European Journal of Taxonomy 208: 1-29

http://dx.doi.org/10.5852/ejt.2016.208

This work is licensed under a Creative Commons Attribution 3.0 License.

ISSN 2118-9773

www. europeanj ournaloftaxonomy. eu

2016 • Delicado D. et al.

Research article

urn:lsid:zoobank.org:pub:F807AC79-2615-463B-8272-82523B46210A

Unraveling a new lineage of Hydrobiidae genera (Caenogastropoda:

Truncatelloidea) from the Ponto-Caspian region

Diana DELICADO 1 *, Vladimir PESIC 2 & Peter GLOER 3

1 Justus Liebig University Giessen, Department of Animal Ecology & Systematics,

Heinrich-Buff-Ring 26-32 IFZD-35392, Giessen, Germany.

2 University of Montenegro, Department of Biology, 81000, Podgorica, Montenegro.

3 Biodiversity Research Laboratory, Schulstr. 3, D-25491, Hetlingen, Germany.

"Corresponding author: [email protected]

2 Email: [email protected]

’Email: [email protected]

1 um:lsid:zoobank.org:author:52A050CB-4AE4-4EEF-8D5E-B508A0EC5AB2

2 urn:lsid:zoobank.org:author:B4FDC912-E185-43E0-868D-95BA1918576E

3 urn:lsid:zoobank.org:author:8CB6BA7C-D04E-4586-BAlD-72FAFF54C4C9

Abstract. Phylogenetic analyses of the highly diverse (non-marine aquatic) gastropod family Hydrobiidae

Stimpson, 1865 have revealed seven main lineages, most of which represent subfamilies. The subfamily

Pseudamnicolinae Radoman, 1977, and specifically the genus Pseudamnicola Paulucci, 1878 (mainly

inhabiting western and central Mediterranean regions), contributes substantially to this hydrobiid

richness. Most of its congeners have been described in terms of their shell and penis features, which are

of limited diagnostic value. Hence, the taxonomic status of some Pseudamnicola species needs to be

revised, particularly of those inhabiting marginal regions, such as the Ponto-Caspian domain, largely

occupied by the subfamily Pyrgulinae Brusina, 1882. Here we present a molecular phylogeny including

species of both subfamilies along with extended morphological descriptions to confirm assignments of

the Iranian species Pseudamnicola zagrosensis Gloer & Pesic, 2009; Sarkia kermanshahensis Gloer &

Pesic, 2009 (originally within Pseudamnicola) and P. saboori Gloer & Pesic, 2009. Our COI-based

tree rejects these assignments suggesting a new potential lineage, sister to the pyrgulinid species, and

comprising three genera: Shadinia Akramowski, 1976, Intermaria gen. nov. and Persipyrgula gen. nov.

These genera differ molecularly by 3.6%-8.5%, and are diagnosable by penis, female genitalia and

radula features. Our findings evidence the high morphological variability of pyrgulinid species and

provide insight into the origins and evolution of the freshwater Ponto-Caspian fauna.

Keywords. Freshwater snails, Pseudamnicola , Cryptic taxa. Anatomy, mtDNA.

Delicado D., Pesic V. & Gloer P. 2016. Unraveling a new lineage of Hydrobiidae genera (Caenogastropoda:

Truncatelloidea) from the Ponto-Caspian region. European Journal of Taxonomy 208: 1-29. http://dx.doi.

org/10.5852/eit.2016.208

European Journal of Taxonomy 208 : 1-29 ( 2016 )

Introduction

The family Hydrobiidae Stimpson, 1865 ( sensu Wilke et al. 2013) is thought to be the most diverse

family of non-marine aquatic gastropods, with ca 70 genus-level and 550 species-level taxa. Phylogenetic

analyses have recovered seven main lineages within this family, most of which represent subfamilies

(see Wilke et al. 2013). Among the genera contributing most to this remarkable richness is the genus

Pseudamnicola Paulucci, 1878 (subfamily Pseudamnicolinae Radoman, 1977). Despite the promotion

of its former congeneric subgenus Corrosella Boeters, 1970 to the genus level (see Delicado et al.

2015), Pseudamnicola still comprises around 70 nominal species distributed mainly across the western

and central Mediterranean region (Gloer et al. 2015). However, this richness may be overestimated

due to erroneous assignments to the genus since most of its species have been diagnosed according

only to a few shell features or at most to both shell and penis descriptions. Pseudamnicola species,

like most hydrobiids, are characterized by a minute, unsculpted shell and simple penis, such that only

comprehensive studies including the description of several anatomical structures and molecular data

have been able to distinguish among congeners (Hershler & Ponder 1998; Wilke et al. 2001; Arconada

& Ramos 2003; Strong et al. 2008). Morphological and molecular data may offer varying resolution in

cases of cryptic species (Wilke et al. 2002; Liu et al. 2003; Delicado & Ramos 2012) or when high

morphological disparity exists among close related taxa (Wilke et al. 2007; Delicado et al. 2014). Thus,

unraveling the systematics of the hydrobiids family calls for an integrative approach.

Although a systematic revision of Pseudamnicola is still pending, Delicado et al. (2015) revealed the

monophyly of Pseudamnicola s. str. and proposed a series of diagnostic characters including a simple

ovate-conic shell shape, single elongated seminal receptacle and broadly triangular penis with several

folds across its surface. Hence, we propose the revision of the taxonomic status of some Pseudamnicola

species originally described according to shell features only. These include species inhabiting Ponto-

Caspian regions, marginal areas of the family’s main distribution range, such as Iran (Gloer & Pesic

2009, 2012), Turkey (Yildirim et al. 2006; Gloer & Georgiev 2012; Gloer et al. 2014, 2015) and

Georgia (Badzoshvili 1979). One such species Sarkhia kermanshahensis (Gloer & Pesic, 2009) has been

recently assigned by Gloer & Pesic (2012) to an independent genus of unclear phylogenetic relationship.

Freshwater systems in these areas are mainly occupied by members of the subfamily Pyrgulinae (see

Radoman 1983; Wilke et al. 2007). These share some morphological and anatomical similarities with

Pseudamnicola (e.g., complete ctenidium, yellowish, oval operculum, pigmented renal oviduct and

nervous system), yet they also differ in terms of other features (e.g., shell shape and sculpture, penis

shape, number of seminal receptacles, number of basal cusps in the radular central tooth, etc.) and also

appear relatively distant from Pseudamnicola in the Hydrobiidae phylogeny (Wilke et al. 2013).

Through a molecular/anatomical approach, the present study revises the taxonomic status of the

Pseudamnicola species from Iran described by Gloer & Pesic (2009), i.e., P. zagrosensis Gloer &

Pesic, 2009, Sarkhia kermanshahensis (Gloer & Pesic, 2009) (originally described as Pseudamnicola

kermanshahensis) and P. saboori Gloer & Pesic, 2009. We also examined whether the springsnail genus

Shadinia Akramowski, 1976, cited only from a few localities of Iran’s neighbour Armenia, anatomically

and molecularly resembles the pyrgulinid taxa. So far, three species of this genus have been described

based both on shell characteristics (Shadin 1952; Egorov 2006) and some anatomical structures

(Akramovski 1976: figs 29, 30; Gloer et al. 2016). Their phylogenetic relationships within the family

Hydrobiidae, nevertheless, remain unclear. Accordingly, we here examine several anatomical structures

of the type species of Shadinia , S. terpoghassiani (Shadin, 1952), and obtained partial gene sequences

in this and the recently described species S. bjniensis Gloer et al ., 2016.

Our results indicate that the Pseudamnicola species described by Gloer & Pesic (2009) and the genus

Shadinia are likely pyrgulinid taxa. This information provides clues about the actual biodiversity and

biogeographic patterns of the hydrobiid subfamilies Pseudamnicolinae and Pyrgulinae, with possible

DELICADO D. et al., New hydrobiid genera from the Ponto-Caspian region

implications for on-going and future research efforts targeted at understanding the origins and evolution

of freshwater biodiversity, such as the projects PRIDE (Ponto-Caspian biodiversity Rise and Demise) or

SCOPSCO (Scientific Collaboration On Past Speciation Conditions in Ohrid).

Material and methods

To assess the taxonomic status of the three Pseudamnicola species from Iran described in Gloer & Pesic

(2009), we examined molecular and anatomical data of the original material collected by these authors,

together with a sample of Shadinia terpoghassiani (type species) collected from the type locality (Lake

Aiger-Lich, Metsamor, Armenia, 40.14288° N 44.17117° E) in 2008 as well as a molecular sequence

of Shadinia bjniensis. The Shadinia terpoghassiani sample is preserved in 80% ethanol and deposited

in the biodiversity collection at Justus Liebig University Giessen (UGSB). The third described species,

Shadinia akramowskii (Shadin, 1952), could not be relocated yet.

Total DNA obtained here de novo was isolated from one individual per species following the CTAB

protocol of Wilke et al. (2006). A partial 658-bp of the mitochondrial fragment cytochrome c oxidase

subunit I (COI) was PCR amplified with the primers LCO1490 and HC02198 (Folmer et al. 1994).

Thermal cycling conditions were as described in Delicado et al. (2012) and were conducted with an

annealing temperature of 48° C. Final PCR products were sequenced in an ABI3730 XL sequencer (Life

Technologies, Carlsbad, CA, USA) using a Big Dye Terminator kit v. 3.1 (Life Technologies). The new

sequences were deposited in GenBank under the accession numbers indicated in Table 1.

DNA sequences were edited in Sequencher 4.6 (Gene Codes, Ann Arbor, MI) and aligned manually in

PAUP* 4.0al23 (Swofford 2002) together with the sequences of other related hydrobiid species obtained

from GenBank (Table 1). Phylogenetic reconstruction was conducted using maximum likelihood (ML)

and Bayesian-inference (BI). ML analysis was performed in PHYML v3.0 (Guindon & Gascuel 2003)

using the evolutionary model selected in jModelTest v. 2.1.4 (Darriba et al. 2012) under corrected

Akaike’s information criterion (Akaike 1974; Sugiura 1978; Hurvich & Tsai 1989). The BI was run

through two independent runs of four Metropolis-coupled chains in MrBayes 3.1.2 (Huelsenbeck 2000;

Huelsenbeck & Ronquist 2001), with 5 million generations each and a sample frequency of 1000. The

analysis was terminated when the standard deviation of split frequencies reached values of < 0.01 in

MrBayes 3.1.2. Convergence between runs was in addition monitored by reviewing that each posterior

parameter reached values of effective sample size, estimated in Tracer 1.5 (Rambaut & Drummond

2009), greater than 200. The first 10% sampled trees were discarded as burn-in. The robustness of the

inferred topologies was assessed by bootstrapping (Felsenstein 1985) with 1000 pseudoreplicates in

ML, and by posterior probabilities (BPPs) of Bayesian trees.

The number of the dissected specimens and their respective localities are indicated in Tables 2 and 3.

Dissections and measurements were made with a Keyence VHX-2000E digital microscope in combination

with the program VHX-2000 Communication software version 2.3.5.0 (Keyence Corporation, 2009-

2012). Radulae were extracted from buccal mass by applying the first step of the established Proteinase

K protocol for DNA isolation (Wilke et al. 2006). After mounting on stubs and drying, radulae were

sputter coated with gold (Balter Sputter Coater SCD004) for 50 sec. in order to photograph them with a

field emission scanning electron microscope (FESEM) DSM982 Gemini (Carl Zeiss GmbH, Germany).

Morphological character states are based on the terminology of Hershler & Ponder (1998). Whorls were

counted according to the method of Ramos et al. (2000). The concentration of the nervous system was

calculated as the RPG ratio (Davis et al. 1976) and also characterized using the categories of Davis et

al. (1984, 1986, 1992) as follows: dorsal nerve ring concentrated (< 0.29); moderately concentrated

(0.30-0.49); elongated (0.50-0.67); extremely elongated (> 0.68). Analysis of variance ( ANOVA) was

applied to test for statistical significance among morphological dimensions of the Ponto-Caspian species

studied here. These calculations have been done using the package MBESS (Kelley & Lai 2011) for the

European Journal of Taxonomy 208 : 1-29 ( 2016 )

Table 1 . Locality name, GenBank accession numbers and original references for the species employed

in the molecular study.

Taxon

Locality

GenBank #

COI

Original reference

Outgroup

Mercuria similis

Italy, Friuli-Venetia-Julia, Udine,

Aquileia, Canale Panigai

AF367646

Wilke et al. 2001

Hydrobiinae

Hydrobia acuta

France, Herault, Etang du Prevost

AF278808

Wilke et al. 2000

Peringia ulvae

Russia, Lagoon ‘Levin navololC

AF118302

Wilke & Davis 2000

Salenthydrobia ferrerii

Italy, Lecce, Porto Cesareo, Torre

Lapillo, Bambinello Spring

AF449205

Wilke 2003

Ecrobia ventrosa

Great Britain, Snettisham Lagoon

AF 118335

Wilke & Davis 2000

Pseudamnicolinae

Pseudamnicola lucensis

Italy, Tuscany, Bagni di Lucca,

Bagni Caldi, thermal spring

AF367651

Wilke etal. 2001

Corrosella falkneri

Spain, Granada, Orce, La Armada

spring

JF312224

Delicado et al. 2012

Diegus gasulli

Spain, Almeria, Rambla de

Retamar

KF060743

Delicado et al. 2014

Pyrgulinae

Chilopyrgula sturanyi

Macedonia, Lake Ohrid, S of

Sveti Zaum

EF379284

Wilke et al. 2007

Dianella thiesseana

Greece, Lake Trichonis at Loutres

Mirtias

AY676127

Wilke et al. 2007

Ewcinipyrgula milachevitchi

Russia, Sea of Azov, Miusski

Liman

EF379290

Wilke et al. 2007

Falsipyrgula pfeiferi

Turkey, Isparta, Lake Egirdir

EF379296

Wilke et al. 2007

Ginaia munda munda

Macedonia, Lake Ohrid

JN398637

Schreiber et al.

2012

Ginaia munda sublitoralis

Macedonia, Lake Ohrid

JN398630

Schreiber et al.

2012

Intermaria kermanshahensis

Iran, Kermanshah Province, spring

between Sarab and Sahneh city

KT896670

Present study

Intermaria zagrosensis

Iran, Kermanshah Province, Sar

KT896669

Present study

Pol Kangarar village, Sar Pol

Kangarar stream

DELICADO D. et al. , New hydrobiid genera from the Ponto-Caspian region

Macedopyrgula pavlovici

Macedonia, Lake Ohrid

JN398635

Schreiber et al.

2012

Macedopyrgula wagneri

Macedonia, Lake Ohrid

JN398617

Schreiber et al.

2012

“MicromelanicT lincta

Romania, Lake Razim, Sarichioi

EP379292

Wilke et al. 2007

Ohridopyrgula macedonica

Macedonia, Lake Ohrid, Otesevo

EP379286

Wilke et al. 2007

Persipyrgula saboori

Iran, Khorrasan Province, Zou

Eram village, Zou Eram spring

KT896671

Present study

Pyrgula annulata

Italy, Brescia, Lake Garda,

Desenzano del Garda

AY341258

Wilke et al. 2007

Shadinia terpoghassiani

Armenia, south of Metzamor,

lake Aiger-Lich outflow

KT896672

Present study

Shadinia bjniensis

Armenia, Kotyak province, Bjni,

Hrazdan river

KT896673

Present study

Turricaspia sp.

Ukraine, Kherson, lower Dnieper,

near hydrobiological station

EP379294

Wilke et al. 2007

Xestopyrgula dybowskii

Macedonia, Lake Ohrid, S of

Sveti, Zaum

EP379288

Wilke et al. 2007

R statistical environment (R Development Core Team 2011). Resulting parameter values are shown in

Tables 2 and 3.

The localities are listed according to the code: spring or lake, city, province, country, co-ordinates,

altitude (when measured) and date of collection.

Abbreviations

Shell characters

AH = aperture height

AL = aperture length

AW = aperture width

LBW = length of body whorl

NSW = number of shell whorls

SL = shell length

SW = shell width

WAW — width of the antepenultimate whorl

WBW = width of the body whorl

WPW = width of the penultimate whorl

Anatomical characters

Ag =

albumen gland

Be =

bursa copulatrix

Cg =

capsule gland

Ct =

ctenidium

dBc =

duct of the bursa copulatrix

European Journal of Taxonomy 208: 1-29 (2016)

L = length

Os = osphradium

P = penis

Po = pallial oviduct

Pr = prostate gland

Ro = renal oviduct

SR = seminal receptacle

W = width

Collections

UGSB = Biodiversity collection at Justus Liebig University Giessen

ZMH = Zoologisches Museum Hamburg

Results

Phylogenetic inferences were based on 658 bp of the COI gene under the nucleotide substitution

model TrN (Tamura & Nei 1993) + I (invariable sites) + G (rate variation among sites). Average base

frequencies for the data set were 27.4% A, 14.1% C, 15% G and 43.5% T. Both tree topologies (ML and

BI) indicated that the Pseudamnicola species from the Ponto-Caspian region formed a monophyletic

group independent of the subfamily Pseudamnicolinae (Fig. 1). However, this monophyly was well

supported by ML (bootstrap value = 77%) but less supported by BI (BPP = 0.84). This newly discovered

lineage appeared as sister to the pyrgulinid clade with high support (85% of bootstrap in ML and 1.00 of

PP in BI). Species of Shadinia also grouped within this newly discovered lineage as sister to the species

P. zagrosensis and Sarkhia kermanshahensis. The close relationship between the latter taxa suggests

they could both belong to the same genus. Additionally, Shadinia and P. saboori may constitute two

different genera. Sequence differences between species of this lineage (uncorrected pairwise distance,

p-distance) ranged from 0.1% to 8.5% for COI (Table 4), and mean divergences between this lineage

and the Pseudamnicolinae and Pyrgulinae species were 13.6% and 13%, respectively.

Mercuria simiHs

99/1

69/0 78

83/0.84

69/0.97

88/1

67/0.68

77/ 0.84

85/1

96/1

70/0.94

100/1 I

50/0.91 —

99/1

80/0.90

100/1

0.2

Hydrobia acuta

Peringia ulvae

Salenthydrobia ferrerii

Ecrobia ventrosa

Diegus gasulii

Corrosella falkneri

Pseudamnicola lucensis

Shadinia terpoghassiani

Shadinia bjniensis

intermaria zagrosensis

Intermaria kermanshahensis

Persipyrguia saboori

Macedopyrgula pavlovici

Ginaia munda subiitoralis

Xestopyrgula dybowskii

Ohridopyrguia macedonica

Chilopyrgula sturanyi

Pyrgtila annulate

Macedopyrgula wagneri

Ginaia munda munda

Dianella thiesseana

Falsipyrgula pfeferi

Micromeiania iincta

r Turricaspia sp.

Euxinipyrgula milachevitchi

Hydrobiinae

Pseudamnicolinae

Pyrgulinae

Fig. 1 . Bayesian inference of hydrobiid species based on COI sequences. Values below branches indicate

bootstrap supports for maximum likelihood and BPPs for Bayesian inference. Black bars on the right

denote subfamily assignments. Scale bar: expected change per site.

DELICADO D. et al. , New hydrobiid genera from the Ponto-Caspian region

Morphological data also supported the existence of three different groups, which are hereafter treated

as independent genera within the newly recovered hydrobiid lineage. The inclusion of this group within

one or other subfamily is discussed in the following section.

Class Gastropoda (Cuvier, 1797)

Superorder Caenogastropoda Cox, 1960

Superfamily Truncatelloidea Gray, 1840

Family Hydrobiidae Stimpson, 1865

Genus Intermaria gen. nov.

urmlsid: zoobanlc.org: act: 1 CDF 6CD1-5D3C-447F -B AAE-7197FBEB2614

Diagnosis

Shell ovate-conic, 3 to 4.5 mm high; large and convex body whorl; rest of whorls small and slightly

convex; aperture pyriform, angled on top and often fused to the body whorl. Operculum corneous,

yellowish, thin, pliable, ellipsoidal, paucispiral with submarginal nucleus. Two pairs of basal cusps

on radular central tooth. Ctenidium occupying nearly the entire length of pallial cavity bearing well-

developed gill filaments. Osphradium opposite approximate middle of ctenidium. Bursa copulatrix

lying against the middle section of the albumen gland; pigmented renal oviduct; one elongate seminal

receptacle. Prostate gland bean-shaped, about twice as long as wide; exit of the pallial vas deferens from

the posterior-most section of the prostate gland and seminal vesicle entering the prostate gland in its

middle section; penis simple, gradually tapering, with its distal end tapered and often with a small distal

lobe on the inner edge. Nervous system with black pigmentation typically elongated.

Etymology

From Fatin inter- (between) -maria (seas), referring to the occurrence of the genus in the continental

area between the Mediterranean and the Caspian seas.

Type species

Pseudamnicola zagrosensis Gloer & Pesic, 2009.

Remarks

Intermaria species, like those of Pseudamnicola , have an ovate-conic simple shell, one elongated

seminal receptacle and a pigmented renal oviduct and nervous system. However, Intermaria differs in

having smaller shell dimensions (e.g., when compared with the 5 mm height of P. granjaensis Gloer &

Zettler, 2007 see Delicado et al. 2014), a more conic-shaped shell, two basal cusps on the central radular

tooth (one in Pseudamnicola ), a shorter prostate gland, and a gradually tapering penis with a small distal

lobe on the inner edge (penis is triangular with many surface folds and has a blunt end in Pseudamnicola

see Delicado et al. 2015).

Intermaria zagrosensis (Gloer & Pesic, 2009) comb. nov.

Figs 2-3, Tables 2-3

Pseudamnicola zagrosensis Gloer & Pesic, 2009: 37, pi. 6, figs 4-6.

New diagnosis

Shell ovate-conic, yellowish, with pyriform aperture; protoconch microsculpture wrinkled; central

radular tooth formula (5)4-C-4(5)/2-2; pyriform bursa copulatrix; one seminal receptacle elongate with

a short duct; penis gradually tapering, with a small distal lobe on inner edge, end tapered and grayish

pigmented; nervous system elongated (mean RPG ratio = 0.60) and slightly black pigmented.

European Journal of Taxonomy 208: 1-29 (2016)

Fig. 2. Shell, operculum and radula of Intermaria zagrosensis (Gloer & Pesic, 2009) gen. et comb. nov.

from Sar Pol Kangarar stream, Kermanshah Province, Iran. A-B. Shell in front view. C. Shell in lateral

view. D. Protoconch and microsculpture. E-F. Internal and external side of the operculum. G. Rows of

teeth of the radula. H. Central teeth. I. Detail of outer marginal teeth.

DELICADO D. et al.. New hydrobiid genera from the Ponto-Caspian region

Fig. 3. Anatomy of Intermaria zagrosensis (Gloer & Pesic, 2009) gen. et comb. nov. from Pol Kangarar

stream, Kermanshah Province, Iran. A. Ctenidium and osphradium. B. Partial nervous system. C. Female

genitalia. D. Bursa copulatrix and seminal receptacle. E-F. Head of a male and penis. G. Prostate gland.

Anatomical abbreviations given in the Material and methods section.

European Journal of Taxonomy 208: 1-29 (2016)

Table 2. Shell measurements (in mm) of the species: 1. Intermaria zagrosensis (Gloer & Pesic, 2009).

2. Intermaria kermanshahensis (Gloer & Pesic, 2009). 3. Persipyrgula saboori (Gloer & Pesic, 2009).

4. Shadinia terpoghassiani (Shadin, 1952). ANOVA results are shown through effect size F, degrees

of freedom (df), residuals (r) and the resulting significance (p-value: *** when 0; ** when 0.001;

* when 0.01; NS = no significant).

Mean ± SD; CV

ANOVA

(Max-Min)

(n= 11)

(n = 7)

(n = 20)

3.58 ±0.40; 0.11

3.30 ±0.36; 0.11

2.90 ±0.67; 0.23

3.02 ±0.25; 0.08

F 3,40= 61 ”

(4.50-2.98)

(4.00-2.80)

(4.40-2.48)

(3.61-2.60)

2.28 ±0.33; 0.15

2.00 ±0.13; 0.07

1.84 ±0.52; 0.28

1.68 ±0.14; 0.09

F =10 4***

3,40

(3.00-1.74)

(2.70-1.77)

(3.00-1.48)

(1.96-1.41)

SL/SW

1.58 ±0.10; 0.07

1.58 ±0.05; 0.03

1.59 ±0.08; 0.05

1.80 ±0.10; 0.06

F —IQ 4***

3,40

(1.71-1.45)

(1.66-1.50)

(1.70-1.48)

(2.05-1.63)

1.82 ±0.22; 0.12

1.62 ±0.13; 0.08

1.31 ± 0.15; 0.11

1.45 ±0.11; 0.08

F = 1 R 4***

3,37 •

(2.34-1.58)

(1.71-1.37)

(1.50-1.09)

(1.68-1.25)

SL-LBW

0.83 ±0.08; 0.10

0.75 ±0.08; 0.10

0.66 ±0.05; 0.07

0.84 ±0.11; 0.14

F = S 9**

3,37 ’

(0.97-0.67)

(0.83-0.61)

(0.75-0.62)

(1.08-0.66)

WBW

1.98 ±0.19; 0.09

1.92 ±0.12; 0.06

1.51 ±0.07; 0.04

1.58 ±0.13; 0.08

F =90 9***

^ 3,37

(2.26-1.58)

(2.07-1.76)

(1.62-1.44)

(1.88-1.37)

1.77 ±0.16; 0.09

1.67 ±0.11; 0.07

1.27 ±0.12; 0.09

1.41 ±0.12; 0.08

F 337 = 28.2***

(2.11-1.53)

(1.79-1.54)

(1.39-1.06)

(1.70-1.24)

1.36 ±0.09; 0.07

1.23 ±0.10; 0.08

0.97 ±0.05; 0.07

1.03 ±0.08; 0.08

F = 41 Q***

3,37

(1.50-1.18)

(1.35-1.06)

(1.06-0.90)

(1.13-0.86)

WPW

1.20 ± 0.12; 0.10

1.17 ±0.05; 0.04

0.96 ±0.05; 0.05

1.03 ±0.09; 0.08

F =19 Q***

3,37

(1.41-0.98)

(1.24-1.10)

(1.02-0.90)

(1.24-0.9)

WAW

0.13 ±0.02; 0.18

0.13 ±0.02; 0.19

0.10 ±0.02; 0.20

0.13 ±0.03; 0.24

p = i 4 NS

3,37 ’

(0.19-0.11)

(0.18-0.11)

(0.13-0.07)

(0.23-0.08)

NSW

4.15 ± 0.21; 0.05

4.37 ±0.14; 0.03

4.20 ±0.25; 0.06

4.13 ±0.17; 0.04

F = 2 6 NS

3,37

(4.50M.00)

(4.50M.25)

(4.50M.00)

(4.50-4.00)

Material examined

Holotype

IRAN: ZMH 51406: 4.5 mm height, 3.0 mm width.

Paratypes

IRAN: ZMH 51407 (5 ex.) and P. Gloer’s (68 ex.) collection.

Type locality

IRAN: Sar Pol Kangavar stream, Kangavar city, Kermanshah Province, 34°30' N, 47°55' E, 27 Jun.

2005.

DELICADO D. et al., New hydrobiid genera from the Ponto-Caspian region

Table 3. Ctenidium, osphradium. Female and male genitalia and nervous system measurements (in mm)

of the species: 1. Intermaria zagrosensis (Giber & Pesic, 2009). 2. Intermaria kermanshahensis (Giber &

Pesic, 2009). 3. Persipyrgula saboori (Giber & Pesic, 2009). 4. Shadinia terpoghassiani (Shadin, 1952).

For ANOVA results effect size F, degrees of freedom (df), residuals (r) and the resulting significance

(p-value: *** when 0; ** when 0.001; * when 0.01; NS = not significant) are shown.

Mean ± SD; CV

(Max-Min)

3 ?-2<?

Mean ± SD; CV

(Max-Min)

1 ?-3<?

Mean ± SD; CV

(Max-Min)

2?-2^

Mean ± SD; CV

(Max-Min)

5 $ -3

ANOVA

1.43 ±0.14; 0.09

0.93 ±0.08; 0.08

0.79 ±0.05; 0.07

1.12 ±0.08; 0.08

F 315 = 32.9***

(1.56-1.28)

(1.00-0.84)

(0.85-0.72)

(1.25-1.00)

Os F

0.41 ±0.09; 0.23

0.32 ±0.02; 0.05

0.24 ± 0.06; 0.24

0.32 ±0.06; 0.17

F =4 8*

3,15

(0.40-0.27)

(0.34-0.30)

(0.33-0.20)

(0.40-0.27)

OsW

0.20 ±0.00; 0.00

0.10 ±0.00; 0.00

0.12 ±0.03; 0.26

0.13 ±0.03; 0.21

F = 1 1 8***

3,15

(0.20-0.20)

(0.10-0.10)

(0.16-0.09)

(0.15-0.08)

PoF

2.29 ±0.20; 0.09

1.71

1.20 ±0.11; 0.09

1.66 ±0.22; 0.13

F 37 = 12.6**

(2.44-2.06)

(1.28-1.13)

(2.00-1.40)

PoW

0.71 ±0.08; 0.12

0.5

0.35 ±0.03; 0.10

0.53 ±0.06; 0.11

F 37 = 12.6**

(0.80-0.63)

(0.38-0.33)

(0.57-0.43)

AgP

1.11 ±0.13; 0.09

(1.23-0.98)

0.98

0.60 ±0.01; 0.01

(0.61-0.60)

0.75 ±0.04; 0.06

(0.80-0.68)

F 37 = 22.9***

CgP

0.13 ±0.05; 0.05

(1.17-1.07)

0.69

0.60 ±0.14; 0.23

(0.70-0.50)

0.93 ±0.09; 0.10

(1.00-0.77)

F =14 7**

3,7

BCF

0.71 ±0.11; 0.16

0.4

0.38 ±0.11; 0.27

0.35 ±0.07; 0.21

F 37 = 10.7**

(0.84-0.63)

(0.46-0.31)

(0.42-0.25)

BCW

0.39 ±0.04; 0.10

0.25

0.21 ±0.06; 0.27

0.21 ±0.05; 0.23

F 37 = 10.3**

(0.43-0.36)

(0.25-0.17)

(0.27-0.15)

dBCF

0.40 ±0.05; 0.11

0.45

0.38 ±0.03; 0.07

0.46 ±0.04; 0.10

F,, = 2 NS

(0.43-0.35)

(0.40-0.36)

(0.50-0.40)

SRIF

0.28 ±0.03; 0.11

0.24

0.08 ±0.01; 0.18

0.26 ±0.10; 0.41

F 37 =2.8 ns

(0.31-0.25)

(0.09-0.07)

(0.42-0.15)

SR II F

absent

0.16 ±0.04; 0.26

(0.22-0.12)

—

1.37 ±0.11; 0.08

1.00 ±0.00; 0.00

0.78 ±0.12; 0.15

1.09 ±0.23; 0.21

F =5 5*

A 3,6

(1.45-1.30)

(1.00-1.00)

(0.87-0.70)

(1.27-0.83)

0.30 ±0.00; 0.00

0.30 ±0.02; 0.07

0.17 ±0.03; 0.20

0.42 ±0.06; 0.13

F =17 7**

3,6

(0.30-0.30)

(0.32-0.28)

(0.20-0.15)

(0.47-0.36)

P P/Head

1.18 ± 0.14; 0.11

0.90 ±0.14; 0.16

0.92 ±0.11; 0.12

0.98 ± 0.15; 0.15

F 35 = 1.8 ns

(1.28-1.08)

(1.06-0.80)

(1.00-0.84)

(1.10-0.81)

ProF

1.10

0.84 ±0.11; 0.13

0.68 ±0.01; 0.01

0.76 ±0.03; 0.04

F 35 =7.7*

(0.94-0.72)

(0.69-0.68)

(0.78-0.72)

ProW

0.55

0.38 ±0.04; 0.10

0.34 ±0.01; 0.04

0.37 ±0.03; 0.08

F 3 _= 10.8*

(0.42-0.35)

(0.35-0.33)

(0.40-0.34)

RPG

0.61 ±0.03; 0.05

0.50 ±0.07; 0.13

0.52 ± 0.02; 0.05

0.45 ±0.03; 0.06

F =11***

3,10

(0.64-0.59)

(0.55-0.41)

(0.55-0.49)

(0.46-0.42)

European Journal of Taxonomy 208: 1-29 (2016)

Other localities

IRAN: Darband stream in Darband village (Azna to Dorud road, ca 16 km to Azna city), Lorestan

Province, 33°25' N, 49°17' E, ca 1800 m a.s.l., 23 Jun. 2005.

Description

Shell ovate-conic with 4^1.5 whorls, height 3—4.5 mm (Fig. 2A-C, Table 2); periostracum yellowish;

protoconch approximately 400 pm wide with 1.3 whorls and nucleus around 125 pm long; protoconch

microsculpture wrinkled (Fig. 2D); body whorl about 2 A of total length; rest of whorls slightly convex

with deep sutures; aperture complete, pyriform, often attached to body whorl on the top; thin inner

peristome but thicker than outer lip; peristome margin slightly sinuate (Fig. 2C).

Operculum with ca 2 whorls (Fig. 2E-F) and muscle attachment area oval and located near the nucleus.

Radula intermediate length (25% total shell length) bearing about 45 rows of teeth; central tooth formula

(5)4—C-4(5)/2-2 (Fig. 2G-H); lateral teeth formula 4-C-4; inner marginal teeth having 18-20 sharp

cusps; outer marginal teeth having 15-19 sharp cusps (Fig. 21).

Pigmentation and anatomy

Head dark brown pigmented from snout to neck; tentacles brown pigmented except on ocular lobes;

snout as long as wide, with medial lobation. Ctenidium extended across most of pallial cavity with

18-21 gill filaments; osphradium two times longer than wide and opposite middle of ctenidium (Fig.

3A, Table 3).

Nervous system with black pigmentation and elongate (mean RPG ratio 0.60, Table 3); cerebral ganglia

equal in size (Fig. 3B).

Female pallial oviduct with a capsule gland longer than albumen gland (Fig. 3C, Table 3); pyriform

bursa copulatrix with a duct longer than its length, and lying against the middle section of the albumen

gland; renal oviduct white from the insertion point of bursal duct to the seminal receptacle and hereafter

black making one or two loops; one elongate seminal receptacle with short duct (Fig. 3D).

Male genitalia with penis gradually tapering with a small distal lobe on inner edge; distal end tapered,

and grayish pigmented on the distal section (Fig. 3E-F); prostate gland twice as long as wide (Fig. 3G,

Table 3).

Remarks

This species anatomically resembles I. kermanshahensis, snails of I. zagrosensis , however, are larger

and present more cusps in lateral and marginal radular teeth, nervous system with higher RPG ratio, and

relatively larger bursa copulatrix and penis.

Ecology and distribution

Recorded in streams and springs from the Kermanshah and Forestan provinces (Iran).

Intermaria kermanshahensis (Gloer & Pesic, 2009) comb. nov.

Figs 4-5, Tables 2-3

Pseudamnicola kermanshahensis Gloer & Pesic, 2009: 38, pi. 6, figs 7-10.

Sarkhia kermanshahensis - Gloer & Pesic, 2012: 33, fig. 12h comb. nov.

DELICADO D. et al.. New hydrobiid genera from the Ponto-Caspian region

Fig. 4. Shell, operculum and radula of Intermaria kermanshahensis (Gloer & Pesic, 2009) gen. et

comb. nov. from a spring near Sarabe - Sahneh city, Kermanshah Province, Iran. A-B. Shell in front

view. C. Shell in lateral view. D. Protoconch and microsculpture. E-F. Internal and external side of the

operculum. G. Rows of teeth of the radula. H. Central teeth. I. Detail of outer marginal teeth.

European Journal of Taxonomy 208: 1-29 (2016)

New diagnosis

Shell ovate-conic, yellowish, with pyriform aperture; protoconch microsculpture slightly wrinkled;

central radular tooth formula 4-C-4/2-2; pyriform bursa copulatrix; one seminal receptacle elongate

with short duct; penis gradually tapering with small distal lobe on inner edge, end tapered; nervous

system elongated (mean RPG ratio = 0.50) with slight black pigmentation.

Material examined

Holotype

IRAN: ZMH 51404: 4 mm height, 2.7 mm width.

Paratypes

IRAN: ZMH 51405 (5 ex.) and P. Gloer’s (66 ex.) collection.

Type locality

IRAN: spring between Sarab and Sahneh cities, Kermanshah Province, 34°27' N 47°44' E, 27 Jun. 2005.

Description

Shell ovate-conic with 4.25—4.5 whorls, height 3-4 mm (Fig. 4A-C, Table 2); periostracum yellowish;

protoconch approximately 500 pm wide with 1.4 whorls and nucleus around 135 pm long; protoconch

microsculpture slightly wrinkled (Fig. 4D); body whorl about 2 Z total length; rest of whorls slightly

convex with deep sutures; aperture complete, pyriform, inner lip thicker than outer lip; peristome margin

straight (Fig. 4C).

Operculum with 2 whorls approximately (Fig. 4E-F) and muscle attachment area oval and located near

the nucleus.

Radula intermediate length (25% total shell length) bearing about 55 rows of teeth; central tooth formula

4—C—4/2-2 (Fig. 4G, H); lateral teeth formula 3-C-3; inner marginal teeth having 15-18 sharp cusps;

outer marginal teeth having 12-14 sharp cusps (Fig. 41).

Pigmentation and anatomy

Head light brown pigmented from snout to neck; tentacles also brown pigmented except on ocular lobes;

snout as long as wide, with medial lobation. Ctenidium extended across most of pallial cavity with

18-21 narrow gill filaments; osphradium three times longer than wide and opposite middle of ctenidium

(Fig. 5A, Table 3).

Nervous system with black pigmentation and elongate (mean RPG ratio 0.50); cerebral ganglia equal in

size (Fig. 5B, Table 3).

Female pallial oviduct with a capsule gland slightly shorter than albumen gland (Fig. 5C, Table 3);

pyriform bursa copulatrix with a duct as long as bursa length, and lying against the middle section of

the albumen gland; renal oviduct white from the insertion point of bursal duct to the seminal receptacle

and hereafter black making one or two loops; one elongate seminal receptacle with short duct (Fig. 5D).

Male genitalia with penis gradually tapering bearing a small distal lobe in the inner edge; slightly grayish

pigmented on the distal section in some specimens (Fig. 5E, F); prostate gland about two times longer

than wide (Fig. 5G, Table 3).

DELICADO D. et al ., New hydrobiid genera from the Ponto-Caspian region

Fig. 5. Anatomy of Intermaria kermanshahensis (Giber & Pesic, 2009) gen. et comb. nov. from a spring

near Sarabe - Sahneh city, Kermanshah Province, Iran. A. Ctenidium and osphradium. B. Partial nervous

system. C. Female genitalia. D. Bursa copulatrix and seminal receptacle. E-F. Head of a male and

penis. G. Prostate gland. Anatomical abbreviations given in the Material and methods section, except

DL (distal lobe).

European Journal of Taxonomy 208: 1-29 (2016)

Remarks

Apart from differences in body dimensions and in certain anatomical features (explained above), the

penial distal lobe is more prominent in this species than in I. zagrosensis. Uncorrected genetic distances

are, on the contrary, low between these two species (0.2% for COI fragment. Table 4), though sister taxa

in the subfamily Pyrgulinae are often genetically very close (e.g., COI p-distances of 0.3% between

species of the genus Macedopyrgula Radoman, 1973). Given this minor genetic variation between

congeners, these observed anatomical differences could potentially reflect intraspecific variation.

Ecology and distribution

Known only from a spring in the Kermanshah Province, Iran.

Genus Persipyrgula gen. nov.

urn:lsid:zoobank.org:act:6E9922EE-56CD-4BBC-BF4E-9224C75859C0

Type species

Pseudamnicola saboori Gloer & Pesic, 2009.

Diagnosis

Shell ovate-conic, 2.5 to 4.5 mm high; large and convex body whorl; rest of the whorls small and

slightly convex; aperture complete, pyriform, slightly angled on the top and separated from body whorl.

Operculum corneous, yellowish, thin, pliable, ellipsoidal, paucispiral with submarginal nucleus. From

one to two pairs of basal cusps in radular central tooth. Ctenidium occupying nearly the entire length

of pallial cavity and bearing well-developed gill filaments. Osphradium opposite approximate middle

of ctenidium. Bursa copulatrix posterior positioned relative to albumen gland; pigmented renal oviduct;

one small pyriform seminal receptacle without duct. Prostate gland bean-shaped, about twice as long as

wide; exit of the pallial vas deferens from the posterior-most section of the prostate gland and seminal

vesicle entering the prostate gland in its middle section; penis simple, gradually tapering, with a distal

end tapered. Nervous system with black pigmentation typically elongated.

Etymology

Referring to Persia, the historic name of the region where the genus was found, and to Pyrgula , type

genus of the subfamily Pyrgulinae.

Remarks

Though similar in shell features, this genus differs from Pseudamnicola mainly due to its small and

pyriform seminal receptacle in the female genitalia (long and elongate in Pseudamnicola ), shorter

prostate gland, tapered and simple penis (triangular and folded in the latter), and because of the occasional

possession of two pairs of basal cusps in the central radular tooth. Differences between Persipyrgula

gen. nov. and Intermaria gen. nov. are: shell dimensions (larger in the latter), aperture not fused to body

whorl in Persipyrgula gen. nov., one (occasionally two) vs two pairs of basal cusps in the central radular

tooth, small pyriform vs elongate seminal receptacle, and absence vs presence of a small distal lobe on

the inner edge of the penis, respectively.

Persipyrgula saboori (Gloer & Pesic, 2009) comb. nov.

Figs 6-7, Tables 2-3

Pseudamnicola saboori Gloer & Pesic, 2009: 36, pi. 6, figs 1-3.

DELICADO D. et al.. New hydrobiid genera from the Ponto-Caspian region

Fig. 6. Shell, operculum and radula of Persipyrgula saboori (Gloer & Pesic, 2009) gen. et comb. nov.

from Zou Eram spring in Zou Eram village, Khorrasan Province, Iran. A-B. Shell in front view. C. Shell

in lateral view. D. Protoconch and microsculpture. E-F. Internal and external side of the operculum.

G. Rows of teeth of the radula. H. Central teeth. I. Detail of outer marginal teeth.

European Journal of Taxonomy 208: 1-29 (2016)

New diagnosis

Shell ovate-conic, yellowish, with pyriform aperture; protoconch microsculpture wrinkled; central

radular tooth formula 5-C-5/(2)l—l(2); pyriform bursa copulatrix, with a duct longer than bursa; one

small pyriform seminal receptacle with no distinct duct; penis gradually tapering and simple, with distal

end tapered and grayish pigmented; nervous system elongated (mean RPG ratio = 0.52) with slight black

pigmentation.

Material examined

Holotype

IRAN: ZMH 51402: 4.4 mm height, 3.0 mm width.

Paratypes

IRAN: ZMH 51403 (5 ex.) and R Gloer’s (46 ex.) collection.

Type locality

IRAN: Zou Eram spring in Zou Eram village, Shirvan city, Khorasan Province, 37°20' N, 57°40' E, ca

1600 m, 11 Jun. 2005.

Description

Shell ovate-conic with 4M.5 whorls, height 2.5M.4 mm (Fig. 6A-C, Table 2); periostracum yellowish;

protoconch approximately 350 pm wide with 1.25 whorls and nucleus around 120 pm long; protoconch

microsculpture wrinkled, more intense on apex (Fig. 6D); body whorl about % total length; rest of

whorls slightly convex with a deep suture; aperture complete and pyriform; inner lip thicker than outer

lip; peristome margin straight (Fig. 6C).

Operculum with 2.5 whorls approximately (Fig. 6E-F) and muscle attachment area oval located near

the nucleus.

Radula intermediate length (30% total shell length) bearing about 50 rows of teeth; central tooth formula

5-C-5/(2) 1-1(2) (Fig. 6G-H); lateral teeth formula 3-C-3; inner marginal teeth having 15-18 cusps;

outer marginal teeth having 14-16 cusps (Fig. 61).

Pigmentation and anatomy

Head dark brown pigmented from snout to the penial base; tentacles also brown pigmented except

on ocular lobes; snout as long as wide, with medial lobation. Ctenidium in middle region of pallial

cavity with 18-20 narrow gill filaments; osphradium two times longer than wide and opposite middle of

ctenidium (Fig. 7A, Table 3).

Nervous system with black pigmentation and elongate (mean RPG ratio 0.52, Table 3); cerebral ganglia

equal in size (Fig. 7B).

Female pallial oviduct with a capsule gland and albumen gland similar in size (Fig. 7C, Table 3);

pyriform bursa copulatrix with a duct about 50-100% of bursa length; renal oviduct white straight from

the insertion point of bursal duct to where it begins to fold; hereafter black pigmented making a simple

loop; one small pyriform seminal receptacle with no distinct duct (Fig. 7D).

Male genitalia with penis simple, gradually tapering, with distal end tapered grayish pigmented (Fig. 7E-

F); prostate gland bean-shaped, about two times longer than wide (Fig. 7G, Table 3).

DELICADO D. et al ., New hydrobiid genera from the Ponto-Caspian region

Fig. 7. Anatomy of Persipyrgula saboori (Gloer & Pesic, 2009) gen. et comb. nov. from Zou Eram

spring in Zou Eram village, Khorrasan Province, Iran. A. Ctenidium and osphradium. B. Partial nervous

system. C. Female genitalia. D. Bursa copulatrix and seminal receptacle. E-F. Head of a male and penis.

G. Prostate gland. Anatomical abbreviations given in the Material and methods section.

European Journal of Taxonomy 208: 1-29 (2016)

Remarks

The small pyriform seminal receptacle and the posterior position of bursa copulatrix relative to albumen

gland are the exceptional features of this species related to the species comprising the new pyrgulinid

lineage found in this study. Moreover, P. saboori is the most distantly related taxa of this newly recovered

lineage (COI p-distances from 6.5 to 8.5%, Table 4).

Ecology and distribution

Only recorded in Khorasan and Markazi provinces (Iran).

Genus Shadinia Akramowski, 1976

Type species

Pyrgula terpoghassiani Shadin, 1952.

New diagnosis

Shell ovate-conic, 3 to 5 mm high; large and convex body whorl; rest of the whorls tall and convex;

aperture complete, pyriform, angled on the top and fused to the body whorl. Operculum corneous,

yellowish, thin, pliable, ellipsoidal, paucispiral with submarginal nucleus. From two to three pairs of

basal cusps in radular central tooth. Ctenidium occupying nearly the entire length of pallial cavity and

bearing well-developed gill filaments. Osphradium opposite approximate middle of ctenidium. Bursa

copulatrix lying against the middle section of the albumen gland; pigmented renal oviduct; two opposite

seminal receptacles, SRII smaller than SRI. Prostate gland bean-shaped, about twice as long as wide;

exit of the pallial vas deferens from the posterior-most section of the prostate gland and seminal vesicle

entering the prostate gland in its middle section; penis simple, gradually tapering, with a distal end

tapered dark pigmented. Nervous system with black pigmentation and moderately concentrated.

Shadinia terpoghassiani (Shadin, 1952)

Figs 8-9; Tables 2-3

Pyrgula terpoghassiani Shadin, 1952: 227

Pyrgula terpoghassiani - Akramowski 1952, nom. nud.

New diagnosis

Shell ovate-conic, yellowish, with two weak parallel keels along the body whorl; pyriform aperture;

protoconch microsculpture wrinkled; central radular tooth formula 3-C-3/(2)3-3(2); pyriform bursa

copulatrix; SRI elongate with a short duct and SRII smaller, globular and without duct; penis gradually

tapering and simple, with a distal end tapered and black pigmented; nervous system moderately

concentrated (mean RPG ratio = 0.45) and with slight black pigmentation.

Material examined

Type material not specified.

Type locality

ARMENIA: Take Aiger-Lich, south of Metzamor, Armavia province, 40° 08.573' N, 44°10.270' E,

18 May 2008.

DELICADO D. etal. New hydrobiid genera from the Ponto-Caspian region

Fig. 8. Shell, operculum and radula of Shadinia terpoghassiani (Shadin, 1952) from Lake Aiger-Lich,

Armenia. A-C. Shell in front view. D. Protoconch and microsculpture. E-F. Internal and external side

of the operculum. G. Rows of teeth of the radula. H. Central teeth. I. Detail of outer marginal teeth.

European Journal of Taxonomy 208: 1-29 (2016)

Fig. 9. Anatomy of Shadinia terpoghassiani (Shadin, 1952) from Lake Aiger-Lich, Armenia.

A. Ctenidium and osphradium. B. Partial nervous system. C. Female genitalia. D. Bursa copulatrix and

seminal receptacle. E-F. Head of a male and penis. G. Prostate gland. Anatomical abbreviations given

in the Material and methods section.

DELICADO D. et a!.. New hydrobiid genera from the Ponto-Caspian region

Description

Shell ovate-conic with 4M.5 whorls, height 2.6-3.6 mm (Fig. 8A-C, Table 2); periostracum yellowish;

protoconch approximately 385 pm wide with 1.3 whorls and nucleus around 125 pm long; protoconch

microsculpture wrinkled (Fig. 8D); body whorl about 2 /3 total length and bearing two weak spiral keels;

rest of whorls tall and convex with a deep suture; aperture complete, pyriform, with an inner lip thicker

than outer lip; peristome margin slightly sinuate.

Operculum with 2.5 whorls approximately (Fig. 8E-F) and muscle attachment area oval and located

near the nucleus.

Radula intermediate length (25% total shell length) bearing around 45 rows of teeth; central tooth

formula 3-C-3/(2)3-3(2) (Fig. 8G-H); lateral teeth formula 3-C-3; inner and outer marginal teeth

bearing 17-21 and 19-25 cusps, respectively (Fig. 81).

Pigmentation and anatomy

Head dark brown pigmented from snout to penial base; pigmentation clearer on neck; tentacles also

brown pigmented except on ocular lobes; snout as long as wide, with medial lobation. Ctenidium in

middle region of pallial cavity with 18-20 gill filaments; osphradium two to three times longer than

wide and opposite middle of ctenidium (Fig. 9A, Table 3).

Nervous system with black pigmentation and moderately concentrated (mean RPG ratio 0.45, Table 3);

cerebral ganglia equal in size (Fig. 9B).

Female genitalia with a capsule gland longer than albumen gland (Fig. 9C, Table 3); pyriform bursa

copulatrix lying against the middle section of the albumen gland; bursal duct longer than bursa length;

renal oviduct straight and white from the insertion point of bursal duct to SRII; hereafter black pigmented

making one or two loops; two opposite seminal receptacles; SRI pyriform with short duct and SRII

smaller, black pigmented, globular and sessile (Fig. 9D).

Male genitalia bearing a penis simple, gradually tapering, with a distal end tapered, and black pigmented

on the distal section (Fig. 9E-F); bean-shaped prostate gland about two times longer than wide (Fig. 9G,

Table 3).

Remarks

Specimens from the type locality varied in shell dimensions and whorl convexity (Fig. 8A-C and

Shadin 1952: fig. 51), though they were similar in their anatomical features. So far, Shadinia is the

only pyrgulinid genus bearing two seminal receptacles (for a morphological review of the Pyrgulinae

subfamily see Radoman (1983); the anatomy of Pyrgula Cristofori & Jan, 1832 and Dianella Gude,

1913 is described in Szarowska 2006). Size of seminal receptacles varies slightly between the three

anatomically known species, being smaller in S. terpoghassiani than in S. bjniensis. Moreover, some

specimens of S. terpoghassiani are larger (shell height S. bjniensis 3.6-4.0 mm, S. terpoghassiani

5.2 mm, see Shadin 1952) and often two weak spiral keels are present in the shell body whorl. Three

basal cusps in the central radular tooth are also present in S. akramowskii (Shadin 1952). The species of

Shadinia here analyzed differ from each other by 0.4% COI p-distances (Table 4).

Ecology and distribution

Armenia and Nakhchivan province of Azerbaijan (Akramowski 1976).

Table 4. Uncorrected COI p-distances between species of the subfamilies Pyrgulinae and Pseudamnicolinae.

European Journal of Taxonomy 208: 1-29 (2016)

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DELICADO D. et al., New hydrobiid genera from the Ponto-Caspian region

Discussion

Our results were consistent with our proposed hypothesis of the incorrect assignment of the Pseudamnicola

species from Iran described by Gloer & Pesic (2009). Thus, molecular and morphological analyses

revealed that shell shape and penis morphology alone are not sufficiently informative for genus

assignment. Our phylogenetic data recovered those Iranian species and the genus Shadinia as a potential

monophyletic group appearing outside Pseudamnicola and the subfamily Pseudamnicolinae (Fig. 1).

A sister relationship between this new lineage and other pyrgulinid species was identified (despite the

limited resolution of COI for inferring phylogenetic relationships on the subfamily level). However,

p-distances between the Ponto-Caspian genera and the molecularly analyzed Pseudamnicolinae and

Pyrgulinae taxa proved similar (13.6% and 13%, respectively). In effect, our phylogenetic reconstruction

suggests these new taxa may comprise a different subfamily intermediate between the other two.

Notwithstanding, this would need confirmation through more comprehensive sampling of the area and

examining other potential new representatives of this lineage. As more shared synapomorphies with the

pyrgulinid group were detected (such as a tapered penis, zero to two seminal receptacles, zero to three

pairs of basal cusps in the central radular tooth and spiral keels on the shell, see Radoman 1955, 1983)

than with Pseudamnicola (diagnosed by, one elongate seminal receptacle, one pair of basal cusps in the

central radular tooth, triangular penis with folds and simple shells slightly longer than wide, of which the

latter two are not present in Pyrgulinae, see Boeters 1988; Szarowska et al. 2009; Delicado et al. 2014),

we tentatively consider these taxa as belonging to the subfamily Pyrgulinae.

Despite the low p-distances characterizing these Ponto-Caspian species, morphological evidence

led us to consider them as different genera, here described as Intermaria gen. nov. and Persipyrgula

gen. nov. This pattern of high morphological variability between closely related genera has been also

observed in other pyrgulinid groups (see Wilke et al. 2007). In addition, the discovery of these genera

suggests that other species from the Ponto-Caspian region assigned to Pseudamnicola according to

shell shape and penis morphology could effectively be members of this newly discovered lineage or

even constitute other clades. For instance, P. kayseriensis Gloer, Yildirim & Kebapgi, 2015, P. gullei

Gloer, Yildirim & Kebap£i, 2015 and P. vinarskii Gloer & Georgiev, 2012 from Turkey bear similar

shell and penis morphologies as the genera here described, and despite a more conical shell, the species

Fig. 10. Distal female genitalia and associated structures of the Ponto-Caspian species. A. Intermaria

zagrosensis (Gloer & Pesic, 2009) gen. et comb. nov. B. Intermaria kermanshahensis (Gloer & Pesic,

2009) gen. et comb. nov. C. Persipyrgula saboori (Gloer & Pesic, 2009) gen. et comb. nov. D. Shadinia

terpoghassiani (Shadin, 1952). Anatomical abbreviations given in the Material and method section.

Scale bars: 500 pm.

European Journal of Taxonomy 208: 1-29 (2016)

Sarkhia sarabensis Gloer & Pesic, 2012 from Iran also shows similar penis characteristics. Additional

anatomical descriptions and phylogenetic data might soon clarify this issue.

In conclusion, our morphological and molecular data question the species richness of the genus

Pseudamnicola , and indicate that the morphological characters traditionally used for the recognition

of Ponto-Caspian hydrobiid species (i.e., conchyliologic) need to be complemented with other features

mainly related to their genital and trophic systems. The high morphological disparity observed among

closely related pyrgulinid genera calls for an intensive study of the reason why some hydrobiids are

so similar and others so diverse. Our phylogeny identified a new potential lineage that could represent

a new subfamily between Pseudamnicolinae and Pyrgulinidae, thus contributing to the knowledge of

evolutionary patterns in the family Hydrobiidae. Our findings also provide future direction for research

on the biodiversity, systematics and biogeography of hydrobiid gastropods, particularly those from the

Ponto-Caspian region.

Acknowledgements

The authors thank the German Academic Exchange Service (DAAD) for enabling T. Hauffe and

S. Sereda to collect the material of Shadinia in Armenia, and Barbara Hoenig and Sabine Agel (Imaging

Unit, Biomedical Research Centre Seltersberg, Justus Tiebig University, Giessen) for their assistance

with the ESEM photomicrographs. The English was reviewed by A. Burton. Two anonymous reviewers

contributed to the improvement of this manuscript. This study was supported by a fellowship of the

postdoctoral program Just'us (Junior Science and Teaching Units, Justus Liebig University, Giessen)

granted to D.D.

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Manuscript received: 25 October 2015

Manuscript accepted: 9 March 2016

Published on: 28 June 2016

Topic editor: Rudy Jocque

Desk editor: Kristiaan Hoedemakers

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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

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