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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

Organismal collection

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

                  Cephalodiscus      South           62[degrees]
                  fumosus H5.3       Shetlands,          45.03'S

                  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

                  Saccoglossus sp.   Friday Harbor.  48[degrees]
                  H44.2              Washington,         32.6I'N

                  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

                  Balanoglossus cf.  Horn Island,    30[degrees]
                  aurantiaca H52.1   Mississippi,        14.75'N

                  Glossohalanus      Norway          58[degrees]
                  marginatus H48.2                       55.00'N

                  Glossobalanus      False Bay,      48[degrees]
                  berkeleyi H68.2    Washington,         28.93'N

                  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

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

                  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

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


Cephalodiscidae  Cephalodiscus hodgsoni   KF683576   --
                 HI 1.3
                 Cephalodiscus hodgsoni   EU728441   --
                 Cephalodiscus fumosus    KF83575    --
                 Cephalodiscus            KF683574   --
                 nigrescens H12.3
                 Cephalodiscus gracilis   AF236798   --
                 Cephalodiscus densus     EU728439   --
                 Cephalodiscus            EU728440   --

Rhabdopleuridae  Rhabdopleura sp. 1       KF6K3598   KF683562
                 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
                 Harrimanici              AF236799   EU728421
                 Protoglossus sp.         EU728432   EU728420
                 Meioglossus              JF900488   JX855287
                 Saccoglossus             KF683588   KF683545
                 mereschkowskii H53.1
                 Saccoglossus sp. H44.2   KF683577   KF683544
                 Harrimaniidae H62.1      KF683587   KF683546
                 Saccoglossus             AF236801   L26348
                 Saccoglossus             L28054     NC_007438
                 Saccoglossus pusillus    AF236800   EU728422
                 Saxipendium coronatum    EU728433   EU728423
                 Saxipendium coronatum    EU 520505  EU520493
                 Saxipendium              JN886774   JN886756
                 Harrimaniidae H74.1      KF683589   KF683551
                 Stereobalanus            EU728434   EU728424

Ptychoderidae    Balanoglossus sp. 1      KF683570   KF683557
                 Balanoglossus cf.        KF683S69   KF683555
                 aurantiaca H51.1
                 Balanoglossus cf.        KF683568   KF683556
                 aurantiaca H52.1
                 Balanoglossus            --         EU728425
                 Balanoglossus carnosus   JF900489   --
                 Balanoglossus curnosus   D14359     AF051097
                 Tampa Ptychoderid        AF278685   EU728427
                 Glossobalanus            KF683566   KF683559
                 marginatus H48.2
                 Glossobalanus berkeleyi  KF6K3567   KF683554
                 Glossobalanus            EU728435   EU728426
                 Glossobalanus minutus    AF119089   --
                 Ptychodera bahamensis    KF683571   KF683560
                 Ptychodera bahamensis    JF900485   JX855285
                 Ptychodera bahamensis    JF900486   --
                 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
                 Tergivelum baldwinae     EU520509   EU520497
                 Tergivelum baldwinae     JN866772   EU520495
                 Tergivelum baldwinae     JN886770   JN886753
                 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
                 Yoda purpurata           JN886758   JN886741
                 IFREMER Enteropneust     EU728438   EU728431


Crinoidea        Metacrinus rotundus      AY275898   AY275905

Ophiuroidea      Gorgonocephalus          DQ060790   DQ297092
                 Amphipholis squamata     X97156     NC_013876

Astern idea      Solaster stimpsoni       DQ060819   DQ297113
                 Qdontaster validus       DQ060801   GQ294457

Holothuroidea    Psychropotes             ZS0956     DQ777099
                 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 ( 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).


Cold-water enteropneusts

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@,

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(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
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Date:Dec 1, 2013
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