Using Mitogenomic and Nuclear Ribosomal Sequence Data to Investigate the Phylogeny of the Xiphinema americanum Species Complex

Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex

Using mitogenomic and nuclear ribosomal sequence data to investigate the phylogeny of the Xiphinema americanum species complex.

Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex

HoweDanaZoologyUsingMitogenomicNuclear_SupportingInformation.zip

Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex

HoweDanaZoologyUsingMitogenomicNuclear.pdf

Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex

Maximum likelihood bootstrap tree of aligned ITS-rDNA gene sequences for populations of <i>Xiphinema americanum</i>, presented in 4 parts (A–D).

<p>Trees generated from the analysis of 204 newly generated ITS sequences from <i>X. americanum</i> and 4 sequences previously available from GenBank (2 of <i>X. americanum</i>, 1 <i>X. citricolum</i> and 1 <i>X. peruvianum</i>). <i>X. peruvianum</i> GQ231531.1 was designated as an outgroup. Sequence names consist of a population designation (e.g. WA-1; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090035#pone-0090035-t001" target="_blank">Table 1</a>) followed by a number to indicate the individual and a letter to indicate the clone (e.g. WA-1.1b is clone “b” from individual 1 from population WA-1). From 2 to 6 clones were sequenced for each individual nematode from 12 populations. Bootstrap values for clades supported at greater than 50% are shown. Brackets and V+/− indicate the presence or absence of nematode transmitted plant virus in those populations, excluding GenBank taxa indicated with an * for which virus status is unknown.</p

Maximum likelihood bootstrap tree of aligned ∼1.1 kb of mtDNA for populations of <i>Xiphinema americanum</i>.

<p>Thirty-two nematode sequences were analyzed. Sequence names consist of a population designation (e.g. WA-1; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090035#pone-0090035-t001" target="_blank">Table 1</a>) followed by a number to indicate the individual (e.g. WA-1.1). Presence or absence of nematode transmitted plant virus in an individual nematode indicated by virus (+/−).</p

Schematic of loci of mitochondrial DNA analyzed.

<p>Rectangles with solid colors show mtDNA protein-coding genes, dashed rectangles show ribosomal RNA genes, and white rectangles show tRNA genes (single-letter abbreviations for associated amino acids). The dashed lines on top indicate the regions amplified in the two long PCRs used to generate templates for Illumina sequencing. Information about regions used for phylogenetic analyses is provided at the bottom.</p

<i>Xiphinema americanum</i> species complex populations included in this study.

a<p>Presence of nepovirus was based upon ELISA detection, direct observation of symptoms, or historical data for the site.</p

Maximum likelihood bootstrap tree of aligned ∼2.5 kb of mtDNA for populations of <i>Xiphinema americanum</i>.

<p>Thirteen nematode sequences were analyzed. Sequence names consist of a population designation (e.g. WA-1; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090035#pone-0090035-t001" target="_blank">Table 1</a>) followed by a number to indicate the individual (e.g. WA-1.1). Presence or absence of nematode transmitted plant virus in an individual nematode indicated by virus (+/−).</p