Hemichordate Molecular Phylogeny Reveals a Novel Cold-Water Clade of Harrimaniid Acorn Worms.
Hemichordates, a small marine phylum integral to hypotheses on deuterostome and chordate evolution, occur throughout the world's oceans. At present, 135 hemichordate species are described: 121 are solitary Enteropneusta, or acorn worms; and 24 are colonial, tube-dwelling Ptero-branchia (Appeltans et al., 2012; Halanych and Cannon, unpubl. data). In the last decade, there has been revived interest in hemichordate taxonomy. The number of entero-pneust genera has increased by 40% since 2005, with several papers on the deep-sea family Torquaratoridae (Holland et al., 2005, 2009, 2012a, b; Smith et al., 2005; Osborn et at., 2011; Priede et at., 2012), as well as recent evaluations of Ritter's and Bullock's historical collections leading to the description of 16 new species and three new genera (Deland et al., 2010; Cameron et al., 2010; Cameron and Ostiguy, 2013; Cameron and Perez, 2012). Another novel genus of enteropneust, Meloglossus, has been described on the basis of meiofaunal samples collected in Bermuda and Belize (Worsaae et al., 2012). Evidently, much of the diversity within this group remains undiscovered (Appeltans et at., 2012, supplement).
There are four currently recognized families of entero-pneusts--Harrimaniidae (39 species in 10 genera), Pty-choderidae (38 species in 3 genera), Spengelidae (19 species in 4 genera), and Torquaratoridae (6 species in 4 genera). Molecular phylogenetic work has indicated that Harrima-niidae, Ptychoderidae, and Torquaratoridae are likely to be valid families (Cannon et at., 2009; Osborn et al., 2011; Worsaae et al., 2012). Spengelids, on the other hand, have thus far been represented in phylogenetic analyses by a single species, Glandiceps hacksi, which is recovered as sister to Ptychoderidae + Torquaratoridae (Osborn et al., 2011; Worsaae et al., 2012).
Extant pterobranchs comprise two groups, Cephalodisc-ida (18 species in 1 genus) and Rhabdopleurida (6 species in 1 genus). Recent analyses of morphology from fossil grap- 4 tolites and extant pterobranchs place Cephalodiscida as sister to Crraptolithina, including Rhabdopleurida (Mitchell et al., 2012). Interrelationships between Enteropneusta and Pterobranchia have been controversial. Several 18S rDNA studies have recovered pterobranclas within Enteropneusta sister to Harrimaniidae (Halanych, 1995; Cameron et at., 2000; Bourlat et at., 2003; Cannon et al., 2009; Worsaae et al., 2012), but other 18S analyses do not recover this relationship (Osborn et aL, 2011). By contrast, recent mi-croRNA data (Peterson et at., 2013), morphological cladistic analysis (Cameron, 2005), and 28S rDNA (Winchell et at.. 2002) suggest that Enteropneusta and Pterobranchia are reciprocally monophyletic taxa, although the latter two data matrixes have been criticized for containing few informative characters (Cannon et al., 2009), and the rnicroRNA analysis included only three hemichordate lineages.
Here we present a molecular phylogeny of Hemichordata that includes novel sequence data from 35 taxa representing all hemichordate families, at least 12 of which are distinct genetic lineages not attributable to known species. Our phylogenetic results reveal a previously unknown clade of harrimaniid enteropneusts from cold waters and provide an updated molecular phylogenetic hypothesis for hemichor-dates with broader taxon sampling across all lineages.
Materials and Methods
Figure 1 shows a world map with collection localities for samples in this study, and Table 1 provides more detailed locality information and sampling method used. Eleven specimens sequenced in this study were collected in September 2011 during Senckenberg's German Center for Marine Biodiversity Research (DZMB) IceAGE expedition led by Dr. Saskia Brix aboard the RN Meteor, which circled Iceland, crossing the Mid-Atlantic and Greenland-Scotland ridges. Cephalodiscus specimens were collected during two research expeditions to the Antarctic Peninsula aboard the R/V Lawrence M. Gould in 2001 and 2004. Antarctic enteropneusts were collected in January--February 2013 in the Amundsen and Ross Seas by the R/V Nathaniel B. Palmer. Enteropneusts from Norway were collected on the R/V kon Mosby or RN Aurelia with the aid of Dr. Christiane Todt and the late Dr. Christoffer Schancler. Balanoglossus sp. specimens from Mississippi were collected with the assistance of Dr. Richard Heard. Dr. Jon Norenburg and Dr. Darryl Felder provided material from Rhabclopleura sp. collected in the Gulf of Mexico on the R/V Pelican.
Table 1 Collection and locality information by taxon Family Taxon Locality Latitude Cephalodiscidae Cephalodiscus Elephant 62[degrees] hodgsoni HI 1.3 Island. 44.74'S Antarctica Cephalodiscus South 62[degrees] fumosus H5.3 Shetlands, 45.03'S Antarctica Cephalodiscus Elephant 62[degrees] nigrescens Hi Island. 44.74'S 2.3 Antarctica Rliabdopleuridae Rhabdopleura sp. Iceland 63[degrees] 1 H71.2 56.07'N Rhabdopleura sp. Gulf of 27[degrees] 2 Mexico 36.94'N Harrimaniidae Harrimaniidae Norway 60[degrees] 1-142.1 18.37'N Harrimaniidae Norway 60[degrees] H61.I 16.58'N Harrimaniidae Iceland 69[degrees] 1172.1 6.51 'N Harrimaniidae Iceland 69[degrees] H72.2 6.51 'N Harrimaniidae Iceland 69[degrees] H72.3 6.51 'N Harrimaniidae Iceland 60[degrees] H69.1 20.87'N Harrimaniidae Iceland 69[degrees] H75.1 6.5I'N Harrimaniidae Iceland 67[degrees] H76.1 5.79'N Harrimaniidae Iceland 67[degrees] H77.1 37.39'N Harrimaniidae Wright's Gulf. 73o15.30'S H86.2 Antarctica Harrimaniidae Amundsen Sea. 72[degrees] H83.1 Antarctica 10.64'S Harrimaniidae Ross Shelf. 75[degrees] H93.I Antarctica 19.80'S Harrimaniidae Ross Shelf, 75[degrees] H94.1 Antarctica 19.80'S Harrimaniidae Ross Shelf, 76[degrees] H98.2 Antarctica 20.47'S Harrimaniidae Oregon, USA 43[degrees] HI03.1 50.60'N Saccoglossus White Sea. 66[degrees] rnereschkowskii Russia 33.I9'N H53.1 Saccoglossus sp. Friday Harbor. 48[degrees] H44.2 Washington, 32.6I'N USA Harrimaniidae Norway 60[degrees] H62.1 27.85'N Harrimaniidae Iceland 62[degrees] H74.1 56.46'N Ptychoderidae Balanoglossus sp. Bocas del 9[degrees] 1 H50.4 Toro. Panama 24.3'N Balanoglossus cf. Ship Island, 30[degrees] aurantiaca H51.1 Mississippi, I4.8'N USA Balanoglossus cf. Horn Island, 30[degrees] aurantiaca H52.1 Mississippi, 14.75'N USA Glossohalanus Norway 58[degrees] marginatus H48.2 55.00'N Glossobalanus False Bay, 48[degrees] berkeleyi H68.2 Washington, 28.93'N USA Ptychodera Shelley Bay, 32[degrees] bahamensis H54.2 Bermuda 19.88'N Spengelidae Schizocardium cf. Bay St. Louis, 30[degrees] braziliense Mississippi, 14.09'N H47.5 USA Torquaratoridae Torquaratoridae Iceland 66[degrees] H78.1 18.06'N Torquaratoridae Ross Shelf. 76[degrees] H89.3 Antarctica 20.47'S Torquaratoridae Ross Shelf. 76[degrees] H90.3 Antarctica 20.47'S Family Taxon Longitude Depth Collection (m) method Cephalodiscidae Cephalodiscus 56[degrees] 207 Dredge hodgsoni HI 1.3 44.88'W Cephalodiscus 56[degrees] 220 Dredge fumosus H5.3 46.26'W Cephalodiscus 56[degrees] 207 Dredge nigrescens Hi 44.88'W 2.3 Rliabdopleuridae Rhabdopleura sp. 25[degrees] 209 Agassiz 1 H71.2 56.53'W Trawl Rhabdopleura sp. 83[degrees] 36.5 Hourglass 2 28.02'W Dredge Harrimaniidae Harrimaniidae 5[degrees] 130 Epibenthic 1-142.1 12.07'E Sled Harrimaniidae 05[degrees] 183 Epibenthic H61.I 11.09'E Sled Harrimaniidae 9[degrees] 2177 Epibenthic 1172.1 55.09'W Sled Harrimaniidae 9[degrees] 2177 Epibenthic H72.2 55.09'W Sled Harrimaniidae 9[degrees] 2177 Epibenthic H72.3 55.09'W Sled Harrimaniidae I8[degrees] 2569 Epibenthic H69.1 8.52'W Sled Harrimaniidae 9[degrees] 2177 Epibenthic H75.1 55.09'W Sled Harrimaniidae 13[degrees] 1612 Agassiz H76.1 0.42'W Trawl Harrimaniidae 12[degrees] 1781 Agassiz H77.1 4.06'W Trawl Harrimaniidae 129[degrees] 481 SmithMac H86.2 12.94'W Grab Harrimaniidae J03[degrees] 341 Blake Trawl H83.1 30.84'W Harrimaniidae 176[degrees] 567 MegaCore H93.I 59.26'W Harrimaniidae 176[degrees] 567 MegaCore H94.1 59.26'W Harrimaniidae 170[degrees] 531 MegaCore H98.2 5L02'W Harrimaniidae 127[degrees] 2950 MultiCore HI03.1 34.03' W Saccoglossus 33[degrees] <20 Dredge rnereschkowskii 6.35'E H53.1 Saccoglossus sp. I23[degrees] 10-5 Dredge H44.2 0.68'W Harrimaniidae 05[degrees] 130 Epibenthic H62.1 05.96'E Sled Harrimaniidae 20[degrees] 916 Epibenthic H74.1 44.06'W Sled Ptychoderidae Balanoglossus sp. 82[degrees] <1 Shovel 1 H50.4 19.45'W Balanoglossus cf. 88[degrees] 1 Yabby Pump aurantiaca H51.1 32.4'W Balanoglossus cf. 88[degrees] 1 Yabby Pump aurantiaca H52.1 46.51'W Glossohalanus I0[degrees] 184 Agassiz marginatus H48.2 33.16'W Trawl Glossobalanus I23[degrees] <1 Shovel berkeleyi H68.2 4.23'W Ptychodera 64[degrees] 1 Snorkel bahamensis H54.2 44.36'W Spengelidae Schizocardium cf. 89[degrees] 12.5 Box Core braziliense 20.09'W H47.5 Torquaratoridae Torquaratoridae 12[degrees] 732 Box Core H78.1 22.40'W Torquaratoridae 170[degrees] 531 Blake Trawl H89.3 51.02'W Torquaratoridae 170[degrees] 531 Blake Trawl H90.3 51.02'W
Specimens were collected at depths ranging from the intertidal to over 2500 m, using diverse sampling methods (Table 1). Enteropneusts from Iceland were collected by decanting sediment through a 1-1.5-mm sieve, and were retained on either a 500- or 300-[micro]m sieve; enteropneusts from Antarctica and off Oregon were decanted from sediment directly onto a 250-[micro]m sieve. Freshly collected worms were preserved in 95%-100% ethanol, and when multiple specimens were available, voucher specimens were relaxed in 7.5% magnesium chloride and fixed in 4%-10% formalin for morphological studies.
Fragile and soft-bodied enteropneusts are easily damaged by dredging or sorting, of sediment. Interestingly, all novel taxa reported here were collected using standard sampling techniques, including epibenthic sleds, which are typically used to recover more robust animals. Minuscule acorn worms may be present in samples collected elsewhere using similar methods, but may have been overlooked due to their minimal and nondescript external characters (Fig. 2). During collection, many enteropneusts were passed through a 1-mm mesh, and therefore could be classified as meiofaunal (Higgins and Thiel, 1988). However, most worms were broken during sieving, so their intact length is unknown. Additionally, because worms fragment easily, they are less amenable to detailed morphological analyses. Whereas even very small fragments can be used for DNA or RNA extraction, worms with intact proboscis, collar, and even a small part of the trunk are much more difficult to find (Fig. 2C). This simple fact may have hindered previous discovery of these worms and has delayed morphological characterization of novel worms reported in this study. Complete morphological species descriptions are forthcoming, but herein we focused on molecular data in order to provide a more comprehensive overview of unknown hemichordate diversity.
base, which was searched with a query composed of diverse hemichordate 18S sequences. Fragments with E-values below 1 x [10.sup.-30] were aligned to the partial 18S sequences as a backbone, and then assembled into a full-length 18S rDNA contig using the software package CLC Genomics Workbench ver. 5 (Aarhus, Denmark). As reported previously (Cannon et al. 2009), Stereobalanus canadensis (En-teropneusta. Harrimaniidae) has a highly divergent 18S rDNA sequence. Previously we had sequenced this acorn worm multiple times, but to further verify this sequence, genomic DNA was extracted from S. canadensis and sequenced via an Illumina MiSeq (San Diego, CA) at Auburn University. This work employed a 2 X 150-bp paired end run and the Nextera DNA sample kit. Scaffolds generated using the genome assembler Ray (Boisvert et al., 2010) were formatted into a BLAST database, and queried as above. A single 18S rDNA sequence was recovered, with 99.6% identity to our previously reported sequence; thus we have retained Stereobalanus canadensis in our analyses.
Taxon sampling and NCBI accession numbers for sequences used in phylogenetic analyses are given in Table 2. Representatives of each echinoderm class were used as outgroup taxa (Table 2). Sequences were aligned with MAFFT ver. 6.09b (Katoh et al., 2005) using the L-INS-i method, and uninformative sites were trimmed using the pen l scripts Aliscore and Alicut (Misof and Misof, 2009). Models of evolution were selected under the Akailce Information Criterion (AIC) implemented by MrModelTest (Ny-lander, 2004). The best fitting model for both 16S and 18S rDNA, GTR+I+G, was used in maximum likelihood (ML) and Bayesian inference analyses (BI) of concatenated alignments. MIL analyses were performed with RaxML version 7.3.9 (Stamatakis, 2006) using 5000 bootstrap replicates. Bayesian analyses were conducted using MrBayes ver. 3.2.0 (Ronquist and Huelsenbeck, 2003). Four independent BI analyses were run for each dataset for 5,000,000 generations with trees sampled every 100 generations using three heated and one cold chain. Plotting likelihood values versus generation number revealed that stationarity was reached after approximately 1,000,000 generations, and thus the first 25% of sampled trees was discarded as burn-in. Competing hypotheses of hemichordate phylogeny were evaluated using the SH-test (Shimoclaira, 2002) as implemented in RAxML ver. 7.3.8 with the GTR+I+G model.
Taxa 18S rDNA 16S rDNA HEMICHORDATA Cephalodiscidae Cephalodiscus hodgsoni KF683576 -- HI 1.3 Cephalodiscus hodgsoni EU728441 -- Cephalodiscus fumosus KF83575 -- H5.3 Cephalodiscus KF683574 -- nigrescens H12.3 Cephalodiscus gracilis AF236798 -- Cephalodiscus densus EU728439 -- Cephalodiscus EU728440 -- nigrescens Rhabdopleuridae Rhabdopleura sp. 1 KF6K3598 KF683562 H71.2 Rhabdopleura sp. 2 KF683597 KFA835A3 Rhabdopleura normani KF683596 -- Rhabdopleura compacta -- FN408482 Harrimaniidae Harrimaniidae H42.1 KF683595 KF683548 Harrimaniidae H61.1 KF683581 KF683547 Harrimaniidae H72.1 KF683591 KF683534 Harrimaniidae H72.2 KF683580 KF683533 Harrimaniidae H69.1 KF683594 KF683537 Harrimaniidae H75.1A KF683578 KF683536 Harrimaniidae H75.1B KF683590 KF683540 Harrimaniidae H75.1C KF683579 KF683535 Harrimaniidae H76.1 KF683592 KF683539 Harrimaniidai; H77.2 KF683593 KF683538 Harrimaniidae H83.1 KF683582 KF683549 Harrimaniidae H86.2 KF683583 KF683550 Harrimaniidae H93.1 KF683585 KF683541 Harrimaniidue H94.1 KF683586 KF683543 Harrimaniidae H98.2 KF683584 KF683542 Harrimaniidae H103.1 -- KF683532 Harrimania kupfferi JF900487 JX855256 MCZ Harrimanici AF236799 EU728421 planktophilus Protoglossus sp. EU728432 EU728420 Meioglossus JF900488 JX855287 psammophilus Saccoglossus KF683588 KF683545 mereschkowskii H53.1 Saccoglossus sp. H44.2 KF683577 KF683544 Harrimaniidae H62.1 KF683587 KF683546 Saccoglossus AF236801 L26348 bromophenolosus Saccoglossus L28054 NC_007438 kowalevskii Saccoglossus pusillus AF236800 EU728422 Saxipendium coronatum EU728433 EU728423 A Saxipendium coronatum EU 520505 EU520493 B Saxipendium JN886774 JN886756 implication Harrimaniidae H74.1 KF683589 KF683551 Stereobalanus EU728434 EU728424 canadensis Ptychoderidae Balanoglossus sp. 1 KF683570 KF683557 H50.4 Balanoglossus cf. KF683S69 KF683555 aurantiaca H51.1 Balanoglossus cf. KF683568 KF683556 aurantiaca H52.1 Balanoglossus -- EU728425 clavigerus Balanoglossus carnosus JF900489 -- MCZ Balanoglossus curnosus D14359 AF051097 Tampa Ptychoderid AF278685 EU728427 Glossobalanus KF683566 KF683559 marginatus H48.2 Glossobalanus berkeleyi KF6K3567 KF683554 H68.2 Glossobalanus EU728435 EU728426 berkeleyi Glossobalanus minutus AF119089 -- Ptychodera bahamensis KF683571 KF683560 H54.1 Ptychodera bahamensis JF900485 JX855285 101774 Ptychodera bahamensis JF900486 -- 103686 Plychodera flava EU728436 EU728429 Ptychodera flava AF27K681 EU728428 Spencelidae Schtzocardium cf. KF683572 KF683561 braziliense H47.5 Glandiceps hacksi JN886773 JN886755 Incertae sedis Gulf Stream Tornaria EU728437 EU728430 Enteropneusta incertae KF683573 KF683558 sedis H89.3 Torquaratondae Torquaratoridea H78.l KF683565 KF683553 Torquaratoridea H90.3 KF683564 KF683552 Tergivelum baldwinae EU520506 EU520494 T1076-1 Tergivelum baldwinae EU520509 EU520497 T1094 Tergivelum baldwinae JN866772 EU520495 T1078-1 Tergivelum baldwinae JN886770 JN886753 Il65-24 Genus C T8S6-A4 EU520511 EU5 20499 Genus C D80-A2 JN886768 JN886751 Allapasus aurantiacus JN886767 JN886750 Allapasus isidis JN886766 JN886749 Genus B sp 1 TI76-A1 JN886761 JN886744 Genus B sp 1 t879-A8 JN886760 EU520500 Genus B sp2 T1011 EU520515 EU520503 Yoda purpurata JN886757 JN886740 I171-36a Yoda purpurata JN886758 JN886741 I171-36b IFREMER Enteropneust EU728438 EU728431 ECHINODERMATA Crinoidea Metacrinus rotundus AY275898 AY275905 Ophiuroidea Gorgonocephalus DQ060790 DQ297092 eucnemis Amphipholis squamata X97156 NC_013876 Astern idea Solaster stimpsoni DQ060819 DQ297113 Qdontaster validus DQ060801 GQ294457 Holothuroidea Psychropotes ZS0956 DQ777099 longicauda Apostichopus japonicas AB595140 NC_012616
The final combined 18S rDNA + 16S rDNA alignment was 2776 nucleotides in length (18S = 2053 nucleotides, 16S = 723 nucleotides), with 88 included taxa and 965 parsimony informative sites. Several lengthy indel regions in the 16S of rhabdopleurid pterobranchs account for the long 16S alignment. Table 2 reports GenBank accession numbers for all data used, and aligned data have been deposited to TreeBase (http://www.treebase.org). Maximum likelihood and Bayesian inference analyses yielded topologies with an identical branching pattern (Fig. 3) in which Hemichordata and all currently recognized families were recovered as monophyletic. Notably, unlike previous 18S results (Halanych, 1995; Cameron et al., 2000; Bourlat et al., 2003; Cannon et al., 2009; Worsaae et al., 2012). Enteropneusta and Pterobranchia were recovered as reciprocally monophyletic, with strong support (bootstrap/posterior probability = 98/1.00), although the alternative hypothesis (Pterobranchia + Harrimaniidae) was not rejected by Shimodaira-Hasegawa tests (P value > 0.05).
Within Harrimaniidae. we recovered a well-supported (98/1.00), but hitherto unknown, clade of small (1-3 mm in length) undescribed cold-water harrimanid worms from Antarctica, Norway, Iceland, and Oregon. This clade consists of four distinct and strongly supported subclades (Fig. 3) that likely represent at least four distinct species. Subclade 1 is composed of small worms from Norway and the Amundsen Sea in Antarctica, subclade 2 of Antarctic worms from the Ross Sea, subclade 3 of Icelandic worms, and subclade 4 of Icelandic worms and a single specimen from deep waters off Oregon. Pairwise genetic distances from 16S rDNA sequences were calculated using the K2P model as implemented by MEGA 5 (Tamura et at.. 2011). Nucleotide substitution values within these subclades were 0.0%12.0%, while distances between subclades were 12.6%26.9%. For comparison, species within the harrimaniid genus Saccoglossus showed distances of 0.1%-17.9%, whereas genetic distances between recognized harrimaniid genera were 18.2%-40.0% (not including the highly divergent Stereobalanus canadensis). This places the four subclades at the higher end of the range for species distinction, and taken together they may represent a new harrimaniid genus. The morphological work needed to confirm their taxonomic status relative to current generic diagnosis will be part of a subsequent study. Other novel harrimanids we sequenced include an unidentified species of Saccoglossus from Norway and a single specimen collected at 916 m off Iceland that is most closely related to the deep-sea genus Saxipendium. Saccoglossus was recovered as monophyletic (100/1.0), whereas Harrimania was non-monophyletic, with H. planktophilus sister to Protoglossus and H. kupfferi (Worsaae et al., 2012) recovered within Saxipendium. At present, whether Harrimctnia planktophilus or Harrimania kupfferi were misidentified cannot be determined. Alternatively, Harrimania may be in need of revision. Additionally, Protoglossus deserves further attention, as Cedhagen and Hansson (2013) reported a new species, Protoglossus bocki, which they placed close to Saxipendium. (As their sequences are not publicly available, P. bocki is not included here.) Interestingly, the late Cyril Burton-Jones, an accomplished hemichordate biologist, confirmed the Proto-glossus designation of both the P. bocki and the Protoglos-sus used in Cannon et al. (2009).
Within Ptychoderidae, Ptychodera and Balanoglossus were monophyletic genera (100/1.0, 97/1.0, respectively). Glossobalanus was rendered non-monophyletic because Balanoglossus was nested with Glossobalanus (95/100). Two spengelid species, Schizocardium braziliense and Glandiceps hacksi, formed a clade with strong support (100/1.0). Spengelidae was sister to Ptychoderidae + Torquaratoridae, as in Osborn et at., 2011. Interestingly, even with increased representation of all known families that reproduce via tornaria larvae, a tornaria larva collected in the Gulf Stream (Cannon et al., 2009) was still distinct from all other sequences, and fell sister to Spengelidae + Ptychoderidae + Torquaratoridae.
Within Torquaratoridae, two specimens, one from Iceland and one from Antarctica, both with distinctly torquaratorid morphotypes, were most closely related to undescribed Genus C from Osborn et al., 2012. A single specimen collected in the Ross Sea, Antarctica, was recovered as sister to Ptychoderidae + Torquaratoridae with poor bootstrap support (41/0.98).
Our results greatly increase our knowledge of deep-sea. Arctic, and Antarctic enteropneusts. Within the novel harrimaniid clade, subclades 3 and 4 comprise specimens collected at depths greater than 1500 m, yet this group is phylogenetically distinct from Saxipendium, the only known genus of deep-sea Harrimaniidae. This finding suggests that diversity of enteropneust taxa in the deep sea is underestimated, despite the recent spate of papers on large-bodied deep-sea species (Holland et al., 2005, 2009, 2012a, b; Osborn et al., 2011, Priede et al., 2012). These smaller worms were collected in cold waters either within or just south of the Arctic Circle, in Antarctica, or in the deep sea off the Oregon coast. Connections between deep-sea fauna and polar fauna have been hypothesized for other taxa (e.g., Strugnell et al., 2008), and this clade may be yet another example of this phenomenon.
Within our molecular phylogeny, this clade is distinct from other genera, yet three harrimaniid genera, Horstia, Mesoglossus, and Ritteria, have recently been described solely on the basis of morphology (Deland et al., 2010). The new clades' position in our phylogeny is consistent with the placement of Horstia in the phylogenetic hypothesis of harrimaniid genera by Deland et al. (2010). However, the present specimens do not demonstrate the extremely narrow trunk or conspicuous gonads arranged in a series of protruding nodules indicated in the generic description of Horstia (Deland et al., 2010) (Fig. 2A, B). Therefore, we suggest that the new clade represents a novel genus, although internal morphological characterization will be needed to fully assess its relationship to other harrimaniid taxa. In terms of morphology and apparent feeding strategy, these novel harrimaniids appeared similar to larger enteropneusts rather than to the considerably smaller (<0.6-mm long) Meioglossus psammophilus (Worsaae et al., 2012).
We also collected Arctic and Antarctic members of Torquaratoridae. Two torquaratorids were recovered sister to Genus C from Osborn et al. (2012)--but see Osborn et al. (2013). These specimens were collected from 732 m in the Norwegian Sea and 531 m in the Ross Sea, far shallower than previously reported depths for Torquaratoridae (1600--4000). Another specimen from Antarctica, "Enteropneusta Antarctica H89.3R" in Figure 3, is recovered sister to Ptychoderidae + Torquaratoridae with moderate support. This is an intriguing result, as this specimen may represent a basal member of Torquaratoridae, or possibly a distinct lineage. Enteropneusts have been essentially unknown from Antarctica, demonstrating gaps in our knowledge of Southern Ocean biodiversity. Sea floor imaging from the Ross Sea reveals large numbers of torquaratorids of multiple morphotypes (Halanych et at., 2013). Similarly to the harrimaniids discussed above. Antarctic continental shelf representatives of a predominantly deep-sea group strengthen hypotheses connecting deep-sea and Antarctic fauna (Gage, 2004; Strugnell et at., 2008). With these discoveries, we provide genetic evidence of at least four enteropneust species in Antarctic seas.
Revised molecular hypothesis of hemichordate evolution
Holland et al. (2012h) and Osborn et al. (2011) have hypothesized a shallow-water origin for enteropneusts on the basis of the comparatively few deep-sea species in Harrimaniidae, Ptychoderidae, and Spengelidae. Our results indicate broader diversity of harrimaniids in the deep sea and a close relationship between deep-sea Saxipendium and shallower species (Harrimania kupfferi and our specimen H74.1, collected at 916 m); they also demonstrate the presence of torquaratorids at <1000 m (Halanych et at., 2013; Osborn et al., 2013). In view of these findings, we suggest that statements on biogeographic patterns within this group may be premature. Continued discovery of unknown species and broader taxonomic categories, such as the novel clade of cold-water harrimaniids reported here, indicate that today's view of hemichordate diversity is highly incomplete, as recently described (Appeltans et al., 2012, supplement).
In the context of understanding the origins and diversity of hemichordate clades, the age and diversification times of various lineages are of interest. Fossils of rhabdopleurid and cephalodiscid pterobranchs are known from the Early and Middle Cambrian (Maletz et at., 2005; Rickards and Durman, 2006; Hou et al., 2011; Mitchell et at., 2012). In general, however, soft-bodied enteropneusts have a poor fossil record. The recently described Spartobranchus tenius (Caron et al., 2013) appears similar to modern torquaratorids (Halanych et al., 2013), and Mesobalanoglossus buergeri Bechly and Fricichinger, 1999 (in Frickhinger, 1999) from the Upper Jurassic may be within Ptychoderidae, but most other body fossils are outlines only, precluding family-level identification (Maletz, 2013). Thus, although several lineages do appear to be hundreds of millions of years old, rendering time calibration of lineages on a molecular topology is not possible. Similarly, discerning clear trends in evolution for lineages associated with particular habitats (e.g., deep sea, polar seas) is still difficult. Future efforts that uncover additional novel hem ichordate taxa will help resolve such issues.
Understanding hemichordate interrelationships is critical for inferring hemichordate ancestral states, and thus, further questions of deuterostome evolution. In particular, whether pterobranchs evolved from within enteropneusts or the two groups are reciprocally monophyletic has major bearing on character polarization within Hemichordata. Prior studies using 18S rDNA have recovered a Pterobranchia + Harrimaniidae clade (Halanych, 1995; Cameron et al., 2000; Bourlat et al., 2003; Cannon et at., 2009; Worsaae et at., 2012). This result suggests that early hemichordates were enteropneust-like, with pterobranchs arising from acorn worm ancestors (Rychel and Swalla, 2007; Brown et al., 2008; Cannon et at., 2009; Peterson et al., 2013). In contrast, a recent microRNA study (Peterson et al., 2013) found that Saccoglossus kowalevskii and Ptychodera flava share 12 microRNAs not found in Cephalodiscus hodgsoni. While these authors suggested that enteropneusts are monophyletic to the exclusion of pterobranchs. they examined only the three hemichordate taxa mentioned above, and thus taxon sampling is a major concern. Unfortunately, determination of early hemichordate character states cannot be conducted without further studies of individual homologous characters (Cannon et al., 2009; Peterson et al., 2013).
We were able to include three 16S rDNA sequences from the pterobranch genus Rhabdopleura in our analyses, as well as 18S rDNA data from two additional Rhabdopleura and three additional Cephalodiscus taxa. In individual analyses of 16S rDNA alone (not shown), Rhabdopleura sequences were recovered within the echinoderm outgroup as sister to ophiuroids. This result has previously been observed in analyses using the complete mitochondrial genome of Rhabdopleura compacta (Perseke et al., 2011). However, mitochondrial sequences of Rhabdopleura are extremely AT rich (Perseke et al., 2011; present study) and may produce artifacts due to long-branch attraction. In combined analyses, however, Pterobranchia and Enteropneusta are reciprocally monophyletic sister taxa with good support (98/1.00). Notably, support for enteropneust monophyly is moderate (65/0.99), and SH testing did not reject the alternative hypothesis (enteropneust paraphyly). If enteropneusts and pterobranchs are indeed monophyletic, studies on pterobranch morphology, evolution, and development must advance to the forefront, so that assessments of character evolution across Hemichordata can be made. To further validate these results, additional data, particularly genome-scale information, will be required.
We are indebted to .Saskia Brix, the IceAGE Project research team, and Senckenberg's German Center for Marine Biodiversity Research (DZMB) for support in collecting Icelandic hemichordates. Many thanks to the staff and crews of R/V Meteor, R/V N. B. Palmer, RN L. M. Gould. RN Kit Jones, R/V Pelican, R/V Hakon Mosby, RN Aurelia, and R/V Oceanus for assistance with obtaing specimens. We gratefully acknowledge Jon Norenburg and Darryl Felder for providing Rhabdopleura sp. from the Gulf of Mexico, supported by NSF DEB-0315995 to S. Fredericq and D. Felder at the University of Louisiana--Lafayette. Thanks to Christoffer Schander and Christiane Todt for providing Norwegian enteropneusts, and Richard Heard for assistance in collecting Balanoglossus cf. auranticus. Kevin Kocot, Mike Page, and Andrea Frey provided collection assistance. This work was supported by NSF DEB-0816892 to Ken Halanych and Billie J. Swalla. This material is supported in part by the National Science Foundation under Cooperative Agreement No. DBI-0939454 BEACON for B. J. Swalla. Antarctic collections were supported by NSF grants OPP-9910164 and OPP-0338087 to R. S. Scheltema and K. M. Halanych, and ANT-1043745 to K. M. Halanych, A. R. Mahon, and S. R. Santos. Support for collection of the Oregon worm was provided by NSF OCE-1155188 to K. M. Halanych and C. R. Smith. J. T. C. was supported by the ACHE-GRSP (NSF EPS-0447675), Bermuda Institute of Ocean Sciences' grants-in-aid program, and Friday Harbor Laboratories Patricia L. Dudley Fellowship. This is Molette Biology Laboratory contribution 20 and Auburn University Marine Biology Program contribution 114.
Reference: Biol. Bull. 225: 194-204. (December 2013) [c] 2013 Marine Biological Laboratory
Received 27 September 2013; accepted 12 November 2013.
* To whom correspondence should be addressed. E-mail: cannojt@ tigermail.auburn.edu, firstname.lastname@example.org
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
Appeltans, W., S. T. Ahyong, G. Anderson, M. V. Angel, T. Artois, N. Bailly, R. Bamber, A. Barber, L Bartsch, A. Berta, et at. 2012. The magnitude of global marine species diversity. Curr. Biol. 22: 21892202.
Boisvert, S., F. Laviolette, and J. Corbeil. 2010. Ray: simultaneous assembly of reads from a mix of high-throughput sequencing technologies. J. Comput. Biol. 17: 1519-1533.
Bourlat, S.. C. Nielsen, A. Lockyer, D. Littlewood, and M. Telford. 2003. Xenoturbella is a deuterostome that eats molluscs. Nature 424: 925-928.
Brown, F. D., A. Prendergast, and B. J. Swalla. 2008. Man is but a worm: chordate origins. Genesis 46: 605-613.
Cameron, C. B. 2005. A phylogeny of the hemichordates based on morphological characters. Can. J. Zool. 83: 196-215.
Cameron, C. B., and A. Ostiguy. 2013. Three new species of Glossa-balanus (Hemichordata: Enteropneusta: Ptychoderidae) from western North America. Zootaxa 3630: 143-154.
Cameron, C. B.. and M. Perez. 2012. Spengelidae (Hemichordata: Enteropneusta ) from the Eastern Pacific including a new species, Schizocardium californicum, from California. Zootaxa 3569: 79-88.
Cameron, C. B., J. Garey, and B. J. Swalla. 2000. Evolution of the chordate body plan: New insights from phylogenetic analyses of deuterostome phyla. Proc. Natl. Acad. Sci. USA 97: 4469-4474.
Cameron, C. B., C. Deland. and T. H. Bullock. 2010. A revision of the genus Saccoglossus (Hemichordata: Enteropneusta: Harrimaniidae) with taxonomic descriptions of five new species from the Eastern Pacific. Zootaxa 2483: 1-22.
Cannon, J. T., A. L. Rychel, H. Eccleston, K. M. Halanych, and B. J. Swalla. 2009. Molecular phylogeny of hemichordata. with updated status of deep-sea enteropneusts. Mol. Phylogenet. Evol. 52: 17-24.
Caron, J. B., S. Conway Morris, and C. B. Cameron. 2013. Tubiculous enteropneusts from the Cambrian period. Nature 496: 503-506.
Cedhagen, T., and H. G. Hansson. 2013. Biology and distribution of hemichordates (Enteropneusta) with emphasis on Harrimaniidae and description of Protoglossus bocki sp. nov. from Scandinavia. Helgol. Mar. Res. 67: 251-265.
Deland, C., C. B. Cameron, K. P. Rao. W. E. Ritter, and T. H. Bullock. 2010. A taxonomic revision of the family Harrimaniidae (Hemichordata: Enteropneusta) with descriptions of seven species from the Eastern Pacific. Zootaxa 2408: 1-30.
Frickhinger, K. A. 1999. Die Fossilien von Solnhofen (The Fossils of Solnhofen). Goldschneck Verlag, Korb, Germany.
Gage, J. D. 2004. Diversity in deep-sea benthic macrofauna: the importance of local ecology, the larger scale, history and the Antarctic. Deep-Sea Res. Pt. II 51: 1689-1708.
Halanych, K. M. 1995. The phylogenetic position of the pterobranch hemichordates based on 18S rDNA sequence data. Mol. Phylogenet. Evol. 4: 72-76.
Halanych, K. M., J. T. Cannon, A. R. Mahon, B. J. Swalla, and C. R. Smith. 2013. Modern Antarctic acorn worms form tubes. Nat. Commun. doi: 10.1038/ncomms3738.
Higgins, R. P., and H. Thiel. 1988. Introduction to the Study of Meiofauna. Smithsonian Institution Press, Washington DC.
Holland, N., D. Clague, D. Gordon, A. Gebruk, D. Pawson, and M. Vecchione. 2005. 'Lophenteropneust hypothesis refuted by collection and photos of new deep-sea hemichordates. Nature 434: 374-376.
Holland, N. D., W. J. Jones, J. Ellena, H. A. Ruhl, and K. L. Smith. 2009. A new deep-sea species of epibenthic acorn worm (Hemichordata, Enteropneusta). Zoosystema 31: 333-346.
Holland, N. D., L. A. Kuhnz, and K. J. Osborn. 2012a. Morphology of a new deep-sea acorn worm (class Enteropneusta, phylum Hemichordata): A part-time demersal drifter with externalized ovaries. J. Morphol. 273: 661-671.
Holland, N. D., K. J. Osborn, and L. A. Kuhnz. 2012b. A new deep-sea species of harrimaniid enteropneust (Hemichordata). Proc. Biol. Soc. Wash. 125: 228-240.
Hou, X.-G., R. J. Aldridge, David J. Siveter, Derek J. Siveter, M. Williams, J. Zalasiewicz, and X.-Y. Ma. 2011. An early Cambrian hemichordate zooid. Curr. Biol. 21: 612-616.
Hyman, L. H. 1959. The Invertebrates: Smaller Coe/ornate Groups. McGraw-Hill. New York.
Katoh, K., K.-I. Kuma, H. Toh, and T. Miyata. 2005. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33: 511-518.
Kocot, K. M., J. T. Cannon, C. Todt, M. R. Citarella, A. B. Kohn, A. Meyer, S. R. Santos, C. Schander, L. L. Moroz, B. Lieb, and K. M. Halanych. 2011. Phylogenomics reveals deep molluscan relationships. Nature 477: 452-456.
Maletz, J. 2013. Hemichordata (Pterobranchia, Enteropneusta) and the fossil record. Palaeogeogr. Palaeoclimatol. Palaeoecol. http://dx.doi.org/10.1016/j.palaeo.2013.06.010.
Maletz J., M. Steiner, and 0. Fatka. 2005. Middle Cambrian ptero-branchs and the question: what is a graptolite? Lethaia 38: 73-85.
Misof, B., and K. Misof. 2009. A Monte Carlo approach successfully
identifies randomness in multiple sequence alignments: a more objective means of data exclusion. Syst. Biol. 58: 21-34.
Mitchell, C. E., M. J. Me'chin, C. B. Cameron, and J. Maletz. 2012. Phylogenetic analysis reveals that Rhabdopleura is an extant graptolite. Lethaia 46: 34-56.
Nylander, J. A. A. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
Osborn, K. J., L. A. Kuhnz, L G. Priede, M. Urata, A. V. Gebruk. and N. D. Holland. 2012. Diversification of acorn worms (Hemichordata. Enteropneusta) revealed in the deep sea. Proc. R. Soc. Lond. B Biol. 279: 1646-1654.
Osborn, K. J., A. V. Gebruk, A. Rogacheva, and N. D. Holland. 2013. An externally brooding acorn worm (Hemichordata, Enteropneusta, Torquaratoridae) from the Russian Arctic. Biol. Bull. 225: 113-123.
Perseke, M., J. Hetmank, M. Bernt, P. F. Stadler, M. Schlegel, and D. Bernhard. 2011. The enigmatic mitochondrial genome of Rhabdopkura compacta (Pterobranchia) reveals insights into selection of an efficient tRNA system and supports monophyly of Ambulacraria. BMC Evol. Biol. 11: 134.
Peterson, K. J., Y.-H. Su, M. I. Arnone, B. J. Swalla, and B. L. King. 2013. MicroRNAs support the monophyly of enteropneust hemichordates. J. Exp. Zool. (Mol. Dev. Eva) 320: 368-374.
Priede, I. G., K. J. Osborn, A. V. Gebruk, D. Jones, D. Shale, A. Rogacheva. and N. D. Holland. 2012. Observations on torquaratorid acorn worms (Hemichordata. Enteropneusta) from the North Atlantic with descriptions of a new genus and three new species. Invertebr. Biol. 131: 244-257.
Rickards, R. B.. and P. N. Durman. 2006. Evolution of the earliest graptolites and other hemichordates. Pp. 5-92 in Studies in Palaeozoic Palaeontology, National Museum of Wales Geological Series, Vol. 25,
M. G. Bassett and V. K. Deisler, eds. National Museum of Wales, Cardiff.
Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformaties 19: 15721574.
Rychel, A. L., and B. J. Swalla. 2007. Development and evolution of chordate cartilage. J. Exp. Zool. (Mol. Dev. Evol.) 308B: 325-335.
Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51: 492-508.
Smith. K., N. D. Holland, and H. Ruhl. 2005. Enteropneust production of spiral fecal trails on the deep-sea floor observed with time-lapse photography. Deep-Sea Res. Pt. I 52: 1228-1240.
Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688-2690.
Strugnell, J. M., A. D. Rogers, P. A. Prodohl, M. A. Collins, and A. L. Allcock. 2008. The thermohaline expressway: the Southern Ocean as a centre of origin for deep-sea octopuses. Cladistics 24: 853-860.
Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739.
Winchell, C., J. Sullivan, C. B. Cameron, B. J. Swalla, and J. Mallatt. 2002. Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data. Mol. Biol. Evol. 19: 762-776.
Worsaae, K., W. Sterrer, S. Kaul-Strehlow, A. Hay-Schmidt, and G. Giribet. 2012. An anatomical description of a miniaturized acorn worm (Hemichordata, Enteropneusta) with asexual reproduction by paratomy. PloS ONE 7: e48529.
JOHANNA T. CANNON (1), *, BILLIE J. SWALLA (2), (3), AND KENNETH M. HALANYCH (1), (3), *
(1) Molette Biology Laboratory for Environmental and Climate Change Studies, Department of Biological Sciences, Auburn University, Auburn, Alabama 36849; (2) Biology Department, University of Washington, Seattle, Washington 98195; and (3)Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington 98250
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|Author:||Cannon, Johanna T.; Swalla, Billie J.; Halanych, Kenneth M.|
|Publication:||The Biological Bulletin|
|Date:||Dec 1, 2013|
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