Salenthydrobia Gen. Nov. (Rissooidea: Hydrobiidae): a Potential Relict of the Messinian Salinity Crisis

Blackwell Science, LtdOxford, UKZOJZoological Journal of the Linnean Society0024-4082The Lin- nean Society of London, 2003 137 Original Article RELICT OF THE MESSINIAN SALINITY CRISIST. WILKE

Zoological Journal of the Linnean Society, 2003, 137, 319–336. With 8 figures

Salenthydrobia gen. nov. (Rissooidea: Hydrobiidae): a potential relict of the Messinian salinity crisis

THOMAS WILKE

The George Washington University Medical Center, Department of Microbiology and Tropical Medicine, 2300 Eye Street, N.W., Washington, DC 20037, USA and J.W. Goethe-Universität Frankfurt am Main, Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 Abteilung Ökologie & Evolution, BioCampus, Siesmayerstraße, D-60054 Frankfurt am Main, Germany

Received December 2001; accepted for publication September 2002

The Messinian salinity crisis (MSC) occurred synchronously throughout the Mediterranean basin about 5.96 ± 0.02 Mya and represents one of the most dramatic oceanic changes since the early Miocene. It is thought that the concomitant environmental changes brought about isolation of faunas and the development of endemism. As part of the search for possible speciation events triggered by the MSC, the author studied 38 populations of hydrobiine snails from the Mediterranean and Black Sea, including three populations from the Salentina Peninsula, Italy. Partial sequences (COI, 16S) and anatomical data were used to test the taxonomic and phylogenetic status of the peninsular populations. A maximum likelihood analysis of 11 hydrobiine taxa revealed ﬁve clades and lineages, four of which corresponded to previously recognized genera: Adriohydrobia, Hydrobia, Peringia, Ventrosia. The ﬁfth clade was formed by haplotypes of the peninsular populations, which are characterized by distinct male and female reproductive systems. Based on molecular and anatomical data, these populations are considered to represent a new species, Salenthydrobia ferrerii, belonging to a new genus, Salenthydrobia. Ecological and biogeographical data for S. ferrerii strongly support a correlation between its origin and the MSC. Based on an island age of 5.33 Myr and a population divergence of 0.0973 ± 0.0114, the COI molecular clock rate for the Salenthydrobia and Peringia clades would be 1.83 ± 0.21% population divergence per Myr. The genetic diversity of S. ferrerii, its phylogenetic relationships, and the validity of the proposed local molecular clock rate are discussed. © 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336.

ADDITIONAL KEYWORDS: COI mtDNA – 16S rRNA – Hydrobiinae – phylogeography.

INTRODUCTION It is thought that the concomitant environmental changes during the MSC included a reorganization of Major geological events play a key role in speciation the drainage system in Europe and the introduction of scenarios and have affected groups of organisms in all Paratethyan taxa into the Mediterranean basin (Hsü ecological settings (e.g. Avise, 2000). An event with et al., 1977). Relic Paratethyan faunas still can be substantial implications for evolutionary changes is found in endemic circum-Mediterranean areas (e.g. the Messinian salinity crisis (MSC), considered to be Lake Ohrid; Stankovic, 1960). Another implication of one of the most dramatic oceanic changes since the the extensive Pliocene ﬂooding is the isolation of fau- early Miocene (Hsü et al., 1977). The MSC occurred nas and development of endemism on newly created synchronously throughout the Mediterranean basin islands (Hsü et al., 1977). about 5.96 0.02 Myr ago (Mya) and caused a large ± A paradigm for testing speciation events caused by fall in sea level (more than 1000 m) with the subse- the MSC is the minute brackish-water mudsnail taxa quent deposition of nonmarine sediments in a large of the subfamily Hydrobiinae (sensu Radoman, 1977), ‘Lago Mare’. The MSC ended with the Pliocene ﬂood- benthic organisms with a low capacity for active dis- ing 5.33 Mya (Krijgsman et al., 1999). persal and restricted to a narrow contact zone of marine and freshwater habitats. Phylogeographical studies of amphi-Atlantic hydro- E-mail: [email protected] biine taxa (Davis, McKee & Lopez, 1989; Wilke &

320 T. WILKE

Davis, 2000; Wilke, Rolán & Davis, 2000; Wilke & As part of the search for possible speciation events Falniowski, 2001; Wilke & Pfenninger, 2002; Wilke, triggered by the MSC, the author studied 38 mudsnail Pfenninger & Davis, 2002) have revealed discrete pat- populations from the Mediterranean and the Black terns of genetic diversity (in terms of populations and Sea. Three unusual ‘Hydrobia’ populations were species). In the northern Atlantic only relatively widely encountered in oligohaline and freshwater Karst cav- distributed taxa are found: Hydrobia acuta neglecta, ities and channels along the western and eastern H. glyca, Ventrosia ventrosa, V. truncata, Peringia coasts of the the Salentina Peninsula (Lecce Province, ulvae. The species diversity in the Mediterranean is Italy; (Fig. 1). It should be noted that the peninsula probably higher, comprising both widely distributed was an isolated island from the end of the MSC (see (e.g. H. acuta acuta, V. ventrosa) and more localized ﬁg. 9 in Steininger & Rögl, 1984) until about taxa (e.g. Adriohydrobia gagatinella, H. djerbaensis 800 000 years ago (Anonymous, 1999). The area is

and Hydrobia sp. B; see Wilke et al., 2000). known for its specialized and endemic aquatic species Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 (Pesce et al., 1978). A preliminary survey of external characters and penis morphology revealed features that were distinct from all known Hydrobia species (sensu Giusti, Man- ganelli & Bodon, 1998) and even from all known putative genera of the Hydrobiinae (Hydrobia, Ventrosia, Peringia, Adriohydrobia, see Fig. 2), indicating that these populations possibly represent a new taxon. Due to its uncertain taxonomic status, it is henceforth referred to as ‘taxon A’. Considering that taxon A has a very restricted range and that it occurs in an area that was heavily affected by the MSC, could it be part of an ancient radiation that survived the MSC? Is it a Paratethyan taxon introduced by the Pliocene flooding? Or is it an endemic taxon that evolved on an isolated island after the flooding? In the present study, sequence data of partial mitochondrial genes coding for cytochrome c oxidase subunit I (COI) and large subunit ribosomal RNA (16S) as well as anatomical data are used to test the taxonomic status of taxon A, its phylogenetic position within the Figure 1. Southern Italy with sampling localities on the subfamily Hydrobiinae, and assess the possible Salentina Peninsula. The shaded areas show the land mass impact of the MSC on its phylogeography and after the Pliocene flooding (about 4.5 Mya). evolution.

Figure 2. Head-penis complex in the four known genera of the Hydrobiinae (sensu Radoman, 1977): Hydrobia (A), Per- ingia (B), Ventrosia (C), Adriohydrobia (D), as well as in taxon A (E). Note that the general penis morphology is constant within the putative genera of the Hydrobiinae. All drawings are to scale.

RELICT OF THE MESSINIAN SALINITY CRISIS 321

MATERIAL AND METHODS pairs (excluding 51 bp primer sequence) were LCO1490 and HCO2198, following Folmer et al. TAXA AND POPULATIONS STUDIED (1994). To optimize the performance of the PCR, the Three populations of taxon A were studied: reverse primer was modiﬁed at position 24 (C Æ Y). (1) Idume Creek (Torre Chianca, Lecce) [40.466∞N, The primers used to amplify a 16S rRNA fragment 18.183∞E]. Karst channel, 0.5–2.0 m deep. Associ- with a target length of 500–502 bp (excluding 42 bp ated ﬂora: Phragmites australis, Ruppia sp., primer sequence) were 16Sar-L and 16Sbr-H, follow- Potamogeton sp. and green algae. Substratum: cal- ing Palumbi et al. (1991). Each PCR reaction mixture, careous rocks. Only a few snails were found in the in a total volume of 50 mL, contained 20–100 ng of oligohaline part of the creek, sympatric with the genomic DNA, 5 mL of 10X reaction buffer, 1 mM hydrobiine species Ventrosia ventrosa. The snails MgCl2, 200 mM of each dNTP, 300 nM of each primer,

were more abundant in the internal part of the 2 mL of 100X BSA solution, 0.5 mL TMAC and 2.5 units Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 creek containing fresh water and with few or no of polymerase. The PCR reactions were performed specimens of V. ventrosa. with annealing temperatures of 48∞C and 46∞C for the (2) Torre Castiglione (Torre Lapillo, Porto Cesareo, COI and 16S fragments, respectively. Amplified DNA Lecce) [40.291∞N, 17.809∞E]. The snails were found products were separated by electrophoresis through a in four small Karst cavities (‘spondulates’) with 1% LMP agarose gel. Bands corresponding to frag- oligohaline or fresh water. The cavities are up to ments of the correct size were cut out, the slices 3 m deep. Associated flora: Ruppia sp. or Juncus melted and the DNA purified with Wizard PCR preps acutus. Substratum: calcareous rocks. (Promega, Madison, WI). Sequences (forward and (3) Bambinello Spring (Torre Lapillo, Porto Cesareo, reverse) were determined using the LI-COR (Lincoln, Lecce) [40.283∞N, 17.838∞E]. Mesohaline Karst NE) DNA sequencer Long ReadIR 4200 and the cavity, up to 0.8 m deep. Associated flora: incrusted Thermo Sequenase fluorescent labelled primer cycle brown algae. Substratum: calcareous rocks. sequencing kit (Amersham Pharmacia Biotech, Ten additional representatives of the subfamily Piscataway, NJ) according to the manufacturer’s Hydrobiinae were included in the study to infer phy- protocols. logenetic relationships (Table 1): Hydrobia acuta The protein-coding COI sequences were aligned acuta (Draparnaud, 1805); Hydrobia acuta neglecta unambiguously by eye using XESEE (Cabot & Muus, 1963; Hydrobia glyca (Servain, 1880); Hydrobia Beckenbach, 1989). The initial alignment of 16S djerbaensis (Wilke, Pfenninger & Davis, 2002) Hydro- sequences using default settings in ClustalX version bia sp. B (see Wilke et al., 2000); Adriohydrobia 1.81 (Thompson et al., 1997) was refined manually, gagatinella (Küster, 1852); Peringia ulvae (Pennant, using the 16S secondary structure model of De Rijk 1777); Ventrosia ventrosa (Montagu, 1803); Ventrosia et al. (1999). Within the ingroup taxa, there is a total pontieuxini (Radoman, 1973); and Ventrosia truncata of only eight single base pair insertions/deletions. (Vanetta, 1924). For most of these taxa, material from The first 2–10 base pairs behind the 3¢ end of each or near the respective type localities was studied. The primer are often difficult to read. Therefore the first type species of the genus Pseudamnicola, P. lucensis and last 10 bp of each sequence were uniformly (Issel, 1866), and the hydrobiid taxon Mercuria similis excluded, leaving a 638-bp-long completely overlap- (Draparnaud, 1805) were used as outgroups (Table 1). ping fragment for the COI gene and a 489-bp-long Pseudamnicola lucensis was chosen based on the sis- fragment (after alignment) for the 16S gene. All ter-group relationship of the putative subfamilies sequences are available from GenBank (see Table 1). Pseudamnicolinae and Hydrobiinae (Radoman, 1977). Mercuria similis was selected as a second outgroup- taxon among 10 hydrobiid outgroup-candidates (Gra- ziana, Belgrandia, Horatia, Sadleriana, Hauffenia, SEQUENCE ANALYSES Mercuria, Fissuria, Alzoniella, Islamia, Avenionia; see In order to test whether there were significant differ- Wilke et al., 2001) based on the result of a likelihood ences in incongruence length between the COI and ratio test (Felsenstein, 1981; Huelsenbeck & Rannala, 16S data sets, the HOMPART command in PAUP was 1997). used to perform a homogeneity-partition test (Farris et al., 1995). As the test did not reveal significant differences (P = 1; 100 replicates), the two data sets were DNA ISOLATION AND SEQUENCING used in a combined analysis. The methods of Spolsky, Davis & Zhang (1996) and To verify that the combined COI + 16S data set as a Davis et al. (1998) were used for isolating DNA from whole exhibited phylogenetic signal, the relative individual snails. The primers used to amplify a frag- apparent synapomorphy analysis (RASA 3.04T; ment of the COI gene with a target length of 658 base Lyons-Weiler, 2001) was used. The data set showed a

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 322 T. WILKE d to GenBank as part AF449206/AF478405 AF449207/– AF449208/– AF449209/– AF449210/– AF449211/– AF449212/– AF449213/AF478406 AF449214/– AF449200/AF478407 AF449201/AF478408 AF449202/– AF449203/– AF449204/AF478409 AF449205/AF478410 Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 306500 AF449216/AF47840# AF449217/AF478404 717 AF118335*/AF478402 719 AF118288*/AF478401 692 AF449215/AF478398 567 AF467640*/AF478397 656848 AF278809*/AF478395 AF467604*/AF478396 2453 2455 2457 2459 2226 2227 2258 2260 2261 2230 2231 2233 2266 2267 2268 2111 AF317843*/AF478400 1198 AF278821*/AF478399 DNA isolation #2551 GenBank accession # COI/16S 2537 AF367646*/AF478393 AF367651*/AF478394 E N ∞ ∞ N N N E N E ∞ ∞ ∞ ∞ ∞ ∞ N E N E N E N E N N E W N ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ ∞ E E N E E N E ∞ ∞ ∞ ∞ ∞ ∞ ∞ N W ∞ ∞ 18.183 40.291 17.809 40.283 17.838 27.513 39.5 74.7 42.430 0.460 52.863 52.957 0.930 16.69385 3.0820 43.44867 33.82 11.07 39.8987 6.1375 3.897 56.68 10.22 36.3937 13.3458 10.5856 Longitude, Latitude 45.7470 44.0113 orre Castiglione T Bambinello Spring Stone Harbor Strandscha and Kraimorie Snettisham lagoon RSPB bird reserve Natural Reserve, Stiffkey Saltmarsh Natural Reserve, near Omis Sebkha de Sidi Garous Puerto de Pollença Ajstrup Bugt Asís lagoon San Francisco de Aquileia, Canale Panigai near Panigai Canale Panigai Aquileia, thermal spring Bagni Caldi, 2002)Tazdaine, Djerba, Médenine, Tunisia, , ., 2000)., Mallorca, Balearic Islands, Spain, et al. (Küster, 1852)(Küster, Cetina River estuary Croatia, et al

Muus, 1963Muus, Mariager Fjord, Jutland, Denmark, (Issel, 1866)(Issel, Bagni di Lucca, Lucca, Toscana, Italy,

(Draparnaud, 1805)(Draparnaud, Etang du Prévost Hérault, France, 43.513 (Wilke (Wilke (Radoman, 1973)(Radoman, between bay Burgas, Bulgaria,

(Vanetta, 1924)(Vanetta, Cape May County, New Jersey, USA, (Montagu, 1803)(Montagu, Wash, The Norfolk, United Kingdom,

(Draparnaud, 1805)(Draparnaud, Udine, Friuli-Venezia-Giulia, Italy,

(Servain 1880) San Fernando, Cádiz, Spain,

(Pennant, 1777)(Pennant, Holkham Norfolk, United Kingdom,

sp. B (see Wilke B (see Wilke sp. Locality information, DNA isolation and GenBank accession numbers for the taxa studied.Locality information, Sequences that were previously submitte

able 1. entrosia truncata entrosia pontieuxini entrosia ventrosa axon Aaxon Aaxon Lapillo, Torre Cesareo, Porto Lecce, Italy, Lapillo, Torre Cesareo, Porto Lecce, Italy, axon Aaxon Idume Creek Chianca, Torre Lecce, Italy, 40.466 eringia ulvae T T V T V V P Adriohydrobia gagatinella Hydrobia Hydrobia djerbaensis Hydrobia acuta neglecta Hydrobia glyca T of related studies are marked with an asterisk SpeciesMercuria similis Pseudamnicola lucensis Hydrobia acuta Location

significant phylogenetic signal of tRASA = 4.44 (P < 0.05; performed with the parameters of sequence substitu- b observed = 5.49; b null = 4.59). Acknowledging tion suggested by Modeltest. In order to assess the recent criticism of the RASA approach (Simmons robustness of the tree topology, 100 bootstrap repli- et al., 2002), the result was verified with an indepen- cates were generated with a heuristic search and TBR dent approach, the permutation tail probability test branch-swapping. (PTP) (Archie, 1989). The PTP test showed a highly Parameters of DNA sequence polymorphism includ- significant cladistic covariance (P = 0.0001), indicating sequence diversity (average number of nucleotide ing that the cladogram could not have arisen by differences per site within groups; p) and sequence chance alone. Based on the results of the RASA and divergence (average number of nucleotide differences

PTP tests, the combined COI + 16S data set was con- per site between groups; dxy) were calculated using sidered to be suitable for phylogenetic analyses. DnaSP 3.0 (Rozas & Rozas, 1999). The standard error

A power test (Walsh et al., 1999) was used to deter- for pairwise sequence comparisons was calculated in Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 mine whether the combined COI + 16S data set with MEGA 2 (Kumar et al., 2000). 1127 bp was sufficient for resolving possible polytomies among clades resulting from divergence events occurring within the relative short time interval of the ANATOMICAL WORK MSC. Based on equation 3 in Braun & Kimball (2001) Dissections were done using a Wild M-5 dissecting and equation 2 in Walsh & Friesen (2001), the length microscope equipped with an ocular micrometer based of an internode (t) in years that can be detected with a on the methods of Davis et al. (1992) and Wilke et al. given data set (resolving a soft polytomy) becomes: (2002). Twelve living specimens from Bambinello - ln(b ) Spring and three from Idume Creek were used for t = Ll anatomical work. where b is the type II error (e.g. 0.05 is the 95% probability of observing one or more substitutions, 0.20 is the 80% probability), L is the sequence length in bp, RESULTS and l is the substitution rate (substitutions/site/year). SEQUENCE DATA Based on a highly conservative substitution rate of 1 ¥ 10-8 for mitochondrial genes and a power of 80% The ML analysis of the combined COI + 16S data set (b = 0.20) set by convention (see Walsh & Friesen, yielded a single tree with a log likelihood of -4260.69 2001), a 1127-bp long data set should be sufficient to (Fig. 3). The tree comprises the two outgroup taxa resolve internodes of about 143 000 years or less. A Mercuria similis and Pseudamnicola lucensis as well more conservative estimate of t based on a power of as five major Hydrobiinae clades and lineages. The 95% (b = 0.05) yields an internode length of about two lineages correspond to the monotypic genera Adri- 266 000 years. For the 638 bp long fragment of the ohydrobia and Peringia, and two clades to the poly- COI gene alone, the internode lengths are typic genera Hydrobia, and Ventrosia. The third clade 252 000 years (b = 0.20) and 470 000 years (b = 0.05). is formed by the three haplotypes of taxon A. The boot- Therefore a sufficient number of substitutions would strap analysis (see Fig. 3) indicates that most of the be expected to resolve the 630 000-year time interval clades and subclades are rather well supported. How- of the MSC. ever, a consensus tree of 100 bootstrap replicates had Prior to the phylogenetic analysis, Modeltest 3.06 three unresolved trifurcations of clades and lineages (Posada & Crandall, 1998) was used in order to find (indicated by arrows in Fig. 3) that apparently the optimal model of DNA substitution. It performs diverged within a relative short time interval: (a) Ven- hierarchical likelihood ratio tests among 56 possible trosia clade, Adriohydrobia lineage, and the major models. The model selected was TVM + I + G (Trans- clade comprising taxon A, Hydrobia and Peringia version model with invariable sites and gamma (bootstrap support for the trifurcation is 87%); (b) distribution) with base frequencies of A = 0.2958, taxon A clade, Peringia lineage, and Hydrobia clade C = 0.1582, G = 0. 1719; a rate matrix of [A- (71% bootstrap support); and (c) Hydrobia glyca lin- C] = 0.8863, [A-G] = 10.8950, [A-T] = 1.0327, [C- eage, H. djerbaensis lineage, and the clade comprising G] = 0.8350, [C-T] = 10.8950, [G-T] = 1.0000; a Hydrobia sp. B, H. acuta acuta, and H. a. neglecta proportion of invariable sites of 0.6992; and a gamma (74% bootstrap support). distribution shape parameter of G = 1.0111. The sequence divergence (dxy) between the five A maximum likelihood (ML) tree was constructed hydrobiine clades and lineages ranges from 0.1038 from all haplotypes using PAUP 4.0b8 (Swofford, 1998). (between Peringia and taxon A) to 0.1483 (between The analysis (heuristic search, TBR branch-swapping, Hydrobia and Adriohydrobia) for the COI gene, and 20 random-addition-sequence replications) was from 0.0228 (between Peringia and taxon A) to 0.0611

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 324 T. WILKE Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020

Figure 3. Maximum likelihood tree for hydrobiine taxa based on 1127 bp of the combined COI and 16S genes. Mercuria similis and Pseudamnicola lucensis were used as outgroups. The scale bar indicates the expected number of substitutions according to the model of sequence evolution applied. For taxon A, individual DNA isolation numbers are given. Bootstrap values (in percentage) are provided (for details see text).

Table 2. Sequence divergence (dxy) between Adriohydrobia, Hydrobia, Peringia, Ventrosia, and taxon A for the COI gene (below diagonal) and for the 16S gene (above diagonal). The number of individuals studied for each gene is given in paren- theses

COI/16S Adriohydrobia (n = 1) Hydrobia (n = 5) Peringia (n = 1) Ventrosia (n = 3) Taxon A (n = 6)

Adriohydrobia (n = 1) – 0.0380 0.0290 0.0576 0.0456 Hydrobia (n = 5) 0.1483 – 0.0255 0.0523 0.0338 Peringia (n = 1) 0.1442 0.1128 – 0.0542 0.0228 Ventrosia (n = 3) 0.1411 0.1373 0.1338 – 0.0611 Taxon A (n = 15) 0.1198 0.1124 0.1038 0.1344 –

(between Ventrosia and taxon A) for the 16S gene latter four were also homogeneous in the 16S gene. (Table 2). They differed from specimens 2231 and 2268 each by

The sequence diversity (p) within the 15 taxon A- dxy = 0.00415. specimens studied was 0.0047 based on the COI gene. Interestingly, 13 of the 15 specimens (all of those from Idume Creek and Torre Castiglione, and 4/6 of those ANATOMY from Bambinello Spring) were homogeneous, whereas the remaining two from Bambinello Spring (individu- A detailed anatomical description of taxon A is given als 2231 and 2268) were rather distinct. Both differed in the Appendix. In brief, two organ/organ systems dis- from the 13 homogeneous specimens by dxy = 0.0188. tinguish taxon A from all other known hydrobiine taxa Note that the 16S gene was studied in only six indi- - the penis and the bursa copulatrix-complex, both viduals of taxon A: in the two distinct specimens 2231 with a unique combination of anatomical character and 2268, and in the (based on the COI gene) homo- states. The penis is large with a very wide base, lacks geneous specimens 2230, 2260, 2267, and 2453. The appendices and has a short but tapered end with a dis-

Table 3. Anatomical characters and their states that differentiate among hydrobiine taxa. The list is based on the characters and states proposed by Hershler & Ponder (1998). Autapomorphies of taxon A are underlined, synapomorphies of taxon A and other taxa are in bold

Character/Character states Hydrobia Adriohydrobia Peringia Ventrosia Taxon A

Penis overall shape 0 1 0 11 0 strap like; 1 tapering Penis base 0 1 0 11 0 not widened; 2 widened Shape of distal end 0 1111 0 blunt; 1 tapered Distal papilla 0 0 1 0 1 Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 0 absent; 1 present Nonglandular processes or lobes (inner edge) 00 12 0 0 absent; 1 present (distal); 2 present (medial) Nonglandular processes or lobes (outer edge) 1 0000 0 absent; 1 present (distal); 2 present (medial) Penial duct (coiling in base) 0 0 0 2 1 0 staight; 1 weakly undulating; 2 strongly undulating Penial duct (coiling in medial penis) 0 0 2 2 1 0 staight; 1 weakly undulating; 2 strongly undulating Penial duct (coiling in distal penis) 00 2 00 0 staight; 1 weakly undulating; 2 strongly undulating Swelling of coiled oviduct and bursa 1 0000 0 absent; 1 present Shape of bursa 0 0 0 11 0 ovate; 1 hammer-shape Length of bursal duct 1 221 2 0 shorter than bursa; 1 same length; 2 longer than bursa Origin of bursal duct 1 0 1 00 0 anterodorsal; 1 anterior Length of seminal receptacle 1 0000 0 shorter than bursa; 1 same length

tinct penial papilla. The penial duct is weakly undu- dom permutations). Therefore no attempt was made to lated in the base and medial penis, and straight in the use the anatomical data for cladistic analyses or, in distal part of the penis (Figs 2E, 7). The bursa copul- combination with the molecular data, for a total atrix is large and hammer-shaped; its duct is longer evidence approach. than the bursa itself. The seminal receptacle is elon- gated and shorter than the bursa. The total number of loops of the pigmented part of the oviduct is relatively DISCUSSION constant at 2.0–2.5 (Fig. 8). PHYLOGENETIC SIGNAL IN THE DATA SETS Table 3 lists all characters and their states (slightly modified from Hershler & Ponder, 1998) that differen- Despite the distinctness of the male and, to a lesser tiate among hydrobiine taxa. The only character extant, female reproductive systems in the hydrobiine states used were those that do not show intraspecific groups, the anatomical data set did not show a signif- variations. Shell and other external characters as well icant phylogenetic signal. Although the result, based as characters from the digestive, nervous, and respi- on only 14 characters, may not be representative for ratory systems therefore had to be excluded. all taxa of the Hydrobiidae, it could explain why pre- A RASA analysis based on the 14 anatomical char- vious cladistic applications of anatomical data in acters in Table 3 indicated that the data set did not hydrobiids have yielded poorly resolved phylogenies have a significant phylogenetic signal (tRASA = -1.32; (e.g. Falniowski & Szarowska, 2000; Bodon, P > 0.05; b observed = 1.31; b null = 1.64). This result Manganelli & Giusti, 2001). The primary problem is could be confirmed with the PTP test; there is no sig- the considerable convergent evolution of many char- nificant cladistic covariance (P = 0.4504; 10 000 ran- acters and character states, and the paucity of unique

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 326 T. WILKE characters to serve as synapomorphies (Wilke et al., PHYLOGENETIC IMPLICATIONS AND ORIGIN OF TAXON A 2001). The different reproductive systems in hydrobi- Taxon A is closely related to the putative genera ine radiations are often uniquely evolved complex Hydrobia and Peringia and, based on both COI and structures with a unique combination of character 16S data, Peringia appears to be its sister group states. Considering the penis in taxon A, it is clearly (Figs 3, 4; Table 2). Preliminary sequence data from distinct from other penis-types in the Hydrobiinae 492 bp of the conservative nuclear 18S rRNA gene (Fig. 2). The same applies to the bursa copulatrix com- (Wilke, unpublished data) supports the distinctness of plex. However, scoring 14 individual character states the five hydrobiine groups and confirms that taxon A results in apparently random synapomorphies is more closely related to Hydrobia and Peringia than (Table 3). Moreover, taxon A is characterized by only to Adriohydrobia and Ventrosia. two autapomorphies. In comparison, Hydrobia is char- The available ecological and biogeographical data acterized by four, Peringia and Ventrosia by two, and for taxon A strongly support a correlation between its Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 Adriohydrobia by none. The low number of autapo- origin and the MSC. Studies on gobies (Pisces: Perci- morphies does not reflect insufficient anatomical dif- formes: Gobiidae) suggest that the disappearance of ferentiation among these taxa. Rather, it indicates marine habitats during the MSC forced some species that scoring characters, without establishing homol- to adapt to freshwater environments, giving rise to the ogy, is inappropriate. The amount of homoplasy is evi- freshwater endemism today (Miller, 1990; but see dent from the data in Table 3; the results indicate a Penzo et al., 1998). Taxon A is the only hydrobiine high degree of ‘noise’ and the lack of phylogenetic sig- taxon that adapted to freshwater habitats. It is possi- nal. From a cladistic standpoint, it may be better to ble that this adaptation was a response to the ecolog- score these complex anatomical structures as single ical changes induced by the MSC. Moreover, it is characters to reduce the degree of homoplasy. How- generally assumed that the Pliocene flooding that ever, given the paucity of these characters, this ended the MSC triggered endemic speciation and radi- approach is likely impractical, too. ation events on newly isolated Mediterranean islands An alternative for phylogenetic reconstruction is the (e.g. in the frog genus Rana; Beerli et al., 1994). The use of molecular data. Of course, the COI and 16S area in which taxon A occurs today became an isolated genes used here may be subject to homoplasy too, island with the onset of the flooding (see Fig. 1). particularly when working at a higher systematic The Salentina Peninsula probably remained isolated level. Considering the relatively high mutation rate in from the mainland for about 4.5 Myr. Based on mitochondrial genes, one can expect a certain number these distinct ecological and biogeographical charac- of random mutations (which may be the reason why teristics, it is possible that taxon A originated at some of the bootstrap values are below 80%). the end of the MSC (5.33 Mya) with the isolation of Nevertheless, within the Hydrobiinae the highly the peninsula. variable COI sequences do not approach transitional saturation (see Wilke et al., 2001) and the same can be assumed for the more conservative 16S gene. Moreover, the phylogenetic tree presented in Figure 3 CALIBRATION OF THE COI MOLECULAR CLOCK RATE is rather robust. Therefore, the rate of homoplasy in The knowledge of the precise island age for the pen- the molecular data set is likely too low to have a seri- insula provides a good basis for calibrating the molec- ous effect on the phylogeny presented here. ular clock rate for taxon A. Unfortunately, it is

Table 4. Previous estimates of molecular clock rates (in percentage sequence divergence per Myr) for the COI gene in Pro- tostomia lineages separated by less than 10 Myr

Classiﬁcation Lineages Molecular clock rate Reference

Archaeogastropoda Tegula verrucosa spp. 2.4 Hellberg & Vacquier, 1999 Decapoda Eurypanopeus spp. 1.7 Schubart et al., 2000 Decapoda Sesarma spp. 2.3 Schubart, Diesel & Hedges, 1998 Hemiptera Maoricicada campelli (central 1.7 Buckley, Simon & Chambers, 2001 Otago/northern lineages) Lepidoptera Heliconius erato (14 races) 2.3 Brower, 1994* Decapoda Alpheus spp. 1.4 Knowlton & Weigt, 1998

*COI + COII

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 RELICT OF THE MESSINIAN SALINITY CRISIS 327 relatively difficult to obtain exact and comparable ancestral polymorphism and therefore likely overesti- molecular clock rates for the nonprotein-coding 16S mated (see Edwards & Beerli, 2000). gene. The gene contains variable regions that can dif- Another important event in hydrobiine phylogeny is fer considerably between closely related groups. These the split of the Adriohydrobia lineage and the taxon A/ regions are sometimes difficult to align and the clock Peringia/Hydrobia clade (Fig. 4). Adriohydrobia is rate can vary with the model of secondary structure similar to taxon A, a taxon with a restricted distribu- applied. Moreover, alignment gaps can be treated as tion area. The monotypic genus has been reported fifth character state or as missing data, possibly exclusively from the middle-eastern Adriatic Sea resulting in different clock rates. (Wilke & Falniowski, 2001).

In contrast, estimates of clock rates for the protein- The sequence divergence (dxy) for the split between coding COI gene are not affected by alignment strat- the Adriohydrobia lineage and the taxon A/Peringia/ egies. A comparison of Protostomia lineages separated Hydrobia clade is 0.1393 ± 0.0137. This value can Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 by less than 10 Myr or 20% sequence divergence (the be corrected with the average within-population frames in which the COI gene is presumably not sat- sequence diversity for Adriohydrobia of p= 0.0026 urated; Brown, George & Wilson, 1979; Avise, 2000) (taken from Wilke & Falniowski, 2001), for Peringia of indicate relative coherent rates of about 2% p= 0.0097 (see above), and for Hydrobia of p= 0.0034 (1.97 ± 0.42%) sequence divergence per Myr between (based on 22 populations studied in Wilke et al., 2000). pairs of lineages (Table 4). The resulting population divergence (t) is A likelihood ratio test (Huelsenbeck & Rannala, 0.1341 ± 0.0137 (Fig. 4). Under the assumption that 1997) of the COI data set did not reject the molecular the COI molecular clock rate of 1.83% population clock null hypothesis (log L0 = -2920.91, log L1 = divergence per Myr applies to all hydrobiine taxa, the -2914.30, -2 log L = 13.22, P > 0.05), indicating that time estimate for its split with the taxon A/Peringia/ the rates among lineages are equal (also see Fig. 4). Hydrobia clade would be 7.33 ± 0.75 Myr. This pre- Therefore, it can be assumed that the COI fragment is dates the MSC by about 1.37 Myr. By that time, the suitable to estimate time of divergence among hydro- eastern Paratethys had disintegrated, forming several biine lineages. basins, each with an own endemic faunal evolution

The average pairwise sequence divergence (dxy) for (Steininger & Rögl, 1984). It is possible that the genus the split between the taxon A clade and the Peringia Adriohydrobia evolved in one of those basins and that lineage is 0.1038 with a SE of 0.0114 (based on aver- it was later introduced to the eastern Adriatic Sea age pairwise standard errors calculated in MEGA). with the Pliocene flooding. In fact, Hsü et al. (1977: However, in order to calibrate the molecular clock fig. 3) suggested drainage from the isolated Pannonian rate, it is necessary to distinguish between genetic Basin via a chain of lakes in the Dinaric Province, end- divergence (here expressed as sequence divergence, ing in the Mediterranean exactly in the area where dxy) and population divergence (t), which is corrected Adriohydrobia occurs today. for ancestral polymorphism (for details see Edwards Of course, considering the limitations of the molec- & Beerli, 2000). Under the assumption that the ular clock approach (e.g. Ayala, 1997) and the SE for sequence diversity in populations today is similar to the sequence divergence, there is the possibility that that at the time of the population splitting, a simple the COI molecular clock in taxon A and Adriohydrobia estimate of population divergence can be obtained by runs at a faster or slower rate, or that the clock rates subtracting the average within-population sequence in the two taxa are significantly different (see Fig. 4 diversity (p) from the average sequence divergence for alternative calibrations based on molecular clock between lineages (dxy) (Fleischer, McIntosh & Tarr, rates from 1 to 3% population divergence per Myr). 1998). Based on p= 0.0034 for taxon A and p= 0.0097 Nonetheless, the proposed COI clock rate is the first for Peringia ulvae (taken from Wilke & Davis, 2000), attempt in hydrobiid phylogeny to estimate such a the population divergence (t) for the split of taxon A rate based on genetic data from endemic radiations, and the genus Peringia would be 0.0973 ± 0.0114 calibrated with major geological events. As more taxa (t = [dxy Peringia/taxon A] - 0.5 [ptaxon A + pPeringia]) (Fig. 4). are studied, a longer gene fragment is used, and With an island age of 5.33 Myr and a population improvements are made in inferring models of divergence of 0.0973 ± 0.0114, the molecular clock rate sequence evolution, higher resolutions obtained (l) for the taxon A clade and the Peringia lineage thereby will better evaluate the validity of the result would be 1.83 ± 0.21 ¥ 10-8 (1.83 ± 0.21% population and interpretation of the COI molecular clock. divergence per Myr). This corresponds closely to the average clock rates for the taxa listed in Table 4, though these are on average slightly higher than that COI GENETIC DIVERSITY OF TAXON A found for taxon A and Peringia. This could be due to The COI population structure of taxon A, though the fact that the rates in Table 4 are not corrected for inferred from only 15 individuals, is interesting

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Figure 4. Time scale for cladogenic events in hydrobiine taxa based on a ML analysis of the COI gene under the constraint of the molecular clock hypothesis. The outgroup taxa were removed a postori. The thick bars represent population divergence (±SE) for the two nodes involving the splits of taxon A as well as Adriohydrobia. Rates of evolution (1–3% population divergence per Myr) are shown above the time scale. relative to the distinctness of haplotypes 2231 and V. pontieuxini; 0.0408 between Hydrobia a. acuta and 2268 from Bambinello Spring (Fig. 5). Hydrobia sp. B). But of course, a genetic distance per Based on the published intra- and interspecific data se does not define taxa. Figure 5 clearly highlights this for hydrobiine taxa, the observed haplotype differ- problem. It shows COI haplotype-trees for three ences probably reflect intraspecific relationships. The hydrobiine taxa: Adriohydrobia, Peringia, and taxon COI divergence of the two haplotype groups in taxon A A. All three are presumably monotypic groups. In is, with dxy = 0.0188, considerably smaller than the addition, Adriohydrobia has, like taxon A, a restricted smallest divergences of putative hydrobiine sister spe- distribution area (see above). Peringia is relatively cies (e.g. 0.0517 between Ventrosia truncata and widely distributed and its phylogeography may not be

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Figure 5. Unrooted COI ML trees for populations of taxon A (three populations, 15 specimens), Adriohydrobia gagatinella (four populations, 40 specimens; adapted from Wilke & Falniowski, 2001), and Peringia ulvae (three populations, 14 specimens; modified from Wilke & Davis, 2000; see text for details). Circle sizes are proportional to the observed number of individuals with each haplotype. Missing haplotypes are indicated by small black circles. All individuals of taxon A are area- coded (Ba = Bambinello Spring, Id = Idume Creek, TC = Torre Castiglione). directly comparable to those of the other two taxa. genetic approach. Future studies with an optimized Therefore, the Peringia haplotype tree presented here sampling design would therefore have to show (1) is modified from Wilke & Davis (2000) following the whether there are intermediate haplotypes between removal of all haplotypes from individuals outside the the two groups in taxon A, (2) whether there is sec- North Sea (which presumably is the geographical ori- ondary contact of formerly isolated lineages, or (3) gin of the extant Peringia lineages; Wilke & Davis, whether there is perhaps a case of sympatric 2000). Comparison of the three trees shows that speciation. closely related groups with similar ecology and bioge- ography may display very different population structures and that species-level decisions are difficult to TAXONOMIC IMPLICATIONS make based on a few sequences alone. The anatomical and genetic data clearly show that The fact that the distinct haplotype groups in taxon taxon A is different from all other taxa of the A were found in sympatry raises questions about Hydrobiinae studied here. In fact, comparison of taxon reproductive isolation and secondary contact. Unfor- A and previously described genera (Hydrobia, Ventro- tunately, the present study was not designed a priori sia, Peringia and Adriohydrobia) indicates a genus- as a population genetics study, but rather as a phylo- level relationship. Accordingly, taxon A is considered

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 330 T. WILKE to be a new genus, Salenthydrobia, with S. ferrerii sp. Davis GM, Chen CE, Wu C, Kuang TF, Xing XG, Li L, Liu nov. its type species (for a detailed description see WJ, Yan YL. 1992. The Pomatiopsidae of Hunan, China Appendix). (Gastropoda: Rissoacea). Malacologia 34: 143–342. Davis GM, Forbes V, Lopez G. 1988. Species status of north- eastern American Hydrobia (Gastropoda: Prosobranchia): ACKNOWLEDGEMENTS ecology, morphology and molecular genetics. Proceedings of The study was supported by a grant of the German the Academy of Natural Sciences of Philadelphia 140: 191– Science Foundation (WI 1902/1–1) and aided by 246. National Science Foundation Major Research Instru- Davis GM, McKee M, Lopez G. 1989. The identity of Hydro- bia truncata (Gastropoda, Hydrobiinae) – comparative anat- mentation Grant no. 9871363 to the Laboratory for omy, molecular-genetics, ecology. Proceedings of the Academy Molecular Systematics and Evolution at the Academy of Natural Sciences of Philadelphia 141: 333–359. of Natural Sciences, Philadelphia. I am grateful to Dr Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 Davis GM, Wilke T, Spolsky C, Qui C-P, Qui D-C, Xia Marco Bodon for directing my attention to the popu- M-Y, Zhang Y, Rosenberg G. 1998. Cytochrome oxidase lations from the Salentina Peninsula and to Dr Dario I-based phylogenetic relationships among the Pomatiop- Ferreri for collecting the material and for providing sidae, Hydrobiidae, Rissoidae and Truncatellidae (Gas- detailed ecological information. I also thank Dr tropoda: Caenogastropoda: Rissoacea). Malacologia 40: Markus Pfenninger and two anonymous referees for 251–266. reviewing a previous version of this paper. De Rijk P, Robbrecht E, de Hoog S, Caers A, Van de Peer Y, De Wachter R. 1999. 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Schubart CD, Diesel R, Hedges SB. 1998. Rapid evolution APPENDIX to terrestrial life in Jamaican crabs. Nature 393: 363–365. SALENTHYDROBIA GEN. NOV. Simmons MP, Randle CP, Freudenstein JV, Wenzel JW. 2002. Limitations of relative apparent synapomorphy anal- Type species. Salenthydrobia ferrerii sp. nov. ysis (RASA) for measuring phylogenetic signal. Molecular Biology and Evolution 19: 14–23. Etymology. Named after the Salentina Peninsula in Spolsky C, Davis GM, Zhang Y. 1996. Sequencing method- Italy, the region where the genus was first found. ology and phylogenetic analysis: cytochrome b gene sequence Distribution Salentina Peninsula, Lecce Province, Italy reveals significant diversity in Chinese populations of Oncomelania (Gastropoda: Pomatiopsidae). Malacologia 38: Description (based on fresh specimens of Salenthydro- 213–221. bia ferrerii n.sp.). Shell and operculum: shells of adult

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Figure 6. Salenthydrobia ferrerii sp. nov., individuals preserved in ethanol. A, holotype (ANSP, A19754). B-D, paratypes (ANSP, A19755). E, F, paratypes (SMF 323031/2).

Figure 7. Salenthydrobia ferrerii sp. nov., penis morphology.

individuals are about 2.8–3.6 mm high and 1.4– parts of the operculum are opaque white; the central 1.7 mm wide with 5.0–6.0 whorls. The shell shape is part has a characteristic orange-brown colour. ovate-conic (Fig. 6). The whorls are relatively ﬂat and External features: head-foot morphology similar to smooth with distinct growth lines. The aperture is that described for Ventrosia truncata by Hershler & ovate to ovate-pyriform. The outer lip is straight and Davis (1980). The snout is only lightly pigmented thin. The inner lip is only slightly arched. Shells of liv- centrally and has a clear tip. Concentrations of ing individuals are opaque cream-coloured to light bright granules posterior to the eyes and, less brown. The protoconch is often eroded. The sculpture densely, laterally on snout and neck. The tentacles of the protoconch, only seen in juveniles, is granular. are free of granules but have a dark pigmented cen- The operculum is corneous and paucispiral. The outer tral bar extending from the base of the tentacle

© 2003 The Linnean Society of London, Zoological Journal of the Linnean Society, 2003, 137, 319–336 RELICT OF THE MESSINIAN SALINITY CRISIS 333 almost to the tip (Fig. 2E). The distinctly orange- (2-4)-1-(2-4) brown coloured buccal mass is visible through the (1-2)-(1-2). snout. The eyes are in weak bulges at the outer base The moderately large stomach is unpigmented and of the tentacles. very similar to that shown in Davis et al. (1988) for Mantle cavity: general anatomy, including ctenid- Ventrosia truncata, except that Salenthydrobia does ium and osphradium, is similar to that of V. truncata not have a caecal appendix. (see Davis, Forbes & Lopez, 1988). Size varies between Female reproductive system: the anterior lobes of the 1.88 mm to 2.44 mm and the number of gill filaments gonad cover the posterior part of the stomach and the between 21 and 30. gonad extends almost to the posterior tip of the diges- Alimentary canal: the radula is typically taenioglo- tive gland. The pallial oviduct is divided into a glan- ssate and very similar to those found in other hydro- dular posterior albumen gland and into a slightly biine taxa (e.g. Giusti & Pezzoli, 1984). The general Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 longer anterior capsule gland. The large bursa formula for the central tooth is: copulatrix is located dextro-lateral to the style sac and is not or only partially covered by the posterior end of the albumen gland. It is hammer-shaped with the relatively long duct of the bursa extending from the dor- sal or anterodorsal end. The single seminal receptacle is elongate, usually with a slight constriction in the middle. The duct of the seminal receptacle is relatively long. It joins the oviduct just before the latter joins the duct of the bursa. A section of the oviduct is black pigmented and coiled with a total number of 2.0–2.5 loops (Fig. 8). Male reproductive system: the yellow gonad extends from the posterior part of the stomach almost to the tip of the digestive gland. The vas efferens leaves the gonad about 10–20% posterior to its anterior end, loops before reaching the anterior end of the gonad and runs back about 60–80% of the length of the gonad. It then returns as the coiled seminal vesicle. From the seminal vesicle the white vas deferens (beginning before the anterior end of the digestive gland) runs ventrally to the stomach, becomes transparent and enters the large prostate just anterio- ventral of its posterior tip and leaves the prostate pos- terio-ventral of the anterior tip. The penis is large and tapering with a wide basis. There are no appendices at the inner or outer edges. The penial duct is weakly undulated in the basal and medium parts and almost straight in the distal part. It emerges as a distinct papilla at the distal end of the penis. Glandular fields are mostly present in the medial part of the penis, close to the outer edge, but also near the inner edge as well as the tip and the base of the penis (Figs 2E, 7). Nervous system: very similar to that described for V. truncata (see Hershler & Davis, 1980: fig. 5) and other hydrobiids. Detailed measurements are given in Table 5.

SALENTHYDROBIA FERRERII SP. NOV. Etymology. Named in honour of Dr Dario Ferreri of Lecce, Italy in recognition of his contribution to Figure 8. Salenthydrobia ferrerii sp. nov., bursa copu- malacology in general and to the discovery of latrix complex. Salenthydrobia in particular.

8) = SD Range

0.32 5.00–6.00 0.180.10 2.76–3.32 1.44–1.68 0.180.10 2.52–3.00 0.72–1.00 0.14 1.68–2.12 0.060.12 0.46–0.60 1.00–1.32 0.080.08 0.96–1.24 0.64–0.88 0.60 3.84–5.16 0.28 1.90–2.50 0.21 1.40–2.26 0.250.300.12 1.24–1.90 0.90–1.56 0.76–1.20 0.04 0.30–0.46 n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 1.61 1.24 0.94 0.39 Salenthydrobia ferrerii (2002: ﬁg. 2). ﬁg. (2002: . et al 7) Male ( =

n SD Range Mean 0.37 5.00–6.00 5.56 0.190.12 3.04–3.60 1.48–1.84 3.08 1.56 0.390.09 1.96–3.20 0.76–1.02 2.76 0.84 0.13 1.84–2.20 1.91 0.030.10 0.48–0.56 1.08–1.36 0.52 1.15 0.080.05 1.00–1.24 0.68–0.84 1.09 0.74 0.70 3.78–5.16 4.64 0.35 1.80–2.50 2.22 0.40 1.50–2.24 1.90 0.070.03 0.50–0.70 0.30–0.50 0.07 0.30–0.44 0.25 0.54–0.90 0.01 0.26–0.30 0.02 0.26–0.52 0.01 0.06–0.08 0.05 0.36–0.50 0.05 0.12–0.24 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± emale ( F Mean shell 1.61 third whorl 0.87 penultimate whorl 1.20 aperture 0.75 prostate albumen gland 0.40 capsule gland 0.39 bursa copulatrix 0.27 duct bursa copulatrix 0.07

L. shellL. 3.24 W. last three whorlsL. 2.77 W. body whorlL. 1.99 L. penultimate whorlL. 0.54 W. apertureL. 1.14 W. L. digestive glandL. 2.17 L. penis L. L. seminal vesicle L. prostate L. W. L. albumen glandL. 0.58 W. capsule glandL. 0.70 L. bursa copulatrixL. 0.41 W. L. duct bursa copulatrixL. 0.44 W. L. seminal receptacleL. 0.17 W. Measurements (in mm) and counts of characters of shell, soft body structures, and organs in 15 fresh specimens of Measurements (in mm) and counts of characters soft body structures, of shell, able 5. the Idume Creek and Torre Castiglione populations. For an illustration of the measurements see Wilke For Castiglione populations. Torre the Idume Creek and T Shell whorls No. 5.54 Soft body soft body L. 4.56 Reprod. systemReprod. gonad L. 1.99

0.27 1.40–2.44 0.25 1.26–2.30 0.010.01 0.40–0.50 0.15–0.18 3.67 21.00–30.00 0.05 0.24–0.38 0.100.06 0.30–0.60 0.26–0.40 0.020.02 0.16–0.20 0.10–0.14 0.010.01 0.04–0.06 0.02–0.06 0.010.01 0.08–0.12 0.06–0.08 0.040.00 0.18–0.28 0.02–0.03 0.020.01 0.08–0.14 0.05–0.07 0.020.01 0.08–0.14 0.06–0.08 0.020.01 0.08–0.14 0.06–0.08 n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020 7) Male ( =

n SD Range Mean 0.02 0.03–0.07 0.00 0.02–0.02 0.04 0.02–0.10 0.10 1.94–2.44 1.88 0.06 1.86–2.30 1.75 0.100.03 0.42–0.70 0.14–0.24 0.47 0.17 1.26 24.00–29.00 25.13 0.03 0.28–0.36 0.28 0.070.01 0.44–0.56 0.30–0.32 0.49 0.33 0.020.03 0.18–0.22 0.10–0.18 0.18 0.12 0.010.01 0.06–0.08 0.02–0.04 0.05 0.04 0.010.01 0.10–0.11 0.05–0.08 0.10 0.07 0.040.00 0.20–0.30 0.02–0.03 0.23 0.02 0.010.01 0.09–0.12 0.05–0.08 0.11 0.06 0.030.01 0.06–0.13 0.06–0.08 0.10 0.06 0.030.00 0.10–0.16 0.07–0.08 0.11 0.07 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± emale ( F Mean seminal receptacle 0.06 duct seminal receptacle 0.02 osphradium 0.19 buccal mass 0.31 cerebral ganglion 0.13 cerebral commissure 0.03 right pleural ganglion 0.06 pleurosupraoesophageal connective 0.02 supraoesophageal ganglion 0.07 suboesophageal ganglion 0.07 left pleural ganglion 0.07

L. duct seminal receptacleL. 0.06 W. W. L. gillL. 1.97 L. osphradiumL. 0.57 W. gill ﬁlamentsNo. 26.29 L. gill ﬁlamentsL. 0.32 W. W. cerebral commissureL. 0.07 W. right pleural ganglionL. 0.11 W. pleurosupraoesophageal connectiveL. 0.27 W. supraoesophageal ganglionL. 0.10 W. suboesophageal ganglionL. 0.10 W. left pleural ganglionL. 0.13 W. Mantle cavity mantle cavity L. 2.09 Buccal mass buccal mass L. 0.48 Nervous system cerebral ganglion L. 0.21

Type material. Idume Creek (Torre Chianca, Lecce) [40.283∞N, 17.838∞E], 11 April 2000, Dario Fer- Lecce) [40.466∞N, 18.183∞E], 12 April 2000, Dario reri, ANSP, A19757. Ferreri. Holotype (Fig. 6A) ANSP, A19754. Description. The description for the new species is the Paratypes ANSP, A19755 (three wet specimens, same as for the new genus above. Measurements of Fig. 6B-D); SMF 323031/2 (two wet specimens, shell, soft body structures, and organs are given in Fig. 6E, F). Table 5. Photographs of shells are provided in Other material examined. Torre Castiglione (Torre Figure 6. For external features and pigmentation of Lapillo, Porto Cesareo, Lecce) [40.291∞N, 17.809∞E], 25 the head see Figure 2E. Variations in morphology of August 2000, Dario Ferreri, ANSP, A19756; the penis and the bursa copulatrix complex are shown Bambinello Spring (Torre Lapillo, Porto Cesareo, in Figures 7 and 8, respectively. Downloaded from https://academic.oup.com/zoolinnean/article/137/2/319/2632295 by guest on 23 November 2020