with a rounded distal end usually wider than the middle part. Zenker organ with seven spine whorls. Other species: K. deformis (Klie, 1940), K. longiseta (Klie, 1930), K. schubarti (Farkas, 1958), K. xanabanica (Furtos, 1936). Distribution: NT. Remarks: Karanovic (2011) included in Keysercypria nine species previously assigned to Physocypria and Cypria. Meisch et al. (2019) returned these species to their original genera, but Almeida et al. (2023) reestablished the genus with a more restricted diagnosis based mainly on the chaetotaxy of T2 and T3 (see discussion). They kept in the genus Keysercypria some species of the Physocypria s.l. group: the type species K. affinis, plus K. deformis and K. schubarti. However, we consider that also two more Physocypria species assigned by Karanovic (2011) to Keysercypria do merit this genus-level distinction (K. longiseta and K. xanabanica), mostly because of their hemipenis morphology. Three former Physocypria species (P. circinata Würdig and Pinto, 1993, P. sanctaeannae Margalef, 1961 and P. ivanae (Díaz and Lopretto, 2011)) assigned to Keysercypria by Karanovic (2011) or by Díaz and Lopretto (2011) are excluded because their hemipenis morphology is not congruent with the others or cannot be confirmed due to lack of males. We decide to maintain C. obtusa and C. pellucida in the genus Cypria because their hemipenes are clearly distinct from Keysercypria affinis (type species) and because of the absence of RV marginal tubercles, as pointed out by Almeida et al. (2023). Mecynocypria Rome, 1962 Physocypria (partim) Karanovic, 2011: 42. Type species: Mecynocypria obtusa (Sars, 1910); Rome 1962. Diagnosis (after Rome (1962)): Elongated carapace, height equal or less than half the length. RV margin without tubercles, LV overlap RV. A1 7-segmented, A2 with long natatory setae reaching beyond tip of terminal claws. Penultimate segment of male A2 divided, t2 and t3 setae transformed into sexual bristles. Terminal segment of Md palp elongated (> 2x longer than wide). Terminal segment of Mxl palp squared. Third endite of the Mxl with bristles. Male T1 with asymmetrical prehensile palps. T2 with basal d1-seta. Terminal segment of T3 subquadrate, g-seta short or absent. CR well-developed, with sp-seta long or short, claws well-developed. Hemipenis with lobes a and b well-developed; a-lobe elongated, usually longer than b-lobe, and bent towards b-lobe. Zenker organ with seven spine whorls. Other species: The list of Mecynocypria species and their synonyms can be found in Meisch et al. (2019). Distribution: AT. Remarks: Karanovic (2011) synonymized the genus Mecynocypria with Physocypria because of the position of the ovaries and the presence of the d1seta on T2. However, following Meisch et al. (2019), Mecynocypria differs from Physocypria because of the absence of marginal tubercles on the valves. We have page 5 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan provided here a new diagnosis for the genus based on Rome’s first description of the genus Mecynocypria but updating appendage nomenclature. Physocypria Vávra, 1897 Physocypria Vávra, 1897: 7. Keysercypria (partim) Karanovic, 2011: 23, fig. 11–16. Type species: Physocypria bullata (Vávra, 1897) G. W. Müller, 1912. Diagnosis: Carapace ovoid in lateral view. RV margin tuberculated anteriorly and posteriorly, sometimes LV tuberculated. LV overlaps RV ventrally, anteriorly, and posteriorly, sometimes RV overlapping LV. Surface of carapace smooth, sometimes with some setae. A1 7-segmented, long natatory setae of A2 reaching beyond tip of terminal claws. Penultimate segment of male A2 divided, t2 and t3 setae transformed into sexual bristles. Terminal segment of Md palp elongated (not more than two times longer than wide). Terminal segment of Mxl palp squared. Third endite of the Mxl with bristles. Male T1 with asymmetrical prehensile palps. T2 with basal d1-seta. Terminal segment of T3 subquadrate, g-seta very short. CR well-developed, with sp-seta usually long, claws welldeveloped. Hemipenis lobes well-developed and long, a-lobe usually longer and curved towards b-lobe. The latter usually curved at the base towards a-lobe, but distally pointed and curved towards the opposite direction (i.e., sinuous shape). Zenker organ with seven spine whorls. Other species: The list of Physocypria species and their synonyms can be found in Meisch et al. (2019), with some exceptions: P. affinis, P. deformis, P. longiseta, P. schubarti and P. xanabanica have been transferred to Keysercypria either by Almeida et al. (2023) or by this work (see above); P. crenulata and P. dumonti have been transferred by us to Dentocypria (see above); and P. granadae has been reallocated to the new genus Vizcainocypria gen. nov. (see below). Distribution: AT, PA, NA, NT and OL. Remarks: Physocypria nipponica and P. biwaensis are closely related taxa (Smith and Janz 2008; Karanovic 2015). The former lacks d1-seta on T2, so it should not be considered a Physocypria species. Nevertheless, we decided to keep P. biwaensis and P. nipponica in Physocypria since their hemipenis morphology is very similar to other Physocypria species (see below). Physocypria pustulosa, P. inflata and P. kerkyrensis present RV marginal tubercles, but lack d1seta on T2 and have short sp-seta (d1-seta present and sp-seta long in Physocypria). However, incomplete original descriptions or hemipenis morphology (see below) suggests these species to be kept in Physocypria. As indicated in the remarks section for other genera, some species might be allocated to other genera rather than Physocypria, but the information available in the bibliography is often incomplete. We strongly recommend a careful revision of the genus. Other genera The genera Allocypria, Brasilocypria, Claudecypria, Cyclocypris, Kempfcyclocypris and Namiotkocypria are not presented here with full diagnoses (also for Dentocypria, see above) because we consider that the original descriptions or their redescription by Karanovic (2011) are sufficient to verify their taxonomic affinities. Nevertheless, we present here a short differential diagnosis for these genera. Allocypria was described by Rome (1962) from Lake Tanganyika. Karanovic (2011) provided an updated diagnosis, and the list of species and their synonyms can be found in Meisch et al. (2019). This genus differs from Dentocypria, Keysercypria, Brasilocypria, Claudecypria and Vizcainocypria gen. nov. by the absence of RV marginal tubercles; from Cypria by the reniform carapace shape and the presence of d1-seta on T2 (absent in Cypria but present in Physocypria too). Allocypria can be differentiated from Mecynocypria by the usually squared terminal segment of the Md palp (elongated, 2x times longer than wide, in Mecynocypria); by bearing bristles on the second and third endites of the Mxl (only in the third endite in Mecynocypria), and by presenting ovaries curved upwards (curved downwards in Mecynocypria). However, the last two traits (Mxl bristles and ovaries curvature) must be taken with caution (see discussion). The two recently raised genera from Brazilian floodplains (Almeida et al. 2023), Brasilocypria and Claudecypria, share with Keysercypria (another Neotropical genus) the absence of the short seta accompanying the five natatory setae on A2, and the absence of the d2-seta on T3; setae which are present in Cypria, Dentocypria, Physocypria, and Vizcainocypria gen. nov. Furthermore, Cypria lacks RV marginal tubercles (present in Brasilocypria and Claudecypria); Physocypria presents d1-seta on T2 (absent in both new Neotropical genera); and Vizcainocypria gen. nov. presents a short sp-seta on CR, which is long in Brasilocypria and Claudecypria. The Md palp terminal segment is very elongated (3x longer than wide) in Brasilocypria, and elongated (2x longer than wide) in Claudecypria; while it is extremely elongated in Keysercypria (5x longer than wide). Brasilocypria can also be differentiated from Claudecypria by page 6 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan the presence of dp-seta on T3 in males, which is absent in Claudecypria. The hemipenis can be used for differential diagnoses of these genera too (see discussion). Allocypria and Mecynocypria differ from the recently raised Neotropical genera by their reniform carapace shape and by the presence of d1-seta on T2. Cyclocypris, the type genus of family Cyclocyprididae, and Kempfcyclocypris, described from Australian groundwaters, are two related genera that share the absence of sexual bristles on male A2 (t2 and t3 not transformed), what makes them different from other Cyclocyprididae genera. Moreover, Cyclocypris has a very elongated T3 terminal segment, and Kempfcyclocypris has a 6-segmentend A1 (7-segmented for other genera) and it lacks natatory setae on A2. Both genera also lack RV marginal tubercles, present a wider carapace in dorsal view, have an elongated (2x longer than wide) terminal segment of the Md palp, and bear d1-seta on T2. Namiotkocypria is a North American groundwater genera that lacks the d1-seta on T2 and RV marginal tubercles. The natatory setae on A2 are reduced and the Md palp terminal segment is elongated (2x longer than wide), which makes this genus different from the Cyclocyprididae genera mentioned above. Vizcainocypria gen. nov. Bisquert-Ribes et al. urn:lsid:zoobank.org:act:B9CBEF1C-0880-4C29-822ABA20245FE678 Type species: Vizcainocypria viator gen. nov. sp. nov. Etymology: Named after Mr. Antonio Vizcaino in recognition of his significant contributions to the management and protection of the Albufera Natural Park (Valencia, Spain), type locality of this new genus. Diagnosis: Carapace ovate or subovate in lateral view; elliptical, subelliptical or subovate and laterally compressed in dorsal view. RV margin tuberculated anteriorly and posteriorly, LV overlapping RV anteriorly, ventrally, and posteriorly, usually with an internal tooth on antero-ventral part. A1 7-segmented, long natatory setae of A2 reaching beyond tip of terminal claws. Penultimate segment of male A2 divided, t2 and t3 setae transformed into sexual bristles. Terminal segment of Md palp very elongated (> 2x longer than wide). Terminal segment of Mxl palp quadrate. Third endite of Mxl with bristles. Male T1 with asymmetrical prehensile palps, first segment of right prehensile palp usually with a spine-like or finger-like protrusion. Basal d1-seta of T2 absent. Terminal segment of T3 subquadrate, g-seta short. CR well-developed, with short sp-seta. Hemipenis with well-developed lobes, subequal or b-lobe slightly longer; a-lobe subtriangular, b-lobe distally thin, elongated and with a rounded tip. Zenker organ with seven spine whorls. Differential diagnosis: Vizcainocypria gen. nov. can be clearly distinguished from other Cyclocyprididae genera. The hemipenis presents a subtriangular a-lobe, shorter than b-lobe, whereas Allocypria and Mecynocypria have elongated, digitiform a-lobes, usually longer than b-lobe. Vizcainocypria gen. nov. also lacks the elongate carapace shape of both genera, d1-seta on T2 and elongated (twice longer than wide) terminal segment of Md palp of Mecycnocypria, and bristles in the second Mxl endite present in Allocypria. Cyclocypris differs from Vizcainocypria gen. nov. by its more globose carapace in dorsal view and by holding not transformed sexual bristles of t2 and t3 on male A2, while Kempfcyclocypris is unique by its 6-segmented A1. The smooth RV margin and reduced A2 natatory setae of Namiotkocypria are not observed in the new genus. Vizcainocypria gen. nov. is closest to Cypria, Dentocypria, Physocypria, Keysercypria, Brasilocypria and Claudecypria. It differs from Cypria by the RV margin (smooth in Cypria and tuberculated in Vizcainocypria gen. nov.), from Physocypria by the d1-seta on T2 (absent in Vizcainocypria gen. nov. and present in Physocypria), from Dentocypria by the short sp-seta on CR (long in Dentocypria), and from Keysercypria, Brasilocypria and Claudecypria by the presence of a short accompanying natatory seta on A2 (absent in the three Neotropical genera). Moreover, the length of the terminal segment of the Md palp is very elongated in Vizcainocypria gen. nov. (and Brasilocypria), extremely elongated in Keysercypria and elongated in Claudecypria. The new genus can be differentiated from Brasilocypria by the sp-seta on CR (very long in Brasilocypria and short in Vizcainocypria gen. nov.). Furthermore, in most species of these genera (including their type species), the a-lobe of the hemipenis is elongated and longer than b-lobe, while it is the opposite in the new genus (b-lobe elongated and longer than a-lobe, which is subtriangular). Other species: V. granadae (Hartmann, 1959) comb. nov. Distribution: PA, NA and NT. Remarks: We have allocated V. granadae comb. nov. to this new genus because of its sexual and nonsexual characters. This species presents marginal tubercles on the RV, does not have d1-seta on T2, the terminal segment of the Md palp is very elongated and the sp-seta on the CR is short. These are the nonsexual characters that differentiate Vizcainocypria gen. nov. from other genera of the family. Moreover, the hemipenis morphology of V. granadae comb. nov. is very similar to the type species of the genus. The a-lobe is subtriangular and slightly shorter than the page 7 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan b-lobe, which is elongated and thin, and with a rounded tip. Finally, the right prehensile palp of the male has a spine-like protrusion, similar to the type species. Nevertheless, new specimens of this species need to be collected and studied to corroborate its position in the new genus due to the lack of information of many traits in its first description by Hartmann (1959). Physocypria inflata and P. pustulosa are two species that could belong to Vizcainocypria gen. nov. because some of the non-sexual and sexual characters are similar to those of the genus. Both species lack the d1-seta on the basal segment of T2, and the spseta of the CR is short. The right prehensile palp of the two species seems to present a spine-like protrusion; however, it looks bigger than in the other Vizcainocypria gen. nov. species. We take in these cases a conservative approach not allocating these two species to Vizcainocypria gen. nov. because the morphology of the hemipenis is not as similar as that of V. granadae comb. nov., and some non-sexual characters cannot be confirmed with the original descriptions, such as the length of the Md palp. Other species also present a spine-like protrusion on the right prehensile palp, including C. lacrima, C. subsalsa or P. gibbera, among others. In some of these cases, the non-sexual characters are not congruent with the genus Vizcainocypria gen. nov., like the absence of the tubercles on the RV margin. However, especially relevant is the hemipenis morphology for determining the genera position of the species. For example, C. lacrima has an a-lobe shorter than b-lobe, as in Vizcainocypria gen. nov., however, the general morphology of the hemipenis is different from Vizcainocypria gen. nov. (see below). Vizcainocypria viator gen. nov. sp. nov. Bisquert-Ribes et al. (Figs. 1–4) urn:lsid:zoobank.org:act:40734E2C-60B9-40B8-9FC3C88E7D13F341 Physocypria sp.: Bou et al., 2019: 41. Dentocypria sp.: Giménez et al., 2020: Fig. 29. Type materials: Holotype: Soft parts of a dissected ♂ preserved in HydroMatrix® on a slide, and valves stored dry in a micropalaeontological slide (MUVHNZY0011). Allotype. ♀ stored like the holotype (MUVHNZY0012). Paratypes. Two dissected ♂ (MUVHNZY0013 and MUVHNZY0014) and three dissected ♀ (MUVHNZY0015, MUVHNZY0016 and MUVHNZY0017) stored like the holotype. Repository: The holotype, allotype and paratypes are deposited in the Museum of Natural History of the University of Valencia (MUVHN), Burjassot, Spain. Type locality: Albufera lake, Albufera Natural Park, Valencia, Comunitat Valenciana, Spain. Material collected in 2014, coordinates: 39°19'10"N, 0°19'27"W. Accompanying ostracod fauna: Cypridopsis vidua (Müller, 1776). Other localities: 1) Rice field, Albufera Natural Park, Valencia, Comunitat Valenciana, Spain. Material collected on 14 December 2020, coordinates 39°16'32"N, 0°18'54"W. 2) Rice field, Marjal PegoOliva Natural Park, Pego, Comunitat Valenciana, Spain. Material collected on 30 May 2018, coordinates 38°53'8"N, 0°3'59"W. 3) Rice field, Girona, Catalunya, Spain. Material collected on 17 July 2018, coordinates 41°58'14"N, 3°8'48"E., Leg: J. Sala. Etymology: The new species is named in relation to the putative geographical dispersal of the species to large distances (see discussion below). The specific epithet means “traveler” in Latin. Diagnosis: Carapace in lateral view ovate, valve surface smooth with some setae, RV anterodorsal margin with a slight depression. RV margin tuberculated anteriorly and posteriorly. LV with an internal tooth on antero-ventral part. Rome organ on A1 large and mushroom-like, penultimate segment of A1 with a dorso-apical claw, terminal segment of Md palp very elongated (c.3 times longer than wide), T2 with g-seta and accompanying-seta subequal, and long e-seta. CR with short sp-seta. Right prehensile palp of male T1 with a spine-like protrusion, left prehensile palp with two subapical spines, one long and one short. Hemipenis with b-lobe slightly longer than a-lobe and a-lobe subtriangular (shark fin shaped), distinctly wider than b-lobe. Differential diagnosis: Vizcainocypria viator gen. nov. sp. nov. is characterized by a spine-like protrusion in the right prehensile palp, subtriangular a-lobe of hemipenis and dorso-apical claw in the penultimate segment of A1. In addition, the length of the e-seta on T2 is different from other species. V. granadae comb. nov. can be distinguished by its more subrectangular carapace (more ovate in V. viator gen. nov. sp. nov.), more elongated male clasping organs and a narrower a-lobe of the hemipenis. The a-lobe of V. granadae comb. nov. also has more parallel margins (wider and more divergent in V. viator gen. nov. sp. nov.), and the margin of a-lobe closer to b-lobe bearing a bump (straight in V. viator gen. nov. sp. nov.). However, a revision of the type material of V. granadae comb. nov. and of new specimens of this species are needed to update the original description of Hartmann (1959). Description of female (Fig. 1): Measurements (mean, in μm): LV (n = 6), L = 578.7, H = 374.4; RV (n = 6), L = 541.9, H = 354.8; Carapace (n = 6), L = page 8 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan Fig. 1. Vizcainocypria viator gen. nov. sp. nov., female (A, C–G, I), male (B, H). A: Mature female specimen. B: Mature male specimen. C: CpL from right side (MUVHNZY0020). D: CpF (MUVHNZY0019). E: CpD (MUVHNZY0018). F: LVi (MUVHNZY0016). G: Detail of the internal tooth (MUVHNZY0016). H: RVi (MUVHNZY0011). I: Detail of the tubercles on RV margin (MUVHNZY0017). Scale bars: A–F, H = 200 µm; G = 10 µm; I = 5 µm. I A B CDE F G H page 9 of 24 Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan (Fig. 5B; Table 1). Vizcainocypria viator gen. nov. sp. nov. clustered significantly (97% bootstrap) with four putative “Candonidae” sequences from the BOLD database (which seem to be in fact Cyclocyprididae specimens collected in US and Canada, see Discussion). Our D. smithi sequence clustered with Physocypria sequences from Eurasia. It should be pointed out that GenBank sequences assigned to Cypria formed two clusters (Fig. 5B). COX1 genetic distances between genera are shown in table 1, and between species in table S1. K2P distances between the four main clusters of Cyclocyprididae were very similar, and mean distance between genera was 0.240 ± 0.025. The lowest K2P distances were observed among Cyclocyprididae gen. and Dentocypria (0.188 ± 0.025), between Physocypria and Dentocypria (0.202 ± 0.024) and then between Physocypria and Vizcainocypria (0.216 ± 0.022). Distances between the Cypria 2 clade and Vizcainocypria (0.235 ± 0.023) were lower than distances between Cypria 2 and Physocypria (0.241 ± 0.022) or between Cypria 2 and Dentocypria (0.266 ± 0.029). Finally, K2P distances between sequences assigned to Vizcainocypria are similar or even lower than distances between Physocypria species (Table S1). DISCUSSION The historical accumulation of species within two Cyclocyprididae genera (Cypria and Physocypria) has resulted in a wastebasket of taxa with highly diverse soft parts, including sexual and non-sexual characters. Nevertheless, the recent allocation of some Physocypria species into new genera (Karanovic 2011) and the description of new genera (Savatenalinton 2017; Külköylüoğlu 2018; Almeida et al. 2023) have improved our understanding of this family. In the present study, a new genus and species (Vizcainocypria viator gen. nov. sp. nov.) is erected to host specimens showing affinities with the most controversial group of cyclocypridid genera (Brasilocypria, Claudecypria, Cypria, Dentocypria, Keysercypria and Physocypria). Sexual and non-sexual characters are proposed to characterize them, describing their hemipenis morphology and comparing them with the type genus of the family, Cyclocypris. Our taxonomical results are expected to be particularly useful for discriminating cyclocypridids in future studies. Furthermore, we provide new molecular information, contributing to a preliminary phylogeny for the most controversial Cyclocyprididae genera, which unfortunately still remains to be fully resolved. After the establishment of the new genus, the family Cyclocyprididae is composed of the following 12 recent genera: Allocypria, Brasilocypria, Claudecypria, Cyclocypris, Cypria, Dentocypria, Kempfcyclocypris, Keysercypria, Mecynocypria, Namiotkocypria, Physocypria, and Vizcainocypria gen. nov. The redescription of the type species of Physocypria by Karanovic (2011) allowed us to confirm the taxonomic position of some species, but the genus assignment of many others could not be verified due to incomplete descriptions. For those genera, a careful revision of museum material or new specimens from type localities is needed to strengthen our current understanding of the group. On the validity of Keysercypria Karanovic (2011) erected Keysercypria by uniting species of Cypria and Physocypria from the Neotropical region, prioritizing soft parts over valve characters (RV marginal tubercles). However, Meisch et al. (2019) argued that shell traits can be easily checked in both living and fossil specimens and defended a more conservative approach, reassigning Keysercypria species to Cypria or Physocypria. Recently, Almeida et al. (2023) re-established Keysercypria with a more restricted diagnosis than Karanovic (2011). The authors agreed with Karanovic (2011) that three Neotropical Physocypria s.l. species that she had classified as Keysercypria were different from Physocypria s.str. However, Almeida et al. (2023) based their diagnoses of the genus mainly on the chaetotaxy of T2 (male), A2 and T3. We agree with Karanovic (2011) and Almeida et al. (2023) on the validity of Keysercypria. However, we included two more species (see above) of Physocypria s.l. originally allocated by Karanovic (2011) to the genus Keysercypria, but which Almeida et al. (2023) did not include in their revision. The main criterion we followed for including the species in Keysercypria was their hemipenis morphology (see below), which is very similar in all the species that we assigned to Keysercypria, and distinct enough from other cyclocypridids to support the monophyly of Keysercypria. New specimens of Keysercypria species, where valves and soft parts can be accurately described, should be collected before a final consensus is achieved. Non-sexual characters Some non-sexual characters, such as the length of the Md palp terminal segment or the length of the sp-seta on CR, can be very useful for disentangling cryptic genera. Here, we discuss the validity of these morphological characters and their diagnostic utility to differentiate between the problematic genera page 16 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan (Brasilocypria, Claudecypria, Cypria, Dentocypria, Keysercypria, Physocypria and Vizcainocypria gen. nov.). RV marginal tubercles Savatenalinton (2017) already noted the utility of RV marginal tubercles as a diagnostic character. In fact, marginal tubercles are also used to discriminate among Cyprinotinae Bronstein, 1947 genera: while Hemicypris Sars, 1903 has marginal tubercles on LV, Heterocypris Claus, 1892 and Cyprinotus Brady, 1886 present tubercles on the RV instead. This trait seems to participate in the enclosure of the valves, and it is related to the valve overlap because tubercles usually appear on the smallest valve. In the Cyclocyprididae, marginal tubercles are present in members of some genera (Dentocypria, Keysercypria, Physocypria and Vizcainocypria gen. nov.), but not others (Allocypria, Cypria, Cyclocypris, Kempfcyclocypris, Mecynocypria and Namiotkocypria). For those genera with tubercles, the tubercles always appear on the RV margin, except in P. bullata (the type species of the genus) and P. ivanae, both of which have tubercles on the two valves, and P. dentifera, which has tubercles on the LV. Following Meisch et al. (2019), we recommend using the presence/absence of RV marginal tubercles as one of the key characters. Our preliminary molecular results do not allow us to conclude that this character defines a single monophyletic clade. The largely unresolved 28S tree suggests a basal ancestor with tubercles and their disappearance in the Cypria clade, but the COX1 tree rather suggests that tubercles may have appeared repeatedly in different clades (assuming an ancestor with no tubercles) or their independent disappearance in different lineages. Karanovic (2011) and Meisch et al. (2019) agree on the utility of another carapace trait, the curvature of the ovaries. However, this trait cannot be observed in fossil specimens, and it can be difficult to see in living animals, so we recommend only using the curvature of the ovaries as a key character in the diagnoses of the genera if it is absolutely necessary. Morphology of T2 Karanovic (2011 2012) and Savatenalinton (2017) supported the utility of the presence or absence of the d1-seta on T2 for generic characterization. The partial redescription of P. bullata by Karanovic (2011) clarified the presence of the d1-seta on T2 in this genus. Whenever possible, we have allocated some species to Dentocypria or Vizcainocypria gen. nov. (both without d1-seta on T2) that had been previously assigned to Physocypria only because of their RV marginal tubercles. Species assigned to Cypria, Dentocypria, Keysercypria and Vizcainocypria gen. nov., as well as the recently raised genera Brasilocypria and Claudecypria, consistently lack the d1-seta on T2. Another character of the T2 that has been considered as a generic character for Cyclocyprididae is the appearance of the e-seta. Meisch (2000) already mentioned this character this character as an important diagnostic trait to distinguish Cypria and Physocypria, as did Savatenalinton (2017), since all Dentocypria species present exceedingly long e-setae. Nevertheless, Almeida et al. (2023) suggested that the length of the e-seta on T2 could be relevant at the species rather than genus level, which is consistent with our rearrangement of species for Dentocypria, Keysercypria, Physocypria, and Vizcainocypria gen. nov. Morphology of the Md palp Savatenalinton (2017) discussed the length of the Md palp terminal segment and its use as a diagnostic character at the genus level, and Almeida et al. (2023) noted that closely related cyclocypridid genera (Brasilocypria, Claudecypria and Keysercypria) present differences in this trait. The Md-palp terminal segment is very elongated in Vizcainocypria gen. nov. (3x–4x longer than wide), distinguishing it from Physocypria (2x longer than wide) and Keysercypria (5x longer than wide). However, obtaining information on this character from original descriptions is often impossible because the Md palp had been widely overlooked. We would like to emphasize here the potential of some commonly overlooked characters, such as the Md palp, as useful guides for the classification of Cyclocyprididae ostracods, taking into account that some features of this palp, such as the number and morphology of a variety of setae, are important for distinguishing between Candonidae genera (Meisch 2000). Morphology of CR The morphology of the caudal ramus is usually described or illustrated in most original descriptions, so we could evaluate this appendix in most taxa. The spseta length is used here to differentiate genera with short sp-seta (Cypria and Vizcainocypria gen. nov.) from those with long sp-seta (Claudecypria, Brasilocypria, Dentocypria, Keysercypria and Physocypria). However, this trait alone cannot be used to characterize genera because some genera present different morphologies. Most Cyclocypris species have a short sp-seta, but others, such as C. scrobiculata Klie, 1936, present a long sp-seta. Similarly, some Cypria s.l. species, such as page 17 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan C. bicolor or C. javana, also present a long sp-seta. Problematic cyclocypridid genera can be disentangled and identified using a combination of non-sexual characters. Nevertheless, more research is needed on Physocypria and Cypria because they have accumulated many species over the years, partly becoming wastebasket genera for any slender Cyclocyprididae with or without marginal tubercles, respectively. The new information provided here allows us to better define the generic position of many species, not just based on RV marginal tubercles, but with the combination of characters discussed. Different traits can be used for classifying cyclocypridid genera. For example, the spined bristles present on the second and third endite of the Mxl palp are outstanding characters for Allocypria, as the remaining genera of the family only have bristles on the third endite of the Mxl palp. However, we should point out that the number of setae, claws and bristles of the Mxl could be difficult to interpret, and are therefore obviated in many descriptions. In addition, as most species of this family have bisexual populations, the particular morphology of the male copulatory organ should also be taken into account, as discussed below. Male copulatory organ morphology Non-sexual characters are useful for distinguishing between genera, especially when ostracod populations are parthenogenetic. However, when males are present, hemipenis morphology has been considered an important character in ostracod systematics, used to highlight differences among Cypridoidea genera (Danielopol 1969), differentiate taxa within the Cytheroidea (Martens 2000; Dung and Tsukagoshi 2014) or even within Candonidae s.s., the closest group to Cyclocyprididae (Danielopol 1969; Smith and Kamiya 2006; Iepure et al. 2007). In this framework, copulatory organs can be very useful for clarifying and distinguishing seven cyclocypridid genera with similar morphologies (i.e., Brasilocypria, Claudecypria, Cypria, Dentocypria, Keysercypria, Physocypria and Vizcainocypria gen. nov.). However, the internal parts of the hemipenis have not always been drawn, especially in the early descriptions. Our revision of the external morphology of the hemipenis in this group, focusing on the appearance of the aand b-lobes and their relative size and position, allowed us to distinguish eight general morphotypes corresponding to Cyclocypris, the type genus of the family, plus the seven aforementioned genera (Fig. 6). Cyclocypris type (Fig. 6A): The hemipenis of the type species C. globosa (Sars, 1863) has three lobes, the inner (b), the outer (a) and the medial (h), but sometimes only lobes a and b are observed or remarked in taxonomic works for other species of the genus. In any case, these lobes are usually wide (not elongated) and broadly rounded or with a distal flat plateau, as in the a-lobe of C. ovum (Jurine, 1820). The Cyclocypris type hemipenis, in terms of the morphology of its lobes, is closest to candonids than to other Cyclocyprididae genera (except Kempfcyclocypris), suggesting Cyclocypris could be an ancient representative of the group. Cypria type (Fig. 6B): The lobes are subequal in length and always distinctly elongated in comparison with those of Cyclocypris. The a-lobe is usually broad with rounded distal end, sometimes pointed, whereas the b-lobe can be thinner or as broad as the a-lobe, but almost always pointed. The b-lobe is usually curved towards the a-lobe, which is also slightly curved towards the b-lobe. Both lobes are of similar length, but the a-lobe is usually slightly longer than the b-lobe. Cypria exsculpta, the type species of Cypria, presents a unique hemipenis morphology among the members of the genus, and it is also different from most other genera. This species has two lobes that are broader, shorter and with more rounded tips than other taxa, particularly regarding the b-lobe which is wide and similar to those of the genus Cyclocypris. In fact, hemipenis morphology is very variable among Cypria species, with some taxa such as C. javana showing hemipenis lobes closer to Physocypria or Dentocypria types. It must be pointed out that Savatenalinton (2017) has noted the possible existence of at least two genera within Cypria and hemipenis morphology could help to distinguish them. Physocypria type (Fig. 6C): Both lobes are elongated, a-lobe longer or as long as b-lobe, with distal end curved towards the inner part of the hemipenis. The b-lobe is subelongated, broader at the base and with thin distal end, usually sinuous or S-shaped. The a-lobe is elongated, its tip usually wider than that of the b-lobe and rounded (more pointed in the b-lobe). Some Physocypria species, such as P. nipponica and P. biwaensis, do not seem to have d1-seta on T2, a distinctive character of Physocypria, but are here considered Physocypria because they present the characteristic male copulatory organ of the genus. Nevertheless, other species within Physocypria s.l. may not belong to the genus because they lack d1-seta on T2, but also present a hemipenis morphology different from the Physocypria type described here. In those cases, we decided to maintain the species in the genus because we take a conservative approach, but further information should be gathered to confirm their genus-level position. Dentocypria type (Fig. 6D): The hemipenis has both lobes elongated. The b-lobe is subtriangular, with page 18 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan H Vizcainocypria ab V. viator a b V. granadae C. rochei b a C. mesquitai b a G Claudecypria b a B. alisonae b a B. pea F Brasilocypria E Keysercypria ab a b K. affinis K. deformis D Dentocypria D. smithi a b D. mesquitai a b C Physocypria P. biwaensis a b P. nipponica a b P. kraepelini a b a b P. bullata B Cypria ab a b ab C. ophtalmica C. subsalsa C. matzkeae a b C. exsculpta ab h C. globosa C. ovum ab A Cyclocypris Fig. 6. Male copulatory organs (hemipenes) of different species of Cyclocyprididae. A: Cyclocypris, B: Cypria, C: Physocypria, D: Dentocypria, E: Keysercypria, F: Brasilocypria, G: Claudecypria, H: Vizcainocypria. Redrawn from Almeida et al. (2023): F, G; Karanovic (2011): C (P. bullata), E; Hartmann (1959): H (V. granadae); Meisch (2000): A, B (C. exsculpta, C. ophtalmica), C (P. kraepelini); Savatenalinton (2017): D; Smith and Janz (2008): B (C. matzkeae), C (P. nipponica, P. biwaensis); Wouters (1984): B (C. subsalsa). Scale bars are shown when available: D. smithi = 46 µm; C. ovum, C. ophtalmica, C. subsalsa, P. nipponica, P. biwaensis, D. mesquitai, B. pea, B. alisonae, C. mesquitai, C. rochei, V. viator = 50 µm; C. matzkeae, P. bullata, K. affinis, K. deformis = 100 µm. page 19 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan a broad base and a thinner distal end, but it can also be similar to that of Physocypria, i.e., with a double curve, sinuous and with a pointed tip. The a-lobe is elongated and with a more or less constant width, and its tip is rounded. The a-lobe is longer than the b-lobe, and straight, or slightly curved towards b-lobe. Keysercypria type (Fig. 6E): The lobes are elongated, but shorter than in the other genera. The lobes are positioned with some separation from each other. The a-lobe presents a rounded, boxing glovelike distal end, usually wider than the central trunk of the lobe. The b-lobe is subtriangular, with a thin and pointed distal end. The a-lobe is slightly longer than the b-lobe. Brasilocypria type (Fig. 6F): Species of this genus have a quite variable hemipenis morphology. However, they share an elongated a-lobe, broader at the base and with a thinner distal end. The b-lobe is usually shorter than the a-lobe and subtriangular in shape. The distal end of the b-lobe could be rounded or pointed. Claudecypria type (Fig. 6G): The two lobes are elongated and subequal in length, with the a-lobe being slightly longer than the b-lobe. Both lobes are distinctly broad throughout their length, but with the distal part broader than the base. The a-lobe presents a big rounded distal end, similar to Keysercypria distal end, but markedly larger. The b-lobe presents a flattered distal end in comparison to the a-lobe. Vizcainocypria type (Fig. 6H): The lobes are elongated and subequal in length or with b-lobe longer than the a-lobe; the latter being subtriangular and presenting a pointed distal end the b-lobe is elongated and thinner than the a-lobe. The distal end of the b-lobe is rounded and slightly curved towards the a-lobe. Both lobes are longer than in Keysercypria or Brasilocypria types. Our results show clear differences between hemipenis morphotypes, which are also congruent with the other morphological traits and with molecular data. For example, the most similar hemipenis morphology between Physocypria and Dentocypria is congruent with the shortest K2P distances in the molecular analysis. Hemipenis morphology could also be helpful when non-sexual characters are incongruent or incomplete. This is the case of some Physocypria species, like P. biwaensis or P. nipponica, that have a Physocypria-type hemipenis, while some non-sexual characters differ from the type specie of the genus. In those cases, hemipenis morphology should be favored as the main diagnostic character, following previous studies that proved its relevance for ostracod classification and evolutionary pathways related to sexual selection (Danielopol 1969; Martens 2000). Molecular analyses Many Cyclocyprididae sequences stored in public databases like NCBI or BOLD seem to be misidentified or left in open nomenclature at the genus, family or even order level, most likely because there is a shortage of ostracod taxonomists working on the genetics of living taxa. Therefore, we had to cross-check some unreliable sequences with those identified by renowned ostracodologists, taking also into account the region in which the specimens were collected, and reviewing specimen photographs when available. For the 28S gene, the Cyclocypris sp. (AB674982) specimen from Hiruta et al. (2016) may belong to Physocypria because it grouped with P. cf. biwaensis (KX940935) from Yoo et al. (2017), a publication coauthored by the ostracod specialist Dr. Ivana Karanovic (Karanovic 2011). A similar problem may apply to P. nipponica (AB674984) from Hiruta et al. (2016), which clusters with other Cypria species, rather than with Physocypria. The putative Cypridopsis sp. (AF363321) from Oakley and Cunningham (2002) was obtained from a commercial company and its true specific identity was not fully checked by a non-marine ostracod taxonomist (Todd Oakley, personal communication), and so most likely belongs to a Cyclocyprididae species. In fact, both sequences clustered with our C. ophtalmica (P522) specimens, which were carefully checked using soft parts, including hemipenis, following Meisch (2000) monograph, and came from a locality where the species had been recorded before (Rueda et al. 2013). Nevertheless, we should also take into account the short sequence available for comparison in this gene, and its potentially highly conserved sequence, which may make an otherwise sound comparison of these molecular data unreliable and consequently, our phylogenetic tree for this gene should be taken with caution, notwithstanding the high bootstrap value in our Cypria clade. The locality for some COX1 sequences of Cypria (MG449500, KT706311, MG448778 and MF744511) corresponded to areas surrounding lakes Ontario or Erie in Canada, while others (BBCRT134-12 to BBCRT137-12 and BBCRT090-12) came from the southern U.S. (Texas and Florida). We decided to keep the original identification as Cypria because the most abundant genera of the family Cyclocyprididae in southern Canada is Cypria, with C. ophtalmica being the most abundant representative of the family in the area (Delorme 1970b). However, some of these specimens might have been incorrectly identified, as the picture provided in BOLD in some cases (e.g., BBCRT134-12) seems to allow for an unclear, faint observation of pustules on the right valve, suggesting the specimen may belong to Physocypria s.l., rather than page 20 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan Cypria. In this context, we would like to remark on the importance of properly identifying the material that is used and uploaded to public databases. In addition, the COX1 sequences from BOLD belonging to specimen codes from BBCRT001-12 to BBCRT006-12 were included in our analyses as Physocypria s.l.? because we could observe tubercles on the RV margin when checking their photographs, even though they were identified in the repository just as candonids. Other specimens, sampled from North America, clustered with what we called “Cypria 2?”, and their pictures showed valves pigmented with a pattern very similar to other Physocypria s.l., which could be another indicator that the unknown Cypria sequences actually belong to Physocypria or even to Vizcainocypria or other similar genera, in case they were misidentified. Although cyclocypridid genera with tubercles on the RV margin (Dentocypria, Physocypria and Vizcainocypria gen. nov.) appear separated from those (supposedly) without tubercles (i.e., identified as Cypria in the repositories), our phylogenetic analyses should be considered just as preliminary results. The COX1 gene tree shows a polytomy, indicating that affinities between different cyclocypridid genera could not be fully resolved. In other words, molecular data cannot confirm whether Vizcainocypria gen. nov. is more closely related to Physocypria than to Cypria. Nevertheless, our molecular analyses do support genuslevel differentiation between Cypria, Dentocypria, Physocypria and Vizcainocypria gen. nov. because average K2P distances between those cyclocypridid taxa (0.240 ± 0.025), agree with average K2P distances observed by Nigro et al. (2016) between different marine ostracod genera (0.260 ± 0.080). Several COX1 sequences from public databases (BBGEN061-15, HM883981, HM883998 and RBNII303-13) were very close to V. viator gen. nov. sp. nov. and show K2P distances similar or even lower than those calculated between different species of Physocypria. This suggests that those unidentified specimens, collected from Ontario (Canada) (BBGEN061-15 and RBNII303-13) or Oklahoma (U.S.) (HM883981, HM883998) could belong to Vizcainocypria gen. nov. The new species described from Iberian waters might therefore belong to an ostracod lineage originated in the Nearctic that later colonized the Palearctic. This is in agreement with the high number of exotic ostracods in the sampled area (Valls et al. 2014) and the absence of micropaleontological remains of any morphologically similar ostracod during the Holocene in the Eastern Iberian Peninsula (Marco-Barba et al. 2013a b 2019) or elsewhere in Europe (Griffiths 1995; Fuhrmann 2012). Another piece of evidence supporting the Nearctic or Neotropical origin of the genus is the geographic distribution in Central America of the only other known species that can be included in the genus, V. granadae comb. nov. Two other species present in the Nearctic, P. inflata and P. pustulosa, could be allocated to Vizcainocypria gen. nov. as well, considering their shell and soft part anatomy, but type material or new specimens from type localities should be reviewed to confirm their allocation. Finally, considering the wide distribution of similar genera and the high number of worldwide human-induced invasions in aquatic organisms (Gherardi 2007), and ostracods in particular (Mckenzie and Moroni 1986), alternative origins such as eastern Asia or Southern Africa cannot be completely discarded. CONCLUSIONS This work stressed the importance of integrative taxonomy in Ostracoda, especially when congeners have very similar morphological traits and molecular data are limited. While classical taxonomy helps identifying diagnostic characters at a generic level, molecular information supports the genus-level distinction and allows for detection of misidentifications in public databases. Further integrative studies are needed, not only for Cyclocyprididae, but for any group of Ostracoda where taxa are difficult to disentangle based on morphology alone. On the other hand, an increasing number of molecular sequences in public databases may become senseless without proper morphological assessment. Acknowledgments: This work and the new species name were registered with ZooBank under urn:lsid:zoobank.org:pub:18A831EC-30BE-4F22B836-0D3305D3E84C. This research was supported by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Comunidad Valenciana Government), though the project EXOCRUST (code AICO/2020/182), by Ministerio de Economía, Industria y Competitividad (Spanish Government), through project METACOM-SET (code CGL2016-7826-P) and by MCIN/AEI/10.13039/501100011033 through project CRUSTRESS (code PID2020-112959GB-I00). M. Bisquert-Ribes has a Predoctoral grant (FPU19/02264) funded by Ministerio de Educación y Formación Profesional (Spanish Government). FP acknowledges the project “CIDEGENT/2019/028 - BIOdiversity PAtterns of Crustacea from Karstic Systems (BIOPACKS): molecular, morphological, and functional adaptations” funded by the Conselleria d'Innovació, Universitats, Ciència i Societat Digital. We wish to page 21 of 24Zoological Studies 62:40 (2023) © 2023 Academia Sinica, Taiwan thank Dr. Sergio Cohuo for facilitating microscope photographs of the limbs of the species V. granadae comb. nov., to Jordi Sala for sending photographs and individuals of V. viator gen. nov. sp. nov. from the Empordà wetland and to the personnel of the SCSIE Microscopy Unit at the University of Valencia for their help during SEM photograph sessions. Special thanks to Koen Martens for his comments and suggestions to an earlier version of this manuscript. Authors’ contributions: MBR worked on illustrations, descriptions and first draft of the manuscript; JR worked on collecting the specimens; FP worked on molecular analyses; SS worked on description of specimens; FMJ designed and supervised the research approach, got funds for this work and identified the first specimens found. All authors contributed to the revision of the manuscript and approved the final manuscript and consent to publication. Competing interests: All authors declare that they have no competing interests. Availability of data and materials: DNA sequences generated in the study have been deposited in GeneBank. Consent for publication: All the authors consent to the publication of this manuscript. Ethics approval consent to participate: This research followed the guidelines specified by Conselleria d’Agricultura, Desenvolupament Rural, Emergència Climàtica i Transició Ecològica (Generalitat Valenciana) of the Government of the Comunitat Valencia (Spain) for collection permission. REFERENCES Almeida NM, Ferreira VG, Martens K, Higuti J. 2023. Seven new species and two new genera of Physocypria sensu latu (Crustacea, Ostracoda) from Brazilian floodplains. Zootaxa 5237:001–088. doi:10.11646/zootaxa.5237.1.1. Baird W. 1845. Arrangement of the British Entomostraca, with a list of species, particularly noticing those which have as yet been discovered within the bounds of the Club. Transactions Berwicksh Nat Club 2:145–158. Bou J, Sala J, Boix D, Gascón S, Vilar L, Quintana X. 2019. Avaluació de l’estat ecològic en arrossars i closes als aiguamolls de l’Empordà. University of Girona. Brady GS. 1886. Notes on Entomostraca collected by Mr. A. Haly in Ceylon. 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