Current surveys of nematode diversity frequently apply DNA barcoding or metabarcoding as a method of assessment (Floyd et al., 2002; Treonis et al., 2018; Schenk and Fontaneto, 2020). The objective of the survey generally dictates the assessment method of choice. If the objective is to assess functional diversity, e.g., trophic groups, molecular methods may not be required and traditional morphological evaluation may be adequate (Hodda and Wanless, 1994; Bloemers et al., 1997). If taxon/species diversity is a goal, then a method of greater taxonomic resolution, such as DNA barcoding, may be necessary. And, if the scope of the survey requires multiple comparisons across broad geographic regions and scales, then a metabarcoding approach may be optimal (Porter and Hajibabaei, 2018; Brunbjerg et al., 2019; Arribas et al., 2021). Each of these methods has advantages and disadvantages, but one common feature of the molecular methods is the need for referral to a DNA database. In this study we chose a single taxon,
Most known
There were two objectives for this
To determine the level of taxonomic resolution provided by each assessment method as it applies to
To molecularly compare
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.
Map of Nebraska and location of the 16-km-long Lancaster County Prairie Corridor. Orange dots indicate the 15 remnant prairie sites.
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 (
Specimen collection information.
Group | NID | Species | Site | Location | Marker | GB Accession # |
---|---|---|---|---|---|---|
1 | N11040 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208981 | |
1 | N11213 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208968 | |
1 | N11461 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208969 | |
1 | N11211 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208970 | |
1 | N11110 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208971 | |
1 | N11042 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208973 | |
1 | N11146 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208976 | |
1 | N11041 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208977 | |
1 | N11526 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208978 | |
1 | N11142 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208979 | |
1 | N11215 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208980 | |
1 | N11161 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208982 | |
1 | N11035 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208983 | |
1 | N11034 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208984 | |
1 | N11109 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208985 | |
1 | N11121 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208986 | |
1 | N11140 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208987 | |
1 | N11216 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208989 | |
1 | N11227 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208990 | |
1 | N11218 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208992 | |
1 | N11473 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208993 | |
1 | N11209 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208988 | |
1 | N12079 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208991 | |
1 | N11107 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP208972 | |
1 | N11033 | Lake Bonney | McMurdo Dry Valleys, Antarctica | COI | OP208974 | |
1 | N11047 | Garwood Valley | McMurdo Dry Valleys, Antarctica | COI | OP208975 | |
2 | N11873 | Barley field | Hall County, MT | COI | OP208994 | |
2 | N11874 | Barley field | Hall County, MT | COI | OP208995 | |
3 | N13530 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209006 | |
3 | N13357 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209005 | |
3 | N13255 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209004 | |
3 | N13045 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209003 | |
3 | N11765 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209001 | |
3 | N13543 | Becker Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209000 | |
3 | N11764 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209002 | |
3 | N13030 | Semani Prairie | Prairie Corridor, Lancaster County, NE | COI | OP208999 | |
3 | N12485 | Eggerling Soybean Field | Prairie Corridor, Lancaster County, NE | COI | OP208998 | |
3 | N13239 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP208996 OP205456 | |
3 | P194018 | Konza Prairie | Riley County, KS | COI | OP208997 | |
a | N11865 | Arctic Preserve | Utqiaghk, AK | COI | OP209007 | |
4 | N13608 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209009 | |
4 | N13280 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209008 OP205457 | |
5 | KU759331.1 | GenBank | COI | KU759331.1 | ||
5 | KU759327.1 | GenBank | COI | KU759327.1 | ||
5 | KU759330.1 | GenBank | COI | KU759330.1 | ||
6 | N13176 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209010 OP205453 | |
6 | N13287 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209011 | |
6 | N13425 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209012 | |
6 | N13614 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209013 | |
a | N8809 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209014 | |
7 | N1023 | LeConte ATBI Site | Great Smoky Mountains Nat’l Park | COI | OP209016 | |
7 | N4342 | Brushy Mountain ATBI Site | Great Smoky Mountains Nat’l Park | COI | OP209015 | |
8 | N8808 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209017 | |
8 | N8817 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209018 | |
8 | N8828 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209019 | |
9 | N4244 | Canyonlands South | Big Thicket National Preserve, TX | COI | OP209020 | |
9 | N9213 | Maddron Bald | Great Smoky Mountains Nat’l Park | COI | OP209021 | |
9 | N9237 | Maddron Bald | Great Smoky Mountains Nat’l Park | COI | OP209022 | |
9 | N8797 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209023 | |
10 | N8706 | Lesund | Møre og Romsdal, Norway | COI | OP209024 | |
a | N9166 | Brendan C. Byrne State Forest | Burlington County, NJ | COI | OP209025 | |
11 | P87048 | Haughton Crater | Devon Island, Canada | COI | OP209026 | |
11 | P88010 | Haughton Crater | Devon Island, Canada | COI | OP209027 | |
a | P89090 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209028 | |
a | P86098 | Konza Prairie | Riley County, KS | COI | OP209029 | |
a | P89089 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209030 | |
12 | KU759349.1 | GenBank | COI | KU759349.1 | ||
12 | KU759350.1 | GenBank | COI | KU759350.1 | ||
12 | KU759348.1 | GenBank | COI | KU759348.1 | ||
13 | N11013 | Lake Hoare | McMurdo Dry Valleys, Antarctica | COI | OP209035 | |
13 | N11018 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP209036 | |
13 | N11028 | Lake Fryxell | McMurdo Dry Valleys, Antarctica | COI | OP209037 | |
13 | N11185 | Taylor Valley | McMurdo Dry Valleys, Antarctica | COI | OP209038 | |
13 | N11199 | Scott Base | Botany Bay, Antarctica | COI | OP209039 | |
13 | N11719 | Lake Miers Valley | McMurdo Dry Valleys, Antarctica | COI | OP209040 | |
13 | N9870 | Scott Base | Botany Bay, Antarctica | COI | OP209031 | |
13 | N9873 | Scott Base | Botany Bay, Antarctica | COI | OP209032 | |
13 | N9880 | Taylor Valley | McMurdo Dry Valleys, Antarctica | COI | OP209033 | |
13 | N9892 | Hjorth Hill | McMurdo Dry Valleys, Antarctica | COI | OP209034 | |
a | N9135 | Tipple Trail | Medicine Bow-Routt Nat’l Forest, WY | COI | OP209041 | |
14 | N8916 | Snowy Range | Medicine Bow-Routt Nat’l Forest, WY | COI | OP209042 | |
14 | N8869 | Happy Jack Trail | Medicine Bow-Routt Nat’l Forest, WY | COI | OP209043 | |
14 | N8874 | Happy Jack Trail | Medicine Bow-Routt Nat’l Forest, WY | COI | OP209044 | |
15 | N12416 | Island Lake | Nebraska Sandhills, Garden County, NE | COI | OP209045 | |
15 | N12419 | Island Lake | Nebraska Sandhills, Garden County, NE | COI | OP209046 | |
15 | N12414 | Island Lake | Nebraska Sandhills, Garden County, NE | COI | OP209047 | |
15 | N12402 | Island Lake | Nebraska Sandhills, Garden County, NE | COI | OP209048 | |
16 | N12101 | Border Lake | Nebraska Sandhills, Garden County, NE | COI | OP209049 | |
16 | N12128 | Gimlet Lake | Nebraska Sandhills, Garden County, NE | COI | OP209050 | |
16 | N12228 | Bean Lake | Nebraska Sandhills, Garden County, NE | COI | OP209051 | |
a | N8576 | El Yunque National Forest | Puerto Rico | COI | OP209052 | |
a | N8582 | El Yunque National Forest | Puerto Rico | COI | OP209053 | |
a | N9089 | Henning Conservation Area | Taney County, MO | COI | OP209054 | |
a | P30012 | Konza Prairie | Riley County, KS | COI | OP209055 | |
17 | N13180 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209056 OP205467 | |
17 | N13460 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209057 | |
a | N11872 | Arctic Preserve | Utqiaghk, AK | COI | OP209058 | |
a | N8374 | Dombas/Hjelle Seter | Oppland, Norway | COI | OP209060 | |
a | N9370 | Happy Jack Trail | Medicine Bow-Routt Nat’l Forest, WY | COI | OP209059 | |
a | N8689 | Hjerkinn | Oppland, Norway | COI | OP209061 | |
a | N9059 | Łukęcin | West Pomerania, Poland | COI | OP209062 | |
a | N11847 | Arctic Preserve | Utqiaghk, AK | COI | OP209063 | |
18 | P89028 | Konza Prairie | Riley County, KS | COI | OP209066 | |
18 | P89030 | Konza Prairie | Riley County, KS | COI | OP209064 | |
18 | N13465 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209065 | |
18 | P117056 | Konza Prairie | Riley County, KS | COI | OP209067 | |
19 | N9339 | Drury-Mincy Conserv. Area | Taney County, MO | COI | OP209068 | |
19 | N12733 | Twin Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209069 | |
19 | P89081 | Konza Prairie | Riley County, KS | COI | OP209070 | |
20 | P90058 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209071 | |
20 | P90062 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209072 | |
20 | P90057 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209073 | |
20 | N13403 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209074 | |
20 | N13479 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209075 | |
20 | N13486 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209076 | |
20 | N13444 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209077 | |
20 | N13440 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209078 | |
20 | N13329 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209083 OP205458 | |
20 | N13344 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209084 OP205459 | |
20 | N13225 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209082 OP205455 | |
20 | N13203 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209081 OP205454 | |
20 | N13133 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209080 | |
20 | N13118 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209079 OP205452 | |
20 | N13369 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209085 OP205460 | |
20 | N13471 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209086 | |
20 | N13491 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209087 | |
20 | N13587 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209088 | |
20 | N13598 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209089 | |
20 | N13601 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209090 | |
20 | N13622 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209091 | |
20 | N13628 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209092 | |
20 | N13638 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209093 | |
20 | N13641 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209094 | |
20 | P86052 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209095 | |
20 | P89091 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209096 | |
20 | P90059 | Nine-Mile Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209097 | |
20 | N12697 | Honvlez Prairie | Prairie Corridor, Lancaster County, NE | COI | OP209098 | |
a | P89023 | Haughton Crater | Devon Island, Canada | COI | OP209099 | |
a | P87033 | Konza Prairie | Riley County, KS | COI | OP209100 | |
a | N12491 | Eggerling Soybean Field | Prairie Corridor, Lancaster County, NE | COI | OP209101 | |
a | N12114 | Border Lake | Nebraska Sandhills, Garden County, NE | COI 18S | OP209102 OP205470 | |
a | N13101 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | COI 18S | OP209103 OP205471 | |
a | N12159 | Kokjohn Lake | Nebraska Sandhills, Garden County, NE | COI | OP209104 | |
a | N9790 | Formerly cultivated land | Volcanoes Nat’l Park, Rwanda | COI | OP209105 | |
a | P195005 | Konza Prairie | Riley County, KS | COI | OP209106 | |
N13211 | Pioneers Park Prairie | Prairie Corridor, Lancaster County, NE | 18S | OP205461 | ||
N13250 | Spring Creek Prairie | Prairie Corridor, Lancaster County, NE | 18S | OP205462 | ||
N13370 | Eggerling Prairie | Prairie Corridor, Lancaster County, NE | 18S | OP205469 | ||
P133013 | La Selva Biological Station | Costa Rica | 18S | OP205466 | ||
P134037 | La Selva Biological Station | Costa Rica | 18S | OP205463 | ||
P137018 | La Selva Biological Station | Costa Rica | 18S | OP205450 | ||
P140029 | La Selva Biological Station | Costa Rica | 18S | OP205451 | ||
P151063 | La Selva Biological Station | Costa Rica | 18S | OP205468 | ||
P183015 | Heredia Province | Costa Rica | 18S | OP205464 | ||
P183018 | Heredia Province | Costa Rica | 18S | OP205465 |
aSpecimen not assigned to a group.
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 (
To perform phylogenetic analyses and assess haplotype relationships among barcoded sequences of
Two general
Prairie Corridor specimen measurements from images.
Group | NID | Taxon | Stage | Initial characterization |
Voucher measurements in micrometer |
||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Amphid | Tail | Tail length | Anal body width | c´ ratio | Amphid width | Neck width at amphid | Amphid/ neck width (%) | ||||
3 | NID | F | Small, | Short | 65 | 26.7 | 2.4 | 2 | 19 | 0.11 | |
12485 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 35 | 15 | 2.3 | 2.5 | 17.5 | 0.14 | |
13239 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 62 | 28 | 2.2 | NA | NA | NA | |
13543 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 70 | 29.4 | 2.4 | 2.5 | NA | NA | |
13030 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 61 | 28.9 | 2.1 | 2.5 | 16.6 | 0.15 | |
13045 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 65 | 27.5 | 2.4 | NA | NA | NA | |
13255 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 66 | 28.9 | 2.3 | 2 | 17.5 | 0.11 | |
13357 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 63 | 25.2 | 2.5 | 2 | 16.2 | 0.12 | |
13530 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 63 | 28.9 | 2.2 | 2 | 17 | 0.12 | |
13538 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 70 | 29 | 2.4 | 2.5 | 12 | 0.21 | |
11764 | sp. 3 | round | |||||||||
3 | NID | J | Small, | Short | 74 | 34 | 2.2 | 2 | 12 | 0.17 | |
11765 | sp. 3 | round | |||||||||
4 | NID | J | Small, | Short | 77 | 25 | 3.1 | 2.5 | 17 | 0.15 | |
13280 | sp. 4 | round | |||||||||
4 | NID | F | Small, | Short | 94 | 33.6 | 2.8 | 2 | 20.4 | 0.10 | |
13608 | sp. 4 | round | |||||||||
6 | NID | J | Small, | Short | 72 | 27.6 | 2.6 | 3 | 15.7 | 0.19 | |
13176 | sp. 6 | round | |||||||||
6 | NID | J | Small, | Short | 75 | 27.2 | 2.8 | 2.5 | 17 | 0.15 | |
13287 | sp. 6 | round | |||||||||
6 | NID | J | Small, | Short | 92 | 33.6 | 2.7 | 3 | 18.3 | 0.16 | |
13425 | sp. 6 | round | |||||||||
17 | NID | F | Large, | Long | 129 | 33.2 | 3.9 | 4 | 17.9 | 0.22 | |
13180 | sp. 17 | round | |||||||||
17 | NID | F | Large, | Long | 120 | 28.5 | 4.2 | 5 | 20.4 | 0.25 | |
13460 | sp. 17 | round | |||||||||
18 | NID | F | Large, | Long | 91.6 | 17.4 | 5.3 | 3.5 | 13.6 | 0.26 | |
13465 | sp. 18 | round | |||||||||
19 | NID | F | Large, | Long | 105 | 18 | 5.8 | 3.3 | 12.5 | 0.26 | |
12733 | sp. 19 | round | |||||||||
20 | NID | F | Large, | Long | 92.5 | 17 | 5.4 | 3.5 | 14 | 0.25 | |
13403 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 94 | 18.3 | 5.1 | 3.5 | 15.3 | 0.23 | |
13479 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 85 | 18.3 | 4.6 | 4 | 14.5 | 0.28 | |
13486 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 88 | 16.2 | 5.4 | 4 | 14.9 | 0.27 | |
13444 | sp. 20 | round | |||||||||
20 | NID | J | Large, | Long | 102 | 20 | 5.1 | 4 | 15.3 | 0.26 | |
13440 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 94 | 18.7 | 5.0 | 3 | 14.9 | 0.20 | |
13118 | sp. 20 | round | |||||||||
20 | NID | J | Large, | Long | 110 | 20.8 | 5.3 | 4.5 | 16.2 | 0.28 | |
13133 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 97 | 19.6 | 4.9 | 3.5 | 14.9 | 0.23 | |
13203 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 98 | 17 | 5.8 | 4.5 | 14.9 | 0.30 | |
13225 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 96 | 20.4 | 4.7 | 4 | 14.9 | 0.27 | |
13329 | sp. 20 | round | |||||||||
20 | NID | J | Large, | Long | 78 | 13.6 | 5.7 | 3.5 | 11.5 | 0.30 | |
13344 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 100 | 17 | 5.9 | 4 | 14.5 | 0.28 | |
13369 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 94 | 17.9 | 5.2 | 4 | 13.6 | 0.29 | |
13471 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 99 | 19.6 | 5.0 | NA | NA | NA | |
13491 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 93 | 19.1 | 4.9 | 3.5 | 15.7 | 0.22 | |
13587 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 78 | 17.9 | 4.4 | 4 | 13.6 | 0.29 | |
13598 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 91 | 18.3 | 5.0 | 3.5 | 14 | 0.25 | |
13601 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 88 | 16.2 | 5.4 | 4.5 | 14 | 0.32 | |
13622 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 108 | 20 | 5.4 | 3.5 | 14.9 | 0.23 | |
13628 | sp. 20 | round | |||||||||
20 | NID | F | Large, | Long | 92 | 18.3 | 5.0 | 3.4 | 14.9 | 0.23 | |
13641 | sp. 20 | round |
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
ML COI phylogenetic tree of
A representation of body length and overall morphology from select haplotype groups. NID and group numbers correspond to placement on the COI tree.
Select anterior body morphology of different haplotype groups.
Representative cephalic regions and stomas of seven haplotype groups. Groups 3, 4, and 6 display offset cephalic regions and relatively small diameter amphids, compared to the continuous head regions and larger amphid diameters of groups 8, 9, 17, and 20. Heavier anterior sclerotization of the stoma is seen in groups 3 and 4 compared to the long, tapered stomas of 8, 9, 17, and 20.
Relative tail lengths and anal body widths in different haplotype groups.
There were 16 COI
Measurements of non-Prairie Corridor Plectus specimens.
Taxon (n) | Group 16 (3) keys to Plectus aquatilis | Group 14 (3) keys to P. cirratus | Group 9 (4) keys to P. cirratus | Group 8 (3) keys to Plectus longicaudatus | Group 13 (5) keys to P. murrayi | Group 15 (4) keys to P. palustris |
---|---|---|---|---|---|---|
L | 1,394.9 ± 191.0 | 1,170.6 ± 78.4 | 1,032.7 ± 111.3 | 696.8 ± 35.1 | 883.8 ± 25.7 | 1,642.0 ± 141.5 |
(1,183–1,646) | (1,071–1,262) | (874–1,176) | (654–740) | (840–917) | (1,411–1,778) | |
Tail | 156.7 ± 15.3 | 123.8 ± 10.1 | 104.1 ± 10.8 | 100.2 ± 6.9 | 101.3 ± 5.2 | 224.7 ± 17.8 |
length | (145–178) | (110–135) | (88–117) | (91–107) | (94–109) | (200–246) |
21.5 ± 0.7 | 27.3 ± 3.4 | 24.6 ± 2.5 | 26.7 ± 1.9 | 23.0 ± 0.8 | 34.2 ± 3.0 | |
(20.8–22.5) | (22.7–30.7) | (20.6–27.2) | (24.5–29.2) | (22.0–24.2) | (31.0–38.9) | |
4.5 ± 0.3 | 3.7 ± 0.1 | 4.0 ± 0.2 | 3.6 ± 0.0 | 4.1 ± 0.1 | 4.8 ± 0.3 | |
(4.2–4.9) | (3.7–3.8) | (3.6–4.1) | (3.6–3.7) | (4.0–4.2) | (4.3–5.0) | |
8.9 ± 0.5 | 9.5 ± 0.5 | 9.9 ± 0.1 | 7.0 ± 0.2 | 8.7 ± 0.3 | 7.3 ± 0.2 | |
(8.2–9.2) | (8.8–10.0) | (9.8–10.1) | (6.7–7.2) | (8.3–9.1) | (7.1–7.6) | |
4.8 ± 0.1 | 5.0 ± 0.5 | 4.2 ± 0.3 | 8.1 ± 0.9 | 4.8 ± 0.3 | 8.1 ± 0.4 | |
(4.6–4.9) | (4.4–5.5) | (4.0–4.7) | (6.9–8.7) | (4.1–5.1) | (7.6–8.7) | |
V% | 49.6 ± 0.9 | 49.8 ± 0.7 | 49.9 ± 0.9 | 47.9 ± 0.5 | 48.1 ± 0.4 | 46.2 ± 0.5 |
(48.4–50.5) | (48.8–50.6) | (49.0–51.3) | (47.2–48.5) | (47.8–48.9) | (45.7–47.0) | |
V–A/T | 3.5 ± 0.3 | 3.8 ± 0.3 | 4.0 ± 0.1 | 2.6 ± 0.1 | 3.5 ± 0.2 | 2.9 ± 0.1 |
(3.1–3.8) | (3.4–4.1) | (3.8–4.1) | (2.6–2.8) | (3.3–3.8) | (2.8–3.1) | |
LR W/H | 2.5 ± 0.3 | 2.6 ± 0.3 | 2.7 ± 0.1 | 2.0 ± 0.4 | 2.3 ± 0.3 | 2.0 ± 0.2 |
(2.0–2.8) | (2.3–3.0) | (2.6–2.8) | (1.5–2.3) | (2.1–2.7) | (1.6–2.2) | |
rec/abw | 0.8 ± 0.1 | 1.2 ± 0.1 | 1.0 ± 0.2 | 1.9 ±0.2 | 1.0 ± 0.2 | 1.2 ± 0.1 |
(0.7–0.9) | (1.1–1.3) | (0.8–1.3) | (1.7–2.2) | (0.7–1.1) | (1.1–1.3) | |
Ceph | 3.5 ± 0.6 (3–4) | 3.5 ± 0.9 (3–5) | 3.7 ± 0.8 (3–5) | 3.2 ± 0.1 (3–3) | 3.4 ± 0.5 (3–4) | 3.0 ± 0.2 (3–3) |
set L | ||||||
Ceph | 5.9 ± 0.6 (5–7) | 5.8 ± 0.8 (5–7) | 6.5 ± 1.0 (5–8) | 5.1 ± 0.5 (4–6) | 5.5 ± 0.8 (5–7) | 6.0 ± 1.0 (5–7) |
set pos | ||||||
Set L/ | 26.5 ± 5.5 | 25.5 ± 3.1 | 29.1 ± 5.3 | 38.7 ± 9.4 | 31.3 ± 6.1 | 24.5 ± 1.9 |
lrw% | (19.3–32.7) | (22.3–29.7) | (22.4–35.2) | (31.6–52.0) | (21.2–39.5) | (22.9–27.7) |
Amphid | 3.5 ± 0.4 (3–4) | 3.3 ± 0.2 (3–3) | 3.5 ± 0.5 (3–4) | 3.1 ± 0.1 (3–3) | 2.5 ± 0.2 (2–3) | 3.4 ± 0.3 (3–4) |
W | ||||||
Amp | 0.9 ± 0.1 | 0.9 ± 0.0 | 0.8 ± 0.1 | 0.7 ± 0.1 | 1.0 ± 0.1 | 1.0 ± 0.2 |
L/W | (0.8–1.0) | (0.9–0.9) | (0.6–0.9) | (0.6–0.9) | (0.9–1.2) | (0.8–1.3) |
Amphid | 12.3 ± 0.5 | 10.3 ± 1.4 | 13.0 ± 1.1 | 8.4 ± 0.6 (8–9) | 11.2 ± 1.1 | 14.2 ± 0.5 |
pos | (12–13) | (8–12) | (12–15) | (10–13) | (13–15) | |
Amp W/ | 19.9 ± 2.8 | 18.6 ± 2.5 | 19.8 ± 2.8 | 26.6 ± 0.9 | 15.5 ± 2.0 | 18.5 ± 2.1 |
head% | (17.5–23.9) | (15.2–20.9) | (15.8–23.3) | (25.5–27.7) | (13.2–18.5) | (16.0–21.7) |
Stoma L | 24.3 ± 0.1 | 24.3 ± 3.5 | 24.1 ± 4.3 | 18.3 ± 2.2 | 21.4 ± 3.8 | 27.0 ± 2.1 |
(24–24) | (19–28) | (20–31) | (17–21) | (16–27) | (24–30) | |
Stoma | 7.4 ±1.2 | 6.7 ± 2.0 | 7.3 ± 2.1 | 5.5 ± 0.7 | 8.7 ± 3.5 | 9.5 ± 1.3 |
L/W | (6.1–8.9) | (4.6–9.5) | (4.2–9.5) | (4.8–6.2) | (6.0–15.4) | (7.5–10.9) |
Sto W/ | 21.8 ±1.8 | 26.7 ± 7.8 | 24.6 ±7.3 | 31.9 ± 2.2 | 21.3 ± 4.8 | 20.8 ± 2.1 |
head% | (19.3–23.5) | (16.2–34.7) | (19.7–37.2) | (29.7–34.1) | (12.3–26.7) | (18.3–24.1) |
Prosto/ | 30.3 ± 2.0 | 24.8 ± 7.0 | 32.1 ± 4.1 | 31.2 ± 1.0 | 22.8 ± 4.4 | 30.8 ± 2.7 |
sto% | (27.8–32.7) | (18.3–34.6) | (27.8–38.8) | (29.9–32.4) | (15.7–28.6) | (27.8–35.1) |
Exc | 56.3 ± 0.8 | 54.1 ± 0.8 | 53.9 ± 3.5 | 54.1 ± 1.6 | 57.2 ± 0.7 | 56.2 ± 0.7 |
Pore% | (55.6–57.4) | (53.1–55.1) | (48.3–57.1) | (52.5–55.7) | (56.4–58.5) | (55.4–57.0) |
Cerv | 208.9 ± 50.2 | 192.1 ± 27.3 | 147.2 ± 13.6 | 90.0 ± 0.0 | 130.9 ± 3.2 | 202.3 ± 8.3 |
papilla | (167–279) | (165–229) | (134–170) | (90–90) | (126–134) | (191–214) |
Body | 1.7 ± 0.3 | 1.2 ± 0.0 | 1.6 ± 0.1 | 1.0 ± 0.0 | 1.3 ± 0.1 | 1.5 ± 0.2 |
ann W | (1.3–2.0) | (1.1–1.2) | (1.4–1.7) | (0.9–1.1) | (1.2–1.4) | (1.2–1.7) |
G’1% | 25.0 ± 1.7 | 19.6 ± 2.9 | 20.5 ± 2.7 | 14.0 ± 0.7 | 16.6 ± 2.2 | 20.0 ± 2.0 |
(23.3–26.7) | (15.9–23.0) | (16.5–23.3) | (12.9–14.6) | (13.5–19.8) | (17.9–22.4) | |
G’2% | 25.5 ± 2.8 | 18.7 ± 3.1 | 18.2 ± 3.2 | 17.1 ± 2.6 | 18.0 ± 3.4 | 20.1 ± 2.0 |
(22.7–28.2) | (14.7–22.4) | (13.4–21.3) | (14.3–20.5) | (14.4–22.3) | (17.0–22.2) | |
Spin | 1.5 ± 0.1 | 1.7 ± 0.2 | 1.2 ± 0.1 | 1.1 ± 0.5 | 1.3 ± 0.4 | 2.0 ± 0.6 |
L/W | (1.3–1.6) | (1.5–2.0) | (1.1–1.4) | (0.6–1.6) | (0.8–1.9) | (1.3–2.8) |
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.
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.
Within-a and between-group distances.
Plectus frigophilus | Plectus sp. 2 | Plectus sp. 3 | Plectus sp. 4 | Plectus sp. 6 | Plectus sp. 7 | Plectus sp. 8 | Plectus sp. 9 | Plectus sp. 10 | Plectus sp. 11 | |
---|---|---|---|---|---|---|---|---|---|---|
0.145469 | ||||||||||
0.132330 | 0.105149 | |||||||||
0.144087 | 0.152672 | 0.153299 | ||||||||
0.164902 | 0.144402 | 0.142419 | 0.136768 | |||||||
0.161954 | 0.152672 | 0.162706 | 0.153944 | 0.169847 | ||||||
0.147804 | 0.152248 | 0.148707 | 0.148007 | 0.155428 | 0.094996 | |||||
0.158646 | 0.164758 | 0.172045 | 0.123410 | 0.152672 | 0.133588 | 0.147371 | ||||
0.146456 | 0.148855 | 0.149938 | 0.115776 | 0.157125 | 0.124682 | 0.118745 | 0.086514 | |||
0.146744 | 0.150763 | 0.146703 | 0.131043 | 0.163486 | 0.141221 | 0.154368 | 0.113868 | 0.124682 | ||
0.147902 | 0.166545 | 0.155100 | 0.138186 | 0.175413 | 0.156327 | 0.163139 | 0.100769 | 0.123120 | 0.100899 | |
0.142599 | 0.149703 | 0.147779 | 0.139525 | 0.155428 | 0.141645 | 0.139949 | 0.118533 | 0.106022 | 0.114080 | |
0.173392 | 0.161578 | 0.172686 | 0.151399 | 0.174936 | 0.145038 | 0.153520 | 0.128499 | 0.114504 | 0.133588 | |
0.169633 | 0.170059 | 0.168288 | 0.152248 | 0.166455 | 0.143342 | 0.146735 | 0.132740 | 0.116200 | 0.120865 | |
0.165902 | 0.176845 | 0.164923 | 0.137405 | 0.144402 | 0.153944 | 0.172604 | 0.145038 | 0.138677 | 0.131679 | |
0.163522 | 0.174936 | 0.170885 | 0.146310 | 0.157761 | 0.152672 | 0.158609 | 0.148855 | 0.131679 | 0.132316 | |
0.176055 | 0.180662 | 0.169356 | 0.152248 | 0.160517 | 0.146735 | 0.153520 | 0.151612 | 0.134012 | 0.133164 | |
0.175147 | 0.172392 | 0.166065 | 0.148219 | 0.160760 | 0.156398 | 0.165243 | 0.157397 | 0.130225 | 0.128862 | |
0.130518 | 0.148551 | 0.143198 | 0.107337 | 0.163043 | 0.163043 | 0.150060 | 0.156703 | 0.142210 | 0.151721 | |
0.147765 | 0.161232 | 0.156868 | 0.132699 | 0.161232 | 0.154891 | 0.162138 | 0.110960 | 0.134964 | 0.101449 |
Plectus murrayi | Plectus sp. 14 | Plectus sp. 15 | Plectus sp. 16 | Plectus sp. 17 | Plectus sp. 18 | Plectus sp. 19 | Plectus sp. 20 | Plectus sp. 5 | Plectus sp. 12 | |
---|---|---|---|---|---|---|---|---|---|---|
0.0012 | ||||||||||
0.120908 | 0.00339 | |||||||||
0.114939 | 0.118745 | 0 | ||||||||
0.122602 | 0.121289 | 0.045802 | 0.00679 | |||||||
0.141001 | 0.138253 | 0.146310 | 0.148855 | 0.01018 | ||||||
0.145731 | 0.144614 | 0.136768 | 0.118957 | 0.136768 | 0.00382 | |||||
0.134193 | 0.133164 | 0.147583 | 0.137970 | 0.124258 | 0.108355 | 0.05513 | ||||
0.146096 | 0.131376 | 0.134224 | 0.128287 | 0.138041 | 0.088877 | 0.088422 | 0.00837 | |||
0.137681 | 0.147645 | 0.132246 | 0.137379 | 0.164855 | 0.157835 | 0.170290 | 0.169999 | 0.01993 | ||
0.100543 | 0.121679 | 0.123188 | 0.128623 | 0.132246 | 0.143569 | 0.136775 | 0.142048 | 0.149457 | 0.06612 |
awithin-group distance values in bold.
bNot able to be calculated.
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
ML tree of 18S sequences. OTUs from metabarcoding are highlighted in yellow. Sanger sequences from the prairie corridor are highlighted in green. Numbers following the genus name correspond to COI clade. GenBank accessions are included with their full species name. ML, maximum likelihood; OTUs, operational taxonomic units.
There were 19 OTUs representing Plectida produced by the NF1 metabarcoding analysis. Not all of these could be classified as members of the genus
The North American Prairies are known for their rich plant and animal diversity (Savage, 2011). Surveys of prairie nematodes have supported the idea of high biotic diversity (Orr and Dickerson, 1966; Todd et al., 2006). Since the initial, historical nematode surveys were conducted using traditional morphological approaches as visualized by light microscopy, it could be expected that molecular approaches will provide equivalent, or even higher, diversity estimates. This expectation was met, although determination of specimen species identity in the seven prairie clades and additional prairie singletons on the COI tree was difficult.
We selected
Morphologically, Prairie Corridor specimens in Clades 3, 4, and 6 resemble
Scanning electron micrograph of short-tailed specimen exhibiting caudal setae near tail terminus on dorsal surface.
The most common
Scanning Electron Micrograph of corresponding heads and tails of long-tailed morphotype (A, B) and short-tailed morphotypes (C, D and E, F).
Species identification for specimens outside the Prairie Corridor could be considered educated guesses at best. There are two clades that are distinctive based on morphology and location. The two Antarctic clades represent well-studied, homogeneous species known only from specific habitats on the Antarctic continent (Kito et al., 1991).
The uniformity in 18S sequence in the NF1 region indicates that neither DNA barcoding nor metabarcoding analysis based on this portion of 18S alone will allow discrimination among
COI haplotype groups may provide insight into