Physocypria Vávra, 1897

Bisquert-Ribes, Maria, Rueda, Juan, Palero, Ferran, Savatenalinton, Sukonthip & Mesquita-Joanes, Francesc, 2023, Fig. 1 in Fig. 4 in Identification of Sexually Dimorphic Genes in Pectoral Fin as Molecular Markers for Assessing the Sex of Japanese Silver Eels ()., Zoological Studies 62 (40), pp. 1-24 : 6-16

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 T 1 with asymmetrical prehensile palps. T 2 with basal d1-seta. Terminal segment of T 3 subquadrate, g-seta very short. CR well-developed, with sp-seta usually long, claws well-developed. 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 T 2, 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 d1- seta on T 2 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 the presence of dp-seta on T 3 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-0880-4C29-822A-BA20245FE678

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 non-sexual 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 non-sexual 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 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 sp-seta 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-60B9-40B8-9FC3-C88E7D13F341

Physocypria sp. : Bou et al., 2019: 41.

Dentocypria sp. : Giménez et al., 2020: Fig. 29.

Type materials: Holotype: Soft parts of a dissected ò preserved in HydroMatrix ® on a slide, and valves stored dry in a micropalaeontological slide (MUVHNZY0011). Allotype. ñ stored like the holotype (MUVHNZY0012). Paratypes. Two dissected ò (MUVHNZY0013 and MUVHNZY0014) and three dissected ñ (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 Pego- Oliva 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 antero-dorsal 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), T 2 with g-seta and accompanying-seta subequal, and long e-seta. CR with short sp-seta. Right prehensile palp of male T 1 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 T 2 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 = 579.2, H = 374.5, W = 277.2. Valves not homogeneously pigmented ( Fig. 1A, B ); one large dorsal dark patch between mid-length and the eye spot, and between dorsal margin and mid height of valve (muscle scars); another distinctive patch in the marginal anterior and central part of the valve; one smaller posterodorsal dark patch and another posterior marginal patch, less intense, at mid height. Carapace in lateral view ( Fig. 1C ) ovate, anterior and posterior margins rounded, LV overlapping RV anteriorly, ventrally and posteriorly, valve surface smooth with some short setules, with small tubercles on the right valve margin anteriorly and posteriorly.

CpF ( Fig. 1D ) ovate, widest at mid-height.

CpD ( Fig. 1E ) elliptical, laterally compressed, widest at or slightly behind mid-length.

LVi ( Fig. 1F ) selvage anteriorly wider than posteriorly, internal tooth ( Fig. 1G ) on antero-ventral part, calcified inner lamella anteriorly wider than posteriorly, small pits complementary of right valve tubercles anteriorly and posteriorly.

RVi ( Fig. 1H ) selvage anteriorly wider than posteriorly, complementary pit of internal tooth on antero-ventral part, slight depression of the valve margin on antero-dorsal part, valve margin tuberculated ( Fig. 1I ) anteriorly and posteriorly, posterior tubercles more prominent than anterior ones, calcified inner lamella anteriorly wider than posteriorly.

A1 ( Fig. 2A ) 7-segmented, first segment with small proximal Wouters organ, one long dorso-subapical seta (reaching beyond tip of next segment), and two long ventro-apical setae, reaching terminal segment. Second segment more than twice as wide as long, with one dorso-apical seta (reaching tip of next segment) and large mushroom-like Rome organ. Third segment bearing two setae: one long dorso-apical seta (reaching tip of fifth segment) and one shorter ventro-apical seta (reaching beyond tip of next segment). Fourth segment with two very long dorsal setae (reaching far beyond tip of terminal segment, but one more than twice as long as the other) and two ventral setae (shortest one not reaching half of sixth segment, longer one reaching half of sixth segment). Fifth segment with two very long-setae (longer than entire limb) dorsally, and with two setae ventrally (short one reaching tip of terminal segment and long one reaching beyond the terminal segment and about one third longer than the short seta). Penultimate segment with four (two dorsal and two ventral) very long apical setae and one dorsal claw (about three times the length of the terminal segment). Terminal segment with two very long apical setae, one apical claw-like seta and one aesthetasc ya, length of the latter c. 1/2 of the claw-like seta.

A2 ( Fig. 2B ) exopod with three (one long, two short) setae, long one reaching beyond tip of first endopodal segment. First endopodal segment with five very long (reaching far beyond tip of terminal claws) natatory setae and one short accompanying-seta, length of the shortest seta less than half of penultimate segment, aesthetasc Y long (almost reaching tip of corresponding segment), ventro-apical seta long, reaching tip of terminal claws. Penultimate segment undivided, distally with three serrated claws (G1, G2, G3), G2 shorter (length of G2 c. 1/2 that of G1), aesthetasc y2 reaching beyond tip of terminal segment, three subapical setae: seta z1 claw-like, short (length of z1 c. 1/2 of that of z2) and wide; z2–z3 setae long, z3 longer than z2; medially with two long dorsal setae, four ventral setae of unequal length (t1–t4) and aesthetasc y1, the latter long (reaching tip of corresponding segment). Terminal segment distally with two serrated claws (GM and Gm), length of Gm c. 4/5 of that of GM; medially with ventral aesthetasc y3, length of aesthetasc y3 c. 2/3 that of accompanying-seta.

Md palp ( Fig. 2C–E ) first segment with two large hirsute setae, one long and slender seta, and a very short, smooth α-seta. Second segment dorsally with two unequal long apical setae (long one reaching beyond half of penultimate segment, length of the short one c. 2/3 of the long one); ventrally with group of two long hirsute setae, one long smooth seta and a small, plumose, dome-shaped β-seta. Penultimate segment consisting of two groups of four setae each and one isolated seta. Dorsally with a group of four similar, long, subapical setae; laterally with apical γ-seta and three unequal apical setae: the longest one slightly beyond tip of γ-seta, the most dorsally one with length 2/3 of γ-seta and the most ventral one with length 1/2 of γ-seta; in the middle part with one isolated, long subapical seta (length similar to terminal segment). Terminal segment very elongated (c. 3 times as long as wide, length similar to penultimate segment) bearing three claws and one longseta.

Mx1 ( Fig. 2F ) with 2-segmented palp, three endites and large branchial plate; basal segment of palp dorsally with two groups of setae, one group with two apical setae (one plumose and another smooth) and another group of four long subapical setae (reaching beyond tip of terminal segment); in the middle part of the palp with one plumose subapical seta. Terminal segment subquadrate, apically with three claws and three setae (two long claws and one slightly longer than the setae). Two unequally long bristles on third endite, length of short one c. 3/4 times that of long one. This endite with four short apical setae, one ventro-apical short seta and one long ventral seta. Second endite with four long and one short apical setae, and one short ventro-apical seta. First endite with two long and seven shorter apical setae, shorter ones about 2/3 the length of the longer ones. Ventrally with two long-setae. Branchial plate with c. 19 ventral rays, the two most basal of which being half the length of the longer ones, and six reflexed dorsal rays.

T 1 ( Fig. 3A ) protopodite with two subequal short a-setae, long b-seta and long d-seta and additional long and hirsute seta, and distally with c. 13 hirsute apical setae of unequal length. Endopod weakly built palp with three short and unequal apical setae. Branchial plate with five rays.

T 2 ( Fig. 3B ) without d1 and d2 basal setae. Second segment with long e-seta (reaching tip of penultimate segment). Penultimate segment divided, proximal segment bearing normally developed f-seta (length 3/4 of length of distal segment), distal segment with apical g and accompanying-setae (both subequal in length). Terminal segment with two (one dorsally, one ventrally) apical h1 and subapical h3 setae and apical serrated claw (h2), as long as the penultimate and antepenultimate segments together.

T 3 ( Fig. 3C ) first segment with long d1, d2 and dp setae, d1 and d2-setae subequal in length. Second segment with subapical e-seta (length c. 1/2 of penultimate segment). Third segment with sub-medially f-seta (reaching tip of corresponding segment), g-seta short. Terminal segment subquadrate, with three setae, two short h1 and h2 setae (subequal in length with h2 slightly longer), and one reflexed subapical h3 seta, length of the latter equal to that of three last segments.

CR ( Fig. 3D ) well-developed, robust, ventrally with three parallel rows of setules, Ga and Gp large, serrated, length of Ga c. half that of ramus, length of Gp c. 2/3 that of Ga. Sa short (less than half of Ga), sp-seta short (length c. 1/3 of Gp).

CR attachment ( Fig. 3E ) with two distal branches.

Description of male: Measurements (mean, in μm): LV (n = 6), L = 527.5, H = 336.1; RV (n = 6), L = 527.7, H = 314.3; Carapace (n = 6), L = 528.4, H = 338.2, W = 275.3. Carapace ( Fig. 1B ) and valves as in female, but somewhat smaller. All limbs as in the female (not illustrated), except for last two segments of A2 and T 1 (illustrated).

A2 ( Fig. 4A ) with penultimate segment divided and t2 and t3 setae transformed into sexual bristles, long (reaching slightly beyond tip of terminal segment) and subequal. Setae z1 and z2 transformed into claws, length of z1 half of z2; claw G1 reduced, appearing smaller and shorter than that of female; claw G3 reduced to seta (length about twice the length of terminal segment).

T 1 with asymmetrical prehensile palps (endopods). Right prehensile palp ( Fig. 4B ) with first segment bearing one short apical spine, distal margin with large spine-like protrusion, distal part wider than basal part; second segment large, subtriangular. Left prehensile palp ( Fig. 4C ) with elongated first segment bearing two subapical spines, one long and the other shorter; second segment narrow, hook-like and pointed tip.

Hemipenis ( Fig. 4D ) b-lobe elongated and thinner than a-lobe, slightly bent towards a-lobe, and with a rounded tip; a-lobe wide, subtriangular, with a shark fin shape and slightly shorter than b-lobe.

Zenker organ ( Fig. 4E ) sub-elongated, length c. 2 times width, set with seven chitinous spiny whorls, five along the central tube, two at the extremes.

Key to the genera of the family Cyclocyprididae

1a. RV margin smooth ...................................................................... 2

1b. RV margin tuberculated .............................................................. 7

2a. d1-seta on T2 present ................................................................. 3

2b. d1-seta on T2 absent ................................................................... 6

3a. A1 7-segmented; A2 natatory setae well-developed .................. 4

3b. A1 6-segmented; A2 natatory setae reduced; t2 and t3 on male A2 not transformed into sexual bristles ........... Kempfcyclocypris

4a. Carapace subovate in lateral view; terminal segment of T3 very elongated (3x–4x longer than wide); g-seta on T3 well-developed; t2 and t3 on A 2 male not transformed into sexual bristles ....................................................................... Cyclocypris

4b. Carapace elongated in lateral view; terminal segment of T3 subquadrate (<3x longer than wide); g-seta on T3 short or absent; t2 and t3 on A 2 male transformed into sexual bristles ... 5

5a. Imprints of ovaries curved upwards; bristles on 2nd and 3rd endites of Mxl present; terminal segment of Md palp usually squared ........................................................................ Allocypria

5b. Imprints of ovaries curved downwards; bristles present only on 3rd endite of Mxl; terminal segment of Md palp elongated (2x longer than wide) ................................................... Mecynocypria

6a. T3 terminal segment elongated; A2 natatory setae reduced; terminal segment of Md palp elongated (2x longer than wide) .... ............................................................................. Namiotkocypria

6b. T3 terminal segment subquadrate; A2 natatory setae well-developed; terminal segment of Md palp very elongated (> 2x longer than wide) ............................................................... Cypria

7a. A2 short accompanying natatory seta absent ............................. 8

7b. A2 short accompanying natatory seta present .......................... 10

8a. Terminal segment of Md palp elongated (2x longer than wide) or very elongated (3x–4x longer than wide) ................................... 9

8b. Terminal segment of Md palp extremely elongated (5x longer than wide); lobe-a of the hemipenis with a boxing glove-like distal end ................................................................. Keysercypria

9a. Terminal segment of Md palp very elongated (3x longer than wide); presence of dp-seta on T3 ........................... Brasilocypria

9b. Terminal segment of Md palp elongated (2x longer than wide); absence of dp-seta on T3 ........................................ Claudecypria

10a. Terminal segment of Md palp elongated (2x longer than wide); d1-seta on T2 usually present ................................... Physocypria

10b. Terminal segment of Md palp very or extremely elongated (3–5x longer than wide); d1-seta on T2 absent .................................. 11

11a. sp-seta on CR long; 1st segment of right prehensile palp with apical subtriangular protrusion and without finger-like protrusion; a-lobe of hemipenis slender and longer than b-lobe ..................... ................................................................................... Dentocypria

11b. sp-seta on CR short; 1st segment of right prehensile palp with apical spine or finger-like protrusion; a-lobe of hemipenis wide and shorter than b-lobe ......................................... Vizcainocypria

Molecular analyses

New COX1 gene sequences have been obtained for D. smithi (GenBank accession number: OP208069) and V. viator gen. nov. sp. nov. (OP208070) and new 28S rDNA sequences for D. smithi (OP216521), V. viator gen. nov. sp. nov. (OP216522) and C. ophtalmica (OP216523). After trimming the alignments, the final COX1 and 28S rDNA datasets had 438 bp and 234 bp, respectively. The best DNA substitution models selected were TPM1uf+I+G4 (lnL: -3413.67) for COX1 and TPM3uf+G4 (lnL: -670.17) for 28S rDNA. Phylogenetic trees for the mitochondrial (lnL: -3418.29) and nuclear markers (lnL: -666.44) were both congruent, showing representatives of Cyclocyprididae s.str. (i.e., Physocypria , Cypria , Dentocypria and Vizcainocypria ) to form a monophyletic clade despite no significant (> 70) bootstrap support. The new C. ophtalmica 28S rDNA sequence and C. exsculpta from GenBank clustered with putative Cypridopsis sp. and P. nipponica sequences ( Fig. 5A ; see Discussion). Despite internal nodes not showing significant bootstrap values, most likely because ribosomal genes are highly conserved, V. viator gen. nov. sp. nov. appeared as an early-splitting lineage within Cyclocyprididae ( Fig. 5 ). Groups of taxa appear to be better defined for the COX1 gene tree, with Physocypria sequences clustering together. Phylogenetic affinities between Cyclocyprididae genera could not be resolved, but genetic differentiation among the four main genera was high for the COX1 gene ( 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).

Figures

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.

Fig. 2. Vizcainocypria viator gen. nov. sp. nov., female (MUVHNZY0012).A: A1 (arrow pointing to apical claw on penultimate segment). B: A2. C: Md coxa. D: Md palp. E: Detail of α and β setae. F: Mxl. Scale bars:A–F = 50 µm.

Fig. 3. Vizcainocypria viator gen. nov. sp. nov., female (MUVHNZY0012). A: T1. B: T2. C: T3. D: CR. E: Caudal attachment. Scale bars: A–E = 50 µm.

Fig. 4. Vizcainocypria viator gen. nov. sp. nov., male (MUVHNZY0011). A: A2. B: Right prehensile palp. C: Left prehensile palp. D: Hemipenis. E: Zenker organ. Scale bars: A–E = 50 µm.

Fig. 5. Maximum likelihood tree for 28S (A) and COX1 (B) genes. Red branches indicate the presence of tubercles on the RV margin.