Zooid Morphology and Molecular Phylogeny of the Graptolite Rhabdopleura Annulata (Hemichordata, Pterobranchia) from Heron Island, Australia

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Canadian Journal of Zoology

Zooid morphology and molecular phylogeny of the graptolite Rhabdopleura annulata (Hemichordata, Pterobranchia) from Heron Island, Australia

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2020-0049.R2

Manuscript Type: Article

Date Submitted by the 16-Oct-2020 Author:

Complete List of Authors: Ramirez Guerrero, Greta; Université de Montréal, Sciences biologiques Kocot, Kevin; The University of Alabama System Cameron, DraftChristopher; Université de Montréal, Sciences biologiques Is your manuscript invited for consideration in a Special Zoological Endeavors Inspired by A. Richard Palmer Issue?:

Rhabdopleura annulata, Pterobranchia, graptolite, rhabdopleurid, Keyword: Australia, PHYLOGENY < Discipline, HEMICHORDATA < Taxon

Zooid morphology and molecular phylogeny of the graptolite Rhabdopleura annulata

(Hemichordata, Pterobranchia) from Heron Island, Australia1

Greta M. Ramírez-Guerrero*, Kevin M. Kocot+, and Christopher B. Cameron*

* Université de Montréal, Département de sciences biologiques, C.P. 6128, Succ. Centre-ville, Montréal,

QC, H3C 3J7, Canada. [email protected]; [email protected]

+ The University of Alabama and Alabama Museum of Natural History, 500 Hackberry Lane, Tuscaloosa,

AL 35487, USA. [email protected]

Draft

1This article is one of a series of invited papers arising from the symposium “Zoological En- deavours Inspired by A. Richard Palmer” that was co-sponsored by the Canadian Society of Zo- ologists and the Canadian Journal of Zoology and held during the Annual Meeting of the Cana- dian Society of Zoologists at the University of Windsor, Windsor, Ontario, 14–16 May 2019.

1 © The Author(s) or their Institution(s) Canadian Journal of Zoology Page 2 of 21

Ramírez-Guerrero, G.M., Kocot, K., and Cameron, C.B. Zooid morphology and molecular phylogeny of Rhabdopleura annulata from Heron Island, Australia.

ABSTRACT

Rhabdopleura is one of the longest surviving animal genera. The five-known species are the only living Graptolithina, a group well known from their diverse Paleozoic fossil record. Here we add information on the soft bodied zooids and molecular phylogenetics of Rhabdopleura annulata

Norman, 1921, which was previously only known from its tubes. Tubes and zooids were collected from Heron Island, Australia. Zooids have a single pair of tentaculated arms. Dark pigment granules are found throughout the body, and particularly dense in the pair of arms and the anterior lip of the cephalic shield. ColoniesDraft grow encrusted in and on coral debris. The tubes are either creeping or erect, but no stolon has been found. Inside of the coral matrix lacunae, the tube cortex formed a parchment-like wallpaper. Phylogenetic analysis based on combined

18S+16S rRNA sequences placed R. annulata as sister to the remaining rhabdopleurids, albeit with weak support. The biogeographic range of R. annulata extends from Indonesia to Tasmania, and New Zealand. Its occurrence on Heron Island does not extend this range, but highlights that rhabdopleurids may be more common, and in shallower waters, than previously appreciated, permitting further studies that may shed light on graptolite paleobiology.

Key words: Rhabdopleura annulata, pterobranch, graptolite, rhabdopleurid, Australia.

2 © The Author(s) or their Institution(s) Page 3 of 21 Canadian Journal of Zoology

INTRODUCTION

Pterobranchs represent one of the two clades that form the phylum Hemichordata. This class of

tube-dwelling organisms includes all fossil forms known as graptolites, as well as living

representatives from the genera Atubaria, Cephalodiscus and Rhabdopleura. The first report of

Rhabdopleura was made in the description of the living species R. normani by Allman (1869),

who collected it from dredging in an archipelago in Scotland. Originally considered a bryzoan,

Rhabdopleura is now recognized as an extant member of the subclass Graptolithina (class

Pterobranchia), and includes five known living species and several fossil forms. The most

recently described living species include R. annulata Norman, 1921 and R. recondita Beli,

Cameron and Piraino, 2018.

The taxonomic description of R.Draft annulata is based on the tubarium structure. Nothing

was previously known about its zooid morphology, or its molecular sequences, limiting our abil-

ity to understand the evolution of the species and rhabdopleurids in general. Additional

rhabdopleurid sequence data are particularly needed because the clade has long branch length in

18S, 16S, mitochondrial, and phylogenomic data sets (Halanych 1995; Cameron et al. 2000;

Cannon et al. 2013; Beli et al. 2018; Li et al. 2019). To contribute to the knowledge of this ge-

nus, here we describe the tubes and zooids of R. annulata from the Great Barrier Reef and in-

clude a new sequence for this species to construct a phylogeny of the genus.

MATERIALS AND METHODS

Samples were collected by snorkeling at ~50 m from shore in less than 1 m of water off Heron

Island, Queensland, Australia (approximately 23° 26.43' S, 151° 54.70' W) in February 2014.

Colonies were found growing in a calcareous matrix where the algae Amphiroa was dominant,

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some colonies were possible to isolate from the substratum. Specimens containing tubes and zooids were fixed in 95% ethanol and 2.5% glutaraldehyde in filtered sea water (with the latter transfer to 70% ethanol) and stored at room temperature. Other zooids were preserved in

RNAlater or placed directly into Ambion RNAqueous lysis buffer for RNA extraction and stored at -80°C. RNA extraction, transcriptome library preparation and sequencing, and mining of sequence regions of interest from the transcriptomes was performed as described in Li et al.

(2018). The raw transcriptome reads are available on NCBI SRA under accession number

SRR11101525.

Rhabdopleura sequences from other species were obtained from GenBank (Table 1).

These sequences include R. normani from Bermuda (Halanych 1995; Worsaae et al. 2012), R.

compacta Hincks, 1880 from the UnitedDraft Kingdom (Perseke et al. 2011), R. recondita from Italy

(Beli et al. 2018), and two Rhabdopleura sp. collected from Florida and Iceland (Cannon et al.

2013). Other hemichordates were sampled including Cephalodiscus fumosus John, 1931 from

Antarctica, and Saccoglossus pusillus Ritter, 1902 and Harrimania planktophilus Cameron, 2002

from British Columbia, Tergivelum cinnabarinum Priede, Osborn, Gebruk, Jones, Shale,

Rogacheva and Holland, 2012 and Yoda purpurata Priede, Osborn, Gebruk, Jones, Shale,

Rogacheva and Holland, 2012 from the Mid-Atlantic Ridge, Balanoglossus carnosus Müller in

Spengel, 1893 from Japan, and Ptychodera flava Eschscholtz, 1825 from French Polynesia. An

echinoderm (Odontaster validus Koehler, 1906) was also sampled and the cephalochordate

(Branchiostoma lanceolatum (Pallas, 1774)) was used to root the tree.

Sequences for 16S and 18S from all those species were aligned with MAFFT 7.407 using the default settings. Ambiguously aligned regions were trimmed with Gblocks 0.91b (b1 = half the number of sequences plus one, b2 = b1, b3 = 8, b4 = 2, b5 = a). Phylogenetic analyses were

4 © The Author(s) or their Institution(s) Page 5 of 21 Canadian Journal of Zoology

conducted in RAxML 8.2.4 with the “-f a” flag, which specifies a search for best scoring ML tree

and a rapid bootstrap analysis in one program run. Each matrix was partitioned by gene and

analyzed with the GTRGAMMA model. Nodal support was assessed with the appropriate

number of rapid bootstraps (-N autoMRE). For the combined analysis of 16S+18S, the matrix

was partitioned by gene.

TAXONOMY

Class Pterobranchia Lankester, 1877

Subclass Graptolithina Bronn, 1849

Family Rhabdopleuridae Harmer, 1905

Genus Rhabdopleura Allman, 1869 Draft

Type species: Rhabdopleura normani Allman, 1869

Diagnosis: Tubarium consist of basal creeping tubes with regular zigzag sutures, and parallel

erect tubes with irregular fusellar rings that slightly widen in length. A black stolon system

connects the tripartite zooids allowing movement along the erect tubes. Zooids possess a single

pair of arms bearing ciliated tentacles and a U-shaped gut.

Rhabdopleura annulata Norman, 1921

Fig. 1.

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Neotypes. Field numbers KK254.2G, KK254.3G and KK254.5E from Heron Island, Australia, deposited in the Alabama Museum of Natural History.

Diagnosis. Encrusting colonies with isolated erect tubes with irregular fuselli and zigzag patterned creeping tubes. Pronounced external projections of fusellar rings frequently show a T- shape. Zooids with brown pigments, especially abundant in the pair of tentacled arms.

Description. Colony consists of isolated tubes and tubes of varying length embedded within a

calcium carbonate coral matrix. Erect tubes with up to 45 irregular full fusellar rings with an

average distance of 40 μm (ranging from 30 to 50 μm). Creeping tubes with regularly spaced

fuselli forming a zig-zag pattern. Tubes Draftslightly conical with internal diameter from 150-140 μm

at the base and 170-190 μm distally. Erect tube lengths and the corresponding number of fusellar

rings varies considerably. Erect tubes grow at right angles from the creeping tube and do not

branch. Rough external projections of the fusellar rings very clearly marked measuring up to 35

μm in thickness from the smooth internal surface, resulting in a maximal external tube diameter

of 220 μm.

Isolated brownish zooids measure 650 μm long. Cephalic shield has dark pigment

granules especially at the anterior margin, and some lighter pigments. Dorsal collar has two arms

that bear numerous tentacles with dark pigment granules. Metasome is oblong and contains

sporadic pigments, slightly bigger than those of the tentacles. Intestine is a typical U-shape and

the anus appears as a black spot at the anterior dorsal metasome, near the collar (Fig 1D).

Contractile stalk extends posteriorly from the anterior half of the ventral trunk, it is pigmented

along the margin closest to the body. Clear evidence of soft or sclerotized stolons were not

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observed. One tube contained a retracted zooid with a total length of 570 μm, of which 420 μm

was the arms and the remainder was the trunk. Arms were compressed inside of the tube and no

detailed anatomy was observed but for numerous tentacles with red pigments. Isolated erect

tubes were tanned light brown, and longer than those encrusting the CaCO3 substrate. Encrusted

tubes were transparent, shorter and numerous, encrusting over one surface of coral fragment,

possibly indicating young colonies. Tube colouration may darken, and fusellar ring number

increases with age and length. Basal end of the tubes may have complex interconnections, but

this could not be determined precisely because tubaria could not be isolated from the substrate

without damaging the material.

Remarks Draft

The Heron Island specimens are Rhabdopleura annulata. For practical purposes, most of the

measurements were taken from specimens found isolated that were likely adult tubes and zooids.

Measurements closely agree with Rhabdopleura annulata originally described by Norman

(1921) for specimens collected in Three Kings Island, New Zealand during the Terra Nova

expedition (tube diameter = 160-200 μm; maximum external diameter = 265 μm; minimum and

maximum wall thickness = 9-13 μm and 30-60 μm, respectively; separation between fusellar

rings = 50 μm). Based on the internal diameter and total thickness of the tubarium, Norman

(1921) also suggested that a rhabdopleurid collected by Harmer (1905) in Indonesia during the

Siboga expedition, was R. annulata. Measurements of our Australian specimens are particularly

consistent with those from the north of New Zealand. This species was originally erected based

on the extensively projecting fuselli, and the erect tubes that projected upright from the creeping

stem, differentiating it from the other known Rhabdopleura species.

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Some of the morphological characteristics that distinguish these specimens from other

Rhabdopleura species are related to dimensional features. R. compacta zooids and tubes are

small relative to the other four Rhabdopleura species. The tubes have a distal diameter of 183

μm and the fusellar rings are more compacted with an average of 30 μm between them (Stebbing

1970a). R. annulata and R. recondita zooids are similar in size. R. striata Schepotieff, 1909 from

Sri Lanka has the biggest colonies (7-8 cm long), and erect tubes with a length of 11 mm and

diameter of 1 mm, with particular longitudinal striations for which the species is named. The

dark brown zooids are the largest recorded for a rhabdopleurid, measuring around 1-1.5 mm

(Schepotieff 1909). R. normani shares with R. annulata the presence of densely pigmented

zooids but differs in coloration of the zooids and pigments, which tends to be lighter and forming

clusters in R. normani. This species coloniesDraft are usually several centimetres long (Stebbing

1970a).

R. annulata, R. recondita, and R. compacta zooids have several dark granules, most abundant in

the cephalic shield and tentacles. Zooid colour differences are present but are not significant,

because colour could be related to aging. R. recondita also presents smooth creeping-like tubes

lacking the typical zigzag fuselli, because these species occupy interstitial CaCO3 habitats.

Norman (1921) is the only description of the species, though it is based solely on tubaria because

the zooids were poorly preserved. The holotype of Rhabdopleura annulata is unknown since no

material was listed by Norman (1921). No type material has been designated ever since,

therefore our specimens represent a complete re-description for the species. However, based on this re-description, we recognize R. annulata as a valid species, that may be local to the southwest Pacific but show distinctive features that differentiate it from other rhabdopleurids, in morphology, distribution and phylogeny. Also, it is possible that R. annulata has an extended

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distribution. For example, the pterobranch collected from Fiji (Dilly and Ryland 1985) was not

assigned to a specific species. Based on distribution, it would be logical to classified as R.

annulata, but when considering its morphology and size it resembles more R. normani. This

raises the question of the level of plasticity and mobility that the genus may have, and highlights

the importance of phylogenetic analyses to try to conceal these discrepancies.

MOLECULAR PHYLOGENY

Phylogenetic trees were constructed using 18S (Fig. 2A) and combined 16S+18S rRNA

sequences Fig. 2B). There was no 16S sequence available for R. normani and therefore it is

absent from Figure 2A. In the 18S tree, R.Draft annulata from Heron Island forms a clade with R.

normani from Bermuda, which is sister to R. recondita from Italy. The combined sequences tree

places R. annulata as sister to all other rhabdopleurids, albeit with weak support. This, and the

branch lengths reinforce the morphological evidence that R. annulata is a distinct and unique

species. Both trees found Cephalodiscus as sister to the rhabdopleurids, supporting the

monophyly of the rhabdopleurids and the class Pterobranchia; this clade remains when more

cephalodiscids are considered in the analysis (Li et al. 2019).

e Th combined 16S+18S tree (Fig. 2B) shows the Caribbean R. normani and

Rhabdopleura sp. 1 from Iceland as the sister group of the Mediterranean R. recondita, and

together are the sister group to R. compacta from the UK. R. annulata branches basal to all other

rhabdopleurids. This tree indicates monophyly of the Pterobranchia and Enteropneusta, which

groups ptychoderids and torquaratorids as the sister group of the harrimaniids, and agrees with

the latest phylogeny (Cannon et al. 2013; Simakov et al. 2015; Li et al. 2019). Cannon et al.

9 © The Author(s) or their Institution(s) Canadian Journal of Zoology Page 10 of 21

(2013) identified Rhabdopleura sp. 2 as possible colonies of R. normani, since this sequence always groups with those from Bermuda, which is conceivable based on distribution. This is supported by the low 18S rRNA genetic distance obtained by Beli et al. (2018) among these two.

DISCUSSION

The Graptolithina are a diverse and important component of Early–Middle Palaeozoic fossil

biotas. The subdivision Eugraptolithinia is comprised of the well-known planktonic and benthic

graptolites, and they appear to be derived from a Rhabdopleura‐like species (Mitchell et al.

2013). Graptolites, including Rhabdopleura, have their origin in the Middle Cambrian (Durman

and Sennikov 1993; Sennikov 2016), about 520 million years ago, and disappeared in the

Carboniferous Period, around 320 millionDraft years ago, with the exception of Rhabdopleura, the

only living graptolite (Mitchell et al. 2013). Rhabdopleura is, geologically speaking, one of the

oldest genera of animal (Durman and Sennikov 1993) and so studies of living Rhabdopleura may

allow one to infer the zooid morphology, variation and precise measurements of tubes, molecular

systematics, and palaeobiology of fossil graptolites in direct relation to this living member of the

clade.

An outcome of this study is that we now have a morphological description of the zooids

of all five living Rhabdopleura species. With the notable exception of R. striata, Rhabdopleura zooids are smaller than Cephalodiscus, with a single pair of arms, and they show little colour

variation. The arms and the anterior cephalic shield have dark, decay-resistant pigment granules

of undermined function (Beli et al. 2018), but the anterior locations suggest a role in light detection, or as a sunblock. The arms have serotonin and dopamine reactive cells and the tentacles have serotonin and GABAergic cells (Strano et al. 2019). Paralleling the low taxonomic

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diversity for the genus (Chapman et al. 1995; Sennikov 2016), is a low zooid disparity, which is

remarkable considering their existence for over 500 million years.

The tubarium of R. annulata had been previously characterized (Norman 1921), and

those measurements match the ranges of the Heron Island specimens. Rhabdopleurid tubes are

benthic, and typically include an encrusting creeping tube with zig-zag shaped fuselli, and erect

tubes that branch from the creeping tube, with complete ring fuselli, and contain a single zooid.

Colonies are established by a planula larva that settles and secretes a dome shaped prosicula,

where they undergo metamorphosis to a zooid that then emerges from a small hole and begins

secreting a tube (Stebbing 1970b; Dilly 1973; Strano et al. 2019). R. annulata and R. recondita

are unusual in that the larva settles in the lacunae of coral debris, or dead bryozoan, respectively.

The creeping tube then takes the form ofDraft a thin parchment that lines the walls of the lacunae.

This tube form is difficult to observe, even following removal of the CaCO3 matrix with an

acidic solution. It is unlikely that this parchment-like form would be found in graptolite fossils.

A well-developed creeping stolon with a pectocaulus/ gymnocaulus was not found in our limited

specimens. A reason why stolons were not observed may include the nature of the colony

growth. Colonies grow deeply embedded in the calcareous matrix, which was difficult to

dissolve or break without damaging the tubes. Traces of black filaments that resemble black

stolons were noticed, however, a significant feature that indicates the existence of stolons is the

contractile stalk preserved in the zooid (Fig. 1D ). Asexually budded rhabdopleurid zooids

emerge from the pectocaulus/ gymnocaulus stolon, a plesiomorphic character of graptolites

(Mitchell et al. 2013).

Our molecular phylogenetic analyses for R. annulata bolster the morphological data that

supports that it is a distinct species, based on tubes and zooids. Figure 2 includes four of the five

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described living species of Rhabdopleura. There is no sequence data for R. striata. There is sequence data for an undescribed species from Iceland (Cannon et al. 2013, Fig. 2). The combined

18S+16S tree place R. annulata as the sister to the remaining rhabdopleurids, suggesting that this species may be particularly germane to understanding the biology of the extinct graptolites, though the support for relationships among Rhabdopleura species is poor. The topology of the

remaining hemichordate species is congruent with previous studies. The 18S tree finds entero- pneust paraphyly whereas the combined tree agrees with the more taxon and data rich studies that place the Enteropneusta and Pterobranchia as monophyletic sister groups (Priede et al. 2012;

Cannon et al. 2013; Simakov et al. 2015).

A review of the biogeography of rhabdopleurids (Beli et al. 2018) hypothesized that ocean basins may structure species distributions.Draft The five species of Rhabdopleura have a global occurrence mainly at higher latitudes. R. compacta is found in the UK in the North and English

Channels (Hincks 1880; Jullien 1890; Stebbing 1970a). R. recondita is found in shallow water in the Mediterranean Sea, and R. striata from coral reefs in Sri Lanka (Schepotieff 1909). Two other taxa, R. manuabialis and R. grimaldi from the Azores, are only known from their original descriptions by Jullien (1890) and Jullien and Calvet (1903), respectively, but are likely R. normani (Beli et al. 2018). R. normani, is unusual in that it is reported to have a global distribution. It is found in North Atlantic basins including the North Sea, Labrador Sea,

Norwegian Sea, from Bermuda and the Azores, and from the Argentine Sea, and the Antarctic

Ocean (Burdon-Jones 1954; Dilly and Ryland 1985). R. annulata is restricted to the southwest

Pacific Ocean, where it is found in Indonesia (Norman 1921), northern New Zealand, Heron

Island (north east Australia), St Vincent Gulf (south Australia), and east of Tasmania where it was collected from 183 to 549 meters depth (Norman 1921; Johnston 1937). Our finding of R.

12 © The Author(s) or their Institution(s) Page 13 of 21 Canadian Journal of Zoology

annulata from Heron Island does not expand the range as much as it demonstrates that these

animals may be more abundant, and in shallower water, than previously appreciated.

Rhabdopleura, regardless of species or biogeography, are frequently found adherent to

calcium carbonate debris of bryozoan, bivalves, brachiopods or coral (Stebbing 1970a; Dilly

1971; Sato et al. 2008; Beli et al. 2018). They require a hard substrate to attach, grow and feed.

This, and the short lived, non-feeding planaria larva, may limit rhabdopleurids ability to

distribute across large, sedimentary, hadal planes. This observation can be extended to the fossil

Rhabdopleura species, which come from single localities (Chapman et al. 1995). This is in sharp

contrast to the planktonic graptolites. On their death, planktonic forms would sink to the deep sea

and fossilize in the fine sediments, providing a rich cosmopolitan fossil record that makes them

indicators of disjunct, global sediment layersDraft (Goldman et al 2013). Rhabdopleurid fossils are

likely rare because they reside on hard substrate, have a limited biogeography (Durman and

Sennikov 1993; Chapman et al. 1995) and perhaps also because the zooids may have resided in

the lacunae of calcium carbonate debris, where they lack well developed creeping tubes.

ACKNOWLEDGEMENTS

This Festschrift paper is dedicated to CBC’s PhD supervisor A.R. Palmer. Rich encouraged CBC

to ask the big questions and to balance assured and daring projects. At a time when no lab had a

program investigating Hemichordata, Rich provided the opportunity, scientific independence, en-

thusiastic guidance and commiseration when experiments failed. It was Bermuda, autumn 1996,

when Rich and CBC first encountered the pterobranchs Rhabdopleura and Cephalodiscus, and

the fascination with these creatures remains. We thank the Heron Island Research Station, and

the United States National Science Foundation and International Postdoctoral Fellowship to

KMK that supported the collection of these specimens.

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FIGURE CAPTIONS

Table 1. NCBI accession numbers for sequences used in phylogenetic analyses.

Figure 1. Rhabdopleura annulata Norman, 1921. A) Adult colony encrusted in matrix before their isolation. B) Small transparent tubes showing fuselli preserved all over a calcareous substrate. C) Fragment of creeping tube showing a regular zigzag pattern. D) Zooid showing protosoma with cephalic shield (cs); mesosome with collar (c) and their two arms (a) with several pigmented tentacles (t); and trunk (t) containing the U-shaped digestive system, particularly the intestines (i) and the anusDraft (n), connected to the contractile stalk (cst). E) Isolated erect tubaria with a brownish coloration and irregular full rings, fusellar projections can be observed at the exterior part of the tube. T-shaped projections are particularly notorious in the basal right part of the tube. F) Tube containing a retracted pigmented zooid. G) Colony extracted from substrate showing three erect tubes with different lengths and number of fusellar rings.

Figure 2. Molecular phylogenetic trees constructed using alignments of A) 18S rRNA sequences, and B) combined 16S+18S rRNA sequences. Values at each node indicate bootstrap values.

18 © The Author(s) or their Institution(s) Page 19 of 21 Canadian Journal of Zoology Table 1. NCBI accession numbers for sequences used in phylogenetic analyses.

Accession numbers Taxa 18S rDNA 16S rDNA Cephalodiscus fumosus John, 1931 KF683575 — Rhabdopleura annulata Norman, 1921 SRR11101525 — Rhabdopleura recondita Beli et al., 2018 LT714192 LT714188 Rhabdopleura recondita LT714193 LT714189 Rhabdopleura recondita LT714194 LT714190 Rhabdopleura recondita LT714195 LT714191 Rhabdopleura recondita KU873083 KU873084 Rhabdopleura compacta Hincks, 1880 DRR198815 FN908482 Rhabdopleura normani Allman, 1869 JF900483 — Rhabdopleura normani U15664 — Rhabdopleura sp. 1 KF683598 KF683562 Rhabdopleura sp. 2 KF683597 KF683563 Saccoglossus pusillus Ritter, 1902 AF236800 EU728422 Harrimania planktophilus Cameron, 2002 AF236799 EU728421 Ptychodera flava Eschscholtz, 1825 EU728436 EU728429 Balanoglossus carnosus Müller in Spengel, 1893 D14359 AF051097 Tergivelum cinnabarinum Priede et al. 2012 JN886770 JN886753 Yoda purpurata Priede et al., 2012 JN886757 JN886740 Odontaster validus Koehler, 1906 DraftDQ060801 GQ294457 Branchiostoma lanceolatum (Pallas, 1774) AY428817 NC001912

Rhabdopleura annulata Norman, 1921. A) Adult colony encrusted in matrix before their isolation. B) Small transparent tubes showing fuselli preservedDraft all over a calcareous substrate. C) Fragment of creeping tube showing a regular zigzag pattern. D) Zooid showing protosoma with cephalic shield (cs); mesosome with collar (c) and their two arms (a) with several pigmented tentacles (t); and trunk (t) containing the U-shaped digestive system, particularly the intestines (i) and the anus (n), connected to the contractile stalk (cst). E) Isolated erect tubaria with a brownish coloration and irregular full rings, fusellar projections can be observed at the exterior part of the tube. T-shaped projections are particularly notorious in the basal right part of the tube. F) Tube containing a retracted pigmented zooid. G) Colony extracted from substrate showing three erect tubes with different lengths and number of fusellar rings.

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Molecular phylogenetic trees constructed using alignments of A) 18S rRNA sequences, and B) combined 16S+18S rRNA sequences. Values at each node indicate bootstrap values.

149x222mm (600 x 600 DPI)