Phylogenetic analysis of a group of palaeonemerteans (Nemertea) including two new species from Queensland and the Great Barrier Reef, Australia Blackwell Science, Ltd PER SUNDBERG, RAY GIBSON & URBAN OLSSON Accepted 8: March 2002 Sundberg, P., Gibson, R. & Olsson, U. (2003). Phylogenetic analysis of a group of palaeonemerteans (Nemertea) including two new species from Queensland and the Great Barrier Reef, Australia. — Zoologica Scripta, 32, 279 – 296. Based on 18S rDNA nucleotide sequences and morphological characters, we reconstruct the phylogeny for a group of palaeonemerteans estimated to be monophyletic. Two new palaeonemertean species from Queensland and the Great Barrier Reef, Australia are included in the phylogenetic analysis. The results confirm that one of the species, Cephalothrix queenslandica sp. n., is part of the Cephalothrix−Cephalotrichella−Procephalothrix group. These genera are redefined phylogenetically under the name Cephalothrix based on the cladistic analysis. The other species, Balionemertes australiensis gen. et sp. n., is placed in a new genus which forms a sister taxon to Cephalothrix. The morphology of both new species is described in detail. Per Sundberg & Urban Olsson, Department of Zoology, Göteborg University, PO Box 463, SE405 300 Göteborg, Sweden. E-mail: p.sundberg@zool.gu.se. Ray Gibson, School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK. Introduction As currently classified, the phylum Nemertea contains four major taxa: Palaeo-, Hetero-, Bdello- and Hoplonemertea. Sundberg & Hylbom (1994) carried out a cladistic analysis of the hitherto described palaeonemertean species for which sufficient characters could be identified from the literature and/or type material. They showed that some of the long established groups within Palaeonemertea, for example the genera Cephalothrix, Cephalotrichella and Procephalothrix, are paraphyletic. A more recent study (Sundberg et al. 2001) (Fig. 1) has shown that Palaeonemertea as a whole (rather than just groups within the taxon) is paraphyletic and that Hoplo- and Bdellonemertea should be combined within a monophyletic taxon. Since Sundberg & Hylbom were unaware in 1994 of the palaeonemerteans being non-monophyletic, their results should be interpreted with care since their analysis was based on this assumption. Two new species recently collected from locations around Queensland, Australia, have been recognized as being palaeonemertean in the broader sense. Taxonomic studies on the marine nemertean fauna of Australia in recent decades have mainly concerned species from this region (Gibson 1979a,b, 1981a,b, 1982a,b, 1983) or from Western Australia (Campbell et al. 1989; Gibson 1990a, 1999; Gibson & Jones 1990; © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 Sundberg & Gibson 1995), although a few taxa have been recorded from other parts of eastern Australia (Gibson 1974, 1978, 1979c; Moore & Gibson 1981). In this paper we describe the morphology of the new species and assess their phylogenetic position in two separate analyses. The first analysis was based on sequences of the 18S rDNA gene from a limited number of species (from Sundberg et al. 2001) together with sequences from the new species. Its purpose was to provisionally estimate the phylogenetic position of the new taxa in order to determine which group (from Sundberg & Hylbom 1994) among the palaeonemerteans (in the broad sense) would benefit from a more detailed analysis. The second analysis was performed based on the morphological characters used by Sundberg & Hylbom (1994). Our results support the view that Cephalothrix, Cephalotrichella and Procephalothrix are paraphyletic and we combine them in the redefined, more inclusive, taxon Cephalothrix. Materials and methods Specimens for histological examination and DNA extraction Specimens of the two species described in this paper were collected by P.S. in July 1995 from locations along the coast of Queensland and in the Great Barrier Reef area. The specimens were anaesthetized in MgCl2 after in vivo examination. 279 Palaeonemertean phylogeny and two new species • P. Sundberg et al. 2.0 µM MgCl2, 0.2 µM of each primer (PCRA and PCRB), 200 µM of each dNTP, 1 × reaction buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl) and 2 U of Taq polymerase (Perkin Elmer). The PCR cycling parameters for double stranded amplification were 2 min 30 s at 95 °C for initial denaturation, followed by 60 cycles of 30 s at 95 °C, 30 s at 45 °C, and 2 min at 72 °C. The cycle concluded with a 7 min sequence extension at 72 °C. Amplified products were purified by QIAquick PCR Purification Kit (Qiagen, Inc). Sequencing was performed using Cy5-labelled primers on an ALFExpress Automatic Sequencer (Pharmacia). ThermoSequenase (Amersham) was used for the sequencing reactions, applying a two-step cycle 2 min denaturation at 96 °C, followed by 20 cycles of 30 s at 95 °C and 40 s at the annealing/ extension temperature. Sequences, read in both directions, are deposited with GenBank (accession numbers AY238988, AY238989). Fig. 1 Strict consensus tree of the two most parsimonious trees from the phylogenetic analysis in Sundberg et al. (2001) based on entire 18S rDNA gene sequences, showing that Palaeonemertea is paraphyletic. Palaeonemertean species are in boldface. Whole animals, or parts thereof, were preserved in Bouin’s fluid for later histological examination on 7 µm sections stained using the Mallory trichrome method. Specimen tissue was placed in 70– 80% ethanol and stored for later DNA extraction using DNeasy (QIAgen, Inc.) following the protocol recommended by the manufacturer. Phylogenetic analysis based on nucleotide sequences DNA amplification and sequencing. A region of approximately 1900 bp from the 5′ end of the 18S rDNA gene was amplified by Polymerase Chain Reaction (PCR) based on eukaryotic specific primers (Medlin et al. 1988) using a PTC-100 (MJ Research, Inc.). Amplifications were performed in 50 µL volume of a solution containing 5 –100 ng template DNA, 280 Sequence analysis and phylogeny reconstruction. Sequences were edited and aligned with the CLUSTAL-V (Higgins et al. 1992) algorithm in LASERGENE (DNASTAR Inc.), using the gap penalty Scheme 60/10 (number of gaps / length of gaps) thus penalizing the number, rather than the size, of gaps. This scheme was employed because it provided a better similarity match between sequences (as judged by eye). Phylogeny was reconstructed using parsimony (branchand-bound search) carried out with PAUP* ver 4.0b2a (PPC) (Swofford 1998) and included the two new species described here and sequences from an additional four palaeonemertean species from Sundberg et al. (2001): Hubrechtella dubia Bergendal, 1902; Carinoma tremaphoros Thompson, 1900; an unidentified palaeonemertean species (possibly cephalothricid); Cephalothrix rufifrons (Johnston, 1837). In accordance with the phylogeny in Sundberg et al. (2001) we have used heteronemerteans as outgroup: Lineus sp. (from Winnepenninckx et al. 1995); Micrura fasciolata Ehrenberg, 1828; Lineus ruber (Müller, 1774). The final alignment contained in total 1769 characters of which 235 were parsimony-informative. It contained just a few, short, ambiguous areas and we used all regions in the analysis. Gaps were treated as missing values. Bootstrap support was estimated from 5000 replicates using full heuristic search, TBR branch swapping, and stepwise addition of taxa. Phylogenetic analysis using morphological characters Based on the above analysis and the results from Sundberg et al. (2001), we selected a subset of the species analysed in Sundberg & Hylbom (1994: Fig. 9). This subset was chosen because we believe that these species form a monophyletic group and consider it likely that the two new species belong in this clade. Four of the included species were referred to by Sundberg & Hylbom as HK sp1–4. They were subsequently Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species described in Gibson & Sundberg (1999) as follows: HK sp1 = Carinina sinensis; HK sp2 = Parahubrechtia jillae; HK sp3 = Hubrechtella sinimarinus; HK sp4 = Callinera bergendali (underlining indicates species used as an outgroup following Sundberg & Hylbom (1994: Fig. 3)). The remaining species included in the analysis were: Carinina arenaria Hylbom, 1957; Carinina atavia (Bergendal, 1902); Carinina coei Hylbom, 1957; Carinina grata Hubrecht, 1887; Carinina remanei (Nawitzki, 1931); Carinina wijnhoffae Kulikova, 1984; Callinera buergeri Bergendal, 1900; Callinera monensis Rogers, Gibson & Thorpe, 1992; Tubulanus lucidus Iwata, 1952; Carinesta orientalis Punnett, 1900; Carinesta tubulanoides Gibson, 1990; Hubrechtella dubia Bergendal, 1902; Hubrechtella malabarensis Gibson, 1979; Hubrechtella queenslandica Gibson, 1979; Hubrechtella sarodravayensis Kirsteuer, 1967; Tetramys ramicerebrum Iwata, 1957; Procephalothrix arenarius Gibson, 1990; Procephalothrix fasciculus Iwata, 1952; Procephalothrix filiformis ( Johnston, 1828); Procephalothrix hermaphroditicus Gibson, Sánchez & Méndez, 1990; Procephalothrix oestrymnicus Junoy & Gibson, 1991; Procephalothrix orientalis Gibson, 1990; Procephalothrix simulus Iwata, 1952; Cephalothrix linearis (Örsted, 1844); Cephalothrix atlantica Gerner, 1969; Cephalothrix notabilis Iwata, 1954; Cephalothrix rufifrons ( Johnston, 1837); Cephalotrichella signata (Hubrecht, 1879); Cephalotrichella alba Gibson & Sundberg, 1992; together with the two new species described below, Cephalothrix queenslandica sp. n., and Balionemertes australiensis gen. et sp. n. Cephalothrix linearis was not included in the Sundberg & Hylbom analysis but included here for definition purposes (below) since it is the type species of the genus Cephalothrix. We have furthermore been able to decide the character states for most of the characters assigned a question mark in Sundberg & Hylbom (1994: Table 2). The morphological phylogenetic analysis was based on 48 characters (Tables 1, 2) using parsimony (heuristic search, random addition (10 replicates) of taxa, TBR swapping) and carried out using PAUP* 4.0b2a (PPC) (Swofford 1998). Bootstrap support was estimated from 5000 replicates using fast stepwise addition. Phylogenetic analyses Nucleotide sequence characters The phylogenetic analysis using nucleotide sequence data resulted in one most parsimonious tree (Fig. 2) (length 728, CI = 0.84, RI = 0.77). This demonstrates that the two new species cluster with cephalothricids (the unidentified palaeonemertean species from Sundberg et al. (2001) is believed to be from this taxon); it is also supported by the bootstrap analysis (Fig. 2). Hubrechtella dubia is in a sister position to the cephalothricids in the tree. If we relate this result to the cladograms of Sundberg & Hylbom (1994: Fig. 9) and Sundberg et al. (2001), we conclude that the extended analysis, © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 Fig. 2 Most parsimonious tree (length 728, CI = 0.84, RI = 0.77) for the six (ingroup) plus three (outgroup) nemertean species based on 18S rDNA sequences with all regions included. The two new species are in boldface. Numbers above branches are bootstrap percentiles from 5000 replicates (‘full heuristic’, TBR branch swapping, stepwise addition). based on the morphological characters, should include the species forming a sister group to the clade in Sundberg & Hylbom formed by Hubrechtella spp.−Tubulanus lucidus− Tetramys ramicerebrum. That is, species from the following genera: Cephalothrix, Procephalothrix, Cephalotrichella, Carinina, Callinera, Carinesta, and Parahubrechtia. Morphological characters Based on 48 characters (Table 1; characters 11 and 47 were excluded due to a high degree of unknown states) we found six equally most parsimonious trees (length 208, CI = 0.34, RI = 0.65). The strict consensus tree (Fig. 3) shows that Procephalothrix, Cephalothrix and Cephalotrichella are paraphyletic, as pointed out by Sundberg & Hylbom (1994). It furthermore indicates that Parahubrechtia jillae (HK sp4 in Sundberg & Hylbom) is not part of the Hubrechtella clade but instead places as a sister taxon to the Carinina−Callinera− Carinesta clade. Gibson & Sundberg (1999) pointed to a number of characters that, according to their interpretation (though without an explicit phylogenetic analysis), should place P. jillae in a separate group, but named the genus Parahubrechtia to indicate a relationship with hubrechtid genera based on the phylogenetic analysis in Sundberg & Hylbom (1994). That relationship is not supported by the present analysis. Moreover, P. jillae did not cluster with the remaining outgroup species, and neither did Carinesta tubulanoides (Fig. 3). 281 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Table 1 The characters and their states used in the phylogenetic analysis based on morphological characters. See Sundberg & Hylbom (1994) for further details on characters. ICM = inner circular muscle layer; OCM = outer circular muscle layer; OLM = outer longitudinal muscle layer; ILM = inner longitudinal muscle layer; CM = circular muscles; LM = longitudinal muscles. Some characters are constant within the taxa analysed but still reported for consistency with the character matrix in Sundberg & Hylbom (1994). However, character states not applicable have been deleted, so the coding does not entirely correspond to Sundberg and Hylbom. Characters 11 and 47 were excluded in the analysis due to the high number of missing entries (> 10). Character and character coding 1 Length at maturity 2 Colour 3 4 5 6 7 8 9 10 11 12 13 14 15 Shape of head Head furrows Height of epidermis/body diameter in brain region Height of epidermis/body diameter in midgut region Processes into epidermis Mesh-like structure in the inner layer of the basement membrane Muscle layers in the body wall Muscle plate between rhyncocoel and intestine Muscle-crosses between outer circular and inner circular muscle layers Radial fibers or muscles in oesophagus region Basement membrane/epidermis in mouth region Diagonal muscles Splanchnic musculature in oesophagus region 16 Excretory organs 17 Cerebral sense organs 18 19 20 21 22 23 24 25 26 Eyes Side organs Sensory pits in head region Statolith Cephalic glands Nervous layer in head region Two nerves in rhynchodaeal epithelium: Proboscis nerves Position of brain and lateral nerves 27 28 29 30 31 32 33 34 Number of dorsal nerves Number of dorsal commissures in brain Position of dorsal commissure in brain Relative size of brain commissure Neurochord Buccal nerves Four large nerves in head region Gonads 35 Width of epithelium in rhynchodaeum 36 37 38 39 40 Glands in epithelium Musculature in posterior part of rhynchodaeum Muscular layers in proboscis sheath Extension of proboscis sheath Caeca on rhynchodaeum or proboscis sheath 282 (0) < 2 cm; (1) 2–10 cm; (2) > 2 cm. (0) uniform white; (1) coloured, without pattern; (2) coloured, with conspicuous pattern. (0) bluntly rounded; (1) sharply pointed. (0) absent; (1) present; (2) present, weakly developed and difficult to observe. (0) ≥ 0.1; (1) < 0.1 0. (0) ≥ 0.05; (1) < 0.05. (0) absent; (1) present. (0) absent; (1) present. (0) OCM + OLM + inner circular; (1) OCM + OLM; (2) OCM + OLM + ICM + ILM, and ILM in anterior part of body; (3) OCM + diagonal muscles + circular + longitudinal + inner circular. (0) absent; (1) present; (2) LM surrounding rhynchocoel. (0) absent; (1) present. (0) absent; (1) present. (0) < 0.1; (1) ≥ 0.1 0. (0) absent; (1) present. (0) absent; (1) CM layer surrounding intestine; (2) LM layer surrounding intestine; (3) both LM and CM layers surrounding intestine. (0) absent; (1) present, canal inside inner circular muscle layer; (2) present, canal outside inner circular muscle layer; (3) present, several systems (‘Cephalothrix-type’ (Coe 1930)). (0) missing; (1) ciliated pits or ciliated canals in epidermis; (2) inner parts of the organ in the lateral blood vessel. (0) absent; (1) present. (0) absent; (1) present. (0) absent; (1) present. (0) absent; (1) present. (0) absent; (1) limpid Tubulanus type; (2) granular cephalothricid type. (0) absent; (1) present. (0) absent; (1) present. (0) absent; (1) present. (0) in epidermis; (1) between basement membrane and outer circular muscles; (2) lateral nerves between basal membrane and outer circular muscles, posteriorly shifting position into longitudinal muscles; (3) in longitudinal muscles; (4) as in Balionemertes. (0) upper dorsal nerve only; (1) both upper and lower dorsal nerves. (0) one; (1) two; (3) none. (0) in front of ventral; (1) in equal position; (2) behind ventral. (0) ventral wider than dorsal; (1) equal. (0) absent; (1) present. (0) absent; (1) present, unpaired; (2) present, paired. (0) absent; (1) present. (0) in a single row on each side of body; (1) packed above each other, on each side of body. (0) greater anteriorly than posteriorly; (1) greater posteriorly than anteriorly; (2) equal throughout rhynchodaeum. (0) absent; (1) present. (0) absent; (1) present. (0) circular muscles; (1) outer circular and inner longitudinal muscle layers. (0) throughout entire animal; (1) not entire animal. (0) absent; (1) present. Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species Table 1 Continued. Character and character coding 41 Muscle layers in proboscis 42 Position of proboscis insertion 43 Rhynchocoel bodies 44 Circular muscle (CM) layers in rear end of proboscis sheath, or in nephridial region 45 Proboscis morphology 46 Alimentary canal caeca 47 Position of lateral bloodvessel in relation to inner circular muscle (ICM) layer 48 Rhynchocoel blood vessels 49 Blood vesels in the mouth-foregut region 50 Cephalic lacunae In the present analysis C. tubulanoides is closer to the other two Carinesta species, in contrast to the analysis in Sundberg & Hylbom where it appeared to be related to Hubrechtella. One of the two new species described below, Cephalothrix queenslandica sp. n., is placed as sister to two Procephalothrix species. The analysis in this paper, as in Sundberg & Hylbom (1994) and Sundberg et al. (2001), suggests that the genus Cephalothrix (this name having nomenclatural priority over Procephalothrix) should be redefined phylogenetically to accommodate species previously named as Procephalothrix and Cephalotrichella. Although clade support (Fig. 3) is weak in general as judged by the bootstrap values, we still propose that these three genera should be combined, and will formally define the taxon Cephalothrix below. There are also three synapomorphies (Fig. 3) supporting this conclusion. The other new species, Balionemertes australiensis gen. et sp. n., is in a sister position to Cephalothrix in both sets of analyses (Figs 2 and 3). It could be placed in Cephalothrix, but it lacks the three supporting synapomorphies (Fig. 3). In addition, its nervous system has a number of unique complexities, including the presence of extraganglionic tissues in the body wall muscle layer; we consider this to be a synapomorphy for the taxon. (0) outer circular and inner longitudinal; (1) outer and inner circular; (2) outer longitudinal and inner circular; (3) arrangement differs along proboscis. (0) in front of brain; (1) in brain region; (2) behind brain region. (0) absent; (1) present. (0) not strongly developed; (1) inner CM strongly developed; (2) CM proboscis sheath extremely well developed (muscle bulb). (0) same overall; (1) different in different regions. (0) absent; (1) present. (0) first inside, then outside; (1) inside; (2) outside. (0) absent; (1) two lateral; (2) one median. (0) two lateral vessels, not surrounding oesophagus; (1) vessels clearly surrounding foregut ventrolaterally in the lacunae system; (2) four lateral vessels, not surrounding oesophagus. (0) absent; (1) present. muscle bundle of proboscis; LD, lower dorsal nerve; LM, body wall longitudinal muscle layer; LN, lateral nerve cord, LV, lateral blood vessel; MT, mouth; NR, peripheral neural ring of proboscis; PC, proboscis circular muscle layer; PG, proboscis epithelial gland cell; PL, proboscis inner lining; PN, proboscis nerve; PR, proboscis; RC, rhynchocoel; UD, upper dorsal nerve; VC, ventral cerebral commissure; VX, ventral extraganglionic tissue. Taxonomy Type specimens of the new taxa are deposited at the Townsville branch of the Queensland Museum (MTQ) and the 18S rDNA gene sequences in GenBank. Balionemertes gen. nov. The taxon is identified as monophyletic based on the autapomorphy ‘presence of extraganglionic tissues, located in the body wall longitudinal muscle layer, surrounding the brain’. This character has not been described for any other palaeonemertean (in the broad sense) species. Linnean definition. The genus is defined by its type species Balionemertes australiensis sp. n. Abbreviations used in figures BE, buccal epithelium; CG, cerebral ganglion; CM, body wall circular muscle layer; CV, cephalic blood vessel; DC, dorsal cerebral commissure; DE, dermis; DG, dorsal ganglion; DX, dorsal extraganglionic tissue; EE, epidermal eyespot; EP, epidermis; FN, foregut nerve; IP, inner longitudinal muscle layer of proboscis; LB1, major outer longitudinal muscle bundle of proboscis; LB2, minor outer longitudinal © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 Phylogenetic definition. The clade stemming from the first species to possess the the autapomorphic character described above which is synapomorphic with this character in the holotype (MTQ G20021) of Balionemertes australiensis sp. n. Diagnosis. Palaeonemertean, without cephalic furrows; body wall musculature comprises outer circular and inner 283 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Table 2 Character matrix for the species included in the morphological analysis. Character numbers correspond to Table 1; ‘?’ denotes missing or inapplicable data. Outgroup species are marked with an asterisk. The species sampling is based on the results in Sundberg & Hylbom (1994). Character Carinina arenaria Carinina atavia Carinina coei Carinina grata Carinina remanei Carinina sinensis Carinina wijnhoffae Callinera bergendali Callinera buergeri Callinera monensis Carinesta orientalis Carinesta uchidai Procephalotrix arenarius Procephalotrix fasciculus Procephalotrix filiformis Procephalotrix hermaphroditicus Procephalotrix oestrymnicus Procephalotrix orientalis Procephalotrix simulus Cephalotrix arenaria Cephalotrix atlantica Cephalothrix queenslandica sp. n. Cephalotrix notabilis Cephalotrix rufifrons Cephalotrichella signata Cephalotrichella alba Balionemertes australiensis sp. n. Carinesta tubulanoides* 284 1 26 2 27 3 28 4 29 5 30 6 31 7 32 8 33 9 34 10 35 11 36 12 37 13 38 14 39 15 40 16 41 17 42 18 43 19 44 20 45 21 46 22 47 23 48 24 49 25 50 0 0 1 0 0 0 0 0 1 0 0 0 1 0 0 1 1 1 1 1 2 1 2 1 1 3 1 3 ? 3 1 3 1 3 2 3 2 3 0 3 0 2 1 3 2 3 1 3 1 3 0 3 1 4 0 1 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 0 1 1 0 0 0 0 0 0 2 0 0 0 0 0 2 0 0 0 2 1 2 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 1 1 1 0 1 1 1 2 0 0 1 0 1 0 0 0 0 0 0 ? 1 0 1 0 1 0 0 0 1 0 1 0 1 0 0 0 0 1 0 0 1 1 1 1 1 1 1 0 0 0 0 2 1 0 0 ? 0 ? 0 0 0 ? 0 ? 0 ? 0 2 0 0 0 ? 0 ? 0 ? 0 1 0 0 0 0 0 1 0 1 0 0 0 0 0 ? 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 1 0 0 ? 1 0 1 0 1 0 0 0 0 0 1 ? ? 0 0 0 ? 0 0 0 0 0 1 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 1 1 0 0 0 0 2 1 2 1 2 0 2 1 2 0 2 1 0 1 1 1 1 1 1 0 2 0 2 0 1 0 1 0 1 0 1 ? 1 1 2 0 1 0 1 0 ? 0 1 0 2 0 1 0 1 0 1 0 2 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 1 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 1 0 1 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 1 0 1 0 0 1 0 1 0 1 0 1 ? 1 0 1 ? 1 ? 1 0 0 0 1 0 2 0 2 0 1 2 2 0 1 0 1 2 1 2 2 2 2 2 2 0 1 1 1 2 1 2 1 2 1 2 1 ? 1 2 ? 2 1 2 1 ? 1 2 1 2 1 1 2 2 1 2 1 2 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 1 1 1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 ? 0 ? ? 0 0 ? 0 ? 0 ? 0 ? 0 0 0 1 1 1 1 1 1 1 1 ? ? 0 1 1 1 1 1 0 1 0 1 0 1 0 0 ? 0 ? 1 ? 0 0 0 0 1 0 1 ? 1 0 0 0 0 0 0 0 1 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 ? 1 ? 1 1 1 1 0 0 1 0 1 1 1 0 0 1 0 0 1 1 1 1 1 0 0 1 1 ? 1 1 1 1 1 1 1 0 1 1 1 0 1 1 1 1 0 0 1 ? 1 0 1 0 0 0 1 0 1 0 1 0 ? 0 1 0 1 ? 1 0 ? 0 1 0 1 0 0 0 1 0 0 0 0 3 0 0 0 3 0 ? 0 3 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 2 3 2 3 2 3 2 0 2 0 3 0 ? 0 3 3 ? 0 3 0 3 0 3 0 0 0 3 3 3 0 ? 1 3 0 0 0 0 3 0 0 2 0 1 2 1 2 1 2 1 ? 1 2 1 2 1 2 0 2 0 2 0 2 0 2 0 ? 0 ? 0 1 0 1 0 1 0 1 0 ? 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 1 0 1 0 1 0 1 0 0 0 0 0 0 0 0 ? 0 1 0 0 0 0 1 2 1 2 ? 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 1 0 1 0 ? 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 ? 0 1 0 0 0 1 1 0 0 0 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 ? 0 0 0 0 0 1 0 ? 0 1 0 1 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 2 1 2 0 2 0 2 0 0 ? 2 2 2 1 2 ? 2 ? 2 ? 2 ? 2 ? ? ? 0 ? 0 1 0 2 1 0 1 0 1 1 1 0 1 0 1 0 ? 0 1 0 1 0 1 0 1 0 0 0 ? 0 1 0 0 0 1 0 1 0 ? 0 1 0 0 0 0 0 0 0 0 0 ? 0 ? 0 0 0 0 0 ? 0 1 0 1 1 1 1 ? 0 1 2 1 2 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 1 1 ? 0 0 0 0 0 0 1 1 1 1 0 1 0 0 0 0 1 0 ? 0 1 0 0 0 1 ? ? 1 1 1 1 0 0 0 Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species Table 2 Continued. Character Parahubrechtia jillae* Tetramys ramicerebrum* Tubulanus Iucidus* Hubrechtella dubia* Hubrechtella malabarensis* Hubrechtella queenslandica* Hubrechtella sarodravayensis* Hubrechtella sinimarinus* 1 26 2 27 3 28 4 29 5 30 6 31 7 32 8 33 9 34 10 35 11 36 12 37 13 38 14 39 15 40 16 41 17 42 18 43 19 44 20 45 21 46 22 47 23 48 24 49 25 50 0 1 1 1 2 1 0 1 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 1 0 ? ? 2 0 0 0 1 0 0 0 0 0 2 1 0 1 ? 1 0 1 0 0 1 1 0 0 0 1 0 0 0 0 0 0 1 1 1 0 1 1 1 ? 1 0 1 0 2 0 2 0 1 0 2 0 ? 0 2 0 ? 0 2 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 ? 1 0 1 ? 1 ? 1 0 1 1 1 2 0 ? 1 2 0 2 0 2 0 2 ? 2 1 ? 1 0 0 1 0 1 ? 1 ? 1 ? 1 ? 0 0 ? 0 0 1 1 0 0 1 1 0 0 1 0 1 0 1 ? 0 0 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 0 0 1 1 1 1 1 1 1 0 1 1 1 0 0 2 0 0 ? 3 0 ? 0 3 0 ? 0 0 0 2 1 0 1 0 2 0 2 0 2 0 1 0 2 0 1 0 0 1 0 2 1 2 0 2 0 2 0 2 0 2 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 1 0 0 0 2 1 2 0 2 0 ? 0 ? 0 ? 0 ? 0 ? 1 0 1 2 ? 2 1 2 1 2 1 2 1 2 1 2 0 0 0 1 0 0 0 1 0 1 0 1 0 0 ? 0 1 0 0 0 ? 0 1 0 0 0 1 0 0 0 0 1 longitudinal layers; no muscle crosses in body wall; neither cerebral sensory nor lateral sensory organs; cephalic region without nerve layer; proboscis with outer longitudinal, middle circular and inner longitudinal muscle layers, outer layer incomplete and restricted to three longitudinal bundles of fibres; proboscis nerves present; brain situated internal to body wall musculature but extraganglionic tissues and lateral nerve cords situated in body wall longitudinal muscle layer; nervous system without neurochord cells; buccal nerves paired; intestine without lateral diverticula; rhynchocoel wall with circular and longitudinal muscle layers; blood vascular system simple, with neither mid-dorsal nor rhynchocoelic vessels; eyes present, epidermal; with no frontal organ, frontal glands or cephalic glands; sexes separate. Etymology. Based on the Greek balios (= swift or nimble); refers to the animal’s rapid movement when disturbed. Balionemertes australiensis sp. n. (Figs 4–20) Holotype. Mature male, complete set of transverse sections of anterior two-thirds of body, 17 slides, MTQ G20021. Sequence for the 18S rDNA gene from this specimen is deposited with GenBank (Accession number AY238988). Type locality. Great Barrier Reef, south-west part of Rib Reef (18°29.6′ S, 146°52.8′ E), from a depth of ca. 10 m, among coral rubble and red algae, 1 specimen. Etymology. Specific epithet indicates that the species was first discovered in Australian waters. Additional material. Great Barrier Reef, Great Palm Island, Coolgaree Bay (18°42.6′ S, 146°35.0′ E), 5 specimens from a © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 depth of 2−3 m, among sea grass covered with algae; Bullumbooroo Bay (18°41.8′ S, 146°35.8′ E), 1 specimen from a depth of 2 m, among algae. Description External features (Fig. 4). Largest specimen about 90 mm long, 4 –5 mm wide. Coloration dark brown with whiter areas forming poorly defined, somewhat irregularly spaced bands with scattered small patches in between. Pigment unevenly distributed dorsally, with appearance of aggregations of brown speckles under close examination. Ventral surface a more homogeneous colour. Bluntly pointed head paler than remainder of body, its white ‘neck band’ extending around body. Body tapers, ending in blunt tip; brown pigmentation becomes paler in more posterior regions. Body wall, musculature and parenchyma. Throughout length of body, glandular epidermis attains a maximum thickness of 30 –35 µm. At posterior brain level it is dominated by dense accumulations of finely particulate acidophilic glands extending to full epidermal height and encircling the body; in precerebral region, glands with an identical histological appearance form ring around the proboscis pore; in other body regions they are not as abundant and restricted to the distal epidermal surface. Epidermis bordered internally by distinct connective tissue dermis (Figs 5 and 6); in most parts of body this is 6 – 7 µm wide, but in cerebral and precerebral regions it rarely exceeds 3– 4 µm. Below dermis, body wall musculature comprises outer circular and inner longitudinal layers (Fig. 6), 6 –7 µm and 60 –75 µm wide respectively, throughout the postcerebral regions of body. Both layers extend to tip of 285 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Fig. 3 Strict consensus tree of six equally most parsimonious trees (length 208, CI = 0.34, RI = 0.65) based on the 48 characters described in Table 1 and listed in Table 2. Heuristic search with random addition (10 replicates) and TBR branch swapping. Numerals above branches denote bootstrap support estimated from 5000 bootstrap replicates using fast stepwise addition. Synapomorphies and character states mentioned in Table 1 are indicated as follows: a (character 26, state 3); b (26, 4); c (26, 2); d (32, 1); e (32, 2); f (33, 1); g (33, 0). *outgroup species. head but are thinner anteriorly, particularly the inner longitudinal layer, which is largely replaced by extraganglionic nerve tissues in this region. Dorsoventral muscles and additional body wall muscle layers not found in any part of body; plate of longitudinal fibres, up to 15 µm wide, extends between gut and rhynchocoel walls throughout most of body length. Parenchymatous connective tissues poorly developed. They are most evident in foregut region adjacent to, sometimes surrounding, blood vessels. 286 Proboscis apparatus. Proboscis pore opens ventrally and subterminally a short distance behind tip of head. Rhynchodaeum with spacious, thin-walled, epithelium, neither ciliated nor glandular. Proboscis insertion situated short distance in front of brain. Rhynchocoel extends to posterior end of body, its wall principally composed of circular muscle fibres; isolated longitudinal fibres, not forming a distinct layer, also distinguishable. Structure of rhynchocoel wall resembles that described for Parahubrechtia by Gibson & Sundberg (1999). Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species epithelium on opposite side mostly only 15 –20 µm wide, completely lacking both acidophilic glands and barbs. Three muscle layers in proboscis below epithelium; outer longitudinal layer incomplete. In thicker epithelial regions, muscle layer restricted to two large longitudinal bands or tracts of fibres running next to pair of large proboscis nerves; on opposite side of proboscis a single, smaller longitudinal fibre bundle runs adjacent to a small, third nerve. The three nerves are linked by a delicate, mostly inconspicuous, peripheral nerve ring running between circular and outer longitudinal muscle layers. Middle circular and inner longitudinal muscle layers completely encircle proboscis; both are better developed on thicker epithelial side of proboscis where they are, respectively, 3–4 µm and 14–18 µm wide. Inner lining of proboscis thin and indistinct. Alimentary canal. Mouth ventral (Fig. 6), located behind brain. It opens into thick-walled buccal chamber whose anterior wall bulges forward toward brain, extending some distance in front of mouth (Fig. 9). Epithelial lining of buccal chamber varies in width, depending upon degree of folding, attaining a maximum of 50–55 µm. Main foregut wall mostly about 30 µm wide, deeply folded, containing both acidophilic and basophilic glands. No somatic muscles associated with the foregut. Intestine leads directly back from foregut, forming simple dorsoventrally compressed tube arching below rhynchocoel, with no evidence of either lateral pouches or diverticula. Fig. 4 Balionemertes australiensis gen. et sp. nov. Drawing of a complete specimen (taken from a colour transparency) illustrating pigmentation pattern and general body shape, viewed from the dorsal aspect. Scale bar, 2 cm. Proboscis (Figs 7 and 8), 260 –290 µm in diameter in retracted position, more or less uniformly but asymmetrically developed throughout its length. Epithelium folded; along one side it is 45–50 µm wide, containing large acidophilic gland cells, distally bearing large numbers of rhabditoid ‘barbs’ about 3–4 µm long loosely arranged into pads; © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 Blood system. Cephalic blood supply consists of pair of vessels (Fig. 10) that join near tip of head. Vessels spacious in front of rhynchodaeum and proboscis pore, but farther back become compressed and reduced in size alongside rhynchodaeum. Near cerebral ring they form small, compressed channels passing between anterior regions of dorsal and ventral ganglia before moving downwards to run close to each other (Fig. 11) between ventral cerebral commissure and rhynchocoel wall; they do not join. Behind the brain the vessels move apart to lateral positions adjacent to lateral nerve cords (Fig. 12), continuing in this position to posterior end of body. Although running close to rhynchocoel wall in many places, the blood vessels do not give off rhynchocoelic vessels, as found in some palaeonemertean genera. Blood system thus represents simple palaeonemertean type, consisting of a pair of spacious lateral longitudinal vessels with neither mid-dorsal vessel nor pseudometameric transverse connectives. Nervous system. Complex. Brain lobes situated internal to body wall muscle layers (Fig. 5); longitudinal nerve cords run throughout length of body within body wall longitudinal muscle layer (Figs 6 and 12). Extraganglionic tissues (Figs 5, 287 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Fig. 5 Balionemertes australiensis gen. et sp. n. Camera lucida drawing of section of the cerebral region, showing the organization of the brain and extraganglionic tissues. Scale bar, 250 µm. Fig. 6 Balionemertes australiensis gen. et sp. n. Camera lucida drawing of section of the mouth region, showing the organization of the various body structures. Scale bar, 250 µm. 13, 14, 15) surround the brain, running in the longitudinal muscle layer; they extend both in front of and behind brain lobes. Brain lobes possess both inner and outer neurilemma. Dorsal lobes wider-set than ventral but of similar size. There 288 are two dorsal cerebral commissures, both about 10 –12 µm wide; anterior situated above ventral commissure, posterior behind it. Ventral commissure (Fig. 15) about 60 µm wide. Thick mid-dorsal nerve extends from rear of anterior dorsal Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species Fig. 7 Balionemertes australiensis gen. et sp. n. Camera lucida drawing of section of the proboscis, showing its structure. Scale bar, 100 µm. commissure through dorsal ganglionic tissues of brain to posterior dorsal commissure, continuing back behind brain (Fig. 13) to run medially in body wall longitudinal muscle layer just below dermis. Here it branches to form upper and lower mid-dorsal nerves (Fig. 16). Upper mid-dorsal nerve continues posteriorly between dermis and body wall circular muscle layer. Lower mid-dorsal nerve extends back between rhynchocoel wall and body wall longitudinal musculature. Both nerves well developed and distinct throughout most of body length. Four additional ‘blocks’ of extraganglionic nerve tissue present around periphery of brain lobes, each enclosed by its own outer neurilemma, run in body wall longitudinal muscle layer (Fig. 5); dorsolateral blocks larger than ventrolateral. Just behind level of ventral cerebral commissure thick nerve tracts lead directly upwards and downwards from each dorsal fibre core of brain (Fig. 17) to dorsolateral and ventrolateral extraganglionic tissues, respectively; extraganglionic tissues on each side of head also linked by slender, lateral, dorsoventral nerve commissures. All four ‘blocks’ extend well into head, reaching in front of proboscis pore. For most of their anterior extent, dorsal extraganglionic tissues run above cephalic blood vessels, ventral on either side of rhynchod- © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 aeum; in front of proboscis pore they lie above and below cephalic blood supply and fill most of cephalic space internal to body wall muscles. Extraganglionic tissues also extend behind brain in longitudinal musculature but do not reach as far back as buccal region. Single giant cell in inner neuroganglionic tissues of each ventral brain lobe (Fig. 18), short distance behind ventral commissure; whether or not these cells represent neurochord cells uncertain, but there are no neurochords in lateral nerves. Thick mid-ventral nerve emerges from rear of ventral commissure to run posteriorly close below rhynchocoel. Where ventral brain lobes begin to separate posteriorly, mid-ventral nerve runs downwards between them, then turns posteriorly to extend for short distance before dividing to form origin of two stout buccal nerves. These nerves pass on either side of buccal region (Figs 6 and 9) behind mouth moving ventromedially to meet via thick postoral commissure. Frontal organ, frontal glands and cephalic glands. No evidence of frontal organ, frontal glands or cephalic glands in any part of head. Sense organs. There are 25 –30 similar-sized pigment-cup ocelli on each side of head, all located in epidermis (Figs 5 289 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Figs 8–14 Balionemertes australiensis gen. et sp. n. —8. Transverse section (TS) of the proboscis; cf. Fig. 7. —9. TS between the brain and mouth showing the two buccal nerves (asterisks) passing on either side of the preoral buccal region. —10. TS of the head showing the cephalic blood supply. —11. TS of the brain region showing the two blood vessels (arrowed) running close together. —12. Part of the foregut region in TS, showing a lateral blood vessel, asterisked, running close to a lateral nerve cord. —13. TS of the brain region showing the dorsal extraganglionic tissues. Arrow indicates the mid-dorsal nerve. —14. Enlargement of the posterior brain region showing the dorsal extraganglionic tissues. Arrow indicates the outer neurilemma of the brain lobes, which separates the brain and extraganglionic tissues. All photomicrographs of sections stained using the Mallory trichrome method. Scale bars: 8–10 and 13, 100 µm; 11, 12 and 14, 50 µm. 290 Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species Figs 15–20 Balionemertes australiensis gen. et sp. n. —15. Transverse section (TS) of the ventral cerebral commissure region, showing dorsal and ventral extraganglionic tissues which enclose the brain lobes. —16. TS of the dorsal body wall showing the upper (large arrow) and lower (small arrow) mid-dorsal nerves. —17. TS of a dorsal cerebral ganglion showing the dorsal (starred) and ventral (asterisked) nerve tracts leading to the extraganglionic tissues. —18. TS of part of a ventral cerebral ganglion showing a giant cell (arrowed). —19. Part of the body wall in TS showing two of the epidermal eyes (arrowed). —20. TS of part of the intestinal region showing a testis containing mature spermatozoa. All photomicrographs of sections stained using the Mallory trichrome method. Scale bars: 15, 100 µm; 16 – 20, 50 µm. and 19). Each ocellus is about 15 µm in diameter and appears as aggregation of dark reddish-brown pigment granules surrounding central clear area. Ocelli distributed irregularly, from ventrolateral margins of head almost to mid-dorsal region, either singly or in groups, between level of proboscis pore and brain region. They reach as far back as about midbrain region, close to posterior dorsal commissure, but not behind it. There are no cerebral sensory organs, and no other type of sensory structures, such as lateral sensory organs, could be distinguished. Excretory system. No evidence of an excretory system could be discerned in any part of body. Reproductive system. Sexes separate. Gonads arranged in © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 single dorsolateral row either side of body above lateral blood vessels, immediately internal to body wall longitudinal musculature (Fig. 20). Each ovary contains several eggs in similar state of development, ovaries being much more tightly packed together than testes. Open gonoducts lead to dorsolateral body surface (Fig. 20). Systematic discussion. Both phylogenetic analyses place Balionemertes australiensis gen. et sp. n. in a sister position to Cephalothrix−Cephalotrichella−Procephalothrix and it could therefore be included as part of this clade. There are, however, a number of synapomorphies for the sister clade (see below) while there are none for a combined clade. In view of both this and the nerve system characteristics we have described above (which are unique among palaeonemerteans), 291 Palaeonemertean phylogeny and two new species • P. Sundberg et al. we prefer to place it in its own taxon Balionemertes, with Balionemertes australiensis sp. n. as the only known species. Genus Cephalothrix (converted clade name) The genus Cephalothrix was erected by Örsted (1843) with Cephalothrix linearis as the type species. Later, Hylbom (1957: 553) defined the genus Cephalothrix as palaeonemerteans with ‘Mouth situated far behind the brain. No inner circular muscle layer. No cerebral sense organs. Nervous system situated in the longitudinal muscle layer of the body. Buccal nerve unpaired’. According to our phylogenetic analysis (Fig. 3), the first three characters are plesiomorphies, while the states for last two (placement of nervous system, and buccal nerves) are synapomorphies for Cephalothrix− Cephalotrichella−Procephalothrix and thus do not support Cephalothrix in the strict sense as a monophyletic group. Neither Cephalotrichella nor Procephalothrix (both Wijnhoff 1913) were defined within a phylogenetic framework and their monophyletic status is not supported in the present study. Instead, our analysis, together with those of Sundberg & Hylbom (1994) and Sundberg et al. (2001), supports the view that the three current genera Cephalothrix, Cephalotrichella and Procephalothrix should be combined. This clade is further supported by the following synapomorphies: character 26, state 3; character 32, state 1; character 33, state 1 (Table 1). Based on the phylogeny in Fig. 3 we define a taxon name Cephalothrix (name chosen for reason of priority) to include also species in the previous genera Procephalothrix and Cephalotrichella. We delineate the taxon to reflect tree topology in accordance with the suggestion in De Queiroz & Gauthier (1990). This new definition of Cephalothrix, including previous Procephalothrix species, will make Procephalothrix arenarius Gibson, 1990 and Cephalothrix arenaria Hylbom, 1957 homonyms, the latter name having priority. We therefore rename Procephalothrix arenarius as Cephalothrix hongkongiensis (replacement name). Definition. Cephalothrix is the most inclusive clade containing Cephalothrix (Cephalotrichella) alba Gibson & Sundberg, 1992 (holotype in Natural History Museum, London 1990.7.1), Procephalothrix arenarius Gibson, 1990 (Natural History Museum, London 1987.2.47), Procephalothrix hermaphroditicus Gibson Sánchez & Méndez, 1990 (Museo Nacional de Historia Natural, Santiago N-10008), Procephalothrix oestrymnicus Gibson & Junoy, 1991 (Natural History Museum, London 1990.6.3), Cephalothrix queenslandica sp. n., Procephalothrix orientalis Gibson 1990 (Natural History Museum, London 1987.2.33), Cephalothrix linearis (Rathke 1799) (previous type species for Cephalothrix), but not Balionemertes australiensis sp. n. (MTQ G20021). 292 Cephalothrix queenslandica sp. n. (Figs 21–28) Holotype. Mature female, series of transverse sections of anterior half of body, 5 slides, MTQ G20022. Sequence for the 18S rDNA gene from this specimen is deposited with GenBank (Accession Number AY238989). Type locality. Great Barrier Reef, Rib Reef, among algae collected from the flat of Rib Reef (18°28.8′ S, 146°52.4′ E) north-east of the Palm Islands, from a depth of 2 m, 1 specimen. Etymology. Specific epithet refers to the Australian state where the species was first found. Additional material. Two specimens from east side of Pelorus Island (18°41.9′ S, 146°30,4′ E) from a depth of 2 m, among algae; 2 specimens from east side of Fantome Island (18°33.5′ S, 146°29.2′ E) from a depth of 2 m, among algae; 1 specimen from Lizard Island, Watson Bay (14°40.0′ S, 145°26.7′ E), from a depth of 3 m, among coral rubble. Description External features (Fig. 21). Slender species which, when disturbed, readily coils up. Longest specimen about 90 mm long but less than 1 mm wide. Background colour pale, transparent yellow, marked with transverse bands of brown, more regularly distributed anteriorly. On dorsal surface brown pigmentation also extends between bands to form vaguely defined but interrupted broad longitudinal stripe, pigmentation appearing situated deep in body tissues in gut. Body tapers posteriorly to end in pointed tail, brownish hue fading toward posterior tip which is almost colourless. No eyes visible in life. Body wall, musculature and parenchyma. Epidermis in brain and cephalic region 30 µm or more wide, posteriorly reducing to a maximum of 20–25 µm. Three gland types distinguishable: orange-staining, oval-shaped with homogeneous contents mainly in distal half of epidermis, few precerebrally, more abundant posterior to mouth; slender basophilic glands with finely granular contents, more abundant in head than elsewhere in body, extending full epidermal height; similar acidophilic glands with more coarse contents, more numerous posteriorly. Seem to correspond with Hylbom’s (1957: 555) Types 1, 2 and 3, respectively. Dermis distinct, mostly 7– 8 µm wide. Body wall musculature consists of outer circular and inner longitudinal layers, former extremely slender, at most 4 –5 µm across and often only one or two fibres thick, latter up to 30 µm or more postcerebrally (Fig. 22). Both muscle layers extend to tip of head. No trace of additional body wall muscle layers, though delicate longitudinal muscle plate extends between rhynchocoel and gut walls (Fig. 22). Parenchymatous connective tissues sparsely developed throughout body. Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species 2–3 µm across, usually less, inner longitudinal muscle layer 5 –6 µm wide. Proboscis nerve supply comprising two neural swellings situated between epithelium and circular muscle layer. Alimentary canal. Small mouth situated behind brain, but not as far back as in several cephalothricids. Hylbom (1957: 556) used quotient of distance between tip of head to brain and brain to mouth, giving value of 1 : 4 for Cephalothrix arenaria. Comparable value in present species is about 1 : 1.5. Foregut epithelium folded, width 15–45 µm, with acidophilic and basophilic glands, former more abundant in anterior regions. Buccal epithelium bulges anteriorly toward but does not reach brain region. Intestinal gastrodermis maximum width of 45 µm, with typical cephalothricid histological appearance. In males there are no lateral intestinal diverticula, but in mature females unbranched, shallow pouches extend dorsolaterally between ovaries (Fig. 24). No dorsoventral muscles in intestinal region. Fig. 21 Cephalothrix queenslandica sp. n. Drawing of complete specimen, taken from a colour transparency, showing the colour pattern and general body shape viewed from the dorsal aspect. Scale bar, 10 mm. Proboscis apparatus. Proboscis pore opens at tip of the snout. Rhynchodaeal wall thin, mostly with neither gland cells nor musculature; just in front of proboscis insertion weak layer of circular muscles not forming obvious sphincter. Basement layer thin but distinct. Proboscis insertion at level of ventral cerebral commissure in the cerebral ring, formed by deeper fibres of body wall longitudinal muscle layer turning inwards to form origin of proboscis. Rhynchocoel does not extend to posterior end of body. Wall with slender separate outer circular and inner longitudinal muscle layers. Proboscis (Fig. 23) diameter about 130 µm (around 1/3 of body width) in retracted position; uniform construction for most of its length. Outer glandular epithelium maximum width of 30–35 µm, outer circular muscle layer at most © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 Blood system. Cephalic blood supply consists of two spacious vessels extending on either side of rhynchodaeum (Fig. 25). Vessels meet close behind proboscis pore via supra-rhynchodaeal connective. After passing back through cerebral ring, vessels expand to form pair of spacious ventrolateral channels running between lateral nerves and rhynchocoel wall, internal to body wall musculature. For most of body length, vessels situated adjacent to gut wall in dorsolateral position. No pseudometameric transverse connectives between vessels and no mid-dorsal vessel, blood system thus being of simple palaeonemertean arrangement. Nervous system. Brain and lateral nerve cords situated in body wall longitudinal muscle layer. Fibrous and neuroganglionic components of brain separated by thin but distinct inner neurilemma, but no outer neurilemma enclosing brain lobes as a whole. Single dorsal and ventral cerebral commissures situated at about same level in cerebral ring, respectively, some 12 µm and 40 µm wide. Dorsal brain lobes not posteriorly divided. In head, two cephalic nerves extend forward, meeting by supra-rhynchodaeal connective short distance behind tip of head. Nerves do not run in rhynchodaeal epithelium; similar condition prevails in other cephalothricids (Sundberg & Hylbom 1994). From rear of dorsal commissure thick nerve leads posteriorly; at first it runs back in body wall longitudinal muscle layer but, behind mouth, moves outwards to continue between dermis and body wall circular musculature, thus forming equivalent of upper mid-dorsal nerve (Fig. 26). Additional peripheral nerves also found on ventrolateral 293 Palaeonemertean phylogeny and two new species • P. Sundberg et al. Figs 22–28 Cephalothrix queenslandica sp. n. —22. Transverse section (TS) of the body wall in the foregut region. Arrow indicates fibres of the longitudinal muscle plate between the rhynchocoel and gut walls. —23. TS showing the structure of the proboscis. —24. TS of the intestinal region showing a group of ova and a dorsolateral intestinal diverticulum. —25. TS of the head showing the cephalic blood vessels (arrowed). —26. Part of the dorsal body wall in TS, showing the mid-dorsal nerve (arrowed). —27. Part of the ventrolateral body wall in TS, showing one of the additional peripheral nerves (arrowed). —28. TS of a lateral blood vessel showing a terminal ‘nephrostome’ of the excretory system (arrowed) protruding into the blood vessel lumen. All photomicrographs of sections stained using the Mallory trichrome method. Scale bars: 22, 23, 26 and 27, 100 µm; 24 and 25, 150 µm; 28, 50 µm. 294 Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters P. Sundberg et al. • Palaeonemertean phylogeny and two new species body margins (Fig. 27), these too running immediately below dermis. Single large median ventral nerve extending back from rear of ventral commissure forms origin of foregut or buccal nerves. Foregut nerve does not branch until just in front of mouth and is thus initially unpaired, similar condition occurring in most cephalothricid palaeonemerteans. these character states are symplesiomorphies and hence do not identify the palaeonemerteans as a monophyletic group. Before proposing a phylogenetic hypothesis for the species traditionally contained in Palaeonemertea we have to await further analyses based on more species and a combination of morphological and nucleotide characters. Frontal organ, frontal glands and cephalic glands. No evidence of frontal organ or frontal glands found in any of specimens examined. Basophilic, finely granular, cephalic glands present in anterior portion of head, mainly located dorsally and ventrally above rhynchodaeum. Glands have a ‘cephalothricid’ appearance. Acknowledgements Sense organs. No evidence of any sensory structures found in any part of body. Excretory system. Excretory system extends between foregut and anterior intestinal body regions. It closely corresponds to metanephridial arrangement described by Coe (1930) in comprising irregularly distributed nephridia with globular or mushroom-shaped terminal portions, 15 –20 µm in diameter, which protrude into lumen of lateral blood vessels (Fig. 28), mostly single, occasionally two together. Coe called these terminal portions ‘nephrostomes’. No evidence of efferent ducts found. Reproductive system. Sexes separate, gonads arranged in double row on each side of body throughout intestinal region. In females, several ova contained within each ovary (Fig. 24). Systematic discussion. Gibson (1995) lists 12 species of Cephalothrix, four of which were included in Sundberg & Hylbom’s cladistic analysis of the Palaeonemertea. The Queensland species differs significantly from all previously known species of Cephalothrix in possessing a distinct colour pattern consisting of transverse brown bands on a pale yellow background; all other members of the genus are either white or more or less uniformly coloured with no colour pattern. Morphologically it also differs from one or more of the other species. For example, Cephalothrix germanica Gerner, 1969, unlike the present form, has no muscle plate between its rhynchocoel and gut walls. Furthermore, the new taxon has only two cephalic nerves whereas most other cephalothricids have four. The phylogenetic analysis in Sundberg et al. (2001) is a strong indication that the palaeonemertean species do not form a monophyletic group and exemplifies the problem of interpreting morphological characters as homologies. The group has been defined based on the position of the longitudinal nerves and the arrangement of body muscular wall. Present evidence (Sundberg et al. 2001: fig. 2) indicates that © The Norwegian Academy of Science and Letters • Zoologica Scripta, 32, 3, May 2003, pp279 – 296 This research was financially supported by the Swedish Research Council and the Erna and Victor Hasselblad Foundation to P. S. We are grateful to the Jubileumsfonden at Göteborg University for a travel grant to R.G., and to Mikael Härlin and Thomas Dahlgren for helpful comments and suggestions. References Campbell, A., Gibson, R. & Evans, L. H. (1989). A new species of Carcinonemertes (Nemertea: Carcinonemertidae) ectohabitant on Panulirus cygnus (Crustacea: Palinuridae) from Western Australia. Zoological Journal of the Linnean Society, 95, 257 –268. Coe, W. R. (1930). Unusual types of nephridia in nemerteans. Biological Bulletin, 58, 203–216. De Queiroz, K. & Gauthier, J. (1990). Phylogeny as a central principle in taxonomy: phylogenetic definitions of taxon names. Systematic Zoology, 39, 307 –322. Gibson, R. (1974). 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Phylogeny of protostome worms derived from 18S rRNA sequences. Molecular Biology and Evolution, 12, 641– 649. Zoologica Scripta, 32, 3, May 2003, pp279– 296 • © The Norwegian Academy of Science and Letters