Rubyspira, new genus and two new species of bone-eating deep-sea snails with ancient habits.
Gastropods of the superfamily Abyssochrysoidea (as currently constructed, Abyssochrysidae + Provannidae) commonly occur in deep-sea environments including hydrother-mal vents, hydrocarbon seeps, and sites of organic deposition (e.g., wood- and whale-falls). These snails represent a relatively ancient lineage that has persisted for at least 131 to 145 million years (MY) (Kaim et al., 2008a). The recent discovery of fossil provannid-like shells associated with pleisosaurid carcasses from the late Cretaceous suggests that these large marine reptiles were capable of supporting a diverse community of decomposers much like those found today on whale-falls (Kaim et al., 2008b). The source of nutrition for these provannid-like snails is unknown, although evidence for microbial sulfide production suggests that chemosynthetic communities might have been supported. Two provannid species found only at hydrother-mal vents, Alviniconcha spp. and Ifremeria nautilei, rely predominantly on endosymbiotic sulfur-oxidizing bacteria for their nutrition. Other vent, seep, and wood- and whale-fall-associated species graze on bacterial biofilms. Although the family Provannidae sensu stricto is only known to occur in marine reducing environments, at least one genus, Provanna, has various species that are capable of exploiting vents, seeps, and food-falls.
Here, we report the discovery of a new genus of provannid-like snails, Rubyspira (Caenogastropoda: Abyssochrysoidea), with two species found living on a gray whale carcass at 2893-m depth in the Monterey Submarine Canyon, California. Rubyspira osteovora sp. nov. is the larger and more abundant of the two species. It was first observed during January 2004 on sediment surrounding the carcass of a whale that had died about 2 years previously (Braby et al., 2007). By May 2004 the snail was one of the most abundant inhabitants of the carcass, with numerous empty shells littering the sediment, and it still flourished in March 2009 after most of the large whalebones had been consumed (Fig. 1A). Rubyspira goffrediae sp. nov. is smaller and exceedingly rare in comparison, and was primarily found on the surface of bones (Fig. 1A, arrows). Despite many years of sampling, only a few individuals of R. goffrediae have been collected. Visual examinations of gut contents revealed a high abundance of bone fragments and stones, with little other organic material. Stable isotope analyses on tissue and gut contents showed that the snail's main source of nutrition was whalebone. Visual inspections of the gut contents of other provannid-like snails that inhabit whale-falls produced no evidence of a whale-bone diet.
Morphologically, neither of the Rubyspira species resembles recent abyssochrysoid snails. Instead, R. osteovora shells closely resemble the fossils of Atresias liratus Gabb, 1869, that occurred at Cretaceous hydrocarbon seeps (Kiel et al., 2008). To assess evolutionary relationships of the new species, we conducted a molecular phylogenetic analysis involving other living representatives of provannids from hydrothermal vents, seeps, and wood- and whale-falls (Fig. 1). We also included abyssochrysid gastropods, long thought to be close relatives to the Provannidae, and used a littorinid and two buccinids as outgroup taxa. Phylogenetic analyses were based on three nuclear genes encoding his-tone-3 and 18S and 28S rRNAs, and three mitochondrial genes encoding cytochrome-c-oxidase subunit 1 and 16S and 12S rRNA. To calibrate a molecular clock for the abyssochrysoids, we relied on a vicariant event (closure of the Isthmus of Panama) and fossil calibrations from the Provannidae and Buccinidae.
Materials and Methods
Rubyspira specimens were sampled from two depths in Monterey Bay, California (Table 1), with the robotic sub-mersibles ROV Tiburon and ROV Doc Ricketts. Samples were obtained with a suction sampler, which deposits samples in discrete canisters with varying mesh sizes, and as by-catch on bones. They were stored in an insulated "biobox," which maintains bottom temperatures and isolates specimens from turbulence during recovery. Upon recovery, specimens were preserved in 95%-99% ethanol or frozen at -80 [degrees]C for molecular work. For morphology, specimens were dissected from their shells with electronic-style wire cutters or by decalcification in 5% HC1 in 70% ethanol. For scanning electron microscopy (SEM), parts of bodies were critical-point-dried with 99.5% ethanol. Radulae were prepared by dissolving buccal masses in 20% KOH at 20-30 [degrees]C, rinsing in distilled water, and mounting on a bed of PVA glue. Provenance of comparative materials is given in Table 1.
Table 1 Samples, geographical coordinates, Swedish Museum of Natural History (SMNH) voucher information, and GenBank accession numbers Genus and Locality Lat. Long. species Rubyspira Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 osteovora n. sp. CA Rubyspira Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 goffrediae n. CA sp. Provanna n. sp. Monterey Bay. 36[degrees]36.80 -122[degrees]26.13 1 CA Provanna n. sp. Monterey Bay. 36[degrees]42.73 -122[degrees]6.55 2 CA Provanna macleani Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 Waren & Bouchet, CA 1989 Provanna ios EPR, 12[degrees]48.15 -103[degrees]56.31 Waren & Bouchet, 13[degrees]N 1986 EPR, 20[degrees]49.81 -109[degrees]08.96 21[degrees]N Provanna aff. Costa Rica 08[degrees]59.58 -084[degrees]43.70 ios Provanna aff. Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 pacifica (Dall. CA 1908) Provanna laevis Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 Waren & Ponder, CA 1991 EPR, 27[degrees]34.58 -111[degrees]26.90 27[degrees]N Provanna Kilo Moana, -20[degrees]03.18 -176[degrees]08.02 buccinoides Waren Lau Basin & Bouchet, 1993 Provanna aff. N. Gorda 41[degrees]0.85 -127[degrees]31.65 variabilis Waren Ridge & Bouchet, 1986 Provanna sculpta Upper 28[degrees]03.70 -089[degrees]42.60 Waren & Ponder, Louisiana 1991 Slope Debruyeresia TowCam, Lau -20[degrees]18.99 -176[degrees]08.21 melanoides Waren Basin & Bouchet, 1993 Debruyeresia n. Mantis Basin -03[degrees]47.19 152[degrees]05.39 sp. Alviniconcha aff. Manus Basin -03[degrees]47.19 152[degrees]05.39 hessleri Okutani et at.. 1988 Ifremeria Manus Basin -03[degrees]47.19 152[degrees]05.39 nautilei Bouchet & Waren, 1991 Abyssochrysos Amami Island, 28[degrees]32.72 127[degrees]02.09 sp.[paragraph] Japan Abyssochrysos N. New -20[degrees]17.7 163[degrees]49.3/4 melvilli Caledonia (Schepman, 1909)[paragraph] Neptunea amianta Monterey Bay, 36[degrees]36.80 -122[degrees]26.13 (Dall, CA 1890)[paragraph] 36[degrees]46.29 -122[degrees]4.96 Neptunea antiqua Denmark 56[degrees]01.10 012[degrees]37.10 (L., Oresund 1758)[paragraph] Littorina littorea (L., 1758)[paragraph] Genus and species Locality Depth (m) Dive #* Rubyspira osteovora n. Monterey Bay, 2893 T617, T671, T769, sp. CA T932, T1162 Rubyspira goffrediae Monterey Bay, 2893 T932, TU62 n. sp. CA Provanna n. sp. 1 Monterey Bay. 2893 T932 CA Provanna n. sp. 2 Monterey Bay. 1820 T990 CA Provanna macleani Monterey Bay, 2893 DR010 Waren & Bouchet, 1989 CA Provanna ios Waren & EPR, 2621 V156 Bouchet, 1986 13[degrees]N EPR, 2553 T556 21[degrees]N Provanna aff. ios Costa Rica 1917 ROV 78 Provanna aff. pacifica Monterey Bay, 2893 T991, T1162 (Dall. 1908) CA Provanna laevis Waren Monterey Bay, 2893 T671 & Ponder, 1991 CA EPR, 1755 T548 27[degrees]N Provanna buccinoides Kilo Moana, Lau 2618 J2 140 Waren & Bouchet, 1993 Basin Provanna aff. N. Gorda Ridge 3277 T888 variabilis Waren & Bouchet, 1986 Provanna sculpta Waren Upper Louisiana 620 JSL 4569 & Ponder, 1991 Slope Debruyeresia TowCam, Lau 2699 J2 142 melanoides Waren & Basin Bouchet, 1993 Debruyeresia n. sp. Mantis Basin 1516 N7 Alviniconcha aff. Manus Basin 1516 N7 hessleri Okutani et at.. 1988 Ifremeria nautilei Manus Basin 1516 Bouchet & Waren, 1991 Abyssochrysos Amami Island, 616 sp.[paragraph] Japan Abyssochrysos melvilli N. New 572-1200 CP3028,CP3030 (Schepman, Caledonia 1909)[paragraph] Neptunea amianta Monterey Bay, 2893 T777, T916/V2858 (Dall, CA 1890)[paragraph] 1018 Neptunea antiqua (L., Denmark 82 1758)[paragraph] Oresund Littorina littorea (L., 1758)[paragraph] Genus and species Locality Hab. Vouchers (SMNH) Rubyspira osteovora n. Monterey Bay, CA B 103978, 84724, 96389 sp. Rubyspira goffrediae n. Monterey Bay, CA B 84726, Not SMNH sp. Provanna n. sp. 1 Monterey Bay. CA B 84725 Provanna n. sp. 2 Monterey Bay. CA B 84729 Provanna macleani Waren Monterey Bay, CA W 84723 & Bouchet, 1989 Provanna ios Waren & EPR, 13[degrees]N V 81974 Bouchet, 1986 EPR, 21[degrees]N V 84691 Provanna aff. ios Costa Rica S 82451 Provanna aff. pacifica Monterey Bay, CA W 84734, 103570 (Dall. 1908) Provanna laevis Waren & Monterey Bay, CA W 84712 Ponder, 1991 EPR, 27[degrees]N V 84701 Provanna buccinoides Kilo Moana, Lau V 78511 Waren & Bouchet, 1993 Basin Provanna aff. N. Gorda Ridge V 105346 variabilis Waren & Bouchet, 1986 Provanna sculpta Waren Upper Louisiana S MCZ DNA 102470 & Ponder, 1991 Slope Debruyeresia melanoides TowCam, Lau Basin V 78553 Waren & Bouchet, 1993 Debruyeresia n. sp. Mantis Basin V 84859 Alviniconcha aff. Manus Basin V hessleri Okutani et at.. 1988 Ifremeria nautilei Manus Basin V Bouchet & Waren, 1991 Abyssochrysos Amami Island, S 105372 sp.[paragraph] Japan Abyssochrysos melvilli N. New Caledonia S IM-2009-5180/81 (Schepman, 1909)[paragraph] Neptunea amianta (Dall, Monterey Bay, CA B 84719 1890)[paragraph] Neptunea antiqua (L., Denmark Oresund M 51826 1758)[paragraph] Littorina littorea (L., 1758)[paragraph] GenBenk Accession Number [double dagger] Genus and species Locality COI 16S 18S (GTR + SS) (HKY + G) (SYM + I + G) Rubyspira osteovora Monterey Bay, GQ290568-73 GQ290477 GQ290530 n. sp. CA Rubyspira Monterey Bay, GQ290574-76 GQ290478 GQ290531 goffrediae n. sp. CA Provanna n. sp. 1 Monterey Bay. GQ290577 GQ290479 GQ290532 CA Provanna n. sp. 2 Monterey Bay. GQ290578 GQ290480 GQ290533 CA Provanna macleani Monterey Bay, GQ290579-83 GQ290481 GQ290534 Waren & Bouchet, CA 1989 Provanna ios Waren EPR, GQ290584 GQ290482 GQ290535 & Bouchet, 1986 13[degrees]N EPR, GQ290585 21[degrees]N Provanna aff. ios Costa Rica GQ290586-87 GQ290483 GQ290536 Provanna aff. Monterey Bay, GQ290588-93 GQ290484 GQ290537 pacifica (Dall. CA 1908) Provanna laevis Monterey Bay, GQ290485-6 GQ290538 Waren & Ponder, CA 1991 EPR, GQ290594 27[degrees]N Provanna Kilo Moana, GQ290487 GQ290539 buccinoides Waren & Lau Basin Bouchet, 1993 Provanna aff. N. Gorda GQ290488 GQ290540 variabilis Waren & Ridge Bouchet, 1986 Provanna sculpta Upper GQ290595 GQ290489 GQ290541 Waren & Ponder, Louisiana 1991 Slope Debruyeresia TowCam, Lau GQ290596 GQ290490 GQ290542 melanoides Waren & Basin Bouchet, 1993 Debruyeresia n. Mantis Basin GQ290543 sp. Alviniconcha aff. Manus Basin GQ290597-602 GQ290491 GQ290544 hessleri Okutani et at.. 1988 Ifremeria nautilei Manus Basin GQ290603-608 GQ290492 GQ290545 Bouchet & Waren, 1991 Abyssochrysos Amami Island, GQ209609-10 GQ290493 GQ290546 sp.[paragraph] Japan Abyssochrysos N. New GQ209611-12 GQ290494 GQ290547 melvilli (Schepman, Caledonia 1909)[paragraph] Neptunea amianta Monterey Bay, GQ209613-15 GQ290495 GQ290548 (Dall, CA 1890)[paragraph] Neptunea antiqua Denmark GQ209616 GQ290496 GQ290549 (L., Oresund 1758)[paragraph] Uittorina littorea EU876332 DQ093481 DQ093437 (L., 1758)[paragraph] GenBenk Accession Number [double dagger] Genus and species Locality 12S 2SSD1 (GTR + G) (F81 + 1) Rubyspira osteovora n. Monterey Bay, CA GQ290497 GQ290515 sp. Rubyspira goffrediae n. Monterey Bay, CA GQ290498 GQ290516 sp. Provanna n. sp. 1 Monterey Bay. CA GQ290517 Provanna n. sp. 2 Monterey Bay. CA GQ290499 Provanna macleani Waren & Monterey Bay, CA GQ290500 GQ290518 Bouchet, 1989 Provanna ios Waren & EPR, 13[degrees]N GQ290501 GQ290519 Bouchet, 1986 EPR, 21[degrees]N Provanna aff. ios Costa Rica GQ290502 GQ290520 Provanna aff. pacifica Monterey Bay, CA GQ290503 GQ290523 (Dall. 1908) Provanna laevis Waren & Monterey Bay, CA GQ290504 GQ290521 Ponder, 1991 EPR, 27[degrees]N Provanna buccinoides Waren Kilo Moana, Lau Basin GQ290522 & Bouchet, 1993 Provanna aff. variabilis N. Gorda Ridge GQ290505 GU808565 Waren & Bouchet, 1986 Provanna sculpta Waren & Upper Louisiana Slope GQ290506 GU808566 Ponder, 1991 Debruyeresia melanoides TowCam, Lau Basin GQ290507 GQ290524 Waren & Bouchet, 1993 Debruyeresia n. sp. Mantis Basin GQ290508 GQ290525 Alviniconcha aff. hessleri Manus Basin GQ290509 GQ290526 Okutani et at.. 1988 Ifremeria nautilei Bouchet Manus Basin GQ290510 GQ290527 & Waren, 1991 Abyssochrysos Amami Island, Japan GQ290511 GU808567 sp.[paragraph] Abyssochrysos melvilli N. New Caledonia GQ290512 GU808568 (Schepman, 1909)[paragraph] Neptunea amianta (Dall, Monterey Bay, CA GQ290513 GQ290528 1890)[paragraph] Neptunea antiqua (L., Denmark Oresund GQ290514 GQ290529 1758)[paragraph] Uittorina littorea (L., AJ488754 DQ279985 1758)[paragraph] GenBenk Accession Number [double dagger] Genus and species Locality 28SD6 H3 (GTR + I (GTR + I + G) + G) Rubyspira osteovora n. Monterey Bay, CA GQ290550 GQ290617 sp. Rubyspira goffrediae n. Monterey Bay, CA GQ290551 GQ290618 sp. Provanna n. sp. 1 Monterey Bay. CA GQ290552 GQ290619 Provanna n. sp. 2 Monterey Bay. CA GQ290553 GQ290620 Provanna macleani Waren & Monterey Bay, CA GQ290554 GQ290621 Bouchet, 1989 Provanna ios Waren & EPR, 13[degrees]N GQ290555 Bouchet, 1986 EPR, 21[degrees]N Provanna aff. ios Costa Rica GQ290556 GQ290622 Provanna aff. pacifica Monterey Bay, CA GQ290557 GQ290623 (Dall. 1908) Provanna laevis Waren & Monterey Bay, CA GQ290558 GQ290624 Ponder, 1991 EPR, 27[degrees]N Provanna buccinoides Waren Kilo Moana, Lau Basin GQ290559 GQ290625 & Bouchet, 1993 Provanna aff. variabilis N. Gorda Ridge GQ290560 GQ290626 Waren & Bouchet, 1986 Provanna sculpta Waren & Upper Louisiana Slope GQ29056I GQ290627 Ponder, 1991 Debruyeresia melanoides TowCam, Lau Basin GQ290562 GQ290628 Waren & Bouchet, 1993 Debruyeresia n. sp. Mantis Basin GQ290563 GQ290629 Alviniconcha aff. hessleri Manus Basin GQ290630 Okutani et at.. 1988 Ifremeria nautilei Bouchet Manus Basin GQ290564 GQ290631 & Waren, 1991 Abyssochrysos Amami Island, Japan GQ290565 GQ290632 sp.[paragraph] Abyssochrysos melvilli N. New Caledonia GQ290633 (Schepman, 1909)[paragraph] Neptunea amianta (Dall, Monterey Bay, CA GQ290566 GQ290634 1890)[paragraph] Neptunea antiqua (L., Denmark Oresund GQ290567 GQ290635 1758)[paragraph] Uittorina littorea (L., N/A DQ093507 1758)[paragraph] * Dive numbers: T = ROV Tiburon, V = ROV Ventana, DR = ROV Doc Ricketts, N = ROV Nautilus Mining, JSL = DSV Johnson Sea Link, and J2 = ROV Jason 2. [dagger] Habitats: B = bone, V = hydro thermal vent, S = seep, W = wood, M = mud. [double dagger] Nucleotide substitution models are given in parentheses for each gene partition, [paragraph] Outgroup taxa.
DNA extractions, polymerase chain reaction (PCR) protocols, and sequencing involved previously described methods for multi-gene analyses of deep-sea invertebrates (Jones et al., 2006; Vrijenhoek et al., 2009). DNA isolations were diluted 1:100 with sterile water for amplification. Here we used primers that amplified [approximately equal to]700 bp of COI mtDNA (Folmer et al., 1994), [approximately equal to]500 bp of 16S rRNA (Palumbi, 1996), [approximately equal to]350 bp of 28S rRNA-D1 (Colgan et al., 2000), [approximately equal to]450 bp of 28S rRNA-D6 (McArthur and Koop, 1999; Colgan et al., 2003), [approximately equal to]440 bp of 12S rRNA (Kocher et al., 1989), [approximately equal to]550 bp of 18S rRNA (Giribet et al., 1996), and [approximately equal to]300 bp of histone-3, H3 (Colgan et al., 2000). The most appropriate outgroup taxa, Abyssochrysos sp., A. melvilli, Littorina littorea, Neptunea amianta, and N. antiqua, were chosen on the basis of a supposed close relationship, similar habitats, and available fossil evidence. All new sequences were obtained bidirectionally and deposited in GenBank (Accession Numbers. in Table 1).
Assembled sequences were aligned using ProAlign ver. 0.5 (Loytynoja and Milinkovitch, 2003). The ProAlign application aligns DNA sequences and then eliminates ambiguously aligned sites for succeeding phylogenetic analyses. To determine which sites are ambiguous and therefore better eliminated, we used posterior probability scores calculated in ProAlign of 60% and above. Appropriate evolutionary models for each gene (Table 1) were selected by AIC, Akaike Information Criteria (Akaike, 1974) in MrModel-Test (Maddison and Maddison, 1992; Nylander, 2004). The program DAMBE ver. 5.0.25 (Xia and Xie, 2001) was used to examine saturation of the mitochondrial COI and H3 sequences for the operational taxonomic units (OTUs) and outgroup taxa. The genes COI and H3 were then partitioned by codon position, and parameters were estimated separately for each position.
Phylogenetic analyses were conducted with MrBayes ver. 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). First, each gene partition was analyzed separately a minimum of three times, each involving 6 chains for 15-50 X [10.sup.6] generations. The graphical system AWTY Online 1/2009 (Nylander et al., 2008) was used to assess convergence of Markov chain Monte Carlo (MCMC) chains and determine burn-in of at least [10.sup.6] generations. Samples were taken at 1000-generation intervals. FigTree ver. 1.3.1 (Rambaut, 2009) was used to display resulting trees. We used the incongruence length difference (ILD) function implemented in Paup* ver. 4.0b10 (Swofford, 2002) to assess congruence among individual gene partitions. ILD analyses were run for 1000 replicates with 10 random additions of gene sequences and were conducted with and without outgroup taxa. Second, a combined analysis of the concatenated sequences modeled each partition separately according to the previously determined parameters.
Calibrating molecular clocks
The dating of internal nodes in the combined MrBayes tree was conducted with BEAST ver. 1.5.4 (Drummond and Rambaut, 2007). We used a relaxed, uncorrelated lognormal clock with the general-time-reversible + gamma + invariant sites substitution model, rate heterogeneity, and unlinked base frequencies. The resulting Newick tree from MrBayes analyses was used for a starting topology. The Yule-process tree-prior was specified with dates based on normal distributions of inferred ages from both a biogeographical vicariance event and fossil evidence. The vicarianee event caused by the closure of the Central American Isthmus separated Provanna ios from seeps off the Pacific margin of Costa Rica and hydrothermal vents at 13[degrees]N along the East Pacific Rise; and P. sculpta from seeps on the Louisiana slope. The closure was completed nearly 3 million years ago (MYA), reproductively isolating Caribbean and Pacific fauna. Deep-sea taxa were reproductively isolated long before, approximately during the mid-Miocene (15-16 MYA) (Lessios, 2008). Independent of the biogeographical calibration, fossil calibrations were also used to calibrate trees: Neptunea amianta (Dall, 1890) and N. antiqua (Linnaeus, 1758) have probably been separated since the earliest appearance of the genus in the Late Eocene 33-37 MYA (Amano, 1997); Provanna and Desbruyeresia are known from Cenomanian (Cretaceous) seep deposits in Japan, 93-100 MYA (Kaim et al., 2008a), which thus can be considered a minimum age of the split between the genera. Results of both analyses are portrayed in Figure 1. Analyses using a normal distribution of the calibration range were initially run 1 X [10.sup.6] generations and were sampled and logged every 1000 generations, then after tuning, were run 1 X [10.sup.8] generations for three independent runs. Results were visualized in Tracer ver. 1.5 (Rambaut and Drummond, 2007) and Figtree ver. 1.3.1 (Rambaut, 2009).
To assess nutritional origin, snail tissues and gut contents were subjected to stable isotope analysis as well as SEM examination and direct observation. For the isotope analysis, the contents were flushed from gut tissues, dried, then fractionated through Prufsieb sieves of mesh sizes 1, 0.5, and 0.25 mm. Fibrous material (> 1 mm in length) was manually removed from the dried contents. All gut contents were treated with 0.2 mol [l.sup.-1] phosphoric acid to remove carbonate, and the dried samples were placed in tin capsules and combusted in a Costech (Valencia, USA) elemental analyzer. Resulting [N.sub.2] and [CO.sub.2] were separated by gas chromatography and examined with a GV instruments (Manchester, UK) Isoprime isotope ratio mass spectrometer (IRMS). Typical differences between replicate analyses were <0.5[per thousand] for [.sup.15]N and <0.2[per thousand] for [delta][.sup.13]C, where [delta] = 1000 X [R.sub.sample]/[R.sub.standard]) - 1[per thousand], where R = the ratio of heavy to light isotopes. The standards were atmospheric nitrogen for [delta][.sup.15]N and Peedee belemnite (PDB) for [delta][.sup.13]C. Egg albumin was used as a daily reference material.
For direct observation the stomach and gut content was critical-point-dried or rinsed with water on a 0.25-mm-mesh sieve and examined with SEM or a dissecting microscope. Organic material was not digested or removed for direct observations of gut contents. Mineral particles from the sediment were extracted from the guts of several R. osteovora specimens by digesting most of the organic material with bleach and dissolving bone with concentrated HC1. Randomly drawn sediment particles were mounted for SEM examinations.
To characterize bacterial associates, we extracted genomic DNA from the gut contents and lining and from gill tissues of four R. osteovora individuals sampled during submersible dives T617, T671, T917, and T1162 (Table 1). Eubacterial primers 27F/1492R were used to amplify a 1465-bp fragment of 16S rRNA (Lane et al., 1991). Three PCR reactions were conducted per individual snail, and the PCR products were pooled, concentrated, cloned using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA), and sequenced bi-directionally. Edited sequences were identified by comparing them against GenBank records (http://www.ncbi.nlm.nih.gov).
Initially, we conducted separate phylogenetic analyses for each of the seven gene partitions (Fig. 1A). A sample of 24 Rubyspira osteovora and 3 Rubyspira goffrediae individuals differed by 17% for COI, and representative samples of the two species also differed consistently by at least a single base-pair (bp) difference for each of the other six gene partitions. Alignments and sequences were deposited in GenBank (Table 1). Tests for the coding loci COI and H3 showed only slight levels of saturation for transitions (>30% F-84 distance) and none for transversions for third-position codons in COI. Saturation was not detected in H3 (data not shown). All codon positions of both fragments were included in analyses. Bayesian trees representing the individual gene partitions are illustrated in an online data supplement at http://www.biolbull.org/supplemental/. Pair-wise incongruence length difference (ILD) tests of homogeneity among the gene partitions revealed significant differences between 16S and H3 and between H3 and 12S when the outgroup taxa were included in the analysis. However, individual gene trees were congruent to one another in regard to branches with posterior probabilities (PP) > 90%. After excluding the outgroup taxa, no significant differences remained between these partitions.
We subsequently conducted a combined Bayesian analysis involving concatenated sequences from the seven gene partitions, which included 3049 bp of data (Fig. 1E). A discrete, young clade composed of Provanna species is strongly supported (green in tree). Next, a phylogenetically more diverse clade composed of the genera Desbruyeresia, Rubyspira, and Abyssochrysos also is well supported (red shades). Finally, two genera, Alviniconcha and Ifremeria, cluster together (blue) and fall basal to the previous groupings. The present multi-gene analysis indicates that the family Provannidae (including Provanna, Desbruyeresia, Alviniconcha, and Ifremeria) is paraphyletic. Until a family-level revision is undertaken, we treat these genera as members of the superfamily Abyssochrysoidea.
BEAST analyses allowed us to estimate a minimal evolutionary age for Rubyspira. Nodes with less than 0.9 PP values were compressed into basal polytomies. Calibrations were combined on one tree. Light grey bars indicate estimates derived from analyses where the closure of the Isthmus of Panama separated Provanna ios and P. sculpta. Dark grey bars indicate results from analyses using fossil calibrations based on Neptunea species separating in the Late Eocene, 33-37 MYA (Amano, 1997) and the split of Provanna and Desbruyeresia known from Cenomanian (Cretaceous) seep deposits in Japan, 93-100 MYA (Kaim et al., 2008a) (Fig. 1E). The node representing the split between Neptunea species was somewhat offset from the fossil calibrations because of the use of the Bayesian starting topology. Abyssochrysos melvilli is also known from Miocene deposits in Fiji (Ladd, 1977); however, this estimate was not used in analyses because we found that multiple calibrations distorted the topology of the tree. Accordingly, the abyssochrysoids diverged 50-158 MYA (vicariance calibration) or 93-228 MYA (fossil calibrations) (95% HPD [highest posterior density] range) into three well-supported clades: (1) the genus Provanna; (2) a group containing the genera Rubyspira, Abyssochrysos, and Desbruyeresia; and (3) the genera Alviniconcha and Ifremeria. Clade 1 radiated [approximately equal to]15-35 MYA (vicariance calibration) or [approximately equal to]17-45 MYA (fossil calibrations) into morphologically diverse species that inhabit the four types of reducing environments. Clade 2, however, diversified earlier, [approximately equal to]26-83 MYA (vicariance calibration) or [approximately equal to]51-90 MYA (fossil calibrations). Within clade 2, the two Rubyspira species appear to have split about 26 MYA (95% HPD range: 10-42 MYA, vicariance calibration) and 31 MYA (95% HPD range: 14-48 MYA, fossil calibrations) (Fig. 1E). Clade 3 diversified [approximately equal to]19-75 MYA (vicariance calibration) or [approximately equal to]31-99 MYA, (fossil calibrations).
On the basis of differences in size and morphology of radulae and observations of animals in situ, the two Rubyspira species appear to differ in how they feed. Rubyspira osteovora was observed living on sediments immediately adjacent to whalebones (Fig. 1A) or in sediment-filled crevices on bone. The radula of R. osteovora is conspicuously short and broad (Fig. 2A-C), not longer than that of an average-sized specimen of Provanna and smaller than that of R. goffrediae (Fig. 2B vs. E) despite the snail being much larger than R. goffrediae (Fig. 1) and typical members of the genus Provanna. Consequently, we assume that the radula is used to aid the mouth in grabbing and swallowing bone fragments from the sediments. In contrast, R. goffrediae was mainly observed living on the surface of bones, where it may use its relatively long radula with enlarged lateral teeth (Fig. 2E, F) to break or scrape fragments from intact bones.
The stomachs of both Rubyspira species are filled with a mixture of bone fragments, sediment, and mineral particles with little other organic matter (Fig. 3). Adults of R. osteovora have a large stomach ([approximately equal to]1 [cm.sup.3]) containing numerous bone splinters (Fig. 3A, B) [less than or equal to]5 mm long. Fecal pellets contain finely ground bone fragments embedded in mucus. Adults of R. goffrediae have a proportionally smaller stomach ([approximately equal to]15 [mm.sup.3]) containing numerous small bone splinters (Fig. 3C) [less than or equal to]1 mm long. Scanning electron microscopy of the mineral fraction from R. osteovora revealed a composition typical of Monterey Bay sediments (Fig. 1A, background), which is consistent with the hypothesis that this species feeds primarily from the sediments surrounding bones. A similar mineralogical analysis of the guts from the small sample of R. goffrediae was not possible because this species is far less abundant and too few specimens were collected. Examination of the gut contents from two co-occurring Provanna species at the same whale-fall (Table 1) did not reveal bone fragments, which is consistent with their habits of grazing on bacterial communities and surface film (Waren and Ponder, 1991).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
A dietary reliance on bone is supported by elevated [delta][.sup.13]C and [delta][.sup.15]N values in R. osteovora (Table 2). Isotopic ratios were similar to those found in bone-eating Osedax worms. The [delta][.sup.13]C and [delta][.sup.15]N values obtained from larger bone particles removed from R. osteovora guts also had relatively high % [.sup.13]C and %[.sup.15]N values, a consequence of the organic content of the bones. Smaller particles were composed mostly of minerals and had lower % [.sup.13]C and % [.sup.15]N.
Table 2 Stable isotope composition and gut contents of Rubyspira osteovora Sample n [[delta].sup.13]C [[delta].sup.15]N (%o) (%o) Rubyspira osteovora Soft tissue 5 -14.5 [+ or -] 1.1 15.7 [+ or -] 1.0 Gut contents Total 3 -14.5 [+ or -] 0.6 15.3 [+ or -] 0.7 Fibrous material 4 -14.2 [+ or -] 0.4 13.0 [+ or -] 0.4 1-mm mesh * 4 -14.8 [+ or -] 0.5 14.2 [+ or -] 0.6 0.5-mm mesh 4 -14.2 [+ or -] 1.4 14.4 [+ or -] 0.4 0.25-mm mesh 4 -15.7 [+ or -] 0.5 14.4 [+ or -] 0.1 Whale bone * 3 -14.2 [+ or -] 0.1 12.8 [+ or -] 0.3 Osedax frankpressi [dagger] Ovisac/root 9 -13.5 [+ or -] 0.2 16.0 [+ or -] 0.1 Crown/trunk 4 -12.5 [+ or -] 0.8 16.6 [+ or -] 0.4 Sample n %C %N Rubyspira osteovora Soft tissue 5 Gut contents Total 3 Fibrous material 4 17.5 [+ or -] 3.3 4.9 [+ or -] 1.1 1-mm mesh * 4 28.3 [+ or -] 4.9 7.7 [+ or -] 1.2 0.5-mm mesh 4 21.4 [+ or -] 3.1 5.3 [+ or -] 0.8 0.25-mm mesh 4 9.5 [+ or -] 4.8 2.0 [+ or -] 1.0 Whale bone * 3 Osedax frankpressi [dagger] Ovisac/root 9 Crown/trunk 4 Enrichments of 0-l%o for [[delta].sup.13]C and 3%-4%o for [[delta].sup.15]N are generally observed relative to diet. Values are given as mean [+ or -] standard deviation. %C and %N values are grams C or N per 100 grams dry material. * Mesh size refers to the selectivity of filters. [dagger] Data from Goffredi et al. (2005).
The composition of 16S ribosomal RNA clone libraries constructed from gill tissues revealed the presence of a microbial community common to Monterey benthic sediments (Goffredi et al., 2005) in addition to [gamma]-Proteobacteria that were related to the thiotrophic gill symbiont found in the gills of deep-sea mussels (Bathymodiolus), GenBank Accession Numbers HM441247-51. Clone libraries generated from gut contents resembled that of the microbial community found in benthic sediments from these depths (Goffredi et al., 2007): Bacteroides, [beta]-(Ralstonia), [delta]- and [gamma]-Proteobacteria (Psychromonas and Sphingomonas). We also found bacteria that were closely related to the Oceano-spirillales endosymbionts hosted by Osedax boneworms.
Mollusca Linnaeus, 1758; Gastropoda Cuvier, 1797; Caenogastropoda Cox, 1959; Superfamily Abyssochrysoidea Tomlin, 1927; Family not settled.
Rubyspira gen. nov.
Type species. Rubyspira osteovora sp. n.
Diagnosis. Abyssochrysoids with tall shell, predominating spiral sculpture, multispiral, probably planktotrophic, multispiral protoconch; aperture with no trace of siphonal canal. Periostracum forming small bristles along the spiral ridges. One large pallial tentacle on right, a single small one on left corner. Larval shell, partly preserved on a specimen of R. osteovora (Fig. 4A) as well as one empty protoconch (Fig. 4B) and resembling that of the provannids Alviniconcha, Desbruyeresia, and Cordesia; thought to reflect planktotrophic development (Kaim et al., 2008a; Waren and Bouchet, 2009). Before settlement the apical whorls are autotomized and the top of the remaining 2-2.5 whorls are sealed with a calcareous plug. The size of the settling Rubyspira larvae is [approximately equal to]0.75 mm, excluding the discarded 0.3-0.5 mm, and it is thus at the upper range of larval shell sizes for abyssochrysoids.
[FIGURE 4 OMITTED]
Etymology. Named after Ruby, the Monterey Bay Aquarium Research Institute's (MBARI) 2893-m deep whale carcass; and spira (Greek and Latin), coil or spiral.
Rubyspira osteovora sp. nov.
Type material & locality. Holotype Swedish Museum of Natural History (SMNH) #4176 and numerous paratypes SMNH #4178 from Monterey Submarine Canyon, California, 36[degrees]36.8'N, 122[degrees]26.0'W, 2893 m depth, at whale-2893 (Braby et al., 2007), Tiburon dive 917.
Material examined. Numerous additional specimens from the type locality were collected on dives during 2004-2009 and stored at SMNH and MBARI. Two specimens were collected at whale-1820 (Braby et al., 2007), "Patrick" whale-fall, 13 March 2009, 36[degrees]42.50'N, 122[degrees]06.31'W, 1820 m depth.
Etymology. From osteon (Greek), bone and vorator (Latin), eater.
Diagnosis. Shell (Figs. 1A, B; 4C) large and [less than or equal to]58 mm in height, Turritella-like, with obliquely teardrop-like aperture, dominating spiral sculpture, and thin brownish-greenish periostracum. Protoconch (Fig. 4A, B) brownish, multispiral with two finely reticulate whorls; apical whorls autotomized. Teleoconch sculpture starts (Fig. 4B) with 3 larger and scattered, much smaller, spiral ridges on first 1.5-2.5 whorls; ribs soon disappear and surface is almost smooth except basally where 10-15 strong, flat spiral ridges remain. Surface of abapical 3-4 whorls covered by much finer secondary sculpture of 3-6 thin, irregularly disposed elevated lines per major ridge and an irregular incremental sculpture. On top of minute lines, periostracum forms small tufts. Columella covered by very thin parietal callus, stronger in old specimens; narrow umbilical chink in some. Aperture 1.8 times as high as broad, lower part evenly rounded, no trace of siphonal canal. Outer lip thin, almost perpendicular, in profile distinctly sinuated at apical 1/5. Soft parts. One large pallial tentacle on the right, one on the left corner of the pallial margin (Fig. 5A) Head-foot (Fig. 5B) comparatively small with short, stubby cephalic tentacles without eye lobes; large, flat snout with ventro-apical mouth; short broad and fleshy foot with a distinct lateral groove paralleling edge of sole. Male aphallic. Operculum (Fig. 2D) distinctly yellowish brown, 1.5 times as high as wide, last half whorl 71% of its height, no conspicuous muscle attachments. Radula (Fig. 2A-C) taenioglossate, 3 mm long and 0.7 mm broad in adult specimen; central tooth simplified, apically rounded and drawn out to a small triangular cusp; laterals slightly taller than central, flat and hook-like, with indistinct, small lateral supports; both marginals of same size and shape, hook-like.
[FIGURE 5 OMITTED]
Remarks. No known gastropod species from the American West Coast resemble R. osteovora. Its abrupt appearance in large numbers at the 2893-m whale-fall suggests that the larvae are common in the deep sea off California, but perhaps restricted to great depths since only two specimens were found at the 1820-m whale-fall. The radula of R. osteovora is remarkably short and broad, actually similar in size to that in an 8-12 mm specimen of the related genus Provanna Dall, 1918 (see Waren and Bouchet, 2009). Fig. 2B shows the radula for comparison with R. goffrediae (Fig. 2E) corrected for the difference in body size.
Rubyspira goffrediae sp. nov.
Type material & type locality. Holotype SMNH #4179 and two paratypes SMNH #4180 from Monterey Submarine Canyon, California, 36[degrees]36.8'N, 122[degrees]26.0'W, 2893 m depth, at whale-2893, Tiburon dive 991 (Braby et al., 2007).
Etymology. Named after Dr. Shana Goffredi, collaborator and friend.
Diagnosis. Shell (Fig. 1C) [less than or equal to]14 mm in height, ovate, with almost round aperture, dominating spiral sculpture and well-developed, slightly bristly, brownish-greenish periostracum. Protoconch lost in all specimens. At diameter 3.2 mm, whorls have 7 low spiral ridges, separated by narrow interstices and crowned by simple series of periostracal tufts. On body, whorl 12 spirals above level of suture, 14 below. No axial sculpture except numerous fine, irregular incremental lines. Columella covered by very thin parietal callus, stronger in old specimens; no umbilical chink. Aperture 1.3 times as high as broad; lower part evenly rounded with no trace of siphonal canal. Outer lip thin, straight, distinctly prosocline. Soft parts. Head-foot (Fig. 5C) with short, stubby cephalic tentacles without eye lobes; large snout with ventro-apical mouth; short, broad, and fleshy foot with a distinct lateral groove paralleling edge; pallial tentacles uncertain, at least one in right corner of pallial margin (Fig. 5D). Male aphallic. Operculum (Fig. 2G) 1.3 times as high as wide and with indistinct incremental sculpture; last half whorl corresponding to 57% of its height. No conspicuous muscle attachments. Radula (Fig. 2E-F) taenioglossate, slender, 5 mm long and 0.5 mm broad in an average-sized specimen; simplified and reduced central tooth; almost scale-like, with one small central cusp and two smaller, blunt cusps at each side. Laterals at least 5 times taller, very sturdily built with a large and conspicuous central and a much smaller lateral cusp at each side; no lateral supports. Marginal teeth rake-like; inner one with 9 denticles, outer one with 18.
Remarks. Adult specimens of R. goffrediae (Fig. 1C) have an ovate shell shape, whereas R. osteovora (Fig. 1B, D) has a tall, slender shell. The radula of R. osteovora is conspicuously short and broad, whereas the radula of R. goffrediae is [approximately equal to]10 times as long as broad, with large and sturdy lateral teeth and reduced central teeth.
The two Rubyspira species have large stomachs filled with a mixture of bone fragments and marine sediments. All R. goffrediae individuals sampled in this study were found living directly on bones. The species possesses a relatively large file-like radula that appears to be used for scraping the very fine bone fragments found in its guts. In contrast, most R. osteovora individuals occurred on sediments immediately adjacent to highly degraded bones. The relatively small scoop-like radula and coarse bone fragments found in its stomach suggest that the snails graze on "bone-dust" generated by decapod crustaceans as they feast on degraded bones full of Osedax. Paralomas crabs can be been observed actively devouring bones occupied by Osedax (Braby et al, 2007). The worms produced complex proliferative "root" systems that penetrate and quickly degrade bones. They also produce a large ovisac full of eggs that appear to provide nutrition for crabs. Our analyses of stable isotopes suggest that Rubyspira obtain their nutrition from the whalebones. Snail tissues exhibited [[delta].sup.13]C and [[delta].sup.15]N signatures that were very similar to those of the whalebones from the same locality and to the isotopic signatures of Osedax, which is known to obtain its nutrition from whalebones (Goffredi et al., 2005, 2007). The present analyses could not, however, distinguish whether the snails obtained their carbon and nitrogen directly from whalebones or from the copious bacteria growing on whalebones. Too few specimens of R. goffrediae were collected to conduct isotopic analyses. Additional support for the hypothesis that Osedax and decapods may be needed to facilitate bone-eating by Rubyspira derives from time-series studies of whale-fall degradation (Braby et al., 2007; L. Lundsten et al. (Monterey Bay Aquarium Research Institute, unpubl. data). Rubyspira osteovora was first observed at the 2893-m whale-fall 2 years after its discovery during March of 2002, and only became abundant 3 years later (Braby et al., 2007). This species was first observed at the 1820-m whale-fall 5 years after deployment and still is not abundant there (L. Lundsten et al., unpubl. data).
Symbiotic microbes might also contribute to nutrition of these snails. At least two species of provannid gastropods host sulfide-oxidizing symbiotic bacteria within their gills. Decaying whalebones generate sufficient sulfides to support bacterial mats and an invertebrate fauna composed of symbiont-bearing mussels and clams (Smith and Baco, 2003). Rubyspira osteovora has enlarged gill leaflets coated with many small (2-3 [micro]m) ovate bacteria. Clone libraries of 16S rRNA derived from gill extracts revealed the presence of [gamma]-Proteobacteria, very closely related to the thiotrophic gill symbiont associated with deep-sea mussels of the genus Bathymodiolus (Duperron et al., 2006). Sulfate-enriched sediments under the whale-fall carcass support a diverse microbial community (Goffredi et al., 2008); consequently, Rubyspira snails might also rely on the thiotrophic bacteria to augment nutrition or as an alternative source of nutrition once the bones are consumed. Although these preliminary results are suggestive, more research on the bacteria associated with these snails is needed to clarify potential roles of various microbes as symbionts and a primary food source.
Abyssochrysoid snails began to diversify during the Mesozoic. The earliest convincing abyssochrysoid fossils were associated with Upper Cretaceous cold seeps (Kaim et al., 2008a), but good evidence exists for an Early Cretaceous origin, 131-145 MYA. Molecular evidence supports a rapid radiation of abyssochrysoids during the mid-Cretaceous (Fig. 1E). The recent discovery of abyssochrysoid shells associated with a plesiosaurid carcass from Late Cretaceous deposits suggests that the carcasses of large marine reptiles supported communities similar to those found at modern whale-falls (Smith and Baco, 2003; Kaim et al., 2008a, b). Fossil abyssochrysoids also were associated with Eocene cold seeps, wood-falls, and bones of odontocete whales (Squires, 1995: Kiel and Goedert, 2007), and with Miocene cold seeps and the bones of mysticete whales (Amano and Little, 2005).
No living abyssochrysoids resemble R. osteovora. Conchologically this species bears a remarkable resemblance to the fossil Atresius liratus Gabb, 1869 (Fig. 4D), from Early Cretaceous (Valanginian to Hauterivian) cold seeps in California (Kiel et al., 2008). The present molecular estimates suggest that the evolutionary lineages leading to the present Rubyspira species split during the upper Eocene/lower Oligocene (Fig. 1E), subsequent to the occurrence of Atresius. The mean time to the most recent common ancestor of R. osteovora and R. goffrediae is slightly more recent than the origins of large archeocete whales (Thewissen and Williams, 2002) and the putative origins of Osedax boneworms (Vrijenhoek et al., 2009; Kiel et al., 2010). Our phylogenetic analyses clearly indicate that living Abyssochrysos species are Rubyspira's closest known living relatives (Fig. 1E). This relationship is corroborated by similarities in the presence of a pedal furrow, a similar arrangement of pallial tentacles, and similar eu- and paraspermatozoa (Waren and Ponder, 1991). Detailed morphometric comparisons with Atresius fossils were not feasible because the aperture of the fossils was missing and other diagnostic features were poorly preserved. Nevertheless, Rubyspira appears to be an extant representative of the lineage containing Atresius.
Inhabiting a variety of deep-sea environments (hydrothermal vents, cold seeps, large food-falls) and deriving nutrition from varied sources (grazing, scavenging, and chemo-autotrophy) may have allowed abyssochrysoids to diversify and escape the extinction events that affected many shallow-water taxa during the Late Cretaceous and Early Cenozoic eras (Jacobs and Lindberg, 1998; Little and Vrijenhoek, 2003). Dietary flexibility and radiation into a variety of chemosynthetic environments are also hypothesized to have contributed to the evolutionary persistence of deep-sea bathymodiolin mussels (Jones et al., 2006; Won et al., 2008).
We thank Shana Goffredi, Eric Cordes, Cindy Van Dover, Stephanie Aktipis, Jun Hashimoto, Michel Segonzac, and Tina Schleicher for their help with this study. S. Kiel graciously supplied the photo of the fossil Atresius. Funding provided by the David and Lucile Packard Foundation and NSF (OCE0241613). AW was funded by Riksmusei Vanner and Bergvalls stiftelse. We thank the MBARI ROV pilots and the RV Western Flyer crew for their hospitality and support.
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S. B. JOHNSON (1), *, A. WAREN (2), R. W. LEE (3), Y. KANO (4), A. KAIM (5), A. DAVIS (1), E. E. STRONG (6), AND R. C. VRIJENHOEK (1)
(1) Monterey Bay Aquarium Research Institute, Moss Landing, California 95039; (2) Swedish Museum of Natural History, Box 50007, SE-10405 Stockholm, Sweden; (3) Washington State University, Pullman, Washington 99164; (4) Department of Marine Ecosystems Dynamics, Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan; (5) Instytut Paleobiologii PAN, ul. Twarda 51/55, PL-00-818 Warszawa, Poland; and (6) National Museum of Natural History, Department of Invertebrate Zoology, MRC 163, PO Box 37012, Washington DC 20013-7012
Received 5 February 2010; accepted 20 July 2010.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
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|Author:||Johnson, S.B.; Waren, A.; Lee, R.W.; Kano, Y.; Kaim, A.; Davis, A.; Strong, E.E.; Vrijenhoek, R.C.|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 2010|
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