Taxonomy of Tobrilidae species from the Alkaline Lakes of the western Nebraska Sandhills

Soil collection and nematode isolation: Nematodes were collected from lake sediment and shoreline soil in 2019, 2020, and 2021 from five lake sites ranging in alkalinity within the Sandhills region of western Nebraska: Bean Lake, Border Lake, Gimlet Lake, Island Lake, and Kokjohn Lake. Additional samples were collected in 2022 from Johnson Creek near Mead, NE, a contaminated stream at an agricultural site approximately 400 miles east of the Alkaline Lakes (Fig. 1). From the Sandhills lakes in 2019, we collected three replicate samples per lake at about 10 meters from shore and in 2020 and 2021 collected four replicate samples per lake with a dredge from a kayak. For shoreline samples, 12–15 soil cores were taken along a 40-m transect at a depth of approximately 20 cm using an Oakfield Soil Corer with a 2.5-cm diameter (Gattoni et al., 2022, 2024). From Johnson Creek, we collected 500 ml of composite stream sediment by wading into the stream and taking 5–10 shovelfuls per site from five sites along 5 miles of the stream nearest the AltEn facility. All samples were stored at 8°C until nematodes were extracted from 200 cm³ of soil via the sieving and sugar centrifugation method (Jenkins, 1964).

Figure 1.

Map of Nebraska with the Sandhills region outlined. Area of research on the alkaline lakes in this region is indicated in an enlarged map inset. A second enlarged map inset shows the area of research in eastern Nebraska on an agrichemical contamination site. Red circles indicate the location of sample collections from the stream sediment.

Lake sediments and shoreline soils were analyzed for biogeochemistry at Ward Laboratories, Inc. (Kearney, Nebraska). The summary is presented in Supplementary Tables S1 and S2.

Nematode community analysis and morphological characterization: Abundance of tobrilid nematodes was estimated as a portion of the entire community, and if available, 25 individual specimens per sample were hand-picked, mounted on glass slides, heat-relaxed, and photographed. Measurements were recorded at ×400 and ×1,000 magnifications (Table 3a). Supplement numbering system follows the convention of Tsalolikhin and Shoshin (2009) (Fig. 2). Following microscopic analysis, slides were dismantled and immediately processed for DNA barcoding to preserve the linkage between DNA and morphology. Nematode images were taken of the full body, head, and tail with a Leica DC300 video camera mounted on a Leica DMLB light microscope with differential interference contrast. DNA was extracted from the photographed specimens by rupturing the nematode in an 18 mL droplet of sterilized water, which was stored at −20°C until PCR (Powers et al., 2014).

Figure 2.

llustration of selected characters in Tobrilidae. A) Female heads (NID 12586, 13994) with teeth and tooth distance annotated. B) Female heads with differing tooth pocket arrangements. C) Male (NID 12533) posterior illustrating the numbering of supplements from the cloacal opening forward, with I designating the most posterior supplement and VII the most anterior.

The taxonomic keys, compendia, and references used to infer genus and species identities by morphology included Bongers (1989); Decraemer et al. (2019); Ebsary (1982); Gagarin and Naumova (2016); Holovachov and Shoshin (2014); Naumova and Gagarin (2017); Tsalolikhin and Shoshin (2009); and Zullini (2006).

DNA barcoding, phylogenetic tree construction, and reverse taxonomy: We applied “Reverse taxonomy” (Markmann and Tautz, 2005; Kanzaki et al., 2012), an approach that initially groups specimens based on position on a phylogenetic tree, followed by morphological assessment of nematodes that formed groups in the phylogenetic analysis. Importantly, each specimen on the COI phylogenetic tree was measured and photographed, and all light micrograph specimen images in the manuscript figures are represented on the COI tree. Nematode specimens were DNA barcoded, targeting the COI mitochondrial protein-coding gene and the 18S ribosomal DNA. The COI primers used were COI-JB3-Tob1 (JB3Tob1) –5′-TTTGGGCATCCTGAGGTTTATATTTTRA-3′(TSH, 2021 design based on JB3 (Bowles et al., 1992) and COI-R9-Tob1 (R9Tob1) – 5′-TGAAAATGAGCWACWACATAATAWGTRTC-3′(TSH, 2021 design based on COI-R9 (Powers et al., 2014)), which produce a 365-bp product once primers are trimmed. PCR was conducted in 0.5-mL thin-wall microcentrifuge tubes containing 30 μL of total volume consisting of 9 μL of the ruptured nematode template, 1.2 μL of double distilled water, 2.4 μL of both forward and reverse 20 μM primer solutions for a 1.6 μM final primer concentration, and 15 mL of JumpStart RED Taq ReadyMix (Sigma-Aldrich) at a 0.05U/mL final enzyme concentration. The initial hot start at 94°C for 5 min was followed by 35 cycles of 30 sec of denaturation at 94°C, annealing at 48°C for 30 sec, and extension at 72°C for 90 sec. The final extension occurred once at 72°C for 5 min. Successful PCR products were extracted with X-Tracta Tools (USA Scientific) prior to DNA sequencing from a 0.7% 1X TAE agarose gel, cleaned using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific), and sent to Eton BioSciences, San Diego, CA for Sanger sequencing in both directions.

The 18S primers were 18s1.2a (5′-CGATCAGATACCGCCCTAG-3′) and 18sr2b (5′-TACAAAGGGCAGGGACGTAAT-3′), which produce a 593-bp product once primers are trimmed. 18s1.2a is the slightly re-designed 18s1.2 primer that was originally designed using consensus arthropod sequences (Mullin et al., 2003), while 18sr2b is a slightly redesigned reverse complement of primer rDNA2 from Vrain et al. (1992). This primer set amplifies approximately 630 bp of the 3’ portion of the 18S ribosomal DNA. PCR amplification of 5 uL of ruptured nematode template was conducted using the same conditions as the COI genetic marker except for annealing at 52°C, and 18S amplicon verification, cleaning, and sequencing were as described above. Forty-six of the 87 specimens used in the 18S phylogeny for this study are also represented on the COI phylogenetic tree.

To perform phylogenetic analyses and assess haplotype relationships among barcoded sequences of Tobrilidae, traces of barcode sequences of nematode specimens were edited using CodonCode Aligner Version 9.0 (http://www.codoncode.com). DNA sequences from this study were aligned with MEGAX v.10.2.6 to produce two separate (COI and 18S) alignments. Both alignments were subjected to analysis using a character-based maximum likelihood (ML) approach. ML trees were built using GTR+G (COI) and K2 + G + I (18S) models, both with 2,000 boot strap repetitions, and both with gap treatments using “Use all sites.” Each initial MEGAX alignment used MUSCLE with gap opening (−1,000) and gap extend (−500) penalties and UPGMB clustering method parameters. COI haplotype groups were generally defined by bootstrap values, a within-group distance that did not exceed 5%, and a distance to the nearest neighbor being greater than any within-group distance. Within- and between-COI clade genetic distances were calculated in MEGAX using p-distance with the assumption of Gamma Distributed Rates among Sites for 124 sequences, each with 393 nucleotide positions.

Discriminant Function Analyses: Discriminant Function Analysis (DFA) is a statistical method used to classify unknown individuals and the probability of their classification into pre-defined groups (Fisher, R. A., 1936; Lachenbruch and Goldstein, 1979; Jombart et al., 2010). Using DFA, we tested how well morphological characteristics or ecological (soil chemistry) attributes, or both morphological and soil chemistry attributes combined, classified female and male Tobrilidae into their respective COI haplotype groups (HGs).

Three datasets were created for both female and male Tobrilidae that were identified molecularly as belonging to specific COI HGs (Fig. 3) for use in our discriminant function analyses (DFA). The first dataset for both genders was soil chemistry attributes and the second dataset for both genders was morphological characters (Tables S1 and S2). The third dataset for both genders was a combination of both morphological characters and soil chemistry attributes.

Figure 3.

A maximum likelihood COI phylogenetic tree of 90 Nebraska specimens and 21 GenBank accessions. Highlighting in labels indicates lake pH. Green equals neutral, orange equals pH >8.5. Blue boxes around labels identify three specimens that display incongruence between COI and 18S trees. Nematode Identification numbers (NID) start with the prefix N followed by the number of the specimen.

Table 3a describes the morphological characters examined for females only, males only, and both males and females. The following soil chemistry attributes are described in Table 3b: soil pH, % OM, NO³, K, SO⁴, Zn, Fe, Mn, Cu, Ca, Mg, Na, B, CEC, % H, % K, % Ca, % Mg, % Na, Cl-, and P.

For the three female and three male datasets, the variables (the morphological characters or soil chemistry attributes) were initially inspected for missing values. If a variable was missing in more than 40% of the observations, such as when a morphological character was obscured, or the specimen was not in the appropriate developmental stage, those variables were dropped from further analysis. If the missing percentage was less than 40%, the missing values were replaced with the average of the remaining values. Correlations were then checked for the remaining variables using Pearson's Correlations Coefficient. Correlations were checked for elimination of highly correlated variables from the discriminant analysis. When two variables were highly correlated (greater than 0.8 or lower than −0.8), only one of the variables was retained for further analysis. The variables selected included a consideration of morphological characteristics specific to sex and important soil attributes such as K for the alkaline lake sites.

For each DFA analysis, the list of variables was determined by the correlation matrix. The dataset was normalized and then used in a stepwise variable selection using Akaike's Information Criterion (AIC) (Akaike, 1973). The stepAIC function in R v.4.1.2 (R Core Team, 2021) determined the final set of variables that can distinguish between the HGs from the model with the lowest AIC. Finally, using the selected features from the stepAIC method, linear discriminant analysis (LDA) was used to segment the HGs based on the continuous variables with equal prior probabilities (otherwise known as ‘priors’) for each HG. Three specimens that exhibited COI-18s incongruities were excluded from the morphological analyses used as the basis for the discriminant function analysis.

Scanning Electron Microscopy (SEM) preparation: Living nematodes were initially fixed in cold 4% glutaraldehyde for 24 hours, then rinsed in 0.1 M sodium cacodylate buffer before fixation in 2% osmium tetroxide for 8–12 hours. Nematodes were again rinsed in cacodylate buffer, followed by dehydration in a progressively increasing concentration of cold ETOH solutions until the nematodes were in 100% ETOH. Nematodes were critical point-dried, coated with silver, and mounted on an SEM stub before viewing on a Hitachi S4700 field-emission scanning electron microscope (SEM) at the Morrison Microscopy Core Research Facility of the Nebraska Center for Biotechnology http://biotech.unl.edu/microscopy.

A total of 226 tobrilid specimens were microscopically examined during this study, 184 from the Alkaline Lake region and 42 from Johnson Creek in eastern Nebraska. Both eastern and western regions of Nebraska contained members of the subfamilies Tobrilinae and Neotobrilinae. Depending on specimen quality, sex, and stage, detailed morphometrics were recorded for a subset of these specimens.

Phylogenetic trees and genetic distances: A maximum likelihood COI phylogenetic tree of 90 Nebraska specimens and 21 GenBank accessions places the Alkaline Lake and Johnson Creek Tobrilidae into 6 haplotype groups (Fig. 3). Haplotype groups (HGs) 1 through 4 exclusively included specimens collected in the Alkaline Lakes, while HGs 5 and 6 are comprised of specimens collected in Johnson Creek. The three high-pH lakes, Bean, Border, and Kokjohn, contained only members of HG 2. The two lakes with more neutral pH, Gimlet and Island Lakes, each have a haplotype group found exclusively in their waters. Gimlet Lake was the only lake with HG 1, and HG 3 was only collected in Island Lake. HG 4 was found in both Island and Gimlet Lakes. The Sandhills Alkaline Lake locality and Johnson Creek in eastern Nebraska had representative specimens of the subfamilies Tobrilinae (HGs 1 and 5) and Neotobrilinae (HGs 2, 3, 4, and 6). Each of the six haplotype groups was supported by a combination of: a) medium to high bootstrap values (e.g., 65–100), b) low within-group mean genetic distance estimates (0.0%–0.54%), and c) relatively high between-group pairwise distance measurements that ranged from 6.04% to 16.44% (Table 4). Included in the COI tree were three clades of GenBank sequences identified as Tobrilus gracilis from Europe (Ristau et al., 2013). These European specimens comprised three haplotype groups on the tree that were well-supported by bootstrap values (88–99), had low levels of within-group mean genetic distances (0.46%–2.74%), and exhibited large between-group distances when compared among the three European populations (11.76%–18.36%). The between-group mean distance estimates for European versus Nebraskan haplotype groups ranged from (10.71%–17.34%). None of the three European haplotype groups identified as Tobrilus gracilis exhibited a genetically close relationship with the Nebraskan COI haplotype groups.

Morphological characteristics of haplotype groups: Measurements were obtained from most of the adult specimens in COI HGs 1–6 from Nebraska (Table 5). Notable diagnostic characters and observations include the following:

Tooth distance and location of “pockets”. HGs 1 and 5 have the typical characteristics of the subfamily Tobrilinae, with two overlapping or adjacent pockets located just posterior to the relatively large, sclerotized buccal cavity. Each pocket contains a single small tooth (Figs. 2, 4). Distance between the teeth in HG 1 averages 5.7 μm in females and 3.0 μm in males, while in HG 5, it averages 2.0 μm in females and 2.1 μm in males. Teeth in HGs 2, 3, 4, and 6 are located in distinctly separate non-overlapping pockets one behind the other, posterior to the stoma base, characteristic of the subfamily Neotobrilinae. Tooth distances average 12.4 μm (females) and 11.8 μm (males) in HG 2.

Figure 4.

Comparison of the heads of specimens from each of the six Haplotype Groups, with their identification number and lake from which they were collected. Stoma shape, pockets, and tooth distance are featured. Blue box indicates incongruent specimen between COI and 18S trees.

Figure 5.

Comparison of male spicules and supplements from haplotype groups one through six. The nematode identification (NID) number and collection location are shown for each. Groups 1, 4, and 5 have supplements that do not conspicuously protrude from the cuticle surface. Groups 2, 3, and 6 have protruding supplements. Group 4 has notably long spicules compared to other groups. The blue box around the label for NID 12595 denotes a specimen incongruent for COI and 18S trees.

Figure 6.

Scanning electron micrographs of the male posterior region showing the intersupplementary structures known as micropapillae. A, B) NID 4829 from Border Lake, micropapillae in ventral view appear as four ridges between supplements; C) NID 4841 from Border Lake, micropapillae in lateral view appear as small bumps or papillae.

Figure 7.

Scanning electron micrographs of Group 4 male spicules, all from Gimlet Lake, displaying possible pores at the tip. A) NID 4913 tail and close-up of spicules; B) NID 4926 posterior and close-up of spicules; C) NID 4899 spicules.

Figure 8.

Comparison of the musculature associated with the vaginal canal in haplotype groups one through six. The Tobrilinae, Groups 1 and 5, display weak or poorly developed vaginal musculature. Group 3 appears to have moderately developed musculature, and Groups 2, 4, and 6 are all characterized by strongly developed muscles arranged in a multi-layered, bulb-like configuration. Blue box around NID 12574 indicates incongruent specimen with respect to the COI and 18S trees and has a strong, bulb-like musculature characteristic of Group 2.

Figure 9.

Scanning electron micrographs of the head, amphid and cephalic setae of selected specimens from alkaline lakes in the Nebraska Sandhills. Amphid shapes were categorized as tiny, oval or slit-like. Slit-like amphids were positioned closest to cephalic setae. Inner labial setae are notably shorter than the outer labial and cephalic setae, which are of similar lengths.

Figure 10.

Entire body images of female and male specimens from each of the six haplotype groups. The nematode identification (NID) number and collection location are shown for each. Blue box indicates incongruent specimen.

Figure 11.

Comparison of female tails from each of the six haplotype groups. The nematode identification (NID) number and collection location is shown for each. Blue box in Group four indicates specimen, NID 12574, considered incongruous between the COI and 18S phylogenies, and which displays differing morphology from the other two individuals.

Combined morphological and ecological analysis of haplotype groupings as determined by discriminant function analysis:

Females

Female Combined – 14 variables were selected using the AIC method and then used for LDA analysis. Six of these variables included morphological characters, and the remaining eight variables were soil chemistry attributes. The most significant morphological characters and soil attributes are presented in Table S1. All 14 variables were used for LDA with almost equal priors. There were 6 groups, the first 4 with a prior of 0.17 and the remaining two groups with a prior of 0.16. There were five linear discriminants (LD) needed to properly discriminate among the haplotype groups, although the first two discriminants accounted for almost 95% of the variation. All the groups were classified correctly into their haplotype groups using the five LDs except 1 specimen in HG 4, which was classified as HG 2 instead (Fig. 12A).

Female Morphological – The seven variables selected using the AIC method for LDA analysis were L, b, c, V, VA/T, L set/lrw, and Dist. All seven variables were used for LDA with almost equal priors. Since there were 6 groups, the first 4 groups had a prior of 0.17, and the remaining two groups had a prior of 0.16. Five linear discriminants (LD) were needed to properly discriminate among the haplotype groups. At least three LDs were needed to account for 90% of the variation in the data. The groups were classified fairly correctly except for a few specimens in HG 2 and HG 4. For HG 1, out of 6, one specimen was classified as HG 5 and the remaining as HG 1. For HG 4, out of 15, one specimen was classified as HG 2 and the remaining as HG 4.

Female Soil Chemistry – The four variables selected using the AIC method for LDA analysis were K, Cu, Mg, and P (Table S1). All four variables were used for LDA with almost equal priors. Since there were 6 groups, the first 4 groups had a prior of 0.17, and the remaining two groups had a prior of 0.16. Five linear discriminants (LD) were needed to properly discriminate among the groups. The first two discriminants accounted for almost 95% of the variation. The haplotype groups were classified correctly only for HG 1 and HG 3; all remaining HGs had some misclassifications. Specimens in HG 2 were classified as HG 3 and HG 4, specimens in HG 4 were classified as HG 3 and HG 6, specimens in HG 5 were classified as HG 1 and HG 6, and finally, specimens in HG 6 were also classified as HG 1 and HG 5.

Figure 12.

Linear discriminant analysis (LDA) of haplotype groupings using combined morphological and ecological attributes for A) females; B) males.

Males

Male Combined – 16 variables were selected using the AIC method for LDA analysis. Nine of these variables were morphological characters, and the remaining seven were soil chemistry attributes. The significant morphological characters were L, a, c, C set/lrw, Stoma L, Dist, Cl-III, SR, and SR/L, and important soil attributes were Soil pH, K, Mg, CEC, % Mg, % OM, and P (Table S2). All 16 variables were used for LDA with almost equal priors. Since there are 6 groups, the first 4 groups have a prior of 0.17, and the remaining two groups have a prior of 0.16. Five linear discriminants (LD) were needed to properly discriminate between the haplotype groups. The first two LDs account for almost 98% of the variation. All the groups were classified correctly into their haplotype groups using the five LDs (Fig. 12B).

Male Morphological - Three variables selected using the AIC method for LDA analysis were c, C set/lrw, and Cl-III (Table S2). All three variables were used for LDA with almost equal priors. Since there are 6 groups, the first 4 groups have a prior of 0.17, and the remaining two groups have a prior of 0.16. Three linear discriminants (LD) were needed to properly discriminate between the haplotype groups. The first two LDs accounted for 85% variation in the data. The groups are classified fairly correctly except for one specimen in HG 2, which is classified as HG 3.

Male Soil Chemistry - Four variables selected using the AIC method for LDA analysis were % OM, K, Mg, and % Mg. All four variables were used for LDA with almost equal priors. Since there are 6 groups, the first 4 groups have a prior of 0.17, and the remaining two groups have a prior of 0.16. Four linear discriminants (LD) were needed to properly discriminate between the haplotype groups. The first two discriminants accounted for almost 98% of the variation. The groups were classified correctly only for HG 1, HG 3, and HG 6. The remaining HGs 2, 4, and 5 have some misclassifications. A specimen in HG 2 was classified as HG 3, in HG 4 as HG 1, and in HG 5 classified as HG 6.

Relationships inferred from 18S Ribosomal DNA: In the 18S tree (Fig. 13), HGs 2, 3, and 6 were well supported as a clade (86 bootstrap support), with HG 3 recognized as a subgroup embedded within HG 2. HGs 1, 4, and 5 were more strongly supported as part of a larger clade with 99 bootstrap support. The 18S sequences from HGs 1 and 5, representative of specimens from western and eastern Nebraska, respectively, were identical. HG 4 was a discrete subgroup within this clade.

Figure 13.

A maximum likelihood 18S phylogenetic tree of 88 Nebraska specimens and 19 GenBank accessions. Tobrilus Groups 1 and 5 are identical for the 18S barcode, and they are united in a strongly supported clade (99 bootstrap value) with Semitobrilus Group 4 and GenBank accessions identified as Tobrilus and Semitobrilus. Blue boxes identify specimens incongruous between COI and 18S trees.

A nonconformity between COI and 18S trees: Three of the specimens in the tobrilid dataset examined with both COI and 18S markers revealed a nonconformity between the two genetic markers. Each of the three nonconforming specimens were members of the COI HG 4 (Fig. 3). Two of the specimens, NID 12595 and 12596, both males, were collected from Gimlet Lake, and the third specimen, NID 12574, a female, was collected from Bean Lake. NID 12574 was the only member of HG 4 collected from a high pH lake. It also stood out among other members of HG 4 in possessing a short tail (198 μm), which conforms to members of HG 2 (154–210 μm), in contrast to the long tails characteristic of HG 4 (350–414 μm) (Fig. 11). Similarly, other morphological measurements of NID 12574 such as body and stoma length, V, b, c, tooth distance and a bulb-like vaginal musculature (Fig. 8) all aligned with HG 2 and not HG 4. Its placement on the 18S tree was firmly within a clade populated by HG 2 specimens (Fig. 13). The two male specimens, NID 12595 and 12596, were grouped together with HG 1 and 5 in the 18S tree. Their morphological measurements, however, were entirely consistent with other HG 4 specimens.

Relationship to described species of tobrilids from North America: The study of Abebe et al., 2013 provides a comparison of the four Neotobrilus species known from North America. The HG 2 Neotobrilus most closely compares to Neotobrilus nicsmolae Abebe et al., 2013. The body lengths of both males and females are larger in HG 2. In N. nicsmolae, the amphid diameter appears larger, the amphid is positioned more posteriorly on the body, and somatic setae appear more prominent and numerous than in HG 2. Two Semitobrilus species, S. pellucidus (Bastian, 1865) Tsalolikhin, 1981 and S. ebsaryi Tsalolikhin, 2000, have been reported from North America. Semitobrilus pellucidus has spicules that range between 80–90 μm versus 130–143 for HG 4. Semitobrilus ebsaryi, formerly Tobrilus longicaudatus (Schneider, 1923) Andrassy, 1959, is morphologically the closest to HG 4, with a shorter spicule of 108–122 μm and a slightly sigmoid vaginal lumen, which is a variable character in HG 4. The genus Tobrilus, according to Zullini (2006), is restricted to Europe and Asia. The Nebraskan Tobrilus species, HGs 1 and 5, clearly belong to the genus Tobrilus and likely represent new species.

The Alkaline Lakes of the western Sandhills region of Nebraska constitute an extreme environment. The three Alkaline Lakes at the upper end of the pH and potassium alkalinity spectrum (Bean Lake, Border Lake, and Kokjohn Lake) contain no vertebrate organisms, few macroinvertebrates, and diatoms which, based on their presence in tobrilid guts, are the primary food source of the nematodes. Members of Tobrilidae were prominent in each of the five Alkaline Lakes, with a single COI haplotype group found in the three high pH lakes, and three additional COI haplotype groups distributed between the two neutral pH lakes.

Sediment samples collected in eastern Nebraska from Johnson Creek provide evidence that tobrilid nematodes can survive in different types of extreme environments. In 2021, Johnson Creek was the site of a major contamination event that washed 4 million gallons of highly contaminated sludge containing concentrated seed coat pesticides directly into the creek. The resulting aquatic nematode community was reduced to species mixtures of Tobrilidae and Monhysteridae. Attempts to identify the tobrilid genera and species from the Alkaline Lakes and Johnson Creek have proved challenging.

The most comprehensive morphological key to the genera in Triplonchida is by Zullini (2006). All the specimens examined in this study readily key to Tobrilidae based on the characteristics of the wide or funnel-shaped stoma with two teeth at its base (Figs. 2, 4). The first couplet within Tobrilidae splits the family into Tobrilinae and Neotobrilinae based on fused or adjacent pockets at the stoma base with two teeth 0–8 μm apart (Tobrilinae), versus well-separated, discrete pockets, 6–25 μm apart, posterior to the buccal cavity, each with a single tooth (Neotobrilinae) (Fig. 4). The COI HGs 1 and 5 morphologically key to the genus Tobrilus Andrássy, 1959 based on overlapping stomatal pockets with teeth no more than 0–6 μm apart, relatively short cephalic setae and small supplements in the male that do not protrude above the cuticle surface (Fig. 5). All of the cephalic setae we observed would be considered relatively short, with a length that rarely exceeds 20–30% of the body width at their location. SEM images of the cephalic region of specimens from the Alkaline Lakes show a general pattern of short labial papillae and more prominent cephalic setae and outer labial setae (Fig. 9).

Taxonomic uncertainty increases among the specimens morphologically identified as members of the subfamily Neotobrilinae, HGs 2, 3, 4, and 6. HGs 2 and 6 most closely conform to the genus Neotobrilus Tsalolikhin, 1981 based on the strong, bulb-like vaginal musculature, protruding echinate supplements, and tooth distances exceeding 11 μm. HG 3 has tooth distances of approximately 10 μm but lacks strong vaginal musculature, and the protruding supplements are not echinate. In most characteristics, it conforms to the genus Brevitobrilus Tsalolikhin, 1981. HG 4 appears to combine characteristics of both subfamilies. In respect to its heavily muscular vagina and well-separated pockets, it conforms to Neotobrilinae. The supplements, however, do not protrude from the cuticle, and the tooth distance of 6–12 μm is intermediate and overlapping with both subfamilies. Notably in most HG 4 female specimens, the vagina is slightly anteriorly directed, and males have exceptionally long spicules. Collectively these features best fit the genus Semitobrilus Tsalolikhin, 1981 (Tsalolikhin, 2009).

Characterization of the haplotype groups by partial COI and 18S sequences provided additional phylogenetic insight and some surprises. The COI gene sequences, due to a relatively rapid mutation rate, strongly supported the existence of haplotype groups, but relationships among haplotype groups remained obscure. The 18S sequences did permit grouping of COI-derived haplotype groups but also raised the possibility of past hybridization between nematodes with distinctly different mitochondrial lineages. Three specimens, one female and two males, had the COI sequence indicative of mitochondrial haplotype HG 4, but were members of different 18S clades and did not cluster with the other HG 4 members in the 18S phylogeny. The incongruence was further supported by morphological evidence, in which the female (NID 12574) with the mitochondrial HG 4 haplotype, had the morphological features of HG 2 (Fig. 11). Both male specimens had the morphological features of HG 4, which matched their COI haplotype group but fell into the 18S clade of Tobrilus Group 1.

Discriminant function analysis was most successful when male or female morphological characteristics were used in combination with the environmental chemistry data. Morphology alone or chemistry alone did not achieve the level of discrimination of the combined analyses.

Given the strong signal provided by the environmental chemistry data, it is reasonable to speculate that tobrilid haplotypes may have value as environmental indicators. Improved taxonomy will aid the effort, with a needed emphasis on improved keys for genus and species diagnosis. This study identified some unique characteristics relative to the spicules, inter-supplement cuticles, and amphids. These observations need to be extended across additional species and linked to diagnostic molecular markers. It should be noted that while the COI marker strongly delineates 6 distinct Nebraskan tobrilid groupings, relationships based on 18S suggest that the conventional subfamily groupings may require reconsideration. Specifically, Haplotype group 4, identified as belonging to the genus Semitobrilus, clusters with the Tobrilinae, and is supported by a trio of GenBank accessions of Semitobrilus specimens. Traditionally, Semitobrilus is classified in the subfamily Neotobrilinae (Zullini, 2006).