Plectus of the Prairie: A Case Study of Taxonomic Resolution from a Nematode Biodiversity Survey

Fifteen sites were sampled within the 16-km-long Prairie Corridor (Fig. 1). The 15 sites were selected because, as remnant prairies, they had never been plowed or converted to agricultural production. Each site ranged from 0.8 ha to 1.2 ha in size, and soil cores were taken from a 40 m × 40 m square in the center of each site following a protocol to ensure standardized soil sampling at each location (Neher et al., 1995). Soil cores were taken at regular intervals throughout the 40 m × 40 m grid at a depth of approximately 20 cm using an Oakfield Soil Corer (Oakfield Apparatus, Oakfield, Wisconsin) with a 2.5-cm diameter. All soil cores from a single 40 m × 40 m square were bulked and stored at 8°C prior to nematode extraction.

Figure 1

Nematodes were extracted from 200 cc of soil via the sieving and sugar centrifugation method (Jenkins, 1964). Nematodes were counted, and a total of 150 nematodes was identified to genus using morphological characters. Twenty-five of the 150 nematodes were photographed at ×200 and ×400 magnifications to serve as photographic vouchers, and immediately processed for DNA barcoding to preserve the linkage between DNA and morphology. Voucher images were taken of the full body, head, and tail with a Leica DC300 video camera (leica-microsystems.com) mounted on a Leica DMLB light microscope with differential interference contrast. The taxonomic keys and compendia of Andrassy (1985), Bongers (1989), Ebsary (1985), Holovachov and De Ley (2006), Holovachov (2014), and Maggenti (1961) were used for a morphology-based identification of Plectus species. DNA was extracted from the photographed specimens by rupturing the nematode in an 18-mL droplet of sterilized water, which was stored at -22°C until PCR (Powers et al., 2014). Plectus specimens from additional sites (a total of 95 specimens from 28 sites) including two well-studied tallgrass prairies, Konza and Nine-Mile Prairies, were added to expand taxonomic breadth and facilitate comparisons across broader geographic regions and ecosystems (Table 1).

The 25 selected nematode specimens from each site were DNA barcoded targeting specific DNA regions of two different genetic loci, the COI mitochondrial protein coding gene and the 18S ribosomal DNA. The COI primers used were JB3 (5¢-TTTTTTGGGCATCCTGAGGTTTAT-3¢) (Bowles et al., 1992) and JB5 (5¢-AGCACCTAAACTTAAAACATAATGAAAATG-3¢) (Derycke et al., 2005), which produce a 393-bp product once primers are trimmed. PCR was conducted in 0.5-mL thin-wall microcentrifuge tubes containing 30 mL of total volume consisting of 9 mL of the ruptured nematode template, 1.2 mL of double distilled water, 2.4 mL of the forward primer, 2.4 mL of the reverse primer, and 15 mL of JumpStart RED Taq ReadyMix (Sigma-Aldrich, Inc. St. Louis, Missouri, U. S. A.) at a 0.05 U/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 50°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 prior to DNA sequencing from a 7% 1X TAE agarose gel, cleaned using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific, Dubuque, Iowa, U.S.A.), and sent to Eton BioSciences for 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 the somewhat 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 mL of ruptured nematode template was conducted using the same conditions as the COI genetic marker, and 18S amplicon verification, cleaning, and sequencing was as described above.

Nematodes were extracted from a separate 100-cc subsample using soil via sieving and sugar centrifugation method (Jenkins, 1964). Once extracted, all nematodes in each sample were counted under an inverted microscope. Counted nematodes were reduced to 0.5-mL volume, transferred to bead-beating tubes of the PowerSoil DNA Isolation kit (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.), and processed according to the manufacturer’s instructions. Extracted DNA of nematodes was processed for amplicon sequencing using the V6–V8 region with NF1-18sr2b primers (Porazinska et al., 2009) producing a 360-bp product using the same PCR protocols. PCR conditions followed protocols of the Earth Microbiome Project (http://www.earthmicrobiome.org/protocols-and-standards/) (Amaral-Zettler et al., 2009, Bellemain et al., 2010, Caporaso et al., 2012). Three technical replicates were amplified for each sample, visualized with gel electrophoresis, pooled, and sent for multiplexing, library preparation, and sequencing (HiSeq 300, paired-end) at the Hubard Center for Genome Sequencing at the University of New Hampshire, Durham, NH. Qiime2 was used to remove the primer regions of the demultiplexed sequences using cutadapt (Martin, 2011). In vsearch, forward and reverse reads were joined with join-pairs (Rognes et al., 2016), joined sequences were filtered for quality with quality-filter q-score-joined, and chimeras were checked with uchime-denovo (Bokulich et al., 2013). Sequences were clustered into operational taxonomic units (OTUs) at 99% similarity. An in-house curated ARB-SILVA SSU database v.111 (Quast et al., 2012; Yilmaz et al., 2014) was used to assign taxonomy to all OTUs with BLAST. Nematode identified sequences were reassigned taxonomy against a nematode curated database based on ARB-SILVA v.138 (Gattoni et al. in review) and further processed using head–tail patterns (Porazinska et al., 2010) to generate “species-equivalent” OTUs and filtered again to retain only OTUs with a match to Plectus. All OTUs identified as Plectus were further verified with BLAST at NCBI.

To perform phylogenetic analyses and assess haplotype relationships among barcoded sequences of Plectus, traces of barcode sequences of nematode specimens were edited using CodonCode Aligner Version 9.0 (http://www.codoncode.com). Sixteen published COI and 29 18S sequences of Plectus were downloaded from GenBank. All DNA sequences from this study (from COI and 18S barcoding and 18S metabarcoding), NCBI, and other prairie sites 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 + I (COI) and K2 + G + I (18S) models, both with 1,000 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) and UPGMB clustering method parameters. COI haplotype groups were generally defined by the bootstrap values of ≥99, a within-group distance that did not exceed 6%, and the distance to the nearest neighbor was greater than the within-group distance. Within- and between-COI clade genetic distances were calculated in MEGAX using p-distance with the assumption of uniform rates for 124 sequences, each with 393 nucleotide positions.

Plectus was recovered from all 15 of the remnant prairie sites investigated in this study using morphology and DNA barcoding, and from only 12 sites using metabarcoding. On an average, there were seven Plectus specimens per 150 nematodes at each site. In total, 101 Plectus corridor specimens were recorded from the sites and 44 of these were analyzed using a combination of morphological and molecular approaches. All Plectus observed were either females or juveniles. Of the 44 specimens that were analyzed by a combination of morphology and molecular approaches, 40% were juveniles and 60% females. Two general morphological phenotypes were observed during the microscopic sorting of corridor specimens: one with a short-tailed body, a c´ ratio (tail length divided by anal body width) <3.0, and an amphid diameter relative to neck width ratio average of 0.14; and one with a relatively long tail with a c´ ratio >5.0 and an amphid diameter to neck width ratio average of 0.26 (Fig. 2 and Table 2).

Figure 2

To extend comparisons beyond morphology, a second morphological assessment was conducted based on the results of DNA barcoding and phylogenetic analysis of COI sequences of voucher specimens. In this “reverse taxonomy” approach (Kanzaki et al., 2012), a phylogenetic tree of 20 clades of Plectus-derived COI haplotypes were recognized and subjected to further analysis (Fig. 3). Five of the 20 clades contained two or more Corridor specimens in both maximum likelihood and neighbor joining analyses. Two clades, referred to as Clades 3 and 20, contained a majority of specimens from the Corridor. Clade 3, a short-tailed morphotype, contained 10 Prairie Corridor specimens plus 1 specimen from Konza Prairie. Clade 20, a long-tailed morphotype, contained 24 Prairie Corridor specimens and six specimens from Nine-Mile Prairie. The remaining clades that contained two or more Prairie Corridor specimens were Clades 4, 6, and 17, comprised of two, four, and three specimens, respectively. Images of representative specimens from each of the corridor clades plus selected specimens from clades outside the Prairie Corridor are presented in Figures 4–7. Figure 4 illustrates a comparison of entire bodies and shows dramatic body length differences among the clades. Notable in the body length comparisons is the indication that there appear to be large and small versions of each of the two identified morphotypes. For example, the Clade 3 female in Figure 4 is 3/4ths the length of the female in Clade 4. However their c´ ratios and amphid width to neck width ratios are very similar (Table 2). In Clade 20, the female body length is <2/3rds of the female body length in Clade 17. Again, the c´ ratios and amphid/ neck width ratios between the females in Clades 17 and 20 are similar. Figures 5 and 6 illustrate in profile the cephalic region, stoma, and pharynx. In both figures the more heavily expanded sclerotized anterior portion of the stoma is seen in Clades 3, 4, and 6, whereas a longer, more tapered stoma is seen in Clades 17 and 20. Higher magnification in Figure 6 provides a clearer indication of amphid aperture size and amphid location on the anterior body region. The smaller amphid aperture relative to neck width is seen in Clades 3, 4, and 6. Clades 17 and 20 are characterized by larger amphid apertures relative to neck width. Figure 7 illustrates relative tail shape and length. Shorter tails relative to anal body width are evident in Clades 3, 4, and 6. Longer tails relative to anal body widths can be seen in both Clades 17 and 20. Table 2 presents measurements of tails and amphids for individual specimens in the aforementioned Prairie Corridor clades.

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Figure 7

There were 16 COI Plectus accessions retrieved from GenBank that were included in the phylogenetic analyses. Additionally, 95 specimens from a University of Nebraska repository, representing collections from 1998 to 2021, were amplified and barcoded to provide additional systematic and biogeographic context for the Plectus COI phylogenetic tree. Two Plectus species from Antarctica, Clade 1 and Clade 13, as well as international specimens from Rwanda, Poland, Ireland, and Canada, were included in the phylogenetic tree. Clade 3 included one additional member from outside the corridor, a specimen from Konza Prairie in Kansas (Knapp, 1998). Clade 20 had six additional members from outside the corridor, all from 9-Mile Prairie in Nebraska. Table 3 presents the morphometrics from select non-corridor clades used for comparative purposes in the phylogenetic trees.

The seven clades with the Prairie Corridor species (Clades 3, 4, 6, 17, 18, 19, and 20) were exclusively comprised of specimens collected from native prairie habitats. Plectus specimens from the New Jersey Pine Barrens (Clade 8), Great Smoky Mountains (Clade 7), Medicine Bow National Forest in Wyoming (Clade 14), Devon Island, Canada (Clade 11), the Antarctic Dry Valleys (Clades 1, 13), and the Alkaline Lakes in the western Sandhills of Nebraska (Clades 15, 16) all exhibited location-specific Plectus taxa. Only a single clade, Clade 9, had well-supported, closely related sequences collected from geographically distant sites. This clade included a mountaintop heath bald in the Great Smoky Mountains, the New Jersey Pine Barrens, and Big Thicket National Preserve in Texas.

Genetic distances within and between the 20 COI Clades are presented in Table 4. Although sample sizes varied, the two Antarctic clades were notable for low levels of within-group polymorphism. Between-group mean pairwise genetic distance varied from 0.0458 to 0.1806 for the 20 clades. The lower value represents mean genetic distance between specimens from different lakes in the western Nebraska Sandhills that were initially separated due to non-overlapping morphological characteristics (Table 3). The highest distance value is between specimens from a wheat field in Montana and Clade 19, a geographically heterogeneous group of grassland specimens.

Figure 8 displays the 18S tree with individual barcodes generated by Sanger sequencing, GenBank accessions, and sequences generated by metabarcoding. Sequences from split template specimens that were also barcoded with COI are identified by their Clade number following their genus name as designated on the COI phylogenetic tree. There was little nucleotide sequence differentiation among Prairie Corridor Plectus specimens for the 18S NF1 region, with only two specimens (NID 13101 and NID 13180) exhibiting sequence variation when compared to other Prairie Corridor specimens. The COI barcoded specimens of Clades 2, 3, 6, 17, and 20 all produced identical 18S sequence for the NF1 region (Fig. 8). GenBank accessions of seven different named species also exhibited identical 18S sequence in the NF1 region.

Figure 8

There were 19 OTUs representing Plectida produced by the NF1 metabarcoding analysis. Not all of these could be classified as members of the genus Plectus, but could be assigned to a higher taxon within the order Plectida. Plectus OTU1 matched the 18S sequence that characterized the majority of specimens sequenced by Sanger sequencing and 12 GenBank accessions. OTU 6 matched N13101, a Plectus singleton on the COI tree. Other OTU sequences matched different plectid genera such as Anaplectus and Tylocephalus, in addition to other unidentified taxa.