A BOLD Reference Library for Plant-Parasitic Nematodes in the Superfamily Criconematoidea
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Genome Nematode biodiversity assessments need vouchered databases: A BOLD reference library for plant-parasitic nematodes in the superfamily Criconematoidea Journal: Genome Manuscript ID gen-2019-0196.R2 Manuscript Type: Article Date Submitted by the 31-Mar-2020 Author: Complete List of Authors: Powers, Thomas; University of Nebraska-Lincoln, Plant Pathology Harris, Timothy ; University of Nebraska-Lincoln, Plant Pathology Higgins, Rebecca; University of Nebraska-Lincoln, Plant Pathology Mullin, Peter;Draft University of Nebraska-Lincoln, Plant Pathology Powers, Kirsten; University of Nebraska-Lincoln, Plant Pathology Keyword: Nematoda, DNA barcoding, Species distribution Is the invited manuscript for consideration in a Special Trends in DNA Barcoding and Metabarcoding 2019 Issue? : https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 28 Genome 1 Nematode biodiversity assessments need vouchered databases: A BOLD reference library for 2 plant-parasitic nematodes in the superfamily Criconematoidea 3 Thomas O. Powers, Timothy S. Harris, Rebecca S. Higgins, Peter G. Mullin, Kirsten S. Powers, 4 University of Nebraska-Lincoln 5 6 Corresponding author: Thomas O. Powers [email protected] Draft https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 28 7 Abstract 8 Nematodes are frequently cited as underrepresented in faunistic surveys using DNA barcoding 9 with COI. This underrepresentation is generally attributed to a limited presence of nematodes in 10 DNA databases which, in turn, is often ascribed to structural variability and high evolutionary 11 rates in nematode mitochondrial genomes. Empirical evidence, however, indicates that many 12 taxa are readily amplified with primer sets specifically targeted to different nematode families. 13 Here we report the development of a COI reference library of 1,726 specimens in the terrestrial 14 plant parasitic nematode superfamily Criconematoidea. Specimens collected during an ecoregion 15 survey of North America were individually photographed, measured, and PCR amplified to 16 produce a 721 bp region of COI for taxonomicDraft analysis. A neighbor-joining tree structured the 17 dataset into 179 haplotype groups that generally conformed to morphospecies in traditional 18 analysis or Barcode Index Numbers (BINs) in the BOLD system, although absent formal BIN 19 membership due to insufficient overlap with the Folmer region of COI. Approximately one-third 20 of the haplotype groups could be associated with previously described species. The geographic 21 distribution of criconematid nematode species suggests a structure influenced by the major 22 habitat types in the United States and Canada. All sequences collected in the ecoregion survey 23 are deposited in BOLD. 24 Key words 25 Nematoda, DNA barcoding, Species distribution 26 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 28 Genome 27 Introduction 28 A taxonomic reference library can provide essential information for studies of diversity, 29 distribution, and taxon identity. Now, as investigations using environmental and community 30 DNA are generating vast quantities of nucleotide sequences from prokaryotes and eukaryotes 31 alike, reference libraries will help establish taxonomic context for meaningful interpretation of 32 the sequence data (Miller et al., 2016; Richards et al., 2018; DeSalle and Goldstein, 2019; Ekrem 33 et al., 2007). Nematodes are frequently considered to be neglected in “all-taxa” surveys and are 34 underrepresented in searchable nucleotide sequence databases (McGee et al., 2019; Brunbjerg et 35 al., 2019; Rodrigues da Silva et al., 2010; Ahmed et al., 2019; Creer et al., 2010, Holovachov et 36 al. 2017; Schenk and Fontaneto, 2019). DraftIn particular, COI representation in GenBank and 37 Barcode of Life Data Systems (BOLD) lags far behind other metazoans, especially considering 38 estimates of total species in the phylum, which may exceed 5 million (Van Den Hoogen et al., 39 2019; Schenk and Fontaneto, 2019; Larsen et al., 2017). Their abundance in soil typically range 40 from 1-8 million per m2 and may include over 200 species in the top 20 cm of soil (Yeates, 41 2010). A reference library that links nematode morphology with diagnostic genetic markers and 42 environmental variables should accelerate studies of comparative morphology, stimulate the 43 neglected fields of nematode biogeography, and serve as a preliminary outline of taxon diversity 44 (Zaharias et al., 2020). The urgency of documentation is underscored by the diminishing number 45 of museums with dedicated curators that currently maintain classical specimen repositories for 46 nematodes. These repositories typically catalogue formalin-fixed nematodes preserved on slides 47 or in vials. Unfortunately, despite the best efforts to preserve type and voucher species, fixed 48 nematodes mounted in glycerin on microscope slides lose detail with time, eventually obscuring 49 key morphological characters. Linking genetic and morphological data of individual specimens, https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 28 50 while retaining a physically intact specimen, is not practical due to the small size and hydrostatic 51 pressure that causes nematodes to collapse when punctured. However, nematodes are excellent 52 candidates for digital imaging given their small size, transparent cuticle, and their limited 53 number of organ systems (De Ley and Bert, 2002). Once imaged, unfixed nematode specimens 54 can be removed from a slide, crushed, and DNA barcoded. BOLD provides an excellent platform 55 and repository for image vouchers and their associated sequence plus metadata. Here we report 56 on the development of a reference library of mitochondrial COI sequences obtained from 57 Criconematoidea, a globally distributed superfamily of plant-parasitic nematodes. 58 Our goal is to 1. Illustrate the challenges associated with creating a reference library for a 59 terrestrial nematode taxon with a cosmopolitanDraft distribution and many undescribed species, and 2. 60 Populate a nematode reference library with entries sufficient in geographic range and numbers to 61 establish a framework to address questions of nematode identity, diversity, and distribution. 62 Evidence already suggests that COI barcodes can recognize the taxonomic boundaries of 63 traditionally described morpho-species, and that geographically these species exhibit nonrandom 64 patterns of distribution (Powers et al., 2014; 2016a; 2016b; Olson et al., 2017; Van den Berg et 65 al., 2018). Herein, we use the BOLD platform to organize records of approximately 1,700 66 nematode specimens in the plant-parasitic superfamily Criconematoidea. 67 68 Materials and Methods 69 Collection data 70 North American collection sites are indicated on Figure 1. A majority of the specimens in this 71 1,726-sequence dataset were obtained from soil within a 40 x 40m grid in which each of the https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 28 Genome 72 corners were georeferenced. Field notes and digital imaging recorded the vegetation and 73 topography of the grid. These metadata can be found in two separate complementary archives. 74 Table S1*, (also available at University of Nebraska-Lincoln Data Repository 75 https://doi.org/10.32873/unl.dr.20200330) provides the individual specimen’s ID number, 76 collection location information, GenBank accession number and other genetic marker accession 77 numbers associated with that specimen. On the public BOLD site 78 (http://www.barcodinglife.org/), the project ‘CRICO’ (dx.doi.org/10.5883/DS-NEMACRIC) 79 yields much of the same information as above, as well as specimen images, habitat images, 80 morphometrics and DNA sequence data. One-hundred and five GenBank sequences of 81 specimens not observed by the authors were included in the neighbor-joining tree of the 82 superfamily (Figure 2, and Figure S1† Draft(also available at University of Nebraska-Lincoln Data 83 Repository https://doi.org/10.32873/unl.dr.20200330). Ninety-six of these GenBank sequences 84 were reported in recent taxonomic studies of two genera, Hemicycliophora and 85 Hemicriconemoides, which employed the COI-F5/ COI-R9 primer set discussed below (Maria et 86 al., 2019; Van den Berg et al., 2018). These GenBank accessions were not added to the dataset of 87 COI sequences submitted to BOLD, but are listed in Table S2‡ (also available at University of 88 Nebraska-Lincoln Data Repository https://doi.org/10.32873/unl.dr.20200330). 89 To obtain nematode samples, a standard soil corer with a 3 cm diameter was used to extract cores 90 to a depth of 20 cm if possible. Depending on the depth of the soil core, 15-25 cores would 91 constitute a single sample. The soil cores were mixed and stored in plastic bags at 15°C prior to 92 processing. A second sampling strategy consisted of focal samples in which all cores were 93 centered on a single plant species, in an effort to determine nematode-host plant associations. In https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 28 94 other cases, approximately 9% of specimens for this dataset were obtained from agricultural 95 surveys of plant pests or sent by colleagues and clients requesting specific identification. 96 Sampling locations were guided by a goal to sample a majority of the ecoregions representing 97 the Major Habitat Types in North America north of Mexico (Olson et al., 2001). Several 98 ecoregions were sampled intensely. For example, Central Tall Grasslands, which have been 99 largely converted to agricultural