An integrative approach to the study of Helicotylenchus (Nematoda: Hoplolaimidae) Colombian and Brazilian populations associated with Musa crops

Abstract In total, 10 populations of Helicotylenchus associated with Musa spp., six from Colombia, and four from Brazil were identified to the species level. Morphological and morphometric data were recorded for each population, performed a principal components analysis (PCA), a conglomerate, along with consensus sequences of D2 to D3 expansion segment of the large subunit of ribosomal DNA (28S) for different populations. Identify of species were performed using the basic local alignment search tool (BLAST), and the evolutionary relationships of species were explored using a phylogeny study. Four species of Helicotylenchus were identified based on morphological and morphometric data: H. multicinctus, H. dihystera, H. erythrinae, and H. californicus. PCA and conglomerate analysis clearly separated these species. BLAST and phylogenetic analysis confirmed the presence of these species associated with Musa spp. crops. This is the first report of these species in Colombia through integrative taxonomy.

Production losses in plantain and banana by Helicotylenchus spp. depend on two main factors. On the one side, the susceptibility of planting material and nematode density, because differences in the population level of H. multicinctus have been registered among Musa spp. cultivars with a high correlation between its density and the necrotic and dead roots percentage (Speijer and Ssango, 1999). On the other hand, losses depend on environmental conditions, since Helicotylenchus spp. are predominant in crops cultivated between 1,000 and 1,350 meters above sea level in andisols and vertisols soils with high contents of Ca, P, Mn and reduced organic matter (Speijer and Ssango, 1999;Karakas, 2007;Araya et al., 2011).
Although Helicotylenchus has been reported in the banana and plantain crops of Colombia and Brazil, morphological, morphometric, and molecular data are scarce for these geographical regions (Zuñiga et al., 1979;Villegas, 1989;Guzmán-Piedrahita and Cataño-Zapata, 2004;Torrado-Jaime and Castaño-Zapata, 2009). In order to contribute to the knowledge of the taxonomic identity of the species of Helicotylenchus associated with Musaceae in Colombia and Brazil, the present study has the following objectives: (i) to identify the species of Helicotylenchus that are associated with banana and plantain crops, using a combination of morphological, morphometric, and molecular analysis, and (ii) to elucidate the phylogenetic relationships of Helicotylenchus species that are associated with banana and plantain crops in the departments of Quindío, Risaralda y Valle del Cauca in Colombia and Minas Gerais in Brazil.

Materials and methods
Area of study, sampling, and nematodes extraction Root and soil samples were collected from banana and plantain rhizosphere crops in the departments of Quindío (Calarcá and Córdoba municipalities), Risaralda (La Celia municipality), and Valle del Cauca (Palmira and Buenaventura municipalities) in Colombia and in Janauba, Minas Gerais state, Brazil during 2015 to 2018. In each crop, compound samples were collected, comprising 15 to 20 plants/ ha. Nematodes were extracted from roots and soil using a modification of Cobb's method (Ravichandra, 2014). Afterwards, representative populations of Helicotylenchus were selected for posterior analysis in the microbiology and molecular biology laboratories of the National University of Colombia (Palmira, Valle del Cauca, Colombia) and the Laboratory of Nematology of the Biological Institute (Campinas, São Paulo, Brazil).

Morphologic and morphometric analysis
Nematodes extracted and identified as Helicotylenchus were killed with heat at 60°C for 4 min and fixated in 2% formalin. Semipermanent preparations were made and morphometric data were registered following Boag and Shamim Jairajpuri (1985) and Uzma et al. (2015). Microphotographs were taken using a compound microscope equipped with differential interference contrast-DIC (DM2500, Leica, Germany).

Statistical analysis:
Morphometric data were analyzed using the Community Analysis Package (PISCES Conservation Ltd, Lymington UK, 1995) with principal components analysis (PCA) and conglomerate analysis to determine groupings and evaluate those characters that could discriminate species.

Phylogenetic analysis
Consensus sequences were edited using the software Geneious (Kearse et al., 2012) and BLAST (Basic Local Alignment Search Tool) at NCBI (National Center for Biotechnology Information) was used to confirm the species' identity of the sequences. To estimate the phylogenetic history of Helicotylenchus, D2 to D3 sequences of others specimens of the species were downloaded from GenBank, including a sequence of Rotylenchus magnus, which was used as outgroup. Sequence alignment was performed using MAFFT v7 (Katoh et al., 2002) (protocol Q-INS-i), and jModelTest v2.1.7 (Posada, 2008) to find the best nucleotide substitution model, based on the Akaike information criterion corrected for small sample sizes. Afterwards maximum likelihood (ML) was used to estimate a tree with 250 bootstraps and the general time reversible model with allowance for gamma distribution of rate variation (GTR+Γ ) in RAxML v8 (Stamatakis, 2014). The phylogeny of Helicotylenchus was inferred using MrBayes v3.2.6 (Ronquist et al., 2012) with the GTR + Γ model. Two independent Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were performed for 2 million generations sampling every 2,000 steps. Convergence was assessed using Tracer v1.5 (burn-in = 20% of the samples), and by examining the average standard deviation of split frequencies among parallel chains. The consensus tree was calculated from the posterior distribution of 1,600 phylogenies. The Bayesian analysis was performed in the CIPRES Science Gateway (Miller et al., 2010). Helicotylenchus multicinctus populations were identified in plantain crops of Colombia (Córdoba, Quindío and Rozo, Valle del Cauca) and banana crops of Brazil (Minas Gerais). Morphologically, these populations show a habitus post-mortem open C form, a hemispherical cephalic region, the shape of the stylet knobs flattened anteriorly and rounded posteriorly, a rounded tail, functional spermatheca and males present ( Fig. 1, Table 1).

Morphological and morphometric identification of nematodes
Populations of H. dihystera were founded in plantain and banana crops from Colombia (Córdoba, Quindío). Morphologically, the populations of this species show a habitus post-mortem with a spiral shape, a hemispherical cephalic region with the shape of stylet knobs indented or flattened anteriorly and rounded posteriorly, a conoide tail and males absent (Fig. 2, Table 2).
The populations identified as H. erythrinae occurred in Colombian plantain and banana crops (Calarcá, Quindío and La Celia, Risaralda). Morphologically, the populations of this species show a habitus postmortem of a loose spiral, a hemispherical cephalic region with the shape of stylet knobs indented or flattened anteriorly and rounded posteriorly, a tail with a long ventral projection, females with functional spermatheca and males present (Fig. 2, Table 3).
The species H. californicus was identified in a plantain crop from Colombia (Delfina-Buenaventura, Colombia) and a banana crop from Brazil (Minas Gerais). Morphologically, these populations presented a habitus post-mortem in a spiral, a hemispherical cephalic region with the shape of stylet knobs flattened anteriorly and rounded posteriorly, an irregular tail with a short sharply pointed or blunt ventral projection and males present ( Fig. 2; Table 4).
PCA and conglomerate analysis separated the species into four groups (H. multicinctus, H. dihystera, H. erythrinae, and H. californicus) (Figs. 3,4). The principal components 1 to 4 have eigenvalues greater than or equal to 1 and explain 91.39% of variance (Table 5). However, PC1 and PC2 axes better separated the species. In PC1, the most discriminating variables were maximum body diameter, the number of tail annuli, and ratio c′, while in PC2 they were vulva position, anal body diameter, and tail length (Table 6).

Molecular identification of nematodes
In relation to the D2 to D3 expansion segment of ribosomal DNA, four consensus sequences were obtained for H. multicinctus with similarity of 99% with different sequences of this species deposited in the NCBI (KF443214, DQ328745, DQ328746, HM014290, HM014291, and HM014292). On the other hand, two consensus sequences were obtained for H. dihystera, which presented similarity of 99% with other sequences of this species already deposited in NCBI         are proposed as standard and reference populations until topotype specimens become available and were molecularly characterized. All sequences obtained in the present study were deposited in NCBI with accession numbers MT321729-MT321739 (Table 7).

Phylogenetic relationships of nematodes
The evolutionary relationships of 51 sequences of the D2 to D3 expansion segment of ribosomal DNA for Helicotylenchus, including those obtained in the present study (        morphometric data but also of multivariate statistical analysis to discriminate among species of the genus (Fortuner and Maggenti, 1991;Subbotin et al., 2015;Uzma et al., 2015). In accordance with PCA, the variables associated with the tail are diagnostic characters powerful enough to discriminate between or to separate among H. multicinctus, H. dihystera, H. californicus, and H. erythrinae, which could be useful for preparing a dichotomous key for identification of Helicotylenchus species in Colombia (Mizukubo et al., 1992;Uzma et al., 2015). The diagnostic characters: number of tail annuli, ratio c′, maximum body diameter, tail length, anal body diameter, and vulva position separated the species identified in this study in accordance with PCA and conglomerate analysis. In various publications, tail length and DGO have been suggested by their discriminate values among Helicotylenchus species (Perry et al., 1959;. Additionally, intraspecific variability at the morphometric level was observed in the four species identified in this study, due to the various morphological and morphometric unconstant characters with a coefficient of high variability within of Helicotylenchus species (Fortuner et al., 1981;. Different morphological and morphometric characters were constants within the species identified, as habitus, ratio a and V as reported for various species of Helicotylenchus . Variation in the shape of the tail was registered in females of the Colombian population of H. californicus. This species is characterized by showing individuals with short ventral projections and sharply pointed or blunt tails (Van Den Berg and Heyns, 1975;Krall, 1990). Variability in the shape of the tail to the intraspecific level has been recorded for various species of Helicotylenchus (Fortuner, 1979 (Subbotin et al., 2011;Xiao et al., 2014). The sequences obtained for H. californicus grouped with an isolate of Helicotylenchus from Calathea not identified to the species level and labeled as CD761 in Subbotin et al. (2015) (Table 7). Recently, Fortuner et al. (2018) identified isolate CD761 as H. pseudorobustus using morphological and morphometric analysis. However, our populations are similar to preliminary reports of H. californicus (Van Den Berg and Heyns, 1975;Krall, 1990).
Unfortunately, there are no sequences of reference for H. erythrinae deposited in any of the databases of genes in the world for comparison. Therefore, the present study is reporting the first sequences of H. californicus and H. erythrinae associated to Musa spp. in a public database.
The four species identified in this study, H. multicinctus, H. dihystera, H. californicus, and H. erythrinae, have been reported in Musa spp. crops around the world (Campos et al., 1987;Araya and De Waele, 2004;Karakas, 2007;Dias-Arieira, 2010;Ravichandra, 2014;Xiao et al., 2014;Lara et al., 2016;Tzortzakakis et al., 2017). However, this is the first report of these species for Musaceae crops in Colombia through integrative taxonomy. Additionally, this research confirmed that Helicotylenchus species occur individually or in a mixture in Musaceae crops in the studied zones, which has been documented in the past in other production zones of the world (Araya and De Waele, 2004;Dias-Arieira, 2010;Roy et al., 2014;Khan and Hasan, 2010;Ravichandra, 2014;Daneel et al., 2015).
In relation to H. multicinctus, it is considered the most limiting species of Helicotylenchus in plantain and banana. It was present in the Musa crop production of both Colombia and Brazil, confirming the wide distribution of this plant-parasitic nematode, which also has been previously registered in Puerto Rico, Vietnam, India, Mexico, and Greece (Ngoc Chau et al., 1997;Ravichandra, 2014;Lara et al., 2016;Tzortzakakis et al., 2017). The presence of H. multicinctus in the different production zones, Quindío and Valle del Cauca, suggests a wide spread of this nematode in Colombia, directly related with the dispersion of contaminated seedlings between production zones, but also to environmental conditions that have favored the establishment of this species (altitude, soil type, and nutrient availability for the plant) (Nath et al., 1998;Speijer and Ssango, 1999;Araya et al., 2011;Godefroid et al., 2017).
reagents. The Molecular Biology Laboratory of the Universidad Nacional de Colombia in Palmira, Valle del Cauca -Colombia and Nematology Laboratory of Instituto Biológico in Campinas, São Paulo -Brasil for the scientific support for the register of molecular, morphological, and morphometric data. Universidad del Pacífico in Buenaventura, Colombia. Peter Ramley for his collaboration in the grammar checking of manuscript. To PhD Angel Vale and PhD Danny Rojas for their assistance with the bioinformatics analyses and manuscript revision and CIPRES by bioinformatics support.