Figure 1.
Meioglossus psammophilus gen. et sp. nov., light- and scanning electron micrographs (LM and SEM).
A. Overview of specimen, lateral view, LM (bright field, DIC). B. Cross section of anterior trunk region showing dorsal position of sperm, LM (bright field). C. Dorsal live view, paratomy (transverse fission, two individuals) and sperm, LM (bright field). D. Semithin sagittal section, toluidine blue stained, LM. E. Specimen showing paratomy, lateral view, SEM (ZMUC-ENP-6). F. Close up from E of anterior end, dorsal view, SEM. G. Specimen showing paratomy, dorsal live view, LM (dark field). H. Different shapes and sizes of sperm heads from paratomy specimen in G, LM (phase contrast). I. Sperm heads and flagella, LM (bright field, DIC). J. Close up of sperm heads from I. Abbreviations: an, anus; cb1, cb2, 1. & 2. ciliary band; co, collar region; en, endodermal cell; ep, epidermis; es, esophagus; gl, glomerulus; gp, gill pore; hg, hindgut; lm, longitudinal muscles; mc, mesocoel; mec, metacoel; mg, midgut; MHC, midgut-hindgut constriction; mo, mouth opening; nt, nerve tissue; pb, proboscis; pbgl, proboscis gland; pc, protocoel; ph, pharynx; phf, pharyngeal fold; phm, perihaemal muscles; plm, proboscis longitudinal muscles; sf, sperm flagellum; sh, sperm head; sp, sperm; TF, transverse fission zone; tr, trunk; tg, tail glands.
Table 1.
Type material and no. of specimens examined with different methods in the presented study.
Figure 2.
Meioglossus psammophilus gen. et sp. nov., 3D-reconstruction of the anatomy of the main organ systems.
Rows from left to right: dorsal, ventral, left and right view. Columns from top to bottom: The merge row (A–D) shows the adult with all reconstructed structures. Note that blood vessels and septa separating the paired coelomic cavities are not shown. Epidermis row (E–H) shows the external shape of M. psammophilus and condensed nerve structures including the collar cord and proboscis stem. Endoderm row (I–L): The digestive tract is subdivided into five regions, i.e. mouth opening, pharynx, esophagus, midgut and hindgut. Anus not shown. Mesoderm row (M–P) shows the position of the anterior protocoel (blue) and the paired meso- (pink) and metacoelic (red) compartments. Perihaemal diverticula not shown. The glomerulus is visible and occupied at least a quarter of the protocoelomic cavity. The pericardium is situated dorsal to the glomerulus. Click on image in Figure S2 to activate 3D model (follow link to full interactive PDF version of Figure 2). Abbreviations: cc, collar cord; co, collar region; es, esophagus; gl, glomerulus; gp, gill pore; hg, hindgut; mg, midgut; mo, mouth opening; pb, proboscis; pd, pericardium; ph, pharynx; ps, proboscis stem; tr, trunk.
Figure 3.
Meioglossus psammophilus gen. et sp. nov., light- and transmission electron micrographs of cross sections (A–D, G), and transmission electron micrographs of sagittal sections (E, F, H).
A. Cross section of posterior pharynx region. B. Oblique cross section of anterior pharynx region showing mouth opening at the bottom right. No stomochord or proboscis skeleton is present. C. Cross section of proboscis region showing glomerulus and pericardium. D. Cross section of anterior proboscis region showing the arrangement of the muscle fibers. E. Sagittal section of pericardium-glomerulus complex, anterior is to the left. F, G close-ups of the subepidermal collar cord that is constituted of numerous neurites. H. The glomerulus is lined by podocytes from the protocoelic side. Abbreviations: arrowheads, extracellular matrix; bv, blood vessel; cm, circular muscle fibers; ep, epidermis; gl, glomerulus; gls, glomerular sinus; lm, longitudinal muscle fibers; mc, mesocoel; nc, neurocord; ne, neurites; nt, nerve tissue; pc, protocoel; pd, pericardium; ph, pharynx; po, podocytes; pst, proboscis stem.
Figure 4.
Meioglossus psammophilus gen. et sp. nov., sagittal sections, transmission electron micrographs.
A. Median section of collar region, showing pharyngeal lumen and esophagus. B. Close up of gill pore. C. Close up of ventral side showing middle groove and ciliary band lined by glandular cells. D. Dorsal trunk, epidermis, sperm and flagella. E. Sperm flagella (double cilia in left flagellum?). F. Close up of sperm, transverse and sagittal view. G. Flagella and sperm heads in metacoel. H. Sperm head, sagittal, median view. I. Close up from A of dorsal pharyngeal foldings and basiepidermal nerve tissue. J. Flagellae and sperm head, transverse view. Abbreviations: aa, anchoring fibre apparatus; ba, electron dense cytoplasma basally; c1, c2, double cilia of flagella; cy, cytoplasma; cb1, cb2, 1. & 2. ciliary band; ce, centriole; ci, cilium; cm, circular muscles; en, endodermal cell; ep, epidermis; es, esophagus; glc, glandular cells; gp, gill pore; hg, hindgut; lm, longitudinal muscles; mec, metacoel; mg, midgut; mo, mouth opening; mv, microvillum; n, nucleus; nt, nerve tissue; nm, nuclear membrane; ph, pharynx; phf, pharyngeal fold; sf, sperm flagellum; sh, sperm head; sp, sperm; va, acrosomal vesicles of anterior cytoplasma.
Figure 5.
Meioglossus psammophilus gen. et sp. nov., confocal laser scanning microscopy, Z-stack projection images showing musculature (phalloidin staining), cilia (anti acetylated α-tubulin IR), nerves (anti α-tubulin and serotonin IR).
A. Two zooids of specimen showing paratomy, paratype ZMUC-ENP-3, dorso-lateral view. Triple staining of cilia, muscles and nervous system showing F-actin (green) and anti acetylated α-tubulin IR (blue) and anti-serotonin IR (red). B. Proboscis and collar cilia, paratype ZMUC-ENP-43, anti acetylated α-tubulin IR, depth coded. Depth scale follows the colors of the spectral light (blue to red). C. Anterior part of specimen from A, musculature, depth coded Z-stack. D. Proboscis, serotonergic nervous system (SNS) of A, depth coded Z-stack. E. Whole single specimen, ZMUC-ENP-47, double staining, anti acetylated α-tubulin IR (blue) and anti serotonin IR (red). Same specimen used in 3D isosurface reconstruction in attached Movie S3. F. Close up of serotonergic perikarya from proboscis of G, depth coded. G. Whole specimen, ZMUC-ENP-47, SNS, depth coded. Abbreviations: apvn, anterior perikarya of ventral nerve net; cb1, cb2, 1. & 2. ciliary band; cm, circular muscles; co, collar region; es, esophagus; lm, longitudinal muscles; map, muscular anchoring point; mg, midgut; MHC, midgut-hindgut constriction; mo, mouth opening; mpvn, median perikarya of ventral nerve net; nc, neurochord (collar); npl, nerve plexus; om, oblique muscles; pb, proboscis; pcb2, perikarya of second ciliary band nerves; pcm, proboscis circular muscles; ped, perihaemal diverticula; pnc1–4, posterior perikarya 1 to 4 of neurochord; plm, proboscis longitudinal muscles; pln, nerves innervating proboscis longitudinal muscles; ppb, perikarya of proboscis; ppvn, posterior perikarya of ventral nerve net; TF, transverse fission zone; vn, ventral nerve net (forms two ventral ‘cords’).
Figure 6.
Meioglossus psammophilus gen. et sp. nov., confocal laser scanning microscopy, Z-stack projection images showing cilia or glands (anti acetylated α-tubulin IR or tyrosinated tubulin IR) and nerves (anti serotonin- and FMRFamide-like IR).
A. Whole specimen, holotype ZMUC-ENP-1, ventral view, double staining of cilia and nervous system showing anti-tyrosinated tubulin IR (blue) and anti serotonin IR (red). B. Proboscis and collar of ZMUC-ENP-49, dorsal view, anti FMRFamide-like IR, depth coded. Depth scale follows the colors of the spectral light (blue to red). C. Proboscis of paratype ZMUC-ENP-5, lateral view, anti FMRFamide-like IR, depth coded Z-stack projection. D. Anterior, dorsal view of ZMUC-ENP-51, anti tyrosinated tubulin IR (blue) and anti serotonin IR (red). E. Collar region of ZMUC-ENP-52, dorsal view, anti tyrosinated tubulin IR (blue) and anti serotonin IR (red). F. Anterior end of paratype ZMUC-ENP-4, lateral view, anti-acetylated α-tubulin IR (blue) and anti FMRFamide-like IR (red). G–H. Proboscis and collar of ZMUC-ENP-50 dorsal view; G. Anti acetylated α-tubulin IR (blue) and anti FMRFamide-like IR (red); H. Anti FMRFamide-like IR, depth coded Z-stack. Abbreviations: apvn, anterior perikarya of ventral nerve net; cb1, cb2, 1. & 2. ciliary band; co, collar region; es, esophagus; gc, glandular cell; mg, midgut; mo, mouth opening; mpvn, median perikarya of ventral nerve net; nc, neurochord (collar); pb, proboscis; pcb2, perikarya of second ciliary band nerves; pln, nerves innervating proboscis longitudinal muscles; ppb, perikarya of proboscis; ppvn, posterior perikarya of ventral nerve net; tun, tubulinergic nerve; vn, ventral nerve net (forms two ventral ‘cords’).
Figure 7.
Optimal maximum likelihood tree obtained in RaxML under GTR+Γ for the 18S rRNA data set (−logL = −8584.713814).
Values on nodes indicate bootstrap support values above 50%; an asterisk indicates a value of 100%.
Figure 8.
Optimal tree at 4456 weighted steps based on the analysis of 18S rRNA under direct optimization in POY (parameter set 3221).
Values above nodes indicate jackknife support values above 50%.
Table 2.
Taxon sampling and sequences included in phylogenetic analyses.