High genetic diversity and geographic subdivision of three lance nematode species (Hoplolaimus spp.) in the United States Claudia M. Holguin1, Juan A. Baeza2, John D. Mueller3 & Paula Agudelo1 1Department of Agricultural and Environmental Sciences, Clemson University, Clemson, South Carolina 29634 2Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634 3Edisto Research and Education Center, Clemson University, Blackville, South Carolina 29817 Keywords Abstract Barcoding, genetic variability, Hoplolaimus spp., lance nematodes, phylogeny. Lance nematodes (Hoplolaimus spp.) feed on the roots of a wide range of plants, some of which are agronomic crops. Morphometric values of amphimic- Correspondence tic lance nematode species overlap considerably, and useful morphological char- Paula Agudelo, Department of Agricultural acters for their discrimination require high magnification and significant and Environmental Sciences, Clemson diagnostic time. Given their morphological similarity, these Hoplolaimus species University, Clemson, SC 29634. provide an interesting model to investigate hidden diversity in crop agroecosys- Tel: 864 656-5741 tems. In this scenario, H. galeatus may have been over-reported and the related Fax:864-656-4960 E-mail: [email protected] species that are morphologically similar could be more widespread in the Uni- ted States that has been recognized thus far. The main objectives of this study Funding Information were to delimit Hoplolaimus galeatus and morphologically similar species using Research was supported by Cotton morphology, phylogeny, and a barcoding approach, and to estimate the genetic Incorporated, Project No. 14-506SC and the diversity and population structure of the species found. Molecular analyses were United States Department of Agriculture performed using sequences of the cytochrome c oxidase subunit 1 (Cox1) and USDA-NIFA, Contract Number: 2010-51101- the internal transcribed spacer (ITS1) on 23 populations. Four morphospecies 21785. were identified: H. galeatus, H. magnistylus, H. concaudajuvencus, and H. steph- Received: 22 December 2014; Revised: 5 anus, along with a currently undescribed species. Pronounced genetic structure May 2015; Accepted: 18 May 2015 correlated with geographic origin was found for all species, except for H. galea- tus. Hoplolaimus galeatus also exhibited low genetic diversity and the shortest Ecology and Evolution2015; 5(14): genetic distances among populations. In contrast, H. stephanus, the species with – 2929 2944 the fewest reports from agricultural soils, was the most common and diverse species found. Results of this project may lead to better delimitation of lance doi: 10.1002/ece3.1568 nematode species in the United States by contributing to the understanding the diversity within this group. Introduction damage to agronomic crops, including cotton (Gossypium hirsutum L.), corn (Zea mays L.) and soybean (Glycine Lance nematodes (Hoplolaimus spp.) are relatively long max L.) (Fassuliotis, 1974; Nyczepir and Lewis, 1979; and robust vermiform nematodes with a distinct cephalic Robbins et al., 1987, 1989; Henn and Dunn, 1989; Noe, region and massive well-developed stylets (Sher 1963; 1993). Ma et al. (2011) suggest that a fourth species, H. Fortuner 1991) that feed on the roots of a diversity of stephanus, may be of economic importance on grasses. monocotyledonous and dicotyledonous plants. Hoplolai- Of the twenty-nine species described in the genus mus species reported in the southeastern United States (Handoo and Golden 1992), H. galeatus is the most com- include H. columbus Sher, 1963; H. galeatus (Cobb, 1913) monly reported in the United States (Lewis and Fassulio- Thorne, 1935, H. magnistylus Robbins, 1982; H. stephanus tis 1982). Morphologically, Hoplolaimus galeatus belongs Sher, 1963; H. seinhorsti Luc, 1958, and H. tylenchiformis to a group of species that Fortuner (1991) called “ances- von Daday, 1905 (Lewis and Fassuliotis, 1982). Hoplolai- tral”, characterized by three esophageal gland nuclei, four mus columbus, H. galeatus, and H. magnistylus are consid- incisures in the lateral field, excretory pore posterior to ered to be economically important and can cause serious the hemizonid, and the presence of abundant males. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 2929 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. lus, H. concaudajuvencus, olaimus Siddiqi (2000) classifies this group as the subgenus Genetic Diversity of 2930 (Table 2). Nematodesand were extracted lawns fromcollected of soil in by 2011 different sugar Nematode regions populations were in obtained theDNA from isolation soil United samples Nematode States sampling, identification, and Materials and Methods species, which constitutebody the length subgenera (Sherspicules, 1963) (Table less 1, areolation Fig.the of 1). lip The region the “derived” comparedthe lateral to 24 field, 32 andmus stephanus shorter second-stage juvenile with conicallyvencus pointed tail. longer stylet (Tablevencus 1, Fig. 1). other species. morphological similarity,nification and these ical significant characters diagnostic for their1991; time. discrimination Handoo require and Given high Goldenconsiderably their 1992), mag- (Table and 1, useful morpholog- Fig. 1) (Sherabsent (Fortuner 1963; 1991). Vovlasduction et is al. mostlypore is parthenogenetic, anterior to withfewer the than hemizonid, males and four the incisures rareH. mode in of or the seinhorsti repro- lateralEthiolaimus field, the excretory scenario, galeatus considering the wideto host investigate range hiddenwithin and the diversity distribution “ancestral” of in clade provide crop an agroecosystems, interesting model States. delimitation of speciesthis of lance group nematodesthis in and the study, United contribute wesity expect to and to population the understandcoding structure the of approach elucidation diversity the and of within similar species (2) species found. the to using With (1) estimate morphology, to phylogeny, the delimit and geneticognized a diver- thus bar- far.more The widespread main in objectives(Siddiqi the of United 2000) this States that studythe that are were has morphologically related been similar rec- could species be within the subgenus Morphometric values of the “ancestral” species overlap – has more definitely tulip-shaped stylet knobs and a . This “ancestral” subgenus includes differ from 28 longitudinal striations on the basal annule of in the United States (Fortuner 1991). In this H. galeatus and include species such as , possess six esophageal gland nuclei and Hoplolaimus magnistylus Hoplolaimus galeatus can be distinguished from – Hoplolaimus 2013 in agricultural fields, golf courses, H. galeatus may have been over-reported and and spp. H. stephanus – 36 in Hoplolaimus concaudaju- by the possession of a Hoplolaimus and morphologically H. galeatus and Basirolaimus H. columbus H. concaudaju- , among a few H H. magnisty- . Hoplolaimus galeatus Hoplolai- , shorter species Hopl- and and H. by ª 2015 The Authors. Table 1. Published data for morphological characters and morphometrics of Hoplolaimus concaudajuvencus, Hoplolaimus galeatus, Hoplolaimus magnistylus, and Hoplolaimus stephanus. Longitudinal Ecology and Evolution striae on Species, (source), and Length Lateral Esophageal Stylet Labial basal lip Position of excretory Phasmids in Tail Spicule (number of specimens) (mm) incisures gland nuclei length (lm) annules annule pore relation to vulva annules length (lm) H. concaudajuvencus (Golden 1.12–2.04 4 3 50.4–56.6 5–6 36 Posterior to hemizonid 1 anterior 7–14 45.0–56.0 and Minton 1970) (n = 20 ♀,20♂) 1 posterior published by John Wiley & Sons Ltd. H. galeatus (Sher 1963) (n = 20 ♀,10♂) 1.24–1.94 4 3 43.0–52.0 5 32–36 Posterior to hemizonid 1 anterior 10–16 40.0–52.0 1 posterior H. magnistylus (Robbins 1982) (n = 30 ♀,20♂) 1.36–1.97 4 3 52.0–61.0 4–622–340 Posterior to hemizonid 1 anterior 12–17 52.0–58.0 1 posterior H. stephanus (Sher 1963) (n = 20 ♀,10♂) 1.01–1.45 4 3 43.0–50.0 4 24–28 Posterior to hemizonid 1 anterior 12 30.0–38.0 C. M. Holguin 1 posterior et al. C. M. Holguin et al. Genetic Diversity of Hoplolaimus spp. (A) (B) (C) (D) Figure 1. Head regions of the four Hoplolaimus species collected in this study. (A) H. magnistylus, (B) H. galeatus, (C) H. concaudajuvencus, and (D) H. stephanus. centrifugal flotation (Jenkins 1964). Lance nematode MgCl2 (Sigma, St. Louis, MO) (20 mmol/L Tris–HCl pH specimens from each soil sample were morphologically 8.3, 100 mmol/L KCl, 3 mmol/L MgCl2, 0.002% gelatin, identified using the key by Handoo and Golden (1992) 0.4 mmol/L dNTP mixture (dATP, dCTP, dGTP, and and the original descriptions of the species (Sher 1963; dTTP), and 0.06 units of Taq DNA polymerase/mL), Golden and Minton 1970; Robbins 1982). DNA from 0.5 lL of each primer (20 lmol/L), and 1 lL of DNA individual nematodes was extracted using the Sigma template. Thermal cycling conditions for the ITS marker Extract-N-Amp kit (XNAT2) (Sigma, St. Louis, MO) as included: initial denaturation at 95°C for 3 min, 33 cycles reported by Ma et al. (2011), and DNA was stored at of 95°C for 45 sec, 59°C for 1 min 15 sec, 72°C for À20°C until use. 2 min, and final extension at 72°C for 10 min. For the COI portion, the initial denaturation was set at 95°C for 3 min, followed by 33 cycles of 95°C for 45 sec, 50°C for PCR amplification and sequencing 1 min 15 sec, 72°C for 2 min, and final extension at The ITS region was amplified using forward primer Hoc- 72°C for 10 min. The amplified products were loaded 1f (50-AACCTGCTGCTGGATCATTA-30) and reverse onto a 1.5% agarose gel and visualized using GelRedTM primers: Hoc-2r (50-CCGAGTGATCCACCGATAA-30) (Biotium, San Francisco, California, United States.). PCR (Bae et al. 2008) and LSUD-3r (50 TATGCTTAAGTTCA products were purified using magnetic beads and GCGGGT-30) (Bae et al. 2009), which amplify the ITS1 sequenced in both directions with the ABI 3730 capillary and the entire ITS region, respectively.