bs_bs_banner Zoological Journal of the Linnean Society, 2012, 166, 530–558. With 10 figures ENRIQUE MARTÍNEZ-ANSEMIL1, MICHEL CREUZÉ DES CHÂTELLIERS2, PATRICK MARTIN3* and BEATRICE SAMBUGAR4 1 Departamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Facultade de Ciencias, Universidade da Coruña, Campus da Zapateira s/n, E-15071 A Coruña, Spain 2 Université de Lyon, UMR5023 Ecologie des Hydrosystèmes naturels et anthropisés, Université Lyon 1, ENTPE, CNRS, 6 rue Raphaël Dubois, 69622 Villeurbanne, France 3 Institut royal des Sciences naturelles de Belgique, Biologie des Eaux douces, Rue Vautier 29, B-1000 Brussels, Belgium 4 Museo Civico di Storia Naturale, Lungadige Porta Vittoria 9, I-37126 Verona, Italy Received 29 November 2011; revised 2 July 2012; accepted for publication 7 July 2012 The Parvidrilidae Erséus, 1999 constitute the most recently described family of oligochaete microdriles. Prior to this study, Parvidrilus strayeri Erséus, 1999, and Parvidrilus spelaeus Martínez-Ansemil, Sambugar & Giani, 2002, found in groundwaters of the USA (Alabama) and Europe (Slovenia and Italy), respectively, were the only two species in this family. In this paper, six new species – Parvidrilus camachoi, Parvidrilus gianii, Parvidrilus jugeti, Parvidrilus meyssonnieri, Parvidrilus stochi, and Parvidrilus tomasini – and Parvidrilus gineti (Juget, 1959) comb. nov. are added to the family. With all species being stygobiont, the Parvidrilidae is unique in being the only family of oligochaetes worldwide comprising taxa that are restricted to groundwater habitats. Parvidrilids are exceedingly small worms whose principal morphological characteristics are the presence of hair setae in ventral bundles, the markedly posterior position of setae within the segments, the presence of mid-dorsal glandular pouches in mesosomial segments, the lateral development of the clitellum, the presence of a single male pore in segment XII, and the presence (or absence) of a single spermatheca. The phylogenetic relationships of the Parvidrilidae within the Clitellata were investigated using the nuclear 18S rRNA gene, and the most representative and taxonomically balanced data set of clitellate families available to date. The data were analysed by parsimony, maximum likelihood, and Bayesian inference. Irrespective of the method used, Parvidrilidae were placed far from Capilloventridae, one family once suggested to be closely related to parvidrilids. Although closer to Enchytraeidae than Phreodrilidae, two other suggested putative sister families, the exact position of Parvidrilidae within Clitellata still remained uncertain in the absence of branch support. The examination of reproductive structures, together with the similarity of other important anatomical traits of the new species herein described, reinforced the idea that phreodrilids were the best candidate to be the sister group to parvidrilids on morphological grounds. A fragment of the mitochondrial cytochrome oxidase I gene, used as a barcode, also genetically characterized a few Parvidrilus species. The observation that two species diverge from each other by high genetic distances, even though their type localities are more or less only 100 km apart, is interpreted in the context of low dispersal abilities of inhabitants of the subterranean aquatic ecosystem, and habitat heterogeneity. The Parvidrilidae appear to be a diversified, Holarctic, and probably widely distributed family in groundwater, but very often overlooked because of the small size and external similarity with the polychaete family Aeolosomatidae of its members. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558. doi: 10.1111/j.1096-3642.2012.00857.x ADDITIONAL KEYWORDS: aquatic oligochaete, molecular systematics, phylogeny. *Corresponding author. E-mail: patrick.martin@naturalsciences.be 530 © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 The Parvidrilidae – a diversified groundwater family: description of six new species from southern Europe, and clues for its phylogenetic position within Clitellata (Annelida) NEW EUROPEAN SPECIES OF PARVIDRILIDAE INTRODUCTION particularly arrangement, number, and types of setae: absence of setae in segment II (dorsal bundles in phreodrilids, dorsal and ventral bundles in capilloventrids), presence of hair setae in dorsal and ventral bundles in capilloventrids, and a similar set of needle and hair setae in the dorsal bundles of phreodrilids and parvidrilids. In parvidrilids, however, dorsal needles emerge from the body wall and do not look like support setae as in phreodrilids – species in which such setae are always contained within the setal sac (Pinder & Brinkhurst, 1997). During this work, we found new specimens of P. spelaeus and six new species of Parvidrilus. In addition, our re-examination of specimens convinced us that Aeolosoma gineti Juget, 1959, should be transferred to Parvidrilidae. The availability of so many new species for description gave us a morphological framework to compare the Parvidrilidae in depth with other oligochaete families. A few specimens of two new Parvidrilus species from France, and of previously described P. spelaeus, were suitable for molecular analyses. This additional molecular facet provided the opportunity to (1) characterize these three species with a fragment of the mitochondrial cytochrome oxidase I (COI) gene, used as a barcode; (2) assess the genetic divergence between them, and to put these data in a biogeographical context; and (3) investigate the phylogenetic relationships of the Parvidrilidae within the Clitellata using the nuclear 18S rRNA gene. The accumulation of new data on the distribution and habitat of parvidrilids resulting from this large amount of material has provided us with a more comprehensive understanding of the biogeography and ecology of this unique family of microdrile oligochaetes. MATERIAL AND METHODS MORPHOLOGICAL STUDY The material presently studied results from different sources: three collecting campaigns in Sardinian caves (Italy) organized by our colleague Fabio Stoch, a study of the groundwater oligochaete fauna of the French Jura, and a large survey of the groundwater fauna of six European regions in the course of the PASCALIS project (Gibert, 2001; Gibert & Culver, 2009), which enabled us to study material from the Italian Lessinian Mountains, Slovenian Krim Massif, and Spanish Cordillera Cantábrica. The PASCALIS sampling protocol was designed to account for habitat heterogeneity, with the primary objective being evaluation of the biodiversity of European groundwater habitats. One advantage of this approach was to characterize habitat preferences of parvidrilid species sampled in this framework. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 The Parvidrilidae Erséus, 1999, constitute the most recently described family of oligochaete microdriles. Previously, only two species had been described in this family – Parvidrilus strayeri Erséus, 1999 from Alabama (USA), and Parvidrilus spelaeus MartínezAnsemil, Sambugar & Giani, 2002 from Slovenia and Italy (southern Europe). Both are exceedingly small worms (around 1 mm long, 50 mm wide) and were collected in groundwater environments. Groundwater habitats are important centres of biodiversity, serving as refugia for relictual species (Gibert & Deharveng, 2002). During the last two decades, and especially as a result of the European Protocols for the Assessment and Conservation of Aquatic Life in the Subsurface (PASCALIS) project (Gibert, 2001; Gibert & Culver, 2009), the study of groundwater oligochaete biodiversity has benefited from a renewed interest from the present authors and a few other colleagues. To date, more than 300 nominal species have been already found in groundwaters all over the world, although the present knowledge is almost limited to the karst of southern Europe (Martin et al., 2008; Martínez-Ansemil & Sambugar, 2010). Some of these species should be considered as incidentals or waifs – taxa most likely carried along by surface waters flowing into subterranean environments where they were unable to sustain viable populations (Creuzé des Châtelliers et al., 2009); however, about one third of them have been found exclusively in this environment (stygobionts; Martin et al., 2008; Creuzé des Châtelliers et al., 2009; Martínez-Ansemil & Sambugar, 2010). The observation of an important species richness of the marine subfamily Phallodrilinae (Naididae) in groundwaters of southern Europe (B. Sambugar, N. Giani & E. Martínez-Ansemil, 1999; unpubl. data) and the discovery of two new species of the hitherto Baikalian genus Rhyacodriloides Chekanovskaya (Naididae, Rhyacodriloidinae) in subterranean water bodies of the eastern Alps (Martin, Martínez-Ansemil & Sambugar, 2010) appear amongst the most outstanding discoveries. Phallodrilines and rhyacodriloidines have also a very small size in common with parvidrilids, although the latter are even smaller. As a result of peculiar morphological features, the Parvidrilidae is thought likely to be an ancient family, and to hold a key phylogenetic position within the Clitellata and vis-à-vis the Annelida. Each time the systematic position of the Parvidrilidae was discussed on morphological grounds (Erséus, 1999; MartínezAnsemil et al., 2002), the attention was focused primarily on Capilloventridae and Phreodrilidae as the closest relatives. These two aquatic families share additional morphological traits with the parvidrilids, 531 532 E. MARTÍNEZ-ANSEMIL ET AL. MOLECULAR STUDY Taxa For phylogenetic relationships of the Parvidrilidae within the Clitellata, we analysed a data set, partly from the EMBL databank, consisting of the nuclear 18S rRNA gene fragments – for a total of 48 annelid taxa representing 14 clitellate and three polychaete families. This data set basically corresponds to the sequence matrix used by Erséus & Källersjö (2004) for investigating basal nodes of the clitellate phylogenetic tree, and is still the most representative and taxonomically balanced data set of clitellate families available to date. It is also the only data set that includes the Enchytraeidae, the Phreodrilidae, and the rare Capilloventridae – three families once assumed to be potential sister groups to the Parvidrilidae (Erséus, 1999; Martínez-Ansemil et al., 2002). Nine polychaete outgroups, identified in Erséus & Källersjö (2004) as the most distantly related taxa to the clitellate ingroup, were removed from the original data set, in order to reduce sources of bias owing to great evolutionary distances (substitutional saturation, sequence alignment ambiguity – see Abouheif, Zardoya & Meyer, 1998; Adoutte et al., 2000; Löytynoja & Goldman, 2008), potentially acute in this gene fragment because of hypervariable regions (Van de Peer et al., 1997). Owing to uncertainties about the taxonomic status of Haplotaxis in North America (Kathman & Brinkhurst, 1998), and its sensitivity to variations in the alignment parameters, the specimen of Haplotaxis cf. gordioides used in Erséus & Källersjö (2004) was removed from this data set and replaced with one specimen of Haplotaxis from France. Another representative of the Haplotaxidae, Delaya bureschi, was also included to assist in resolving the phylogenetic position of the family in the clitellate tree, and to minimize a possible misleading attraction between taxa that are not well fixed within the trees. Other additions to this data set included two species in the new naidid subfamily Rhyacodriloidinae (Martin et al., 2010), Rhyacodriloides abyssalis and Rhyacodriloides latinus (EMBL accession nos: FN796452 and FN796453, respectively), the tubificine Baikalodrilus digitatus, and the two new Parvidrilus species from France, Parvidrilus jugeti and Parvidrilus meyssonnieri (Table 1). Because of the limited material available, the genetic characterization of Parvidrilus taxa – based on a fragment of the mitochondrial COI gene – was only possible for three species: P. spelaeus, P. jugeti, and P. meyssonnieri. Specimens of P. spelaeus were from a sample collected in Pisoliti cave (Trieste, Italy; one specimen successfully sequenced). All specimens of P. meyssonnieri and P. jugeti were collected from their type localities (P. meyssonnieri: three and two specimens from ‘La Martinière’ and ‘Navogne’ galleries, respectively; P. jugeti: two specimens from Corveissiat cave – Table 1). All live specimens collected in the field were immediately preserved in 95 or 90% ethanol, and then stored at -20 °C after return to the laboratory. Owing © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Samples were taken in different groundwater habitats including the unsaturated and saturated zones of karst systems as well as the hyporheic and phreatic zones of alluvial groundwater. In springs and caves, the fauna was sampled by sieving sediments through a hand net (100 mm mesh); in the hyporheic zone, the Bou-Rouch pump or the Karaman-Chappuis methods were used (Malard et al., 2002). The phreatic zone was investigated in sampling wells by means of the Cvetkov net (Malard et al., 2002). Samples were fixed in the field using either a 4% formaldehyde solution or a Bouin-Hollande solution (Hollande, 1918; Laurylab, Elvetec, France <http://www. laurylab.com/>; French material), sorted out in the laboratory under a stereomicroscope, and subsequently transferred to 70% ethanol. Microdriles were either mounted whole on microscope slides or serially sectioned. Whole-mounted specimens were temporarily mounted on slides in glycerine during initial observation, and then returned to alcohol. These specimens were subsequently stained with Mayer’s paracarmin, differentiated in 70% acid alcohol, dehydrated in ethanol, cleared in xylene, and then permanently mounted on slides in Canada balsam. Serially sectioned specimens were embedded in Spurr’s resin, sectioned on an ultramicrotome stained with toluidine blue, then permanently mounted on slides in Canada balsam. The specimens were observed with a light microscope equipped with differential interference contrast (DIC) optics. Sediments containing specimens of parvidrilids destined for in vivo observations were transported to the laboratory in an icebox for processing. Specimens in the sediment samples were then sorted from the samples in a cold room kept at 11 °C. Some specimens in the samples collected from localities in France were observed and filmed in vivo in a drop of water, gently pressed under a coverslip on a microscope slide. All measurements, drawings, and photographs refer to these mounts. The type material for the new species described in this paper has been deposited in the following institutions: MHNL, Muséum d’Histoire naturelle de Lyon, Lyon (France); IRScNB, Institut royal des Sciences naturelles de Belgique, Brussels (Belgium); MCSNVR, Museo Civico di Storia naturale di Verona, Verona (Italy); and MNCN, Museo Nacional de Ciencias Naturales, Madrid (Spain). to their small sizes, complete specimens of each of the parvidrilid species were used for DNA extraction. Sections of the posterior ends of specimens representing species in the other families were used for DNA sequencing, with their anterior sections stored in 95% ethanol and deposited as voucher material in the collection of the IRScNB, Brussels. DNA extraction, PCR amplification, and sequencing followed protocols detailed in Martin et al. (2010). HE800203 HE800205 HE800204 HE800202 HE800209 HE800210 M. Creuzé des Châtelliers M. Creuzé des Châtelliers M. Creuzé des Châtelliers B. Sambugar Corveissiat cave (Ain (01), France; 2007 Gallery of Navogne, Bas-en-Basset (43), France; 2008 Gallery of ‘La Martinière’, Thurins (69), France, 2007 Pisoliti cave, Trieste, Italy; 2000 COI, cytochrome oxidase I. Parvidrilus spelaeus Martinez Ansemil et al., 2002 – HE800208 P. Martin Frolikha Bay, Lake Baikal, Russia; 1995 – – HE800206 HE800207 M. Creuzé des Châtelliers B. Sket Azergues River, Rhône (69), France; 2009 Mrzla jama cave, near Loz, Slovenia; 2007 Haplotaxidae Haplotaxis cf. gordioides (Hartmann, 1821) Delaya bureschi (Michaelsen, 1825) Naididae, Tubificinae Baikalodrilus digitatus Holmquist, 1979 Parvidrilidae Parvidrilus jugeti sp. nov. Parvidrilus meyssonnieri sp. nov. 18S rDNA Collector Collection site and year Taxon 533 Alignment and phylogenetic inference Alignments were carried out manually and by PRANK v. 081202 (default options – Löytynoja & Goldman, 2005; Löytynoja & Goldman, 2008) for COI and 18S, respectively. PRANK is a multiple sequence alignment method that recognizes insertions and deletions as distinct evolutionary events, preventing systematic errors associated with traditional methods. We determined PRANK to be the most suitable alignment method for dealing with 18S as this gene is well known for its hypervariable regions (Van de Peer et al., 1997), which might potentially cause important errors in alignment if insertions and deletions are not dealt with appropriately. PRANK was used in combination with PRANKSTER v. 100701 (default options – Löytynoja & Goldman, 2010): only sites that have a reliability of greater than or equal to 95% (posterior probabilities) in all the pairwise alignments were kept. In addition to providing a potential ‘DNA barcode’ for three parvidrilid species, the COI sequences were used to estimate the genetic divergence between these species, and to relate it to their biogeographical distribution. The genetic divergence was assessed with MEGA v. 5 (Tamura et al., 2011); pairwise distances were computed using all codon positions and the Kimura two-parameters (K2P) model of nucleotide substitutions (Kimura, 1980). The position of the Parvidrilidae within the Clitellata was assessed with phylogenetic analyses conducted on 18S sequences. The data set was analysed by parsimony, maximum likelihood, and Bayesian inference. Parsimony analyses were carried out in PAUP* 4.0b10 (Swofford, 2003). All characters were equally weighted and unordered. Alignment gaps were treated as a new state (fifth base) or as missing data. Heuristic searches were carried out with random sequence addition (ten replicates) and using tree-bisectionreconnection branch swapping. Branch support was evaluated using nonparametric bootstrapping (1000 replicates). When the alignment with gaps treated as missing data was analysed, the number of trees retained dramatically increased. In that case, the bootstrapping procedure was carried out using the MaxTrees option limited to 1000. No limit was set when gaps were treated as a new state. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Table 1. Clitellate taxa (Annelida) used in the analyses (newly sequenced specimens), and EMBL accession number for the respective sequences COI NEW EUROPEAN SPECIES OF PARVIDRILIDAE 534 E. MARTÍNEZ-ANSEMIL ET AL. SYSTEMATICS PARVIDRILUS MEYSSONNIERI DES CHÂTELLIERS & MARTIN SP. NOV. (FIGS 1, 2) Types: Holotype. MHNL 44003358, slide 6, mature specimen, stained in paracarmine and wholemounted in Canada balsam. Gallery of ‘La Martinière’ (45°40′51′′N, 04°38′51′′E, 350 m asl), Thurins (69), France, 28.ix.2000. Paratypes. MHNL 44003359, slide 1, six specimens, four immature, one mature and one fragment; MHNL 44003360, slide 3, four specimens, two mature and two immature; slide 6, one immature specimen; all specimens stained with paracarmine and wholemounted in Canada balsam; 28.ix.2000; gallery of ‘La Martinière’, Thurins (69), France. Other material examined: Apart from the type material, 11 specimens were observed and mounted on five scanning electron microscope (SEM) stubs, IRScNB, 28.i.2008; stub 1, three specimens, type locality; stubs 2–5, eight specimens, gallery of ‘Navogne’ (45°17′16′′N, 04°05′58′′E, 520 m asl), Bas-en-Basset (43), France. Thirty-six specimens were wholemounted in Canada balsam or in Amman’s lactophenol, in the collection of one of the authors (MCdC): ‘La Martinière’ (seven specimens, four mature and three juvenile, 14.ix.1999; 24 specimens, ten mature and 14 juvenile, 28.ix.2000); ‘Navogne’ (three immature specimens, 8.x.1999; one mature specimen, 28.i.2008). Etymology: Named after Marcel Meyssonnier, as a tribute to this outstanding and tireless speleologist, and long-time friend and team member of one of the authors (MCdC), and whose involvement and support were instrumental in the discovery of this new species. Description: Length 1.3-1.6 mm, 21–30 segments (seven complete mature specimens). Width 55–85 mm at V (mean = 59 mm ± 11, N = 24), 50–120 mm at XII (mean = 72 mm ± 19, N = 24). Prostomium rounded, 35–50 mm long, 20–38 mm wide at base. Body wall not papillate, cuticle smooth. Clitellum poorly developed, limited to large, swollen, transparent cells, only visible on living animals (Fig. 1C, c), budding on a mid-ventral depression of body wall (Fig. 2D, vdp) that extends behind genital pores, between ventral setae in XII and XIII. Dorsal (dorsolateral) and ventral (ventrolateral) setae present from III, posteriorly situated in each segment (Figs 1A, B, 2B, ds, vs). Dorsal bundles with two (three) straight, thin single-pointed needles (15– 28 mm long), distal end bluntly pointed, alternating with (one) two thin and flexible hair setae (length 150–230 mm at V, 200–260 mm at VIII, 170–225 mm at XI). Hair setae densely pilose (with soft hairlets) on one side of the shaft (pilosity mostly observed on specimens during study using SEM). Ventral bundles with (two) three (four) sigmoid, bifid crotchets with upper tooth slightly thinner and shorter than lower tooth, and accompanied by one (two) pilose hair setae in preclitellar segments (80–170 mm long); ventral hair setae absent from XII; crotchets without nodulus, 17–28 mm long, shaft out of the body wall faintly pilose, with distal half enlarged on the convex side. Within ventral bundles, the uppermost seta bifid, followed by one hair seta; these two setae separated from the other two bifid setae by a distance sometimes greater than that between them. One modified genital seta per bundle in XII, 55–60 mm long, straight with enlarged, spearhead ectal tip, the latter with a spoon-like curvature (Figs 1A, B, 2A, C, D, gs). No eyes. Brain long, extending into IV, posteriorly indented. Ventral nerve cord in contact with epidermis, beneath muscles of the body wall. Digestive tract complete; eversible pharynx with dorsal pad, sinuous, ciliated oesophagus, with pharyngeal glands present in IV-VI, followed by a simple intestine, slowly dilat- © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 For maximum likelihood and Bayesian analyses, the best-fit model was selected using JModel-Test 0.1.1 (Posada, 2008) by estimating and comparing maximum-likelihood scores for different substitution models. Models were selected according to the Akaike information criterion (Akaike, 1973) and its corrected variant, and the Bayesian information criterion (Schwarz, 1978). All criteria identified the general time-reversible substitution model with invariable sites and a discrete G correction for amongst-site variation (GTR + I + G model) as the best-fit substitution model, or one of its variant. Accordingly, this model was used to conduct maximum likelihood and Bayesian analyses. Maximum likelihood analyses were performed with PHYML v3.0 (Guindon & Gascuel, 2003), using default parameters and a subtree pruning and regrafting strategy for the exploration of the space of tree topologies. Bootstrapping was performed on 1000 generated pseudo data sets, using the same parameters. Bayesian analyses were conducted using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). Two parallel runs with four Markov chains each were run for two million generations and every 100th generation was sampled (resulting in 20 000 trees). The level of convergence was monitored on the .p files using TRACER 1.5.0 (Rambaut & Drummond, 2009), and the ‘burn-in’ value was set accordingly. The first 25% of the trees were discarded and the last 15 000 trees were used to reconstruct a consensus tree and estimate Bayesian posterior probabilities. NEW EUROPEAN SPECIES OF PARVIDRILIDAE 535 ing from X-XI and ending in a terminal anus. Layer of chloragogen cells surrounding digestive tract possibly present; details not observable in specimens thus far examined. Coelomocytes absent. Dorsal glandular body-wall pouches very small and difficult to observe, but observed in VIII and XII in one specimen), opening mid-dorsally at about the intersection with setal line. Voluminous glands associated with genital setae (20–48 mm long; 20–32 mm wide), bulging at the body surface; fine internal ducts running inside the glands and converging on the shaft of genital setae; setal glands asymmetrically arranged on each lateral side of the body, the left gland anteriad to the right one (Figs 1A–C, 2B, sg). Clearly delimited testes not present; however, a lump of cells with small nuclei observed dorsally in XI, in posterior end of coelom of XI, is probably unpaired male protogonia (Fig. 1A, t). Diffuse formation of male gametes in XI with many morulae (or ‘cysts’ according to Ferraguti, 1997), 2.8–4.0 mm wide, freely floating inside the coelomic cavity although usually lying ventrally in XI (Fig. 1A, mo); a few globular cytophores in a more dorsal and posterior position in XI, 8.0–11.3 mm wide, sometimes in a stage of disintegration with the detachment of spermatozoa in the coelom. Sperm funnels and vasa deferentia not seen. Atria elongate (90–190 mm long, 7–10 mm wide), either extending into XIII or restricted to XII; in the latter case, atria folded in different configurations: one atrium bent anteriad, with distal end folded up posteriad, the other atrium (or if present, both atria), bent posteriad, with distal end folded up anteriad (Fig. 1A, B, a). Atria merging below the nerve cord (Fig. 1A, B, nc) into a common ejaculatory duct, and opening at the tip of a mid-ventral, conical porophore (Figs 1A, B, 2A, C, mp), in posterior part of XII, between setal glands and in front of a median ventral depression of body wall in XII-XIII (Fig. 2D, vdp). Atrial ampullae © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Figure 1. Parvidrilus meyssonnieri sp. nov. A, left lateral view of genitalia in segments X-XII (holotype). B, ventral view of genitalia in segments XI-XIII (paratype MHNL 3.2). C, hand drawings of the unpaired spherical ampulla and associated cell cluster (from living animal). Abbreviations: a, atrium; am, spherical ampulla; c, clitellum; ccl, cell cluster; ds, dorsal seta; gs, genital seta; mo, morula; mp, male pore; nc, nerve cord; ov, oocyte; sg, setal gland; sp, spermatozoa; st, septum; t, testis (or germ cells); vs, ventral seta. Scale bars = 50 mm. 536 E. MARTÍNEZ-ANSEMIL ET AL. longitudinally striated, with large lumen filled with sperm, mature spermatozoa more concentrated near the distal part of ampullae (Fig. 1A, B, sp); no peculiar aggregate of sperm in the ampullae. An (unpaired?) ovary, apparently developing on one side, observed in anterior part of XII, with a ventral attachment on 11/12 (Fig. 1A, B, ov). Specimen ovigerous with large mature eggs filling coelom in XII, and extending through XIII. Female funnels not observed. No typical spermatheca observed. However, an unpaired, spherical ampulla in XII, 10 mm in diameter, ventrally located, ahead of the point of union of atrial ampullae, usually on the right side of the ventral nervous chord but sometimes on the left side in some specimens (Fig. 1A–C, am); ampulla opening, through a duct, 14 mm long, mid-ventrally, close to the basis of male porophore (Fig. 2C, amo); duct surrounded by a muscular ring near opening. Spherical ampulla filled with cell material (sperm?) and surrounded by a cell cluster, clearly delimitated, noticeable on living worms but difficult to resolve on mounted slides, possibly glandular (Fig. 1B, C). © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Figure 2. Parvidrilus meyssonnieri sp. nov.; scanning electron micrographs. A, left lateral view of anterior part of body showing dorsal and ventral setal bundles, and the absence of setae in II. B, ventral and dorsal setae. C, left lateral view of genital region showing a genital seta and its associated gland (damaged), the single male pore, and the opening of the spherical ampulla. D, left lateral view of genital region showing the left voluminous setal gland, the median ventral depression behind the porophore (not visible on the picture), and the left genital seta in XII (gs). Specimens from ‘Navogne’ (A–C) and ‘La Martinière’ (D) sites. Abbreviations: amo, spherical ampulla opening; ds, dorsal setae; gs, genital seta; mp, male pore; sg, setal gland; vdp, ventral depression; vs, ventral setae. Scale bars: A = 100 mm; B–D = 20 mm. NEW EUROPEAN SPECIES OF PARVIDRILIDAE COI sequences: EMBL accession numbers: HE800204 (gallery of ‘La Martinière’); HE800205 (gallery of ‘Navogne’). (see below). In addition, when copulatory organs are present in microdriles – whether in the form of porophores or penes – they are always associated with the male aperture (Stephenson, 1930), thus making the latter hypothesis less convincing. Distribution and habitat: Parvidrilus meyssonnieri is known only from the ‘La Martinière’ and ‘Navogne’ galleries in the Rhône department, France, in a region lying on metamorphic bedrock. The ‘La Martinière’ gallery opens in the upper alteration layer of a crystalline craton, which forms granitic sand with scarce, particulate organic matter. A rivulet runs along the gallery and passes slowly through a thin layer of coarse sand. Historically (between 1846 and 1919) the Navogne gallery was dug in arkoses and ferruginous sandstone for iron ore exploitation. The water is present in the gallery as isolated puddles with gravelly soil, rich in organic matter. PARVIDRILUS JUGETI DES CHÂTELLIERS & MARTIN SP. NOV. (FIG. 3) Holotype: MHNL 44003361, 07.07.1998, mature specimen, stained in paracarmine and whole-mounted in Canada balsam. Corveissiat Cave (46°14′34′′N, 05°29′01′′E, 378 m asl), Ain (01), France, 7.vii.1998. Paratypes: MHNL 44003364, one mature specimen; MHNL 44003365, one mature specimen; all specimens stained in paracarmine and whole-mounted in Canada balsam. Corveissiat Cave, France, 20.xii.2007. Figure 3. Parvidrilus jugeti sp. nov. Right lateral view of genitalia in segments XI-XIII (holotype). Abbreviations: a, atrium; ds, dorsal seta; mp, male pore; nc, nerve cord; omo, ovarian morula; ov, oocyte; sp, spermatozoa; t, testis; vs, ventral seta. Scale bar = 50 mm. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Remarks: Within the parvidrilid species known so far (including all new species being described herein plus the reassigned Parvidrilus gineti – see below), the presence of genital setae is unique to P. meyssonnieri sp. nov. – clearly separating this species from its congeners. The most outstanding feature in P. meyssonnieri sp. nov. is the presence of an enigmatic unpaired, spherical ampulla in XII – the function of which remains obscure. There is a possibility that this structure is used for storing sperm from a concopulant, which would imply that it is a spermatheca. However, the ampulla is surrounded by a cell cluster, which seems more glandular (prostate-like cells) than shaped in order to play the role of a bag for sperm storage. In this respect, such a structure is somewhat similar to the glandular, atrium-like organ in the pre-atrial segment of some Rhynchelmis (Rhynchelmis) species (Lumbriculidae), referred to as ‘accessory organ’ (formerly ‘rudimentary atrium’), whose function and derivation are unknown (Fend & Brinkhurst, 2010). Some material has been observed in the spherical ampulla but it does not resemble mature spermatozoa. Hence, the absence of spermathecae in this species seems plausible. Conversely, considering the ampulla and its surrounding cell cluster as an atrium and a prostate is another possibility – implying that the structures described above as ‘atria’ are in fact the spermathecae and the porophore is spermathecal. However, prostates seem to be absent in Parvidrilus 537 538 E. MARTÍNEZ-ANSEMIL ET AL. Other material examined: Six other specimens, wholemounted in Canada balsam, in the collection of one of the authors (MCdC): Corveissiat cave (one immature specimen, 7.vii.1998; three mature specimens, 20.xii. 2007; two mature specimens, 10.iii.2008). Description: Length 1.96 mm, 28 segments (one complete mature specimen). Width 68 mm at V, 85 mm at XII. Prostomium bluntly conical, 25 mm long, 40 mm wide at base; prostomium epidermis with numerous stained cell nuclei, indicating a possible sensory or glandular function. Body wall not papillate, cuticle smooth. Clitellum not seen. Dorsal (dorsolateral) and ventral (ventrolateral) setae present from III, posteriorly situated in each segment. Dorsal bundles with two (three) simplepointed needles (10–20 mm long), alternating with one (two) thin and flexible hair setae (length 80 mm at V, 110 mm at VIII, 200 mm at XI) (Fig. 3, ds). Hair setae apparently smooth when observed on permanent mount using an oil immersion lens. Ventral bundles with two (three) sigmoid, bifid crotchets with upper tooth slightly thinner and smaller than lower tooth, and accompanied by one hair seta in preclitellar segments (90 mm long); from XII, ventral hair setae absent, two crotchets per bundle (Fig. 3, vs). Crotchets without nodulus, 20–25 mm long, and shaft out of the body wall, with distal half enlarged on the convex side. Within ventral bundles in preclitellar segments, the uppermost seta bifid, followed by one hair seta; these two setae separated from the other two bifid setae by a distance greater than that between them. No modified genital setae. No eyes. Brain short, limited to III. Ventral nerve cord in contact with epidermis, beneath muscles of the body wall. Digestive tract complete; eversible pharynx with dorsal pad, sinuous, ciliated oesophagus, with pharyngeal glands present in III-IV, followed by a simple intestine, dilating in from X and ending in a terminal anus. Layer of chloragogen cells surrounding digestive tract possibly present; details not observable. Coelomocytes absent. Middorsal glandular pouches not observed. Testes in XI, as an unpaired, dorsal lump of cells with small nuclei in posterior end of coelomic cavity (Fig. 3, t); numerous spermatozoa below this cellular mass, with well-defined tails, detached in the coelom (Fig. 3, sp). Sperm funnels and vasa deferentia not seen. Atria elongate, tubular (265 mm long, 11 mm COI sequence: EMBL accession number: HE800203 (Corveissiat cave, France). Remarks: The long, narrowly tubular atria of P. jugeti sp. nov., which extends to XIV, are quite distinctive amongst the Parvidrilidae known to date. The high refringence of the atrial walls is unique. The outer atrial surface is striated all along the ampullae. Parvidrilus jugeti (and possibly P. meyssonnieri) is (are) the only parvidrilid(s) known thus far that lack spermathecae. The exact location of ovaries (paired or unpaired?) in P. jugeti remains obscure. A mature oocyte was observed ventrally in XII, on the right side of the specimen, close to 11/12, which suggests the existence of an unpaired, ventral ovary. In this specimen, it is likely that this female gonad has disappeared after having produced the few morulae observable in the coelomic cavity of XII, as documented previously in naidids (Chekanovskaya, 1962). Distribution and habitat: Parvidrilus jugeti is known only from the type locality in the Corveissiat cave, Ain (01), France. Sedimentary rock formation (karstic area); in a little pond, very finely clayey sediment, with little organic matter. PARVIDRILUS CAMACHOI MARTÍNEZ-ANSEMIL & SAMBUGAR SP. NOV. (FIG. 4) Holotype: MNCN 16.03/3070, mature specimen, longitudinal sectioned at 0.4 mm, stained with toluidine blue and mounted in Canada balsam. Estaragüeña cave (43°17′58.2′′N, 4°36′23.5′′W, Z 53 m asl), Puentellés, Cantabria, Spain, 4.ix.2002, leg. Ana Camacho. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Etymology: Named after Jacques Juget (1928–2006), mentor to one of us (MCdC), and friend and colleague to all of us, as a tribute to his lifelong contribution to the knowledge of the Clitellata, and particularly subterranean oligochaetes (see Creuzé des Châtelliers, Lafont & Giani, 2007). wide), one atrium entering and restricted to XIII, with distal end folded up anteriad, the other one extending straight into XIV (Fig. 3, a). Atria proximally merging below the nerve cord into a common ejaculatory duct, and opening ventrally in anterior part of XII, in front of setal line, through a penis-like structure, surrounded by a thick muscular ring (Fig. 3, mp). Atrial ampullae longitudinally striated, with large lumen filled in with undefined material (cellular? secretory?), different from mature spermatozoa visible in XI; atrial walls very thick, highly refringent under DIC. One mature oocyte (Fig. 3, ov), full of vitellus, ventrally, in anterior part of XII, near 11/12, suggesting the location of an unpaired ovary; a few small ovarian morulae freely developing into the coelomic cavity. Female funnels and spermathecae not observed. 539 NEW EUROPEAN SPECIES OF PARVIDRILIDAE a gp a a b ne pg sd sa vd mt XII p A C gp sv D cl oe nc B cr E g sa ee XIII cr XII VIII sa F g pg VII a XI a o nc o VI sv G sa XIII g sp mo vd XII a gm XI I g XIII sp XII p XI a H gm t ed XIII XII p XI a J g ed sa XIII sd t g p XII XI XIII XII mp XI Figure 4. Parvidrilus camachoi sp. nov. A, main components of the genitalia (schematic view). B–C, sections in anterior body region. D–J, sections in genital region. Abbreviations: a, atrium; b, brain; cl, clitellum; cr, crotchet; e, egg; ed, ejaculatory duct; g, gut; gm, granular material; gp, glandular pouch; mo, morula; mp, male pore; mt, mouth; nc, nerve cord; ne, neuropile; o, ovary; oe, oesophagus; p, porophore; pg, pharyngeal gland; sa, spermathecal ampulla; sd, spermathecal duct; sp, spermatozoa; sv, seminal vesicle; t, testis; vd, vas deferens; VI–XIII, segments. Scale bars = 10 mm. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 ed XIII 540 E. MARTÍNEZ-ANSEMIL ET AL. Etymology: Named after Ana Camacho, responsible for the PASCALIS project for the Spanish partners, in honour of her important contribution to the knowledge of European biospeleology. Remarks: The description of external anatomy cannot be given in detail as the individual was sectioned. Nevertheless, prior to sectioning, we noted that the external anatomy of this specimen agrees in all respects with that of P. spelaeus, including size of the worm, absence of modified genital setae (Fig. 4A, E, cr), and shape and number of dorsal and ventral somatic setae. Parvidrilus camachoi sp. nov. is one of three Parvidrilus species known so far that have a single spermatheca in segment XIII. The narrow and moderately elongated, tubular, bent atria and the roundish spermathecal ampulla filed with spermatozoids attached around the wall are diagnostic traits for this new species. The closest relative to P. camachoi sp. nov. is P. spelaeus but in the latter, atria are pyriform, the spermatheca is very large and irregular in shape, and sperm is arranged in large masses of agglutinated spermatozoids into the spermatheca. Distribution and habitat: Parvidrilus camachoi is known only from the type locality in the Estaragüeña cave Puentellés, Cantabria, Spain. Estaragüeña corresponds to a resurgence of the Diva River. The cave is hardly accessible, as there is a siphon a few metres from the entrance of the gallery. The species was found in the entrance hall, along the edge of the siphon, in wet sands with abundant organic matter. PARVIDRILUS GIANII MARTÍNEZ-ANSEMIL & SAMBUGAR SP. NOV. (FIG. 5) Types: Holotype. MNCN 16.03/3071, mature specimen, stained in paracarmine and whole-mounted in Canada balsam. Seldesuto cave (43°18′21.0′′N, 3°37′34.5′′W, 192 m asl), Matienzo, Cantabria, Spain, 2.ix.2002, leg. Ana Camacho. Paratype. MNCN 16.03/3072, immature specimen from type locality, 2.ix.2002, leg. Ana Camacho, stained in paracarmine and whole-mounted in Canada balsam. Etymology: Named after Narcisse Giani ‘maître et ami’, to whom many European oligochaetologists are very much indebted. Description: Entire mature worm, length 1.2 mm, 26 segments, width 40 mm at V, 42 mm XII. Prostomium rounded, 15 mm long, 25 mm wide at base. Body wall thin (especially in dorsal part), unpapillated; foreign © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Description: Body wall thin (especially in dorsal part), and unpapillated. Large globular clitellar cells observed on lateral sides of XI–XIII (Fig 4D, cl); porophore and surrounding area glandular. Brain long, extending into segment IV, deeply incised posteriorly (Fig. 4B, b). Ventral nerve cord in contact with epidermis, beneath muscles of the body wall (Fig. 4B, C, nc). Most setigeral segments have a mid-dorsal glandular pouch opening posteriorly on each segment, at or near the transversal setal line (Fig. 4B, C, gp); glandular pouches absent from IV-VI. Digestive tract complete, entirely ciliated, and ending in a terminal anus. Eversible pharynx, with small dorsal pad set off from oesophagus, followed by a narrow winding tube extending into VIII, with thick muscular walls at about VII–VIII (Fig. 4C, oe); alimentary canal completed by a gut, clearly enlarged from segment XIII (Fig. 4E–J, g). Compact pharyngeal glands present in IV–VI (Fig. 4B, C, pg). Digestive tract from V, surrounded by a well-developed layer of chloragogen cells. Coelomocytes and nephridia not observed. Two narrow testes attached to septum 10/11 (Fig. 4H–I, t). Free germ cells and morulae floating in the coelomic cavity of segment XI (Fig. 4G, mo). A small seminal vesicle in XII (Fig. 4E, G, sv). Sperm funnels not observed. A piece of vas deferens (about 1.5 mm wide) observed near the basal part of an atrium (Fig. 4A, F, vd). Atria tubular, somewhat curved, moderately elongated (about 32 mm long, 8–10 mm wide), extending into the beginning of XIII (Fig. 4A, F–J, a) merging, below the nerve cord, into a common ejaculatory duct which opens at the tip of a mid-ventral porophore located on the transversal setal line of segment XII (Fig. 4A, G–J, ed, mp, p). Atrial wall thin (less than 2 mm thick) and muscular (?); ejaculatory duct surrounded by a thin layer of muscles. Atrial lumen filled in with a granular material (secretions?, or remains of old epithelial cells?), and with a large amount of spermatozoids, their heads distally attached to atrial wall, and their long flagella orientated to the proximal atrial end, with a clear flamigerous appearance (Fig. 4H–I, gm, sp). Prostate glands absent. Two ovaries attached to septum 11/12 (Fig. 4E, o). A large vitellogenic mature egg in segment XII, slightly protruding into XIII (Fig. 4E, e). Oviducts not observed. A single spermatheca is present in XIII. Spermathecal ampulla round (about 18 mm in diameter), with a fine epithelial wall (1–3 mm thick); spermathecal duct (about 10 mm long) as a loosely defined structure, hollowed by a very narrow duct (?), ending near the septum 12/13 in a somewhat lateral position, at the left side of the worm (Fig 4A, E–H, sa, sd). Ampulla filled with spermatozoids, attached around the wall, and with a fine secretion. 541 NEW EUROPEAN SPECIES OF PARVIDRILIDAE A B h vd f h n o mo h n cr a2 XIII a1 XII od III XI a1 a2 cr sd ma sa h X mp cr cr IV sd D C a1 sa cr a1 a1 XII XII a2 a1 a2 XIII a1 a1 E XIII F g g sa g a1 a2 o vd XII p XII ma a2 g a2 XIII nc a2 g c XIII Figure 5. Parvidrilus gianii sp. nov. A, reconstruction of genitalia. B, setae. C–F, genital region. Abbreviations: a1-a2, atria; c, cuticle; cr, crotchet; f, sperm funnel; g, gut; h, hair; ma, muscular arch; mo, morula; mp, male pore; n, needle; nc, nerve cord; o, ovary; od, oviduct; p, porophore; sa, spermathecal ampulla; sd, spermathecal duct; vd, vas deferens; III–XIII, segments. Scale bars = 10 mm. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 cr 542 E. MARTÍNEZ-ANSEMIL ET AL. eggs attached to septum 11/12 (Fig. 5A, F, o). Two small oviducts seemingly attached to septum 12/13 (Fig. 5A, od). A single spermatheca present in XIII. Spermathecal ampulla (empty) ovoid (30 mm long, 9 mm high), with a nucleated epithelial wall surrounded by a thick muscular lining; spermathecal duct about 13 mm long, as a loosely defined structure, without lumen, basally enlarged, in continuity with the ventral body wall at the most anterior part of segment XIII, in a somewhat lateral position, at the left side of the worm (Fig. 5A, D–E, sa, sd). Remarks: The combination of long atria, a single spermatheca in segment XIII, and the cuticular wall of the common ejaculatory duct characterize P. gianii sp. nov. Amongst the three Parvidrilus species that have a single spermatheca in segment XIII, P. gianii is easily distinguishable from the others by its ovoid spermathecal ampulla surrounded by a thick muscular lining, and elongated atria (ten times longer than wide). The cuticular wall of the ejaculatory duct of this new species is unique amongst the genus. Distribution and habitat: Parvidrilus gianii is known only from the type locality, the Seldesuto cave, Matienzo, Cantabria, Spain. The species was sampled at 100 m from the entrance, along the shore of the lake, by stirring up rocks and sand covered by about 25 cm of water. The depth of the lake deepens very quickly a short distance from the shore; the gallery ends in a siphon. PARVIDRILUS STOCHI SAMBUGAR & MARTÍNEZ-ANSEMIL SP. NOV. (FIG. 6) Types: Holotype. VRO1003, mature specimen unstained, whole-mounted in Canada balsam. Monte Majore cave (40°30′47′′N, 8°36′33′′E), Thiesi, Sardinia, Italy, 26.vi.2008, leg. Fabio Stoch, Gianfranco Tomasin, Beatrice Sambugar, Paolo Marcia. Other material examined: One partially mature specimen, stained in paracarmine and whole-mounted in Canada balsam, from type locality, 17.iii.2005, leg. Fabio Stoch, Gianfranco Tomasin, Jos Notenboom. Two immature specimens, stained in paracarmine and whole-mounted in Canada balsam, from type locality, 8.ix.2006, leg. Fabio Stoch, Paolo Marcia. Etymology: Named after our friend Fabio Stoch, for his important contributions to the knowledge of European subterranean fauna. Description: Entire mature worms, length 1.2 mm, 28 segments, width 45 mm at V, 55 mm at XII. Prostomium rounded, 15 mm long, 29 mm wide at base. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 particles adhering to cuticle here and there along the body. Numerous transversal rows of thin cutaneous glands per segment. Clitellum not elevated, occupying at least XII-XIII. Dorsal (dorsolateral) and ventral (ventrolateral) setae present from III and posteriorly situated in each segment. Dorsal bundles with two (three) straight, thin single-pointed needles (20–28 mm long) and one or two long, thin, and flexible hair setae (105–190 mm long) (Fig. 5A, B, h, n). Ventral bundles composed of (one) two–three (four) strongly curved crotchets, 20–26 mm long, with enlarged distal half and doublepronged tip with minute distal tooth (Fig. 5A–C, cr); one thin hair seta (60–90 mm long) in preclitellar ventral bundles of III–VI. No modified genital setae; a single bifid crochet in ventral bundles of segment XII. No eyes. Brain long, extending into segment IV, deeply incised posteriorly. Ventral nerve cord in contact with epidermis, beneath muscles of the body wall. Most setigeral segments have a mid-dorsal glandular pouch opening posteriorly on each segment, at about the transversal setal line; glandular pouches absent from IV–VI. Digestive tract complete, entirely ciliated and ending in a terminal anus. An eversible pharynx, with small dorsal pad set off from oesophagus, followed by a narrow, thick walled winding tube extending into IX, with thick muscular walls at about VII–IX; alimentary canal completed by a gut clearly enlarged and filled in with sediment posteriorly from segment XIII (Fig. 5E, F, g). Compact pharyngeal glands present in IV–VI. Digestive tract surrounded from V by a well-developed layer of chloragogen cells. Coelomocytes and nephridia not observed. No compact testes attached to septum but free germ cells and morulae floating in the coelomic cavity of segment XI (Fig. 5A, mo). Two small sperm funnels opening in ventral part of septum XI/XII and continuing into very thin vasa deferentia (1.5-2 mm wide), very likely to be long and tightly folded, entering atria basally (Fig. 5A, F, f, vd). Atria elongate (about 150 mm long, 14–16 mm wide), extending in segments XII-XIII, curling anteriad (Fig. 5A, C–F, a1, a2), merging below the nerve cord and a thick muscular arch (Fig. 5A, E, ma, nc) into a common ejaculatory duct with cuticular walls, and opening on tip of a mid-ventral porophore located somewhat anterior to the transversal setal line of segment XII (Fig. 5A, F, c, mp, p). Atria made up by outer thin muscular layer (< 1 mm thick) and thick lining of vacuolated tissue, with indistinct lumen, except at the most basal portion, where the lumen is large and epithelial cells are finely granulated. Sparse unordered sperm embedded into vacuolated cells all along atria. Prostate glands absent. Two small ovaries with maturing 543 NEW EUROPEAN SPECIES OF PARVIDRILIDAE A sd a1 a2 sa B h h n n mo ma p cr a2 e XIII XII cr XI XIV h C D sa sd g g a1 a1 cr a1 XIII cl XI XII E ed p mo mp XII XIII XIV F g g g a1 a2 a2 a2 g XIII XIII p XI XII XII a1 XIV Figure 6. Parvidrilus stochi sp. nov. A, reconstruction of genitalia. B, setae. C–F, genital region. Abbreviations: a1-a2, atria; cl, clitellar cell; cr, crotchet; e, egg; ed, ejaculatory duct; g, gut; h, hair; ma, muscular arch; mo, morula; mp, male pore; n, needle; o, ovary; p, porophore; sa, spermathecal ampulla; sd, spermathecal duct; XI–XIV, segments. Scale bars = 10 mm. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 o 544 E. MARTÍNEZ-ANSEMIL ET AL. by sperm (about two thirds of atrial length of the holotype and only a very small part of atria of the maturing specimen collected in March 2005). Prostate glands absent. Two ovaries attached to septum 11/12 (poorly visible) (Fig. 6A, o). A single egg sac containing mature eggs extending into segment XV (Fig. 6A, e). Two small oviducts seemingly attached to septum 12/13. A single spermatheca present in XII. Spermathecal ampulla ovoid (33 mm long, 10 mm high), thin walled, followed by a conical, bent duct (about 20 mm long), without clear cut off from ampulla, thin walled distally, and proximally constituted by a loosely defined tissue ending close to male pore, in an anterior and somewhat lateral position, on left side of the worm. Ampulla and distal part of spermathecal duct full of spermatozoids (Fig. 6A, D, sa, sd). Remarks: Parvidrilus stochi sp. nov. is one of the three Parvidrilus species known so far that have a single spermatheca in segment XII and are devoid of genital setae. The most outstanding features of P. stochi sp. nov. are the very long, wide, and twisted atria (15 times longer than wide), combined with a spermatheca in the atrial segment that has a large spermathecal duct distally constituted by a loosely defined tissue. The ovoid shape of the spermatheca of this new species is only comparable to that of P. gianii. Distribution and habitat: Parvidrilus stochi is known only from the type locality in the Monte Majore cave (Sardinia, Italy). The cave opens in a small, isolated Miocene limestone outcrop rising from a volcanic plateau dating from the Oligocene-Miocene volcanism. The upper level of the cave is fossilized and percolating waters are collected in pools on clay and rock; the lower gallery is crossed by a small brook, which collects the waters sinking from a small valley at the entrance of the cave. PARVIDRILUS TOMASINI SAMBUGAR & MARTÍNEZ-ANSEMIL SP. NOV. (FIG. 7) Types: Holotype. MCSNVRO1004, entire mature specimen, stained in paracarmine and mounted in Canada balsam. Sa Ucca de su Tintirriolu cave (40°27′08′′N, 8°39′15′′E) Siniscola, Sardinia, Italy, 17.iii.2005, leg. Gianfranco Tomasin, Fabio Stoch, Jos Notenboom. Etymology: Named after Gianfranco Tomasin, for his important contribution to the knowledge of Italian cave ecosystems. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Body wall thin (especially in dorsal part), unpapillated; foreign particles adhering to cuticle along the body, but neither dense nor continuous. Numerous transversal rows of thin cutaneous glands per segment. Clitellum weakly developed (about XI-XIII), but some large cells observed in XII and XIII (Fig 6C, cl). Dorsal (dorsolateral) and ventral (ventrolateral) setae present from III, posteriorly situated in each segment. Dorsal bundles with two (three) straight, thin single-pointed needles (20–28 mm long) and one (two) long, thin and flexible hair setae (115–200 mm long) (Fig. 6A–C, h, n). Ventral bundles with (one) two–three (four) strongly curved crotchets, 20–27 mm long, with enlarged distal half and double-pronged tip with minute distal tooth (Fig. 6A–C, cr), and accompanied by one thin hair seta (95–110 mm long) only present in preclitellar ventral bundles III-VIII. No modified genital setae; ventral bundles of segment XII with three bifid crochets. No eyes. Brain long, extending into segment IV, deeply incised posteriorly. Ventral nerve cord in contact with epidermis, beneath muscles of the body wall. Most setigeral segments with a mid-dorsal glandular pouch opening posteriorly on each segment, on or immediately adjacent to the transversal setal line; glandular pouches absent from IV-VI. Digestive tract complete, entirely ciliated, ending in a terminal anus. An eversible pharynx, with small dorsal pad set off from oesophagus, followed by a narrow, winding tube, extending into IX, with thick muscular walls at about VII-IX; alimentary canal completed by a gut beginning in X, enlarged and filled in with sediment from segment XV (Fig. 6D–F, g). Compact pharyngeal glands present in IV-VI. Digestive tract surrounded by a well-developed layer of chloragogen cells from V. Coelomocytes and nephridia not observed. No compact testes attached to septum, but many free germ cells and morulae floating in the coelomic cavity of segment XI and in the most posterior and anterior parts of X and XII, respectively (Fig. 6A, E, mo). Sperm funnels and vasa deferentia not observed. Atria very elongate (about 250 mm long), twisted, tubular, with a diameter slightly decreasing from the proximal to the distal end (20–15 mm), extending in segments XII–XIV (Fig. 6A, D–F, a1, a2), merging below the nerve cord and opening on the tip of a mid-ventral porophore located between the two ventral setal bundles of segment XII (Fig. 6A, D, E, ed, ma, mp, p). Atria made up by extremely thin (muscular?) outer layer and thick lining of vacuolated tissue, with indistinct lumen, except at the most basal portion, where the lumen is large and the epithelial cells are finely granulated. Space occupied by vacuolated cells seemingly progressively replaced 545 NEW EUROPEAN SPECIES OF PARVIDRILIDAE h n A vd h a2 a1 cr B h mo sa f t e o cr XI cr sd XII h a1 XIII X XIV a2 C D sa sa a2 h a1 a1 n sp sp XIII gm XIII cr gm ed XII XII sp sd E g o F a1 vd a2 XIII a1 sp g gm XII cl XIII XII G cr nc XIII XII Figure 7. Parvidrilus tomasini sp. nov. A, reconstruction of genitalia. B, setae. C–G, genital region. Abbreviations: a1-a2, atria; cl, clitellum; cr, crotchet; e, egg; ed, ejaculatory duct; f, funnel; g, gut; gm, granular material; h, hair; mo, morula; n, needle; nc, nerve cord; o, ovary; sa, spermathecal ampulla; sd, spermathecal duct; sp, spermatozoa; t, testis; vd, vas deferens; X–XIV, segments. Scale bars = 10 mm. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 n 546 E. MARTÍNEZ-ANSEMIL ET AL. granular material (secretions?, remains of old epithelial cells?), and with a large amount of spermatozoids, their heads orientated towards the distal atrial end, and their long flagella orientated towards the proximal atrial end, with a clear flamigerous aspect (Fig. 7C–F, gm, sp). Prostate glands absent. Two small ovaries with maturing eggs attached to septum 11/12 (Fig. 7A, F, o). A mature egg observed in XIV (Fig. 7A, e). Oviducts not observed. A single spermatheca present in XII. Spermathecal ampulla tubular (35 mm long, about 10 mm wide), thin walled, and obliquely orientated towards the posterodorsal part of the segment; ampulla followed by a conical, bent duct (about 20 mm long), beginning with a thick epithelium delimiting a wide lumen, and then tapering, with the lumen becoming very narrow, and ending in a minute pore in an anterior and somewhat lateral position, on left side of the worm. Ampulla full of spermatozoids (Fig. 7A, C, D, sa, sd). Remarks: Parvidrilus tomasini sp. nov. is a species with long atria (about eight times longer than wide), and a single spermatheca located in the atrial segment. The most characteristic anatomical features of this species are the tubular shape of the spermathecal ampulla and the well-defined, narrow spermathecal duct. Compared with the other two species that have a spermatheca in segment XII, the atria of P. tomasini are considerably shorter than those of P. stochi and longer than those of P. strayeri (see Erséus, 1999, and remarks below). Distribution and habitat: Parvidrilus tomasini is known only from the type locality in the Sa Ucca de su Tintirriolu cave (Sardinia, Italy). The cave occurs in an isolated Miocene limestone area surrounded by volcanic rocks of the same age. A small brook runs through the lower gallery in contact with the basaltic floor. PARVIDRILUS SPELAEUS MARTÍNEZ-ANSEMIL, SAMBUGAR & GIANI, 2002 New material: Mine of Ponte Vajo Falconi, Italy (PASCALIS LES 060; 10°59′07′′E; 45°39′36′′N), pools of percolating water, 2002, leg. Stoch F., Tomasin G., one specimen; Bus del Cao cave, Italy (PASCALIS LES 001; 10°54′48′′E; 45°35′36′′N), subterranean brook, 2002, leg. Stoch F., Tomasin G., two specimens; Buso del Progno cave, Italy (PASCALIS LES 003; 10°55′02′′E; 45°36′28′′N), subterranean brook, 2002, leg. Stoch F., Tomasin G., one specimen; Covolo della Croce cave, Italy (PASCALIS LES 099; 11°07′16′′E; 45°36′43′′N), rimstone pools, 2002, leg. Stoch F., Tomasin G., one specimen; Monte Capriolo cave, Italy © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Description: Entire mature worm, length 1.2 mm, 24 segments, width 45 mm at VI, 55 mm at XII. Prostomium rounded, 15 mm long, 29 mm wide at base. Body wall thin (especially in dorsal part), unpapillated; foreign particles adhering to cuticle here and there along body. Numerous transversal rows of thin cutaneous glands per segment. Clitellum weakly developed (about XI-XIII), except for two prominent pads comprising a few cells on lateral sides in XII, just anterior to the transversal setal line (Fig. 7G, cl). Dorsal (dorsolateral) and ventral (ventrolateral) setae present from III and posteriorly situated on each segment. Dorsal bundles with two (three) straight, thin single-pointed needles (22–29 mm long) and one long, thin, and flexible hair setae (120– 200 mm long) (Fig. 7A–C, h, n). Ventral bundles composed of two-three strongly curved crotchets, 20–26 mm long, with enlarged distal half and doublepronged tip with minute distal tooth, and accompanied by one thin hair seta (95–135 mm long) only present in III-XI (Fig. 7A–C, G, h, cr). No modified genital setae; ventral bundles of segment XII with a single bifid crochet. No eyes. Brain long, extending into segment IV, deeply incised posteriorly. Ventral nerve cord in contact with epidermis, beneath muscles of the body wall. Most setigeral segments with a mid-dorsal glandular pouch opening posteriorly on each segment, on about the transversal setal line; glandular pouches absent from IV-VI. Digestive tract complete, entirely ciliated. An eversible pharynx, with small dorsal pad set off from oesophagus, followed by a narrow, winding tube extending into IX, with thick muscular walls at about VII-VIII; alimentary canal completed by a gut containing sediment, clearly enlarged from segment XIV (Fig. 7E, g). Compact pharyngeal glands present in IV-VI. Digestive tract surrounded by a welldeveloped layer of chloragogen cells from V. Coelomocytes and nephridia not observed. Two somewhat disaggregated testes attached to septum 10/11 (Fig. 7A, t). A few free germ cells and morulae floating in the coelomic cavity of segment XI and in the most anterior part of XII (Fig. 7A, mo). Sperm funnels hardly observed (Fig. 7A, f). Vasa deferentia seemingly present as very thin (about 1.5 mm wide), long and tightly folded ducts in XII (Fig. 7A, E, vd). Atria elongate (about 150 mm long), tubular (15–22 mm wide), extending into segments XII-XIII, merging below the nerve cord and opening on the tip of a mid-ventral pore (in a small porophore?) located between ventral setal bundles of segment XII (Fig. 7A, D, a1, a2, ed). Atria made up by an outer thin muscular layer < 1.5 mm thick, and a nucleated epithelial layer, limited to the proximal and most distal parts of atrial wall. Atrial lumen filled in by a NEW EUROPEAN SPECIES OF PARVIDRILIDAE described by Erséus (1999) to be the two atria merging into a common ejaculatory duct and the spermatheca, respectively. Distribution and habitat: Known only from the type locality in Alabama, USA. Interstitial water. [A subsequent visit to the type locality by C. Erséus and M. J. Wetzel in March 2008 failed to collect additional specimens of P. strayeri for study (M. J. Wetzel, pers. comm.).] SPECIES PARVIDRILUS (JUGET, 1959) COMB. NOV. Aelosoma Gineti Juget, 1959: 397–399, figure 3a. Aelosoma gineti Juget (1959) – Ginet, 1961: 310. Aeolosoma gineti Juget, 1959 – Brinkhurst, 1967: 112; Bunke, 1967: 266; Brinkhurst & Jamieson, 1971: 695; Seyed-Reihani, 1980: 57, table VI; Dole, 1983a: 227; Dole, 1983b: 82, 110, 113, ann. 8; Juget & Dumnicka, 1986: 234, 239; Dole-Olivier et al., 1993: 457, Table 2; Dole-Olivier et al., 1994: 321; Ferreira et al., 2003: 17; Artheau & Giani, 2006: 230; Ferreira et al., 2007: 585; Timm, 2009: 18, 132. Description: The new material of P. spelaeus enables us to confirm the attachment of the spermathecal duct to the anterior part of segment XIII, and very thin vasa deferentia joining atria proximally. The so-called diffuse prostate surrounding the atria referred to in Martínez-Ansemil et al. (2002: fig. 12) is now interpreted as a large peritoneal layer, not observed in fully developed individuals. Types: Lectotype. MHNL 44003362, ‘Aeolosoma gineti, Lac souterrain La Balme, février 1959’, fragment (first 11 segments), whole-mounted in an unspecified, slightly yellow liquid medium (Amman’s lactophenol?), sealed with red lute. La Balme Cave (45°51′10′′N, 5°20′21′′E, 220 m asl), Isère (38), France. Paralectotype. MHNL 44003363, ‘Aeolosoma gineti, Lac souterrain La Balme, février 1959’, wholemounted in an unspecified, slightly yellow liquid medium, sealed with red lute. COI sequence: EMBL accession number: HE800202 (Pisoliti cave, Trieste, Italy). Distribution and habitat: Presently, the known distribution of P. spelaeus is limited to the Alpine district of northern Italy and extends into the Slovenian Dinaric region, where it was found in several phreatic and hyporheic habitats: from small pools of percolating water to streamlets and in cave lakes, springs, and rock aquifers (see also Martínez-Ansemil et al., 2002). STRAYERI GINETI INQUIRENDA Other material examined: UCBLZ 1.011.1-1.011.3, ‘Aeolosoma cf. gineti; Ph 7 1, 20/10/76’, alluvial plain of the Haut Rhône, phreatic and hyporheic waters (45°48′50′′N, 05°04′48′′E, 179 m asl; see Artheau & Giani, 2006), Rhône (69), France; three specimens whole-mounted in Canada balsam. ERSÉUS, 1999 Amended description: In the light of the atrial appearance, and the preatrial location of the spermatheca in some of the new species, we can now interpret the so-called ‘copulatory organ’ and ‘genital body’ Table 2. Matrix of evolutionary divergence between cytochrome oxidase I sequences of three Parvidrilus species. Values in the upper right corner are p-distances. Values in the lower left corner are distances estimated according to the Kimura two-parameter model Taxon P. spelaeus P. meyssonnieri (La Martinière) P. meyssonnieri (Navogne) P. jugeti Parvidrilus spelaeus Parvidrilus meyssonnieri sp. nov. (La Martinière) Parvidrilus meyssonnieri sp. nov. (Navogne) Parvidrilus jugeti sp. nov. – 0.21 0.20 0.23 0.18 – 0.09 0.22 0.17 0.09 – 0.23 0.19 0.19 0.19 – © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 (PASCALIS LES 195; 11°04′52′′E; 45°35′38′′N), rimstone pools, 2002, leg. Stoch F., Tomasin G., four specimens; Buso della Volpe cave, Italy (PASCALIS LES 149; 11°12′52′′E; 45°36′48′′N), small pools in gravel, 2002, leg. Stoch F., Tomasin G., two specimens; Montorio, via del Lanificio, Italy (PASCALIS LES 131; 11°03′59′′E; 45°27′34′′N), hyporheic site, Bou-Rouch pump, 2002, leg. Stoch F., Tomasin G., two specimens; spring near Jelenska jama, Slovenia (PASCALIS KRI 097; 14°21′19′′E; 45°55′05′′N) 2002, leg. Brancelj A., one specimen; spring near Žumerju, Slovenia (PASCALIS KRI 005; 14°35′47′′E; 45°53′16′′N), 2002, leg. Brancelj A., one specimen; Pajsarjeva cave, Slovenia; (14°15′56′′E; 45°59′52′′N), subterranean brook, 2009, leg. Gasparo F., Sambugar B., two specimens. PARVIDRILUS 547 548 E. MARTÍNEZ-ANSEMIL ET AL. Remarks: According to the original description by Juget (1959), it seems likely that a few specimens were observed alive, which would explain the mention of chains of zooids of two to five specimens, although they were absent in the material available to us for observation. In all probabilities, the specimen shown in the original illustration (Juget, 1959: 398, fig. 3a) corresponds to the lectotype. It is similar in external appearance, and in the number and arrangement of setal bundles, except that the first setigeral segment is not shown on the figure – suggesting that there are eight setigeral segments instead of nine. In this specimen, the so-called ‘zooid’ does not show any clear zone of scission and cannot be considered as such. The combination of setal features (e.g. shape, number, and location of setae) is typical of Parvidrilus, supporting its placement within the family Parvidrilidae. In fact, Bunke (1967: 266) early considered ‘Aeolosoma gineti’ as a species dubia and suggested that the species should be excluded from the polychaete genus Aeolosoma because of setal features, the absence of coloured glands in the tegument, the small and unciliated prostomium, and the organization of intestinal tract. A possible alignment with the oligochaete family, Naididae, was also suggested by Bunke, an opinion that was later restated by others (Brinkhurst & Jamieson, 1971; Artheau & Giani, 2006). More recently, Timm & Veldhuijzen van Zanten (2002) and Timm (2009) suggested that the species might well be a parvidrilid although no paratomy was yet recorded in this group. The external morphology of P. gineti is similar to other Parvidrilus species. However, in the absence of properly preserved internal organs – and in particular, the genitalia – no unambiguous description of the species can be provided. Even if new material from the type locality could be collected and properly preserved, there would be no guarantee of having sampled this same taxon; thus we must consider P. gineti to be a species inquirenda. Distribution and habitat: ‘La Balme’ cave, Isère (38), France, in a subterranean lake, heterogeneous sediment (rough sand and fine clay, with organic remains, mostly of plant origin); alluvial plain of the Haut Rhône, France, phreatic and hyporheic waters (Miribel canal and ‘Lône du Grand Gravier’; Artheau & Giani, 2006). OTHER PARVIDRILIDAE Several other specimens from three localities (below) appear to be parvidrilids and have unique characters supporting their consideration as new species. Unfortunately, they cannot be identified or classified until additional material has been collected. • Slovenia, phreatic zone of the Podlipščica valley, (PASCALIS KRI 187; 45°59′34″N, 14°15′01″E). One single specimen. Atria and spermatheca in XII, atria very large and globular, filling whole of segment. • Slovenia, Pajsarjeva cave, 1997. One specimen characterized by very long atria reaching 13/14, with a thin wall and full of spermatozoids. Spermathecae not seen because of the poor preservation of the specimen. • Italy, Tondello spring, Verona (PASCALIS LES 129: 45°27′52″N, 11°03′55″E). One broken specimen with very long atria. RESULTS MOLECULAR ANALYSES Genetic divergence The COI sequences of five specimens of P. meyssonnieri (three and two specimens from the galleries of ‘La Martinière’ and ‘Navogne’, respectively), and two specimens of P. jugeti (both from the Corveissiat cave) were successfully obtained. Specimens from the same locality have identical haplotypes. In contrast, the COI sequences of P. meyssonnieri from the Gallery of ‘La Martinière’ and from the Gallery of ‘Navogne’ differ from each other by 9%. At the specific level, the genetic divergence amongst P. jugeti, P. meyssonnieri, and P. spelaeus ranges from 17–20% at the minimum to 19–23% at the maximum (p- and K2P distances, respectively; Table 2). Phylogenetic position within the Clitellata The final PRANK alignment of the 18S data contained a total number of 1802 characters, 350 parsimony-informative characters when gaps were © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Emended description: Lectotype: length 435 mm, 11 segments (fragment); paralectotype: length 1450 mm, 21 segments (uncertain as specimen badly damaged as a result of stretching into two parts). Breadth 115 mm at widest (IV-IX). Prostomium rounded to conical, 40 mm long, 45–55 mm wide at base; ciliation absent. Dorsal and ventral setae present from III, posteriorly situated in each segment, absent in II. Dorsal bundles with one or two straight, singlepointed needles (15–30 mm), and one hair seta (length 150–237 mm). Ventral bundles with one (lectotype) to two–three (paralectotype) sigmoid, bifid crotchets (15–18 mm) with upper tooth slightly thinner and shorter than lower tooth, accompanied by one smooth hair seta (length 95–133 mm); ventral hair setae absent from XI. Internal organs mostly destroyed in the mounting medium used. Progressive intestinal dilation from VII partially visible. NEW EUROPEAN SPECIES OF PARVIDRILIDAE DISCUSSION AUTAPOMORPHIES AND OTHER RELEVANT CHARACTERS OF THE PARVIDRILIDAE The six new species of Parvidrilus described in this work support the soundness of the character choice by Martínez-Ansemil et al. (2002), considered as the most reliable traits supporting the monophyly of the family from the morphological point of view: the presence of singular segmental glandular pouches in the mid-dorsal line of the body, the lateral development of the clitellum with cells that are large in relation to the body diameter, the specific set of anterior ventral setae (bundles composed of bifid crotchets and hair setae), and the markedly posterior position of setae within the segments. The glandular pouches were not observed in P. jugeti, and only sporadically in P. meyssonnieri – perhaps a negative consequence attributable to the fixative (Bouin-Hollande solution) used for the specimens in the French collections. The unique spermatheca attached to the ventral body wall (when present) is another distinctive character of the family. As all parvidrilids presently known have a single male pore, this feature also becomes an important diagnostic character for the family. In fact, apart from the hirudineans, acanthobdellids, and branchiobdellids (Sawyer, 1986; Brinkhurst, 1999), the confluence of paired male ducts into a single mid-ventral pore is quite uncommon within clitellates. Interestingly, the male ducts of some parvidrilids look similar to that of most phreodrilids: long atria, with a thick and glandular inner epithelial lining, leaving only a narrow lumen, and vasa deferentia entering the ectal part of the atria. No prior attention has been directed towards spermatogenesis of parvidrilids, which seems to begin very early, free in the coelomic cavity, when germ cells come off septum 10/11. In oligochaetes, testes never grow very large because their activity is restricted to the formation of reproductive cells in their early stages of development (Chekanovskaya, 1962). The parvidrilids seem to be an extreme case, only comparable to what occurs in some small naidids, where the testes disappear very early (Sperber, 1948). It is possible that the quick release of germ cells, developing free in the coelomic cavity of parvidrilids, is related in some way to the minute size of these worms. The presence of spermatozoids with their heads embedded into the atria body wall is also an uncommon phenomenon in oligochaetes that warrants further study. As neither spermatophores nor spermatozeugmata were observed in parvidrilids, we suggest the possibility that the secretions provided by the epithelial glandular wall feed the spermatozoa at the end of spermiogenesis, or (at a minimum) supply abundant fluid to sperm. In their review on sexual and parthenogenetic planarians, D’Souza & Michiels (2009) emphasized the fact that parthenogens seem to reduce the proportion of sperm and transfer more accessory fluid. Interestingly, some of the parvidrilid species presently known are either devoid of spermathecae or have a single spermatheca that is empty. Although a leech-like impregnation is not excluded, sperm-dependent parthenogenesis – frequently associated with hermaphroditism (Beukenboom & Vrijenhoek, 1998) – could be also expected as a mechanism of reproduction in parvidrilids. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 coded as missing data, and 389 parsimonyinformative characters when gaps were coded as a fifth character state. A total of 11 025 trees was obtained in parsimony analyses when gaps were coded as missing data, resulting in a consensus tree with most basal nodes unresolved. Only 174 trees were produced when gaps were coded as a fifth character, yielding a consensus tree that is nearly completely resolved (Fig. 8). Bayesian and maximum likelihood analyses returned trees that are quite similar, except for two conflicting nodes, owing to a switched position of the Parvidrilidae and the Propappidae according to the analysis considered (Fig. 9). Parsimony (MP), maximum likelihood (ML) and Bayesian (B) analyses produced trees that are congruent to a great extent when only supported branches (posterior probabilities > 90%, or bootstrap values > 70; Hillis & Bull, 1993) are considered (Figs 8, 9). All methods gave a similar branching pattern (except for MP with gaps as missing data), even if many nodes, mostly basal, were not supported. The monophyly of all families are recovered and supported, but Haplotaxidae – of which the sister relationship of Delaya bureschi was found to be with Crassiclitellata, instead of Haplotaxis gordioides – makes the family paraphyletic. At a higher taxonomic level, the monophyly of Crassiclitellata and of the clade (Lumbriculidae, Branchiobdellida, Acanthobdellida, Hirudinea) is confirmed and supported, although – within this last assemblage – the position of Branchiobdellida vis-à-vis Lumbriculidae and clade (Acanthobdellida, Hirudinea) remains unresolved. As to Parvidrilidae, the three methods (MP, ML, B) gave congruent, although unsupported results, placing the family well within the Clitellata – far from Capilloventridae and Phreodrilidae, but close to Enchytraeidae. The ML analysis only differed from MP and B in a switched position of Parvidrilidae with Propappus: Enchytraeidae was placed as the sister group to either Propappus (ML) or Parvidrilidae (MP, B). 549 550 E. MARTÍNEZ-ANSEMIL ET AL. 94/80 95/96 85/85 77/72 91/79 100/100 56/- 94/84 71/61 92/86 81/75 99/98 100/100 100/100 53/* 100/100 100/100 100/100 100/100 96/96 80/71 61/64 92/91 74/63 100/100 93/91 58/53 54/100/99 82/81 100/100 100/100 Polychaeta Capilloventridae Phreodrilidae Naididae (naidids + tubificids) Enchytraeidae Propappidae Parvidrilidae Crassiclitellata Haplotaxidae Lumbriculidae Branchiobdellida Acanthobdellida Hirudinea -------------- Figure 8. Strict consensus of 174 most parsimonious trees resulting from a cladistic analysis of the 18S rDNA of 48 species of Clitellata (gaps coded as fifth character). Grey lines represent outgroup families. Numbers next to nodes correspond to nonparametric bootstrap values > 50% based on 1000 pseudoreplicates. From left to right, the two values refer to bootstrap when gaps are treated as a fifth character or missing data, respectively. Conflicting nodes between both analyses are asterisked. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 56/- Notomastus latericeus Loimia medusa Arenicola marina -------------Capilloventer australis -------------Antarctodrilus proboscidea Insulodrilus bifidus -------------Rhyacodriloides abyssalis Rhyacodriloides latinus Nais communis Pristina longiseta Baikalodrilus digitatus Thalassodrilus gurwitschi Smithsonidrilus humilis Tubificoides bermudae Heteridrilus decipiens Pectinodrilus molestus Heronidrilus heronae Bathydrilus litoreus Bothrioneurum vejdovskyanum Buchholzia fallax -------------Fridericia tuberosa Grania variochaeta Grania americana Marionina sublitoralis -------------Propappus volki Parvidrilus jugeti sp.-------------n. Parvidrilus meyssonnieri sp. n. Dichogaster saliens -------------Pontodrilus litoralis Lumbricus castaneus Lumbricus terrestris Eisenia andrei Criodrilus lacuum Dendrobeana clujensis -------------Delaya bureschi Haplotaxis cf. gordioides Eclipidrilus frigidus -------------Stylodrilus heringianus Rhynchelmis tetratheca Lumbriculus variegatus -------------Cirrodrilus sapporensis Branchiobdella parasita Cambarincola pamelae -------------Acanthobdella peledina -------------Glossiphonia complanata Helobdella stagnalis Erpbdella japonica Haemopis caeca NEW EUROPEAN SPECIES OF PARVIDRILIDAE Notomastus latericeus 53/- -------------------------------------------------------Polychaeta Capilloventridae Phreodrilidae Naididae (naidids + tubificids) Enchytraeidae Propappidae Parvidrilidae Crassiclitellata Haplotaxidae Branchiobdellida Lumbriculidae Acanthobdellida Hirudinea ----- Figure 9. Maximum likelihood tree resulting from an analysis of the 18S rDNA of 48 species of Clitellata (gaps treated as unknown characters). Grey lines represent outgroup families. From left to right, numbers next to nodes correspond to nonparametric bootstrap values > 50% based on 1000 pseudoreplicates, and to posterior Bayesian probabilities, respectively. Conflicting nodes between both analyses are asterisked. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Loimia medusa Arenicola marina ----------------------------------------------------Capilloventer australis 95/1.00 Antarctodrilus proboscidea -----------------------------------------------------------Insulodrilus bifidus --------------------------------------------------------------------88/1.00 Rhyacodriloides abyssalis 91/1.00 -/0.79 Rhyacodriloides latinus 89/1.00 Nais communis Pristina longiseta 65/0.94 Baikalodrilus digitatus Thalassodrilus gurwitschi -/0.87 99/1.00 Smithsonidrilus humilis 70/1.00 Tubificoides bermudae Heteridrilus decipiens 94/1.00 Pectinodrilus molestus Heronidrilus heronae Bothrioneurum vejdovskyanum Bathydrilus litoreus -------------------------------------------------------98/1.00 Buchholzia fallax -/0.78 71/0.89 Fridericia tuberosa Grania variochaeta 100/1.00 100/1.00 Grania americana * Marionina sublitoralis --------------------------------------------------------------Propappus volki --------------------------------------------------------------Parvidrilus jugeti sp. n. 100/1.00 Parvidrilus meyssonnieri sp. n. 100/1.00 Dichogaster saliens ---------------------------------/0.91 Pontodrilus litoralis Lumbricus castaneus 95/1.00 Lumbricus terrestris Eisenia andrei * 100/1.00 88/1.00 Criodrilus lacuum Dendrobeana clujensis 62/1.00 ------------------------------Delaya bureschi Haplotaxis cf. gordioides Cirrodrilus--------------------------------------------------sapporensis 60/100/1.00 Branchiobdella parasita Cambarincola pamelae 67/1.00 -------------------------------------------------------Eclipidrilus frigidus 92/1.00 Stylodrilus heringianus 79/0.98 Rhynchelmis tetratheca 70/0.90 Lumbriculus variegatus --------------------------------------------Acanthobdella peledina --------------------------------------86/1.00 Glossiphonia complanata 97/1.00 Helobdella stagnalis 100/1.00 Erpobdella japonica 100/1.00 Haemopis caeca 0.05 551 552 E. MARTÍNEZ-ANSEMIL ET AL. PHYLOGENETIC PARVIDRILIDAE CLITELLATA Morphology From the morphological point of view, the most relevant characters currently used in oligochaete taxonomy at the family level are the number and location of gonads, the location of male pores with respect to male funnels, the location of spermathecae, and the structure of male ducts (e.g. Stephenson, 1930; Brinkhurst & Jamieson, 1971; Brinkhurst, 1984; Erséus, 2005). The recent merging of the former family Tubificidae Vejdovský with the Naididae Over the last few years, DNA sequences have been increasingly employed in taxonomy and the COI gene has been considered as an important marker that helps species delimitation (Lefébure et al., 2006b; Erséus & Gustafsson, 2009; Grant & Linse, 2009; Trontelj et al., 2009; Havermans et al., 2011). The efficiency of this approach depends on the separation between intra- and interspecific divergences (Meyer & Paulay, 2005; Waugh, 2007). In several animal taxa, DIVERGENCE BETWEEN SPECIES © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 DNA Based on a data set mostly similar to the one analysed by Erséus & Källersjö (2004), it is reassuring that our analysis produced nearly the same relationships, amply discussed by those authors, in spite of other methods as to alignments (PRANK vs. ClustalX) and phylogenetic reconstructions (ML, B, in addition to MP), and despite lack of support for basal nodes of phylogenies. A major discrepancy was, however, noted for the position of Haplotaxidae and Propappidae vis-à-vis other clitellate families. The most striking difference lies in the fact that Haplotaxidae, although as a paraphyletic assemblage, are now the closest taxon to the Crassiclitellata, as suggested by morphology-based evolutionary relationships (Brinkhurst, 1994). As a result, the Diplotesticulata are restored as a monophyletic group, in conformity with the classification structured according to a morphocladistic analysis by Jamieson (1988) (see also Jamieson & Ferraguti, 2006). As for Propappidae and Parvidrilidae, nothing conclusive can be reached in the absence of support for most basal branches. At best, we emphasize that a suggested yet still unsupported sister relationship between Propappidae and Enchytraeidae in the ML analysis is in agreement with their morphology (Coates, 1986, 1987) and evolutionary relationships suggested by morphology (Brinkhurst, 1994). Such a Propappidae-Enchytraeidae sister relationship was also recently obtained by Marotta et al. (2008) in a phylogenetic analysis of Clitellata using a combination of molecular (18S rDNA) and morphological data (somatic and spermatozoal characters). All analyses are congruent in suggesting that Parvidrilidae are nested well within clitellates, namely far from Capilloventridae, a family that was once suggested to be a potential sister taxon (Erséus, 1999; MartínezAnsemil et al., 2002). Although closer to Enchytraeidae than Phreodrilidae, another suggested putative sister family, the exact position of Parvidrilidae within Clitellata still remains uncertain in the absence of branch support. Ehrenberg, following phylogenetic assessments using DNA data (see Erséus & Gustavsson, 2002; Erséus, Wetzel & Gustavsson, 2008), seemed to weaken the traditional view that considers the location of gonads as a primary taxonomical character. However, the forward position of the gonads in the former Naididae is probably related with the emergence of paratomy as their main reproductive mechanism. Hence, the traditional view remains acceptable until there is evidence to the contrary. The presence of testes in XI and ovaries in XII was already stated to be a character shared by the Parvidrilidae, the Enchytraeidae, the monotypic Tiguassidae, most Phreodrilidae, the monotypic opistocystid genus Crustipellis Harman & Loden, and perhaps most Capilloventridae (Erséus, 1999; Martínez-Ansemil et al., 2002). Although sharing a plesioporous condition (the male pores are in the segment directly behind the testes), the male ducts of the parvidrilids were considered quite different from those of the enchytraeids, capilloventrids, and tiguassids (e.g. all the latter three families lack atria). Moreover, these three families have their spermathecae located in segments anterior to the gonadal ones (the prosothecous condition). Apart from the location of testes (segment XI) and male pores (segment XII), the absence of nodulus on setae in Parvidrilidae and many Enchytraeidae is the only distinctive character shared between these two families, which deserves to be mentioned. Close phylogenetic relationships between opistocystids and parvidrilids have also been discarded in the light of other anatomical and biological traits (Martínez-Ansemil et al., 2002). Recent molecular studies led Erséus et al. (2010) to consider that Opistocystidae are likely to be a group within Naididae. As a result of the present study, the anatomy of several reproductive structures has become clearer. The male ducts of some parvidrilids resemble those of most phreodrilids. This, together with the similarity of other important anatomical traits (see MartínezAnsemil et al., 2002), reinforce the idea that phreodrilids are the best candidate to be the sister group to parvidrilids on morphological grounds. POSITION OF WITHIN THE NEW EUROPEAN SPECIES OF PARVIDRILIDAE are presumably highly isolated, geologically, from each other. ECOLOGY AND BIOGEOGRAPHY Since the description of P. strayeri from groundwater in Alabama (USA), and the subsequent erection of the new oligochaete family Parvidrilidae (Erséus, 1999), eight new species – all from Europe, have been added to this family (P. camachoi, P. gianii, P. gineti, P. jugeti, P. meyssonnieri, P. spelaeus, P. stochi, P. tomasini), making this family much more diverse than initially expected. Recent investigations in Slovenian groundwaters will probably result in the description of two new Parvidrilus species, and expand the known distribution of P. spelaeus, a species already reported from that country (Giani et al., 2011), thus suggesting an even wider diversity of the genus in European groundwaters. To date, five of the Parvidrilus species each appear to be strictly endemic to a single cave: in Spain, P. camachoi to Estaragueña cave and P. gianii to Seldesuto cave; in Italy, P. stochi to Monte Maiore cave and P. tomasini to Sa Ucca de su Tintirriolu cave; in France, P. jugeti to Corveissiat cave (Fig. 10). Parvidrilus meyssonnieri was found in two galleries mined in consolidated granitic sands of the Rhône department: La Martinière and Navogne (same geological formation, located only 58 km from each other). Parvidrilus gineti was present in La Balme cave and possibly in the hyporheic zone in the alluvial plain of the Rhône river. Parvidrilus spelaeus proved to be the most widely distributed species in the Alpine Arc, from Italy to Slovenia. Our data on Parvidrilidae emphasize the wide ecological range of this family, found in both karstic and granite districts, and on unconsolidated as well as consolidated substrata. Hence, it is likely that Parvidrilidae could colonize the entire hypogean environment. Considering the data related to the parvidrilid fauna, the efficiency of the PASCALIS sampling strategy, which focused equally on both the unconsolidated and karstic aquifers (Dole-Olivier et al., 2009), is herein confirmed. During this study, Parvidrilidae were found in each of the three studied southern European karst units – Cordillera Cantábrica, Lessinian mountains, Krim Massif. These discoveries support the presence of parvidrilids in Spain (from which they had not been reported prior to this present study), and contribute new data for P. spelaeus, thus expanding both the known distribution and habitat preferences of this species in both kinds of aquifer type. We can confirm the stygobiont character of Parvidrilidae and infer that Parvidrilus is probably widely distributed throughout underground waters, yet may have been unreported in past studies owing © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 the effectiveness of DNA taxonomy (in the sense of prediction and classification of new taxa; Lefébure et al., 2006b) has been confirmed, although its accuracy seems to depend on the taxonomic knowledge and the sample coverage of the group (e.g. Meyer & Paulay, 2005). In Clitellata, an accumulation of evidence suggests that most congeneric species differ from each other by about 10% (or more) COI divergence (p-distance) (Erséus & Gustafsson, 2009; Zhou et al., 2010). In this respect, the high genetic divergence amongst P. jugeti, P. meyssonnieri, and P. spelaeus (17–19%, p-distances; 20–23%, K2P distances) is in good accordance with their specific status. Furthermore, such high divergences may even justify considering the erection of different genera in order to accommodate the species diversity of this family. However, the 9% divergence between specimens of P. meyssonnieri from two different localities is intriguing, and even more so because (1) morphological scrutiny of these specimens did not reveal any significant difference, except, perhaps, in hair setae (slightly more plumose in specimens from the ‘Navogne’ population, by comparison with the ‘La Martinière’ population), and (2) both populations are located in stations that belong to the same hydrogeological basin, and are distant from each other by not more than 60 km. Recent molecular studies of groundwater crustaceans have shown that extreme conditions of life in groundwater promote cryptic diversity by inducing convergent morphological evolution (Lefébure et al., 2006a; Lefébure, Douady & Gibert, 2007). Accordingly, we cannot eliminate the possibility that the ‘Navogne’ population and the ‘La Martinière’ population may in fact represent two different species. Conversely, the subterranean aquatic ecosystem is well known for its habitat heterogeneity (Gibert & Deharveng, 2002). Therefore, it is plausible to expect a substantial range of intraspecific genetic variation in stygobiont species because of low dispersal abilities of inhabitants of such an environment – and even within the same hydrogeological basin. For instance, Lefébure et al. (2006b) considered that, in groundwater crustaceans, two monophyletic groups have a strong probability of belonging to different species, but only when they diverge by more than 0.16 substitutions/site in the COI gene (patristic distances). More studies are clearly needed to address this issue in the future. In this respect, it is worth noting that P. jugeti and P. meyssonnieri diverge from each other by 19% (p-distance) and 23% (K2P). Although the known locations of these two species are relatively close to each other (~100 km or less), they belong to two different bedrock formations (granitic vs. karstic), and thus 553 554 E. MARTÍNEZ-ANSEMIL ET AL. to its exceedingly small size, or more likely missed during sampling because of abrasive sample processing and coarse mesh size of nets. Moreover, the apparent morphological similarity of Parvidrilidae and Aeolosomatidae may have caused confusion between the two taxa groups, as illustrated by P. gineti, leading to the oversight of parvidrilid taxa present in collections. With respect to the issue of morphological adaptations of oligochaetes to the subterranean environment, the Parvidrilidae show interesting features (e.g. very tiny transparent bodies, presence of epidermal glands, and presence of simple-pointed setae) that are usually considered to be adaptations to subterranean life in rock microcrevices and interstices (Sambugar et al., 1999; Giani et al., 2001). However, such an issue remains controversial and it is still speculative to categorize these characters as pre-adaptations, or adaptations to life in groundwaters (Creuzé des Châtelliers et al., 2009). The presence of Parvidrilus in Sardinia is quite noteworthy. Previously, a few specimens of Parvidrilus, seemingly attributable to P. spelaeus, were noted from Bue Marino cave (Martínez-Ansemil et al., 2002). Recently, additional specimens referred as P. cf. spelaeus were discovered in this same cave (E. Martínez-Ansemil & B. Sambugar, unpubl. data). The discovery of two new species (P. stochi and P. tomasini, described herein) broadens the diversity of the genus in Sardinia and highlights the endemicity of the stygobiotic fauna of this ancient Mediterranean island. The Sardo-Corsican system, in fact, was contiguous to the Pyrenees and became an independent microplate in the late Oligocene. It drifted until it reached the present position, carrying with it a sample of the Palaeogene fauna. The affinities between the fauna of Catalonia and of Sardinia-Corsica are in agreement with this palaeogeographical reconstruction (e.g. the distribution of Trichodrilus angelieri Giani & Rodríguez, 1994, in groundwaters of the south © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 04 June 2020 Figure 10. Geographical distribution of the family Parvidrilidae in Europe. The shaded patterns indicate the areas of carbonate rock outcrops (exposed karst terrains; Williams & Fong, 2010). NEW EUROPEAN SPECIES OF PARVIDRILIDAE underwent radiation and niche diversification. Such an evolutionary trajectory might have led to cryptic biodiversity and the occurrence of congeners that are morphologically very similar, i.e. the Sardinian P. spelaeus and P. cf. spelaeus. Future molecular studies are clearly needed to address this issue. Besides revealing potential cryptic diversity within the genus, such studies would provide important clues as to relationships and affinities between Pyrenean and Sardinian species, as well as times of colonization of the genus. In number of species, the genus Parvidrilus constitutes an appreciable fraction of the known stygobiont oligochaete fauna (nine of 111 species, 8%). With all these species being stygobiont, the Parvidrilidae is unique in being the only family of oligochaetes, worldwide, comprising taxa that are restricted in distribution to groundwater habitats, and as such, can be considered the most representative oligochaete family in this environment. KEY TO THE PARVIDRILID SPECIES 1. – 2. – 3. – 4. – – 5. – Genital setae present (one per bundle in XII) ......................................................... Parvidrilus meyssonnieri Genital setae absent .......................................................................................................................... 2 Spermatheca absent.....................................................................................................Parvidrilus jugeti Spermatheca present..........................................................................................................................3 Spermatheca in XII (atrial segment).....................................................................................................4 Spermatheca in XIII (postatrial segment) .............................................................................................. 5 Spermathecal ampulla tubular; atria elongate (about 150 mm long) .................................. Parvidrilus tomasini Spermathecal ampulla ovoid; atria very elongate (about 250 mm long) .................................. Parvidrilus stochi Spermathecal ampulla bilobed (‘U’ shaped); atria moderately long (up to 60 mm)..................Parvidrilus strayeri Spermathecal ampulla ovoid; atria elongate (about 150 mm long).........................................Parvidrilus gianii Spermathecal ampulla irregular in shape, very large (up to 45 mm wide); atria short, pyriform ........................ ............................................................................................................................ Parvidrilus spelaeus – Spermathecal ampulla round, rather small (about 18 mm wide); atria tubular, somewhat curved, moderately elongate (about 32 mm) ............................................................................................ Parvidrilus camachoi Note: Parvidrilus gineti species inquirenda is excluded from the present key because of the lack of sufficient description of the species. ACKNOWLEDGEMENTS REFERENCES This work was partially supported by the European project PASCALIS (no. EVK2-CT-2001-00121) under the EU Fifth Framework Programme: global change, climate and biodiversity. We thank Zohra Elouaazizi (IRScNB, Belgium) for her assistance in processing the DNA material, and Mark J. Wetzel (Illinois Natural History Survey, USA) for his comments and linguistic improvements. We are very much indebted to Fabio Stoch, Gianfranco Tomasin, and Ana Camacho for collecting and placing the oligochaete material at our disposal. Abouheif E, Zardoya R, Meyer A. 1998. Limitations of metazoan 18S rRNA sequence data: implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the Cambrian explosion. Journal of Molecular Evolution 47: 394–405. Adoutte A, Balavoine G, Lartillot N, Lespinet O, Prud’homme B, de Rosa R. 2000. The new animal phylogeny: reliability and implications. Proceedings of the National Academy of Sciences, USA 97: 4453–4456. 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