Journal of Nematology 49(1):42–66. 2017. Ó The Society of Nematologists 2017. Species Delimitation and Description of Mesocriconema nebraskense n. sp. (Nematoda: Criconematidae), a Morphologically Cryptic, Parthenogenetic Species from North American Grasslands 1 1 1 1 1 2 MAGDALENA OLSON, TIMOTHY HARRIS, REBECCA HIGGINS, PETER MULLIN, KIRSTEN POWERS, SEAN OLSON, 1 AND THOMAS O. POWERS Abstract: Nematode surveys of North American grasslands conducted from 2010 to 2015 frequently recovered a species of crico- nematid nematode morphologically resembling Mesocriconema curvatum. These specimens were recovered from remnant native prairies in the central tallgrass ecoregion of North America, and not from surrounding agroecosystems. Historical records indicate that M. curvatum is a cosmopolitan species feeding on a wide range of agronomic and native plants. DNA barcoding indicates North American grasslands contain at least 10 phylogenetically distinct lineages of Mesocriconema that resemble, but are not, M. curvatum. Analysis of the two most common lineages reveals two distinctly different population structures. The variation in population structure suggests unique evolutionary histories associated with their diversification. These two major lineages share a sympatric distribution and their slight morphological differences contrast with a high level of genetic separation. Based on their genetic divergence, fixed diagnostic nucleotides, population structure, species delimitation metrics, and a sympatric distribution, we believe that one of these distinct lineages warrants formal nomenclatural recognition. Herein, we provide formal recognition for Mesocriconema nebraskense n. sp. and discuss its relationship to other Mesocriconema lineages discovered in native North American grasslands. Key words: biogeography, cryptic species, nematode distribution, network analysis, plant-parasitic nematodes, tallgrass prairies, taxonomy, phylogeny. During the course of soil surveys conducted to assess In a previous taxonomic analysis of species of nematode biodiversity in native grasslands of North Mesocriconema Andrassy, 1965, from North America, America, a single criconematid morphospecies was molecular and morphological analyses were used to found to be widespread and abundant. Using di- differentiate formally described members of the genus chotomous and synoptic keys (Hoffmann, 1974; Brzeski, as well as lineages lacking a formal description (Powers 2002;Geraert,2010),thismorphospecieswasidenti- et al., 2014). With few exceptions, the specimens col- fied as Mesocriconema curvatum (Raski, 1952) Loof and lected from native grasslands were determined to be De Grisse, 1989. This plant-parasitic taxon has pre- phylogenetically distinct from specimens found in viously been recognized as a component of the nem- agroecosystems, with the agricultural group of speci- atode community in central tallgrass prairies (Norton mens judged to most closely conform to the original and Ponchillia, 1968; Schmitt and Norton, 1972; description of M. curvatum (Powers et al., 2014). The Norton, 1978; Powers et al., 2010). Historically, it was question remained, however, as to the taxonomic status recorded as associated with a broad range of hosts of the grassland specimens. In this study, we use a spe- from monocots to dicots, annual herbaceous plants to cies concept that recognizes species as separately conifer and hardwood trees, and in agroecosystems as evolving metapopulation lineages (De Queiroz, 2007) well as native plant communities. Geographically, M. and apply species delimitation methods that in- curvatum has been reported from six continents in corporate character state and genetic distance analyses both tropical and temperate climates. Judging from (DeSalle et al., 2005; Pons et al., 2006; Wiens, 2007; these reports, either this species is an extreme gener- Puillandre et al., 2012). The lineages discovered during alist, adapted to a multitude of environments and these analyses appear to represent members of a group plant hosts, or M. curvatum as recorded in the litera- of morphologically similar, parthenogenetic species ture is actually a composite of morphologically similar endemic to North American grasslands. Two of the but genetically distinct lineages, possibly cryptic spe- lineages were geographically widespread across the cies (Fontaneto et al., 2008; Nadler and De Leon, central grasslands and were usually abundant when 2011; Ristau et al., 2013; Palomares-Rius et al., 2014). found in remnant prairies. In an earlier study, these two lineages were referred to as haplotype group (hg) 18 Received for publication January 25, 2017. and hg 24 (Powers et al., 2014). In the current study, the 1Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, number of grassland collection sites with positive finds NE 68583-0722. 2Department of Statistics, University of Nebraska-Lincoln, Lincoln, NE has been expanded to 35, and the number of speci- 68583-0963. mens studied of hg 18 and hg 24 has been increased to We acknowledge funding from the National Science Foundation DEB- 1145440, Big Thicket National Preserve study no. BITH-000103 including The 98 and 100 specimens, respectively. Negative collection Big Thicket Association ‘‘Thicket of Diversity,’’ Great Smoky Mountains Na- sites helped define the geographic limits of these tional Park no. GRSM-01076, and Discover Life in America. We would like to thank Lisa Sutton of University of Nebraska-Lincoln for processing soil samples groups. A statistical parsimony network analysis pro- and all the organizations and individuals that aided in our sampling efforts in vided information about the population structure and the unique grasslands of North America. E-mail: [email protected]. haplotype relationships for both lineages. In this study, This paper was edited by Zafar A. Handoo. hg 18 is deemed deserving of a unique, formal species 42 Mesocriconema nebraskense n. sp.: Olson et al. 43 description based on phylogenetic clade support, re- 5) A 36-specimen dataset of internal transcribed spacer 1 ciprocal monophyly, genetic distance metrics, species (ITS1) DNA representing members of hg 18 to 27 as delimitation analyses, and discriminant function ana- determined by COI DNA analysis (Fig. 4). lyses (DFA) that provide evidence of a degree of mor- 6) A 161-specimen dataset of all female specimens in hg phological distinction. Haplotype group 24, while 18, 19, 24, and 25. This dataset was used in morpho- exhibiting some of the same characteristics suggesting logical analyses including the DFA (Fig. 5). lineage distinction, is not as readily delineated and re- Microscopic analysis and documentation: Nematodes quires additional study before formal nomenclatural isolated from soil were first examined using a dissecting recognition. stereomicroscope, and specimens recognized as be- longing to Criconematina were handpicked for com- MATERIALS AND METHODS pound light microscope analysis. Individual nematodes were mounted on glass slides, measured, and digitally Nematode collection: Nematodes were obtained from photographed using a Leica DMLB light microscope soil samples collected using a standardized collection with differential interference contrast optics and procedure to facilitate consistent and optimal recovery a Leica DC300 video camera. A set of 16 standard between sites (Neher et al., 1995). Soil cores were taken measurements were taken on individual specimens randomly within a 40 3 40 m grid using Oakfield tubes allowing for the combined retention of morphological with a 3-cm diameter at a maximum depth of 30 cm. characters and molecular data. Both adult females and Samples consisted of approximately 1,000 cc of bulked juveniles were subjected to morphological and molec- soil from a single grid and were stored at 88C until ular analyses, but only adult females were included in nematodes from a 200-ml subsample were isolated us- morphological comparisons for species delimitation. ing a modified flotation-sieving and centrifugation No male Mesocriconema specimens were encountered in method ( Jenkins, 1964). this study. Nematodes were prepared for scanning Samples included in this study were collected from electron microscopy (SEM) by fixation in 4% formalin 35 sites recorded in Table 1 and Fig. 1. The collection followed by dehydration in a graded series of alcohol to sites represent 13 ecoregions as designated by the 100% ethyl alcohol, critical point drying, mounting on World Wildlife Fund (Olson et al., 2001). Twenty-eight SEM specimen stubs, and coating with gold. Images of these sites were native prairie remnants managed to were obtained on a Hitachi S-3000N scanning electron maintain native plant diversity. The most intensively microscope. Microscopic images of all specimens were sampled ecoregion was the central tallgrass prairie with stored in an in-house database in the Department of 14 sites. Other grassland ecoregions were the Flint Hills Plant Pathology at University of Nebraska-Lincoln, and tall grasslands, Texas Blackland prairies, and central images of specimens with DNA barcodes have been and southern mixed grasslands. Two of the study sites, deposited in the Barcode of Life Database (http://v4. Nine-Mile Prairie and Spring Creek Prairie of the cen- boldsystems.org/). tral tallgrass ecoregion, were sampled intensively over Polymerase chain reaction amplification: Following doc- multiple years and seasons during the survey. Several umentation, nematodes were removed from slides other sampling sites were