Table 1.
List of protocols for fixation and GenBank accession numbers of the sequences of materials.
Table 2.
List of primers used in this study.
Table 3.
List of species used for the molecular phylogenetic analysis, and their respective collection localities and habitats, GenBank accession numbers, and references.
Fig 1.
Boninia uru sp. nov., ICHUM 8278: Photographs of living and fixed specimens.
(A) Entire animal, living state, dorsal view; (B) entire animal, living state, ventral view; (C) magnification of anterior body, living state; (D) posterior body showing reproductive organs, fixed state, stained with acid fuchsin and cleared in xylene, ventral view. Abbreviations: ad, adhesive organ; ce, cerebral eyespots; fg, female gonopore; me, marginal eyespot; mg, male gonopore; ph, pharynx; t, tentacles; ut, uterus. Scale bars: A–C, 1 mm; D, 0.5 mm.
Fig 2.
Schematic diagrams of Boninia uru sp. nov. (anterior to the left).
(A) ICHUM 8278 (holotype), copulatory complex; (B) ICHUM 8283 (paratype), pharynx, male and female copulatory apparatuses. Abbreviations: au, anterior dilations of uteri; cg, cement glands; cp, cement pouch; fa, female atrium; fg, female gonopore; it, intestine; Lv, Lang’s vesicle; ma, male atrium; mg, male gonopore; ph, pharynx; po, prostatoid organ(s); pp, penis papilla; pu, posterior dilations of uteri; pv, prostatoid vesicle; sd, sperm duct; st, stylet; sv, seminal vesicle; uc, uterine canal; uv, uterine vesicle; va, vagina. Scale bars: A, 100 μm; B, 300 μm.
Fig 3.
Boninia uru sp. nov.: Photomicrographs of sagittal (A, B, H, I) and horizontal (C–G) sections. (A, B) ICHUM 8278 (holotype), male copulatory apparatus, anterior to the left; (C, D) ICHUM 8279 (paratype), male copulatory apparatus, with stylet of the prostatoid organ indicated by arrowheads; (E–G) ICHUM 8283 (paratype), connection between uterine canal and Lang’s vesicle; (H) ICHUM 8278 (holotype), female copulatory complex; (I) ICHUM 8278 (holotype), adhesive organ. Abbreviations: ad, adhesive organ; cg, cement glands; cp, cement pouch; fa, female atrium; fg, female gonopore; Lv, Lang’s vesicle; ma, male atrium; mf, muscle fiber; po, prostatoid organ(s); pp, penis papilla; pu, posterior dilations of uteri; pv, prostatoid vesicle; st, stylet; uc, uterine canal; uv, uterine vesicle. Scale bars: A, B, 100 μm; C–G, 50 μm; H, I, 200 μm.
Table 4.
Comarison of selected characters among Boninia species.
Fig 4.
Boninia yambarensis sp. nov.: Photographs taken in the living state.
(A) ICHUM 8284, dorsal view; (B) ICHUM 8284, magnification of the anterior body, dorsal view; (C) ICHUM 8288, ventral view; (D) ICHUM 8289, magnification of anterior body (squeezed); (E) ICHUM 8289, arrangement of prostatoid organs (squeezed). Pointed tentacles are shown by arrowheads (A, B). Abbreviations: ad, adhesive organ; br, brain; ce, cerebral eyespots; fg, female gonopore; mg, male gonopore; ph, pharynx; sd, sperm duct; st, stylet; ut, uteri. Scale bars: A, C, 5 mm; B, 1 mm; D, 500 μm; E, 300 μm.
Fig 5.
Schematic diagrams of Boninia yambarensis sp. nov. (anterior to the right).
(A) ICHUM 8284 (holotype), sagittal view of copulatory complex; (B) ICHUM 8285 (paratype), histological view of copulatory complex. Abbreviations: au, anterior dilation of uterus; cg, cement glands; cp, cement pouch; fa, female atrium; fg, female gonopore; it, intestine; Lv, Lang’s vesicle; ma, male atrium; mg, male gonopore; po, prostatoid organ; pog, prostatoid organ glands; pp, penis papilla; pu, posterior dilation of uterus; pv, prostatoid vesicle; sd, sperm duct; st, stylet; sv, seminal vesicle; uc, uterine canal; uv, uterine vesicle; va, vagina. Scale bars: A, 100 μm; B, 300 μm.
Fig 6.
Boninia yambarensis sp. nov.: Photomicrographs of sagittal (A, B, D–G) and horizontal sections (C). (A) ICHUM 8284 (holotype), male copulatory apparatus, anterior to the right; (B) ICHUM 8284 (holotype), prostatoid organ, anterior to the right; (C) ICHUM 8285 (paratype), arrangement of prostatoid organs; (D) ICHUM 8284 (holotype), uterine vesicles, anterior to the right; (E) ICHUM 8284 (holotype), female copulatory apparatus, anterior to the right; (F) ICHUM 8284 (holotype), adhesive organ. Abbreviations: ad, adhesive organ; au, anterior dilation of uterus; cg, cement glands; cp, cement pouch; fa, female atrium; fg, female gonopore; it, intestine; Lv, Lang’s vesicle; ma, male atrium; mg, male gonopore; po, prostatoid organ; pog, prostatoid organ glands; pp, penis papilla; pu, posterior dilation of uterus; pv, prostatoid vesicle; sd, sperm duct; st, stylet; sv, seminal vesicle; uc, uterine canal; uv, uterine vesicle; va, vagina. Scale bars: A, C–F, 100 μm; B, 50 μm.
Fig 7.
Maximum likelihood phylogenetic tree based on a concatenated dataset of partial 18S and 28S.
Numbers near nodes are posterior probability and bootstrap values, respectively.
Fig 8.
Ancestral reconstruction of habitats produced using Bayesian Binary Markov chain Monte Carlo analysis.
Nodes are numbered. Pie charts on nodes show the probabilities of possible ancestral states with numbers representing the highest probabilities (%). *, ancestral state with a relative probability <5%.