The Parvidrilidae a Diversified Groundwater Family

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Zoological Journal of the Linnean Society, 2012, 166, 530–558. With 10 ﬁgures

The Parvidrilidae – a diversiﬁed groundwater family: description of six new species from southern Europe, and clues for its phylogenetic position within Clitellata (Annelida) Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019

ENRIQUE MARTÍNEZ-ANSEMIL1, MICHEL CREUZÉ DES CHÂTELLIERS2, PATRICK MARTIN3* and BEATRICE SAMBUGAR4

1Departamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Facultade de Ciencias, Universidade da Coruña, Campus da Zapateira s/n, E-15071 A Coruña, Spain 2Université de Lyon, UMR5023 Ecologie des Hydrosystèmes naturels et anthropisés, Université Lyon 1, ENTPE, CNRS, 6 rue Raphaël Dubois, 69622 Villeurbanne, France 3Institut royal des Sciences naturelles de Belgique, Biologie des Eaux douces, Rue Vautier 29, B-1000 Brussels, Belgium 4Museo 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.

ADDITIONAL KEYWORDS: aquatic oligochaete, molecular systematics, phylogeny.

*Corresponding author. E-mail: [email protected]

INTRODUCTION particularly arrangement, number, and types of setae: absence of setae in segment II (dorsal bundles The Parvidrilidae Erséus, 1999, constitute the most in phreodrilids, dorsal and ventral bundles in capill- recently described family of oligochaete microdriles. oventrids), presence of hair setae in dorsal and Previously, only two species had been described in ventral bundles in capilloventrids, and a similar set of this family – Parvidrilus strayeri Erséus, 1999 from needle and hair setae in the dorsal bundles of phreo- Alabama (USA), and Parvidrilus spelaeus Martínez- drilids and parvidrilids. In parvidrilids, however, Ansemil, Sambugar & Giani, 2002 from Slovenia and dorsal needles emerge from the body wall and do not Italy (southern Europe). Both are exceedingly small look like support setae as in phreodrilids – species in worms (around 1 mm long, 50 mm wide) and were which such setae are always contained within the Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 collected in groundwater environments. setal sac (Pinder & Brinkhurst, 1997). Groundwater habitats are important centres of During this work, we found new specimens of biodiversity, serving as refugia for relictual species P. spelaeus and six new species of Parvidrilus.In (Gibert & Deharveng, 2002). During the last two addition, our re-examination of specimens convinced decades, and especially as a result of the European us that Aeolosoma gineti Juget, 1959, should be trans- Protocols for the Assessment and Conservation of ferred to Parvidrilidae. The availability of so many Aquatic Life in the Subsurface (PASCALIS) project new species for description gave us a morphological (Gibert, 2001; Gibert & Culver, 2009), the study of framework to compare the Parvidrilidae in depth with groundwater oligochaete biodiversity has beneﬁted other oligochaete families. A few specimens of two from a renewed interest from the present authors new Parvidrilus species from France, and of previ- and a few other colleagues. To date, more than 300 ously described P. spelaeus, were suitable for molecu- nominal species have been already found in ground- lar analyses. This additional molecular facet provided waters all over the world, although the present the opportunity to (1) characterize these three species knowledge is almost limited to the karst of southern with a fragment of the mitochondrial cytochrome Europe (Martin et al., 2008; Martínez-Ansemil & oxidase I (COI) gene, used as a barcode; (2) assess the Sambugar, 2010). Some of these species should be genetic divergence between them, and to put these considered as incidentals or waifs – taxa most likely data in a biogeographical context; and (3) investigate carried along by surface waters ﬂowing into sub- the phylogenetic relationships of the Parvidrilidae terranean environments where they were unable to within the Clitellata using the nuclear 18S rRNA sustain viable populations (Creuzé des Châtelliers gene. The accumulation of new data on the distribu- et al., 2009); however, about one third of them have tion and habitat of parvidrilids resulting from this been found exclusively in this environment (stygo- large amount of material has provided us with a bionts; Martin et al., 2008; Creuzé des Châtelliers more comprehensive understanding of the biogeogra- et al., 2009; Martínez-Ansemil & Sambugar, 2010). phy and ecology of this unique family of microdrile The observation of an important species richness oligochaetes. of the marine subfamily Phallodrilinae (Naididae) in groundwaters of southern Europe (B. Sambugar, N. Giani & E. Martínez-Ansemil, 1999; unpubl. data) MATERIAL AND METHODS and the discovery of two new species of the hitherto Baikalian genus Rhyacodriloides Chekanovskaya MORPHOLOGICAL STUDY (Naididae, Rhyacodriloidinae) in subterranean water The material presently studied results from different bodies of the eastern Alps (Martin, Martínez-Ansemil sources: three collecting campaigns in Sardinian & Sambugar, 2010) appear amongst the most out- caves (Italy) organized by our colleague Fabio Stoch, standing discoveries. Phallodrilines and rhyacodri- a study of the groundwater oligochaete fauna of the loidines have also a very small size in common with French Jura, and a large survey of the groundwater parvidrilids, although the latter are even smaller. fauna of six European regions in the course of the As a result of peculiar morphological features, the PASCALIS project (Gibert, 2001; Gibert & Culver, Parvidrilidae is thought likely to be an ancient family, 2009), which enabled us to study material from the and to hold a key phylogenetic position within the Italian Lessinian Mountains, Slovenian Krim Massif, Clitellata and vis-à-vis the Annelida. Each time the and Spanish Cordillera Cantábrica. The PASCALIS systematic position of the Parvidrilidae was discussed sampling protocol was designed to account for habi- on morphological grounds (Erséus, 1999; Martínez- tat heterogeneity, with the primary objective being Ansemil et al., 2002), the attention was focused pri- evaluation of the biodiversity of European groundwa- marily on Capilloventridae and Phreodrilidae as the ter habitats. One advantage of this approach was to closest relatives. These two aquatic families share characterize habitat preferences of parvidrilid species additional morphological traits with the parvidrilids, sampled in this framework.

Samples were taken in different groundwater habi- taxa representing 14 clitellate and three polychaete tats including the unsaturated and saturated zones of families. This data set basically corresponds to the karst systems as well as the hyporheic and phreatic sequence matrix used by Erséus & Källersjö (2004) zones of alluvial groundwater. In springs and caves, for investigating basal nodes of the clitellate phylo- the fauna was sampled by sieving sediments through genetic tree, and is still the most representative a hand net (100 mm mesh); in the hyporheic zone, the and taxonomically balanced data set of clitellate Bou-Rouch pump or the Karaman-Chappuis methods families available to date. It is also the only data set were used (Malard et al., 2002). The phreatic zone that includes the Enchytraeidae, the Phreodrilidae, was investigated in sampling wells by means of the and the rare Capilloventridae – three families once Cvetkov net (Malard et al., 2002). Samples were assumed to be potential sister groups to the Parvid- Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 fixed in the field using either a 4% formaldehyde rilidae (Erséus, 1999; Martínez-Ansemil et al., 2002). solution or a Bouin-Hollande solution (Hollande, Nine polychaete outgroups, identified in Erséus & 1918; Laurylab, Elvetec, France ; French material), sorted out in the the clitellate ingroup, were removed from the original laboratory under a stereomicroscope, and subse- data set, in order to reduce sources of bias owing to quently transferred to 70% ethanol. great evolutionary distances (substitutional satura- Microdriles were either mounted whole on micro- tion, sequence alignment ambiguity – see Abouheif, scope slides or serially sectioned. Whole-mounted Zardoya & Meyer, 1998; Adoutte et al., 2000; Löy- specimens were temporarily mounted on slides tynoja & Goldman, 2008), potentially acute in this in glycerine during initial observation, and then gene fragment because of hypervariable regions (Van returned to alcohol. These specimens were subse- de Peer et al., 1997). quently stained with Mayer’s paracarmin, differenti- Owing to uncertainties about the taxonomic status ated in 70% acid alcohol, dehydrated in ethanol, of Haplotaxis in North America (Kathman & cleared in xylene, and then permanently mounted Brinkhurst, 1998), and its sensitivity to variations in on slides in Canada balsam. Serially sectioned speci- the alignment parameters, the specimen of Haplo- mens were embedded in Spurr’s resin, sectioned on taxis cf. gordioides used in Erséus & Källersjö (2004) an ultramicrotome stained with toluidine blue, then was removed from this data set and replaced with one permanently mounted on slides in Canada balsam. specimen of Haplotaxis from France. Another repre- The specimens were observed with a light microscope sentative of the Haplotaxidae, Delaya bureschi, was equipped with differential interference contrast (DIC) also included to assist in resolving the phylogenetic optics. Sediments containing specimens of parvidril- position of the family in the clitellate tree, and to ids destined for in vivo observations were transported minimize a possible misleading attraction between to the laboratory in an icebox for processing. Speci- taxa that are not well fixed within the trees. Other mens in the sediment samples were then sorted additions to this data set included two species in the from the samples in a cold room kept at 11 °C. Some new naidid subfamily Rhyacodriloidinae (Martin specimens in the samples collected from localities in et al., 2010), Rhyacodriloides abyssalis and Rhyaco- France were observed and filmed in vivo in a drop of driloides latinus (EMBL accession nos: FN796452 water, gently pressed under a coverslip on a micro- and FN796453, respectively), the tubificine Baikalo- scope slide. drilus digitatus, and the two new Parvidrilus species All measurements, drawings, and photographs from France, Parvidrilus jugeti and Parvidrilus mey- refer to these mounts. The type material for the new ssonnieri (Table 1). species described in this paper has been deposited in Because of the limited material available, the the following institutions: MHNL, Muséum d’Histoire genetic characterization of Parvidrilus taxa – based naturelle de Lyon, Lyon (France); IRScNB, Institut on a fragment of the mitochondrial COI gene – was royal des Sciences naturelles de Belgique, Brussels only possible for three species: P. spelaeus, P. jugeti, (Belgium); MCSNVR, Museo Civico di Storia naturale and P. meyssonnieri. Specimens of P. spelaeus were di Verona, Verona (Italy); and MNCN, Museo Nacio- from a sample collected in Pisoliti cave (Trieste, Italy; nal de Ciencias Naturales, Madrid (Spain). one specimen successfully sequenced). All specimens of P. meyssonnieri and P. jugeti were collected from their type localities (P. meyssonnieri: three and two MOLECULAR STUDY specimens from ‘La Martinière’ and ‘Navogne’ galler- Taxa ies, respectively; P. jugeti: two specimens from Cor- For phylogenetic relationships of the Parvidrilidae veissiat cave – Table 1). within the Clitellata, we analysed a data set, partly All live specimens collected in the field were imme- from the EMBL databank, consisting of the nuclear diately preserved in 95 or 90% ethanol, and then 18S rRNA gene fragments – for a total of 48 annelid stored at -20 °C after return to the laboratory. Owing

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). Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019

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).

Corveissiat cave (Ain (01),Gallery France; of 2007 Navogne, Bas-en-BassetGallery (43), of France; ‘La 2008 Martinière’,Pisoliti Thurins cave, (69), Trieste, France, Italy; M. 2007 2000 Creuzé des Châtelliers M. Creuzé des HE800210 Châtelliers HE800205 M. Creuzé des Châtelliers HE800209The HE800203 HE800204 position B. Sambugar of the Parvidrilidae – within HE800202 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 sp. nov. as a new state (ﬁfth base) or as missing data. Heuristic (Hartmann, 1821) Azergues River, Rhône (69), France; 2009 M. Creuzé des Châtelliers HE800206 –

Holmquist, 1979 Frolikha Bay, Lake Baikal, Russia; 1995 P. Martinsearches were HE800208 carried out with random sequence Martinez Ansemil

sp. nov. addition (ten replicates) and using tree-bisection- reconnection branch swapping. Branch support was (Michaelsen, 1825) Mrzla jama cave, near Loz, Slovenia; 2007 B. Sketevaluated using HE800207 nonparametric – bootstrapping (1000 gordioides replicates). When the alignment with gaps treated cf. as missing data was analysed, the number of trees Clitellate taxa (Annelida) used in the analyses (newly sequenced specimens), and EMBL accession number for the respective sequences

., 2002 retained dramatically increased. In that case, the bootstrapping procedure was carried out using the et al MaxTrees option limited to 1000. No limit was set Haplotaxis Delaya bureschi Baikalodrilus digitatus Parvidrilus jugeti Parvidrilus meyssonnieri Parvidrilus spelaeus Table 1. TaxonHaplotaxidae COI, cytochrome oxidase I. Collection site and year Collector 18S rDNA COI Naididae, Tubiﬁcinae Parvidrilidae when gaps were treated as a new state.

For maximum likelihood and Bayesian analyses, (43), France. Thirty-six specimens were whole- the best-fit model was selected using JModel-Test mounted in Canada balsam or in Amman’s lactophe- 0.1.1 (Posada, 2008) by estimating and comparing nol, in the collection of one of the authors (MCdC): ‘La maximum-likelihood scores for different substitu- Martinière’ (seven specimens, four mature and three tion models. Models were selected according to the juvenile, 14.ix.1999; 24 specimens, ten mature and 14 Akaike information criterion (Akaike, 1973) and its juvenile, 28.ix.2000); ‘Navogne’ (three immature corrected variant, and the Bayesian information crite- specimens, 8.x.1999; one mature specimen, 28.i.2008). rion (Schwarz, 1978). All criteria identified the general time-reversible substitution model with invariable Etymology: Named after Marcel Meyssonnier, as a sites and a discrete G correction for amongst-site tribute to this outstanding and tireless speleologist, Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 variation (GTR + I + G model) as the best-fit substitu- and long-time friend and team member of one of the tion model, or one of its variant. Accordingly, this authors (MCdC), and whose involvement and support model was used to conduct maximum likelihood and were instrumental in the discovery of this new Bayesian analyses. species. Maximum likelihood analyses were performed with PHYML v3.0 (Guindon & Gascuel, 2003), using Description: Length 1.3-1.6 mm, 21–30 segments default parameters and a subtree pruning and (seven complete mature specimens). Width 55–85 mm regrafting strategy for the exploration of the space at V (mean = 59 mm±11, N = 24), 50–120 mm at XII of tree topologies. Bootstrapping was performed on (mean = 72 mm ± 19, N = 24). Prostomium rounded, 1000 generated pseudo data sets, using the same 35–50 mm long, 20–38 mm wide at base. Body wall not parameters. Bayesian analyses were conducted using papillate, cuticle smooth. Clitellum poorly developed, MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). Two limited to large, swollen, transparent cells, only parallel runs with four Markov chains each were run visible on living animals (Fig. 1C, c), budding on a for two million generations and every 100th genera- mid-ventral depression of body wall (Fig. 2D, vdp) tion was sampled (resulting in 20 000 trees). The level that extends behind genital pores, between ventral of convergence was monitored on the .p files using setae in XII and XIII. TRACER 1.5.0 (Rambaut & Drummond, 2009), and Dorsal (dorsolateral) and ventral (ventrolateral) the ‘burn-in’ value was set accordingly. The first 25% setae present from III, posteriorly situated in each of the trees were discarded and the last 15 000 trees segment (Figs 1A, B, 2B, ds, vs). Dorsal bundles with were used to reconstruct a consensus tree and esti- two (three) straight, thin single-pointed needles (15– mate Bayesian posterior probabilities. 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 SYSTEMATICS at XI). Hair setae densely pilose (with soft hairlets) PARVIDRILUS MEYSSONNIERI DES CHÂTELLIERS & on one side of the shaft (pilosity mostly observed on MARTIN SP. NOV. (FIGS 1, 2) specimens during study using SEM). Ventral bundles with (two) three (four) sigmoid, bifid crotchets with Types: Holotype. MHNL 44003358, slide 6, mature upper tooth slightly thinner and shorter than lower specimen, stained in paracarmine and whole- tooth, and accompanied by one (two) pilose hair setae mounted in Canada balsam. Gallery of ‘La Martinière’ in preclitellar segments (80–170 mm long); ventral (45°40′51′′N, 04°38′51′′E, 350 m asl), Thurins (69), hair setae absent from XII; crotchets without nodulus, France, 28.ix.2000. 17–28 mm long, shaft out of the body wall faintly Paratypes. MHNL 44003359, slide 1, six specimens, pilose, with distal half enlarged on the convex side. four immature, one mature and one fragment; MHNL Within ventral bundles, the uppermost seta bifid, 44003360, slide 3, four specimens, two mature and followed by one hair seta; these two setae separated two immature; slide 6, one immature specimen; all from the other two bifid setae by a distance some- specimens stained with paracarmine and whole- times greater than that between them. One modified mounted in Canada balsam; 28.ix.2000; gallery of ‘La genital seta per bundle in XII, 55–60 mm long, Martinière’, Thurins (69), France. straight with enlarged, spearhead ectal tip, the latter with a spoon-like curvature (Figs 1A, B, 2A, C, D, gs). Other material examined: Apart from the type mate- No eyes. Brain long, extending into IV, posteriorly rial, 11 specimens were observed and mounted on five indented. Ventral nerve cord in contact with epider- scanning electron microscope (SEM) stubs, IRScNB, mis, beneath muscles of the body wall. Digestive tract 28.i.2008; stub 1, three specimens, type locality; complete; eversible pharynx with dorsal pad, sinuous, stubs 2–5, eight specimens, gallery of ‘Navogne’ ciliated oesophagus, with pharyngeal glands present (45°17′16′′N, 04°05′58′′E, 520 m asl), Bas-en-Basset 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 NEW EUROPEAN SPECIES OF PARVIDRILIDAE 535 Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019

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. ing from X-XI and ending in a terminal anus. Layer ‘cysts’ according to Ferraguti, 1997), 2.8–4.0 mm wide, of chloragogen cells surrounding digestive tract pos- freely floating inside the coelomic cavity although sibly present; details not observable in specimens usually lying ventrally in XI (Fig. 1A, mo); a few thus far examined. Coelomocytes absent. Dorsal glan- globular cytophores in a more dorsal and posterior dular body-wall pouches very small and difficult to position in XI, 8.0–11.3 mm wide, sometimes in a stage observe, but observed in VIII and XII in one speci- of disintegration with the detachment of spermatozoa men), opening mid-dorsally at about the intersec- in the coelom. Sperm funnels and vasa deferentia not tion with setal line. Voluminous glands associated seen. Atria elongate (90–190 mm long, 7–10 mm wide), with genital setae (20–48 mm long; 20–32 mm wide), either extending into XIII or restricted to XII; in the bulging at the body surface; fine internal ducts latter case, atria folded in different configurations: one running inside the glands and converging on the shaft atrium bent anteriad, with distal end folded up pos- of genital setae; setal glands asymmetrically teriad, the other atrium (or if present, both atria), bent arranged on each lateral side of the body, the left posteriad, with distal end folded up anteriad (Fig. 1A, gland anteriad to the right one (Figs 1A–C, 2B, sg). B, a). Atria merging below the nerve cord (Fig. 1A, B, Clearly delimited testes not present; however, nc) into a common ejaculatory duct, and opening at a lump of cells with small nuclei observed dorsally the tip of a mid-ventral, conical porophore (Figs 1A, B, in XI, in posterior end of coelom of XI, is probably 2A, C, mp), in posterior part of XII, between setal unpaired male protogonia (Fig. 1A, t). Diffuse forma- glands and in front of a median ventral depression of tion of male gametes in XI with many morulae (or body wall in XII-XIII (Fig. 2D, vdp). Atrial ampullae

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 536 E. MARTÍNEZ-ANSEMIL ET AL. Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019

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. longitudinally striated, with large lumen filled with located, ahead of the point of union of atrial ampullae, sperm, mature spermatozoa more concentrated near usually on the right side of the ventral nervous chord the distal part of ampullae (Fig. 1A, B, sp); no peculiar but sometimes on the left side in some specimens aggregate of sperm in the ampullae. An (unpaired?) (Fig. 1A–C, am); ampulla opening, through a duct, ovary, apparently developing on one side, observed in 14 mm long, mid-ventrally, close to the basis of male anterior part of XII, with a ventral attachment on porophore (Fig. 2C, amo); duct surrounded by a mus- 11/12 (Fig. 1A, B, ov). Specimen ovigerous with large cular ring near opening. Spherical ampulla filled with mature eggs filling coelom in XII, and extending cell material (sperm?) and surrounded by a cell through XIII. Female funnels not observed. No typical cluster, clearly delimitated, noticeable on living worms spermatheca observed. However, an unpaired, spheri- but difficult to resolve on mounted slides, possibly cal ampulla in XII, 10 mm in diameter, ventrally glandular (Fig. 1B, C).

COI sequences: EMBL accession numbers: HE800204 (see below). In addition, when copulatory organs are (gallery of ‘La Martinière’); HE800205 (gallery of present in microdriles – whether in the form of poro- ‘Navogne’). phores or penes – they are always associated with the male aperture (Stephenson, 1930), thus making the latter hypothesis less convincing. Remarks: Within the parvidrilid species known so far (including all new species being described herein plus the reassigned Parvidrilus gineti – see below), the Distribution and habitat: Parvidrilus meyssonnieri presence of genital setae is unique to P. meyssonnieri is known only from the ‘La Martinière’ and ‘Navogne’ galleries in the Rhône department, France, in a sp. nov. – clearly separating this species from its Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 congeners. The most outstanding feature in P. meys- region lying on metamorphic bedrock. The ‘La Mar- sonnieri sp. nov. is the presence of an enigmatic tinière’ gallery opens in the upper alteration layer of unpaired, spherical ampulla in XII – the function of a crystalline craton, which forms granitic sand with which remains obscure. There is a possibility that this scarce, particulate organic matter. A rivulet runs structure is used for storing sperm from a concopu- along the gallery and passes slowly through a thin lant, which would imply that it is a spermatheca. layer of coarse sand. Historically (between 1846 and However, the ampulla is surrounded by a cell cluster, 1919) the Navogne gallery was dug in arkoses and which seems more glandular (prostate-like cells) than ferruginous sandstone for iron ore exploitation. The shaped in order to play the role of a bag for sperm water is present in the gallery as isolated puddles storage. In this respect, such a structure is somewhat with gravelly soil, rich in organic matter. similar to the glandular, atrium-like organ in the pre-atrial segment of some Rhynchelmis (Rhynchel- PARVIDRILUS JUGETI DES CHÂTELLIERS & mis) species (Lumbriculidae), referred to as ‘accessory MARTIN SP. NOV. (FIG.3) organ’ (formerly ‘rudimentary atrium’), whose function and derivation are unknown (Fend & Brinkhurst, Holotype: MHNL 44003361, 07.07.1998, mature 2010). Some material has been observed in the spheri- specimen, stained in paracarmine and whole-mounted ′ ′′ cal ampulla but it does not resemble mature sperma- in Canada balsam. Corveissiat Cave (46°14 34 N, ′ ′′ tozoa. Hence, the absence of spermathecae in this 05°29 01 E, 378 m asl), Ain (01), France, 7.vii.1998. species seems plausible. Conversely, considering the ampulla and its surrounding cell cluster as an atrium Paratypes: MHNL 44003364, one mature specimen; and a prostate is another possibility – implying that MHNL 44003365, one mature specimen; all speci- the structures described above as ‘atria’ are in fact the mens stained in paracarmine and whole-mounted spermathecae and the porophore is spermathecal. in Canada balsam. Corveissiat Cave, France, However, prostates seem to be absent in Parvidrilus 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.

Other material examined: Six other specimens, whole- wide), one atrium entering and restricted to XIII, mounted in Canada balsam, in the collection of one of with distal end folded up anteriad, the other one the authors (MCdC): Corveissiat cave (one immature extending straight into XIV (Fig. 3, a). Atria proxi- specimen, 7.vii.1998; three mature specimens, 20.xii. mally merging below the nerve cord into a common 2007; two mature specimens, 10.iii.2008). ejaculatory duct, and opening ventrally in anterior part of XII, in front of setal line, through a penis-like Etymology: Named after Jacques Juget (1928–2006), structure, surrounded by a thick muscular ring mentor to one of us (MCdC), and friend and colleague (Fig. 3, mp). Atrial ampullae longitudinally striated, to all of us, as a tribute to his lifelong contribution with large lumen filled in with undefined material to the knowledge of the Clitellata, and particularly (cellular? secretory?), different from mature sperma- Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 subterranean oligochaetes (see Creuzé des Châtelli- tozoa visible in XI; atrial walls very thick, highly ers, Lafont & Giani, 2007). refringent under DIC. One mature oocyte (Fig. 3, ov), full of vitellus, ventrally, in anterior part of XII, near Description: Length 1.96 mm, 28 segments (one 11/12, suggesting the location of an unpaired ovary; complete mature specimen). Width 68 mmatV,85mm a few small ovarian morulae freely developing into at XII. Prostomium bluntly conical, 25 mm long, the coelomic cavity. Female funnels and spermath- 40 mm wide at base; prostomium epidermis with ecae not observed. numerous stained cell nuclei, indicating a possible sensory or glandular function. Body wall not papillate, cuticle smooth. Clitellum not seen. COI sequence: EMBL accession number: HE800203 Dorsal (dorsolateral) and ventral (ventrolateral) (Corveissiat cave, France). setae present from III, posteriorly situated in each segment. Dorsal bundles with two (three) simple- Remarks: The long, narrowly tubular atria of P. jugeti pointed needles (10–20 mm long), alternating with one sp. nov., which extends to XIV, are quite distinctive (two) thin and flexible hair setae (length 80 mmatV, amongst the Parvidrilidae known to date. The high 110 mm at VIII, 200 mm at XI) (Fig. 3, ds). Hair setae apparently smooth when observed on permanent refringence of the atrial walls is unique. The outer mount using an oil immersion lens. Ventral bundles atrial surface is striated all along the ampullae. with two (three) sigmoid, bifid crotchets with upper Parvidrilus jugeti (and possibly P. meyssonnieri)is tooth slightly thinner and smaller than lower tooth, (are) the only parvidrilid(s) known thus far that lack and accompanied by one hair seta in preclitellar seg- spermathecae. The exact location of ovaries (paired or unpaired?) ments (90 mm long); from XII, ventral hair setae absent, two crotchets per bundle (Fig. 3, vs). Crotch- in P. jugeti remains obscure. A mature oocyte was observed ventrally in XII, on the right side of the ets without nodulus, 20–25 mm long, and shaft out of the body wall, with distal half enlarged on the convex specimen, close to 11/12, which suggests the existence side. Within ventral bundles in preclitellar segments, of an unpaired, ventral ovary. In this specimen, it is the uppermost seta bifid, followed by one hair seta; likely that this female gonad has disappeared after these two setae separated from the other two bifid having produced the few morulae observable in the setae by a distance greater than that between them. coelomic cavity of XII, as documented previously in No modified genital setae. naidids (Chekanovskaya, 1962). No eyes. Brain short, limited to III. Ventral nerve cord in contact with epidermis, beneath muscles of Distribution and habitat: Parvidrilus jugeti is known the body wall. Digestive tract complete; eversible only from the type locality in the Corveissiat cave, pharynx with dorsal pad, sinuous, ciliated oesopha- Ain (01), France. Sedimentary rock formation (karstic gus, with pharyngeal glands present in III-IV, area); in a little pond, very finely clayey sediment, followed by a simple intestine, dilating in from X with little organic matter. and ending in a terminal anus. Layer of chloragogen cells surrounding digestive tract possibly present; details not observable. Coelomocytes absent. Mid- dorsal glandular pouches not observed. PARVIDRILUS CAMACHOI MARTÍNEZ-ANSEMIL & Testes in XI, as an unpaired, dorsal lump of cells SAMBUGAR SP. NOV. (FIG.4) with small nuclei in posterior end of coelomic cavity Holotype: MNCN 16.03/3070, mature specimen, lon- (Fig. 3, t); numerous spermatozoa below this cellular gitudinal sectioned at 0.4 mm, stained with toluidine mass, with well-defined tails, detached in the coelom blue and mounted in Canada balsam. Estaragüeña (Fig. 3, sp). Sperm funnels and vasa deferentia cave (43°17′58.2′′N, 4°36′23.5′′W, Z 53 m asl), Puent- not seen. Atria elongate, tubular (265 mm long, 11 mm ellés, Cantabria, Spain, 4.ix.2002, leg. Ana Camacho.

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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.

Etymology: Named after Ana Camacho, responsible by a very narrow duct (?), ending near the septum for the PASCALIS project for the Spanish partners, in 12/13 in a somewhat lateral position, at the left side honour of her important contribution to the knowl- of the worm (Fig 4A, E–H, sa, sd). Ampulla filled with edge of European biospeleology. spermatozoids, attached around the wall, and with a fine secretion. Description: Body wall thin (especially in dorsal part), and unpapillated. Large globular clitellar cells Remarks: The description of external anatomy cannot observed on lateral sides of XI–XIII (Fig 4D, cl); poro- be given in detail as the individual was sectioned. phore and surrounding area glandular. Brain long, Nevertheless, prior to sectioning, we noted that extending into segment IV, deeply incised posteriorly the external anatomy of this specimen agrees in all Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 (Fig. 4B, b). Ventral nerve cord in contact with epi- respects with that of P. spelaeus, including size of the dermis, beneath muscles of the body wall (Fig. 4B, C, worm, absence of modified genital setae (Fig. 4A, E, nc). Most setigeral segments have a mid-dorsal glan- cr), and shape and number of dorsal and ventral dular pouch opening posteriorly on each segment, somatic setae. at or near the transversal setal line (Fig. 4B, C, gp); Parvidrilus camachoi sp. nov. is one of three glandular pouches absent from IV-VI. Parvidrilus species known so far that have a single Digestive tract complete, entirely ciliated, and spermatheca in segment XIII. The narrow and mod- ending in a terminal anus. Eversible pharynx, with erately elongated, tubular, bent atria and the round- small dorsal pad set off from oesophagus, followed ish spermathecal ampulla filed with spermatozoids by a narrow winding tube extending into VIII, with attached around the wall are diagnostic traits for this thick muscular walls at about VII–VIII (Fig. 4C, oe); new species. The closest relative to P. camachoi sp. alimentary canal completed by a gut, clearly enlarged nov. is P. spelaeus but in the latter, atria are pyriform, from segment XIII (Fig. 4E–J, g). Compact pharyn- the spermatheca is very large and irregular in shape, geal glands present in IV–VI (Fig. 4B, C, pg). Diges- and sperm is arranged in large masses of aggluti- tive tract from V, surrounded by a well-developed nated spermatozoids into the spermatheca. layer of chloragogen cells. Coelomocytes and nephridia not observed. Distribution and habitat: Parvidrilus camachoi is Two narrow testes attached to septum 10/11 known only from the type locality in the Estaragüeña (Fig. 4H–I, t). Free germ cells and morulae floating cave Puentellés, Cantabria, Spain. Estaragüeña cor- in the coelomic cavity of segment XI (Fig. 4G, mo). responds to a resurgence of the Diva River. The cave A small seminal vesicle in XII (Fig. 4E, G, sv). is hardly accessible, as there is a siphon a few metres Sperm funnels not observed. A piece of vas deferens from the entrance of the gallery. The species was (about 1.5 mm wide) observed near the basal part of found in the entrance hall, along the edge of the an atrium (Fig. 4A, F, vd). Atria tubular, somewhat siphon, in wet sands with abundant organic matter. curved, moderately elongated (about 32 mm long, 8–10 mm wide), extending into the beginning of XIII PARVIDRILUS GIANII MARTÍNEZ-ANSEMIL & (Fig. 4A, F–J, a) merging, below the nerve cord, into a common ejaculatory duct which opens at the tip of SAMBUGAR SP. NOV. (FIG.5) a mid-ventral porophore located on the transversal Types: Holotype. MNCN 16.03/3071, mature speci- setal line of segment XII (Fig. 4A, G–J, ed, mp, p). men, stained in paracarmine and whole-mounted Atrial wall thin (less than 2 mm thick) and muscular in Canada balsam. Seldesuto cave (43°18′21.0′′N, (?); ejaculatory duct surrounded by a thin layer of 3°37′34.5′′W, 192 m asl), Matienzo, Cantabria, Spain, muscles. Atrial lumen filled in with a granular mate- 2.ix.2002, leg. Ana Camacho. rial (secretions?, or remains of old epithelial cells?), Paratype. MNCN 16.03/3072, immature specimen and with a large amount of spermatozoids, their from type locality, 2.ix.2002, leg. Ana Camacho, heads distally attached to atrial wall, and their long stained in paracarmine and whole-mounted in flagella orientated to the proximal atrial end, with Canada balsam. a clear flamigerous appearance (Fig. 4H–I, gm, sp). Prostate glands absent. Two ovaries attached to Etymology: Named after Narcisse Giani ‘maître et septum 11/12 (Fig. 4E, o). A large vitellogenic mature ami’, to whom many European oligochaetologists are egg in segment XII, slightly protruding into XIII very much indebted. (Fig. 4E, e). Oviducts not observed. A single spermatheca is present in XIII. Spermathecal ampulla Description: Entire mature worm, length 1.2 mm, 26 round (about 18 mm in diameter), with a fine epithe- segments, width 40 mmatV,42mm XII. Prostomium lial wall (1–3 mm thick); spermathecal duct (about rounded, 15 mm long, 25 mm wide at base. Body wall 10 mm long) as a loosely defined structure, hollowed thin (especially in dorsal part), unpapillated; foreign

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cr n a2 XI III XII a1 a2 od cr XIII a1 sd h sa ma X cr mp cr IV D sd C sa

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a1 XII p XII a2 ma a2 c nc XIII g XIII g

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, 530–558 542 E. MARTÍNEZ-ANSEMIL ET AL. particles adhering to cuticle here and there along the eggs attached to septum 11/12 (Fig. 5A, F, o). Two body. Numerous transversal rows of thin cutaneous small oviducts seemingly attached to septum 12/13 glands per segment. Clitellum not elevated, occupying (Fig. 5A, od). A single spermatheca present in XIII. at least XII-XIII. Spermathecal ampulla (empty) ovoid (30 mm long, Dorsal (dorsolateral) and ventral (ventrolateral) 9 mm high), with a nucleated epithelial wall sur- setae present from III and posteriorly situated in each rounded by a thick muscular lining; spermathecal segment. Dorsal bundles with two (three) straight, duct about 13 mm long, as a loosely defined structure, thin single-pointed needles (20–28 mm long) and one without lumen, basally enlarged, in continuity with or two long, thin, and flexible hair setae (105–190 mm the ventral body wall at the most anterior part of long) (Fig. 5A, B, h, n). Ventral bundles composed segment XIII, in a somewhat lateral position, at the Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 of (one) two–three (four) strongly curved crotchets, left side of the worm (Fig. 5A, D–E, sa, sd). 20–26 mm long, with enlarged distal half and double- pronged tip with minute distal tooth (Fig. 5A–C, cr); Remarks: The combination of long atria, a single one thin hair seta (60–90 mm long) in preclitellar spermatheca in segment XIII, and the cuticular wall ventral bundles of III–VI. No modified genital setae; of the common ejaculatory duct characterize P. gianii a single bifid crochet in ventral bundles of segment sp. nov. Amongst the three Parvidrilus species that XII. have a single spermatheca in segment XIII, P. gianii No eyes. Brain long, extending into segment IV, is easily distinguishable from the others by its ovoid deeply incised posteriorly. Ventral nerve cord in spermathecal ampulla surrounded by a thick muscu- contact with epidermis, beneath muscles of the body lar lining, and elongated atria (ten times longer than wall. Most setigeral segments have a mid-dorsal glan- wide). The cuticular wall of the ejaculatory duct of dular pouch opening posteriorly on each segment, at this new species is unique amongst the genus. about the transversal setal line; glandular pouches absent from IV–VI. Distribution and habitat: Parvidrilus gianii is known Digestive tract complete, entirely ciliated and only from the type locality, the Seldesuto cave, Mat- ending in a terminal anus. An eversible pharynx, with ienzo, Cantabria, Spain. The species was sampled at small dorsal pad set off from oesophagus, followed by 100 m from the entrance, along the shore of the lake, a narrow, thick walled winding tube extending into by stirring up rocks and sand covered by about 25 cm IX, with thick muscular walls at about VII–IX; ali- of water. The depth of the lake deepens very quickly mentary canal completed by a gut clearly enlarged a short distance from the shore; the gallery ends in a and filled in with sediment posteriorly from seg- siphon. ment XIII (Fig. 5E, F, g). Compact pharyngeal glands present in IV–VI. Digestive tract surrounded from V PARVIDRILUS STOCHI SAMBUGAR & by a well-developed layer of chloragogen cells. Coelo- mocytes and nephridia not observed. MARTÍNEZ-ANSEMIL SP. NOV. (FIG.6) No compact testes attached to septum but free Types: Holotype. VRO1003, mature specimen un- germ cells and morulae floating in the coelomic cavity stained, whole-mounted in Canada balsam. Monte of segment XI (Fig. 5A, mo). Two small sperm funnels Majore cave (40°30′47′′N, 8°36′33′′E), Thiesi, Sar- opening in ventral part of septum XI/XII and continu- dinia, Italy, 26.vi.2008, leg. Fabio Stoch, Gianfranco ing into very thin vasa deferentia (1.5-2 mm wide), Tomasin, Beatrice Sambugar, Paolo Marcia. very likely to be long and tightly folded, entering atria basally (Fig. 5A, F, f, vd). Atria elongate (about Other material examined: One partially mature 150 mm long, 14–16 mm wide), extending in segments specimen, stained in paracarmine and whole-mounted XII-XIII, curling anteriad (Fig. 5A, C–F, a1, a2), in Canada balsam, from type locality, 17.iii.2005, leg. merging below the nerve cord and a thick muscular Fabio Stoch, Gianfranco Tomasin, Jos Notenboom. arch (Fig. 5A, E, ma, nc) into a common ejaculatory Two immature specimens, stained in paracarmine duct with cuticular walls, and opening on tip of a and whole-mounted in Canada balsam, from type mid-ventral porophore located somewhat anterior to locality, 8.ix.2006, leg. Fabio Stoch, Paolo Marcia. the transversal setal line of segment XII (Fig. 5A, F, c, mp, p). Atria made up by outer thin muscular Etymology: Named after our friend Fabio Stoch, for layer (< 1 mm thick) and thick lining of vacuolated his important contributions to the knowledge of Euro- tissue, with indistinct lumen, except at the most basal pean subterranean fauna. portion, where the lumen is large and epithelial cells are finely granulated. Sparse unordered sperm Description: Entire mature worms, length 1.2 mm, embedded into vacuolated cells all along atria. Pros- 28 segments, width 45 mmatV,55mm at XII. Pros- tate glands absent. Two small ovaries with maturing tomium rounded, 15 mm long, 29 mm wide at base.

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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.

Body wall thin (especially in dorsal part), unpapil- by sperm (about two thirds of atrial length of the lated; foreign particles adhering to cuticle along the holotype and only a very small part of atria of the body, but neither dense nor continuous. Numerous maturing specimen collected in March 2005). Prostate transversal rows of thin cutaneous glands per glands absent. Two ovaries attached to septum 11/12 segment. Clitellum weakly developed (about XI-XIII), (poorly visible) (Fig. 6A, o). A single egg sac con- but some large cells observed in XII and XIII taining mature eggs extending into segment XV (Fig 6C, cl). (Fig. 6A, e). Two small oviducts seemingly attached Dorsal (dorsolateral) and ventral (ventrolateral) to septum 12/13. A single spermatheca present in setae present from III, posteriorly situated in each XII. Spermathecal ampulla ovoid (33 mm long, 10 mm segment. Dorsal bundles with two (three) straight, high), thin walled, followed by a conical, bent duct Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 thin single-pointed needles (20–28 mm long) and one (about 20 mm long), without clear cut off from (two) long, thin and flexible hair setae (115–200 mm ampulla, thin walled distally, and proximally consti- long) (Fig. 6A–C, h, n). Ventral bundles with (one) tuted by a loosely defined tissue ending close to male two–three (four) strongly curved crotchets, 20–27 mm pore, in an anterior and somewhat lateral position, long, with enlarged distal half and double-pronged tip on left side of the worm. Ampulla and distal part of with minute distal tooth (Fig. 6A–C, cr), and accom- spermathecal duct full of spermatozoids (Fig. 6A, D, panied by one thin hair seta (95–110 mm long) only sa, sd). present in preclitellar ventral bundles III-VIII. No modified genital setae; ventral bundles of segment XII Remarks: Parvidrilus stochi sp. nov. is one of the with three bifid crochets. three Parvidrilus species known so far that have a No eyes. Brain long, extending into segment single spermatheca in segment XII and are devoid IV, deeply incised posteriorly. Ventral nerve cord in of genital setae. The most outstanding features of contact with epidermis, beneath muscles of the body P. stochi sp. nov. are the very long, wide, and twisted wall. Most setigeral segments with a mid-dorsal glan- atria (15 times longer than wide), combined with a dular pouch opening posteriorly on each segment, on spermatheca in the atrial segment that has a large or immediately adjacent to the transversal setal line; spermathecal duct distally constituted by a loosely glandular pouches absent from IV-VI. defined tissue. The ovoid shape of the spermatheca Digestive tract complete, entirely ciliated, ending of this new species is only comparable to that of in a terminal anus. An eversible pharynx, with small P. gianii. 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 Distribution and habitat: Parvidrilus stochi is known completed by a gut beginning in X, enlarged and filled only from the type locality in the Monte Majore cave in with sediment from segment XV (Fig. 6D–F, g). (Sardinia, Italy). The cave opens in a small, isolated Compact pharyngeal glands present in IV-VI. Diges- Miocene limestone outcrop rising from a volcanic tive tract surrounded by a well-developed layer of plateau dating from the Oligocene-Miocene volcan- chloragogen cells from V. Coelomocytes and nephridia ism. The upper level of the cave is fossilized and not observed. percolating waters are collected in pools on clay and No compact testes attached to septum, but many rock; the lower gallery is crossed by a small brook, free germ cells and morulae floating in the coelomic which collects the waters sinking from a small valley cavity of segment XI and in the most posterior and at the entrance of the cave. 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, PARVIDRILUS TOMASINI SAMBUGAR & tubular, with a diameter slightly decreasing from the MARTÍNEZ-ANSEMIL SP. NOV. (FIG.7) proximal to the distal end (20–15 mm), extending Types: Holotype. MCSNVRO1004, entire mature in segments XII–XIV (Fig. 6A, D–F, a1, a2), merging specimen, stained in paracarmine and mounted below the nerve cord and opening on the tip of in Canada balsam. Sa Ucca de su Tintirriolu cave a mid-ventral porophore located between the two (40°27′08′′N, 8°39′15′′E) Siniscola, Sardinia, Italy, ventral setal bundles of segment XII (Fig. 6A, D, 17.iii.2005, leg. Gianfranco Tomasin, Fabio Stoch, Jos E, ed, ma, mp, p). Atria made up by extremely thin Notenboom. (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 Etymology: Named after Gianfranco Tomasin, for his epithelial cells are finely granulated. Space occupied important contribution to the knowledge of Italian by vacuolated cells seemingly progressively replaced cave ecosystems.

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A vd h a1 a2 cr h B

n Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 mo sa f f t e o XI cr cr sd XII h XIII a1 XIV X

a2 C D sa sa a2 h a1 a1 n sp sp XIII XIII gm gm cr XII ed XII sd

sp E o F g g a1 vd a2 XIII gm a1 sp XII g cl XIII G cr XII 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.

Description: Entire mature worm, length 1.2 mm, 24 granular material (secretions?, remains of old epithe- segments, width 45 mm at VI, 55 mm at XII. Prosto- lial cells?), and with a large amount of spermatozoids, mium rounded, 15 mm long, 29 mm wide at base. Body their heads orientated towards the distal atrial wall thin (especially in dorsal part), unpapillated; end, and their long flagella orientated towards the foreign particles adhering to cuticle here and there proximal atrial end, with a clear flamigerous aspect along body. Numerous transversal rows of thin cuta- (Fig. 7C–F, gm, sp). Prostate glands absent. Two neous glands per segment. Clitellum weakly devel- small ovaries with maturing eggs attached to septum oped (about XI-XIII), except for two prominent pads 11/12 (Fig. 7A, F, o). A mature egg observed in XIV comprising a few cells on lateral sides in XII, just (Fig. 7A, e). Oviducts not observed. A single sper- anterior to the transversal setal line (Fig. 7G, cl). matheca present in XII. Spermathecal ampulla Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 Dorsal (dorsolateral) and ventral (ventrolateral) tubular (35 mm long, about 10 mm wide), thin walled, setae present from III and posteriorly situated on and obliquely orientated towards the posterodorsal each segment. Dorsal bundles with two (three) part of the segment; ampulla followed by a conical, straight, thin single-pointed needles (22–29 mm long) bent duct (about 20 mm long), beginning with a and one long, thin, and flexible hair setae (120– thick epithelium delimiting a wide lumen, and then 200 mm long) (Fig. 7A–C, h, n). Ventral bundles tapering, with the lumen becoming very narrow, and composed of two-three strongly curved crotchets, ending in a minute pore in an anterior and somewhat 20–26 mm long, with enlarged distal half and double- lateral position, on left side of the worm. Ampulla full pronged tip with minute distal tooth, and accompa- of spermatozoids (Fig. 7A, C, D, sa, sd). nied by one thin hair seta (95–135 mm long) only present in III XI (Fig. 7A–C, G, h, cr). No modified - Remarks: Parvidrilus tomasini sp. nov. is a species genital setae; ventral bundles of segment XII with a with long atria (about eight times longer than single bifid crochet. wide), and a single spermatheca located in the atrial No eyes. Brain long, extending into segment segment. The most characteristic anatomical features IV, deeply incised posteriorly. Ventral nerve cord in of this species are the tubular shape of the spermath- contact with epidermis, beneath muscles of the body ecal ampulla and the well-defined, narrow spermath- wall. Most setigeral segments with a mid-dorsal glan- ecal duct. Compared with the other two species dular pouch opening posteriorly on each segment, on that have a spermatheca in segment XII, the atria about the transversal setal line; glandular pouches of P. tomasini are considerably shorter than those of absent from IV VI. - P. stochi and longer than those of P. strayeri (see Digestive tract complete, entirely ciliated. An Erséus, 1999, and remarks below). 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 Distribution and habitat: Parvidrilus tomasini is VII-VIII; alimentary canal completed by a gut con- known only from the type locality in the Sa Ucca de taining sediment, clearly enlarged from segment XIV su Tintirriolu cave (Sardinia, Italy). The cave occurs (Fig. 7E, g). Compact pharyngeal glands present in an isolated Miocene limestone area surrounded by in IV-VI. Digestive tract surrounded by a well- volcanic rocks of the same age. A small brook runs developed layer of chloragogen cells from V. Coelo- through the lower gallery in contact with the basaltic mocytes and nephridia not observed. floor. 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 PARVIDRILUS SPELAEUS MARTÍNEZ-ANSEMIL, and in the most anterior part of XII (Fig. 7A, mo). SAMBUGAR &GIANI, 2002 Sperm funnels hardly observed (Fig. 7A, f). Vasa def- New material: Mine of Ponte Vajo Falconi, Italy erentia seemingly present as very thin (about 1.5 mm (PASCALIS LES 060; 10°59′07′′E; 45°39′36′′N), pools wide), long and tightly folded ducts in XII (Fig. 7A, E, of percolating water, 2002, leg. Stoch F., Tomasin G., vd). Atria elongate (about 150 mm long), tubular one specimen; Bus del Cao cave, Italy (PASCALIS (15–22 mm wide), extending into segments XII-XIII, LES 001; 10°54′48′′E; 45°35′36′′N), subterranean merging below the nerve cord and opening on the brook, 2002, leg. Stoch F., Tomasin G., two specimens; tip of a mid-ventral pore (in a small porophore?) Buso del Progno cave, Italy (PASCALIS LES 003; located between ventral setal bundles of segment XII 10°55′02′′E; 45°36′28′′N), subterranean brook, 2002, (Fig. 7A, D, a1, a2, ed). Atria made up by an outer leg. Stoch F., Tomasin G., one specimen; Covolo della thin muscular layer < 1.5 mm thick, and a nucleated Croce cave, Italy (PASCALIS LES 099; 11°07′16′′E; epithelial layer, limited to the proximal and most 45°36′43′′N), rimstone pools, 2002, leg. Stoch F., distal parts of atrial wall. Atrial lumen filled in by a Tomasin G., one specimen; Monte Capriolo cave, Italy

(PASCALIS LES 195; 11°04′52′′E; 45°35′38′′N), rim- described by Erséus (1999) to be the two atria stone pools, 2002, leg. Stoch F., Tomasin G., four merging into a common ejaculatory duct and the specimens; Buso della Volpe cave, Italy (PASCALIS spermatheca, respectively. LES 149; 11°12′52′′E; 45°36′48′′N), small pools in gravel, 2002, leg. Stoch F., Tomasin G., two speci- Distribution and habitat: Known only from the type mens; Montorio, via del Lanificio, Italy (PASCALIS locality in Alabama, USA. Interstitial water. [A sub- LES 131; 11°03′59′′E; 45°27′34′′N), hyporheic site, sequent visit to the type locality by C. Erséus and Bou-Rouch pump, 2002, leg. Stoch F., Tomasin G., two M. J. Wetzel in March 2008 failed to collect additional specimens; spring near Jelenska jama, Slovenia (PAS- specimens of P. strayeri for study (M. J. Wetzel, pers. CALIS KRI 097; 14°21′19′′E; 45°55′05′′N) 2002, leg. comm.).] Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 Brancelj A., one specimen; spring near Žumerju, Slo- venia (PASCALIS KRI 005; 14°35′47′′E; 45°53′16′′N), 2002, leg. Brancelj A., one specimen; Pajsarjeva SPECIES INQUIRENDA cave, Slovenia; (14°15′56′′E; 45°59′52′′N), subterra- PARVIDRILUS GINETI (JUGET, 1959) COMB. NOV. nean brook, 2009, leg. Gasparo F., Sambugar B., two Aelosoma Gineti Juget, 1959: 397–399, figure 3a. specimens. Aelosoma gineti Juget (1959) – Ginet, 1961: 310. Aeolosoma gineti Juget, 1959 – Brinkhurst, 1967: 112; Description: The new material of P. spelaeus enables Bunke, 1967: 266; Brinkhurst & Jamieson, 1971: 695; us to confirm the attachment of the spermathecal Seyed-Reihani, 1980: 57, table VI; Dole, 1983a: 227; duct to the anterior part of segment XIII, and very Dole, 1983b: 82, 110, 113, ann. 8; Juget & Dumnicka, thin vasa deferentia joining atria proximally. The 1986: 234, 239; Dole-Olivier et al., 1993: 457, Table 2; so-called diffuse prostate surrounding the atria Dole-Olivier et al., 1994: 321; Ferreira et al., 2003: 17; referred to in Martínez-Ansemil et al. (2002: fig. 12) Artheau & Giani, 2006: 230; Ferreira et al., 2007: 585; is now interpreted as a large peritoneal layer, not Timm, 2009: 18, 132. observed in fully developed individuals. Types: Lectotype. MHNL 44003362, ‘Aeolosoma COI sequence: EMBL accession number: HE800202 gineti, Lac souterrain La Balme, février 1959’, frag- (Pisoliti cave, Trieste, Italy). ment (first 11 segments), whole-mounted in an unspecified, slightly yellow liquid medium (Amman’s Distribution and habitat: Presently, the known distri- lactophenol?), sealed with red lute. La Balme Cave bution of P. spelaeus is limited to the Alpine district of (45°51′10′′N, 5°20′21′′E, 220 m asl), Isère (38), northern Italy and extends into the Slovenian Dinaric France. region, where it was found in several phreatic and Paralectotype. MHNL 44003363, ‘Aeolosoma hyporheic habitats: from small pools of percolating gineti, Lac souterrain La Balme, février 1959’, whole- water to streamlets and in cave lakes, springs, and mounted in an unspecified, slightly yellow liquid rock aquifers (see also Martínez-Ansemil et al., 2002). medium, sealed with red lute.

Other material examined: UCBLZ 1.011.1-1.011.3, PARVIDRILUS STRAYERI ERSÉUS, 1999 ‘Aeolosoma cf. gineti; Ph 7 1, 20/10/76’, alluvial plain Amended description: In the light of the atrial appear- of the Haut Rhône, phreatic and hyporheic waters ance, and the preatrial location of the spermatheca (45°48′50′′N, 05°04′48′′E, 179 m asl; see Artheau & in some of the new species, we can now interpret Giani, 2006), Rhône (69), France; three specimens the so-called ‘copulatory organ’ and ‘genital body’ whole-mounted in Canada balsam.

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

P. meyssonnieri P. meyssonnieri Taxon P. spelaeus (La Martinière) (Navogne) P. jugeti

Parvidrilus spelaeus – 0.18 0.17 0.19 Parvidrilus meyssonnieri sp. nov. (La Martinière) 0.21 – 0.09 0.19 Parvidrilus meyssonnieri sp. nov. (Navogne) 0.20 0.09 – 0.19 Parvidrilus jugeti sp. nov. 0.23 0.22 0.23 –

Emended description: Lectotype: length 435 mm, 11 preserved, there would be no guarantee of having segments (fragment); paralectotype: length 1450 mm, sampled this same taxon; thus we must consider 21 segments (uncertain as specimen badly damaged P. gineti to be a species inquirenda. as a result of stretching into two parts). Breadth 115 mm at widest (IV-IX). Prostomium rounded to Distribution and habitat: ‘La Balme’ cave, Isère conical, 40 mm long, 45–55 mm wide at base; ciliation (38), France, in a subterranean lake, heterogeneous absent. Dorsal and ventral setae present from III, sediment (rough sand and fine clay, with organic posteriorly situated in each segment, absent in II. remains, mostly of plant origin); alluvial plain of the Dorsal bundles with one or two straight, single- Haut Rhône, France, phreatic and hyporheic waters Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 pointed needles (15–30 mm), and one hair seta (length (Miribel canal and ‘Lône du Grand Gravier’; Artheau 150–237 mm). Ventral bundles with one (lectotype) & Giani, 2006). to two–three (paralectotype) sigmoid, bifid crotchets (15–18 mm) with upper tooth slightly thinner and shorter than lower tooth, accompanied by one smooth OTHER PARVIDRILIDAE hair seta (length 95–133 mm); ventral hair setae Several other specimens from three localities (below) absent from XI. Internal organs mostly destroyed in appear to be parvidrilids and have unique characters the mounting medium used. Progressive intestinal supporting their consideration as new species. Unfor- dilation from VII partially visible. tunately, they cannot be identified or classified until additional material has been collected. Remarks: According to the original description by • Slovenia, phreatic zone of the Podlipšcˇica valley, Juget (1959), it seems likely that a few specimens (PASCALIS KRI 187; 45°59′34″N, 14°15′01″E). One were observed alive, which would explain the mention single specimen. Atria and spermatheca in XII, of chains of zooids of two to five specimens, although atria very large and globular, filling whole of they were absent in the material available to us for segment. observation. In all probabilities, the specimen shown • Slovenia, Pajsarjeva cave, 1997. One specimen in the original illustration (Juget, 1959: 398, fig. 3a) characterized by very long atria reaching 13/14, corresponds to the lectotype. It is similar in external with a thin wall and full of spermatozoids. Sper- appearance, and in the number and arrangement of mathecae not seen because of the poor preservation setal bundles, except that the first setigeral segment of the specimen. is not shown on the figure – suggesting that there are • Italy, Tondello spring, Verona (PASCALIS LES eight setigeral segments instead of nine. In this speci- 129: 45°27′52″N, 11°03′55″E). One broken specimen, the so-called ‘zooid’ does not show any clear zone men with very long atria. of scission and cannot be considered as such. The combination of setal features (e.g. shape, number, and location of setae) is typical of Parvidri- RESULTS lus, supporting its placement within the family MOLECULAR ANALYSES Parvidrilidae. In fact, Bunke (1967: 266) early considered ‘Aeolosoma gineti’asaspecies dubia and Genetic divergence suggested that the species should be excluded from The COI sequences of five specimens of P. meysson- the polychaete genus Aeolosoma because of setal fea- nieri (three and two specimens from the galleries of tures, the absence of coloured glands in the tegument, ‘La Martinière’ and ‘Navogne’, respectively), and two the small and unciliated prostomium, and the orga- specimens of P. jugeti (both from the Corveissiat nization of intestinal tract. A possible alignment with cave) were successfully obtained. Specimens from the the oligochaete family, Naididae, was also suggested same locality have identical haplotypes. In contrast, by Bunke, an opinion that was later restated by the COI sequences of P. meyssonnieri from the Gallery others (Brinkhurst & Jamieson, 1971; Artheau & of ‘La Martinière’ and from the Gallery of ‘Navogne’ Giani, 2006). More recently, Timm & Veldhuijzen van differ from each other by 9%. At the specific level, the Zanten (2002) and Timm (2009) suggested that genetic divergence amongst P. jugeti, P. meyssonnieri, the species might well be a parvidrilid although no and P. spelaeus ranges from 17–20% at the minimum paratomy was yet recorded in this group. to 19–23% at the maximum (p- and K2P distances, The external morphology of P. gineti is similar to respectively; Table 2). other Parvidrilus species. However, in the absence of properly preserved internal organs – and in particu- Phylogenetic position within the Clitellata lar, the genitalia – no unambiguous description of The final PRANK alignment of the 18S data con- the species can be provided. Even if new material tained a total number of 1802 characters, 350 from the type locality could be collected and properly parsimony-informative characters when gaps were

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 NEW EUROPEAN SPECIES OF PARVIDRILIDAE 549 coded as missing data, and 389 parsimony- the body diameter, the specific set of anterior ventral informative characters when gaps were coded as setae (bundles composed of bifid crotchets and hair a fifth character state. A total of 11 025 trees was setae), and the markedly posterior position of setae obtained in parsimony analyses when gaps were within the segments. The glandular pouches were not coded as missing data, resulting in a consensus tree observed in P. jugeti, and only sporadically in P. mey- with most basal nodes unresolved. Only 174 trees ssonnieri – perhaps a negative consequence attri- were produced when gaps were coded as a fifth char- butable to the fixative (Bouin-Hollande solution) acter, yielding a consensus tree that is nearly com- used for the specimens in the French collections. The pletely resolved (Fig. 8). Bayesian and maximum unique spermatheca attached to the ventral body likelihood analyses returned trees that are quite wall (when present) is another distinctive character Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 similar, except for two conflicting nodes, owing to of the family. a switched position of the Parvidrilidae and the As all parvidrilids presently known have a single Propappidae according to the analysis considered male pore, this feature also becomes an important (Fig. 9). diagnostic character for the family. In fact, apart from Parsimony (MP), maximum likelihood (ML) and the hirudineans, acanthobdellids, and branchiobdel- Bayesian (B) analyses produced trees that are con- lids (Sawyer, 1986; Brinkhurst, 1999), the confluence gruent to a great extent when only supported of paired male ducts into a single mid-ventral pore is branches (posterior probabilities > 90%, or bootstrap quite uncommon within clitellates. values > 70; Hillis & Bull, 1993) are considered Interestingly, the male ducts of some parvidrilids (Figs 8, 9). All methods gave a similar branching look similar to that of most phreodrilids: long atria, pattern (except for MP with gaps as missing data), with a thick and glandular inner epithelial lining, even if many nodes, mostly basal, were not sup- leaving only a narrow lumen, and vasa deferentia ported. The monophyly of all families are recovered entering the ectal part of the atria. and supported, but Haplotaxidae – of which the No prior attention has been directed towards sper- sister relationship of Delaya bureschi was found to matogenesis of parvidrilids, which seems to begin be with Crassiclitellata, instead of Haplotaxis gor- very early, free in the coelomic cavity, when germ cells dioides – makes the family paraphyletic. At a higher come off septum 10/11. In oligochaetes, testes never taxonomic level, the monophyly of Crassiclitellata grow very large because their activity is restricted and of the clade (Lumbriculidae, Branchiobdellida, to the formation of reproductive cells in their early Acanthobdellida, Hirudinea) is confirmed and sup- stages of development (Chekanovskaya, 1962). The ported, although – within this last assemblage – the parvidrilids seem to be an extreme case, only compa- position of Branchiobdellida vis-à-vis Lumbriculidae rable to what occurs in some small naidids, where the and clade (Acanthobdellida, Hirudinea) remains testes disappear very early (Sperber, 1948). It is pos- unresolved. As to Parvidrilidae, the three methods sible that the quick release of germ cells, developing (MP, ML, B) gave congruent, although unsupported free in the coelomic cavity of parvidrilids, is related in results, placing the family well within the Clitellata some way to the minute size of these worms. – far from Capilloventridae and Phreodrilidae, The presence of spermatozoids with their heads but close to Enchytraeidae. The ML analysis only embedded into the atria body wall is also an uncom- differed from MP and B in a switched position of mon phenomenon in oligochaetes that warrants Parvidrilidae with Propappus: Enchytraeidae was further study. As neither spermatophores nor sperma- placed as the sister group to either Propappus (ML) tozeugmata were observed in parvidrilids, we suggest or Parvidrilidae (MP, B). the possibility that the secretions provided by the epithelial glandular wall feed the spermatozoa at the end of spermiogenesis, or (at a minimum) supply DISCUSSION abundant fluid to sperm. In their review on sexual and parthenogenetic planarians, D’Souza & Michiels AUTAPOMORPHIES AND OTHER RELEVANT (2009) emphasized the fact that parthenogens seem to CHARACTERS OF THE PARVIDRILIDAE reduce the proportion of sperm and transfer more The six new species of Parvidrilus described in this accessory fluid. Interestingly, some of the parvidrilid work support the soundness of the character choice species presently known are either devoid of sper- by Martínez-Ansemil et al. (2002), considered as the mathecae or have a single spermatheca that is empty. most reliable traits supporting the monophyly of the Although a leech-like impregnation is not excluded, family from the morphological point of view: the pres- sperm-dependent parthenogenesis – frequently asso- ence of singular segmental glandular pouches in the ciated with hermaphroditism (Beukenboom & Vrijen- mid-dorsal line of the body, the lateral development hoek, 1998) – could be also expected as a mechanism of the clitellum with cells that are large in relation to of reproduction in parvidrilids.

94/80 Notomastus latericeus Loimia medusa Polychaeta Arenicola marina ------Capilloventer australis Capilloventridae 95/96 Antarctodrilus proboscidea------Insulodrilus bifidus Phreodrilidae 85/85 Rhyacodriloides abyssalis------Rhyacodriloides latinus

91/79 77/72 Nais communis Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 Pristina longiseta Baikalodrilus digitatus 56/- 100/100 Thalassodrilus gurwitschi Naididae Smithsonidrilus humilis 56/- 94/84 Tubificoides bermudae (naidids + tubificids) 71/61 92/86 Heteridrilus decipiens Pectinodrilus molestus 81/75 Heronidrilus heronae Bathydrilus litoreus Bothrioneurum vejdovskyanum 99/98 Buchholzia fallax ------100/100 Fridericia tuberosa 100/100 Grania variochaeta Enchytraeidae Grania americana 53/* Marionina sublitoralis ------Propappus volki Propappidae 100/100 sp.------n. Parvidrilus jugeti Parvidrilidae Parvidrilus meyssonnieri sp. n. 100/100 Dichogaster saliens ------Pontodrilus litoralis 100/100 Lumbricus castaneus Lumbricus terrestris Crassiclitellata 100/100 96/96 Eisenia andrei Criodrilus lacuum 80/71 Dendrobeana clujensis Delaya bureschi ------Haplotaxis cf. gordioides Haplotaxidae 61/64 Eclipidrilus frigidus ------92/91 Stylodrilus heringianus 74/63 Rhynchelmis tetratheca Lumbriculidae Lumbriculus variegatus------100/100 Cirrodrilus sapporensis 93/91 Branchiobdella parasita Branchiobdellida 58/53 Cambarincola pamelae------54/- Acanthobdella peledina Acanthobdellida 100/99 Glossiphonia complanata------82/81 Helobdella stagnalis Hirudinea 100/100 Erpbdella japonica 100/100 Haemopis caeca ------

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.

53/- Notomastus latericeus ------Loimia medusa Polychaeta Arenicola marina ------Capilloventer australis Capilloventridae 95/1.00 ------Antarctodrilus proboscidea Phreodrilidae Insulodrilus bifidus ------88/1.00 Rhyacodriloides abyssalis 91/1.00 -/0.79 Rhyacodriloides latinus 89/1.00 Nais communis Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 Pristina longiseta 65/0.94 Baikalodrilus digitatus Naididae Thalassodrilus gurwitschi -/0.87 99/1.00 (naidids + tubificids) 70/1.00 Smithsonidrilus humilis Tubificoides bermudae 94/1.00 Heteridrilus decipiens Pectinodrilus molestus Heronidrilus heronae Bothrioneurum vejdovskyanum Bathydrilus litoreus ------/0.78 98/1.00 Buchholzia fallax 71/0.89 Fridericia tuberosa 100/1.00 Grania variochaeta Enchytraeidae 100/1.00 Grania americana * Marionina sublitoralis ------Propappus volki ------Propappidae 100/1.00 Parvidrilus jugeti sp. n. Parvidrilidae Parvidrilus meyssonnieri sp. n. 100/1.00 ------/0.91 Dichogaster saliens Pontodrilus litoralis 95/1.00 Lumbricus castaneus Lumbricus terrestris Crassiclitellata * 100/1.00 88/1.00 Eisenia andrei Criodrilus lacuum 62/1.00 Dendrobeana clujensis ------Delaya bureschi Haplotaxidae Haplotaxis cf. gordioides 60/- Cirrodrilus------sapporensis 100/1.00 Branchiobdella parasita Branchiobdellida 67/1.00 Cambarincola pamelae Eclipidrilus frigidus ------92/1.00 Stylodrilus heringianus Lumbriculidae 79/0.98 Rhynchelmis tetratheca 70/0.90 Lumbriculus variegatus ------Acanthobdella peledina ------Acanthobdellida 86/1.00 97/1.00 Glossiphonia complanata 100/1.00 Helobdella stagnalis Hirudinea 100/1.00 Erpobdella japonica Haemopis caeca 0.05 -----

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.

PHYLOGENETIC POSITION OF PARVIDRILIDAE Ehrenberg, following phylogenetic assessments using WITHIN THE CLITELLATA DNA data (see Erséus & Gustavsson, 2002; Erséus, DNA Wetzel & Gustavsson, 2008), seemed to weaken the Based on a data set mostly similar to the one analy- traditional view that considers the location of gonads sed by Erséus & Källersjö (2004), it is reassuring that as a primary taxonomical character. However, the our analysis produced nearly the same relationships, forward position of the gonads in the former Naididae amply discussed by those authors, in spite of other is probably related with the emergence of paratomy methods as to alignments (PRANK vs. ClustalX) and as their main reproductive mechanism. Hence, the traditional view remains acceptable until there is

phylogenetic reconstructions (ML, B, in addition to Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 MP), and despite lack of support for basal nodes of evidence to the contrary. phylogenies. A major discrepancy was, however, noted The presence of testes in XI and ovaries in XII for the position of Haplotaxidae and Propappidae was already stated to be a character shared by the vis-à-vis other clitellate families. Parvidrilidae, the Enchytraeidae, the monotypic The most striking difference lies in the fact that Tiguassidae, most Phreodrilidae, the monotypic Haplotaxidae, although as a paraphyletic assemblage, opistocystid genus Crustipellis Harman & Loden, are now the closest taxon to the Crassiclitellata, and perhaps most Capilloventridae (Erséus, 1999; as suggested by morphology-based evolutionary Martínez-Ansemil et al., 2002). Although sharing a relationships (Brinkhurst, 1994). As a result, the plesioporous condition (the male pores are in the Diplotesticulata are restored as a monophyletic segment directly behind the testes), the male ducts group, in conformity with the classiﬁcation structured of the parvidrilids were considered quite different according to a morphocladistic analysis by Jamieson from those of the enchytraeids, capilloventrids, and (1988) (see also Jamieson & Ferraguti, 2006). tiguassids (e.g. all the latter three families lack atria). As for Propappidae and Parvidrilidae, nothing con- Moreover, these three families have their spermath- clusive can be reached in the absence of support for ecae located in segments anterior to the gonadal ones most basal branches. At best, we emphasize that a (the prosothecous condition). Apart from the location suggested yet still unsupported sister relationship of testes (segment XI) and male pores (segment XII), between Propappidae and Enchytraeidae in the the absence of nodulus on setae in Parvidrilidae ML analysis is in agreement with their morphology and many Enchytraeidae is the only distinctive (Coates, 1986, 1987) and evolutionary relationships character shared between these two families, which suggested by morphology (Brinkhurst, 1994). Such a deserves to be mentioned. Close phylogenetic rela- Propappidae-Enchytraeidae sister relationship was tionships between opistocystids and parvidrilids have also recently obtained by Marotta et al. (2008) in a also been discarded in the light of other anatomical phylogenetic analysis of Clitellata using a combina- and biological traits (Martínez-Ansemil et al., 2002). tion of molecular (18S rDNA) and morphological data Recent molecular studies led Erséus et al. (2010) to (somatic and spermatozoal characters). All analyses consider that Opistocystidae are likely to be a group are congruent in suggesting that Parvidrilidae within Naididae. are nested well within clitellates, namely far from As a result of the present study, the anatomy of Capilloventridae, a family that was once suggested several reproductive structures has become clearer. to be a potential sister taxon (Erséus, 1999; Martínez- The male ducts of some parvidrilids resemble those of Ansemil et al., 2002). Although closer to Enchy- most phreodrilids. This, together with the similarity traeidae than Phreodrilidae, another suggested of other important anatomical traits (see Martínez- putative sister family, the exact position of Parvidril- Ansemil et al., 2002), reinforce the idea that phreo- idae within Clitellata still remains uncertain in the drilids are the best candidate to be the sister group to absence of branch support. parvidrilids on morphological grounds.

Morphology DIVERGENCE BETWEEN SPECIES From the morphological point of view, the most Over the last few years, DNA sequences have been relevant characters currently used in oligochaete increasingly employed in taxonomy and the COI taxonomy at the family level are the number and gene has been considered as an important marker location of gonads, the location of male pores with that helps species delimitation (Lefébure et al., 2006b; respect to male funnels, the location of spermathecae, Erséus & Gustafsson, 2009; Grant & Linse, 2009; and the structure of male ducts (e.g. Stephenson, Trontelj et al., 2009; Havermans et al., 2011). The 1930; Brinkhurst & Jamieson, 1971; Brinkhurst, efficiency of this approach depends on the separation 1984; Erséus, 2005). The recent merging of the former between intra- and interspeciﬁc divergences (Meyer & family Tubiﬁcidae Vejdovský with the Naididae Paulay, 2005; Waugh, 2007). In several animal taxa,

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 NEW EUROPEAN SPECIES OF PARVIDRILIDAE 553 the effectiveness of DNA taxonomy (in the sense of are presumably highly isolated, geologically, from prediction and classification of new taxa; Lefébure each other. et al., 2006b) has been confirmed, although its accu- racy seems to depend on the taxonomic knowledge and the sample coverage of the group (e.g. Meyer & ECOLOGY AND BIOGEOGRAPHY Paulay, 2005). In Clitellata, an accumulation of evi- Since the description of P. strayeri from groundwater dence suggests that most congeneric species differ in Alabama (USA), and the subsequent erection of from each other by about 10% (or more) COI diver- the new oligochaete family Parvidrilidae (Erséus, gence (p-distance) (Erséus & Gustafsson, 2009; Zhou 1999), eight new species – all from Europe, have been et al., 2010). added to this family (P. camachoi, P. gianii, P. gineti, Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 In this respect, the high genetic divergence P. jugeti, P. meyssonnieri, P. spelaeus, P. stochi, P. to- amongst P. jugeti, P. meyssonnieri, and P. spelaeus masini), making this family much more diverse than (17–19%, p-distances; 20–23%, K2P distances) is in initially expected. Recent investigations in Slovenian good accordance with their specific status. Further- groundwaters will probably result in the description more, such high divergences may even justify consid- of two new Parvidrilus species, and expand the ering the erection of different genera in order to known distribution of P. spelaeus, a species already accommodate the species diversity of this family. reported from that country (Giani et al., 2011), thus However, the 9% divergence between specimens of suggesting an even wider diversity of the genus in P. meyssonnieri from two different localities is intrigu- European groundwaters. ing, and even more so because (1) morphological scru- To date, five of the Parvidrilus species each tiny of these specimens did not reveal any significant appear to be strictly endemic to a single cave: in difference, except, perhaps, in hair setae (slightly Spain, P. camachoi to Estaragueña cave and P. gianii more plumose in specimens from the ‘Navogne’ to Seldesuto cave; in Italy, P. stochi to Monte Maiore population, by comparison with the ‘La Martinière’ cave and P. tomasini to Sa Ucca de su Tintirriolu population), and (2) both populations are located in cave; in France, P. jugeti to Corveissiat cave (Fig. 10). stations that belong to the same hydrogeological Parvidrilus meyssonnieri was found in two galleries basin, and are distant from each other by not more mined in consolidated granitic sands of the Rhône than 60 km. department: La Martinière and Navogne (same Recent molecular studies of groundwater crusta- geological formation, located only 58 km from each ceans have shown that extreme conditions of life in other). Parvidrilus gineti was present in La Balme groundwater promote cryptic diversity by inducing cave and possibly in the hyporheic zone in the alluvial convergent morphological evolution (Lefébure et al., plain of the Rhône river. Parvidrilus spelaeus proved 2006a; Lefébure, Douady & Gibert, 2007). Accord- to be the most widely distributed species in the Alpine ingly, we cannot eliminate the possibility that the Arc, from Italy to Slovenia. Our data on Parvidrilidae ‘Navogne’ population and the ‘La Martinière’ popula- emphasize the wide ecological range of this family, tion may in fact represent two different species. Con- found in both karstic and granite districts, and on versely, the subterranean aquatic ecosystem is well unconsolidated as well as consolidated substrata. known for its habitat heterogeneity (Gibert & Dehar- Hence, it is likely that Parvidrilidae could colonize veng, 2002). the entire hypogean environment. Therefore, it is plausible to expect a substantial Considering the data related to the parvidrilid range of intraspecific genetic variation in stygobiont fauna, the efficiency of the PASCALIS sampling strat- species because of low dispersal abilities of inhabit- egy, which focused equally on both the unconsolidated ants of such an environment – and even within the and karstic aquifers (Dole-Olivier et al., 2009), is same hydrogeological basin. For instance, Lefébure herein confirmed. During this study, Parvidrilidae et al. (2006b) considered that, in groundwater crusta- were found in each of the three studied south- ceans, two monophyletic groups have a strong prob- ern European karst units – Cordillera Cantábrica, ability of belonging to different species, but only when Lessinian mountains, Krim Massif. These discoveries they diverge by more than 0.16 substitutions/site in support the presence of parvidrilids in Spain (from the COI gene (patristic distances). More studies are which they had not been reported prior to this present clearly needed to address this issue in the future. study), and contribute new data for P. spelaeus, thus In this respect, it is worth noting that P. jugeti expanding both the known distribution and habitat and P. meyssonnieri diverge from each other by 19% preferences of this species in both kinds of aquifer (p-distance) and 23% (K2P). Although the known loca- type. We can confirm the stygobiont character of tions of these two species are relatively close to each Parvidrilidae and infer that Parvidrilus is probably other (~100 km or less), they belong to two different widely distributed throughout underground waters, bedrock formations (granitic vs. karstic), and thus yet may have been unreported in past studies owing

© 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166, 530–558 554 E. MARTÍNEZ-ANSEMIL ET AL. Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019

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). to its exceedingly small size, or more likely missed The presence of Parvidrilus in Sardinia is quite during sampling because of abrasive sample process- noteworthy. Previously, a few specimens of Parvidri- ing and coarse mesh size of nets. Moreover, the appar- lus, seemingly attributable to P. spelaeus, were ent morphological similarity of Parvidrilidae and noted from Bue Marino cave (Martínez-Ansemil Aeolosomatidae may have caused confusion between et al., 2002). Recently, additional specimens referred as the two taxa groups, as illustrated by P. gineti, P. cf. spelaeus were discovered in this same cave (E. leading to the oversight of parvidrilid taxa present in Martínez-Ansemil & B. Sambugar, unpubl. data). The collections. discovery of two new species (P. stochi and P. tomasini, With respect to the issue of morphological adapta- described herein) broadens the diversity of the genus tions of oligochaetes to the subterranean environ- in Sardinia and highlights the endemicity of the sty- ment, the Parvidrilidae show interesting features gobiotic fauna of this ancient Mediterranean island. (e.g. very tiny transparent bodies, presence of epider- The Sardo-Corsican system, in fact, was contiguous to mal glands, and presence of simple-pointed setae) the Pyrenees and became an independent microplate that are usually considered to be adaptations to in the late Oligocene. It drifted until it reached the subterranean life in rock microcrevices and inter- present position, carrying with it a sample of the stices (Sambugar et al., 1999; Giani et al., 2001). Palaeogene fauna. The affinities between the fauna However, such an issue remains controversial and it of Catalonia and of Sardinia-Corsica are in agree- is still speculative to categorize these characters as ment with this palaeogeographical reconstruction pre-adaptations, or adaptations to life in groundwa- (e.g. the distribution of Trichodrilus angelieri Giani ters (Creuzé des Châtelliers et al., 2009). & Rodríguez, 1994, in groundwaters of the south

Pyrenees, Corsica, and Sardinia; E. Martínez-Ansemil underwent radiation and niche diversiﬁcation. Such & B. Sambugar, unpubl. data). an evolutionary trajectory might have led to cryptic Although present information on the geographical biodiversity and the occurrence of congeners that coverage of the stygofauna is still patchy (Gibert & are morphologically very similar, i.e. the Sardinian Culver, 2009), the biogeographical distribution of the P. spelaeus and P. cf. spelaeus. Future molecular Parvidrilidae suggests that the family is an archaic studies are clearly needed to address this issue. Holarctic lineage, which could well illustrate the Besides revealing potential cryptic diversity within ‘refuge’ nature of the subterranean environment; this the genus, such studies would provide important concept was again presented (Martin et al., 2010) to clues as to relationships and affinities between Pyre- explain the geographical distribution of new Rhyaco- nean and Sardinian species, as well as times of colo- Downloaded from https://academic.oup.com/zoolinnean/article-abstract/166/3/530/2629179 by guest on 09 October 2019 driloides species, a genus previously only known from nization of the genus. Lake Baikal. In number of species, the genus Parvidrilus consti- The Holarctic distribution of stygobiont taxa tutes an appreciable fraction of the known stygobiont (such as Parvidrilus) lends credence to an ancient oligochaete fauna (nine of 111 species, 8%). With all colonization of groundwaters by the group, which these species being stygobiont, the Parvidrilidae is probably pre-dated the opening of the Atlantic unique in being the only family of oligochaetes, world- Ocean in the late Cretaceous or early Tertiary. Fol- wide, comprising taxa that are restricted in distribu- lowing this scenario, we can consider that Parvid- tion to groundwater habitats, and as such, can be rilidae constituted a part of the drifted fauna of considered the most representative oligochaete family the Sardo-Corsican microplate that subsequently in this environment.

KEY TO THE PARVIDRILID SPECIES 1. Genital setae present (one per bundle in XII)...... Parvidrilus meyssonnieri – Genital setae absent ...... 2 2. Spermatheca absent...... Parvidrilus jugeti – Spermatheca present...... 3 3. Spermatheca in XII (atrial segment)...... 4 – Spermatheca in XIII (postatrial segment) ...... 5 4. 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 5. 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.

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