Nematology 21 (2019) 923-935 brill.com/nemy Morphological and molecular characterisation of Hoplolaimus smokyensis n. sp. (Nematoda: Hoplolaimidae), a lance nematode from Great Smoky Mountains National Park, USA ∗ Xinyuan MA 1, , Robert T. ROBBINS 2,ErnestC.BERNARD 3, Claudia M. HOLGUIN 4 and Paula AGUDELO 1 1 Department of Plant and Environmental Sciences, Clemson University, Clemson, SC 29634, USA 2 Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA 3 Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, USA 4 Corpoica es ahora Agrosavia, Rionegro, 054040, Colombia Received: 25 November 2018; revised: 11 April 2019 Accepted for publication: 11 April 2019; available online: 20 May 2019 Summary – Hoplolaimus smokyensis n. sp. is a new species of lance nematode collected in Great Smoky Mountains National Park, USA. Females of H. smokyensis n. sp. have a labial region characterised by six, occasionally five, annules. The basal lip annule is subdivided by about 24 longitudinal striae. The stylet averages 47 μm long with robust, tulip-shaped stylet knobs bearing anterior projections. The hemizonid is ca 4 μm anterior to the excretory pore. The lateral field is incompletely areolated and has four continuous incisures from the metacorpus region to the tail region. There are three pharyngeal gland nuclei. Vulval epiptygma are absent. The scutellate phasmids are located one anterior and one posterior to the vulva. The male is shorter than the female and the head region is higher and more rounded than that of the female. The bursa extends to the tail tip and the gubernaculum is large and protrusible and has titillae and a capitulum. Morphologically, H. smokyensis n. sp. is most similar to H. galeatus and H. stephanus, but can be distinguished by differences such as the number of annules and longitudinal striae on the lip region and morphometric values. Hoplolaimus smokyensis n. sp. is also genetically distinct from other species according to comparisons of ribosomal and mitochondrial DNA sequences. Phylogenetic analyses based on ribosomal and mitochondrial gene sequences suggest that H. smokyensis n. sp. is a lineage distinct from related Hoplolaimus species. Keywords – Acer sp., Eastern hemlock, Halesia carolina, lance nematode, maple, morphology, morphometrics, new species, phylogeny, plant-parasitic nematode, silverbell, taxonomy, Tsuga canadensis. During a survey of fauna of Great Smoky Mountains idase subunit I (COI) sequences supported this result, National Park, along the Tennessee-North Carolina bor- ensuring that molecular information of this undescribed der in the south-eastern USA, a Hoplolaimus von Daday, lance nematode had not yet been reported. Females, males 1905 species was isolated from a mixed forest sample of and juveniles were examined for morphological character- maple (Acer sp.), Eastern hemlock (Tsuga canadensis (L.) istics, morphometrics and phylogenetic relationships. We Carrière) and silverbell (Halesia carolina L., 1759). This name the species Hoplolaimus smokyensis n. sp. in refer- species was initially distinguished from other reported ence to the locality where it was first found. lance nematodes based on a BLAST (Basic Local Align- Hoplolaimus smokyensis n. sp. is closest to H. galea- ment Search Tool) search result of internal transcribed tus (Cobb, 1913) Thorne, 1935 and H. stephanus Sher, spacer 1 (ITS1) sequences at the NCBI (National Center 1963, but can be distinguished by minor morphological for Biotechnology Information). Its position was isolated differences, such as the numbers of annules and longitu- on a phylogenetic tree composed of all available unique dinal striae in the lip region, morphometric values, and ITS1 sequences of Hoplolaimus species from GenBank. absence of epiptygma. In this study, we applied two ge- A phylogenetic analysis of mitochondrial cytochrome ox- netic markers, mitochondrial (COI) and ribosomal DNA ∗ Corresponding author, e-mail: [email protected] © The authors, 2019 DOI 10.1163/15685411-00003266 This is an open access article distributed under the terms of the CC-BY-NC 4.0 License. Downloaded from Brill.com09/29/2021 05:26:35PM via free access X. Ma et al. (ITS1), to obtain an analysis of higher resolution on tax- was set at 95°C for 3 min, followed by 33 cycles of 95°C onomic relationships, which support the delimitation of for 45 s, 50°C for 1 min 15 s, 72°C for 2 min and final H. smokyensis n. sp. DNA sequences were aligned with extension at 72°C for 10 min (Holguin et al., 2015a). The BLAST in GenBank. The highest similarity (89% on both amplified products were loaded onto a 1.5% agarose gel COI and Internal Transcribed Spacer (ITS1) markers) was and visualised with GelRed™ (Biotium). PCR products with H. magnistylus Robbins, 1982 sequences. Alignment for both regions were purified using magnetic beads and analyses using MUSCLE (Edgar, 2004) showed unique sequenced in both directions with the ABI 3730 capil- molecular characteristics possessed by H. smokyensis n. lary sequencer (Applied Biosystems) in the DNA Labo- sp. Phylogenetic trees generated with MrBayes (Huelsen- ratory (School of Life Sciences) at Arizona State Univer- beck & Ronquist, 2001) indicated H. smokyensis n. sp. as sity. Sequencing results were edited and assembled in Se- a distinct clade. quencher 5.1 (Genes Code). Consensus DNA sequences were searched in GenBank using BLAST, then aligned with MUSCLE (Edgar, 2004). Materials and methods A best-fit model of nucleotide substitution was selected using the GTR + I + G model with the Akaike Infor- NEMATODE ISOLATION mation Criterion (AIC) among 56 different models using ModelTest v 3.7 (Posada & Crandall, 1998). Bayesian in- Female, male and juvenile specimens were sampled ference was implemented for each gene separately using from Great Smoky Mountains National Park in July 2006. the MrBayes 3.1.2 program (Huelsenbeck & Ronquist, Nematodes were extracted from soil with a combina- 2001) running the chain for 1 × 107 generations with tion of sieving-decanting and sucrose centrifugal-flotation the Markov chain Monte Carlo (MCMC) method, a sam- (Jenkins, 1964). Specimens were killed and fixed, pro- ple frequency of 100 and burn-in value of 2500. We esti- cessed to glycerin and permanently mounted on slides as mated the posterior probabilities of the phylogenetic trees described by Ye & Robbins (2003). (Larget & Simon, 1999) using the 50% majority rule. The phylogenetic trees were viewed on phylo.io (Robinson et MORPHOLOGICAL OBSERVATION AND MICROGRAPHY al., 2016) and iTOL (Interactive tree of life v3) (Letunic & Bork, 2016). Permanently mounted specimens were examined and imaged. Some live specimens were imaged as well. Most images were produced with a 14-megapixel Q-camera Results on an Olympus BX-63 differential interference-contrast microscope system. Measurements were made on a Nikon Hoplolaimus smokyensis* n. sp. Optiphot II compound microscope with the aid of a (Figs 1-4) drawing tube or ocular micrometer. Mean annule width was calculated by measuring a series of ten annules at MEASUREMENTS mid-body. See Table 1. MOLECULAR PROFILES AND PHYLOGENY DESCRIPTION DNA was extracted from individual nematodes us- ing Sigma Extract-N-Amp kit (XNAT2) (Sigma). The Female manufacturer’s protocol was modified by reducing vol- Female body generally cylindrical, vermiform, tapering umes to one eighth of the recommended amount (Ma et slightly at each end. Head set off, with massive cephalic al., 2011). The ITS1 was amplified with primers Hoc-1f framework, usually bearing six lip annules and the oral (5 -AACCTGCTGCTGGATCATTA-3 ) and LSUD3r (5 - disc, but sometimes with five annules (six individuals out TATGCTTAAGTTCAGCGGGT-3 ) following Bae et al. of 30 specimens), basal annule tessellated, divided into ca (2008, 2009); and a portion of cytochrome c oxidase 20 equal blocks (Fig. 1A, B). Oral disc surrounded by a subunit I (COI) sequence was amplified with primers lip annule separated into six sections: two subdorsal, two JB3 (5-TTTTTTGGGCATCCTGAGGTTTAT-3) and JB5 (5 -AGCACCTAAACTTAAAACATAATGAAA-3 ) * The specific epithet refers to the locality where the species was (Derycke et al., 2005). For COI, the initial denaturation first found. 924 Nematology Downloaded from Brill.com09/29/2021 05:26:35PM via free access Hoplolaimus smokyensis n. sp. from the USA Table 1. Morphometrics of Hoplolaimus smokyensis n. sp. All measurements are in μm and in the form: mean ± s.d. (range). Character Female Male Holotype Paratypes Paratypes n – 30 17 L 1409 1335 ± 174 (997-1870) 1096 ± 87 (972-1261) a 34.7 30.1 ± 3.3 (23.5-36.9) 30.5 ± 2.3 (26.1-34.5) b9.99.8± 1.6 (7.1-14.9) 8.1 ± 0.7 (7.1-9.3) b’ 7.3 6.8 ± 0.9 (4.9-8.9) 5.8 ± 0.5 (5.2-6.7) c 115.7 114 ± 15.8 (91.3-154) 36.8 ± 3.7 (28.9-42.3) c’ 0.7 0.7 ± 0.1 (0.5-1.0) 1.5 ± 0.2 (1.3-1.9) V 54.4 56.5 ± 2.6 (49.7-62.1) – Max. body diam. 41 44.4 ± 4.1 (34.5-52.8) 36.1 ± 3.4 (30.5-42.6) Lip annulus 6 5.8 ± 0.4 (5-6) 5.8 ± 0.4 (5-6) Lip height 8 8.1 ± 0.3 (7.1-9.1) 8.1 ± 0.2 (7.1-8.7) Lip diam. 16 15.8 ± 1.0 (14.2-18.3) 13.9 ± 0.6 (12.2-14.2) Conus 24 25.8 ± 1.6 (22.3-28.4) 23.8 ± 1.5 (22.3-26.4) Stylet length 47 47.9 ± 1.9 (44.7-50.8) 43.4 ± 2.1 (40.6-48.7) Pharynx 142 138 ± 16.7 (79-183) 136 ± 6.4 (126-152) Anterior end to pharyngeal gland tip 193 199 ± 11.2 (168-223) 189 ± 10.7 (173-207) Tail length 26 24.9 ± 4.0 (20.3-34.5) 30.0 ± 3.4 (24.4-36.5) Anal body diam.