Unraveling a new lineage of Hydrobiidae genera ( Caenogastropoda : Truncatelloidea ) from the Ponto-Caspian region

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 Glöer & Pešić, 2009; Sarkia kermanshahensis Glöer & Pešić, 2009 (originally within Pseudamnicola) and P. saboori Glöer & Pešić, 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.


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 (Glöer 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 (Glöer & Pešić 2009, 2012), Turkey (Yıldırım et al. 2006;Glöer & Georgiev 2012;Glöer et al. 2014Glöer et al. , 2015) ) and Georgia (Badzoshvili 1979).One such species Sarkhia kermanshahensis (Glöer & Pešić, 2009) has been recently assigned by Glöer & Pešić (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 Glöer & Pešić (2009), i.e., P. zagrosensis Glöer & Pešić, 2009, Sarkhia kermanshahensis (Glöer & Pešić, 2009) (originally described as Pseudamnicola kermanshahensis) and P. saboori Glöer & Pešić, 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;Glöer 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 Glöer et al., 2016.Our results indicate that the Pseudamnicola species described by Glöer & Pešić (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 European Journal of Taxonomy 208: 1-29 (2016) 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 Glöer & Pešić (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 HCO2198 (Folmer et al. 1994).Thermal cycling conditions were as described in Delicado et al. (2012) and were conducted with an annealing temperature of 48º C. Final PCR products were sequenced in an ABI 3730 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* 4.0a123 (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. (1984Davis et al. ( , 1986Davis et al. ( , 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) 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.

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

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 welldeveloped 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 Latin inter-(between) -maria (seas), referring to the occurrence of the genus in the continental area between the Mediterranean and the Caspian seas.

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 Glöer & 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).(Glöer & Pešić, 2009) comb.nov.
Operculum with ca 2 whorls (Fig. 2E-F) and muscle attachment area oval and located near the nucleus.

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

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).
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).

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.

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

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).
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).

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).

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.(Shadin, 1952) Figs 8-9; Tables 2-3 Pyrgula terpoghassiani Shadin, 1952: 227 Pyrgula terpoghassiani -Akramowski 1952, nom.nud.

Material examined
Type material not specified.

Type locality
ARMENIA: Lake Aiger-Lich, south of Metzamor, Armavia province, 40° 08.573' N, 44°10.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.
of the antepenultimate whorl WBW = width of the body whorl WPW = width of the penultimate whorl Anatomical characters Ag = albumen gland Bc = bursa copulatrix Cg = capsule gland Ct = ctenidium dBc = duct of the bursa copulatrix

Fig. 1 .
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.

Fig. 2 .
Fig. 2. Shell, operculum and radula of Intermaria zagrosensis (Glöer & Pešić, 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.
Fig. 4. Shell, operculum and radula of Intermaria kermanshahensis (Glöer & Pešić, 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.

Fig. 6 .
Fig. 6.Shell, operculum and radula of Persipyrgula saboori (Glöer & Pešić, 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.

Table 1 .
for the DELICADO D. et al., New hydrobiid genera from the Ponto-Caspian region Locality name, GenBank accession numbers and original references for the species employed in the molecular study.
European Journal of Taxonomy 208: 1-29 (2016) R statistical environment (R Development Core Team 2011).Resulting parameter values are shown in Tables