Adaptations to the hyporheic in Aloninae (Crustacea: Cladocera): allocation of Alona protzi Hartwig, 1900 and related species to Phreatalona gen. nov.

Abstract

Morphological study of Alona protzi Hartwig, 1900, Alona phreatica Dumont, 1983 and Alona smirnovi Petkovski & Flößner, 1972 reveals close affinities with Alona labrosa Vasiljeva & Smirnov, 1969. We separate these four species from the polyphyletic Alona Baird, 1843 (Anomopoda: Chydoridae). United under Phreatalona gen. nov., these taxa share primitive features on the limbs, together with specializations to a rheic life mode. Phreatalona contains some of the only true hyporheic taxa within the Cladocera. Endemism in two ancient lakes (P. smirnovi and P. labrosa) and a preference for river sediments in Europe (P. phreatica and P. protzi) suggest a long isolation from typical littoral/benthic biotopes. We discuss close links with southern vicariant Nicsmirnovius, the position of these (hypo)rheic chydorids within the subfamily and their affinities with Acroperus. We remark an independent evolution of external (habitus, postabdomen) vs. internal (limb) morphology in the protzi-complex. Phreatalona is likely tertiary in origin, evolving from a littoral alonine adapting to rheic and finally hyporheic environments. Baikal endemic P. labrosa is likely the most primitive species of the genus. We discuss adaptations and evolution in the hyporheic and the effect on dispersal and biogeography of Phreatalona.

Introduction

Suggestions of the polyphyletic nature of the freshwater chydorid genus Alona Baird, 1843 were formulated on morphological (e.g. Sinev, 2004; Van Damme & Dumont, 2008, in press) and molecular grounds (Sacherova & Hebert, 2003). Alona consists of several species complexes the classification of which is unstable. Necessity for global revision was stated repeatedly (e.g. Kotov & Sanuamuang, 2004). For a fuller discussion on this subject and a revision of the type species (A. quadrangularis), see Van Damme & Dumont (2008, in press). Taxa were attributed to different genera using limb morphology, a useful tool to unravel affinities between these externally similar micro-crustaceans (e.g. Dumont & Silva-Briano, 2000; Sinev et al., 2004, 2005; Van Damme et al., 2003). Redescriptions of marginal Alona species help to understand their position and delineate natural groups but a large portion still await such ‘treatment’. Even within Europe, several Alonas are rare and poorly described. One of these is the rare Alona protzi Hartwig, 1900. Hartwig (1900) recognized the aberrant morphology within Alona and was careful about its status: “Since I am able to identify this delicate form with none of the 50 more or less well described kinds of the genus Alona, I describe it here as new, leaving it to the person working on this genus in the future to determine whether this is a valid species. I did not want to let the form be lost”. Being the only European “Alona” with denticles in the posteroventral corner of its valves, protzi is unmistakable. It occurs in the whole of Western Europe (Smirnov, 1971; Vranovsky, 1971; Flößner, 2000), but is strongly localized with low densities, few specimens collected per site. Its ecology was poorly known (Vranovsky, 1971). Because of its rarity and low abundances, morphology was not studied and the affinities with other Aloninae are unknown. The stygophilic/stygobiotic mode of life of A. protzi and related species was recognized recently (Dumont, 1983; Brancelj & Dumont, 2007). A superficial similarity between A. smirnovi, A. phreatica and A. protzi was suggested by Dumont (1983) with the description of A. phreatica, and later by Sinev & Kotov (2000) for A. labrosa. Only the latter, an endemic from Lake Baikal, has to date been described in detail, including limb morphology, revealing a set of rare characters for Aloninae. Details of the other three species were unknown, though there are few drawings of limbs of A. smirnovi and A. phreatica in Alonso (1996) and Petkovski and Flößner (1972).

Since 2000, we were able to collect more specimens to check if the above-mentioned taxa are closely related as their external morphological characters suggest. Our preliminary results and distribution of Phreatalona were presented at the Cladocera Symposium in 2005 in Herzberg (Switzerland) (Van Damme et al., 2005). Here, we document comparative morphology of the three taxa and discuss the relationship between and the status of A. protzi Hartwig, 1900 , Alona phreatica Dumont, 1983, Alona smirnovi Petkovski & Flößner, 1972 and Alona labrosa Vasiljeva & Smirnov, 1969. The trigger for this study was new material from the hyporheic realm collected within the PASCALIS project (Protocols for the ASsessment and Conservation of Aquatic Life In the Subsurface), a research project supported by the European Commission under the Fifth Framework Programme. The PASCALIS project spans six study areas, each with four neighbouring small rivers with catchment areas of approximately 100 km² (one each in Spain, Belgium, Italy and Slovenia and two in France). Two types of subterranean aquifers were studied: karstic systems in consolidated, fractured rocks and hyporheic zone in unconsolidated, alluvial sediments (Gibert & Deharveng, 2002; PASCALIS project, 2004). Samples taken under the PASCALIS project in porous aquifers (Brancelj & Dumont, 2007) and subsequent samplings resulted in new records for A. phreatica and A. protzi from Belgium and France. We use topotypical and new material, allocate these species to a new genus and discuss their ecology, morphology (and variability), biogeography and evolution. Study of morphological characters, especially structure and armature of thoracic limbs, enables us to establish a new genus, Phreatalona, presented further on in this paper.

Materials and methods

During the PASCALIS project, the Bou-Rouch method was employed to sample fauna in hyporheic zone (30–60 cm below the river bottom) and the phreatic zone (90–120 cm below the river bottom). Ten sampling locations were set for each river, evenly spaced along the river in the alluvial part. On each location, three to five sub-samples were collected (usually in a form of transect across the river’s profile), and for each sub-sample a water volume of 10 l was pumped by a piston pump. On all sampling locations, nets with mesh size of 100 μm or less were used. On each sub-sampling point, first, the sample for fauna was collected from hyporheic zone, and then temperature, conductivity and oxygen concentration were measured in situ. In the next step, a steel pipe was inserted further down into the phreatic zone, where the procedure was repeated. Fauna samples from each zone were preserved in 4% formaldehyde solution before being sorted out. Final deposition of specimens was in 60% alcohol. (For more details on sampling methods and protocols see: PASCALIS project, 2004.) During an additional sampling campaign (by KVD in 2007), animals were collected using dip nets with 50 μm mesh, and fixed in 4% formaldehyde or 80% ethanol. We collected protzi from Heerenlaak (Belgium) and a side branch of La Lanterne (France) from a gravelly/sandy substrate by gathering the upper 20 cm of gravel by hand, rinsing it in a bucket, pouring water off through a 50 μm net and repeating this procedure. Specimens for permanent storage and dissection were transferred to a formaldehyde–glycerin mixture, mounted on glass slides, dissected under a WILD stereomicroscope at low magnification, and sealed using a rapidly solidifying varnish. Methods and materials for optical microscopic examination, SEM and annotation/numbering of the limb structures are described in Van Damme & Dumont (2007). Enumeration of setae and other limb structures is done from the epipodite towards the gnathobase, without suggestion of homology. Photo of live P. protzi was taken in the same way as Anchistropus in Van Damme & Dumont (2007), using HeliconFocus for combining photo layers.

Cladistic analysis: We used PAUP 4.0b10 (Swofford, 2000) for generating a small dendrogram illustrating morphological similarities. Branch-swapping algorithm: tree-bisection-reconnection (TBR), with random addition sequence (1000 replications). Characters, states and data matrix are given in the Supplemetary material. We performed a heuristic search and parsimony analysis with 100 bootstrap replicates and parsimony as the optimality criterion. We selected a total of 31 morphological characters for this analysis, of which 17 were limb characters. Twelve characters are considered specializations to the rheic life mode, while the function of the remaining characters is unknown. All characters were unordered and of equal weight, and starting tree was obtained via stepwise addition, default settings. We chose 11 Aloninae taxa for comparison: Alona quadrangularis, Alona affinis, Acroperus harpae, four species of Nicsmirnovius and four species of Phreatalona. Character choice for Nicsmirnovius was partly based on previous analysis by Kotov (2004). A wider sampling of Alona is beyond the scope of this paper, a short analysis, and our choice of characters is aimed at illustrating a rheic sub-branch of the Aloninae. Data for morphology are based on Alonso (1996), Van Damme et al. (2003), Kotov & Sanuamuang (2004), this study (Phreatalona) and unpublished data (quadrangularis). We could not perform quantitative analyses to study variability of external character (carapace and postabdomen) for the four Phreatalona species because of the limited number of specimens and populations of phreatica and smirnovi and none of labrosa.

Abbreviations

The following abbreviations, in alphabetical order, are used throughout the manuscript including Figures and Tables. A1: antennule; A2: antenna; ant: anterior; as: accessory seta; en: endite; ep: epipodite; ex: exopodite; fasc: fascicle; fc: filter comb; ft: flaming torch; gn: gnathobase; IDL: inner distal lobe; il: inner lobe; lat: lateral; nat: natatorial; ODL: outer distal lobe; P1-P6: first to sixth trunk limbs; PA: postabdomen; parth: parthenogenetic; pep: pre-epipodite; PvC: posteroventral valve corner; scr: scraper; segm: segment; ss: soft seta; tc: terminal claw. For phylogenetic analysis, CI: consistency index; HI: homoplasy index; RC: Rescaled consistency index; RI: retention index.

Definitions

We use the term “hyporheic” throughout this paper, but definitions vary according to the scientific discipline (Smith, 2005). From ecological viewpoint, the hyporheic zone is delineated by the distribution of its organisms, the hyporheos. They may contain true subterranean freshwater fauna, stygobionts and stygophiles. It is a dynamic ecotone (Sabater & Vila, 1991) intermediate between the river water above and the groundwater from saturated bedrock below, the phreatic (Valett et al., 1993; Boulton et al., 1998). The latter zone is part of the groundwater system, typically >1 m below the riverbed, where strict “subterranean” conditions prevail. There is intense exchange with both surface and underground water and even contains different zones within it (White, 1993). Differences in permeability affect movements of the hypogean fauna and their distribution. Delineation of the hyporheic and an alluvial aquifer in the floodplain, which may extend horizontally up to a few km from the main channel, is not always clear (Stanford & Ward, 1988). To hydrogeologists, the hyporheic zone is part of the groundwater system (Smith, 2005). The special habitat we are interested in for the study of Phreatalona is the water-saturated subsurface zone less than 1 m (typically 30–60 cm) below riverbeds of relatively small streams, where interstitial water and organisms move freely through permeable streambed deposits, between a coarse mixture of sand and gravel. This falls within the hyporheic zone.

Taxonomic account

Family CHYDORIDAE Stebbing, 1902 emend. Dumont and Silva-Briano, 1998

Subfamily Aloninae Dybowski and Grochowski, 1894 emend. Frey, 1967

Tribe Alonini Dybowski and Grochowski, 1894 emend. Kotov, 2000

Phreatalona gen. nov.

Type species. Alona protzi Hartwig, 1900

Etymology. The name “Phreatalona” is composed of “phreat-” (from A. phreatica) and Alona to indicate their adaptations to the subterranean mode of life in the protzi-complex.

Diagnosis

Adult parthenogenetic female. Body rectangular to more elongate in lateral view, with straight ventral margin; small to medium-sized animals (0.2–0.5 mm), in life translucid. Head protruding and rounded with short or no rostrum, three main head pores and two small pores lacking additional structures. Mandible articulation as for subfamily. Carapace lacking a dorsal keel, ornamentation consisting of fine wide lines or absent, no fine striation; posteroventral corner with small notch; marginal setae in posteroventral portion followed by fine spinules and up to three denticles in the posteroventral corner. Postabdomen 2–2.5 times as long as wide, ventral and dorsal margins relatively straight and parallel, postanal and anal portion of same dimensions, sharp preanal angle moderately to well developed; anal margin straight, postanal moderately concave, rounded dorsodistal angle; distal portion protruding, with distal notch. Marginal denticles consisting of groups of small denticles of which distalmost partly merged; lateral fascicles with spines of similar size and thickness. Terminal claw about as long as anal margin, straight to moderately curved, basal spine two to three times as long as width at base, implanted at some distance from claw base and reaching up to half of claw length. First antenna about two times as long as wide, with sensory seta implanted apically; seven apical aesthetascs subequal in length, about half as long as antennular corm and two strongly elongated aesthetascs longer than corm. Second antenna with spinal formula 001/(1)01 and setal formula 113/003. First exopod seta on antenna relatively long and narrow; first endopod spine reduced to fourth to third of second segment length. Labrum long with convex to straight margin, keel with protruding conical posterior portion and rounded tip; keel naked, lacking ventral setules. First maxilla with two setulated setae. Five pairs of limbs. First limb. First endite with two marginal setae (dorsal seta absent), second endite with three setae of which two little longer, third endite with four setae; anterior structures on endites: en1 a long seta, en2 a long seta and minute element. ODL with one slender seta, IDL with three setae of which two implanted with short or long setules in distal half. Accessory seta present and well developed. Anteriorly on corm, six to seven setule groups with more than six long setules in each group; setules in each group not decreasing in size ventrally. Ejector hooks relatively small and subequal; epipodite round, with long projection. Gnathobase a single setulated projection. Second limb. Exopodite with one well-developed seta; endites with eight relatively slender scrapers, first two long and slender, third shorter, last three gradually decreasing in size, all with similar denticles; gnathobasic ‘brush’ elongated triangular, gnathobase with a sensillum and three elements, filter comb with seven setae of which the first much shorter and thicker, brushlike. Third limb. Exopodite with quadrangular corm and seven setae in 2 + 5 arrangement; first longer than second, third exopodite seta not strongly elongated, fourth and fifth may vary in size between species (long or short), fifth and sixth setae with fine plumose setulation. External endite with three setae (1′–3′) of which first two long with fine setulation in distal half and with minute element in between, third thicker; four well-developed plump plumose setae on inner side (1″–4″); one element and four small setae on internal endite preceding gnathobase; gnathobase with a bottle-shaped sensillum, bent plumose seta with two long setae emerging from its base. Filter comb with seven long setae. Fourth limb. Epipodite oval-round, with projection. Exopodite small and square, with six plumose setae of which third relatively longest and fifth and sixth setae relatively narrower than others. Fourth exopodite seta strongly reduced to setulated hillock, sixth with blunt apex and subapical cluster of merged setules (not labrosa). Endite with marginal row of four setae, first short and scraperlike, following three strongly reduced flaming torch setae about as thick as wide and decreasing in size (not labrosa), and one marginal round naked sensillum implanted on the inner side of the endite; gnathobase with one long setae, bent over endite and one reduced naked element; inner side naked, filter comb with five short setae. Fifth limb. Epipodite round, with projection. Exopodite oval to heart shaped, mostly with deep concave margin between setae three and four; four exopodite setae, of which first two longest, same size or shorter than exopodite itself and oriented dorsally; fourth exopodite seta well developed and of similar dimensions as other three; inner lobe elongated with oval apex and long terminal setules; two thick endite setae (1′–2′) of which first little longer than second or both of same size; gnV with a process but no filter comb. Adult male smaller than female, with clear sexual dimorphic postabdomen with gonopores opening ventrally, subapical to terminal claw; marginal denticles consisting of unmerged groups of setules. Male IDL with three setae; thick copulatory hook with terminal rugae (ridges).

Short diagnosis of Phreatalona gen. n.

Small to medium-sized Aloninae with body elongated in lateral view, tapering posteriorly, three main head pores and round small pores (as opposed to Nicsmirnovius); head protruding rostrum short to absent, labrum strongly elongated with blunt tip, second antenna with first endopod spine reduced. Relatively short postabdomen with straight dorsal margin and straight to moderately convex ventral margin; terminal claw long, basal spine up to half its length. Five limb pairs. P1 lacking dorsal seta, IDL with three setae, anterior setae on endites one and two very long, accessory seta present, fine setule groups; P2 with well-developed exopodite seta, scrapers homogeneous in denticulation, additional element at base of scraper 1 and an elongated gnathobasic ‘soft’ brush, P3 with seven setae and short third exopodite seta, full set of setae in endite; P4 with reduced fourth seta (not labrosa). Endite lacking inner structures (autapomorphy) and with shifted receptor; P5 with four exopodite setae, elongated inner lobe and reduced gnathobase (0 setae). Males have marked postanal corner and basal spine as long as in female (Sinev & Kotov, 2000).

• 1. Phreatalona protzi (Hartwig, 1900) comb. nov.

• =Alona protzi Hartwig, 1900: 228–230.

• Type locality. Königsberg, bank of Hellsee by Biesenthai, Germany (Hartwig, 1900).

• Etymology. Named in honour of Dr A. Protz, curator of Crustacea collection at Berlin in 1900 (Hartwig, 1900).

Specimens examined. Five adult parthenogenetic females, Grabensee/Salzburg, Germany, Wiener Coll., Leg. D. Flößner, 28.IV.1984, Flößner Coll., Museum Für Naturkunde, Berlin. One adult male, Hellsee by Biesenthai/Mark Brandenburg, Germany, Hartwig Collection, Leg. A. Protz, October 1889 (Hartwig, 1900), Museum Für Naturkunde, Berlin. Two adult parthenogenetic females, hyporheic Amblève River, Ile de Halleux, Walloon Region, Belgium, Leg. PASCALIS project, 30.VII.2002. One adult parthenogenetic female, hyporheic Oignin River, Charmine, Lyon, France, Leg. PASCALIS project, 18.V.2002. One adult parthenogenetic female, hyporheic Oude River, Roussillon, few kilometres from the Mediterranean coast (elevation of 13 m a.s.l.), France, Leg. PASCALIS project, 24.VII.2003. 50 adult parthenogenetic females, between moss on stones and river gravel, La Lanterne, Haute-Saône, France, 25.X.2007, Leg. K. Van Damme & D. Van Damme. 150 adult parthenogenetic females, gravel quarry Heerenlaak, adjacent to Maas River, Maaseik, Belgium, 15.VIII.2007, Leg. K. Van Damme & D. Van Damme. Two adult parthenogenetic females (in slide) from Abingdon, Berkshire, United Kingdom, 25.V.1966, Ugent Collection. Two adult parthenogenetic females (in slide) from Channel of Egridir, Turkey, Leg. H.J. Dumont, 23.VII.1973, Ugent Collection.

Redescription parthenogenetic female

Habitus (Figs. 1, 2A,B, and 3A,B)

Small to medium-sized animals, 0.32–0.43 mm, mean around 0.35 mm (n = 50, population Maaseik, Belgium), 0.31–0.35 mm in Vranovsky (1971), 0.32–0.42 mm in Flößner (2000). Light brown-yellow in life, colourless and transparent after fixation. Body length about 1.3–1.5 times height (Figs. 1, 2A, 3B). Dorsum strongly arched, body highest in middle, more or less ovoid, not strongly tapering posteriorly (Figs. 1 and 2A,B). Ventral margin slightly convex, with deepest point in middle (Fig. 1). Posteroventral corner with moderate notch close to posterior margin (Fig. 2I–J). In dorsal view, body compressed lacking a keel. Head. Ocellus and eye well developed and of similar size (Figs. 1 and 2A, E). Head shield with blunt and relatively narrow posterior margin (Fig. 2C). Rostrum present, blunt, truncated and short (Fig. 2C), aesthetascs projecting beyond its tip (Figs. 1, 2E, and 3D). In lateral view, rostrum not reaching beyond ventral carapace margin (Fig. 2E). Three main head pores (Fig. 2E) of same size, narrowly connected, PP distance about one IP distance; small pores more than half distance between midline and lateral margin of head pores (Fig. 2E).

Fig. 1

Live adult parthenogenetic females of Phreatalona protzi (Hartwig, 1900) in sediment from hyporheic, their typical environment (from a small stream La Lanterne, Haute-Saône, France). Adult parthenogenetic female processing plant material (arrow). Aesthetascs are visible, projecting beyond rostrum, and typical shape of protzi postabdomen. Inset: same female (arrow), showing dimensions of sand grains vs. animal

Fig. 2

Phreatalona protzi (Hartwig, 1900) = Alona protzi Hartwig, 1900. Habitus and external characters of parthenogenetic females from Belgium, Walloon Region (A,B, D, F–I, K, M, P) and Maaseik (E); Germany, Hellsee (J,K); UK, Berkshire (N) and Turkey, Egredir (O). A Habitus; B Body outline; C Headshield (after Flößner, 2000: Fig. 115c); D Head pores; E Head; F Antennule; G Antenna; H Labrum; I,J Posteroventral valve corner; K,L Postabdomen; M Idem, detail, with indication of distal embayment; N,O Postabdomen; P Idem, detail lateral fascicle and marginal denticle

Fig. 3

Phreatalona protzi (Hartwig, 1900) = Alona protzi Hartwig, 1900. SEM of parthenogenetic females from Amblève River, Belgium. A Habitus, valve removed; B Habitus; C Postabdomen and inside of valve; D Antenna and labral keel; E Inner side of posteroventral valve corner with three denticles; F First and second limb

Carapace

No ornamentation or faint wide striation; no fine striation (Figs. 2A and 3B). Number of lines 12–15. Marginal setae all similar, group in anterior and posterior third slightly longer, median group little shorter (Figs. 1 and 2A). Marginal row of 46–55 setae decreasing in size towards posteroventral corner and followed by one to four short denticles, in majority of specimens three denticles (Figs. 2I–J, 3E). Left and right valve may have a different number of denticles (also in Vranovsky, 1971). Small setules along inner margin, not arranged in groups (Figs. 2I and 3E).

Labrum (Figs. 2H and 3A)

Large, labral keel widely rounded in anterior portion and elongate triangular tip with rounded apex. About two times as long as wide. No ventral setules or denticles on labral keel. Antennules (Fig. 2F). Corm about two times as long as wide, sensory seta long, implanted apically. Three rows of short setules on dorsal margin. All but two aesthetascs about half as long as antennular corm and subequal in length; two aesthetascs strongly developed, longer than the antennular corm itself and reaching far beyond rostrum (Fig. 3D). Second antennae (Figs. 2G and 3D). Coxal setae of moderate size. Exopod without spinules or spines on second segment. Setae: 113/003, spines: 001/(1)01. First endopod spine very small, about a third of second endopod segment. Apical spines well developed about as long as or little shorter than ultimate segments. First exopod seta slender, reaching beyond terminal exopod segment. Apical exopod spine about half as long as apical endopod spine. Terminal setae subequal in length and little longer than antennal segments + coxa. Antennal muscles well developed (Fig. 2E).

Postabdomen (Figs. 2K–O and 3C)

Relatively short, dorsal margin rather straight, length about two times as long as wide (Fig. 2K). Ventral margin shorter than anal and postanal margin. Anal, preanal and postanal margins of similar length. Anal margin straight to slightly concave. Postanal margin straight to slightly tapering distally, distal margin protruding. Distal gap deep and closed (Figs. 2K–L, M and 3C). Preanal corner moderately developed, triangular, not protruding beyond postanal margin (Fig. 2K–L). In few populations, preanal corner deeper (Fig. 2N). Marginal denticles of small spines, arranged in 9–11 postanal groups (Fig. 2K). Distal postanal groups consisting of one larger denticle with nearly merged, smaller adjacent spines; marginal denticle groups closer to the anal margin in groups of 10–13 similar, short denticles. Lateral fascicles five to eight groups postanal portion, consisting of over 15–18 parallel spinules all of similar size (no thicker distal spine) (Fig. 2P). Two to three clusters of marginal denticles and up to three rows of fascicles in anal portion (Fig. 2K,L).

Terminal claw (Fig. 2K–M)

As long as anal margin, rather thick and evenly curved, without strong pecten. Relatively thick basal spine, slightly curved, about two times as long as claw width at base, tip reaching almost half of claw length. Group of six to eight equally long basal spinules, about one-fourth of basal spine length and continuing in setules along its dorsal margin.

Five pairs of limbs. First limb (Figs. 3F and 4A–C)

Epipodite with long projection (Fig. 4A). First endite with two slender marginal setae of similar size, second endite with three setae of which two longer and subequal in size, third endite with four setae of which two posterior most longer (Fig. 3A); anterior setae on en1 and en2 long, the latter accompanied by a small element (Fig. 3B). Anterior seta on second endite more than half as long as that on first endite (Fig. 3B). ODL with one long seta as long as longest IDL seta (Fig. 3C); IDL with three setae, third seta half as long as smallest of two other setae, naked (Figs. 2F and 3C); armature of IDL setae row of equal long denticles implanted unilaterally in distal half (Figs. 2F and 3C). Accessory seta present, longer than half-length of ODL seta and finely plumose (Fig. 3C). Seven to eight anterior setule groups with more than five long slender setules in each group, all groups of similar length (Fig. 4A). Ejector hooks subequal (Fig. 4A).

Fig. 4

Phreatalona protzi (Hartwig, 1900) = Alona protzi Hartwig, 1900. Limb characters of parthenogenetic females from Maaseik, Belgium. A First limb; B Idem, anterior side; C Idem, IDL and ODL; D Second limb; E Idem, exopodite; F Idem, morphology scrapers 1–4; G Third limb, exopodite; H Fourth limb, exopodite; I Idem, setae 5–6; J Idem, endite; K Fifth limb. See separate section for abbreviations, below material and methods

Second limb (Figs. 3F and 4D,F)

Exopodite oval, with one short setulated seta (subapical), shorter than exopodite itself and implanted with short setules (Figs. 3F and 4D,E); endites with eight scrapers of which first two of similar length (Fig. 4D). At base of first seta, an additional naked soft seta is present (Figs. 3F and 4D); third scraper shorter by half of second scraper (Fig. 4F), fourth to sixth scraper of similar length as first and second (scraper five longer), seventh to eight decreasing gradually in size towards gnathobase, all relatively long and slender, increasing in thickness of proximal half towards gnathobase and all with fine denticulation (Fig. 4D); gnathobasic ‘brush’ strongly expanded and triangular, implanted with short setules (Fig. 4D), gnathobase with a sensillum and three elements, of which first a bent seta; filter comb with seven long setae of which the first (a) much shorter, thicker and brushlike with setules implanted around distal half (Fig. 4D).

Third limb (Fig. 4G)

Pre-epipodite round, epipodite oval with fingerlike projection; exopodite with square corm and seven setae in 2 + 5 arrangement; first exopodite seta two times longer than second; third exopodite seta shorter than sixth exopodite seta, fourth and fifth setae short and of similar length, sixth and seventh setae narrow and long, seventh shorter than sixth. Endite as for genus.

Fourth limb (Figs. 4H–J)

Pre-epipodite round, epipodite round, with long fingerlike projection, reaching beyond centre of exopodite. Exopodite (Fig. 4H) square, with six plumose setae (2 + 4) of which first two of similar length, third seta four times as long as first, fourth strongly reduced in size to a round setuled hillock, fifth and sixth well developed. Fifth exopodite seta twice as long as sixth, the latter blunt with merged subapical setules (Fig. 4I). Endite (Fig. 4J) with marginal row of four setae, first scraperlike and shorter than first flaming torch seta. The three flaming torch setae are strongly reduced, with thick base, decreasing in size towards gnathobase, and one marginal round naked sensillum implanted on the inner side of the endite; gnathobase with one long seta, bent over endite and two reduced naked elements; on inner side, no plumose setae; filter comb with five relatively short setae (Fig. 4J).

Fifth limb (Fig. 4K)

Pre-epipodite round; epipodite round, with long fingerlike projection, reaching beyond exopodite margin. Exopodite shape broadly oval, about two times as long as wide, with strongly concave expanded margin between setae three and four; four thick exopodite setae, of which the first is longest, first two oriented dorsally, as long as exopodite length; fourth exopodite seta well developed, little shorter than third. Inner lobe elongated oval with long terminal setules; two slender endite setae (1′–2′) of which first less than twice as long as second and bent towards inner lobe; gnathobase and filter comb absent.

Sixth limb. Absent.

Ephippial female: Unknown.

Male (Fig. 5A–D): Described in Flößner (2000), picture in Nykänen & Sarmaja-Korjonen (2007). Postabdomen and first limb based on specimen of Hartwig (1900) in collection of Flößner (2000). Postabdomen (Fig. 5A) 2.5 times as long as wide, gonopores dorsal and subapical, close to the base of the claws. Terminal claw rather thick, curved and with long basal spine reaching over half of claw; preanal corner not pronounced (Fig. 5A); lateral fascicles six postanal and four anal groups with 12–15 slender spinules each, parallel; marginal spines in groups of small unmerged spinules (Fig. 5B). First limb with IDL with three setae, hook with thick elbow and narrow tip with rugae, distal half parallel to proximal half (Fig. 5C–D).

Fig. 5

Phreatalona protzi (Hartwig, 1900) and Phreatalona phreatica (Dumont, 1983), male morphology, both from type localities (Hellsee, Germany, and Auvergne, France, respectively). A/E Postabdomen; B/F Lateral fascicles and marginal denticles; C/G First limb, IDL and ODL; D/H First limb, copulatory hook

Ecology

The most adaptive species of the genus, with relatively broad ecology. In low abundances in littoral of fast-flowing water between Cordylophora (Gurney, 1921), river edges; littoral in channels between Schoenoplectus (Vranovsky, 1971), lakes, rivers, hyporheic (Brancelj & Sket, 1990; Dumont & Negrea, 1996; Flößner, 2000), springs, quarries and on stones in small streams. The preferred habitats are saturated zones in gravel bars along rivers and hyporheic (upper 60 cm) of clear nutrient-poor streams. P. protzi lives in interstitial spaces of heterogeneous sediment. D.G. Frey found protzi in larger numbers in an underground waterconduct in the UK (Dumont, 1983). In lake littorals rich in macrophyte stands where this species is sporadically found (e.g. Nykänen & Sarmaja-Korjonen, 2007), they are probably connected with a presence of a spring, groundwater feed or inlet of small streams. During PASCALIS project, 124 specimens of P. protzi were collected in total (1 specimen in Belgium—hyporheic of Ourthe river; 120 specimens in Jura region near Lyon in France—hyporheic of rivers Suran and Oignin; and three specimens in Roussillon region in SW France—hyporheic of Aude river). They were all found in hyporheic zone (30–60 cm deep in gravel) with a maximum number of 30 specimens in 10 litres of pumped water. P. protzi appears to have a hypo-epigean life style intermediate between a stygophilic (Dumont, 1987; 1995; Dumont & Negrea, 1996) and a true stygobiont (Brancelj & Dumont, 2007) but relatively frequently washed-out into surface water, where it can extend into the littoral zone but never in high numbers. Surface waters may act as a ‘sink hole’ for this species, which can survive in places where no strong predation or unfavourable conditions appear, but is not their typical environment. Thus, it is rarely recorded via ‘classic’ sampling. It was abundantly found in two sites through aimed sampling which confirms ideas on ecology. Loc. 1: a small side-branch of La Lanterne, an oligotrophic, fast flowing stream in Eastern France (Haute-Saône), one metre deep and two metres wide. Specimens of P. protzi were collected in large numbers (over fifty specimens in October 2007) in shaded areas between moss (Fontinalis sp.) on stones and from washing out gravel (Karaman-Chappuis method). The species is sympatric with the rare Alona intermedia (mentioned also in Hartwig, 1900), which was common between the moss and with Unio crassus. The latter, a bivalve, may be a good indicator of the type of streams where P. protzi is likely to occur (see also ecology of P. phreatica). Observation of live P. protzi from the latter locality showed that these are slow erratic swimmers. Specimens were kept alive at Ugent for several months in small 40 ml containers with Fontinalis moss and produced offspring without extra oxygen addition. Loc. 2: large gravel quarry (Herenlaak) near the River Maas (Maaseik, border Belgium-Germany), wherefrom water percolates into the reservoir through an aquifer. P. protzi was discovered here by G. Louette in the summer of 2007 and sampled subsequently. Hundreds of specimens were washed out from bare shore, from the top 10 cm of gravel or rinsing stones with epiphytes and tree roots from the reservoir’s submerged ‘beach’, together with Monospilus. Within the reservoir itself, only one locality, closest to the river, contained specimens of P. protzi. There were no specimens in the Meuse, which shows signs of eutrophication and pollution.

Distribution

A broad range, but never before recorded in concentrations of three to four specimens per locality before this study (see also Brancelj & Dumont, 2007). Restricted to “Danubian Europe”; The Netherlands: de Molenpolder (Leentvaar, 1978), Finland, including subfossil remains, and Denmark (Silfverberg, 1999; Nykänen & Sarmaja-Korjonen, 2007), Ireland, UK (Gurney, 1921), Turkey (Güher, 2002), Lake Inkit (Behning, 1941), France (e.g. Rhone gravel bars; Dumont, 1987; Brancelj & Dumont, 2007), Germany and Poland (Flößner, 2000), Donau-delta in Rumania (Negrea, 1966), SW-Slowakia (Great Rye Island, Vranovsky, 1971), up to the Kaukasus in Georgia (Schiklejew, 1930). Not reported south of the Pyrenees (only River Ter, Sabater, 1987), Alps, Kaukasus, Taurus Mountains or east of the Ural. Record of P. protzi from India is a result of misidentification (Vranovsky, 1971).

Differential diagnosis

Phreatalona protzi is easily recognized by body shape and postabdomen (Figs. 1 and 2). It has the most ovoid body of the genus (body is high, dorsum arched and not strongly tapering distally, 1.3–1.5 times as long as high), antennal setae short, less than two times the length of the segments + coxa. The eye and ocellus are of similar size, well developed (diameter ocellus 0.8–1 times eye diameter and both as large as antennular width), in comparison to phreatica and smirnovi, which have a reduced eye and generally three denticles in the posteroventral corner. The latter is a good but variable (between two and four, rarely one or no denticles) and asymmetric character (Keilhack, 1911; Gurney, 1921; Vranovsky, 1971). In comparison with phreatica or labrosa,, postabdomen of protzi has a closed distal gap (rarely open), a convex ventral margin of postabdomen and a preanal corner that rarely exceeds the postanal postabdominal margin. There is, however, strong variation between populations of protzi. In comparison with P. smirnovi, P. protzi is larger, with shorter habitus (in smirnovi length to height is 1.6 or more), and the head has a pronounced rostrum. On limbs, P. protzi has modified IDL setae with long-spaced setules and a widened base, a P2 with third scraper half as long as second and a P5 with first seta as long as second.

Note

Brehm (1933) proposed the name Alona protzi schiklejewi Brehm, 1933, retained by Smirnov (1971), who indicated two subspecies of P. protzi. Behning (1941) reported P. p. schiklejewi from Lake Inkit, Georgia (Schiklejew, 1930; Vranovsky, 1971), stating that it differs from P. p. protzi in the presence of lateral fascicles on the postabdomen, six bundles with six to eight spinules each (Schiklejew, 1930; Smirnov, 1971). These bundles are definitely present in true P. protzi too, and this character is therefore inapplicable (Vranovsky, 1971). Published figures, however, show a short terminal claw on the postabdomen in Caucasian specimens (Schiklejew, 1930: Fig. 10). However, two specimens from Turkey, closest to P. p. shiklejewi, fall within the variation of typical P. protzi in this respect. We suggest schiklejewi as a synonym of protzi (Flößner, 2000), and not as a separate subspecies.

• 2. Phreatalona phreatica (Dumont, 1983) comb. nov.

• = Alona phreatica Dumont, 1983

• Type locality. Mountain streamlet Couze Pavin near Besse-en-Chandesse, Auvergne, France (Dumont, 1983).

• Etymology. Name refers to the obligate life mode in groundwater.

Specimens examined. Eight adult parthenogenetic females, Ninglispo, Amblève, Belgium; sixteen adult parthenogenetic females and one male from hyporheic of Oignin river, Charmine, Jura region; Lyon, France, 24.7.2003; two adult parthenogenetic females, hyporheic Tech river; Correc Maureillas, Roussillon region, SW France, 17.5.2003. Three adult parthenogenetic females, hyporheic, opposite to soccer terrain, Aisne river, Bomal, Belgium, 14.8.2003. Two adult parthenogenetic females, hyporheic, Aisne, Roche-a-Frène, Bomal, Belgium; Four adult parthenogenetic females (type series, in slides) from stream Couze Pavin, Auvergne, France (Dumont, 1983), Leg. H.J. Dumont, 21–26.VI.1982, Ugent collection.

Habitus (Figs. 6A,B and 7A,B)

Medium-sized animals, 0.37–0.46 mm (n = 18, populations from Charmine, France) with mean length 0.4 mm, colourless and transparent. Body length 1.47–1.5 times height (Fig. 6A,B). Dorsum arched, body highest in middle, tapering posteriorly, with low posterodorsal angle (Fig. 6B). Ventral margin straight (Fig. 6A). Posteroventral corner with moderate notch close to posterior margin (Fig. 6H). In dorsal view, body compressed lacking a keel. Head. Ocellus and eye reduced, small and of similar size (Fig. 6A). Head shield with wavy and relatively narrow posterior margin (Fig. 6D). Rostrum wide and round without marked tip (Fig. 6C), aesthetascs projecting beyond frontal margin (Fig. 6A). Three main head pores of same size, narrowly connected, PP distance about one IP distance; small pores at about one IP distance from margin (Fig. 6D).

Fig. 6

Phreatalona phreatica (Dumont, 1983) = Alona phreatica Dumont, 1983. External morphology of adult parthenogenetic females from Ninglispo, France. A Habitus; B Body shape; C Head shield (after Alonso, 1996); D Head pores; E–G Labrum; H Posteroventral valve corner; I Antennule; J Second antenna; K,L Postabdomen; M Lateral fascicle and marginal denticle

Fig. 7

Phreatalona phreatica (Dumont, 1983) = Alona phreatica Dumont, 1983. SEM of adult parthenogenetic females from Amblève, Ninglispo, Belgium, and Oignin river, Jura Region, France. A,B Habitus; C Postabdomen; D Head with antennae and labral keel; E Posteroventral valve corner, inner side; F First and second limb

Carapace

Ornamentation is weak, with wide striation most pronounced in posteroventral quarter, no fine striation (Figs. 6A and 7B). Number of lines 13–15. Marginal setae with longer anterior group, median group little shorter (Fig. 6A). In total, marginal setae 50–56, decreasing in size towards posteroventral corner. On inner side of valve in posteroventral corner, setae continue in row of small setules which may be merged into denticles (Fig. 7E). These denticles do not reach beyond the valve (Fig. 7E).

Labrum (Fig. 6E–G)

Large, labral keel slightly convex in anterior portion and elongate triangular tip with rounded apex. Small notch before apex, labral keel about two times as long as wide. No ventral setules or denticles on labral keel. Antennules (Fig. 6I). Corm about two times as long as wide, sensory seta implanted apically and about half the size of longest aesthetasc. Two to three rows of short setules on dorsal margin. All but two aesthetascs about half as long as antennular corm and subequal in length; two aesthetascs strongly developed, longer than the antennular corm itself and reaching far beyond rostrum. Second antennae (Figs. 6J and 7D). Coxal setae of moderate size. Exopod without spinules or spines on second segment. Setae: 113/003, spines: 001/(1)01. First endopod spine very small, less than a third of second endopod segment. Apical spines well developed about as long as or little shorter than ultimate segments. First exopod seta slender, reaching beyond terminal exopod segment. Apical exopod spine longer than half of apical endopod spine. Terminal setae subequal in length and about twice as long as antennal segments + coxa.

Postabdomen (Figs. 6K,L and 7C)

Relatively short, dorsal margin rather straight, length about 2.5 times as long as wide. Ventral margin shorter than anal and postanal margin. Anal, preanal and postanal margins of similar length, anal margin may be little shorter. Anal margin straight to slightly concave. Postanal margin straight, distal margin protruding. Distal gap not closed. Preanal corner well developed, triangular, protruding beyond postanal margin. Marginal denticles of small spines, arranged in 9–11 postanal groups. Distal postanal groups consisting of one larger denticle with parallel adjacent spines; marginal denticle groups closer to the anal margin in groups of 6–10 similar, fine spinules. Lateral fascicles five to seven groups in postanal portion, consisting of over 11–15 spinules all of similar size (no thicker distal spine) but not parallel (Figs. 6M and 7C). Two to three clusters of marginal denticles and up to three rows of fascicles in anal portion.

Terminal claw (Fig. 6K,L)

As long as anal margin, rather thick and straight, without strong pecten. Relatively slender and straight basal spine, about three times as long as claw width at base, tip not reaching half of claw length. Group of four to six long basal spinules, about one-third of basal spine length.

Five pairs of limbs. First limb (Figs. 7F and 8A–C)

Epipodite with long projection (Fig. 8A). First endite with two slender marginal setae of similar size, second endite with three setae of which two longer and subequal in size, third endite with four setae of similar size (Fig. 8A); anterior setae on en1 and en2 long, the latter accompanied by a small element (Fig. 8B). Anterior seta on second endite half as long as that on first endite (Fig. 8B). ODL with one long seta as long as longest IDL seta (Figs. 7F and 8C); IDL with three setae, third seta less than half as long as smallest of two other setae, naked (Figs. 7F and 8C); armature of IDL setae row of equal short denticles in implanted unilaterally in distal half (Fig. 8C). Accessory seta present, more than half length of ODL seta and finely plumose (Fig. 8C). Six to seven anterior setule groups with more than five long slender setules in each group, all groups of similar length (Fig. 8A). Ejector hooks subequal (Fig. 8A).

Fig. 8

Phreatalona phreatica (Dumont, 1983) = Alona phreatica Dumont, 1983. Limb morphology of adult parthenogenetic females from Amblève, Ninglispo, Belgium. A First limb; B Idem, anterior portion; C Idem, ODL and IDL; D Second limb; E Idem, scrapers 1–4; F Idem, exopodite, first scraper and adjacent soft seta (ss); G Third limb, exopodite; H Third limb, endite; I Idem, gnathobase; J Idem, outer endite setae; K Fourth limb, exopodite; L Fourth limb, endite; M Fifth limb

Second limb (Figs. 7F and 8D,F)

Exopodite oval, with one short setulated seta (subapical), about as long as exopodite itself and implanted with short setules (Fig. 8D,F); endites with eight scrapers of which first two of similar length (Fig. 8E). At base of first seta, an additional naked seta is present (Figs. 7F and 8E); third scraper shorter by one-fourth of second scraper, fourth to sixth scraper of similar length as first and second (scraper five longer), sixth to eight decreasing gradually in size towards gnathobase, all still relatively long and slender and of similar morphology, with fine denticulation; gnathobasic ‘brush’ strongly expanded, implanted with short setules, gnathobase with a sensillum and three elements, of which first a bent seta; filter comb with seven long setae of which the first (a) much shorter, thicker and brushlike with setules implanted around distal half (Fig. 8D).

Third limb (Fig. 8G–J)

Pre-epipodite round, epipodite oval with fingerlike projection; exopodite (Fig. 8G) with square corm and seven setae in 2 + 5 arrangement; first exopodite seta little longer than second; third exopodite seta longer than sixth exopodite seta, fourth and fifth setae short and of similar length, sixth and seventh setae narrow and long, seventh shorter than sixth (Fig. 8G). External endite (Fig. 8H) with three setae (1′–3′) of which first two slender, of similar size and with minute element in between, third (3′) short, plump and with long setules; four well-developed and thick-based plumose setae on inner side (1″–4″) of similar length; one short element and four small naked setae on internal endite preceding gnathobase (Fig. 8J); the latter with a bottle-shaped sensillum and large plumose seta with two naked setae (little shorter) at its base (Fig. 8I). Filter comb with seven long setae (Fig. 8H).

Fourth limb (Fig. 8K,L)

Pre-epipodite round, epipodite round, with long fingerlike projection, reaching beyond centre of exopodite. Exopodite (Fig. 8K) square, with six plumose setae (2 + 4) of which first two of similar size (first longer), third seta three times as long as first, fourth strongly reduced in size to a round setulated projection, fifth and sixth well developed. Fifth exopodite seta twice as long as sixth, the latter blunt with merged subapical setules. Endite (Fig. 8L) with marginal row of four setae, first scraperlike and as long as first flaming torch seta, following three ft setae strongly reduced, with thick base, decreasing in size towards gnathobase, and one marginal round naked sensillum implanted on the inner side of the endite; gnathobase with one long setae, bent over endite and two reduced naked elements; on inner side, no plumose setae; filter comb with five relatively short setae.

Fifth limb (Fig. 8M)

Pre-epipodite round; epipodite round, with long fingerlike projection, reaching beyond exopodite margin. Exopodite shape broadly oval, about two times as long as wide, with strongly concave expanded margin between setae three and four; four thick exopodite setae, of which the second is longest, first three oriented dorsally, as long as exopodite length; fourth exopodite seta well developed, little shorter than third. Inner lobe elongated oval with long terminal setules; two slender endite setae (1′–2′) of which first twice as long as second and bent towards inner lobe; gnathobase and filter comb absent.

Sixth limb. Absent

Male. Rare (Brancelj & Dumont, 2007). Described and depicted in Sabater (1987) and Alonso (1996). Postabdomen (Fig. 5E) 2.5–3 times as long as wide, gonopores dorsal and subapical, at some distance from the base of the claws. Terminal claw rather thin, straight and with long basal spine reaching over half of claw; preanal corner pronounced (Fig. 5E); lateral fascicles six postanal and three-four anal groups with 7–12 slender spinules each, widening; marginal spines in groups of small unmerged spinules (Fig. 5F). First limb with IDL with three setae, hook with thick elbow and narrow tip with rugae, distal half not parallel to proximal half (Fig. 5G,H).

Ephippial female. In Alonso (1996). Ephippium with faint yellowish tint.

Ecology

An obligate hyporheic (Dumont, 1983), found in river sediment, incapable of swimming (Dumont, 1983). In sediment of oligotrophic, clean shallow streams on sandy/gravel substrate. In Ter River (Spanish Pyrenees), Phreatalona phreatica reached high densities between March and September, with highest peak (ca. 800 individuals/50 l water) and a gamogenetic population in June (Sabater, 1987). During the PASCALIS project, nearly 500 specimens of P. phreatica were counted from the hyporheic zone at 30–60 cm below river bed. In Belgium, we counted 120 specimens from the hyporheic zone of rivers Ambleve and Ourthe, with a maximum density of 57 specimens per 10 l of pumped water. In SW France, in Rousillion region, seven specimens were collected from the hyporheic zone of river Tech. Streams in Roussillon (France) where phreatica was found in the hyporheic also contain the bivalve Unio crassus. The most numerous (incl. males and ephippial females—with pale ephippia) were in Jura region in Eastern France. More than 350 specimens were counted from hyporheic of rivers Suran, Albarine, Oignin and Valouse with a maximum density of 90 specimens per 10 l of pumped water. Only three specimens were found in the true phreatic zone (i.e. 90–120 cm below river bed). So, despite its name, it is atypical for the true phreatic and actually prefers hyporheic. In Jura region P. protzi and P. phreatica co-occurred on three locations but P. protzi was in lower densities compared to P. phreatica. It is obvious that both species can co-exist and both are tightly connected with hyporheic zone, where intensive exchange between surface and groundwater exists.

Distribution. Western Europe. Northern Spain (Pyrenees, Ter River), eastern France (Auvergne, Roussillon, Charmine), south of Belgium (streams Ourthe, Aisne, Amblève). Main literature: Dumont (1983), Alonso (1996), Sabater (1987), Brancelj & Dumont (2007).

Differential diagnosis. Phreatalona phreatica is close to protzi. Both may be found sympatric, but the body in phreatica is relatively more elongate with length 1.5 times the height and with a relatively lower dorsum (compare habitus of Fig. 1 with Fig. 6). Eye and ocellus are reduced in phreatica, diameter of the eye is maximally half the size of the antennular body. The antennal setae are relatively longer than in protzi, about 2.5 times as long as the antenna segments and coxa. In protzi, these setae are less than two times the length of antennal segments and coxa. P. phreatica lacks a protruding rostrum. This is hard to see from just the habitus, but in general the rostrum reaches less ventral than in protzi. P. phreatica rarely has denticles in the posteroventral corner of the valves and has relatively long anterior marginal setae on the valve in comparison to the other species. On the postabdomen, we listed several differences with protzi in Fig. 9; best characters are the open distal gap in phreatica (Fig. 9; number 4), a straight ventral margin, deep preanal corner reaching more ventral than the postanal margin and spread fine lateral fascicles (compare Fig. 9C with 9G). On limbs, P1 has relatively long and fine ventral setule groups implanted on the limb, and the two larger IDL setae are not modified; they have short setules and a narrow base in comparison to protzi. Third scraper in second limb is two-thirds as long as second scraper and exII seta is shorter than the exopodite itself.

Fig. 9

Comparison of Phreatalona protzi (A–D) postabdomen with P. phreatica (E–H). P. protzi from Amblève, Belgium (A–C), and River Meuse, Maaseik, Belgium (D); P. phreatica from Ninglispo, France (E–G), and type locality (Besse, France) (H). 1. curvature ventral margin (curved in protzi, straight in phreatica); 2. depth of preanal corner (deeper in phreatica); 3. dorsodistal angle (more protruding in phreatica); 4. distal “gap” (closed in protzi, open in phreatica); 5. length and number basal spinules (more and shorter spinules in protzi); 6. curvature basal spine (more curved in protzi); 7. lateral fascicles (thicker and parallel in protzi, finer and divergent in phreatica) and marginal denticles (longer and fewer per group in phreatica)

• 3. Phreatalona labrosa (Vasiljeva & Smirnov, 1969) comb. nov.

• =Alona labrosa Vasiljeva & Smirnov, 1969.

• Synonymy and full description in Sinev and Kotov (2000).

Distribution and ecology. Lake Baikal, between rivers Utulic and Murina; Irkutsk Reservoir (Sinev & Kotov, 2000). Data on ecology is provided in Vasiljeva & Smirnov (1969) and Smirnov (1971). P. labrosa is present on sand and stones, most abundant in the open water of Lake Baikal at different depths in the littoral, between 1 and 10 m, sympatric with endemic Chydoridae Alona setosocaudata, Kozhowia kozhowi and Parakozhowia baicalensis. Highest densities in August; Vasiljeva & Smirnov (1969) collected over 400 specimens/m² between 6 and 10 m depth here in August 1966. There is no vegetation on the shore, the mainly stony bottom is washed by waves, and there is a vertical displacement of the littoral chydorid fauna to the open water in Baikal (Smirnov, 1971).

• 4. Phreatalona smirnovi (Petkovski and Flößner, 1972) comb. nov.

• = Alona smirnovi Petkovski & Flößner (1972)

Type locality. In Petkovski & Flößner (1972): “Geröll des flachen litorals beim Kloster Sv. Naum am Südufer des Sees (Ohrid)” and “Loser Sand an demselben Strand, nahe der Einmündung der dort entspringenden wasserreichen Karstquellen”. On submerged stones and between sand of inlet of karstic spring near Naum Monastry, southern margin of Lake Ohrid.

Etymology. Named after Dr N.N. Smirnov, diligent researcher of Cladocera systematics, Moscow (Petkovski & Flößner, 1972).

Specimens examined. Four adult parthenogenetic females, Lake Ohrid, southern margin near Monastry, Naum, Macedonia, Leg. Petkovski, IX, 1969. Coll. D. Flößner, 1970. Museum für Naturkunde, Berlin, Germany. Thirteen adult parthenogenetic females on ethanol, Lake Ohrid, Naum feeder springs (= type locality), on rocks, Macedonia, Leg. Petkovski, year unknown, Det. Petkovski, specimens provided by G. Kostoski (Director Hydrobiological Institute Ohrid), 20.VII.2007.

Habitus (Fig. 10A,B)

Small animals, 0.28–0.43 mm, colourless and transparent. In Petkovski & Flößner (1972), length 0.33–0.43 mm. Body length 1.6–1.7 times height. Dorsum arched, body highest just before middle, tapering posteriorly, with low posterodorsal angle (Fig. 10B). Ventral margin straight to moderately convex in anterior third (Fig. 10A). Posteroventral corner without notch close to posterior margin (Fig. 10G,H). In dorsal view, body compressed lacking a keel. Head. Ocellus and eye reduced, ocellus much smaller than eye (Fig. 10E). In type specimens from underground, eye is absent (Fig. 10A), but in surface material from type locality, eye is present but contains only few ommatidia (Fig. 10E). Head shield with smooth narrow posterior margin (Fig. 10C). Rostrum wide and round (not protruding) (Fig. 10C), aesthetascs projecting beyond its tip (Fig. 10E). In lateral view, rostrum reaching beyond ventral margin of carapace (Fig. 10E). Three main head pores of same size, narrowly connected and with chitinous thickening (Fig. 10D), PP distance smaller than one IP distance; small pores at about three IP distance from midline and one IP distance from margin.

Fig. 10

Phreatalona smirnovi = Alona smirnovi. Adult parthenogenetic females, underground (from type series, underground of Naum feeder springs, Ohrid; A–D, I,J, H, K–M) and surface population, on rocks of Naum feeder springs, Ohrid (E–G). A Habitus; B Body outline; C Head shield after Petkovski & Flößner (1972); D Head pores; E Head; F Second antenna; G,H Posteroventral valve corner. I,J. Labrum; K,L Postabdomen; M Terminal claw and distal part of postabdomen

Carapace

No striation (Fig. 10A). Marginal setae of similar size. In total, marginal setae 50–56, decreasing in size towards posteroventral corner (Fig. 10A). On inner side of valve in posteroventral corner (Fig. 10H), setae continue in row of small setules, which may be merged into denticles in specimens from surface (Fig. 10G). These one to two denticles may reach beyond the valve (Fig. 10G).

Labrum (Fig. 10I,J)

Large, labral keel straight to slightly convex in anterior portion and elongate triangular tip with rounded apex. No notch before apex, labral keel 1.5–2 times as long as wide. No ventral setules or denticles on labral keel. Antennules as for genus. Second antennae (Fig. 10F). Exopod without spinules or spines on second segment. Setae: 113/003, spines: 001/(1)01. First endopod spine very small, about a fourth of second endopod segment. Apical spines well developed about as long as or little shorter than ultimate segments. First exopod seta slender, reaching beyond terminal exopod segment. Apical exopod spine half of apical endopod spine. Terminal setae subequal in length and longer than antennal segments + coxa. Antennal muscles poorly developed (Fig. 10E). Antennal setae with long setules (Fig. 10F).

Postabdomen (Fig. 10K–M)

Relatively short, dorsal margin rather straight, length 2–2.5 times as long as wide. Anal margin little shorter than postanal margin. Anal margin straight to slightly concave. Postanal margin straight, distal margin strongly protruding with rounded dorsodistal angle. Distal gap deep and closed. Preanal corner short triangular, somewhat protruding beyond postanal margin. Marginal denticles of small spines, arranged in 10–11 postanal groups. Distal postanal groups consisting of one larger denticle with parallel adjacent spines, merged; marginal denticle groups closer to the anal margin in groups of 4–5 similar, fine spinules. Lateral fascicles five to seven groups in postanal portion, consisting of over 9–11 parallel spinules with slightly thicker distal spine. Two to three clusters of marginal denticles and up to three rows of fascicles in anal portion.

Terminal claw (Fig. 10M)

As long as anal margin, rather straight, without strong pecten. Relatively slender and straight basal spine, two to three times as long as claw width at base, tip just before or reaching half of claw length. Group of four to six long basal spinules, about one-third of basal spine length.

Five pairs of limbs. First limb (Fig. 11A,C)

Epipodite with long projection (Fig. 11A). First endite with two slender marginal setae of similar size, second endite with three setae of which two longer and subequal in size, third endite with four setae of similar size (Fig. 11A); anterior setae on en1 and en2 (Fig. 11B) long, the latter accompanied by a small element. ODL with one long seta little longer than longest IDL seta (Fig. 11C); IDL with three setae, third seta half as long as smallest of two other setae, naked; armature of IDL setae row of equal short denticles; accessory seta present, relatively short (less than half of ODL seta) and finely plumose (Fig. 11C). Six to seven anterior setule groups with more than five setules in each group, all groups of similar length (Fig. 11A). Ejector hooks subequal and gnathobase with single short setulated seta on globular process (Fig. 11A).

Fig. 11

Phreatalona smirnovi = Alona smirnovi. Adult parthenogenetic females from Lake Ohrid, Naum feeder springs, on rocks, Macedonia, Leg. Petkovski. Limb morphology. A First limb; B Idem, anterior portion; C Idem, ODL and IDL; D Second limb; E Idem, scrapers 1–4; F Idem, exopodite, first scraper and adjacent soft seta (ss); G Third limb, exopodite; H Idem, exopodite setae 6–7; I Idem, endite; J Idem, inner endite row; K Idem, gnathobase; L Idem, outer endite row; M Fourth limb, exopodite; N Idem, setae 5–6; O Idem, endite; P Fifth limb

Second limb (Fig. 11D–F)

Exopodite oval, with one long setulated seta (subapical), about twice as long as exopodite itself and implanted with short setules (Fig. 11D, F); endites with eight scrapers of which first fine and two of similar length. At base of first seta, an additional naked seta is present (Fig. 11F, ss); third scraper shorter by one-third of second scraper (Fig. 11D–E), fourth to sixth scraper of similar length as first and second (scraper five longer), and last two scrapers shorter, all still relatively long and slender and of similar morphology, with fine denticulation (Fig. 11D); gnathobasic ‘brush’ triangular elongate, implanted with short setules, gnathobase with a sensillum and three elements, of which first a bent seta; filter comb with seven long setae of which the first (a) much shorter and brushlike with setules implanted around distal half (Fig. 11D).

Third limb (Fig. 11G–L)

Pre-epipodite round, epipodite oval with fingerlike projection; exopodite (Fig. 11G) with square corm and seven setae in 2 + 5 arrangement; first exopodite seta two times as long as second; third exopodite seta about as long as sixth exopodite seta, fourth and fifth setae short and of similar length, sixth and seventh setae narrow and long, seventh shorter than sixth (Fig. 11H). External endite (Fig. 11I) with three setae (1′–3′) of which first two slender, of similar size and with minute element in between, third (3′) short, plump and with long setules; four well-developed and stout plumose setae on inner side (1″–4″) of similar length (Fig. 11J); one element and four small naked setae on internal endite preceding gnathobase (Fig. 11I,L); the latter (Fig. 11K) with a bottle-shaped sensillum and large plumose seta with two naked setae (little shorter) at its base. Filter comb with seven long setae (Fig. 11I).

Fourth limb (Fig. 11M–O)

Pre-epipodite round, epipodite oval-round, with long fingerlike projection, reaching beyond centre of exopodite. Exopodite (Fig. 11M) square, with six plumose setae (2 + 4) of which first two of similar size, third longest, fourth strongly reduced in size to a setulated hillock, fifth and sixth well developed. Fifth exopodite seta twice as long as sixth, the latter blunt with merged subapical setules (Fig. 11N). Endite (Fig. 11O) with marginal row of four setae, first scraperlike and as long as first flaming torch seta, following three ft setae strongly reduced, with thick base, decreasing in size towards gnathobase, and one marginal round naked sensillum implanted on the inner side of the endite; gnathobase with one long setae, bent over endite and two reduced naked elements; on inner side, no plumose setae; filter comb with five relatively short setae (Fig. 11O).

Fifth limb (Fig. 11P)

Pre-epipodite round; epipodite round, with long fingerlike projection, reaching beyond exopodite margin. Exopodite shape broadly oval, about two times as long as wide, with strongly concave expanded margin between setae three and four; four exopodite setae, of which the second is longest, first three oriented dorsally and longer than length of exopodite centre; fourth exopodite seta well developed, little shorter than third. Inner lobe elongated oval with long terminal setules; two slender endite setae (1′–2′) of which first longest and bent towards inner lobe; gnathobase with a naked reduced bump and setulated hillock, filter comb absent.

Sixth limb. Absent

Distribution and ecology. Endemic to Lake Ohrid (Albania/Macedonia). Records of A. protzi from this lake may be P. smirnovi. Found in only one locality, Naum Monastry, Macedonia, in hyporheic (mesopsammom) of karstic feeder springs (Petkovski & Flößner, 1972) and on rocks at inlet of this stream, southern margin of Ohrid. The groundwater is most likely the true habitat of this species (Dumont, 1983). Its locality, Lake Ohrid is the oldest lake in Europe, formed 4–10 million years ago, supplied from surface and underwater springs (Spirkovski et al., 2001).

Differential diagnosis

Phreatalona smirnovi is a relatively small species (0.28–0.43 mm), with a mean around 0.33 mm, longest body (1.6–1.7 times as long as wide), lacking a rostrum and with eye absent or with few ommatidia, never black pigmented as in protzi or labrosa. P. smirnovi looks most like a small form of phreatica and lacks a rostrum, but its postabdomen has an arched ventral margin, the most protruding distal portion of all species and closed distal gap. This species has a chitinous thickening around the main head pores (Petkovski & Flößner, 1972). On second antenna, first endopod spine is not longer than one-fourth of the second endopod segment, in phreatica it reaches up to a third of this segment. The limbs (Table 1) have a short accessory seta on P1 in smirnovi (less than half ODL seta), exopodite seta on P2 is as long as the exopodite, third scraper is two-thirds of second scraper and on P3 the third exopodite seta is longer than the fifth.

Table 1 Differences between adult parthenogenetic females of the four Phreatalona species, and ecology and distribution

Results of the cladistic analysis

Heuristic search yielded a single most parsimonious tree with score of best trees 51. The one retained tree was identical in topology to the tree obtained by separate bootstrap analysis (50% majrule shown here). The single tree retained from heuristic search (Fig. 12) had CI = 0.77, HI = 0.22 (excluding uninformative characters), RI = 0.84 and RC = 0.66; two of 31 characters were found parsimony-uninformative (10, 14). The bootstrap analysis produced a 50% majority rule consensus tree of 2872 trees (using tree weights) as shown in Fig. 12. The bootstrap 50% majority rule tree shows separation between true Alona, A. quadrangularis and a “rheic” branch containing Nicsmirnovius and Phreatalona. Acroperus is positioned basal to the latter clade. As in Kotov (2004), Neotropical N. fitzpatricki and Afrotropical N. camerounensis come out as closest relatives within Nicsmirnovius. For Phreatalona, P. labrosa is a basal taxon, followed by protzi, phreatica and smirnovi. The latter two cluster together as closest relatives, with protzi as their basal taxon. To check if the tree is not strongly biased by morphological specializations for the eight rheic taxa, we excluded characters that are related to their mode of life (1–3, 9–10, 16, 20–25) and reran the bootstrap analysis. This resulted in a 50% majority rule consensus tree with same topology as in the previous analysis, but with lower support for the rheic branch (54.3 instead of 65.7) and a polytomy for the four Phreatalona species. The latter is not shown here.

Fig. 12

Dendrogram illustrating morphological similarities of Phreatalona with Nicsmirnovius and a separation of the rheic branch from “true” Alona (quadrangularis). Within Phreatalona, P. labrosa is least specialized. The species may be basal to the protzi-group as suggested here. Single parsimonious tree, bootstrap 50% majority rule consensus of 2872 trees based on 31 morphological characters. For data and characters, see Supplementary material

Discussion

A case study of ongoing radiation: limb morphology vs. habitus and postabdomen

While labrosa is clearly distinct, morphological differences between protzi, phreatica and smirnovi are small (Table 1). Variability between protzi populations is considerable (e.g. postabdomen morphology), and both phreatica and smirnovi could be considered morphological extremes of the former. Adult parthenogenetic females of phreatica and protzi differ in details, but we found specimens with dubious morphology where both occur sympatrically, in Belgium and France, that could not be assigned easily to either. Also, adult males of both are almost identical. Limbs of female protzi, phreatica or smirnovi differ in small details, mainly on P1 and P2 (Table 1). In ‘external features’, however, these taxa clearly differ (e.g. body shape, rostrum, postabdomen; Table 1). We noted a variation for the shape of the postabdomen (protzi) and the number of denticles on the posteroventral corner. A more ‘common’ morphology can be delineated, e.g. phreatica has no denticles and protzi two to four (see later). The same can be said for eye pigmentation, which may vary between taxa (protzi/phreatica) and populations (smirnovi).

Close relationships of smirnovi–protzi–phreatica raise the problem of assigning a taxonomic rank of these ‘protzi-forms’: are they subspecies or full species? We opted for a species rank for three reasons: 1. differences in external characters indicate separation and speciation, although limb morphology and habitat preferences are close; 2. main range of the three differs (see Distribution); and 3. forms with ‘dubious’ morphology occur where distributions overlap (narrow belt from S Belgium into N, E and SW France—for protzi and phreatica), where a cline may exist. The latter needs more detailed study, and we aimed here to provide a detailed description of the “typical” forms for each species.

Limb investigation helps us to discriminate between conservative and more instable characters. In the protzi-complex, structures on P1–P2 show more variation, and therefore seem to evolve faster, than P3–P4 or the exopodite shape of P5 (Table 1). Setulation of the IDL setae, anterior setule groups on the first limb, relative length of the third scraper and exopodite seta on the second limb show more variation between these taxa than structures on the third and fourth limbs, and provide rough estimates of relatedness. Externally, the three forms differ (Table 1) in body shape and size, rostrum, length of swimming setae on second antennae and shape of the postabdomen (and lateral fascicles). The Phreatalona protzi-complex may be undergoing speciation, which is most obvious in external characters. In all limbs, distant position of labrosa is clear. In the protzi-complex, for example, P4 is conserved and separate from the allopatric labrosa, and this Baikal endemic is presumably older (see below).

The protzi-complex shows different speeds of evolution in limbs vs. ‘external’ characters. We think that evolutionary stasis in limbs is not necessarily combined with stasis in habitus and postabdomen. Evolutionary fine-tuning of external morphology and limbs may not run in parallel in these micro-crustaceans. Both may evolve independently. The disparity results from different selection pressures: habitus, antennae and postabdomen have main function in movement (crawling, swimming), whereas limbs serve mainly for food handling (selection, filter feeding, scraping). Selective pressure may not be the same for all limbs or limb characters. The exploration of new niches may be facilitated by a novelty either in limb morphology enabling another food source or in external morphology resulting in changes in mobility. Specializations as a result from different evolutionary pressures on movement vs. food handling are a key to understanding radiation and evolution in the Aloninae. This may explain why limb characters in Aloninae are sometimes so similar between taxa while habitus or postabdomen may differ strongly, or vice versa. For the Phreatalona protzi-complex, selection on food handling may be relatively stronger than on movement, resulting in only minor differences on limbs. Indeed, similar ecological conditions in the hyporheic zone keep limb morphology similar but allow changes in external morphology. The same habitats are frequented, and both protzi and phreatica can be found together.

Position of Phreatalona gen. nov. within the Aloninae

Species related to P. protzi have an unusual morphology for the subfamily. Sinev & Kotov (2000) list nine points of similarity between phreatica and labrosa, which can be used as features for Phreatalona. Superficially they may seem to have a small Alona habitus, but limbs differ from those of most Aloninae. Life in a rheic environment elicited adaptations of the limb structures in a completely different direction as the general littoral–benthic Chydoridae. Phreatalona species live mainly in the deeper part of the hyporheic, a transitional zone between surface water and phreatic zone in rivers, among coarse heterogeneous sediment. The only species not recorded from ‘subterranean’ habitats is P. labrosa, present in the littoral of Lake Baikal (Vasiljeva & Smirnov, 1969; Sinev & Kotov, 2000). Preliminary results of sampling gravel pits in a littoral zone on the NW part of lake Baikal (in the vicinity of limnological station Bolshoy Koty; using Karaman-Chappuis method) indicate that there are some weak subsurface inlets of seeping water, most of them quite restricted, inhabited by exclusively stygobiotic Harpacticoida and Bathynellaceae (AB, pers. observ.).

Both protzi and smirnovi also occur in surface waters, but related to a spring, gravel bed or inflow. Phreatalona differs from other Alona in general morphology and a separate evolution is clear. Main synapomorphic characters of Phreatalona are: 1. first antenna with elongated aesthetascs, second antenna with relatively short swimming setae and spine on first endopod segment reduced in size; 2. elongated labral keel; 3. postabdomen with a deep incision in the distal margin and protruding dorsodistal angle, relatively long basal spine, small marginal denticles arranged in clusters and lateral fascicles of similar size; 4. first limb with two well-developed anterior setae, IDL with three setae; 5. second limb with an extra seta at the base of first scraper (also in A. hercegovinae), all scrapers relatively slender and finely denticulated; elongated gnathobasic region; 6. third limb with seven setae of which the third is not markedly long; 7. fourth limb exopodite with reduced fourth seta and modified sixth seta with blunt apex (not labrosa); endite lacking a row of three inner plumose setae which are present in all Aloninae, round receptor shifted to the inner face of the endite, and a filter comb strongly reduced in size. 8. absence of a filter comb on P5 and of P6. Sinev & Kotov (2000) list characters of the antennule and postabdominal claws of males, which can be seen as additional characters for Phreatalona. Also, the latter authors mention an unpigmented ephippium for P. labrosa, unique within Aloninae. As phreatica also has a pale ephippium (Alonso, 1996), this unusual condition may be typical for the genus. In general, ephippia as well as males are rare in these species (Brancelj & Dumont, 2007).

Similar to many Aloninae, Phreatalona contains a mix of primitive and derived features on the thoracic appendages, while maintaining a general habitus. It differs in limb characters from a group of medium-sized Aloninae with small marginal denticles on the postabdomen, which lack a sixth limb and gnV (e.g. Leberis). For example, Phreatalona contains long anterior setae on the first limb and an extra soft seta basally from the first scraper on the second limb, characters considered as primitive in Aloninae. Long anterior setae on the first limb are absent in all other Alona species with five limb pairs except for Nicsmirnovius and the cave inhabiting A. hercegovinae-group (limbs in Brancelj, 1990; Van Damme et al., 2003; Kotov & Sanuamuang, 2004). The character is however typical for a group of Aloninae with a gnathobase on P5 and a P6 (e.g. A. affinis-group, A. costata-group, Acroperus, Graptoleberis). Retaining these anterior setae, Phreatalona may be derived from the latter group but lost the sixth limb. On the other hand, specific morphological adaptations, discussed below, can be attributed to life in a (hypo)rheic environment. In general, Phreatalona shows remarkable reductions in limb size (exIII-V) and structure. Endite of P4 lacks three inner setae, an autapomorphy for the genus.

Adaptations to (hypo)rheic and affinities with Nicsmirnovius

Phreatalona shares most characters with another (hypo)rheic genus, Nicsmirnovius Chiambeng & Dumont, 1999. Morphology of Nicsmirnovius was described in detail (Van Damme et al., 2003; Kotov & Sanuamuang, 2004). Both genera have a similar mode of life and similarities seem too striking to attribute to convergence. We have listed several characters with comments in Table 2, and discuss the most important here.

Table 2 Comparison of morphology Phreatalona with Nicsmirnovius, with comments on functionality and occurrence of these characters in subfamily Aloninae

Nicsmirnovius and Phreatalona have two long anterior setae on first limb, elongated pre-gnathobasic process on P2 (also in Monospilus), a short third seta on exIII (e.g. in Monospilus, Acroperus) and modifications of exIV setae. The function of these modified setae is unknown, but specializations are very rare in the subfamily and likely related to rheic life mode (Van Damme et al., 2003). The majority of Aloninae have two narrowed setae with pointed apex here instead of blunt setae with subapical group of clusters. Only Alonopsis has similar adaptations on setae of the fourth limb (Van Damme, unpubl.).

Total exopodite surface (exopodites, setae + setules) in relation to the body of Nicsmirnovius and Phreatalona is small compared to majority of Aloninae, especially to benthic Alona (quadrangularis-group). This is understandable from an evolutionary context: benthic Aloninae live in an oxygen-low environment and need a large ‘exopodite pump’, while the rheic species live in relatively oxygen-rich environments. Measurements from the hyporheic habitats sampled in Belgium and France (PASCALIS) where protzi and phreatica were found indicated that the oxygen concentration rarely drops below 50%; in the Pyrenees, phreatica also occurs in very clear, oxygenated interstitial water (Sabater, 1987). In this aspect, the exopodites correspond to a type of leg apparatus described by Smirnov (1971) for Rhynchotalona and Monospilus. Both these genera live on sand in open littoral, for example lake shores, where vegetation may be absent and oxygen relatively high. Both Monospilus and Rhynchotalona have passive filtering where exopodites do not make rhythmic vibrations as in the majority of Chydoridae. Limbs of Phreatalona, which lives in similar conditions, suggest passive filtration as well.

On enIV, Nicsmirnovius has a receptor shifted to the inner side like Phreatalona, whereas in most members of the subfamily the receptor is implanted marginally, except in four genera (Alonopsis, Acroperus, Camptocercus and Graptoleberis). Function of the shift of this sensorial structure is unknown. Both genera share a reduction on enIV of the three inner endite setae (two first strongly reduced in size in Nicsmirnovius, completely absent in Phreatalona) and a small filter comb. The combination of modifications on the fourth limb in Phreatalona is unique within the Chydoridae. In Phreatalona labrosa, the fourth limb (but even the third and fifth exopodites) is less modified than in the three taxa of the protzi-complex (Table 1). For example, apex of exopodite setae is sharp in labrosa, fourth exopodite seta not as reduced, flaming torch setae not as small and fifth exopodite is more typical for the subfamily (Table 1). In short, P. labrosa (Table 1) shows relatively least adaptations. This species is endemic to a lake formed 25–30 Mya ago (Baikal), where it occurs in the littoral (Fig. 13). Phreatalona species evolved from littoral taxa (see below), and therefore ecology of labrosa is relatively closer to a ‘primitive’ state. Morphology may reflect a primitive condition or a reversal, and we think a primitive condition is the most likely.

Fig. 13

Habitats occupied by different Phreatalona species in Western Europe and Baikal. Three species of the Western European protzi-complex belong to the hyporheos. Their typical habitat is the water-saturated subsurface zone of clear oligotrophic streams, in the heterogeneous sediment (gravel/sand) at 30–60 cm below the riverbed. The morphologically and geographically isolated P. labrosa lives in open littoral of lake Baikal. Ecology coincides with a morphological gradient: order of specialization to the hyporheic is labrosa < protzi < smirnovi/phreatica, from right to left in figure. P. smirnovi and phreatica have the strongest adaptations, e.g. a reduced eye and rostrum

Phreatalona and Nicsmirnovius are no typical scrapers, active collectors of large particles or pure filter feeders, but are specialized in handling and processing soft, fine particulate organic matter, mycelia and decaying plant material. We observed protzi actively foraging and feeding on the latter (Fig. 1). Stronger reductions and modifications on limb endites occur in a few Chydoridae specialized in feeding on soft material (e.g. animal tissue feeders Anchistropus and Pseudochydorus; Van Damme & Dumont, 2007). Nicsmirnovius and Phreatalona enter surface waters, e.g. P. protzi, though rarely in high numbers. We found it easy to keep P. protzi in culture, without the need for extra addition of oxygen. Oxygen requirements may not be the main reason for their low abundances, though these species are not adapted to benthic conditions. More likely, due to their adaptations to the interstitial, Phreatalona may have lost the ability to thrive in more eutrophic surface waters. This could be due to competition by other Chydoridae, predation or parasitic pressures, but temperature restraints and sensitivity to UV may also play an important role. These animals are used to relatively more stable waters of the sheltered underground, lacking conditions from surface waters, such as UV-radiation, fluctuating temperatures and subsequent warming of the body. Oscillations and temperatures are still present in the hyporheic, but are less pronounced than in surface water. We think that UV is an important factor. Indications for light sensitivity or loss of UV-protection in Phreatalona are: (1) clear, unpigmented/slightly pigmented ephippia in P. phreatica (Alonso, 1996) and P. labrosa (Sinev & Kotov, 2000) instead of pigmented ephippia like in majority of Aloninae; (2) inability of P. phreatica to survive in daylight (Dumont, 1983); and (3) abundance of P. protzi in littoral only in shaded localities (during our sampling campaign). The interstitium likely acted as a refuge, and today has become their true living environment.

Several Phreatalona species show reduced swimming abilities, observed in life for both phreatica (Dumont, 1983) and protzi (this study). It reflects in morphology of the second antenna: swimming setae in protzi and antennal segments in phreatica are short, antennal muscles are reduced in smirnovi and first endopod spine is reduced in all species. On the other hand, the antennal setae have long setules (depicted for smirnovi), which may act as sensorial equipment. Nicsmirnovius and Phreatalona share a short or no rostrum and elongated aesthetascs. An elongation of sensory equipment is an adaptation in Aloninae for tracing food in a diluted lotic environment (Van Damme et al., 2003), a common adaptation in stygobiont crustaceans.

A closer look at habitus reveals interesting adaptations. General body outline for both genera is elongate, high in anterior half and tapering posteriorly, least pronounced in P. protzi and N. greeni. In both genera, the frontal margin strongly protrudes forward and the posterior head shield margin forms a straight angle with the dorsum. This is highly unusual in Aloninae. Most species have a rectangular or oval body with highest point in the middle. The anterior head margin is not protruding forward in most Aloninae, but is inclined backwards, posterior head shield margin forming an angle of more than 90° with the dorsum. The most anterior point in most Aloninae is the rostrum, but in these rheic species it is the middle of the head. In short, heads of Nicsmirnovius and Phreatalona are built like tiny ‘battering rams’. The shape is an adaptation to life in (hypo)rheic, which exerts high mechanical forces on the frontal region. Phreatalona moves between heterogeneous sediment, more or less loosely distributed. There is some free space here, where particles can be displaced; otherwise animals cannot move in such environment. Only in loose sediment would these animals survive. In homogeneous, densely packed sand they cannot be found. A wide head divides forces over the frontal surface and facilitates movement through both flowing water (reduces lift) and hard substrate. The absence of dorsal keel or strong carapace ornamentation reduces the risk of damaging the carapace. Whether flow or friction, forces at this level are similar. As one of the few Chydoridae, head is strongly protruding in Acroperus, which, considering limb similarities, may not be a coincidence. Also Monospilus has a straight broad front (see Alonso, 1996). Finally, the body may be relatively fusiform in Phreatalona and Nicsmirnovius in comparison to other Aloninae. This is most pronounced in smirnovi, but also occurs in labrosa. This may be another adaptation to the rheic environment as a more fusiform shape reduces drag.

Adaptations in external morphology vary at species level (see earlier): in more typical groundwater species P. phreatica and P. smirnovi, a rostrum is absent in comparison to P. protzi or the littoral P. labrosa (Table 1). Both genera show a reduction of eye pigmentation in some species but not total blindness, a result of life in the interstitial (Dumont, 1995). The loss of eye or ocellus happened several times independently in Aloninae, e.g. in Monospilus, Spinalona or stygobionts like Karualona alsafadii and the Alona hercegovinae-group. In P. labrosa and P. protzi, the eye is well developed, in contrast to P. phreatica and P. smirnovi. In Phreatalona smirnovi, underground and surface morphologies seem to differ: specimens from groundwater (types) lacked eyes and there were no denticles on the carapace (Petkovski & Flößner, 1972), while epigean specimens from the same locality had eyes with few ommatidia and one to two denticles. Maybe, these characters are reversible. Denticles are rare in phreatica while in surface protzi they are common. Such a reduction occurs in Karualona, where groundwater species K. alsafadii lacks the strong denticles typical for the genus (Dumont, 1995). Function of the denticles is unknown in Aloninae. However, several ‘costly’ structures like antennae, pigmentation and carapace outgrowths may be reduced in underground habitats because of energetic limitations. Other shared characters of Nicsmirnovius and Phreatalona, which cannot be attributed to the life mode, are an elongate naked labral keel, postabdomen with long basal claw and an obtuse dorsodistal angle.

Our small dendrogram (Fig. 12) illustrates the morphological similarity between Nicsmirnovius and Phreatalona and the separation of this rheic branch from Alona. A larger analysis of the Aloninae may add more taxa between Alona and this lineage, but Phreatalona and Nicsmirnovius would likely remain near Acroperus in any larger morphological analysis. Inclusion of all Aloninae genera would be premature for this paper and the phylogeny of this subfamily is a complex matter. Departure of Phreatalona from Alona is however clear, and we have no doubt that this is a valid genus, separate from Nicsmirnovius. Existence of the rheic branch yet needs an independent test with molecular data. Robustness of this branch depends on inclusion of specialized characters in phylogenetic analysis, and there is still a possibility that these adaptations occurred twice. Our analysis with the exclusion of the specializations still showed the rheic branch but with lower support. Nicsmirnovius and Phreatalona are likely related within a wider context, retaining some primitive characters unrelated to the life mode (e.g. long anterior setae on first limb, an important character) which are reduced in many of the smaller Aloninae. The Baikal endemic P. labrosa is likely basal to the protzi group, as shown in the dendrogram (Fig. 12). P. labrosa is the least specialized and may therefore be the relatively more primitive species of the genus, closest to a hypothetical Phreatalona ancestor, which is originally a surface form (see below). P. phreatica and smirnovi seem relatively closer related, mainly due to eye reduction and the absence of a rostrum, adaptations to the hyporheic. It is still unclear whether these affinities represent actual phylogenetic relationship.

So, Phreatalona is far from the benthic/littoral, “true” Alona, and closest to Nicsmirnovius. Both are specialized to rheic/ hyporheic, and the affinities may result from common ancestry. Specializations of Phreatalona relate to life in interstitial, including eye reduction in two species, decreased swimming capacity and increase of sensorial equipment (aesthetascs). On limbs, there is a reduction of exopodite surfaces (P3–P5). Several characters, rare within the subfamily, are shared with Acroperus and Alonopsis (Table 2). Morphology suggests Acroperus is actually closest to Alona species groups with setulated labral keel and merged marginal teeth on the postabdomen. Phreatalona and Nicsmirnovius may emerge from this group, sharing an ancestor with Acroperus. The latter has an apparently different habitus and postabdomen, but limbs are nevertheless very close. The link even shows in ecology. Acroperus species enter the rheic, in littoral of oligotrophic rivers (Alonso, 1996) and sporadically into the subterranean (Brancelj & Sket, 1990). Also, Monospilus shares a few characters rare for the subfamily (e.g. elongated aesthetasc, long anterior setae on P1, long gnathobasic process on P2, short third seta of exIII, reduction of eye pigment). Monospilus dispar lives on bare sandy substrate of lake shores (Smirnov, 1971), and its adaptations suggest entering top interstitial in stagnant waters. Position of the latter, which has several adaptations to life in interstitial and primitive external features (e.g. single head pore, postabdomen with two basal spines), is unclear.

Different levels of specialization form a kind of morphological gradient within the genus (Fig. 13). Order of specialization to hyporheic, considering overall morphology, is labrosa < protzi < smirnovi/phreatica; from the littoral lacustrine Phreatalona labrosa over the ‘stygophilic’ P. protzi to the obligate hyporheic P. phreatica. In P. smirnovi, the comparison of two populations suggests that a switch from hyporheic to surface goes combined with phenotypical changes that may be reversible (e.g. eye pigmentation).

Age of Phreatalona

Our morphological analysis shows that Phreatalona (and Nicsmirnovius) separated early from the main Aloninae trend, allowing specialization. A discussion on what is meant here by “early” may help us to estimate of the age of the Aloninae, of which no pre-Pleistocene fossils exist. Adaptations of Phreatalona and Nicsmirnovius, e.g. of limb structures, clearly derive from general alonine morphology. Both genera resemble most in limb characters to Acroperus, which may be related. Without doubt these chydorids originate from an ancestor with general Alona habitus inhabiting the freshwater littoral, exploring rheic niches and finally hyporheic. Within Nicsmirnovius and Phreatalona, surface as well as underground forms are present, with varying degrees of specialization. Adaptations to the rheic in surface waters and exploration of hard substrate in the streambed facilitated the step to hyporheic during their evolution. Several species (e.g. Nicsmirnovius eximius) may be found in the zone in between. Phreatalona shows typical features for subterraneous crustaceans, e.g. eye reduction or increased sensory equipment. Some of these adaptations may happen fast and may be reversible. As stated before, isolation of Phreatalona is enough for species to lose the ability in successfully recolonise surface waters, although P. protzi tries.

Estimated age for underground Cladocera is at least pre-Pleistocene, probably going back to the Miocene (Brancelj & Dumont, 2007). The interstitium and ancient lakes conserve groups that went extinct during glacial periods in Europe. Several stygobiont micro-crustaceans likely derive from late Tertiary surface-dwelling taxa taking shelter in the subterranean against climatic fluctuations and cooling (Brancelj & Dumont, 2007). Phreatalona may fit this hypothesis. Endemism in ancient lakes and the occurrence in river sediments indicate a marked isolation of Phreatalona.

An interesting example, P. labrosa is likely the most primitive member in the genus, in morphology as well as ecology. In comparison with the protzi-complex, the species shows least adaptations to life in the hyporheic. In Baikal, labrosa is found in the open water (1–10 m) and on rocks in a vegetation-free shore. Conditions are similar to those in oxygen-rich sands as with the other species, but labrosa is the only true surface species of a predominantly underground genus. Presence of surface forms of a hypogean group in Lake Baikal resembles a phenomenon observed in Bathynellacea (Syncarida), Mesozoic in origin. Of these micro-crustaceans found in caves and groundwater worldwide, the only two epibenthic species (Bathynella baicalensis and Baicalobathynella magna) live in Lake Baikal, at depths up to 1400 m (Kozhow, 1963). The latter species found refuge in Baikal from changing climatic conditions during ice ages, while disappearing in surface waters. The same may have happened with P. labrosa. Colonization of Baikal, deriving from a widespread surface form, may have happened as early as the formation of the lake (25–30 Mya) from surrounding areas where these species are now extinct (or not found yet?).

Biogeography of the protzi-complex (See further and Fig. 14) could be seen as another argument for pre-Pleistocene age in Phreatalona. Its southern boundary, determined by mountains of the Alpine Orogeny (Palaeo-Eocene, culmination in Miocene), the Pyrenees, Taurus, Caucasus and Alps, may not be coincidental. Glaciations during the Pleistocene certainly pressured colonization of groundwater (Brancelj & Dumont, 2007). Regions covered in ice sheets and several mountain chains likely acted as effective barriers, as with many groups (Hewitt, 2000, 2004). The protzi-complex may have never reached Spain or Italy, cut off by the Alps and Pyrenees, entering Western Europe only after these mountains were well established. Age of Lake Ohrid, home of P. smirnovi, could be an estimate for differentiation within the P. protzi-complex. As minimal estimate, it is possible that the three species of the protzi-complex diverged later as a result of isolations of protzi-like populations during glacial periods.

Fig. 14

World distribution of phreatic chydorids Phreatalona and Nicsmirnovius; inset: Western records of P. protzi-complex. Distribution of Nicsmirnovius after Van Damme et al. (2003), with more recent records in Neotropics (Elmoor-Loureiro, 2007) and Asia (Borneo; Dumont, unp.). For Phreatalona, after Smirnov (1971; protzi), Flößner (2000; protzi), Sinev & Kotov (2000; labrosa), Sabater (1987; phreatica), Dumont (1983; phreatica), Petkovski & Flößner (1972) and this study

To conclude, we think Phreatalona originates from Aloninae of pre-Pleistocene origin. The morphology clearly shows a separate evolution to this environment. Most likely, its ancestors were well spread in surface waters throughout Europe, possibly even before the formation of Baikal, where the only open water/littoral species lives. The Baikal endemic also has the relatively more primitive morphology. Several populations of the protzi-complex likely survived adverse climate conditions in Europe finding refuge in groundwater. Brancelj & Dumont (2007) suggest the groundwater cladocerans to be relatively younger than the true karstic stygobionts (A. hercegovinae-group) because of fewer specializations. However, limb adaptations in Phreatalona are more aberrant for the subfamily than in blind Alona’s and still suggest a strong isolation. There are no morphological affinities with the true stygobiont Alona’s and Phreatalona; both entered underground separately and live in different environments. An important note for the age of Aloninae: because we lack fossils to interpret the age of different groups in this subfamily, Phreatalona is one of the few genera that could be used for calibration of molecular clocks. Morphological separation from their ancestor may date as far back as 30 Mya.

Distribution of Phreatalona and Nicsmirnovius

The majority of Aloninae genera and Alona species groups have a relatively wide cosmopolitan or southern hemisphere distribution. Phreatalona however is restricted to the Palaearctic. The genus is bounded to the south by the Mediterranean (Spain, Turkey) and represented here by protzi and phreatica, of which the main centre of distribution is Western Europe. The genus is so far restricted here, lacking from the Iberian Peninsula, most of Italy and Scandinavia (not S Finland). The west of France is inhabited by P. phreatica, the rest of Europe with P. protzi, but not south of the Pyrenees and Alps. Only a narrow belt of overlap of both species is known so far—along Meuse and Rhône valleys, continuing along the French Mediterranean coast to the Pyrenees. Two are endemic to ancient lakes: P. labrosa to Lake Baikal and P. smirnovi to Lake Ohrid. The former is situated mainly in old volcanic or metamorphic geology, but with some inserts of limestone, providing phreatic groundwater for springs and interstitial in a sandy littoral zone, perfect environment for Phreatalona. There is a “Baikalian disjunction” between this lake and ‘Danubian Europe’ (see below) for the genus. In the vast area in between, records are missing. It is possible that both the Ural and a large discontinuity of subsurface water in Central Asia currently act as effective barriers for these river-bound species.

Phreatalona may not disperse like surface Aloninae. The genus is adapted to permanent riverine conditions and may have lost the ability to withstand desiccation or temperature fluctuations. Furthermore, reduced pigmentation in ephippia (labrosa, phreatica) suggests poor adaptation to UV and no doubt affects the dispersal abilities. Their dispersal may rely predominantly on drainage basin limits and the geomorphological evolution of hydrographical networks. In this way, biogeography of the P. protzi-complex parallels biogeography of a large group of primary freshwater fishes restricted to rivers in ‘Danubian Europe’ and lacking from Peri-Mediterranea (including Spain and Italy) (Reyjol et al., 2007). Besides climatological differences, these two regions have different biogeographical history (Hewitt, 2000). Extinction, dispersal and evolution of riverine groups are strongly affected by the Last Glacial Maximum. For riverine fishes, postglacial colonization was possible from refuge populations in the Danube Basin (Reyjol et al., 2007). Biogeographical history of the P. protzi-complex may be similar. As discussed earlier, Phreatalona is likely pre-Pleistocene in origin. The current distribution pattern of the genus shows a strong influence by the Quaternary ice ages. Disjunction with Baikal suggests that Phreatalona was widespread in the Palaearctic and populations were likely fragmented during glaciations. The ecologically most versatile species, P. protzi, is widespread in Danubian Europe while all others have very limited distributions, and its expansion may be relatively recent.

Population studies of these remarkable Cladocera would be interesting, for example to investigate if separation of protzi-phreatica predates the Pleistocene. This would be a separate work, considering wide geographical coverage of protzi (e.g. UK, Scandinavia, etc.). In any case, because of the peculiar ecology and therefore a limitation to hypothetical scenarios, Phreatalona shows potential for the study of cladoceran biogeography and evolution.

The related Nicsmirnovius inhabits riverine habitats south of Phreatalona’s range: the Afrotropics (N. greeni, N. camerounensis), South East Asia, Australia (N. eximius) and the Neotropics (N. fitzpatricki) (Van Damme et al., 2003). Distributions are complementary and we can consider Phreatalona as a northern vicariant of the tropical Nicsmirnovius. As in Phreatalona, we think Nicsmirnovius largely depends on river systems for distribution. These species may however be less limited in dispersal than Phreatalona. Nicsmirnovius occurs in surface waters, though in low numbers, and ephippia are pigmented. The latter genus shows a relatively old intercontinental distribution, with separate (sub)species in tropical rainforests (e.g. N. camerounensis) or on isolated mountain chains of ancient islands (Socotra). We are tempted to say that the intercontinental distribution of Nicsmirnovius resembles that of ‘Gondwana-relics’.

Gaps in distribution remain, maybe due to sampling bias. In the largest part of the Holarctic (especially North America), no hyporheic chydorids have yet been recorded. There is no reason why these should be lacking from the Nearctic and new taxa may be found here. Also, in the largest part of Inner or Eastern Asia, some remaining permanent river systems may contain surviving hyporheic chydorids. In Australia, records of hyporheic species, most likely Nicsmirnovius, are missing. Targeted efforts sampling interstitial riverine habitats in these regions may reveal a wider distribution or even new species. The species are however rare and may be sensitive (maybe even recently extinct in many places?) due to eutrophication/pollution.

Key to parthenogenetic females of Phreatalona (see also Table 1)

Note: P. protzi has the widest distribution and may be sympatric with phreatica in W Europe.

• 1. Eye and ocellus well developed, black, eye diameter similar to width of antennule. Rostrum short but present……………………………….3

• 2. Eye reduced, its diameter half of antennular width, to absent. If diameter similar to the antennular width, the eye has few ommatidia and is not densely pigmented but more transparent. Rostrum absent, head shield rounded………………………………………….5

• 3. Ocellus large, diameter 0.8–1 that of eye. Denticles in posteroventral corner of valves two to four (mostly three). Body ovoid, dorsum highly arched and mostly short, length about 1.3–1.5 times width. Postabdomen distal gap closed (see Fig. 9), IDL setae on first limb modified, with widened base and long setules, P5 with concave margin……………………………P. protzi

• 4. Ocellus small, diameter 0.5–0.8 times that of eye. No denticles in PvC. Body elongate, not highly arched, length of body 1.5–1.6 times width. Postabdomen with distal gap open. IDL setae on first limb not modified (not with widened base and setules short), P5 not with concave margin. Endemic to Lake Baikal, drawings in Sinev & Kotov (2000)……………………………P. labrosa

• 5. Postabdomen with distal gap open (see Fig. 9). Spinules in lateral fascicles spread, not parallel. Spine on first endopod segment of antenna small, up to one-third of second endopod segment. Body about 1.5 as long as high, not strongly fusiform…………………………P. phreatica

• 6. Postabdomen with closed distal gap, distal portion strongly protruding. Spinules in lateral fascicles parallel. Spine on first endopod segment of antenna up to one-fourth of second endopod segment. Body very long, its shape fusiform (tapering distally and 1.6–1.7 as long as high)……………………………P. smirnovi