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FIGURE 5 in Using Gordiid cysts to discover the hidden diversity, potential distribution, and new species of Gordiids (Phylum Nematomorpha)

FIGURE 5. Maxent model summary of gordiid infected snail geography showing known occurrence points (black circles) and predicted potential geographic distribution (green areas of streams) in Payne County, Oklahoma. Blue areas of streams are identified as unsuitable. Purple and red arrows indicate locations of free-living adults of Gordius n. sp. and Chordodes morgani, respectively. Note that all Gordius n. sp. were collected in the predicted geographic distribution, and C. morgani was collected from a dog water bowl from a home near a region of a stream with predicted distribution for gordiids. Scale bar = 6.7 km.

FIGURE 4 in Using Gordiid cysts to discover the hidden diversity, potential distribution, and new species of Gordiids (Phylum Nematomorpha)

FIGURE 4. Scanning electron and light microscope micrographs of adult free-living gordiids reared from field collected cysts or collected as free-living adults from Payne County, Oklahoma. (A) Dorsal view of the posterior region of a female Paragordius varius. Note the three posterior tail lobes (Tl). Scale bar = 500 Μm. (B) Ventral view of the posterior region of a male Paragordius varius. Note the two long posterior tail lobes (Tl), cloaca (c), and spines on the ventral midline (white arrows). Scale bar = 200 Μm. (C) Midbody cuticular structures of a male P. varius. Note the hemisphirical structures (Hs). Scale bar = 10 Μm. (D) Tapered anterior end and cuticular patern of a male Chordodes morgani. Scale bar = 100 Μm. (E) Midbody region of a male C. morgani showing the characteristic leopard pattern. Scale bar = 300 Μm. (F) Midbody region of a male C. morgani showing the characteristic crown areole pattern. Scale bar = 100 Μm. (G) Ventral view of the posterior region of a male C. morgaini. Note no tail lobes and an oval cloaca (c). Scale bar = 200 Μm. (H) Cloaca opening surrounded by circumcloacal spines. Scale bar = 10 Μm. (I) Cuticular structures on the posterior region of a male C. morgaini. Note the crown areoles (Ca), bulging areoles (BA), and bristles (white arrow). Scale bar = 8 Μm. (J) Tapered anterior end and cuticular patern of a male Gordius n. sp. Scale bar = 10 Μm. (K) Areole pattern on the posterior body region of a male Gordius n. sp. Note the weakly developed areoles (A) and the presence of bristles (white arrows). Scale bar = 20 Μm. (L) Ventral view of the posterior region of a male Gordius n. sp. Note the two posterior tail lobes (Tl), cloaca (c), and post cloacal crescent (Pcc). Scale bar = 120 Μm.

FIGURE 3 in Using Gordiid cysts to discover the hidden diversity, potential distribution, and new species of Gordiids (Phylum Nematomorpha)

FIGURE 3. (A) Female Acheta domesticus releasing a single female Paragordius varius. (B) A gordian knot of male and female P. var ius after emerging from a female A. domesticus. Note that males are the darker and thiner individuals. Scale bars = 1 cm.

FIGURE 1 in Using Gordiid cysts to discover the hidden diversity, potential distribution, and new species of Gordiids (Phylum Nematomorpha)

FIGURE 1. Photomicrographs of gordiid cyst and larval types recovered from Physa acuta snails collected in Payne County, Oklahoma. (A) Cyst of Gordius sp. Note the lack of spines on the preseptum and tighly double folded larva. Scale bar = 25 Μm. (B) Cyst of Paragordius sp. Note the characterisitc spines on the preseptum (black arrow) and double folding of the postseptum (white arrow) never reaching the posterior end of the preseptum. Scale bar = 15 Μm. (C) Cyst of Chordodes/Neochordodes sp. Note the single folding position of the postseptum and relatively small spines on the preseptum. Scale bar = 10 Μm. (D) Larva of Gordius sp. in the process of folding. Note the characteristic single spine on the posterior end of the postseptum (arrow). Scale bar = 15 Μm. (E) Unencysted larva of Paragordius sp. Note the characteristically long spines of the outer hooks of the preseptum (black arrow) and characteristic granules of the pseudointestine (white arrow). Scale bar = 15 Μm. (F) Unencysted larva of Chordodes/Neochordodes sp. Note the equal length ratio of the prespeptum and postseptum and relatively small spines on the preseptum. Scale bar = 10 Μm.

FIGURE 2 in Using Gordiid cysts to discover the hidden diversity, potential distribution, and new species of Gordiids (Phylum Nematomorpha)

FIGURE 2. Positive (black circles) and negative (white circles) localities for (A) Paragordius spp., (B) Gordius spp., and (C) Chordodes/Neochordodes spp. in Payne County, Oklahoma. Numbers represent names of each site (see Table 1). Scale bars = 6.9 km.

FIGURE 6 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 6. Developing and fully formed cysts of Chordodes kenyaensis n. sp. recovered from an experimentally infected Physa gyrina snail. (A) Larva in snail tissue 14 DPE in the process of folding (lateral view). Note the empty pseudointestine (black arrow). Scale bar = 12 µm. (B) Mature cysts (lateral view); note the clear halo surrounding the encysted larvae and single folding pattern. Scale bar = 15 µm.

FIGURE 3 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 3. Adult and areole pattern of deformed Chordodes kenyaensis n. sp. (A) Adult male removed from a cricket while trying to emerge. Note the larval cuticle (arrow). Scale bar = 1.0 mm. (B) DIC photomicrograph of the mid-body cuticle of a female showing tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles scattered among simple (1) areoles. Note that simple areoles are smooth but contain short bristles making them appear bumpy. Scale bar = 30 µm. (C) DIC photomicrograph of the same region of the cuticle in a higher focal plane of the mid-body cuticle showing tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles scattered among simple areoles. Scale bar = 30 µm. (D) DIC photomicrograph of a thorn (3) areole among simple areoles of the mid-body region of an adult deformed female worm. Scale bar = 7 µm.

FIGURE 4 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 4. Mating, oviposition behavior, egg strings, and eggs of Chordodes kenyaensis n. sp. (A) Male and female in a Gordian knot. Note the lighter color and thicker size of the female worm compared to the male. Scale bar = 2.0 mm. (B) Female in the process of depositing egg strings in a zigzag pattern on a stick. Scale bar = 5 mm. (C) Undeveloped egg. Scale bar = 15 µm.

FIGURE 5 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 5. Larval characteristics of Chordodes kenyaensis n. sp. (A) DIC photomicrograph of a live larva (lateral view). Note two of the three stylets (black arrow) and v shaped pseudointestine (white arrow). Scale bar = 15 µm. (B) DIC photomicrograph of anterior end of larva (lateral view), note the everted proboscis (arrow) with three stylets. Scale bar = 13 µm. (C) SEM photomicrograph of an entire larva (lateral view). Note the distinct preseptum (Pre) and postseptum (Pos) Scale bar = 8.0 µm. (D) SEM photomicrograph of the posterior end (ventral side). Note the two anterior spines (AS), two posterior spines (PS) and pseudointestine gland opening (PSGO). Scale bar = 3.0 µm. (E) SEM photomicrograph of the anterior end (en face view). Note the orientation of the proboscis (P) with left lateral spines (LLS) to the ventral hooks (VH). Scale bar = 3.0 µm. (F) SEM photomicrograph of the preseptum (ventral view). Note the orientation of the proboscis (P) to the ventral hooks (VH); median hooks (MH). Scale bar = 3.3 µm. (G) Close up SEM photomicrograph of a ventral view of the proboscis (P). Note the arrangement of the five ventral spines (VS). Scale bar = 1.5 µm.

FIGURE 2 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 2.Allotype of female Chordodes kenyaensis n. sp. (A) SEM photomicrograph showing the tapered anterior end and areole pattern. Note distinct thorn areoles (arrows) among simple areoles. Scale bar = 115 µm. (B) SEM photomicrograph showing higher magnification of the anterior region of the cuticle with simple (1) and tubercle (2) areoles. Note that interareolar furrows are arranged laterally across the cuticle. Scale bar = 10 µm. (C) SEM photomicrograph showing overview of the mid-body cuticle. Note the pattern of crowned areoles. Scale bar = 300 µm. (D) DIC photomicrograph showing two crowned (6) areoles surrounded by circumcluster (5) areoles in the mid-body region. Scale bar = 20 µm. (E) SEM photomicrograph showing simple (1), bulging (4), circumcluster (5) and crown (6) areoles in the mid-body region. Note the long filaments on crown areoles. Scale bar = 20 µm. (F) SEM photomicrograph showing higher magnification of simple (1), tubercle (2), and thorn (3) areoles in the mid-body region. Interareolar furrows are arranged laterally across the cuticle. Note the fine terminal bristles on the simple and tubercle areoles. Scale bar = 10 µm. (G) SEM photomicrograph showing posterior end and cloacal opening (arrow). Note the non-areolated region around the cloacal opening and bristles on the lateral sides of the non-areolated field. Scale bar = 150 µm. (H) SEM photomicrograph showing higher magnification of the posterior region of the cuticle with simple (1), tubercle (2), bulging (4), circumcluster (5) and crowned (6) areoles. Note the variation in simple areoles in this body region compared to the anterior and mid-body region of the cuticle. Scale bar = 40 µm.

FIGURE 1 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 1. Holotype of male Chordodes kenyaensis n. sp. (A) SEM photomicrograph showing the tapered anterior end and areole pattern. Scale bar = 170 µm. (B) SEM photomicrograph showing higher magnification of the anterior region of the cuticle with simple (1), tubercle (2), and two types of thorn (3 and 3*) areoles. Note that interareolar furrows are arranged laterally across the cuticle. Scale bar = 15 µm. (C) SEM photomicrograph showing an overview of the mid-body cuticle. Note the pattern of crowned areoles. Scale bar = 190 µm. (D) SEM photomicrograph showing a higher magnification of the midbody cuticle containing simple (1), tubercle (2), thorn (3), bulging (4), circumcluster (5) and crowned (6) areoles. Interareolar furrows are arranged laterally across the cuticle. Note the long filaments on crowned areoles and short bristles on top of simple smooth areoles which are separated between neighbors by interareolar furrows. Scale bar = 25 µm. (E) SEM photomicrograph showing the posterior end and cloacal opening (arrow). Note the indistinct lobes (thick arrows). Scale bar = 115 µm. (F) SEM photomicrograph showing a higher magnification of the posterior region of the cloacal opening surrounded by circumcloacal spines. Note the non-areolated region and bristles on the lateral regions of the non-areolated field. Scale bar = 40 µm.

FIGURE 7 in Novel techniques for biodiversity studies of gordiids and description of a new species of Chordodes (Gordiida, Nematomorpha) from Kenya, Africa

FIGURE 7. Molecular phylogenetic analysis by maximum likelihood method based on partial cox1 gene from Chordodes kenyaensis n. sp. and 16 other nucleotide sequences from Genbank. Paragordius and Gordius were used as outgroups. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Only bootstrap values with a value of 85% or higher are shown. Note that Chordodes kenyaensis n. sp. is placed within other species of Chordodes.