Zoologica Scripta
Evolution of cave suspension feeding in Protodrilidae (Annelida) ALEJANDRO MARTINEZ,KIRSTEN KVINDEBJERG,THOMAS M. ILIFFE &KATRINE WORSAAE
Submitted: 9 March 2016 Mart ınez, A., Kvindebjerg, K., Iliffe, T. M., Worsaae, K. (2016). Evolution of cave suspen- Accepted: 17 June 2016 sion feeding in Protodrilidae (Annelida). — Zoologica Scripta, 00,1–13. doi:10.1111/zsc.12198 Protodrilidae belongs in a lineage that until now entirely consisted of deposit-feeding, highly adapted interstitial annelids. Except for a pair of anterior palps, all protodrilids lack appen- dages, parapodia and chaetae; and have slender bodies adapted to glide between the sand grains by ciliary motion. The first exception to these characteristics is Megadrilus pelagicus n. sp. inhabiting the water column of the anchialine La Corona cave system in Lanzarote. Its morphology and evolutionary history are here investigated by combining observations from in vivo video recordings and advanced microscopy with phylogenetic analyses. Our studies revealed a unique pelagic, suspension feeding behaviour attained by its long ciliated palps in combination with an autopomorphic dorsal ciliated keel and several longitudinal and trans- verse ciliary bands. Phylogenetic analyses recovered Megadrilus pelagicus n. sp. nested within Protodrilidae indicating that its unique traits are derived within the family. These traits are traced in the tree topologies in correlation to cave colonization. The evolution of these traits can be functionally explained by the different demands of a pelagic suspension feeding strat- egy compared to the ancestral deposit-feeding guild of the family. The origin of this suspen- sion feeding strategy was presumably favoured by the partial isolation of the anchialine ecosystem, connected to the sea only through the highly porous volcanic subterranean bed- rock. This crevicular connection limits the amount of predators and turbulence in the cave, but allows continuous water flow into the system carrying organic particles, which is the main source of food when photosynthetic primary production does not occur and sedimenta- tion is limited. These conditions may select for pelagic suspension feeding as the most feasi- ble life-strategy in anchialine caves, which the dominance of pelagic, suspension feeding crustaceans and annelids in anchialine cave assemblages may also reflect. For species of ancestrally deposit-feeding lineages entering the cave system, such as the annelid families Protodrilidae and Nerillidae, an adaptive-shift from interstitial to crevicular habitats seem- ingly correlates with dramatic morphological changes and speciation. The dramatic changes observed in these primarily interstitial lineages compared to their relatives, point to alterna- tive adaptive evolutionary pathways related to ecological fitness contrary to the previously proposed theories focusing on geological or stochastic processes. Corresponding author: Alejandro Mart ınez, Marine Biological Section, University of Copenhagen, Universitetsparken 4, DK-2100 Copenhagen, Denmark. E-mails: [email protected] Kirsten Kvindebjerg, Marine Biological Section, University of Copenhagen, Universitetsparken 4, DK-2100 Copenhagen, Denmark. E-mails: [email protected] Thomas M. Iliffe, Department of Marine Biology, Texas A&M University at Galveston, Galveston, TX 77553-1675, USA. E-mail: [email protected] Katrine Worsaae, Marine Biological Section, University of Copenhagen, Universitetsparken 4, DK-2100 Copenhagen, Denmark. E-mail: [email protected]
Introduction metazoan phyla occur in the tiny interstices, including Marine interstitial environments constituted by small spaces miniaturized members of macroscopic phyla (Giere 2009; between sand grains are inhabited by the most diverse ani- Worsaae et al. 2012) as well as exclusively meiofaunal mal community on Earth from a phylogenetic point of view lineages, some of them with unclear phylogenetic affinities (Rundell & Leander 2010). Members of most of the extant (e.g. Loricifera, Gastrotricha, Diurodrilus, Lobatocerebrum)
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(Swedmark, 1964; Worsaae & Rouse 2009; Kerbl et al. 2015; absent or limited. Sediments constituting the main habitats Laumer et al. 2015). Despite their paraphyletic origin, inter- of interstitial species are limited to very coarse cinder stitial species share miniaturized and seemingly simple body patches from the erosion of the walls of the cave, which plans (Rundell & Leander 2010) adapted to the size restric- accumulate in certain areas, as well as to a 30 m high tions and physical conditions of the interstitial environment mound of loose sand, Montana~ de Arena, at 750 m from the (Boaden 1964; Webb 1969). Viscous regimen dominates entrance of Tunel de la Atl antida (Wilkens et al. 2009). interstitial water motion, limiting the flow to the upper few Accordingly, the endemic cave fauna is dominated by spe- centimetres of the sediment column (Huettel & Webster cialized swimming or drifting suspension feeders; whereas 2001) and resulting in accumulation of organic matter and marine benthic and interstitial species flourish exclusively often steep vertical gradients of oxygen (Jansson 1967; Webb where benthic trophic resources are available (Wilkens & & Theodor 1968; Huettel et al. 1996; Rusch et al. 2000; Parzefall 1974; Mart ınez et al. 2009). In fact, this is a gen- Burdige 2006). Emergence from the sediment by interstitial eral trend in most studied anchialine caves; all dominated by species is prevented by the exposure to predators and risk of cave endemic suspension feeding faunal assemblages such as being carried away from their habitat by current. Accord- the crustacean remipedes, thaumatocyprid ostracods, ther- ingly, interstitial species are photonegative and exhibit vari- mosbaenaceans and atyid shrimps among others (Iliffe et al. ous adhesive properties and other adaptations to ensure their 1983; Palmer 1985; Humphreys & Eberhard 2001; Alvarez permanence in the sediments (J€agersten 1952, 1954; Gray et al. 2015; Mart ınez et al. 2016). These lineages exhibit a 1967; Jouin 1970; Martin 1978a,b; Palmer 1988). high degree of endemism and disjunct distribution in geo- Protodrilidae Hatschek 1888 is a lineage of marine inter- graphically widely separated caves of Bermuda, Bahamas, stitial annelids distributed worldwide (Westheide, 2009). Yucat an, Canary Islands, Christmas Island and Western All protodrilids are externally simple, highly adapted to Australia. This disjunct distribution and the absence of obvi- deposit-feeding while gliding in the interstices of marine ous marine relatives have attracted the interest of zoologists coastal sediments. The 38 described species of Protodrili- and biogeographers, who have proposed two main hypothe- dae are subdivided into six genera (Mart ınez et al. 2015), ses to explain the origin of these anchialine communities: (i) all sharing the presence of cylindrical and ciliated (sensory a Tethyan origin depending on relictualization and vicari- cilia and motile bands) bodies, without parapodia or chae- ance linked to plate tectonics or changes in the sea level, tae, and paired palps with internal canals connected behind and (ii) active colonization and adaptation of deep sea forms the brain (Jouin 1970; Von Nordheim 1989). Protodrilidae, possibly entering the cave systems through crevicular path- together with Saccocirridae and Protodriloididae constitute ways (Hart et al. 1985; Wilkens et al. 1986, 2009; Iliffe et al. the clade Protodrilida (Purschke & Jouin 1988; Purschke 2000; Koenemann et al. 2009). However, the results from 1990; Di Domenico et al. 2014). This clade, previously molecular phylogenetic analyses on some of these iconic recovered nested within Canalipalpata (Annelida) in light anchialine taxa (Ahyong et al. 2011; Botello et al. 2012; of morphological evidence (Purschke 1993; Worsaae & Hoenemann et al. 2013; Mart ınez et al. 2013b, 2014; Gon- Kristensen 2005), has been recently placed as the sister zalez et al. 2015) do not always substantiate these traditional group of Aciculata based on molecular phylogenomic anal- hypotheses, showing that the origin of this fauna is more yses (Andrade et al. 2015; Struck et al. 2015). complex than what any single model can predict and high- Interstitial fauna, including several protodrilid species, is lighting that cave colonization and speciation might have abundant in the coastal sediments of Lanzarote, a 12 million followed more diverse adaptive pathways, related to ecologi- years old volcanic island arising from the oceanic crust, cal fitness. This emphasizes the necessity for morphological 110 km off NW Africa. Geological diversity of the island and evolutionary studies on new cave-adapted species in includes very permeable vesicular basalts and scoria which order to understand the collective origin of anchialine allows the infiltration of marine waters into La Corona lava communities. tube, a 22,000 years old anchialine cave at the northern end Despite the ecological differences between the anchialine of the island (Carracedo & Rodr ıguez-Badiola 1993; Mari- water column and interstitial environments, a population of noni & Pasquar e 1994; Carracedo et al. 2003). The water Megadrilus (Protodrilidae) was discovered within La Corona column of La Corona lava tube is not stratified and is only lava tube (Iliffe et al. 2000; Mart ınez et al. 2009). We here affected by tidal currents (0.5 – 2.5 m/s), carrying suspended describe its unique morphology and behaviour, combining organic matter and small organisms which constitute the light-, electron- and confocal laser scanning microscopy primary trophic source in most of the cave system (Iliffe techniques. The evolution of this species is discussed in et al. 2000). Primary production is limited to Los Jameos relation to phylogenetic analyses including all species of del Agua anchialine lake and the entrance pool of Tunel de the genus. Our tree topologies are used as a frame to dis- la Atl antida. Strong currents, wave action and predators are cuss the character evolution and the origin of this species.
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Materials and methods stubs, sputter-coated with platinum and examined with a Sample collection and behavioural studies JEOL JSM-6335F Field Emission scanning electron micro- Specimens of Megadrilus pelagicus n. sp. were collected by scope at the Natural History Museum, University of two to four divers along first 250 m of Tunel de la Atl an- Copenhagen (SNM, UC). The two specimens (ZMUC- tida (eight dives), Cueva de los Lagos (one dive) and Los POL 2458-2459) used for TEM were embedded in EPON Jameos del Agua lake (2 dives) (see Wilkens et al. 2009). epoxy resin and then transferred into distilled water. Semi- Specimens of Megadrilus pelagicus n. sp. were collected from thin (1 lm) and ultrathin sections (80 nm) were prepared the water column in numbered bottles, together with infor- with a Leica UltracutS ultramicrotome. Semithin sections mation on depth, distance from bottom and penetration were stained with toluidine-blue and examined with an from the entrance. The significance of the distribution was Olympus DP71 camera mounted on an OlympusBX50 tested using a Pearson’s chi-square test for count data, microscope (MBS, UC). Ultrathin sections were investi- calculated with the function chisq.test from the package gated with a Philips CM100 TEM at Faculty of Life MASS in R (Patefield 1981; R-core team, 2008). Sciences (LIFE; UC), after contrasting with uranyl acetate Underwater video recording in the cave was performed and lead citrate. with an Olympus SP-560 UZ digital camera (15 fps) in a PT-016 camera housing. Laboratory observations on live Confocal scanning microscopy (CLSM). Confocal scanning specimens were performed in a 30 9 20 cm aquarium, pro- microscopy (CLSM) was carried out on six specimens viding a maximal focal distance of 50 mm. Animals were (ZMUC-POL 2445-2450), fixed in 2% paraformaldehyde video recorded with a GigE uEye camera equipped with a in PBS (pH 7.4; 6% sucrose). After 24 h of fixation at macro lens. 5 °C, the animals were rinsed and stored in PBS-buffer fi In order to investigate the presence of Megadrilus pelagi- with 0.05% NaN3. The xed specimens were preincubated cus n. sp. in other habitats within the lava tube, samples in PTA buffer (PBS with 0.5% Triton-X, 0.05% NaN3, from lava cinders and sandy sediments were collected at 0.25% bovine serum albumin (BSA) and 5% sucrose) for different sites within the cave, as well as from several 2 h and then incubated overnight in the primary antibody coastal localities around the island, including sediments on mouse monoclonal acetylated-tubulin (SIGMA T 6793). the sea floor immediately above the cave. Animals were The primary antibody was rinsed in PBS and the speci- extracted from sediment samples using the MgCl2 decanta- mens then transferred to the secondary antibody anti- tion technique through a 63-lm mesh (Higgins & Thiel mouse with the fluorophore CY5 (Jackson Immuno- 1988) and sorted out alive under a SZX16 Olympus Research 115-175-062). Thereafter, the specimens were dissecting microscope. incubated for 1 h in 0.34 M Alexa fluor 488 phalloidin (Invitrogen, A12379) in PTA and mounted in Vectashield Morphological studies with DAPI (Vector Laboratories, Burlingame, CA). The Light microscopy (LM). Light microscopy (LM) examina- specimens were investigated with a Leica TCS SP5 confo- tions were performed on two specimens fixed in 2% glu- cal laser scanning microscope at Faculty of Health Sciences taraldehyde and prepared as permanent whole-mounts in (SUND, KU) and an Olympus FluoView FV1000 confocal glycerol (ZMUC-POL 2443-2444). Contrast of glandular laser scanning microscope at the Marine Biological Sec- structures was increased by staining the specimens with tion (MBS, KU). Imaris v7 (Bitplane AG, Zurich,€ Switzer- mucihaematein for 10–15 s (J€agersten 1952; Mart ınez et al. land) was used to analyse the series and produce maximum 2013a). Whole mounted species were measured and pho- projections of Z-stacks into 2D images. tographed with an Olympus DP71 camera mounted on an OlympusBX50 microscope at the Marine Biological Sec- DNA extraction and amplification tion, University of Copenhagen (MBS, UC). DNA extractions were performed using a Qiagen DNeasy Tissue and Blood kit (Qiagen, Dusseldorf,€ Germany), fol- Scanning and transmission electron microscopy (SEM and lowing the manufacturer’s protocol. The molecular markers TEM). Scanning and transmission electron microscopy included approximately 1800 base pairs (bp) of the nuclear (SEM and TEM) studies were performed on specimens small-subunit ribosomal RNA (18S rRNA), 1000 bp of the fi fi fi xed in 2% glutaraldehyde (in ltered MgCl2), post xed in nuclear large-subunit ribosomal RNA (28S rRNA), 450 bp fi osmium (60 min, 1% in ltered MgCl2) and rinsed in dis- of the 16S ribosomal RNA (16S rRNA), 650 bp of the tilled water. The seven specimens (ZMUC-POL 2451- mitochondrial protein-coding gene cytochrome c oxidase 2457) selected for SEM were then dehydrated through subunit I (COI) and 340 bp of the nuclear protein-coding graded ethanol series, transferred to 100% acetone and gene histone 3 (H3). Polymerase chain reactions (PCRs) critical point dried. Dried specimens were mounted on were performed using Illustra PuReTaq Ready-To-Go
ª 2016 Royal Swedish Academy of Sciences 3 Evolution of cave Protodrilidae A. Mart ınez et al.
PCR beads, including 2 lL of template DNA and 1 lLof Bayesian (BA) and maximum likelihood (ML) analyses each primer (10 lM) (see Mart ınez et al. 2015 for primer were conducted on a combined matrix. Individual genes information) and 21 lL for Milli-Q water. PCRs were car- were concatenated using Sequence Matrix (Vaidya et al. ried out using a Bio-Rad S1000 Thermal Cycler. They 2011). Maximum likelihood analyses were computed using involved a 2-min initial denaturalization at 94 °C, followed RaxML version 7.2.8 (Stamatakis 2006) implemented in the by 40 cycles consisting of a denaturation step (94 °C, dur- Cipres Phylogenetic Portal (Miller et al. 2010). A general ing 30 s), annealing (38–55 °C) for 30 s and extension at time reversible (GTR) model of sequence evolution with 72 °C for 1 min and ended with a final extension at 72 °C corrections for a discrete gamma distribution GTR + Γ, for 7 min. PCR products were resolved by 1% agarose gels was specified for each molecular partition and an Mkv stained with GelRedTM (Hayward, CA, USA) and purified model was implemented for the morphological partition with E.Z.N.A. Cycle Pure kit (Omega Bio-tek). Purified (Lewis 2001). Nodal support was estimated via non-para- products were sent to Macrogen Europe Laboratory (Ams- metric bootstrap (Felsenstein 1985), with 1000 replicates terdam, the Netherlands). Sequences were assembled with and a GTR + Γ model. Sequencer 4.10.1 (GeneCodes Corporation, Ann Arbor, Bayesian analyses were performed using MrBayes version MI, USA). Sequenced taxa, locality information and Gen- 3.12 (Ronquist & Huelsenbeck 2003) implemented at the Bank accession numbers are provided in Table S1. Cipres Phylogenetic Portal (Miller et al. 2010). Best-fit evolutionary models were selected using the Akaike infor- Phylogenetic analyses and character evolution mation criterion (AIC) in jModelTest (Posada 2008). The Taxon sampling was based on previous phylogenetic analy- following models were selected for each gene: GTR + Γ ses of Protodrilidae (Mart ınez et al. 2015), which combined model for 16S rRNA and 28S rRNA; K2 + Γ + I for molecular and morphological data analysed with parsimony 18S rRNA and H3; and HKY + Γ for COI. A Mk1 model under direct optimization as well as statistical methods on was implemented for morphological partition (Lewis 2001). static alignments. The dataset included seven species of We ran four independent analyses, each consisting of four Megadrilus (two newly sequenced). One species of Lindrilus chains (three heated and one cold). The number of genera- and two species of each of the protodrilid genera Astomus, tions was set to 120 000 000 and the chain was sampled Claudrilus, Meiodrilus and Protodrilus were selected as out- every 100 000 generations, with 20 000 000 of the samples groups (Di Domenico et al. 2013; Mart ınez et al. 2013a) discarded as burn-in. The remaining trees were used to (Table S1). Morphological characters were selected based construct the 50% majority-rule consensus and estimate on previous phylogenetic analyses (Mart ınez et al. 2015). posterior probabilities and branch lengths (as mean of the Morphological matrix consisted of 36 characters coded for posterior probability density), after assessing the conver- all the included species. The morphological matrix and the gence of the runs with Tracer 1.4.1 (Rambaut & Drum- character states are summarized in Tables S3 and S4. mond 2007). Character description can be found in Mart ınez et al. Character evolution was reconstructed on the maximum (2015). likelihood and Bayesian combined trees using parsimony The sequences for each gene were aligned indepen- methods with Mesquite (Maddison & Maddison 2007) and dently. The ribosomal gene fragments corresponding to MacClade (Maddison & Maddison 2001) respectively. 16S rRNA, 18S rRNA and 28S rRNA were aligned using MAFFT version 6 (Katoh et al. 2002, 2009) with the Results aligning strategy E-ins-I, optimized for sequences with Brief morphological description multiple conserved domains and long gaps. Each align- Megadrilus pelagicus is a long protodrilid, with reddish phar- ment was treated with GBlocks version 0.91b (Castresana ynx and up to 41 segments (summary of the main measures 2000) to cull positions of ambiguous homology. Smaller included in Table S2). Segments 1–5 are whitish and cylin- final blocks, gap position within final blocks and less strict drical (Fig. 1A,H); subsequent segments are translucent, flanking positions were allowed. Sequences for protein- laterally compressed and bearing a dorsal ciliated keel encoding fragments, COI and H3 were confirmed to be unique from this species (dk, Fig. 1E–G, Figs S2A,B and constant in length with MAFFT using default parameters. S3D,G,J). Salivary glands extend from segment 1-6 (sg, Despite the third codon position for the COI gene frag- Fig. S1A,B). The prostomium is round and possesses two ment was found to be saturated in previous phylogenetic palps, very long for the family, provided with bacillary analyses of Protodrilidae (Mart ınez et al. 2015), we did glands as well as ventral and abfrontal band of cilia (pa, not discard it from our analyses as the third codon posi- Figs 1A,B, 2B,C). Eyes are absent, and unpigmented recep- tion might be informative for the closely related species tors are small and rounded (upr, Fig. S1C–D). Nuchal included here. organs are conspicuous, and extend along dorsolateral
4 ª 2016 Royal Swedish Academy of Sciences A. Mart ınez et al. Evolution of cave Protodrilidae transverse furrows between prostomium and peristomium Only one fragmented specimen was found among lava peb- (no, Figs 2A,B and S2C,E). Peristomium with a densely bles from the entrance slope to Tunel de la Atl antida. The ciliated mouth provided with an expanded anterior lip Pearson’s chi-square tests showed no significant differences (cl, Fig. 2C). The pygidium possesses three lobes (pyd, pyl, in the abundance of M. pelagicus along the first 150 m of the Figs 2F and S2D,H). The trunk is provided with dorsal cave (P = 0.8013); but they show significant differences in and lateral longitudinal ciliary bands (Fig. S3J), as well as the vertical distribution of the species (P = 0.0012), with one transverse band ciliary band per trunk segment, all of individuals preferably occurring more than 1 m above the them presumably involved in swimming (dc, lc, Figs 1F–I bottom (Table 1). Megadrilus pelagicus n. sp. was absent from and S3D,E); and midventral ciliary band involved in gliding the coastal localities where three other species of protodrilids (vc, Figs 1E–G and S3A–B). Males with segmented lateral were recorded, including Megadrilus schneideri (Langerhans, organs on segments 6-12 (lo, Figs 2A and S1I). First lateral 1880), Claudrilus cf. hypoleucus (Armenante, 1903) and Meio- organ small and rounded (lo, Figs S1H, S4F–I). Four pairs drilus n. sp. (in Mart ınez et al. 2015). Despite the abundance of spermioducts with gonopores on segments 9-12 (sp, of other cave endemic species as well as marine meiofauna Fig. S4D,I,J). Females with two pairs of oviducts in seg- (Garc ıa-Valdecasas, 1985; Nu nez~ et al. 2009; Worsaae et al. ments 17–18 and 18–19 (ov, Fig. S4C,E). Further detailed 2009; Mart ınez et al. 2016), M. pelagicus was also absent in descriptions are provided in the supplementary material. the samples collected from Montana~ de Arena, the large, cone-shaped pile of loose sand at 750 m penetration into the Motility Tunel de la Atl antida. Most animals were observed drifting through cave waters in a J-position (Video S1b), with the trunk in vertical posi- Phylogenetic analyses and character evolution tion respect to the bottom and bent at segments 5-6 All phylogenetic analyses resulted in congruent tree topolo- (Fig. 1A). Drifting animals continuously moved the palps gies, regardless their inclusion or not of the morphological around the head intercepting suspended particles and car- partition. The ingroup, consisting of nine species of Mega- rying them towards the mouth (pa, Fig. 1B). Neutral buoy- drilus, was consistently recovered monophyletic in both max- ancy was achieved by ciliary beating, which was also imum likelihood (ML) and Bayesian (BA) analyses (Fig. 3). responsible for the gentle movements of the animals in the Within the ingroup, Megadrilus pelagicus n. sp. always water column of the cave (Video S1b). branched off sister to the remaining species of the genus. In the aquarium, M. pelagicus n. sp. was observed gliding This position received higher support values in the molecu- propelled by the antiplectic metachronal beating of cilia in lar analyses (maximum likelihood bootstrap, MLB = 98; midventral ciliary band (Video S4). Gliding animals change Bayesian Posterior Probabilities, BPP=0.97), than in the direction by asymmetric contraction of the anterior mus- combined analyses including morphology (MLB = 74, cles. Gliding animals also waved the palps around head and BPP = 0.77). The remaining species of Megadrilus split off towards the mouth (Video S4). Undulatory swimming as two clades. One of these clades included all the Atlantic (Videos S1a, S2, S3) was recorded during few seconds (ca. and Mediterranean species of the genus, with M. purpureus 10–20 s), both in animals drifting at the cave or gliding in (Schneider, 1868) from Sweden, sister taxon to M. schneideri the aquarium, sometimes as a response to disturbance (Canary Islands)-M. cf. schneideri (Sardinia, Italy). The sec- (Video S2–S3). During undulatory swimming, the body ond clade included Western Atlantic and Indo-Pacific spe- accommodates 1.5-2 retrograde lateral undulations pro- cies, with Megadrilus n. sp. (Lord Howe, Australia), sister to duced by longitudinal muscles (Fig. 1D). The movement is Megadrilus n. sp. (Brazil)-Megadrilus n. sp. (West Panama) possibly enhanced by the dorsal keel, lateral compression and M. hochbergi (East Panama)-M. cf. hochbergi (Belize). All of the trunk and reduction of the longitudinal muscles in nodal support values for both, molecular and combined anal- the pygidium (Fig. 1E–G). Changes in direction are possi- yses, are summarized on Fig. 3. ble, sometimes as a response to obstacles (Video S3). As a The genus Megadrilus was supported by the presence of response to disturbance, some animals started spasmodic the following synapomorphies: presence of transverse muscular contractions producing random movements that nuchal organs (character, ch. 9, unique apomorphy; led to fragmentation of the body at the level of septa. Fig. 2A), salivary glands extending to segment 7 (ch. 15, with one internal homoplasy; Fig. S1A), pygidium with Ecology three lobes (ch. 16, unique apomorphy; Fig. 2F), first lat- Megadrilus pelagicus n. sp. was very abundant during the ele- eral organ starting on segment 6 (ch. 19, one external ven dives conducted in January 2010 (Table 1). Thirty-six homoplasy; Fig. 2A), palp abfrontal ciliation consisting in specimens were collected from the water column of Tunel motile tufts (ch. 28, one external homoplasy; Fig. 2D). All de la Atl antida and two at Cueva de los Lagos (Video S1b). these features were shared by M. pelagicus, supporting its
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Fig. 1 Megadrilus pelagicus n. sp., live specimen and schematic drawings. —A. Live specimen, dorsal view (photograph by Ulrike Strecker). —B. Head of live specimens, showing the motility of the palps. —C. Pygidium on a live specimen. —D. Snapshots from a video recording showing the undulatory swimming behaviour. —E. Schematic drawing from a semithin section at the segment 3. —F. Schematic drawing from a semithin section at the segment 20. —G. Schematic drawing from a semithin at the segment 30. —H. Drawing showing the j- behaviour of a live species, as typically observed in the water column of the cave. —I. Fragmented specimen showing the body cavity of a posterior segment. Abbreviations: bg, bacillary glands; dc, dorsal ciliary band; dk, dorsal keel; dlm, dorsolateral muscles; ep, epidermis; gn, gonopores; hg, hindgut; lc(1–3), lateral ciliary band; lo, lateral organs; mg, midgut; obm, oblique muscles; pa, palp; pe, peristomium; pr, prostomium; py, pygidium; vc, midventral ciliary band; vlm, ventrolateral muscles.
placement within the genus. Moreover, the species was fur- Protodrilidae and the entire Protodrilida (Mart ınez et al. ther characterized by the following autapomorphic features: 2015). Among the sand grains, short sensory palps facili- presence of ciliated dorsal keel (Fig. 1I,2A), presence of tate the exploration of the tight interstices, whereas the longitudinal ciliary band on the surface of the body (Figs animals glide using midventral ciliary band in this habitat S2B and S3J) and presence of transverse bands of com- governed by viscous forces (Bear 1988; Huettel & Web- pound cilia in all trunk segments (Fig. S3D,E). Of all pro- ster 2001). Conversely, the high porosity of the volcanic todrilids investigated to date, M. pelagicus is the only materials, the absence of photosynthetic primary produc- species presenting a reduction of trunk musculature tion and the scarce sediment in La Corona lava tube towards the pygidium (Figs 1E–G and S5H). favour inertial water flow along the cave and prevent the deposition of organic particles (Bear 1988; Wilkens et al. Discussion 2009; Mart ınez et al. 2014). In absence of deposited food, Morphological features of Megadrilus pelagicus the survival of M. pelagicus relies on the collection of the Megadrilus pelagicus exhibits a highly derived morphology suspended organic particles carried by tidal currents, being compared to any marine protodrilid (Mart ınez et al. 2015). the main trophic resource of the cave (Wilkens et al. The dorsal ciliated keel, formed as an extension of the dorsal 2009). The partial isolation of the cave system, without epidermis of the body, is the most conspicuous autoapomor- major predators or turbulence, reduces the otherwise phy of M. pelagicus, while similar structures have never been harsh selection pressure on this behaviour in open waters reported in annelids. The joint presence of transverse bands (Parzefall 1986). of compound cilia, longitudinal bands of ciliary tufts and long ciliated palps with motile ciliation is also unique in Unique traits correlated to colonization of anchialine caves M. pelagicus, although these features can be interpreted alone The morphological change in M. pelagicus compared to its as homoplastic, since ciliary bands or ciliated palps are found marine relatives reflects the different functional demands of in other Protodrilidae (Mart ınez et al. 2015). However, these its pelagic life style and correlates in our phylogenetic anal- features in M. pelagicus differ in function and arrangement yses with the colonization of the cave crevicular habitat. when compared to other Protodrilidae. Transverse bands in Coordinated beating of the palp ciliation seems to effi- M. pelagicus consist of groups of three ciliary tufts and are ciently gather suspended food particles, which are inter- present on all trunk segments. These bands differ from the cepted by mucous secretions from the bacillary glands and transverse bands present in other species, either consisting transported to the mouth by movements of the long palps. on ciliary tufts beating metachronally to produce water cur- The keel and the trunk ciliation stabilize the animal in the rents (e.g. Protodrilus ciliatus J€agersten 1952) or on joint sen- water column, providing neutral buoyancy at low energetic sory cilia (e.g. Megadrilus schneideri, Protodrilus smithsoni; cost (Iliffe & Kornicker 2009), while mucous secretions Mart ınez et al. 2013a, 2015). Similarly, longitudinal bands from the trunk bacillary gland tether the animals in the are present in Astomus, but they are situated in furrows and water column, avoiding translation while feeding (Fenchel used to produce water currents (Jouin 1979, 1992). Palps in & Ockelmann 2002). M. pelagicus are much longer than in any other protodrilid and are provided with dense and complex ciliary patterns as Ecological conditions in anchialine caves favour suspension well as abundant bacillary glands. feeding strategies The higher fitness of suspension feeding strategies in the Suspension feeding behaviour of Megadrilus pelagicus anchialine cave environments is supported by the domi- Megadrilus pelagicus is the only pelagic suspension feeding nance of pelagic suspension feeders in anchialine cave protodrilid, in contrast to the ancestral interstitial, assemblages worldwide (Iliffe 1992; Sket 1996; Mart ınez deposit-feeding foraging strategy of the family et al. 2009). Most of these cave endemic species are
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Fig. 2 Megadrilus pelagicus n. sp., scanning electron micrographs. —A. Anterior end of body, lateral view. —B. Dorsal view of prostomium and peristomium, showing palps. —C. Ventral view of the prostomium and peristomium. —D. Abfrontal surface of the palp. —E. Ventral surface of the palp, coloured to indicate the different ciliary bands (explanation in the text). —F. Pygidium, dorsal view. Abbreviations, bg, bacillary gland; bgs, bacillary gland secretion; cl, ciliary lip; dc, dorsal ciliary band; dk, dorsal keel; lc, lateral ciliary band; lo1-7, lateral organs 1-7; mcr, multiciliary receptor; mo, mouth opening; mcf, mouth ciliary field; no, nuchal organ; pa, palp; pe, peristomium; pf, palp frontal ciliation; pr, prostomium; pv1-3, palp ventral ciliation; pyd, pygidial dorsal lobe; pyl, pygidial lateral lobe; tc, transverse ciliary band; vc, ventral ciliary band.
Table 1 Vertical and horizontal distribution of Megadrilus pelagicus carbon resources in the water column in most studied n. sp. at La Corona lava tube according to studied specimens. All anchialine ecosystems (Mart ınez et al. 2016), point at sus- “ ” distances in metres. 0m, referring to specimens collected among pension feeding as the most feasible foraging strategy in lava debris. Abbreviations: vert., vertical; hor., horizontal these habitats. Most of these systems are sustained by
Distance from entrance exogenous organic matter carried by tidal currents or by internal chemoautotrophic processes in the water column 0–30 30–60 60–90 90–120 120–150 Total (vert.) (Pohlman et al. 2000; Wilkens et al. 2009; Gonzalez et al. Distance bottom 2011). Most of these suspension feeding strategies evolve 0m 1 0 0 0 0 1 together with secondary pelagicism in the water column of 1m 2 2 1 1 0 6 the caves. This swimming behaviour is further favoured by 1–2m 1 6 5 1 1 14 the low density of predators and the low turbulence in + 2m 2 2 2 6 4 16 Total (hor.) 6 10 8 8 5 37 these habitats. In cave species belonging to ancestrally deposit-feeding lineages, such as M. pelagicus or Longipalpa, the adaptive shift between interstitial and crevicular habi- tats favours morphological divergence and speciation, as a crustaceans, preadapted to suspension feeding (Iliffe & consequence of the different functional demands of a pela- Bishop 2007). However, secondary adaptations to caves gic suspension feeding life style. have been recorded in other primarily deposit-feeding annelids, involving increase in palp length and evolution of The “interstitial shift” and the origin of certain anchialine ciliary structures for swimming and gathering suspended lineages particles (Mart ınez et al. 2013b, 2014). The most remark- An ancient Tethyan origin, as opposed to a more recent, able case is represented by the genus Longipalpa, belonging deep sea origin, are the two main hypotheses that have to the otherwise interstitial meiobenthic lineage Nerillidae been proposed to explain the origin and diversification of (Worsaae et al. 2004). In contrast to other species of the the anchialine lineages (Iliffe & Kornicker 2009). A Teth- family, which inhabit marine and cave benthic habitats yan origin has been suggested for those anchialine groups, (Worsaae et al. 2009), Longipalpa species colonized the such as remipedes or thermosbaenaceans, which are exclu- water column of anchialine caves in the Bahamas, Ber- sively found in anchialine habitats, exhibit disjunct distri- muda, Cuba, Yucat an and Canary Islands (Worsaae et al. bution patterns and lack close extant marine relatives 2004; Mart ınez et al. 2016). They swim by the coordinated (Humphreys 2000). As an alternative, a deep sea origin beating of paired pygidial lobes and transverse ciliary bands has been hypothesized mostly for anchialine endemic spe- on the trunk while collecting suspended particles or bacte- cies that otherwise belong to entirely deep sea lineages ria with unusually long and distinctly ciliated palps. (Hart et al. 1985), such as the squat lobster Munidopsis Derived pelagic behaviour has been reported in other cave polymorpha, or cave polynoid and scalibregmatid annelids species in the families Polynoidae, Scalibregmatidae, (Iliffe et al. 1984; Mart ınez et al. 2013b; Gonzalez et al. Hesionidae and Spionidae (Mart ınez et al. 2013b; Mart ınez 2015). Megadrilus pelagicus is exclusive to caves, but has a et al. 2016; Gonzalez et al. 2015). clear marine shallow water ancestry within an exclusively In summary, (i) the convergent origin of suspension interstitial lineage (Mart ınez et al. 2015). The physical feeding in the water column among independent evolution- continuity between interstitial and crevicular environments ary lineages of cave endemic annelids descending from might favour the colonization of anchialine caves by inter- benthic deposit feeders (Worsaae et al. 2004; Mart ınez stitial species. However, the lack of suitable sandy habitats et al. 2013b; Gonzalez et al. 2015), (ii) the dominance of and deposited organic material in most anchialine systems suspension feeders in cave communities worldwide (Iliffe & prevents their survival. This paradox (high probability of Kornicker 2009) and (iii) the similar main distribution of colonization vs. low probability of survival), might favour
ª 2016 Royal Swedish Academy of Sciences 9 Evolution of cave Protodrilidae A. Mart ınez et al.
Lindrilus rubropharyngeus * Protodrilus pythonius * Protodrilus ciliatus Meiodrilus gracilis * Meiodrilus adhaerens
88/* Claudrilus draco * * Claudrilus hypoleucus Astomus n. sp. * Astomus taenioides Megadrilus
Megadrilus purpureus (Sweden) interstitial species Marine 47/.96 37/.96 Megadrilus schneideri (Canary Islands)
98/.97 * Megadrilus cf. schneideri (N.E. Sardinia, Italy) 74/.77 Megadrilus n. sp. (Lord Howe, Australia)
94/* Megadrilus n. sp. (W. Panama) 88/* * */* 96/* * Megadrilus n. sp. (Brazil) 88/1 * Megadrilus hochbergi (E. Panama) * Megadrilus cf. hochbergi (Belize)
0.05 Anchialine cave species
Megadrilus pelagicus n. sp. Long. bands * Sparse musculature** Transverse bands* ciliary Long palps*** Ciliated dorsal keel
Fig. 3 Phylogenetic position of Megadrilus pelagicus n. sp. within Protodrilidae based on maximum likelihood and Bayesian analyses. Molecular and combined analyses resulted in congruent topologies. Topology from Bayesian combined analyses is shown, including 16S, 18S, 28S, H3, COI gene fragments and 34 morphological characters. Numbers above the nodes correspond to maximum likelihood bootstrap (MLB) values and Bayesian posterior probabilities (BPP) from molecular analyses; numbers below the nodes correspond to MLB values and BPP from combined analyses including morphology. Stars indicate full support (MLB=100, BPP=1). The main unique features of M. pelagicus are schematically represented on the tree and explained in the text. Images correspond to Megadrilus schneideri, from interstitial sediments in Lanzarote; and Megadrilus pelagicus n. sp. from the water column of caves.
the evolution of secondary adaptations in cave species with annelid and crustacean cave lineages point at more diverse interstitial ancestry, as seen in M. pelagicus n. sp., but also pathways, related to ecological fitness, involved in their in the nerillid genus Longipalpa, and within another size origin. These pathways differ from those proposed by range, the scalibregmatid annelids Speleobregma lanzaro- previous theories which instead emphasize the role of his- teum and Axiokebuita cavernicola (Worsaae et al. 2004; torical and stochastic processes in the origin of anchialine Mart ınez et al. 2013b). The strange morphology of remi- cave fauna (Humphreys 2000). pedes, sister to the interstitial cephalocarids, could also be linked to an old ecological shift in these lineages (Regier Acknowledgements et al. 2010). The dramatic changes observed in these pri- This work would not be possible without the support of ~ ı marily interstitial lineages when compared with other our diving team: L. E. Canadas, E. Dom nguez, C. Jorge,
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J. Valenciano R. Schoenemark and, T. Mart ın, as well as Di Domenico, M., Mart ınez, A., Lana, P. & Worsaae, K. (2013). the Uesteyaide Caving Group, specially A. P. Perdomo and Protodrilus (Protodrilidae, Annelida) from the Southern and J. P. Trujillo. Special thanks are extended to C. Dizy and Southeastern Brazilian coasts. Helgoland Marine Research, 67, 733–748. his family from Las Pardelas Park (Orzola), who kindly ı ı Di Domenico, M., Mart nez, A., Lana, P. & Worsaae, K. (2014). hosted us in Lanzarote. Elena Mateo and Agust n Aguilar Molecular and morphological phylogeny of Saccocirridae provided invaluable assistance in obtaining the collecting (Annelida) reveals two cosmopolitan clades with specific habitat permits from Cabildo de Lanzarote and Gobierno de preferences. Molecular Phylogenetics and Evolution, 75, 202–218. Canarias. Thanks to S. Fontes and the staff at Los Jameos Felsenstein, J. (1985). Confidence limits on phylogenies: an del Agua for their help. Additionally, we thank Prof. H. approach using the bootstrap. Evolution, 78,3–791. Wilkens and U. Strecker for their support in the field and Fenchel, T. & Ockelmann, K. W. (2002). Larva on a string. Ophe- lia, 56, 171–178. the photographs of living specimens; TEM work was made Garc ıa-Valdecasas, A. (1985). Estudio faun ıstico de la cueva sub- possible thanks to P. Hyttel and H. M. Hølm from LIFE marina “Tunel de la Atl antida”, Jameos del Agua, Lanzarote. (KU). Research grants to K.W. from the Danish Indepen- Naturalia Hisp anica, 27,1–56. dent Research Council (grant # 272-06-0260); Freja Pro- Giere, O. (2009). Meiobenthology. The Microscopic Motile Fauna gram, University of Copenhagen and Carlsberg of Aquatic Sediments. Berlin: Springer Foundation (grant # 2010_01_0802), founded the labora- Gonzalez, B.C., Iliffe, T.M., Macalady, J., Schaperdoth, I. & tory work and salaries. Field work was further supported Kakuk, B. (2011). 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Megadrilus pelagicus n. sp., j.-behavior and Von Nordheim, H. (1989). Six new species of Protodrilus (Annel- swimming after disturbance in the water column of the ida, Polychaeta) from Europe and New Zeland, with a concise Corona lava tube. presentation of the genus. Zoologica Scripta, 18, 245–268. Video S3. Megadrilus pelagicus n. sp., muscular swimming Webb, J. E. (1969). Biologically significant properties of submerged marine sands. Proceedings of the Royal Society B, 174, 355–402. in an aquarium. Webb, J. E. & Theodor, J. (1968). Irrigation of submerged marine Video S4. Megadrilus pelagicus n. sp., gliding and palp sands through wave action. Nature, 220, 682–683. movements in an aquarium. Wilkens, H. & Parzefall, J. (1974). Die Oekologie der Jameos del Video S5. Megadrilus pelagicus n. sp., gliding and palp Agua (Lanzarote) zur Entwicklung limnischer Hoehlentiere aus movements among the lapilli crevices in an aquarium. – marinen Vorfahren. Annals of Speleology, 29, 419 434. Video S6. Megadrilus pelagicus n. sp., escape reaction and Wilkens, H., Parzefall, J. & Iliffe, T. M. (1986). Origin and age of autotomy, in an aquarium. the marine stygofauna of Lanzarote, Canary Islands. Mitteilungen Hamburgisches Zoologisches Museum und Institut, 83, 223–230. Table S1. Specimen and collection data used in the phy- Wilkens, H., Iliffe, T. M., Orom ı, P., Mart ınez, A., Tysall, T. N. logenetic analyses, with GenBank accession numbers. & Koenemann, S. (2009). The Corona lava tube, Lanzarote: Table S2. Morphological matrix. Geology, habitat diversity and biogeography. Marine Biodiversity, Table S3. Summary of the character coding. – 39, 155 167. Table S4. Meristic and morphometric characters of Worsaae, K. & Kristensen, R. M. (2005). Evolution of interstitial holotype and type material of M. pelagicus n. sp. Polychaeta. Hydrobiologia, 535/536, 319–340.
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