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TAXONOMIC REVISION
Adaptations to the hyporheic in Aloninae (Crustacea:
Cladocera): allocation of Alona protzi Hartwig, 1900
and related species to Phreatalona gen. nov.
Kay Van Damme ÆAnton Brancelj Æ
Henri J. Dumont
Received: 2 April 2008 / Revised: 1 September 2008 / Accepted: 8 September 2008 / Published online: 25 October 2008
Springer Science+Business Media B.V. 2008
Abstract Morphological study of Alona protzi
Hartwig, 1900, Alona phreatica Dumont, 1983 and
Alona smirnovi Petkovski & Flo
¨ßner, 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 affin-
ities 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 hypor-
heic 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.
Keywords Chydoridae Cladocera Hyporheic
zone Morphology Phreatalona gen. n.
Stygobionts Systematics
Introduction
Suggestions of the polyphyletic nature of the fresh-
water 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
Electronic supplementary material The online version of
this article (doi:10.1007/s10750-008-9607-6) contains
supplementary material, which is available to authorized users.
Handling editor: S. I. Dodson
K. Van Damme (&)H. J. Dumont
Department of Biology, Ghent University,
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
e-mail: kay.vandamme@ugent.be
H. J. Dumont
e-mail: henri.dumont@ugent.be
A. Brancelj
National Institute of Biology, Vecna pot 111,
1000 Ljubljana, Slovenia
e-mail: anton.brancelj@nib.si
123
Hydrobiologia (2009) 618:1–34
DOI 10.1007/s10750-008-9607-6
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) recog-
nized 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; Flo
¨ßner, 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 Flo
¨ßner (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 Sympo-
sium in 2005 in Herzberg (Switzerland) (Van Damme
et al., 2005). Here, we document comparative mor-
phology of the three taxa and discuss the relationship
between and the status of A. protzi Hartwig, 1900,
Alona phreatica Dumont, 1983,Alona smirnovi
Petkovski & Flo
¨ßner, 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 km
2
(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 evo-
lution. 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 lmor
less were used. On each sub-sampling point, first, the
sample for fauna was collected from hyporheic zone,
and then temperature, conductivity and oxygen con-
centration 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
2 Hydrobiologia (2009) 618:1–34
123
deposition of specimens was in 60% alcohol. (For more
details on sampling methods and protocols see: PAS-
CALIS project, 2004.) During an additional sampling
campaign (by KVD in 2007), animals were collected
using dip nets with 50 lm 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 lm net and
repeating this procedure. Specimens for permanent
storage and dissection were transferred to a formalde-
hyde–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 (Swof-
ford, 2000) for generating a small dendrogram
illustrating morphological similarities. Branch-swap-
ping 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 har-
pae, 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: postab-
domen; parth: parthenogenetic; pep: pre-epipodite;
PvC: posteroventral valve corner; scr: scraper; segm:
segment; ss: soft seta; tc: terminal claw. For phylo-
genetic 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 disci-
pline (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 stygo-
philes. It is a dynamic ecotone (Sabater & Vila, 1991)
intermediate between the river water above and the
groundwater from saturated bedrock below, the phre-
atic (Valett et al., 1993; Boulton et al., 1998). The latter
zone is part of the groundwater system, typically[1m
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
Hydrobiologia (2009) 618:1–34 3
123
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 (typi-
cally 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 com-
posed 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 rela-
tively 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 setulat-
ed 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 projec-
tion. 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; gnat-
hobasic ‘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
(10–30) 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 (100–400); one element and four small setae on
internal endite preceding gnathobase; gnathobase with
a bottle-shaped sensillum, bent plumose seta with two
4 Hydrobiologia (2009) 618:1–34
123
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 (10–20) 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 termi-
nal 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 Nics-
mirnovius); 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; termi-
nal 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 homoge-
neous 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 (auta-
pomorphy) 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. Ko
¨nigsberg, 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. Flo
¨ßner, 28.IV.1984, Flo
¨ßner Coll., Museum
Fu
¨r Naturkunde, Berlin. One adult male, Hellsee by
Biesenthai/Mark Brandenburg, Germany, Hartwig
Collection, Leg. A. Protz, October 1889 (Hartwig,
1900), Museum Fu
¨r Naturkunde, Berlin. Two adult
parthenogenetic females, hyporheic Amble
`ve River,
Ile de Halleux, Walloon Region, Belgium, Leg.
PASCALIS project, 30.VII.2002. One adult partheno-
genetic 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 Mediterra-
nean coast (elevation of 13 m a.s.l.), France, Leg.
PASCALIS project, 24.VII.2003. 50 adult partheno-
genetic females, between moss on stones and river
gravel, La Lanterne, Haute-Sao
ˆne, 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,
Hydrobiologia (2009) 618:1–34 5
123
Belgium), 0.31–0.35 mm in Vranovsky (1971),
0.32–0.42 mm in Flo
¨ßner (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. 1and 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. 1and
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).
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. 1and 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. Anten-
nal 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
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-
Sao
ˆne, 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
6 Hydrobiologia (2009) 618:1–34
123
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
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; BBody outline; C
Headshield (after Flo
¨ßner,
2000: Fig. 115c); DHead
pores; EHead; F
Antennule; GAntenna; H
Labrum; I,J Posteroventral
valve corner; K,L
Postabdomen; MIdem,
detail, with indication of
distal embayment; N,O
Postabdomen; PIdem,
detail lateral fascicle and
marginal denticle
Hydrobiologia (2009) 618:1–34 7
123
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).
Second limb (Figs. 3F and 4D,F)
Exopodite oval, with one short setulated seta (subapi-
cal), 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
Fig. 3 Phreatalona protzi
(Hartwig, 1900) =Alona
protzi Hartwig, 1900. SEM
of parthenogenetic females
from Amble
`ve River,
Belgium. AHabitus, valve
removed; BHabitus; C
Postabdomen and inside of
valve; DAntenna and labral
keel; EInner side of
posteroventral valve corner
with three denticles; FFirst
and second limb
8 Hydrobiologia (2009) 618:1–34
123
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 exo-
podite. 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
Fig. 4 Phreatalona protzi
(Hartwig, 1900) =Alona
protzi Hartwig, 1900. Limb
characters of
parthenogenetic females
from Maaseik, Belgium. A
First limb; BIdem, anterior
side; CIdem, IDL and
ODL; DSecond limb; E
Idem, exopodite; FIdem,
morphology scrapers 1–4;
GThird limb, exopodite; H
Fourth limb, exopodite; I
Idem, setae 5–6; JIdem,
endite; KFifth limb. See
separate section for
abbreviations, below
material and methods
Hydrobiologia (2009) 618:1–34 9
123
shorter than third. Inner lobe elongated oval with long
terminal setules; two slender endite setae (10–20)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 Flo
¨ßner (2000),
picture in Nyka
¨nen & Sarmaja-Korjonen (2007).
Postabdomen and first limb based on specimen of
Hartwig (1900) in collection of Flo
¨ßner (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).
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 Schoenoplec-
tus (Vranovsky, 1971), lakes, rivers, hyporheic
(Brancelj & Sket, 1990; Dumont & Negrea, 1996;
Flo
¨ßner, 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. Nyka
¨nen & Sarmaja-
Korjonen, 2007), they are probably connected with a
presence of a spring, groundwater feed or inlet of
small streams. During PASCALIS project, 124 spec-
imens of P. protzi were collected in total (1 specimen
in Belgium—hyporheic of Ourthe river; 120 speci-
mens 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-Sao
ˆne), one
metre deep and two metres wide. Specimens of
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/FLateral
fascicles and marginal
denticles; C/GFirst limb,
IDL and ODL; D/HFirst
limb, copulatory hook
10 Hydrobiologia (2009) 618:1–34
123
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 discov-
ered 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 concen-
trations 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;
Nyka
¨nen & Sarmaja-Korjonen, 2007), Ireland, UK
(Gurney, 1921), Turkey (Gu
¨her, 2002), Lake Inkit
(Behning, 1941), France (e.g. Rhone gravel bars;
Dumont, 1987; Brancelj & Dumont, 2007), Germany
and Poland (Flo
¨ßner, 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 Pyre-
nees (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. 1and 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 (Keil-
hack, 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 mar-
gin. 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 postab-
domen 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 (Flo
¨ßner, 2000),
and not as a separate subspecies.
Hydrobiologia (2009) 618:1–34 11
123
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, Amble
`ve, 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, hypor-
heic 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 parthe-
nogenetic females, hyporheic, Aisne, Roche-a-Fre
`ne,
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, pop-
ulations 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 mod-
erate 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).
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 seg-
ment. 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 post-
anal 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
12 Hydrobiologia (2009) 618:1–34
123
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
Fig. 6 Phreatalona
phreatica (Dumont,
1983)=Alona phreatica
Dumont, 1983. External
morphology of adult
parthenogenetic females
from Ninglispo, France. A
Habitus; BBody shape; C
Head shield (after Alonso,
1996); DHead pores; E–G
Labrum; HPosteroventral
valve corner; IAntennule; J
Second antenna; K,L
Postabdomen; MLateral
fascicle and marginal
denticle
Hydrobiologia (2009) 618:1–34 13
123
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).
Second limb (Figs. 7F and 8D,F)
Exopodite oval, with one short setulated seta (sub-
apical), 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 rela-
tively 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
Fig. 7 Phreatalona
phreatica (Dumont,
1983) =Alona phreatica
Dumont, 1983. SEM of
adult parthenogenetic
females from Amble
`ve,
Ninglispo, Belgium, and
Oignin river, Jura Region,
France. A,BHabitus; C
Postabdomen; DHead with
antennae and labral keel; E
Posteroventral valve corner,
inner side; FFirst and
second limb
14 Hydrobiologia (2009) 618:1–34
123
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 (10–30)of
which first two slender, of similar size and with
minute element in between, third (30) short, plump
and with long setules; four well-developed and thick-
based plumose setae on inner side (100–400) 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 fin-
gerlike projection, reaching beyond centre of
exopodite. Exopodite (Fig. 8K) square, with six plu-
mose 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
Fig. 8 Phreatalona
phreatica (Dumont,
1983) =Alona phreatica
Dumont, 1983. Limb
morphology of adult
parthenogenetic females
from Amble
`ve, Ninglispo,
Belgium. AFirst limb; B
Idem, anterior portion; C
Idem, ODL and IDL; D
Second limb; EIdem,
scrapers 1–4; FIdem,
exopodite, first scraper and
adjacent soft seta (ss); G
Third limb, exopodite; H
Third limb, endite; IIdem,
gnathobase; JIdem, outer
endite setae; KFourth limb,
exopodite; LFourth limb,
endite; MFifth limb
Hydrobiologia (2009) 618:1–34 15
123
terminal setules; two slender endite setae (10–20)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 Pyre-
nees), 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. phre-
atica 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, Amble
`ve). 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. 1with 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.
3. Phreatalona labrosa (Vasiljeva & Smirnov,
1969) comb. nov.
=Alona labrosa Vasiljeva & Smirnov, 1969.
Synonymy and full description in Sinev and Kotov
(2000).
16 Hydrobiologia (2009) 618:1–34
123
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 setoso-
caudata, Kozhowia kozhowi and Parakozhowia
baicalensis. Highest densities in August; Vasiljeva
& Smirnov (1969) collected over 400 specimens/m
2
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 Flo
¨ßner,
1972) comb. nov.
=Alona smirnovi Petkovski & Flo
¨ßner (1972)
Type locality. In Petkovski & Flo
¨ßner (1972):
‘‘Gero
¨ll des flachen litorals beim Kloster Sv. Naum
am Su
¨dufer des Sees (Ohrid)’’ and ‘‘Loser Sand an
demselben Strand, nahe der Einmu
¨ndung 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, dili-
gent researcher of Cladocera systematics, Moscow
(Petkovski & Flo
¨ßner, 1972).
Specimens examined. Four adult parthenogenetic
females, Lake Ohrid, southern margin near Monastry,
Naum, Macedonia, Leg. Petkovski, IX, 1969. Coll.
D. Flo
¨ßner, 1970. Museum fu
¨r 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 trans-
parent. In Petkovski & Flo
¨ßner (1972), length
0.33–0.43 mm. Body length 1.6–1.7 times height.
Dorsum arched, body highest just before middle,
Fig. 9 Comparison of Phreatalona protzi (A–D) postabdomen
with P. phreatica (E–H). P. protzi from Amble
`ve, 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)
Hydrobiologia (2009) 618:1–34 17
123
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; BBody outline; C
Head shield after Petkovski
& Flo
¨ßner (1972); DHead
pores; EHead; FSecond
antenna; G,H
Posteroventral valve corner.
I,J. Labrum; K,L
Postabdomen; MTerminal
claw and distal part of
postabdomen
18 Hydrobiologia (2009) 618:1–34
123
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 posta-
nal 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 glob-
ular process (Fig. 11A).
Second limb (Fig. 11D–F)
Exopodite oval, with one long setulated seta (sub-
apical), 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).
Hydrobiologia (2009) 618:1–34 19
123
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
(10–30) of which first two slender, of similar size and
with minute element in between, third (30) short,
plump and with long setules; four well-developed and
stout plumose setae on inner side (100–400) 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
Fig. 11 Phreatalona
smirnovi =Alona smirnovi.
Adult parthenogenetic
females from Lake Ohrid,
Naum feeder springs, on
rocks, Macedonia, Leg.
Petkovski. Limb
morphology. AFirst limb;
BIdem, anterior portion; C
Idem, ODL and IDL; D
Second limb; EIdem,
scrapers 1–4; FIdem,
exopodite, first scraper and
adjacent soft seta (ss); G
Third limb, exopodite; H
Idem, exopodite setae 6–7;
IIdem, endite; JIdem,
inner endite row; KIdem,
gnathobase; LIdem, outer
endite row; MFourth limb,
exopodite; NIdem, setae
5–6; OIdem, endite; PFifth
limb
20 Hydrobiologia (2009) 618:1–34
123
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 devel-
oped, little shorter than third. Inner lobe elongated
oval with long terminal setules; two slender endite
setae (10–20) 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 (mesop-
sammom) of karstic feeder springs (Petkovski &
Flo
¨ßner, 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 & Flo
¨ßner, 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.
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 unin-
formative characters), RI =0.84 and RC =0.66;
two of 31 characters were found parsimony-uninfor-
mative (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.Acrope-
rus 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.ForPhreatalona, 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.
Hydrobiologia (2009) 618:1–34 21
123
Table 1 Differences between adult parthenogenetic females of the four Phreatalona species, and ecology and distribution
Phreatalona protzi-complex Phreatalona labrosa
protzi phreatica smirnovi
Limb morphology
P1 anterior setules Short fine Long fine Short thick Short thick
P1 en2 anterior seta length [en1 ant seta \en1 ant seta [en1 ant seta =en1 ant seta
P1 IDL setae base Thick Parallel Parallel Thick
P1 IDL setulation Long, spaced Short, dense Short, dense Short, dense
P1 naked IDL seta Short IDL seta \Short IDL seta Short IDL seta Short IDL seta
P1 accessory seta (base ODL) Long Long Short Long
P2 exopodite seta Short (*ex) Short (\ex) Long ([ex) Short (*ex)
P2 length scrapers 1–2 Like scr. 4–6 Like scr. 4–6 Like scr. 4–6 Longer than scr. 4–6
P2 scraper 3 length Half scraper 2 Two-thirds of scr 2 Two-thirds of scr 2 Half scr 2
P2 scraper 5 vs. 4–6 Similar length scr 5 longer scr 5 longer Similar length
P3 exopodite seta 3 As long as 5 As long as 5 Longer than 5 As long as 5
P3 exopodite setae 4–5 Short Short Short Long
P4 exopodite seta 4 Reduced Reduced Reduced Present
P4 exopodite seta 6 apex Blunt Blunt Blunt Acute
P4 endite ft setae Reduced Reduced Reduced Developed
P5 exopodite setae 1–2 length \Width of ex \Width of ex [Width of ex [Width of ex
P5 exopodite setae 1–2 length 1 As long as 2 1 Shorter than 2 1 Shorter than 2 1 Longer than 2
P5 exopodite margin Strongly concave Strongly concave Strongly concave Straight
P5 inner (10) seta Long (2 920) Short Short Long (2 920)
‘‘External’’ morphology
Body shape (lateral) Short and high Long, not high Long, not high Long, not high
Body max. length/width 1.3–1.5 *1.5 1.6–1.7 1.5–1.6
Size adult parth. female 0.32–0.42 mm 0.37–0.46 mm 0.28–0.43 mm 0.38–0.48 mm
Eye and ocellus Well developed Present, reduced Eye reduced to absent Well developed
A1 longest aesthetasc length *A1 corm [A1 corm [A1 corm *A1 corm
A2 nat.setae/A2 segm.
?coxa
Setae *segm. Setae [segm. Setae [segm. No data
A2 apical exopod spine *Endop. spine [Endopod spine Endopod spine [Endopod spine
A2 1st exopod seta Long Long Long Short
A2 1
st
endopod spine Small Small Minute Minute
Rostrum Short Absent Absent Short
Carapace PvC denticles 2–3 (0) 0 (–2?) 0–2 0
PA ventral margin Arched Straight Arched Arched
PA distal margin Protruding Protruding Strongly protruding Not protruding
PA distal ‘gap’
(near base tc)
Closed (/open) Open Closed Open
PA lat. fasc. length Short-long Long Short Short
PA lat. fasc. spread Parallel Spread, fine Parallel Parallel
Ecology Hyporheic/littoral
stygophilic/stygobiont
Hyporheic obligate
stygobiont
Hyporheic/(littoral)
stygobiont/(stygophilic)
Littoral not in
interstitial
Distribution ‘Danubian’ Europe BE, France, Spain Lake Ohrid Lake Baikal
Data of Phreatalona labrosa comb.nov. from Sinev & Kotov (2000). For abbreviations, see section below Material and methods
22 Hydrobiologia (2009) 618:1–34
123
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 mor-
phology), 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 postero-
ventral 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 related-
ness. Externally, the three forms differ (Table 1)in
body shape and size, rostrum, length of swimming
setae on second antennae and shape of the postabdo-
men (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
Fig. 12 Dendrogram illustrating morphological similarities of
Phreatalona with Nicsmirnovius and a separation of the rheic
branch from ‘‘true’’ Alona (quadrangularis). Within Phreata-
lona, 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
Hydrobiologia (2009) 618:1–34 23
123
micro-crustaceans. Both may evolve independently.
The disparity results from different selection pres-
sures: 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 fre-
quented, 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 mor-
phology for the subfamily. Sinev & Kotov (2000) list
nine points of similarity between phreatica and
labrosa, which can be used as features for Phreat-
alona. 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 postab-
dominal 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
24 Hydrobiologia (2009) 618:1–34
123
Alona species with five limb pairs except for
Nicsmirnovius and the cave inhabiting A. hercegovi-
nae-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 adapta-
tions, 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.
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 modi-
fications 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 (quadrangu-
laris-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 condi-
tions, 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). Func-
tion 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 modi-
fications 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.
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
Hydrobiologia (2009) 618:1–34 25
123
Table 2 Comparison of morphology Phreatalona with Nicsmirnovius, with comments on functionality and occurrence of these characters in subfamily Aloninae
Character Phreatalona Nicsmirnovius Remarks
P1 anterior setae en1–2 ??Primitive condition; in Alona, Acroperus, Camptocercus, Monospilus etc.
P1 accessory seta (en6) ?-Present in most Aloninae, including Alona
P2 extra soft seta ?-Primitive; soft seta in this position in several Alona, Acroperus, etc.
P2 gnathobasic process Elongate Elongate Unusual for Aloninae; e.g. occurs in Monospilus but not in Alona
P3 exopodite setae Seven Six Seven in Alona quadrangularis, six in more advanced alonines
(like Alona rectangula-group)
P3 third exopodite seta Short Short Similar to penultimate exopodite seta here, much longer
(about twice as long) in most Aloninae. Also short in Acroperus, Monospilus
P4 ex setae 5–6 5 Blunt apex 5–6 Blunt apex Only in Alonopsis (setae 5–6). Sharp apex in rest of subfamily
P4 ex seta 4 Reduced, as short as
wide (not labrosa)
Present, longer than
wide
In other Aloninae never reduced
P4-P5 exopodites Small Small Adaptation to rheic; in majority of Aloninae and especially Alona
quadrangularis, exopodites larger in comparison to other limbs,
where a flow is generated actively
P5 exopodite ‘bilobed’,
with concave margin
?(Not labrosa)-In several Alona (affinis, costata, guttata), Acroperus ‘bilobed’,
not A. quadrangularis
P4 endite, receptor Shifted to inner side Shifted to inner side Rare; only shifted in Acroperus, Camptocercus, Graptoleberis, Alonopsis
P4 endite, inner setae -Reduced in size Always three setae here in subfamily, reduced in size in Acroperus, Alonopsis
P5 filter comb setae 0 0–2 No remarks
P6 --Present in several Alona, absent in true A. quadrangularis
Body high in anterior
portion, tapering
?(Less in protzi)?(Least greeni) Most Aloninae have oval or round body with highest point in middle.
Fusiform shape may help to reduce drag in rheic environments
Head strongly protruding ?(Less protzi)?Adaptation to interstitial/rheic; in Aloninae, head different with rostrum
protruding; present in Acroperus and Monospilus
A1 one or two elongated
aesthetascs
??Subequal in most Aloninae; elongate in Acroperus, Camptocercus, Alonopsis
A1 sensory seta Terminal Terminal Atypical for Aloninae, mostly implanted at 1/2d-1/3d from apex
A2 first endopod spine Reduced Well developed Well developed in majority of Aloninae; longer than half segment; short in
Acroperus
Rostrum Short (less labrosa,
protzi)
Short Related to elongated aesthetascs (detection in flow). Rare in Aloninae
Labral keel Elongate apex Elongate (not eximius
and greeni)
Rare character in Aloninae where labral keel is mostly short
26 Hydrobiologia (2009) 618:1–34
123
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 under-
ground, 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 ephip-
pia in P. phreatica (Alonso, 1996)andP. 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 swim-
ming 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 environ-
ment (Van Damme et al., 2003), a common
adaptation in stygobiont crustaceans.
Table 2 continued
Character Phreatalona Nicsmirnovius Remarks
Eye reduction ?(phreatica, smirnovi)?(camerounensis) Eye reduction rare; related to underground life; in Karualona,
A. hercegovinae-group, Spinalona, Monospilus
Small head pores Simple With 8-like structures Simple round in majority of Aloninae, without underlying structures
PA prominent dorso-distal angle ?(phreatica)?This causes a gap, also in Acroperus and Camptocercus; not in majority Alona
PA marginal postanal denticles Very small Very small Large denticles, spines or serrated teeth in majority of Aloninae including Alona
PA lateral fascicles Fine, similar Fine, similar Mostly distalmost thicker
PA male, basal spine As long as in females As long as in females Rare for Aloninae and not in Alona
Morphology characters of Nicsmirnovius after Van Damme et al. (2003) and Kotov & Sanuamoang (2004), for Phreatalona labrosa after Sinev & Kotov (2000), and Acroperus
and Monospilus after Alonso (1996). All but last characters are for parthenogenetic females
Hydrobiologia (2009) 618:1–34 27
123
A closer look at habitus reveals interesting adap-
tations. General body outline for both genera is
elongate, high in anterior half and tapering posteri-
orly, 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 90with
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 move-
ment 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, consid-
ering 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 com-
parison 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 ground-
water 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.InPhreatalona
smirnovi, underground and surface morphologies
seem to differ: specimens from groundwater (types)
lacked eyes and there were no denticles on the
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 \prot-
zi \smirnovi/phreatica, from right to left in figure. P. smirnovi
and phreatica have the strongest adaptations, e.g. a reduced eye
and rostrum
28 Hydrobiologia (2009) 618:1–34
123
carapace (Petkovski & Flo
¨ßner, 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, postab-
domen 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 indepen-
dent 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. Nicsmirno-
vius 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 special-
ized and may therefore be the relatively more
primitive species of the genus, closest to a hypothe-
tical 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. Acrope-
rus species enter the rheic, in littoral of oligotrophic
rivers (Alonso, 1996) and sporadically into the
subterranean (Brancelj & Sket, 1990). Also, Mono-
spilus 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.InP. smirnovi, the compar-
ison of two populations suggests that a switch from
hyporheic to surface goes combined with pheno-
typical 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
Hydrobiologia (2009) 618:1–34 29
123
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 special-
ization. Adaptations to the rheic in surface waters and
exploration of hard substrate in the streambed
facilitated the step to hyporheic during their evolu-
tion. 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 Phreat-
alona 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 intersti-
tium 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 under-
ground genus. Presence of surface forms of a
hypogean group in Lake Baikal resembles a phe-
nomenon 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 Bai-
calobathynella 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 cer-
tainly 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.
To conclude, we think Phreatalona originates
from Aloninae of pre-Pleistocene origin. The mor-
phology 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. herceg-
ovinae-group) because of fewer specializations.
However, limb adaptations in Phreatalona are more
aberrant for the subfamily than in blind Alona’s and
30 Hydrobiologia (2009) 618:1–34
123
still suggest a strong isolation. There are no morpho-
logical 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. Morpholog-
ical 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 how-
ever 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 Scan-
dinavia (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 Rho
ˆne 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, provid-
ing 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 Aloni-
nae. 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 net-
works. 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-Mediterra-
nea (including Spain and Italy) (Reyjol et al., 2007).
Besides climatological differences, these two regions
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), Flo
¨ßner (2000;
protzi), Sinev & Kotov (2000; labrosa), Sabater (1987;
phreatica), Dumont (1983; phreatica), Petkovski & Flo
¨ßner
(1972) and this study
Hydrobiologia (2009) 618:1–34 31
123
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 coloniza-
tion 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-Pleisto-
cene in origin. The current distribution pattern of the
genus shows a strong influence by the Quaternary ice
ages. Disjunction with Baikal suggests that Phreat-
alona was widespread in the Palaearctic and
populations were likely fragmented during glacia-
tions.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 Pleisto-
cene. 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. fitzpa-
tricki) (Van Damme et al., 2003). Distributions are
complementary and we can consider Phreatalona as a
northern vicariant of the tropical Nicsmirnovius.Asin
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 sam-
pling 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 sam-
pling 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 hasfew 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. Den-
ticles 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. Pos-
tabdomen with distal gap open. IDL setae on first
limb not modified (not with widened base and
setules short), P5 notwith 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
32 Hydrobiologia (2009) 618:1–34
123
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
Acknowledgements Sampling of hyporheic in Belgium and
France of A. Brancelj was carried out within the PASCALIS
Project, in particular sampling by M-J. Dole-Olivier, C. Boutin,
F. Fiers and P. Martin. Our sincere gratitude to Dr Gerald
Louette (INBO, Brussels) for his keen observation of protzi in
Heerenlaak, Belgium, and sharing the exact locality of the
finding, to Dr D. Van Damme for help in sampling and
prospecting in Belgium and France and useful comments
during the course of this manuscript, Dr Goce Kostoski,
Director of Hydrobiological Institute Ohrid, for providing new
specimens of smirnovi and Dr Christian Albrecht for help in
retrieving these specimens. Thanks also to Dr. D. Floessner and
Dr. Charles Oliver Coleman.
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Appendix – Cladistic analysis – data.
Appendix 1. Characters and states selected for analysis.
1. Eye pigmentation; compound eye absent (0); reduced (1); present (2).
2. Head shape; rostrum forms most anterior point in lateral view (0); middle of head most anterior point, not rostrum (1).
3. Rostrum; present (0); absent (1).
4. Head pores; two main pores (0); three main pores, small pores no cosmaria (1); three main pores, small pores with cosmaria (2).
5. Labral keel with elongated apex; present (0); absent (1).
6. Labral keel setule groups; present (0); absent (1).
7. First antenna elongated; present (0); absent (1).
8. First antenna subapical aesthetasc next to sensory seta; present (0); absent (1).
9. First antenna sensory seta; implanted at half to one third of apex (0); terminal (1).
10. First antenna apical aesthetascs; similar length (0); one longer (1) or two longer (2).
11. A2 exopod seta; present, longer than exopod segments (0); present, shorter than exopod segments (1); absent (2).
12. A2 spine on first endopod segment; present and longer than half second segment (0); reduced (1).
13. P1 accessory seta on endite 6 (base of IDL); absent (0); present (1).
14. P1 anterior setae on endites one and two; reduced (0); long (1).
15. P1 dorsal seta on first endite; present (0); reduced (1); absent (2).
16. P2 gnathobasic process; short (0); elongate (1).
17. P2 exopodite seta; present (0); absent (1).
18. P2 soft seta at base of first scraper; present (0); absent (1).
Supplementary Material
19. P3 number of exopodite setae; six (0); seven (1).
20. P3 length second exopodite seta; long, more than twice other setae (0); similar length (1).
21. P4 endite receptor after last flaming torch seta; marginal (0); on inner side (1).
22. P4 endite inner setae (1’–3’); three long plumose (0); three short naked (1);two reduced one long (2); all absent (3).
23. P4 apex of fifth exopodite seta; acute (0); blunt (1).
24. P4 apex of sixth exopodite seta; acute (0); blunt (1).
25. P4 flaming torch setae; well developed (1); reduced, with distal part missing (1).
26. P5 exopodite margin; concave (0); straight (1); convex (2).
27. P5 filter comb; three setae (0); two setae (1); absent (2).
28. P5 first seta of inner portion; twice as long as inner lobe (0); as long as inner lobe or just longer (1).
29. P6; present (0); absent (1).
30. Postabdomen marginal denticles; merged (0); unmerged (1).
31. Postabdomen dorsodistal gap (between basal claw and dorsodistal margin); open (0); closed (1); absent (2).
Appendix 2. Character data matrix. Data for morphology based on Alonso (1996), Van Damme et al. (2003), Kotov
(2004), Kotov & Sanoamuang (2004), this study (Phreatalona) and unpublished data (quadrangularis). Characters 10 and
14 were indicated as parsimony-uninformative by PAUP.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Alona
quadrangularis 2 0 0 1 1 0 1 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 2 2 0 1 0 2
Alona affinis 2 0 0 0 1 0 1 1 0 2 1 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2
Acroperus
harpae 2 1 0 1 1 0 0 0 0 1 1 0 1 1 1 0 0 0 1 1 1 1 0 0 1 0 0 1 0 1 1
Nicsmirnovius
eximius 2 1 0 2 1 1 1 0 0 1 2 0 0 1 2 1 1 1 0 1 1 2 0 1 0 1 2 1 1 1 2
Nicsmirnovius
greeni 2 1 0 2 1 1 1 0 0 1 2 0 0 1 2 1 1 1 0 1 1 2 0 1 0 1 1 1 1 1 2
Nicsmirnovius
fitzpatricki 2 1 0 2 0 1 0 0 0 1 2 0 0 1 2 1 1 1 0 1 1 2 0 1 0 1 1 1 1 1 2
Nicsmirnovius
camerounensis 0 1 0 2 0 1 0 0 0 1 2 0 0 1 2 1 1 1 0 1 1 2 0 1 0 1 ? 1 1 1 2
Phreatalona
labrosa 2 1 0 1 0 1 1 1 1 2 1 1 1 1 2 1 0 0 1 1 1 3 0 0 0 1 2 1 1 1 0
Phreatalona
smirnovi 0&1 1 1 1 0 1 1 1 1 2 0 1 1 1 2 1 0 0 1 1 1 3 1 1 1 0 2 1 1 1 1
Phreatalona
protzi 2 1 0 1 0 1 1 1 1 2 0 1 1 1 2 1 0 0 1 1 1 3 1 1 1 0 2 1 1 1 1
Phreatalona
phreatica 1 1 1 1 0 1 1 1 1 2 0 1 1 1 2 1 0 0 1 1 1 3 1 1 1 0 2 1 1 1 0
2009Hydrobiologia Volume 618 pp. i–iv + 1–204
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Hydrobiologia
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1 February 2009 • Volume 618 • ISSN 0018-8158 • Code: HYDRB88
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