European Journal of Taxonomy 164: 1-28

http://dx.doi.org/! 0.5852/ej t .2015.164

ISSN 2118-9773

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2015 • Brad T. et al.

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Research article

um:lsid:zoobank.org:pub:6EC6F517-B4A2-4221-B8F0-678A59557896

Niphargus dancaui sp. nov. (Amphipoda, Niphargidae) - a new species

thriving in sulfidic groundwaters in southeastern Romania

Traian BRAD 1 *, Cene FISER 2 , .lean-Francois FLOT 3 , Serban M. SARBU 4

1 “Emil Racovifa” Institute of Speleology, str. Clinicilor 5, 400006 Cluj-Napoca, Romania

department of Biology, Biotechnical Faculty, University of Ljubljana,

Vecnapot 111, 1000 Ljubljana, Slovenia.

3 Universite Libre de Bruxelles (ULB), Evolutionary Biology & Ecology, C.R 160/12,

Avenue F.D. Roosevelt 50, 1050 Brussels, Belgium.

4 "Emil Racovifa” Institute of Speleology, str. Frumoasa 31, 010986 Bucure§ti, Romania.

* corresponding author: [email protected]

2 Email: [email protected] .si

3 Email: [email protected]

4 Email: [email protected]

1 urn:lsid:zoobank.org:author:A2A9B356-4C75-4C61-9724-99538B8DF3CA

2 urn:lsid:zoobank.org:author:292536DA-ADC2-4C50-8E6F-DF5CBA2ED4F5

3 um:lsid:zoobank.org:author:D171C848-B6B3-46F8-B422-EBD205C5F347

4 um:lsid:zoobank.org:author:3A7EFBE9-5004-4BFE-A36A-8F54D6E65E74

Abstract. Niphargus dancaui sp. nov., previously referred to as Niphargus cf. stygius , was sampled

from various groundwater sites in and near the town of Mangalia (SE Romania) and described with

Movile Cave (a sulfidic, chemoautotrophically based ecosystem) as type locality. A short comparison

with Niphargus stygius specimens from Slovenia was made, together with a morphological analysis

of interpopulational variability. Males of N. dancaui sp. nov. were relatively large (17 mm), with long

antennae, pereiopods and uropod III. Females were slightly smaller, with shorter antennae, pereiopods

and uropod III. Interpopulational variability was noticed in the chaetotaxy of the telson lobes and uropod

III. N. dancaui sp. nov. is morphologically very close to N. lessiniensis and N. tridentinus , two species

present in northern Italy, but distinct genetically from them based on 28S rRNA sequences. Instead,

the closest relative of N. dancaui sp. nov. sequenced so far for this marker is N. montanarius , which

inhabits a sulfidic cave system in central Italy. The work presented here contributes to our knowledge

of groundwater crustacean biodiversity in general and of the systematics of the genus Niphargus in

particular.

Keywords. 28S phylogeny, hypogenic cave, interpopulational diversity, Movile Cave.

Brad T., FiserC., Flot J.-F. & Sarbu S.M. 2015. Niphargus dancaui sp. nov. (Amphipoda, Niphargidae) - a new

species thriving in sulfidic groundwaters in southeastern Romania. European Journal of Taxonomy 164: 1-28.

http://dx.doi.org/10,5852/eit.2015.164

European Journal of Taxonomy 164 : 1-28 ( 2015 )

Introduction

The amphipod genus Niphargus Schiodte, 1849 is widely distributed in the western Palearctic and

represents the most speciose genus of freshwater amphipods in the world (Vainola et al. 2008). This

genus is taxonomically demanding and its species richness is far from being completely described.

The morphology of niphargids is not a very good proxy for the phylogenetic relationships among these

species (Fiser et al. 2008; Trontelj et al. 2012; Flot et al. 2014), and cryptic species (i.e., species that are

nearly indistinguishable from a morphological point of view but possess distinctive DNA sequences)

appear common in this group (Trontelj et al. 2009).

Most Niphargus species are confined to subterranean waters (Fiser 2012), where they represent the

dominant group of macroinvertebrates. Niphargus thrives in all types of subterranean waters, from shallow

hypotelminorheic ones (i.e., shallow seeps filled with groundwater; Fiser et al. 2010) to epikarst (Sket

1981), sinking streams and phreatic lakes (Trontelj et al. 2012). Niphargid amphipods have also been

noted for their dominant presence in hypogenic caves, i.e., caves that result from limestone dissolution

by hydrogen sulfide arising from deep reservoirs (Latella et al. 1999; Sarbu et al. 2000; Forti et al. 2002;

Flot et al. 2010, 2014). These sulfidic caves are of particular interest from an ecological viewpoint:

instead of sunlight (photoautotrophy) or allochthonous organic material (organoheterotrophy), they

derive their energy from inorganic chemical compounds (chemoautotrophy; Sarbu et al. 1996, 2000).

Although the fauna of these unique ecosystems should be highly protected, not all Niphargus species

present in hypogenic caves have been described. As incomplete taxonomy is a major impediment to

conservation biology, the present study aims to contribute to bridging this gap.

The two best-studied hypogenic cave ecosystems in Europe are the Frasassi cave system in Italy and

Movile Cave in Romania. The niphargid fauna of Frasassi was recently studied molecularly, revealing

the presence of four species (Flot et al. 2010; Karaman et al. 2010). A similar molecular study conducted

in Romania revealed the presence of seven niphargid species in and around Movile Cave: Niphargus cf.

stygius (Schiodte, 1847), N. decui Karaman & Sarbu, 1995, N. dobrogicus Dancau, 1964, N. gallicus

Schellenberg, 1935, N. hrabei Karaman, 1932, Pontoniphargus racovitzai Dancau, 1970, and P. ruffoi

Karaman & Sarbu, 1993 (Flot et al. 2014; Fiser et al. 2015). The sulfidic waters of Movile Cave are

host to two of these species. One of them, P. racovitzai , was originally described as belonging to the

distinct genus Pontoniphargus Dancau, 1970, although molecular analyses show it clearly nested within

the genus Niphargus (Flot et al. 2014). The second species was called Niphargus cf. stygius by Dan

Dancau in the 1980s (in an unpublished determination) as it presented morphological similarities with

Niphargus stygius (Schiodte, 1847). Sarbu & Popa (1992) considered it as a putative new species, and

a recent molecular analysis provided additional support for this hypothesis (Flot et al. 2014). However,

this species was never named nor properly described.

Material and methods

Morphological analysis

Niphargus cf. stygius specimens were sampled from existing wells in the town of Mangalia (SE

Romania), from Movile Cave and from a spring in Hagieni Forest (Table 2, Fig. 1). In Mangalia, we either

descended in the wells and picked specimens directly from the walls; pulled and dragged a planktonic

net through the mass of water in the well; or examined large amounts of water (typically 100-150 liters)

brought up to the surface using a rope and a bucket; in Movile Cave, we picked specimens directly from

the edges of the cave lake; in Hagieni Forest spring, we collected them under the rocks and in detritus.

Niphargids were immediately transferred to 70% or 96% ethanol to allow for both morphological and

genetic analyses. Morphological inspection, drawings and measurements were performed using an MBS-

1 stereo microscope (Lytkarino, USSR) and a Carl Zeiss microscope (Jena, Germany). The terminology

used for body parts and the choice of appendages taken into consideration for measurements were as in

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

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Fig. 1. Map of the research area with indication of sampling locations

European Journal of Taxonomy 164 : 1-28 ( 2015 )

Fiser et al. (2009). True spines, i.e., extrusions of cuticle, are not known in Niphargus (Fiser et al. 2009).

Species of this genus have appendages armed with flexible thin setae, flexible plumose setae and stout

spiniform setae. To simplify descriptions, we refer to the thin flexible setae as ‘setae’ and stout spiniform

setae as ‘spines’ as in Svara et al. 2015.

Molecular analyses

To shed light on the relationships of Niphargus cf. stygius with other species of the genus Niphargus ,

all nuclear ribosomal 28S sequences of members of the Niphargidae available on 28 Jul. 2015 were

downloaded from GenBank (Benson et al. 2015). This dataset of 494 sequences was then completed

with unpublished 28S sequences of topotypes of N. montellianus Stoch, 1998 and A r . tridentinus Stoch,

1998 (sequenced using the same protocol as in Flot et al. 2010; GenBank accession numbers KT878856

and KT878857) and pre-aligned in MAFFT using the FFT-NS-i mode to identify unsuitable sequences

(originating from a different 28S region than our target region). After filtering out these sequences, the

remaining dataset of 422 sequences from Lefebure et al. (2006, 2007), Fiser et al. (2008), Trontelj et al.

(2009), Flot (2010), Flot et al. (2010, 2014), Hartke et al. (2011), Trontelj et al. (2012), Hekmatara et

al. (2013), Fiser et al. (2013), Mclnemey et al. (2014), Altermatt et al. (2014), Ntakis et al. (2015) and

Esmaeili-Rineh et al. (2015) were screened for perfect duplicates using FaBox (Villesen 2007), resulting

in a dataset a 260 unique sequences, to which two outgroup sequences from the genera Synurella

Wrzesniowski, 1877 and Gammarus Fabricius, 1775 were added as in Flot et al. (2014). The sequences

were aligned in MAFFT version 7 (Katoh & Standley 2013) using the E-INS-i option and analyzed

in FastTree 2 (Price et al. 2010) using the GTR model (Lanave et al. 1984; Tavare 1986) with 1000

bootstrap replicates (Felsenstein 1985). The resulting Newick tree was turned into PDF using MEGA6

(Tamura et al. 2013), then beautified using Inkscape (Bah 2011).

The holotype and paratypes are deposited in the collection of the Department of Biology, Biotechnical

Faculty, University of Ljubljana.

Results

Order Amphipoda Latreille, 1816

Family Niphargidae Bousfield, 1977

Genus Niphargus Schiodte, 1849

Niphargus dancaui sp. nov.

urn: lsid: zoobank.org: act: 01A72B96-94E1 -401C-A6D0-4B4DD9C8D5 54

Figs 1-15

Niphargus cf. stygius - Sarbu & Pop a 1992: 651.

Diagnosis

Mid to large-sized Niphargus of robust appearance, with acute to right postero-ventral angle of epimeral

plates. Pleon with at most 4 setae along the posterior margin of each segment and a single tiny seta in

postero-lateral position on urosomite I. Antenna I shorter than half of the total body length. The outer

lobe of maxilla I has 7 spines with 1-3 teeth. The inner lobe of maxilliped has 9 spines. The propodus

of both gnathopods are almost square-shaped, with 5 setae along the outer margins of the dactyli. The

pereopods are shorter than half of the total body length, with one spine at the nail base. The uropods III

are sexually dimorphic and elongated in males. The male telson bears 3 apical spines, 1 lateral and 1

dorsal spine on each lobe, plus 1 subapical spine per lobe for the female telson.

BRAD T. et ah, Niphargus dancaui sp. nov., a new amphipod species from Romania

Etymology

The specific name is derived from the name of the late Dan Dancau (1933-1994), who first studied the

amphipod fauna in the Dobrogea region and described some of its species, notably Pontoniphargus

racovitzai from Doi Mai and Mangalia (Dancau 1970) and Niphargus dobrogicus from Doi Mai, Schitu

and Varna Veche (Dancau 1964).

Material examined

Holotype

ROMANIA: $, Movile Cave, Mangalia (Fig. 1, Table 2). The holotype specimen collected from location

1 in Fig. 1 was not dissected, but was deposited intact in 75% ethanol.

Paratypes

ROMANIA: Mangalia, 1 $, 3 $ $, well 12, Aleea Cetafli 1; 1 $, well 7, str. Avram Iancu 26; 1 $, well

10, str. Crinului 34; 1 §, well 3, str. General Dragalina; \S, 1 §, well 8, str. Horia, Clo§ca §i Cri§an 13.

Remark

The description was performed and species variability was examined on the basis of several paratypes

collected from various hand-dug wells in the town of Mangalia (SE Romania) (Fig. 1, Table 2). The

male and female described here were sampled from a well on Aleea Cetafii 1. The other specimens were

included in order to examine the variability.

Description (male)

The total male body length is 17.4 mm (Fig. 2A). A detailed chart containing the measurements of all

diagnosis-relevant body appendages for both male and female is presented in Table 1.

Head

The head (Fig. 3A) represents 8.5 % of total body length, no rostrum was observed.

Antennae

Antenna I (Fig. 3B) almost half of total body length (Table 1), with a flagellum formed of 32 articles.

Most flagellum articles bear one short aesthetasc (Fig. 3C). Tength of peduncle slightly more than

one third of the total length of antenna I. Accessory flagellum (Fig. 3D) biarticulated, the proximal

article exceeds half of second article of main flagellum, distal article is approximately one fifth of total

length of accessory flagellum. Antenna II (Fig. 3E) with flagellum formed of 13 articles, half as long as

antenna I. Peduncle almost twice as long as flagellum.

Mouthparts

Labium (Fig. 4A) bilobate; length of inn er lobes half length of outer lobes. Both inn er and outer lobes

with distally fine setae. Labium displayed in Fig. 4A from female sampled from str. Horia, Cloaca §i

Cri§an 13; identical to all inspected labia from other specimens, males and females.

Left mandible (Fig. 4B) with five teeth on incisor process, four teeth on lacinia mobilis and a row of

eight serrate spines between lacinia mobilis and molar process (Fig. 4C).

Right mandible (Fig. 4D) with four teeth on incisor process, several small denticles on lacinia mobilis

and a row of five denticulate setae between lacinia mobilis and molar process (Fig. 4E).

Two mandibular palps (Fig. 4B and Fig. 4D), highly similar and of same length. The three articles

represent 21% (article 1), 37% (article 2) and 42% (article 3) of total palp length (Table 1). Proximal

article without setae, article 2 with 8-11 ventral setae and article 3 with one group of 5-6 A setae, three

groups of 3M B setae, approximately 36 D setae and 5 E setae (Fiser et al. 2009).

European Journal of Taxonomy 164 : 1-28 ( 2015 )

Fig. 2. General appearance of Niphargus dancaui sp. nov. A. f. B. $.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Fig. 3. $. A. Head. B. Antenna I. C. Aesthetascs of antenna I. D. Accessory flagellum of antenna I.

E. Ante nn a II.

European Journal of Taxonomy 164: 1-28 (2015)

Table 1. Measurements in mm of the various appendages of male and female Niphargus dancaui sp. nov.

sampled from a well located on Aleea Cetapi 1 in the town of Mangalia (SE Romania).

Appendage

Male

Female

Appendage

Male

Female

Body length

17.4

14.3

PHI

Coxal plate depth

1.4

1.3

Head length

1.5

1.4

Coxal plate width

1.0

Antenna I

Peduncle length

3.1

2.3

Basis

2.0

1.7

Flagellum length

5.2

3.4

Ischium

0.3

0.2

Art 1 FI. ace.

0.2

Merus

1.2

1.0

Antenna II

Peduncle length

2.6

2.0

Carpus

0.7

0.6

Flagellum length

1.3

1.1

Propodus

1.0

0.7

Mandible palp

Art 1

0.3

0.2

Dactylus 1

0.2

Art 2

0.5

0.4

Dactylus 2

0.2

0.1

Art 3

0.6

0.4

PIV

Coxal plate depth

1.4

1.3

Gnathopod I

Propodus length

1.2

0.9

Coxal plate width

1.4

1.0

Propodus width

1.0

0.8

Basis

2.0

1.7

Propodus depth

0.9

0.8

Ischium

0.3

Dactylus 1

0.9

0.6

Merus

1.0

0.8

Dactylus 2

0.3

Carpus

0.8

0.6

Carpus length

0.8

0.5

Propodus

0.9

0.7

Basis width

0.6

0.5

Dactylus 1

0.2

Basis length

1.4

1.1

Dactylus 2

0.2

0.1

Coxal plate depth

1.3

1.1

Basis length

1.7

1.5

Coxal plate width

0.8

0.7

Basis width

1. 3

1.0

Gnathopod II

Propodus length

1.6

1.2

Ischium

0.3

Propodus width

1.5

1.2

Merus

1.0

0.9

Propodus depth

1.2

1.0

Carpus

1.2

1.0

Dactylus 1

1.3

1.0

Propodus

1.3

1.0

Dactylus 2

0.4

0.3

Dactylus 1

0.2

0.1

Carpus length

1.0

0.8

Dactylus 2

0.1

Basis width

0.6

0.5

PVI

Basis length

1.9

1.7

Basis length

1.9

1.7

Basis width

1.3

1.1

Coxal plate depth

1.3

1.0

Ischium

0.3

Coxal plate width

1.1

0.9

Merus

1.3

1.1

Uropod I

Basis length

1.4

1.3

Carpus

1.7

1.5

Endopodite length

1.1

1.0

Propodus

2.1

1.8

Exopodite length

1.1

1.0

Dactylus 1

0.3

0.2

Uropod II

Basis length

0.8

0.6

Dactylus 2

0.2

0.1

Endopodite length

0.7

PVII

Basis length

2.0

1.6

Exopodite length

0.8

0.6

Basis width

1.3

1.1

Uropod III

Basis length

0.9

0.5

Ischium

0.3

0.4

Endopodite length

0.5

0.3

Merus

1.2

1.0

Exopodite art 1

3.4

2.1

Carpus

1.7

1.4

Exopodite art 2

3.2

0.3

Propodus

2.4

2.0

Telson

Cleft length

0.5

0.4

Dactylus 1

0.4

0.2

Telson width

0.6

0.5

Dactylus 2

0.2

0.1

Telson length

0.8

0.7

Longest spine length

0.2

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Table 2. List of sampling locations with their geographic position and physico-chemical characteristics.

The numerals in the first colu mn relate to the location numbers in Fig. 1. The specimens dissected and

analyzed for this study were selected from the locations highlighted in grey.

Nr.

Location

Latitude Longitude

N E

T C

jaS cm 1

h 2 s

mg/1

Movile Cave

43°49 , 36” 28°33’43”

7.4

21.2

1071

8.3

Mangalia, str. Dumitru Ana 13

43°49’23” 28°34 , 01”

7.3

19.1

1052

3.4

Mangalia, str. General Dragalina 10

43°49 , 15” 28°34 , 08”

Mangalia, str. Matei Basarab 74

43°49 , 10” 28°34’06”

7.4

19.5

1550

12.2

Mangalia, str. Gheorghe Netoi 1

43°49 , 10” 28°34 , 12”

7.4

18.6

1078

4.5

Mangalia, str. Matei Basarab 62

43°49 , 09” 28°34 , 15”

Mangalia, str. Avram Iancu 26

43049 ’ 14 ” 28°34’22”

7.5

19.6

1540

6.4

Mangalia, str. Horia, Cloaca §i Crisan 13

43°49 , 18” 28°34’23”

7.7

18.7

1870

Mangalia, str. Ion Mecu 51

43°49 , 25” 28°34 , 29”

7.68

19.9

1135

2.2

Mangalia, str. Crinului 34

43°49 , 13” 28°34 , 4F’

7.43

20.2

1490

Mangalia, str. Pictor Tonitza 1

43°49 , 09” 28°35 , 03”

7.27

19.0

1242

Mangalia, Aleea Cetafii 1

43°48’53” 28°35 , 01”

7.42

19.3

1650

Mangalia, str. Mihai Viteazul 20

43°48 , 49” 28°34 , 50”

7.3

19.5

1770

Hagieni Spring

43°48 , 08” 28°28’29”

7.6

10.5

905

5.2

Maxilla I (Fig. 4F) with 7 apical setae on distal palp article. Outer lobe with 7 spines with 1-3 teeth,

inner lobe with 3 apical setae.

Maxilla II (Fig. 4G) with inner lobe slightly shorter than outer lobe; both lobes with numerous apical

setae.

Maxilliped (Fig. 4H) with palp formed of four articles. Article 2 with numerous setae in approximately

10 groups aligned along inner margin. Article 3 with three groups of 4-5 setae on inner margin, one

group of 3 setae on dorsal margin and one apical group with 5 setae. Article 4 without setae. Outer lobe

of maxilliped with 6 shorter, flattened spines and 7 longer, slightly hairy, apical spines. Inner lobe with

9 setae-like spines.

Gnathopod I

Gnathopod I (Fig. 5A) with relatively ovoid coxal plate with depth greater than its width (ratio

depth:width 1.0:0. 6 ). Basis length:width ratio 1.0:0.4. Ischium with one posteroventral group of 4 setae.

Basis length:carpus length 1.0:0. 6 . Carpus with two groups of 8-10 setae on ventral margin, and one

group of 4 setae located anterodorsally. Length:width ratio of propodus 1.0:0. 8 . Propodus with 7 groups

of 3M setae on ventral margin, one anterodorsal group with 6 setae and one antero-apical group of 4

setae. Two groups with 2-3 setae on lateral surface of propodus close to its ventral side,and two groups

of 4-5 setae closer to propodus dorsal margin. One group of 3 long setae present close to palmar spine.

Strong palmar spine and 4 outer denticulate spines in palmar corner. Dactylus (Fig. 5B) strong, with

claw representing one quarter of total dactylus length and with five setae along outer margin.

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 4. S, mouthparts. A. Labium. B. Left mandible. C. Detail (the incisor process and lacinia mobilis)

of left mandible. D. Right mandible. E. Detail (the incisor process and lacinia mobilis) of right mandible.

F. Maxilla I. G. Maxilla II. H. Maxilliped.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Gnathopod II

Coxal plate (Fig. 5C) with rectangular shape, deeper than wide (ratio depth:width 1.0:0.8). Basis

length:width ratio 1.0:0.3. Ischium with one anteroventral group of 2 setae. Basis length:carpus length

1.0:0.6. Carpus has with two groups of 8-10 setae on ventral margin and one group of 2 setae located

anterodorsally. Propodus almost square-shaped, with length:width ratio of 1.0:0.96. Propodus with 7

groups of 2—4 setae on ventral margin, one anterodorsal group of 2 setae, and one apical group of 4 setae.

Lateral surface of gnathopod II propodus without setae, with only two long setae close to palmar spine.

Fig. 5. <$■ A. Gnathopod I. B. Propodus of gnathopod I. C. Gnathopod II. D. Propodus of gnathopod II.

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 6. <f. A. Pereopod III. B. Dactylus of pereopod III. C. Pereopod IV. D. Dactylus of pereopod IV.

E. Pereopod V. F. Dactylus of pereopod V.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

One strong spine and one outer, smaller spine on palmar comer. Dactylus (Fig. 5D) strong, with claw

representing one quarter of total dactylus length and with five setae along outer margin.

Pereopod III

Coxal plate of pereopod III (Fig. 6A) with rectangular shape, with depth:width ratio of 1.0:0.7. Posterior

margin concave, with three setae. Gill irregularly ovoid. Dactylus (Fig. 6B) robust, with a nail measuring

half of total dactylus length; with one dorsal seta with plumose tip and one spine at nail base. Propodus

length:dactylus length ratio 1.0:0.34. Pereopod III nearly equal in length to pereopod IV (pereopod III

length:pereopod IV length ratio 1.0:0.95).

Pereopod IV

Coxal plate of pereopod IV (Fig. 6C) almost square-like, depth:width ratio 1.0:0.96. Posterior margin

concave, with four setae. Gill irregularly ovoid. Dactylus (Fig. 6D) robust, with nail slightly longer than

half of total dactylus length; with one dorsal seta with plumose tip, one spine and one seta with plumose

tip at nail base. Propodus length:dactylus length ratio 1.0:0.35.

Pereopod V

Coxal plate of pereopod V (Fig. 6E) shape of heart, with one small seta on anterior lobe. Basis with

ovoid-trapezoidal shape, with length:width ratio of 1.0:0.68. Basis with 9 spine-like setae on anterior

margin and 9 small setae on posterior margin. Dactylus (Fig. 6F) with one seta with plumose end on

outer margin and one spine and one smaller seta with plumose end at base of nail. Nail represents 41%

of total dactylus length.

Pereopod VI

Coxal plate of pereopod VI (Fig. 7 A) highly similar to that of pereopod V. Basis with ovoid-trapezoidal

shape, with length:width ratio of 1.0:0.68. Basis with 9 spine-like setae on anterior margin and 9 small

setae on posterior margin. Dactylus (Fig. 7B) with one seta with plumose end on outer margin and one

spine and one smaller seta with plumose end at base of nail. Nail represents 34% of total dactylus length.

Pereopod VII

The pereopod VII (Fig. 7C) is almost half of the total body length. The coxal plate pereopod VII is half-

ovoid, with one small seta on its posterior margin. The basis has a ovoid-trapezoidal shape, with a ratio

length: width of 1.0:0.66. The basis presents 6 spine-like setae on the anterior margin and 11 small setae

on the posterior margin. The dactylus (Fig. 7D) has one seta with a plumose end on the outer margin,

one spine and one smaller seta with a plumose end at the base of the nail. The nail represents 33% of the

total dactylus length.

Pereopods V: VI: VII equal 1.0:1.35:1.40.

Pleopods

Pleopods I—III (Fig. 8 A, Fig. 8B and Fig. 8C) highly similar, with rami of unequal length and 2 retinacles

each.

Uropod I (Fig. 8D) with two dorsolateral spines onto peduncle. Tength of endopodite equal to that of

exopodite, segments with a low number of spines. One strong spine at base of uropod I.

Uropod II (Fig. 8E) with three dorsolateral spines onto peduncle. Exopodite slightly longer than

endopodite, exopodite length:endopodite length ratio 1.0:0.88, both rami with a low number of spines.

Uropod III (Fig. 8F) long (43% of body length) and sexually differentiated. Protopodite with 4-5 small

apical spines. Endopodite as long as protopodite with two apical setae. Proximal segment of exopodite

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 7. $. A. Pereopod VI. B. Dactylus of pereopod VI. C. Pereopod VII. D. Dactylus of pereopod VII.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Fig. 8. $. — A-C. Pleopods with detail of retinacles A. Pleopod I. B. Pleopod II. C. Pleopod III. —

D. Uropod I. E. Uropod II. F. Uropod III. G. Epimeral plates. H. Urosome of $ sampled from str. Horia,

Clo§ca §i Cri§anl3.1. Telson.

European Journal of Taxonomy 164: 1-28 (2015)

almost equal to distal segment (ratio 1.0:0.94). Proximal segment with seven groups of 1-3 spines on

inner margin, and six groups with 1-3 spines on outer margin. Distal segment of exopodite with 8 spines

on inner margin, 6 spines on outer margin and 5 apical setae.

Epimeral plates

Epimeral plate I (Fig. 8G) with acute postero-ventral angle, convex ventral margin with no spines and

straight posterior margin with five setae.

Epimeral plate II (Fig. 8G) with right postero-ventral angle, straight posterior margin and convex ventral

margin. Two spines present along ventral margin, one strong spine in postero-ventral angle and two

shorter setae along posterior margin.

Epimeral plate III (Fig. 8G) slightly different compared to epimeral plate II; postero-ventral angle rather

acute, posterior margin slightly concave, ventral margin convex. Three spines present along ventral

margin and nine setae along posterior margin.

Urosomite I with two dorsolateral spines, whereas urosomite II with four dorsolateral spines of various

lengths. Dorsal margin of urosomite III spineless (Fig. 8H). Urosome on Fig. 8H belonged to S analyzed

from str. Horia, Clo§ca §i Cri§an 13. Number of setae on urosomites identical on all inspected specimens.

Telson

Telson (Fig. 81) slightly longer than wide (widthdength ratio 1.0:1.2). Three apical spines relatively

short, approximately one fifth of telson length. Telson with two fragile, plumose-ended setae along each

side, as well as one lateral spine and one dorsal spine per lobe.

Sexual dimorphism

The female (Fig. IB) is smaller (body length 14.3 mm) compared to the male. The female appendages

(Figs 9-14) are highly similar to those of males, with a few exceptions. Antenna I, with 22 articles,

reaches only one third of the total body length (Fig. 9B). Antenna II has just 9 articles (Fig. 9E). The

female gnathopods I and II (Fig. 11) are similar to those of the male, except that the female propodus

has a more inclined palmar margin, conferring it a rather trapezoidal shape in comparison to the more

rectangular shape of the male propodus (Fig. 5). The female telson (Fig. 14H) is slightly different from

the male one, with one sub-apical spine on each lobe. The uropod III (Fig. 14F) presents a distal segment

of the exopodite shorter than that of the male. For the female, the proximal segment: distal segment ratio

of the exopodite is 1.0:0.17, vs. 1.0:0.94 for the male.

Intraspecific variability

The intraspecific variability of Niphargus dancaui sp. nov. appears to be relatively low. The individuals

sampled from various locations differed mainly in age-related size. The male and female described here

were fully grown adults. These two specimens sampled from the well on Aleea Cetafii 1 were larger

and probably older than the other inspected specimens. However, their appendages, including their

mouth parts, gnathopods, pereopods, pleopods and uropods, were largely similar with a few exceptions.

The number of articles in the flagellum of antennae I and II appears variable, with numbers of articles

ranging from 17 to 24 and from 9 to 11, respectively. Although the telson has always three apical spines

and a pair of lateral spines its number of subapical and dorsal spines is variable (Fig. 141—J). The shape

of the epimeral plates is largely similar but the number of spines along their ventral margins ranges from

1 to 2 and from 1 to 3 in epimeral plates II and III, respectively.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Fig. 9. $. A. Head. B. Antenna I. C. Aesthetascs of antenna I. D. Accessory flagellum of antenna I.

E. Ante nn a II.

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 10. Female mouthparts. A. Left mandible. B. Right mandible. C. Maxilla I. D. Maxilla II.

E. Maxilliped.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Fig. 11. 5- A. Gnathopod L B. Propodus teeth of gnathopod I. C. Gnathopod II. D. Propodus teeth of

gnathopod II.

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 12. §. A. Pereopod III. B. Dactylus of pereopod III. C. Pereopod IV. D. Dactylus of pereopod IV.

E. Pereopod V. F. Dactylus of pereopod V.

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

Fig. 13. $ • A. Pereopod VI. B. Dactylus of pereopod VI. C. Pereopod VII. D. Dactylus of pereopod VII.

European Journal of Taxonomy 164: 1-28 (2015)

Fig. 14. — A-H. 5- A. Pleopod I (with detail of retinacles). B. Pleopod II (with detail of retinacles).

C. Pleopod III (with detail of retinacles). D. Uropod I. E. Uropod IT. F. Uropod III. G. Epimeral

plates. H. Telson. — I. Telson of 3 sampled from str. Avram Iancu 26. J. Telson of $ sampled from str.

Crinului 34.

BRAD T. et ah, Niphargus dancaui sp. nov., a new amphipod species from Romania

Discussion

Morphological affinities of Niphargus dancaui sp. nov.

Niphargus dancaui sp. nov. shares several similarities with A. stygius (Sleet 1974; personal observations).

Both species are mid- to large-sized, robust and have similar lengths of appendages (e.g., pereopods,

antennae) compared to their body lengths. Moreover, both species present similar mouthparts, gnathopods

(notably the shape of the propods), spine patterns on pereopod dactyli, sexually non-dimorphic rami of

uropod I, as well as similar sexually dimorphic traits such as elongated uropod III in males.

Yet, several details help distinguish N. dancaui sp. nov. from N. stygius. As pointed out by S. Karaman

(1952), N. stygius and some related species (A. novomestanus Karaman, 1952, N. likanus Karaman,

1952, N. podpecanus Karaman, 1952, N. kenki Karaman, 1952, N. karamani Schellenberg, 1935) share

four main traits: the outer margins of the dactyls of both gnathopods are armed with groups of setae, at

most 4 setae are present along the posterior margin of the pleon segments, the first urosomite segment

has a single tiny seta in a postero-lateral position, and the uropod III is sexually dimorphic. Some of

these characters are also present in N. dancaui sp. nov. (namely, its sexually dimorphic uropod III and

the fact that the dorso-posterior margin of its pleonites presents less than 4 setae). However, the setae

along the gnathopod dactyls of N. dancaui sp. nov. are not in groups, and the urosomite I of this species

has more than one seta, which sets N. dancaui sp. nov. apart from N. stygius and related species.

In this aspect, N. dancaui sp. nov. resembles some other species from the N. costozzae group living in

northern Italy (A. costozzae Stoch, 1998, A. / ess in ie ns is S to eh, 1998, A. monte llianus and A. tridentinus ;

see Stoch 1998 for a revision) or the A. sphagnicolus-N. plurispinosus group from Slovenia and Slovakia

(Rejic 1956; Hudec & Mock 2014). The latter group differs from A. dancaui sp. nov. by having more

than three dorsal spines per telson lobe. Niphargus costozzae and A. montellianus differ from A. dancaui

sp. nov. by having their setae along the gnathopod dactyli arranged in groups and by having more than

one dorsal spine per telson lobe. Niphargus lessiniensis and A. tridentinus , however, show striking

similarities to A. dancaui sp. nov. as described here. The only difference we found is the number of setal

groups along the proximal exopodite article of uropod III in males: in both Italian species up to five setal

groups can be found along the inner or outer margin of this article, whereas this number is slightly higher

in A. dancaui sp. nov. (up to seven groups of setae).

Position of N. dancaui sp. nov. in the niphargid tree of life

The updated 28S rRNA phylogeny of Niphargidae presented here (Fig. 15) is consistent with the ones

in Flot et al. (2014) and Mclnerney et al. (2014): notably, the position of the two species Niphargus

glenniei and A. irlandicus as a sister group to the rest of the genus is confirmed with a very strong

bootstrap support. This phylogeny shows that A. dancaui sp. nov. (black arrow) is genetically very

distinct from all the species to which it is morphologically similar (white arrows): instead, it may be

related to Niphargus montanarius and Niphargus sp. 4 from the Frasassi Cave system (Flot et al. 2010),

a relationship that only receives low bootstrap support.

Ecological data

Individuals of A. dancaui sp. nov. collected from various wells in the town of Mangalia, in Hagieni

Spring and in Movile Cave were largely similar regardless of the concentration of hydrogen sulfide

in the waters where they were collected (Table 2). These niphargids seem to be highly tolerant of the

presence of hydrogen sulfide in water, but are not dependent on it for their survival.

Niphargus dancaui sp. nov. is probably more widely distributed in the area than our 14 sampled locations

since it was found to occur in Hagieni Spring (Fig. 1), which is located at an aerial distance of 8 km from

the town of Mangalia. However, A. dancaui sp. nov. is probably endemic to the area of Mangalia, given

European Journal of Taxonomy 164: 1-28 (2015)

Niphargus tatrensis

Niphargus aggtelekiensis

- Niphargus cf. tatrensis

,— Niphargus pectinicauda

L Niphargus bajuvaricus

,- Niphargus multipennatus \

■J79

L Niphargus aberrans

— Niphargus labacensis

■ |- Niphargus sp. 2 VZ-2014

4 -- Niphargus strouhali alpinus

H r Niphargus longidactylus

Niphargus cf. aquilex

- Niphargus grandii

Niphargus tamanini

Niphargus pupetta

-^ Niphargus decui

Niphargus transsylvanicus

Niphargus sp. 2 INM-2013

Niphargus sp. 3 INM-2013

- Niphargus andropus

I— Niphargus cf. tauri

son— Niphargus wolfi

u I— Niphargus carniolicus

L Niphargus cf. tauri

Niphargus cf. aquilex

- Niphargus aquilex dobati

- Niphargus cf. tauri

100 pu Niphargus sp. 4 INM-2013

L, Niphargus bihorensis

- Niphargus cf. tauri

Niphargus cf. aquilex

Niphargus ambulator

- Niphargus sp. 12 SER-2013

Pontoniphargus racovitzai/ruffoi

Niphargus dobrogicus

- Niphargus gallicus

- Niphargus kieferi

Niphargus aquilex

^ Niphargus schellenbergi

Niphargus schellenbergi

Niphargus cf. aquilex

Niphargus cf. longidactylus

3 1 Niphargus aquilex

- Niphargus pachypus

- Niphargus delamarei

(I Niphargus virei

- Niphargus laisi

- Niphargus ladmiraulti

100 -^ Niphargus kochianus/dimorphopus

I— Niphargus irlandicus

- Niphargus glenniei

— Synurella sp.

- Gammarus sp.

3 Niphargus rhenorhodanensis

Niphargus rhenorhodanensis

^ Niphargus puteanus

r Niphargus thienemanni

I L Niphargus cf. fontanus

'— Niphargopsis casparyi

( Niphargus kenki

Niphargus rhenorhodanensis

■ Niphargus rhenorhodanensis

^ Niphargus rhenorhodanensis

Niphargus rhenorhodanensis

Niphargus foreli

. Niphargus sp. 4 JFF-2010

so! — Niphargus montanarius

L-j Niphargus dancaui

Niphargus s<p. 1 VZ-2014

r Niphargus frasassianus

gaP— Niphargus pasquinii

1] Niphargus cf. longicaudatus

L Niphargus cvijici

Niphargus longicaudatus

- Niphargus cf. longicaudatus

Niphargus cf. longicaudatus

Niphargus timavi

Niphargus hrabei

jh Niphargus plateaui

>us tridentinus \ <J

Niphargus costozzae <J=

\- \Niphargus montellianus\ ^ ■

¥4 Niphargus sphagnicolus <1=

gel Niphargus dolenianensis

l Niphargus thuringius

Niphargus sp. ROM

^ Niphargus sp. Djevojacka

Niphargus tridentinus

Niphargus lessiniensis <J=

- Niphargus rhenorhodanensis

r Niphargus cf. longicaudatus

Niphargus vinodolensis

Niphargus illidzensis dalmaticus

Niphargus elegans elegans

r Niphargus spoeckeri

^kiphargus hadzii

Niphargus stygius likanus

g0 r Niphargus novomestanus

1 Niphargus sp. N0098

- Niphargus brachytelson

— Niphargus podpecanus

r Niphargus elegans zagrebensis

I Niphargus zagrebensis

L. Niphargus illidzensis

l- Niphargus illidzensis

Niphargus slovenicus

- Niphargus cf. tauri

Niphargus laticaudatus

^ Niphargus sp. 1 INM-2013

Iranian clade (Esmaeili-Rineh et al. 2015)

t Niphargus sanctinaumi

Niphargus maximus

Niphargus cf. aquilex

Niphargus sp. 3 VZ-2014

Niphargus cf. aquilex

— Niphargus auerbachi

I-. Niphargus rhenorhodanensis

1— Niphargus lourensis

Niphargobates orophobata

r Niphargus pachytelson

Niphargus subtypicus

— Niphargus steueri kolombatovici

Niphargus dolichopus

5 t

Niphargus stenopus

Niphargus rejici

l Niphargus cf. arbiter

Niphargus arbiter

I— Niphargus ictus

3S|

Niphargus salonitanus

M— Niphargus cf. salonitanus

L Niphargus longiflagellum

— Niphargus stochi

- Niphargus spinulifemur

Niphargus foreli

gg j- Niphargus sp. N0115

i Niphargus stygius Q ■

Niphargus fontanus

Niphargus vjeternicensis bilecanus

Niphargus vjetrenicensis

r Niphargus balcanicus

Niphargus dabarensis

-Niphargus hercegovinensis

Niphargus vjetrenicensis kusceri

Niphargus trullipes

Niphargus polymorphus

- Niphargus factor

- Niphargus lunaris

2-4 Niphargus hvarensis

— Niphargus boskovici

- Niphargus krameri

- Niphargus cf. krameri

- Niphargus karamani

- Niphargus orcinus

0.01

BRAD T. et al., Niphargus dancaui sp. nov., a new amphipod species from Romania

that it was never found in the wells sampled in the neighboring villages (i.e., Limanu, Varna Veche, Doi

Mai, Arsa, Alberti, Vanatori, Coroana, Pecineaga, Dulce§ti, 23 August - data not shown).

Groundwater crustaceans are in general stenobiontic: they do not tolerate large fluctuations of the abiotic

conditions in their environment (Gibert 2001). In places inhabited by humans, groundwater ecosystems

are in general polluted with various anthropogenic wastes produced by industries, agricultural practices

or household activities. Niphargus dancaui sp. nov. therefore appears vulnerable to extinction according

to IUCN Red List categories and criteria. Apart from the occurrence of this species in Hagieni Spring, all

other recorded sampling locations (Movile Cave and in 12 old hand-dug wells in the town of Mangalia)

are spread over approximately 2 km 2 . The wells were used in the past as drinking water sources but have

now been replaced with a modern water supply system. As a result, most of these wells are abandoned or

are even being used for dumping various wastes, with potentially severe consequences for groundwater

crustaceans inhabiting the aquifer. It is our hope that the description of N. dancaui sp. nov. and its

recognition as a species endemic to Mangalia and its vicinity will lead to conservation measures to

protect the fauna of this unique sulfidic ecosystem.

Acknowledgements

Thanks to M. Baciu, D. Bianco, A. Cohn, S. Dattagupta, A. Hillebrand-Voiculescu, E. Piva and F. Stoch

for assisting with field work and/or collecting some of the specimens that were analyzed in this article.

Cene Fiser was funded by the Slovenian Research Agency, Program PI-0184.

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Manuscript received: 31 July 2015

Manuscript accepted: 22 October 2015

Published on: 16 December 2015

Topic editor: Rudy Jocque

Desk editor: Kristiaan Hoedemakers

Printed versions of all papers are also deposited in the libraries of the institutes that are members of the

EJT consortium: Museum national d’Histoire naturelle, Paris, France; Botanic Garden Meise, Belgium;

Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United

Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of

Denmark, Copenhagen, Denmark.