Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditis tufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus—specifically from non-extreme locales—establishes a comparative system for the study of extremophilic traits in this model.

Light microscopic observation and preparation of type specimens. Adult nematodes were collected from a one-week-old culture, whereafter they were heat-killed and fixed in TAF (triethanolamine:forma lin:distilled water = 2:7:91) for one week. Fixed material was processed to glycerin using a modified Seinhorst's method 18 and mounted in glycerin according to the methods of de Maeseneer and d'Herde 19 . Mounted specimens were used for morphometrics and kept as type material. In addition, live adult nematodes from 1-week-old culture were used for detailed morphological observations following the methods of Kanzaki 20 . All micrographs were obtained using a digital camera system (MC170 HD; Leica, Wetzlar, Germany) and morphological drawings were made using a drawing tube (Eclipse Ni; Nikon, Tokyo, Japan) connected to the microscope.
Scanning electron microscopy. To obtain adult males for scanning electron microscopic (SEM) observation, two or three dauer larvae were transferred onto each of several NGM plates seeded with E. coli OP50 and incubated at 20 °C. In the next generation, males were present at relatively high frequency and were collected into M9 buffer. Males were killed by heating on a hot plate (60 °C for 5 min) and were pre-fixed in 2% formaldehyde plus 2.5% glutaraldehyde in M9 buffer for 15 h at 4 °C. Nematodes were then post-fixed with 1% osmium tetroxide for 1.5 h and then dehydrated with an ethanol series (25,50,70,80,90, and twice in 99.5% ethanol). After dehydration, samples were immersed in 100% tert-butyl alcohol twice (15 min each) and then dried with a freeze dryer (JFD-310; JEOL, Tokyo, Japan). Samples were coated with osmium with an sputter coater (HPC-1SW; Vacuum Device, Ibaraki, Japan), and then observed with a SEM (JSM-6700F; JEOL).

Molecular profiles and phylogeny.
To establish molecular profiles and vouchers for the new species, approximately 4 kb of ribosomal DNA (rDNA), including nearly the full length of small subunit (SSU), intergenic transcribed spacer (ITS), and D1-D4 extension segments of the large subunit (LSU), were sequenced for the new species and another, undescribed species (coded as NKZ329). In addition to these two species, a partial SSU (ca. 0.9 kb of 5′ end) was determined for a second undescribed species (EJR13) for inclusion in phylogenetic analysis. All sequences were derived from bulk DNA samples, which were prepared from cultured nematodes (ca. 20 individuals) following methods described by Kikuchi et al. 21 and Tanaka et al. 22 , and sequences were determined with PCR-direct sequencing according to the method of Ekino et al. 23 , i.e., a long (ca. 4 kb) fragment was amplified with a primer set SSU988F (5′-CTC AAA GAT TAA GCC ATG C-3′) and D4R (5′-GCG GTA TTT GCT ACT ACC AYY AMG ATC TGC-3′), and the sequences determined using amplified primers and internal primers, SSUF22 (5′-TCC AAG GAA GGC AGC AGG C-3′), SSUF23 (5′-ATT CCG ATA ACG AGC  -TCG GAA  GGA ACC AGC TAC TA-3′), D3a (5′-GAC CCG TCT TGA AAC ACG GA-3′), and D3aR (5′-TCC GTG TTT  CAA GAC GGG TC-3′) for the former two species, and only SSU988F and SSU1912R were used for EJR13.
Phylogenetic relationships of the new species to other rhabditid nematodes were inferred from partial SSU sequences by Bayesian analysis. First, sequences were aligned using MAFFT 24 26 . Combined Bayesian analysis was performed using MrBayes 3.2 27,28 ; four chains were run for 4 × 10 6 generations, and Markov chains were sampled at intervals of 100 generations 29 . Two independent runs were performed, and after confirming the convergence of runs and discarding the first 2 × 10 6 generations as burn-in, the remaining topologies were used to generate a 50% majority-rule consensus tree. Species were included in the analysis as informed by Kanzaki et al. 10 . Accession numbers of included sequences are given in Supplementary Table S1.
Fecundity measurements. L4 larvae of the new species were singled onto NGM plates seeded with E.
coli OP50 and allowed to develop for 24 h into day-1 adults. Adults were then transferred daily onto new seeded plates until they died, and the progeny that they laid were counted every day. Ten replicates were tested. Animals were maintained at 22.5 °C, and adults were transferred with an eyelash to minimize damage from picking.
Sex ratios of self-progeny. To study reproductive mode in the new species, we quantified sex ratios among offspring produced by a single hermaphrodite. First, we established that all dauers developed into adult hermaphrodites, as in Auanema species 30 . Therefore, dauer juveniles were selected to become the mothers from which offspring sexes were quantified. Each individual dauer juvenile was placed on an NGM medium (40 mm diam.) seeded with E. coli OP50 and kept in a culture room (approximately 23 °C), after which the nematode was examined for developmental stage and reproduction twice a day (approximately every half-day) until it died. As soon as reproduction commenced, each mother was transferred to a new culture plate, also twice a day. The sex was determined as male vs. morphological female (i.e., female/hermaphrodite) for each offspring left on each plate from which the mother was removed. Specifically, sex was determined once larvae had reached the L3 stage or L4, according to body shape and shape of genital anlagen, as observed under a dissecting microscope. Male juveniles were then removed from the culture medium, while the rest of the juveniles (females/hermaphrodites) were allowed to proceed with development. After at least one day of adulthood, the sex of each individual was distinguished according to the contents of their uteri: individuals carrying only undeveloped oocytes were detetermined to be females, whereas hermaphrodites carried juvenile offspring in their uterus. Once reproduction ceased, the number of male, female, and hermaphrodite offspring were tallied for each half-day time point.
Offspring sexing was performed for six hermaphroditic mothers.

Results
Taxonomic description of Tokorhabditis n. gen.. Etymology: the genus name is derived from the Greek τόκος ("birth") and "Rhabditis, " the type genus of Family Rhabditidae. Rhabditidae. Diagnostic characters of the genus that are in this combination not found in species from other genera: males much smaller than females/hermaphrodites. Stoma (buccal cavity) tube-like, comprising simple short tube-like cheilo-, gymno-, and stegostom, where stegostom occupies about half of stoma (pharyngeal sleeve present), metastegostom possessing two small denticles on each of its three radial sectors; median bulb relatively well developed, round to square. Vagina of young female/hermaphrodite folded, forming "Z' shape in lateral view, albeit folding unclear in mature individuals after laying juveniles, and surrounded by layers of flattened, elongated cells stretched longitudinally and perpendicularly from the vagina. Female tail conical; bursa on male tail open anteriorly with more or less distinctly bilobed posterior margin; eight genital papillae (GP) and pore-like phasmids posterior to genital papillae. One to two GP precloacal, 5th and 7th attached to the dorsal surface of the fan. Anterior pairs forming papilliform GP or short bursal rays. 6th GP separate, 7th and 8th close together. Spicules separate and dagger-shaped; gubernaculum half as long as spicules. Dauer juveniles ensheathed. Three sexes (male, female, self-fertilizing hermaphrodite) present. Males rare, but still found in populations with very low density. Female and hermaphrodite both obligately viviparous (Supplementary Data 1), with embryos growing in size before reaching the L1 stage 8 .

Relationships.
The adult morphology of Tokorhabditis n. gen., including the stoma, male tail characters (papilla configuration and variation therein), and presence of three sexes, overlaps with that of Auanema 10 , the closest genus based on biological and morphological characters. However, the new genus is distinguished from Auanema most prominently by being obligately viviparous. Additionally, Tokorhabditis n. gen. is distinguished by its male phasmid structure, being pore-like vs. papilliform, and by the genitalia of females/hermaphrodites, specifically by the presence of a vaginal folding and by a tissue composed of layered, flattened, elongated cells stretched perpendicularly from the vagina. Diagnostic characters of Tokorhabditis n. gen. with respect to phylogenetically close genera Auanema, Rhabditella, and Cephaloboides, as informed by Kiontke and Sudhaus 31 , Scholze and Sudhaus 32 , Sudhaus 33 and Kanzaki et al. 10 , are summarized in Table 1.
Tokorhabditis is also similar to Haematozoon and Rhabditoides in general morphology, e.g., with a more or less long and narrow stoma and conspicuous pharyngeal sleeve, conical female/hermaphrodite tail, daggershaped male spicule, and a gubernaculum that is wealky arcuate and approximately half of spicule in length 33 . However, Tokorhabditis is readily distinguished from both genera with its biological characters, three vs. two sexes, vivipary vs. ovipary, and the number and arrangement of male genital papillae, namely by having eight pairs with one to two that are precloacal vs. nine pairs with three to four precloacal pairs and by the 5th and 7th pairs being dorsally oriented vs. laterally oriented 33 . Tokorhabditis tufae n. gen., n. sp.. Figures 1, 2 www.nature.com/scientificreports/ The species epithet is the Latin genitive of "tufa, " which describes the carbonate mineral precipitates characteristic of the majestic rock towers of Mono Lake. Table 2.

Measurements. See
Description. Adults. Body cylindrical. Cuticle thick, annulation fine, greatest annule width being 1.5-2.0 μm. Lateral field present, without ridges (alae). Lip region continuous with body contour, separated into six sectors of equal size. Stomatal opening beset by three pairs of lip sectors (two subdorsal sectors; right lateral plus subventral sectors; left lateral plus subventral sectors), forming triangular stomatal (buccal cavity) opening en face. Labial sensilla setiform, one per lip sector. Cephalic sensilla setiform, one per dorsal sector and subventral sector (four total). Amphid openings at the level of margin between cheilo-and gymnostom, forming ovalshaped pores. Stoma cylindrical; cheilostom, gymnostom, and stegostom with approximately 1:2:3 of relative length, respectively such that pharyngeal sleeve (= stegostom) surrounds posterior half of stoma. Cheilostom and gymnostom forming short tube, with arcade syncytia slightly visible where they meet gymnostom. Prostegostom and mesostegostom forming a simple short cylinder surrounded by pharyngeal sleeve, and comprising about 55% of stomatal cylinder. Metastegostom slightly anisotopic and isomorphic with two small denticles on each sector, slightly more posterior on the dorsal side. Procorpus muscular, cylindrical. Metacorpus muscular, forming a well-developed median bulb. Isthmus slender, not muscular. Basal bulb rounded (not polygonal) and possessing weakly-developed duplex haustrulum posterior to valves. Anterior pharynx (Procorpus plus metacorpus) slightly longer than posterior pharynx (isthmus plus basal bulb). Cardia (pharyngo-intestinal junction) well-developed, funnel-shaped. Nerve ring circling middle of isthmus. Secretory-excretory pore conspicuous, slightly variable in position among individuals, mostly overlapping with level of basal bulb. Excretory duct extending anteriad from pore, then reflexing posteriad distally. Excretory cell adjacent to excretory duct, slightly posterior to pore. Deirid on lateral side of body, around level of excretory pore.
Males. Two separate lines of lateral field sometimes observed, i.e., not conspicuous, with visibility depending on preparation of specimens. Tail region ventrally curved when killed by heat. Stoma on average six times as long as wide. Testis single, on the right of intestine; anterior part ventrally reflexed. Distal third of gonad forming vas deferens, either empty or containing small sperm cells. Two subventral glands and one dorsal cloacal (anal) gland visible at level of anterior end of retracted spicules. Spicules paired, arranged as a "V" shape in ventral view; often protracted in heat-killed specimens. In lateral view, spicule with roundish-square manubrium separated from blade with constriction; blade widest at just posterior to constriction, then smoothly tapered to bluntly pointed tip; spicule with small but pronounced dorsal, spike-like projection present at slightly anterior to proximal of spicule tip. Gubernaculum short, narrow, slightly ventrally arcuate in lateral view, approximately 2/3 of spicule in length; thin, flat extensions of both sides cover the dorsal side of spicule blade; elongate oval in ventral view. Bursa present, anteriorly open; leptoderan, somewhat polygonal, with smooth edges; distal end of bursa deeply notched, forming a rounded flap on each sides of a slender tail spike with approximately 1.5 times of cloacal body diam. In length. Eight pairs of genital papillae (GP) arranged as < (GP1, GP2), (GP3, GP4), (GP5d, CO), GP6, (phasmid, GP7d, GP8) > , where CO is the cloacal opening, GP1-GP4 form papilliform genital papillae, and other papillae form bursal rays. First four subventral papillae form two doublets (GP1 + GP2 and GP3 + GP4), where first and second doublets located ca. 2.5 and 0.5 cloacal body diameters (CBD) anterior to CO; GP5d adcloacal, GP6 between CO and root of tail spike; phasmid, GP7d and GP8 close to each other around the slightly anterior to the root of tail spike. Tips of GP5d and GP7d attached to the dorsal side of bursa, all other GP attached ventrally. Pore-like phasmids with ventral openings in a terminal position near tail tip.
Females and hermaphrodites. Females and hermaphrodites are morphologically indistinguishable. Body weakly, smoothly, and ventrally arcuate when heat-relaxed. Vulva located at mid body, forming horizontal slit 1/3 of vulval body diam. In length; cuticle around the vulva lacks annulations. Stoma 3.5-4.7 times as long as wide. Two gonads, one extending anteriad from vulva along right side of body, the other extending posteriad from vulva and along left side, are both dorsally reflexed. Germ cells arranged in multiple (two to three) rows in Table 1. Comparison of key typological characters among four closely related genera.
Sex ratios of self-progeny of T. tufae n. gen., n. sp.. Sexes of offspring from selfing hermaphrodites were determined throughout the mother's lifespan (Fig. 8). We found the first offspring 1 day after an individual dauer juvenile was placed on a E. coli OP50 plate. The sex ratio of an entire brood born from a self-cross was 2.7% male, 8.8% female, and 88.5% hermaphrodite. Further, mothers produced female and male offspring only at the beginning of their reproductive lifespan, such that almost all females and males were produced within Phylogenetic relationships of T. tufae n. gen., n. sp.. Molecular phylogenetic analysis of the partial (ca. 0.9-kb) SSU rDNA gene inferred Tokorhabditis n. gen. to be the sister group of Auanema and that these two genera formed a sister group to Rhabditella (Fig. 9). Furthermore, each of these three genera was fully supported (with 100% posterior probability) as monophyletic. Further, the new genus was exclusive of the typologically similar genera Haematozoon and Rhabditoides (Fig. 9).

Discussion
Our description of Tokorhabditis n. gen. presents a model for extreme physiological resilience, specifically one in a group of nematodes ("Rhabditidae") that are easily manipulated and well used as laboratory organisms. Moreover, the new species provides a new point of comparison for both the evolution of trioecious reproduction and obligate vivipary, life-history characters that may have-as it is easy to speculate, and now feasible to test-fostered the colonization of such extreme niches as those in Mono Lake.
An unusual form of vivipary in nematodes. The majority of nematodes are oviparous, although some viviparous species have been reported, both in "Rhabditidae" and elsewhere. However, in most cases where this feature has been adequately described, it is apparent that this vivipary, which would be described as ovovivipary as defined above, is simply the retention of eggs wherein embryonic development proceeds as in oviparous species. For example, in parasitic females of the entomoparasitic species Deladenus nitobei, a non-rhabditid nematode in which vivipary has been characterized, the physical sizes (volumes) of single-celled embryos are equal to those of juveniles that hatch in utero 36 . In contrast, nematodes of Tokorhabditis n. gen. grow in size during their embryonic development. This key difference suggests that, unlike oviparous and ovoviparous nematodes, whose embryonic development is completed based on the original provisioning of their eggs (lecithotrophy), embryos of Tokorhabditis n. gen. obtain some form of nutrition from their mother. Although unusual among nematodes, obligate vivipary as described here has evolved convergently in the diplogastrid genus Sudhausia 17 . Intriguingly, Tokorhabditis n. gen. and Sudhausia are united by two similarities, www.nature.com/scientificreports/ despite vivipary being the result of two independent evolutionary events. First, both species have self-fertile hermaphrodites, and in Sudhausia this feature has gone to the extreme that the pre-adult stage is already sexually mature and can self-fertilize. Second, the ancestors of both genera were most likely associated with dung, i.e., of mammalian herbivores. Although T. tufae n. gen., n. sp. has thus far only been reported from Mono Lake, two other putative species of the new genus (represented by strains EJR13 and NKZ329) have been isolated from dung beetles, a finding that will be described elsewhere (Ragsdale and Kanzaki, in prep.). Although this similarity www.nature.com/scientificreports/ may be coincidence, it might also be speculated that dung somehow offers a habitat conducive to maternal investment through vivipary, a life-history strategy thought to offer protection from adverse environments 37 . For example, dung is known to host a diverse array of nematode (including predatory) species, some of which have clearly specialized for different growth curves during the cycle of dung decomposition 38 . It is thus possible that competition with, and threat of predation by, such cohabitants might have given such a pressure toward a viviparous lifestyle. The ability to empirically manipulate nematodes in the semi-natural setting of dung offers a route to testing this hypothesis 39 .

Convergent reproductive morphologies correlating with obligate vivipary. Also concomitant
with the convergent evolution of obligate vivipary in Tokorhabditis n. gen. and Sudhausia are striking similarities in vaginal morphology. Specifically, the female and hermaphrodite of T. tufae n. gen., n. sp. has a vagina comprising layers of stretched, elongated cells surrounding a Z-shaped vaginal duct. A similar morphology is found in Sudhausia, suggesting the morphology is functional in vivipary, for example to accommodate the passage of larger, developed juveniles while also being able to seal off the reproductive tract-that is, from environmental insults or natural pathogens-when not in use. Additionally, Tokorhabditis n. gen. shows another reproductive morphology-specifically, its vaginal fold-that is not found in either its sister group Auanema or in the distantly related, viviparous Sudhausia 10,17 .
A comparative model for the evolution of trioecy. As previously described, Auanema comprises multiple species that have a trioecious mating system, which consists of males, females, and self-fertile hermaphrodites 10,30,40 . This mixed mating system has been considered very rare in animal systems 41 , and theory has predicted such a mating system to be transitional, i.e., between predominantly outcrossing and predominantly selfing modes of reproduction 42 . Here, we report a mating polymorphism similar to that of Auanema  Posterior probability values exceeding 50% are given on appropriate clades. www.nature.com/scientificreports/ species, such that the eldest offspring of a sireless brood are males and females, followed by the emergence of self-fertile hermaphrodites. Because this form of trioecy is shared by both Auanema and Tokorhabditis n. gen., our report suggests that this mating system was ancestral to both genera, pushing back the presumptive evolutionary origin in rhabditid nematodes even further. Thus, we present a new point of comparison more precision in reconstructing the evolutionary correlates, for the evolution of this unusual mode of reproduction.
A model for studying maternal care in nematodes. Maternal investment in nematodes, through mechanisms such as nutrient transport and yolk provisioning, can promote the survival of progeny in stressful environments 43 . Since T. tufae n. gen., n. sp. was isolated from the arsenic-rich environment of Mono Lake, we speculate that maternal investment in Tokorhabditis n. gen. was a preadaptation to the extreme stressors of that environment. Our description of this species offers an entry point to testing if, and to what degree, maternal care further facilitated an extremophilic lifestyle where the necessary physiological tolerance was already in place, since physiological tolerance of Mono Lake conditions are shared with Auanema species 8 . Our results suggest a trade-off between obligate vivipary and fecundity: selfing hermaphrodites of T. tufae n. gen., n. sp. produce an average of 115 progeny, which we found to be far less than the mean brood size of C. elegans as well as a representative of Auanema (A. rhodensis). The small brood size of T. tufae n. gen., n. sp. suggests a greater investment toward the development of individual progeny over the production of large broods in this species. However, we cannot rule out the possibility that the laboratory conditions used in this study were sub-optimal for T. tufae n. gen., n. sp. growth and thereby also reduced its brood size.
Clues to the mechanism of viviparity in T. tufae n. gen., n. sp. can be obtained from the mechanisms of maternal care in C. elegans. For instance, C. elegans mothers can secrete into their environment yolk milk that can be used to feed their young 44 . Milk production is controlled by insulin signaling and involves the breakdown of the gut to produce yolk, which contributes to senescence in the mother. This raises the question of whether nutrient transport in T. tufae n. gen., n. sp. viviparity involves similar signaling pathways and shortening of lifespan. Further studies of vivipary in T. tufae n. gen., n. sp. will be useful for comparing how nutrient transport and reproductive aging have diverged between this species and the well-characterized C. elegans. www.nature.com/scientificreports/