Identification of
Morphological features and measurements of
There is consensus that nematode identification today should be supported by molecular analyses so sequence data are rapidly accumulating for multiple gene fragments including the cytochrome c oxidase subunit1 (COI) mitochondrial gene (Janssen et al., 2017), rDNA internal transcribed spacers (De Luca et al., 2011), D2/D3 expansion region of 28S rDNA, and 18S rDNA (Subbotin et al., 2008). Janssen et al. (2017) used a combination of morphological and sequence data of COI to successfully distinguish species among the Penetrans group of
Intraspecific genetic variation among
We recently surveyed Wisconsin for species of
Nematodes were recovered from soil samples collected from 11 farms in nine Wisconsin counties for a commodity-sponsored testing program. The samples were randomly selected from a pool of stored samples that were known to contain
Nematodes from at least three Petri dishes were harvested and pooled for morphological characterization, given the reported sensitivity of some features to environmental conditions (Tarte and Mai, 1976a). Fresh specimens were mounted following the protocol of Eisenback (2012), visualized using differential interference contrast optics, and measured and photographed using NIS-Elements AR Software on a Nikon Eclipse Ti-E inverted microscope (Nikon Instruments, Melville, NY). Tail terminus morphology, tail shape, and spermatheca shape were recorded. Data were collected from a minimum of 25 females of each isolate, except for Chippewa2 (
Intraspecific variation in the morphometrics among the
For HAC, Euclidean distance coefficients were computed on normalized mean values to evaluate the (dis)similarity between each pair of isolates using the ‘dist’ function with the Euclidean method option in the ‘STATS’ R package (R Core Team, 2020). The normalized means were computed by
Canonical discriminant analysis (CDA) of the clusters suggested by HAC was performed using the CANDISC procedure in SAS (SAS Institute, Cary, NC) to determine which variables discriminated clusters. Univariate statistics (ANOVA) were also conducted for each morphometric variable to test equality of means among the 13 isolates.
Nematodes from the 13 cultured isolates and 39 field populations collected from 39 farms in 27 Wisconsin counties were characterized molecularly for mitochondrial cytochrome c oxidase subunit 1 (COI) (Fig. 1). The 52 populations were named for the county of origin and multiple populations from the same country were distinguished by numbers and individual specimens by letters (Table 3). Nematode DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). The DNA extracts were stored at −20°C if PCR was not conducted immediately. Five microliters of DNA were suspended in a 25 μl reaction volume composed of 2.5 μl ddH2O, 2.5 μl of forward and reverse primers, and 12.5 μl of GoTaq Green Master Mix x2 (Promega, Madison, WI). The primer used for COI was forward
Information for origin and hosts of the
GenBank Accession number | |||||
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Locality | Specimen name | Planted crop | Haplotype group | COI | D2/D3 of 28S rRNA |
USA, Wisconsin, Brown Co. | Brown-a | corn | Pp1 | MT527067 | MT528159 |
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Pp1 |
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Pp1 |
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Pp1 |
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USA, Wisconsin, Columbia Co. | Columbia1-a | corn | Pp1 | MT527044 | MT528189 |
USA, Wisconsin, Columbia Co. | Columbia3-a | corn | Pp1 | MT527077 | MT528192 |
USA, Wisconsin, Crawford Co. | Crawford-b | – | Pp1 | MT527055 | MT528196 |
USA, Wisconsin, Dunn Co. | Dunn1-b | soybean | Pp1 | MT527024 | MT528199 |
USA, Wisconsin, Eau Claire Co. | Eau Claire1-a | soybean | Pp1 | MT527073 | MT528203 |
USA, Wisconsin, Eau Claire Co. | Eau Claire2-a | soybean | Pp1 | MT527059 | MT528205 |
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Pp1 |
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Pp1 |
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USA, Wisconsin, Jackson Co. | Jackson1-a | soybean | Pp1 | MT527038 | MT528171 |
USA, Wisconsin, Jackson Co. | Jackson2-a | soybean | Pp1 | MT527053 | MT528208 |
USA, Wisconsin, Jefferson Co. | Jefferson2-a | soybean | Pp1 | MT527015 | MT528173 |
USA, Wisconsin, Jefferson Co. | Jefferson3-a | corn | Pp1 | MT527085 | MT528210 |
USA, Wisconsin, Juneau Co. | Juneau1-a | – | Pp1 | MT527037 | MT528175 |
USA, Wisconsin, Manitowoc Co. | Manitowoc-a | corn | Pp1 | MT527045 | MT528215 |
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Pp1 |
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USA, Wisconsin, Marathon Co. | Marathon3-a | – | Pp1 | MT527035 | MT528176 |
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Pp1 |
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USA, Wisconsin, Marquette Co. | Marquette2-a | corn | Pp1 | MT527090 | MT528216 |
USA, Wisconsin, Monroe Co. | Monroe-b | – | Pp1 | MT527019 | MT528178 |
USA, Wisconsin, Outagamie Co. | Outagamie1-a | soybean | Pp1 | MT527089 | MT528219 |
USA, Wisconsin, Outagamie Co. | Outagamie2-a | soybean | Pp1 | MT527070 | MT528220 |
USA, Wisconsin, Pierce Co. | Pierce-a | soybean | Pp1 | MT527042 | MT528222 |
USA, Wisconsin, Portage Co. | Portage3-a | soybean | Pp1 | MT527074 | MT528223 |
USA, Wisconsin, Portage Co. | Portage4-b | – | Pp1 | MT527021 | MT528182 |
USA, Wisconsin, Sauk Co. | Sauk-a | soybean | Pp1 | MT527048 | MT528227 |
USA, Wisconsin, Sawyer Co. | Sawyer-a | soybean | Pp1 | MT527064 | MT528230 |
USA, Wisconsin, Vernon Co. | Vernon-a | soybean | Pp1 | MT527050 | MT528232 |
USA, Wisconsin, Waushara Co. | Waushara-c | kidney beans | Pp1 | MT527017 | MT528188 |
USA, Nebraska, Otoe Co. | – | apple | Pp1 | MK877987b | – |
USA, Nebraska, Phelps Co. | – | corn | Pp1 | MK877993b | – |
Japan, Chiba | CA193a | wild carrot | Pp1 | KY817024a | EU130857c |
The Netherlands, Zoetermeer | T181a | grasses | Pp1 | KY817007a | |
Colombia, San Vicente | T666a | cape gooseberry | Pp1 | KY816982a | KY828351a |
Colombia, San Vicente | T677a | cape gooseberry | Pp1 | KY816981a | KY828350a |
Colombia, San Vicente | T678a | cape gooseberry | Pp1 | KY816980a | KY828349a |
Colombia, San Vicente | T679a | cape gooseberry | Pp1 | KY816979a | |
The Netherlands, Wemeldinge | T725a | sweet cherry | Pp1 | KY816949a | |
The Netherlands, Nagele | V3Fa | apple | Pp1 | KY916940a | KY828346a |
The Netherlands , Meijel | V4Ba | apple | Pp1 | KY816939a | KY828345a |
The Netherlands, Wemeldinge | V8Aa | apple | Pp1 | KY816936a | KY828342a |
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Japan, Aichi | CA85a | wild cabbage | Pp3 | KY817021a | EU130860c |
USA, Wisconsin, Clark Co. | Clark-b | corn | Pp4 | MT527033 | MT528161 |
USA, Wisconsin, Marquette Co. | Marquette3-a | soybean | Pp4 | MT527079 | MT528218 |
USA, Wisconsin, Outagamie Co. | Outagamie2-b | soybean | Pp4 | MT527071 | MT528221 |
USA, Alaska | – | peony | Pp4 | MK877984b | – |
The Netherlands, Stramproy | T716a | sweet cherry | Pp5 | KY816958a | |
USA, Wisconsin, Monroe Co. | Monroe-a | – | Pp6 | MT527018 | MT528177 |
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Pp6 |
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USA, Minnesota | – | corn | Pp6 | MK877982b | – |
The Netherlands, Stramproy | T715a | sweet cherry | Pp6 | KY816959a | |
USA, Wisconsin, Clark Co. | Clark-c | corn | Pp7 | MT527033 | MT528161 |
USA, Wisconsin, Dunn Co. | Dunn2-a | soybean | Pp7 | MT527054 | MT528202 |
USA, Wisconsin, Racine Co. | Racine-a | soybean | Pp7 | MT527062 | MT528224 |
The Netherlands, Meijel | V1Ba | apple | Pp7 | KY816942a | KY828348a |
USA, Wisconsin, Adams Co. | Adams-a | – | Pp8 | MT527029 | MT528157 |
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Pp8 |
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Rwanda, Nyakirbia | T172a | pear | Pp8 | KY817009a | KY828356a |
The Netherlands, Apeldorn | T293a | pear | Pp8 | KY816992a | KY828353a |
The Netherlands, Sambeek | T686a | apple | Pp8 | KY816976a | |
The Netherlands, Sambeek | T687a | apple | Pp8 | KY816975a | |
The Netherlands, Schimmert | T699a | plum | Pp8 | KY816972a | |
The Netherlands, Kloetinge | T723a | sweet cherry | Pp9 | KY816951a | |
USA, Wisconsin, Clark Co. | Clark-a | corn | Pp10 | MT527034 | MT528160 |
USA, Wisconsin, Columbia Co. | Columbia2-a | soybean | Pp10 | MT527078 | MT528191 |
USA, Wisconsin, Crawford Co. | Crawford-a | – | Pp10 | MT527056 | MT528195 |
USA, Wisconsin, Dodge Co. | Dodge1-a | soybean | Pp10 | MT527025 | MT528162 |
USA, Wisconsin, Dodge Co. | Dodge2-a | soybean | Pp10 | MT527041 | MT528163 |
USA, Wisconsin, Dodge Co. | Dodge2-b | soybean | Pp10 | MT527080 | MT528164 |
USA, Wisconsin, Eau Claire Co. | Eau Claire2-b | soybean | Pp10 | MT527058 | MT528206 |
USA, Wisconsin, Fond du Lac Co. | Fond du Lac-a | wheat | Pp10 | MT527084 | MT528165 |
USA, Wisconsin, Green Co. | Green-a | – | Pp10 | MT527032 | MT528169 |
USA, Wisconsin, Jackson Co. | Jackson1-b | soybean | Pp10 | MT527039 | MT528170 |
USA, Wisconsin, Jefferson Co. | Jefferson1-a | corn | Pp10 | MT527040 | MT528172 |
USA, Wisconsin, Jefferson Co. | Jefferson2-b | soybean | Pp10 | MT527026 | MT528174 |
USA, Wisconsin, Juneau Co. | Juneau2-a | soybean | Pp10 | MT527087 | MT528212 |
USA, Wisconsin, Monroe Co. | Monroe-c | – | Pp10 | MT527051 | MT528179 |
USA, Wisconsin, Portage Co. | Portage4-a | – | Pp10 | MT527020 | MT528183 |
USA, Wisconsin, Sauk Co. | Sauk-b | soybean | Pp10 | MT527047 | MT528228 |
USA, Wisconsin, Trempeleau Co. | Trempeleau-a | – | Pp10 | MT527028 | MT528185 |
USA, Wisconsin, Waushara Co. | Waushara-b | kidney beans | Pp10 | MT527036 | MT528187 |
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Pp10 |
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USA, Idaho | – | potato | Pp10 | MK877988b | – |
USA, Wisconsin, Waushara Co. | Waushara-a | kidney beans | Pp11 | MT527016 | MT528186 |
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USA, Wisconsin, Dunn Co. | Dunn1-a | soybean | Pp14 | MT527082 | MT528200 |
USA, California, Stanislaus Co. | CA91a | cowpea | Pp14 | KY817023a | EU130862c |
USA, Wisconsin, Dane Co. | Dane-b | soybean | Pp15 | MT527068 | MT528198 |
France, Britany | CA192a | apple | Pp15 | KY817022a | KY828242a |
Ethiopia, Shashamane | T225a | corn | Pp15 | KY817001a | KY828354a |
The Netherlands, Hei en Boeicop | T296a | pear | Pp15 | KY816990a | – |
The Netherlands, Vredepeel | T44a | wild carrot | Pp15 | KY817020a | KY828359a |
The Nethelands, Schimmert | T697a | plum | Pp15 | KY816974a | – |
The Netherlands, Kloetinge | T720a | sweet cherry | Pp15 | KY816954 | – |
The Netherlands, St. Oedenrode | T730a | grape vine | Pp15 | KY816944a | – |
The Netherlands, Baarlo | V3Aa | apple | Pp15 | KY816941a | KY828347a |
The Netherlands, Wemeldinge | T726a | sweet cherry | Pp16 | KY816948a | |
USA, Wisconsin, Adams Co. | Adams-b | – | Pp17 | MT527066 | MT528158 |
USA, Wisconsin, Sawyer Co. | Sawyer-b | soybean | Pp17 | MT527065 | MT528229 |
USA, Wisconsin, Columbia Co. | Columbia1-b | corn | Pp18 | MT527043 | MT528190 |
The Netherlands, Stramproy | T714a | sweet cherry | Pp18 | KY816960a | – |
The Netherlands, Stramproy | T713a | sweet cherry | Pp19 | KY816961a | – |
USA, Wisconsin, Dane Co. | Dane-a | soybean | Pp20 | MT527069 | MT528197 |
USA, Wisconsin, Marquette Co. | Marquette2-b | corn | Pp20 | MT527091 | MT528217 |
Rwanda, Nyakirbia | T143a | onion | Pp20 | KY817013a | KY828357a |
Rwanda, Nyakiriba | T144a | onion | Pp20 | KY817012a | |
Rwanda, Nyakiriba | T145a | onion | Pp20 | KY817011a | |
The Netherlands, Arkel | T184a | pear | Pp20 | KY817005a | |
Rwanda, Bushoki | T200a | potato | Pp20 | KY817004a | KY828355a |
The Netherlands, Hei en Boeicop | T295a | pear | Pp20 | KY816991a | KY828352a |
The Nethelands, Schimmert | T698a | plum | Pp20 | KY816973a | |
The Netherlands, Kloetinge | T721a | sweet cherry | Pp20 | KY816953a | |
The Netherlands, Wemeldinge | T724a | sweet cherry | Pp20 | KY816950 | |
The Nethelands, Zoetermeer | T132a | grasses | Pp21 | KY817015a | KY828358a |
The Netherlands, Kloetinge | T722a | sweet cherry | Pp21 | KY816952 |
The DNA sequences were edited with Finch TV (Geospiza Inc, Seattle, WA), and aligned with MUSCLE in MEGA 7 (Kumar et al., 2016) using default parameters with the gap opening penalty set at 15 and the gap extension penalty at 6.66. Alignments were further adjusted by inspection. A complemented sequence was constructed by overlapping sequence reads with forward and reverse primers for each specimen. Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI) was used to compare the complemented sequences to the GenBank sequence database. Sequences were deposited to GenBank and assigned accession numbers (Table 3).
Redundant COI sequences among multiple individuals sequenced from the same population were dropped, leaving 72 sequences for the haplotype analysis, 13 from cultured isolates, and 59 from field populations. In total, 48 published sequences from populations in other USA states (Ozbayrak et al., 2019) and the populations used in the Janssen et al.’s (2017) study were also included for a total of 120 sequences used in the analyses. The haplotype network was calculated and visualized using TCS network in the software PopART Version 1.7. Geographical origin of the sequences was used as trait information in the analysis.
For phylogenetic analysis, maximum-likelihood (ML) trees were constructed based on the aligned sequences of COI using the default program settings in MEGA 7 with 1000 bootstrap replications. The best DNA model of the nucleotide substitution tool option in MEGA 7 selected the Hasegawa-Kishino-Yano model with a gamma distribution and proportion of invariable sites (HKY + G + I) for COI. The COI sequences of all the Wisconsin populations and CA85, T716, T723, T726, T713, T132 as well as the other species in the Penetrans group and
All 13 Wisconsin isolates possessed the diagnostic characters of the species; three lip annuli, round spermatheca filled with sperm, four incisures in the lateral field, and a majority of specimens had a smooth tail tip (Tables 1 and 2). Stylet basal knobs were usually round but sometimes cupped anteriorly. The CV for the pooled data of all isolates was greatest for PUS (24.8%) followed by lip region height (17.4%) and
Mean and range of morphometrics and morphological features of
Portage 1 | Portage 2 | Marathon 1 | Marathon 2 | Chippewa 1 | Chippewa 2 | |
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|
675 (602-832) | 637 (544-748) | 595 (513-682) | 628 (507-702) | 594.8 (494-694) | 630 (564-704) |
V% | 80.3 (77.5-82.5) | 79.0 (75.0-82.0) | 78.8 (75.6-81.1) | 80.9 (78.9-86.6) | 80.2 (77.3-81.9) | 80.7 (78.5-82.3) |
|
26.4 (22.1-33.2) | 27.9 (24.8-32.0) | 25.9 (19.9-30.3) | 23.6 (20.2-25.7) | 26.1 (20.6-28.7) | 23.0 (18.4-28.2) |
|
5.62 (4.37-6.74) | 6.11 (5.11-7.53) | 5.69 (4.81-6.93) | 6.09 (5.14-7.20) | 5.18 (4.15-5.18) | 5.43 (4.89-6.00) |
|
18.7 (15.1-23.1) | 21.4 (17.3-26.7) | 19.2 (16.3-24.8) | 19.8 (14.4-31.2) | 18.8 (15.4-24.2) | 20.4 (16.4-22.8) |
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4.86 (3.89-5.63) | 5.33 (4.39-8.76) | 4.81 (4.17-5.78) | 5.15 (4.34-6.09) | 4.39 (3.64-5.31) | 4.68 (4.26-5.10) |
|
2.44 (1.93-3.06) | 2.39 (1.99-2.88) | 2.43 (1.67-2.94) | 2.09 (1.42-2.86) | 1.67 (1.24-2.90) | 1.97 (1.71-2.31) |
Maximum body width | 25.8 (21.3-29.4) | 22.9 (19.3-27.5) | 23.2 (19.0-29.3) | 26.7 (21.4-33.9) | 22.7 (19.1-27.0) | 27.7 (21.0-33.7) |
Esophageal length | 121 (102-142) | 103 (72-128) | 105 (91-125) | 104 (83-121) | 116 (102-133) | 116 (97-138) |
Head end to posterior gland | 140 (122-160) | 121 (84-145) | 124 (109-144) | 123 (101-141) | 134 (120-152) | 135 (115-157) |
Anterior end to excretory pore | 97 (80-108) | 92 (67-106) | 91 (77-101) | 90 (81-98) | 89 (80-99) | 93 (84-112) |
Lip region height | 2.47 (2.07-3.10) | 2.89 (2.4-4.07) | 2.41 (1.87-2.92) | 2.62 (2.09-3.15) | 2.44 (1.94-3.43) | 2.36 (2.04-2.77) |
Stylet | 14.8 (13.9-16.2) | 14.3 (13.4-15.4) | 15.5 (14.1-17.5) | 14.8 (13.3-16.3) | 16.8 (15.3-17.9) | 15.3 (13.9-16.4) |
DGO | 2.67 (2.13-3.51) | 2.93 (2.26-3.87) | 2.92 (2.08-3.54) | 2.46 (1.75-3.48) | 2.73 (2.15-3.71) | 2.61 (1.89-3.42) |
Body width at vulva | 22.2 (17.6-25.9) | 20.0 (17.0-22.5) | 20.2 (17.8-22.4) | 23.6 (17.9-28.4) | 20.2 (17.9-25.1) | 23.4 (18.9-27.9) |
PUS | 18.5 (15.1-22.4) | 14.4 (9.4-21.7) | 16.4 (12.1-21.8) | 16.5 (12.4-20.8) | 21.4 (14.1-35.0) | 18.3 (14.2-24.1) |
Body width at anus | 15.05 (13.5-17.0) | 14.6 (12.7-16.9) | 13.8 (12.2-15.3) | 15.7 (12.8-18.3) | 14.1 (12.5-16.5) | 15.9 (13.1-19.8) |
Tail length | 36.6 (30.8-52.2) | 34.8 (28.6-40.6) | 33.5 (20.3-44.0) | 35.6 (22.3-41.8) | 31.7 (22.9-42.0) | 31.2 (27.0-45.5) |
Vulva to anus distance | 95 (79-119) | 94 (74-114) | 91 (81-105) | 89 (63-108) | 87 (67-110) | 91(79-107) |
Lateral incisures | 4 with striation | 4 | 4 | 4 | 4 with striation | 4 |
Labial annules | 3 | 3 | 3 | 3 | 3 | 3 |
Tail tipb | SMO | SMO | SMO | SMO | SMO | SMO |
Tail shapec | SHM | SHM | SHM | SHM | SHM | SHM |
Spermatheca | Round | Round | Round | Round | Round | Round |
Spicule | 14.3 (11.7-17.5) | 14.8 (12.0-16.6) | 15.3 (12.4-17.2) | 14.7 (11.7-18.2) | 16.1 (13.2-19.6) | 15.6 (10.9-19.2) |
Gubernaculum | 4.09 (2.71-5.71) | 3.92 (2.94-5.67) | 4.86 (3.87-5.99) | 3.62 (3.02-4.22) | 4.49 (3.50-5.94) | 3.56 (2.43-4.95) |
Mean and range of morphometrics and morphological features of the
Calumet | Sheboygan | Wood | Marquette 1 | Buffalo | Grant | Iowa | |
---|---|---|---|---|---|---|---|
|
603 (497-844) | 624 (556-728) | 605 (541-717) | 603 (518-749) | 548 (465-608) | 588 (495-704) | 701 (631-818) |
V% | 79.9 (76.0-82.4) | 79.7 (77.6-81.7) | 79.2 (74.8-81.0) | 80.0 (77.3-81.8) | 80.9 (78.2-83.9) | 80.8 (78.3-85.2) | 79.7 (77.4-81.9) |
|
28.7 (24.2-38.5) | 28.1 (25.0-30.4) | 26.2 (20.9-30.3) | 24.6 (20.7-29.5) | 25.7 (22.7-29.2) | 28.2 (22.6-31.9) | 24.1 (21.1-27.7) |
|
5.39 (4.44-6.11) | 5.69 (4.77-7.10) | 5.33 (4.59-6.38) | 5.68 (4.42-7.22) | 5.20 (4.46-6.21) | 5.12 (4.34-6.11) | 6.44 (5.36-7.56) |
|
18.8 (13.9-23.7) | 20.9 (16.1-33.2) | 23.0 (19.2-31.0) | 20.1 (16.0-24.8) | 24.7 (19.4-30.5) | 20.9 (15.2-31.2) | 20.3 (17.7-29.1) |
|
4.62 (3.82-5.26) | 4.85 (4.20-5.88) | 4.58 (4.08-5.38) | 4.83 (3.87-5.90) | 4.43 (3.78-5.36) | 4.40 (3.81-5.23) | 5.47 (4.65-6.31) |
|
2.57 (2.02-3.29) | 2.20 (1.43-2.77) | 2.26 (1.79-3.16) | 2.01 (1.66-2.67) | 1.96 (1.62-2.40) | 2.27 (1.61-3.15) | 2.16 (1.82-2.62) |
Maximum body width | 21.0 (19.4-24.9) | 22.3 (20.4-28.6) | 23.2 (20.1-32.4) | 24.6 (19.2-30.6) | 21.3 (19.4-23.4) | 20.9 (18.0-27.3) | 29.2 (24.9-34.8) |
Esophageal length | 112 (92-138) | 110 (87-134) | 114 (109-154) | 107 (88-137) | 106 (89-116) | 116 (95-131) | 109 (86-128) |
Head end to posterior gland | 131 (108-160) | 129 (106-152) | 132 (109-154) | 126 (107-155) | 124 (109-134) | 134 (114-149) | 129 (104-148) |
Anterior end to excretory pore | 94 (85-117) | 92 (79-102) | 90 (81-101) | 86 (75-110) | 87 (77-95) | 88 (78-100) | 95 (84-110) |
Lip region height | 3.17 (2.24-3.87) | 3.02 (2.39-3.88) | 3.02 (2.17-3.89) | 2.37 (1.87-2.99) | 3.04 (2.42-3.51) | 3.29 (2.56-3.96) | 2.43 (1.69-3.08) |
Stylet | 14.8 (13.8-17.7) | 15.3 (14.3-16.6) | 14.9 (13.6-16.6) | 15.8 (14.6-17.7) | 14.8 (14.0-15.9) | 14.9 (13.9-15.8) | 15.9 (14.8-17.4) |
DGO | 3.09 (2.39-4.45) | 2.98 (2.21-3.88) | 2.79 (2.18-3.46) | 2.63 (1.77-3.32) | 2.92 (2.30-3.59) | 2.85 (2.11-3.57) | 2.49 (1.88-3.06) |
Body width at vulva | 19.6 (17.4-22.5) | 19.7 (17.5-24.3) | 20.6 (16.7-28.9) | 21.4 (16.7-25.0) | 18.9 (16.4-21.6) | 18.5 (15.8-22.9) | 25.6 (23.0-29.2) |
PUS | 21.9 (11.8-35.2) | 15.5 (9.2-21.0) | 16.7 (11.7-16.4) | 22.0 (16.3-34.9) | 16.4 (11.1-23.3) | 15.3 (11.3-22.5) | 19.8 (13.9-28.5) |
Body width at anus | 12.6 (11.1-14.2) | 14.0 (12.1-17.5) | 14.3 (11.7-16.4) | 15.1 (11.3-17.5) | 13.5 (11.3-15.3) | 12.8 (9.5-16.0) | 16.2 (11.8-17.8) |
Tail length | 32.4 (26.1-44.7) | 30.8 (19.4-42.6) | 32.1 (27.5-42.7) | 30.3 (23.1-40.3) | 26.4 (21.5-31.9) | 28.9 (20.2-37.9) | 34.9 (25.5-43.2) |
Vulva to anus distance | 91 (62-124) | 96.51 (86-121) | 94 (75-119) | 89 (73-109) | 79 (65-105) | 84 (65-113) | 107 (90-131) |
Lateral incisures | 4 | 4 with striation | 4 with striation | 4 | 4 with striation | 4 with striation | 4 with striation |
Labial annules | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Tail tipb | SMO | SMO, ANN | SMO | SMO | SMO, CFT | SMO, ANN | SMO |
Tail shapec | SHM, TRC, CLA | SHM, SBD, CLA | SHM | SHM | SHM, TRC, CLA | SHM, TRC | SHM |
Spermatheca | Round | Round | Round | Round | Round | Round | Round |
Spicule | 16.2 (12.3-20.1) | 15.4 (12.1-20.0) | 14.4 (11.7-17.9) | 14.9 (13.1-17.3) | 15.3 (11.0-18.5) | 15.4 (12.8-18.7) | 15.2 (13.0-17.5) |
Gubernaculum | 3.87 (2.69-4.88) | 3.98 (2.74-5.18) | 3.99 (2.96-5.59) | 3.75 (3.27-4.09) | 3.56 (2.58-5.73) | 3.60 (2.74-4.28) | 4.17 (3.65-4.47) |
Mean morphometric values for the isolates were all within the range reported for
HAC separated the 13
The clusters determined by HAC were confirmed by the CDA analysis (Wilks’
The 402 bp of nucleotide sequence was read without gaps by the COI primer. The pairwise sequence identity between the 13 Wisconsin isolates ranged from 94.2% (Portage1 and Marathon2) to 100% similarity (Table 4). There were 30 single nucleotide polymorphisms (SNPs) on COI sequences among the 13
Pairwise distance (percent similarity) for CO1 mtDNA between 13
Population | Portage 1 | Portage 2 | Marathon 1 | Marathon 2 | Chippewa 1 | Chippewa 2 | Calumet | Sheboygan | Wood | Marquette | Buffalo | Grant | Iowa |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
|||||||||||||
Portage 1 | |||||||||||||
Portage 2 | 99.5 | ||||||||||||
Marathon 1 | 94.5 | 95.0 | |||||||||||
Marathon 2 | 94.2 | 94.8 | 99.8 | ||||||||||
Chippewa 1 | 95.0 | 95.0 | 95.5 | 95.5 | |||||||||
Chippewa 2 | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | ||||||||
Calumet | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | 100.0 | |||||||
Sheboygan | 95.0 | 95.0 | 95.5 | 95.5 | 99.5 | 95.5 | 95.5 | ||||||
Wood | 95.5 | 95.5 | 96.5 | 96.5 | 98.5 | 96.5 | 96.5 | 98.5 | |||||
Marquette | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | 100.0 | 100.0 | 95.5 | 96.5 | ||||
Buffalo | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | 100.0 | 100.0 | 95.5 | 96.5 | 100.0 | |||
Grant | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | 100.0 | 100.0 | 95.5 | 96.5 | 100.0 | 100.0 | ||
Iowa | 94.5 | 95.0 | 100.0 | 100.0 | 95.5 | 100.0 | 100.0 | 95.5 | 97.6 | 100.0 | 100.0 | 99.9 |
A total of 120 sequences, 72 from Wisconsin, revealed 21 haplotypes for
The ML tree based on COI validated that the 72 Wisconsin populations were conspecific with
Our study demonstrated considerable intraspecific variation in morphological and molecular characters for
Three morphological features used to distinguish
Data for 49 isolates and populations of
We detected enough pattern in morphological variation to assign our 13 Wisconsin isolates of
Our haplotype analysis revealed up to 5.8% divergence in COI DNA among populations confirmed to be
The widespread incidence and high degree of intraspecific variability of