Table 1.
Primers and references for PCR protocols for amplification of the four DNA fragments sequenced in the study.
Table 2.
Sequences used for molecular comparisons and phylogenetic analyses of Macrobiotus shonaicus sp. nov. with all other species of the Macrobiotus hufelandi group for which DNA sequences are currently available.
Fig 1.
Macrobiotus shonaicus sp. nov.–habitus.
A–dorso-ventral projection (holotype, Hoyer’s medium, PCM); B–dorsal view (paratype, SEM); and C–E–cuticular pores seen in SEM on the anterior (C), median (D) and posterior (E) part of the body (paratype). Scale bars in μm.
Fig 2.
Macrobiotus shonaicus sp. nov.–cuticular structures on legs.
A–granulation on leg II (paratype, PCM); B–granulation on leg IV (paratype, PCM); C–granulation on leg II (paratype, SEM); D–granulation on leg IV (SEM); E–cuticular bulge resembling pulvinus like structure and faint cuticular fold on the internal surface of leg II (holotype, PCM); F–cuticular bulge resembling pulvinus like structure and faint cuticular fold on the internal surface of leg III (paratype, SEM); Indented arrowheads indicate pores on legs, filled flat arrowheads indicate the cuticular bulge, whereas empty flat arrowheads indicate faint cuticular fold under the claws. Scale bars in μm.
Fig 3.
Macrobiotus shonaicus sp. nov.–buccal apparatus and the oral cavity armature seen in PCM (all paratypes).
A–dorso-ventral projection with ventral teeth of the third band and ventral placoids, the upper insert shows dorsal placoids (a different individual); B–C oral cavity armature of the maculatus type (only the third band of teeth visible), dorsal and ventral view, respectively; and D–E oral cavity armature of the patagonicus type (both the second and the third band of teeth visible), dorsal and ventral view, respectively. Empty flat arrowhead indicates the second band of teeth in the oral cavity whereas empty indented arrowheads indicate third band of teeth. Fig A assembled from several photos. Scale bars in μm.
Fig 4.
Macrobiotus shonaicus sp. nov.–mouth opening and the oral cavity armature seen in SEM (paratype).
A–B–mouth opening with six peribuccal sensory lobes and ten lamellae; C–D–the oral cavity armature of a single paratype seen in SEM from different angles. Filled arrows indicate pores in the irregular ring of pores surrounding the peribuccal lobes, flat filled arrowheads indicate teeth of the first band, empty flat arrowheads indicate teeth of the second band, the peaks in the ridges of the third band that correspond with teeth of the third band in species with better developed oral cavity armatures are marked “m1-m3” (the dorso-median tooth comprises three small peaks visible as thickenings in PCM), “M” (ventro-median tooth) and “L” (lateral teeth). Scale bars in μm.
Fig 5.
Macrobiotus shonaicus sp. nov.–claws (paratypes).
A–B–claws I and IV seen in PCM, with smooth and slightly dentate lunules, respectively and C–D–claws I and IV seen in SEM, with smooth and slightly dentate lunules, respectively. Figs A and B assembled from several photos. Scale bars in μm.
Fig 6.
Macrobiotus shonaicus sp. nov.–egg seen in PCM.
A–midsection under 400× magnification; B–surface under 400× magnification; C–D–midsection under 1000× magnification; E–surfaces of terminal discs under 1000× magnification; and F–surface of egg between processes under 1000× magnification (note fine dark dots between the processes). Flat filled arrowheads indicate thin flexible filaments whereas empty arrowhead indicate the indentation at the terminal disc edges. All photos show the details of a single egg. Scale bars in μm.
Fig 7.
Macrobiotus shonaicus sp. nov.–egg chorion morphology seen in SEM.
A–B–entire eggs with clearly visible flexible filaments on the egg processes; C–D–processes with filaments of various lengths and the surface between processes; and E–F–zoom on a single-egg process. Scale bars in μm.
Table 3.
Measurements [in μm] and pt values of selected morphological structures of the holotype and paratypes of Macrobiotus shonaicus sp. nov. mounted in Hoyer’s medium (N–number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD–standard deviation).
Table 4.
Measurements [in μm] of selected morphological egg structures of Macrobiotus shonaicus sp. nov. mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; and SD–standard deviation).
Fig 8.
The Bayesian Inference (BI) phylogeny constructed from COI sequences of the Macrobiotus hufelandi group species.
Numbers at nodes indicate Bayesian posterior probability. The hufelandi species clade with modified egg processes is indicated by thicker branches. Please see Table 2 for details on species sequences used in the analysis. Scale bar represents substitutions per position.