Introduction

Plant-parasitic nematodes (PPN) cause crop losses estimated at 12.3 % worldwide, equivalent to 173 US billion dollars (^{Kumar et al., 2020}). One of the major groups of PPN is root-lesion nematode (Pratylenchus spp.), which ranks third as having a great economic impact on crops worldwide (^{Jones et al., 2013}). The first description of the genus was made in 1880 by De Man, who originally called the nematode Tylenchulus pratensis (^{Castillo & Vovlas, 2007}). The current name, Pratylenchus, was given later in 1936 by Filipjev, with the description of the characteristics that make this genus distinguishable from others (^{Filipjev, 1936}).

Pratylenchus is a migratory endoparasite globally distributed that has been reported in more than 400 hosts (^{Castillo & Vovlas, 2007}; ^{Handoo et al., 2008}). It is known as “root-lession nematode” because of the damage caused when enters the roots, where it generates hollow channels when moving and feeding inside the root system (^{Piedrahita et al., 2012}). The number of legitimate Pratylenchus species is still in debate, but it is estimated with around 103 species (^{Nguyen et al., 2019}).

Species identification is valuable for adequate integrated nematode management (^{Al-Banna et al., 2004}; ^{Mokrini et al., 2019}), e.g., to enforce quarantine measures to prevent the entry of species that could have detrimental effects on crops or to avoid the rejection of agricultural exports (^{Blok, 2005}). Additionally, species identification is essential to implement effective nematode management strategies in response to the ban of several nematicides from the market. Among the management alternatives to chemical control, are the use of resistant varieties and crop rotation which requires the identification at the species level (^{Moura Cintra Goulart, 2008}; ^{Starr et al., 2002}).

Many studies involve the use of a taxonomic polyphasic approach (morphological, morphometric, biochemical, and molecular methods) in the characterization of Pratylenchus species (^{Castillo & Vovlas, 2007}). However, the identification of the genus Pratylenchus is complex due to the number of validated species and the variation within the same species (^{Nguyen et al., 2019}). The morphological identification of Pratylenchus is challenging for diagnostics because of the presence of cryptic species (^{Bogale et al., 2021}) that make some of the species identifiable only by DNA analysis (^{Singh et al., 2020}). Molecular tools for species identification can help to overcome these problems and have become essential for accurate classification (^{De Luca et al., 2012}; ^{Janssen, Karssen, Couvreur, et al., 2017}; ^{Janssen, Karssen, Orlando, et al., 2017}; ^{Palomares-Rius et al., 2014}). Besides, more reliable results are obtained in less time with molecular techniques than other methods (^{Donn et al., 2008}). Usually, molecular tools reduce the time for species identification, increase precision, and require less experience (^{Starr et al., 2002}).

Molecular studies of Pratylenchus have been done extensively by the analysis of ITS rDNA, 18S rDNA, D2-D3 of the 28S rDNA and mitochondrial genes (^{Castillo & Vovlas, 2007}; ^{De Luca et al., 2012}; ^{Janssen, Karssen, Couvreur et al., 2017}; ^{Orui & Mizukubo, 1999}). The region D2-D3 of the 28S rDNA of Pratylenchus has been defined as a better molecular marker to identify species within the genus compared to 18S rDNA (^{Subbotin et al., 2008}). Inside the genome region, the D3 subunit fragment of the 28S-rDNA gene has been widely used for the molecular identification of Pratylenchus species (^{Al-Banna et al., 1997}; ²⁰⁰⁴; ^{De Luca et al., 2004}; ^{Hodda et al., 2014}).

In Costa Rica, the Pratylenchus species characterized initially, based on morphological methods were P. zeae, P. brachyurus, P. coffeae, and P. penetrans (^{González-Fernández, 1979}; ^{Guzmán-Hernández et al., 2011}; ^{López et al., 1987}; ^{López & Salazar, 1990}). Later on, P. coffeae and P. brachyurus were analyzed and identified with molecular techniques (^{Humphreys-Pereira et al., 2017}), and P. bolivianus, P. gutierrezi, P. pseudocoffeae, and P. zeae with both morphological and molecular methods (^{Zamora-Araya et al., 2016}). Current information about the Pratylenchus species in Costa Rica is scarce. In this research, we conducted a Pratylenchus survey in different agricultural crops in Costa Rica by the selection of molecular tools. Pitfalls of the molecular-exclusive approach were decreased by the selection of at least one accession per Pratylenchus species from a per-review publication containing information related to the morphological identification of the species. The objective of this study was to identify the Pratylenchus species associated with 12 crops based on the D3 segment of the 28S rDNA gene.

Materials and methods

Nematode populations and hosts

Roots samples from 12 crops were collected, during 2013 to 2015, from five provinces of Costa Rica (Table 1). Three root subsamples (10 plants each) were collected with a shovel from each location site in a systematic pattern in zigzag, from 0 to 30 cm depth. Crops and localities were selected based on samples detected as positives for Pratylenchus spp. when the producers provide them to the Nematology Laboratory of the Universidad de Costa Rica. The number of sequences analyzed per location varied from 2 to 13 (Table 1). The distribution of the Pratylenchus populations used in this study is shown in Figure 1.

Table 1  Pratylenchus spp. populations collected in five provinces of Costa Rica. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. Cuadro 1. Poblaciones de Pratylenchus spp. colectadas en cinco provincias de Costa Rica. Laboratorio de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. 

Figure 1 Geographic localization of the Pratylenchus populations collected in five provinces of Costa Rica. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015.PA: rice (Oryza sativa), PB: banana (Musa paradisiaca); PC1 and PC2: coffee; PE1 and PE2: onion; PF: strawberry (Fragaria x ananassa), PG: gypsophila, PL: lily, PÑ1 and PÑ2: sugarcane (Saccharum officinarum); PM: black pepper (Piper nigrum); PS: aster (Aster sp.); PH: leather-leaf fern (Rumohra adiantiformis); PP: potato (Solanum tuberosum). Figura 1. Localización geográfica de las poblaciones de Pratylenchus colectadas en cinco provincias de Costa Rica. Laboratorio de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015.PA: arroz, PB: banano; PC1 y PC2: café; PE1 y PE2: cebolla; PF: fresa, PG: gyphsophila, PL: lily, PÑ1 y PÑ2: caña de azúcar; PM: pimienta negra; PD: áster; PH: helecho de hoja de cuero; PP: papa. 

Sample preparation

Nematode extractions were done in the Laboratory of Nematology from the Crop Protection Research Center (CIPROC) from the Agronomy School at the Universidad de Costa Rica, San Pedro, San José. Plant roots were washed, cut into 1-3 cm-long pieces and 10 g were selected randomly for processing by flotation-centrifugation method (^{Caveness & Jensen, 1955}) with modifications from the Nematology Laboratory from the Universidad de Costa Rica. The roots were washed and blended for 20 s, the solution was poured into 100 over 500 sieves and was collected in a 50 mL conical tube (Eppendorf). It was centrifuge at 2800 rpm for 3 min. Supernatant was discarded and a sugar solution with a specific gravity of 1,18 was added into the tube. The tube with the sugar solution was agitated and centrifugated at 2800 rpm for 3 min. This was poured into a 500-mesh sieve and washed with abundant water. The remnant was verted into plastic 50 mm Petri dishes (Thermo Fisher Scientific) for the posterior identification. Nematodes were first identified at the genus level using a stereomicroscope Nikon model SMZ754T. The genus was differentiated by the flat cephalic structure, short and wide stylet, ventral overlap with the intestine, in females the vulva is located at 70-85 % of the body length, conical tail rounded in the terminus. Males has terminal bursa, Pratylenchus individuals at the adult stage were picked using a metal surgical needle and placed in a Petri dish with distilled water for DNA extraction.

DNA extraction and PCR amplification

The DNA extraction and PCR amplification were done in the Biotechnology Laboratory of the Agronomic Research Center (CIA) from the Agronomy School at the Universidad de Costa Rica, San Pedro, San José. Five nematodes, per sample, were placed in a coverslip with a drop of Worm Lysis Buffer (WLB, 50 mM KCl, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl₂, 0.45 % Tween 20) (^{Madani et al., 2005}) + 100 µg mL^-1 proteinase K (20 mg mL^-1); Thermo Fisher Scientific). Each nematode was cut with a scalpel into three pieces and the five nematodes per sample were transferred into PCR tubes containing 5 µL of Worm Lysis Buffer + proteinase K. Samples were incubated at 80 °C for 30 min, 65 ºC for 1 h, followed by a proteinase K inactivation step at 95 ºC for 15 min. Primers D3A (5'-GACCCGTCTTGAAACACGGA-3') and D3B (5'-TCGGAAGGAACCAGCTACTA-3') (^{Al-Banna et al., 1997}) were used to amplify the D3 region of the 28S rDNA. PCR was carried out in 25 µL as follows: 2.5 µL of Dream Taq Buffer 10X, 1µL of dNTP's (2 mM), 2 µL of each primer (10 µM), 1 µL of BSA (20 mg mL^-1), 1.5 mM of MgCl2, 0.5 µL of Dream Taq (5 U µL^-1, Thermo Fisher Scientific), and 5 µL of the DNA extract.

The amplification conditions consisted of an initial denaturation at 92 ºC for 5 min, 40 cycles at 94 ºC for 30 s, 55 ºC for 45 s, 72 ºC for 1 min, and a final extension at 72 ºC for 7 min. The PCR products were visualized by electrophoresis in 1.6 % agarose gel (1.6 g of agarose in 100 mL of TRIS-Borate-EDTA 0.5X buffer). PCR products were stained with GelRedTM (Biotium). PCR products were purified with a NucleoSpin® gel and PCR Clean-up Kit (Macherey-Nagel) and sequenced bidirectionally by Macrogen (Seoul, South Korea). The resulting sequences were edited using BioEdit (^{Hall, 1999}) and compared with other sequences of Pratylenchus from the GenBank by BLAST Search (National Center for Biotechnology Information, NCBI, USA).

Phylogenetic analysis

The phylogenetic analysis of the D3 sequences of Pratylenchus spp. generated in this study and sequences recovered from the GenBank was estimated with the Bayesian Inference (BI) analysis with the program MrBayes 3.2 (^{Ronquist & Huelsenbeck, 2003}). The MUSCLE algorithm (^{Edgar, 2004}) implemented in Mega 6 (^{Tamura et al., 2013}) was used for the sequence alignment. The best DNA model of evolution (K80 + G) was selected on JmodelTest (^{Darriba et al., 2012}; ^{Guindon & Gascuel, 2003}) with the BIC criterion. The phylogenetic tree was visualized on FigTree 1.4.3 (^{Rambaut, 2012}). At least one sequence from previous peer-review publications were included per Pratylenchus species used for the phylogenetic analysis (Table 2).

Table 2 GenBank accessions used for the phylogenetic analysis. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. Cuadro 2. Accesiones del GeneBank usadas para el análisis filogenético, Laboratorio de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. 

^{Zamora Araya et al. (2016)}, ^{Subbotin et al. (2008)}, ^{de la Pena et al. (2006)}, ^{Duncan et al. (1999)}, ^{Humphreys-Pereira et al. (2017)}, ^{De Luca et al. (2010)}, ^{Inserra et al. (2007)}, ^{Hajieghrari et al. (2007)}, ^{Wang et al. (2012)}

Results

Molecular identification and phylogenetic analyses

The resulted D3 segment amplification products from all Pratylenchus DNA samples were ca.300 bp (Figure 2). The similarity percentage between sequences obtained in this study and sequences retrieved from the GenBank are shown in Table 3. The phylogenetic relationships based on the D3 region included an alignment of 74 Pratylenchus sequences and one sequence from Zygotylenchus guevarai sequence as an outgroup (Figure 3). Most of the D3 Pratylenchus sequences generated in this study were positioned on highly supported clades (Bayesian Posterior Probability, BPP>90). The tree topology showed in a basal position, a large monophyletic group formed with P. zeae accessions and Pratylenchus sequences obtained from rice (PA) and sugar cane (PÑ2) (BPP=100). Pratylenchus sequences from onion (PE1), potato (PP02), and from a field with both potato and onion (PP-E) were positioned in a clade with P. crenatus, with a high support value (BPP=100), but P. crenatus formed an independent group (BPP=98%).

Figure 2  Pratylenchus spp. rDNA amplification of a segment of the D3 region of the 28S gene. Well 1 and 12: molecular marker 100 bp. Well 2 to 10: amplification using the primers D3A and D3B. Well 11: negative control. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. Figura 2. Amplificación de un segmento de la region D3 del gen 28S del ADNr de Pratylenchus spp. Pozo 1 y 12: marcador molecular 100 pb. Pozo 2 a 10: amplificación del con los primers D3A y D3B. Pozo 11: control negative. Laborario de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. 

Table 3 Similarity of the Pratylenchus sequences generated in this study, with the GenBank accessions based on the D3 region of 28S rDNA gene and number of haplotypes per population. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. Cuadro 3. Similitud de las secuencias de Pratylenchus generadas en este estudio, con las accesiones del GeneBank, basados en la región D3 del gen 28S ADNr y número de haplotipos por población Laboratorio de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro. San José, Costa Rica. 2013-2015. 

Figure 3 Phylogenetic relationships within the genus Pratylenchus as inferred from the Bayesian analysis of the D3 region of the 28S rRNA gene using the GTR+ I+ G model. Laboratory of Nematology, Crop Protection Research Center, Agronomy School, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. Figura 3. Relaciones filogenéticas dentro del género Pratylenchus de acuerdo con el análisis Bayesiano de la región D3 del gen 28SrRNA con el modelo GTR+ I+ G Laboratorio de Nematología, Centro de Investigación en Protección de Cultivos, Escuela de Agronomía, Universidad de Costa Rica. San Pedro, San José, Costa Rica. 2013-2015. 

The Pratylenchus sequence from black pepper formed a clade with sequences of P. brachyurus retrieved from the GenBank (BPP=100). Two sister groups both with high support values were formed with sequences of P. penetrans from the GenBank and sequences from onion (PE2), lily (PL), strawberry (PF), and Gypsophila (PG) for group one (BPP=100), and two sequences of P. dunensis for group two (BPP=93). The Pratylenchus sequences from coffee PC1 and PC2 were grouped with P. gutierrezi (BPP=100), whereas the Pratylenchus sequences from aster (PS01 and PS02) were grouped with sequences from P. pseudocoffeae (BPP=100). The Pratylenchus DNA samples from leather-leaf fern, one from potato, banana, and one from sugarcane did not show a good resolution.

Discussion

The molecular analysis of Pratylenchus sequences based on the D3 region of the 28S rDNA gene allowed the identification of nine different genetic groups. Morphological and morphometric methods of nematodes identification should complement the molecular methods for accurate identification of these organisms, but in genera like Pratylenchus, some species can only be distinguished based on molecular identification (^{De Luca et al., 2012}; ^{Palomares-Rius et al., 2014}; ^{Troccoli et al., 2021}; ^{Wang et al., 2015}). In this research it was corroborated that it was possible to identified different Pratylenchus phylogenetic groups using the molecular approach. However, some of the genetic groups identified need to be validated using a polyphasic approach. The phylogenetic tree recovered in this research for the D3 region presented a similar topology to that published by ^{Subbotin et al. (2008)} for the D2-D3 28S rDNA gene region. The same authors mentioned that despite some exceptions the topology recovered from the larger fragment D2-D3 region of the 28S rDNA is similar to the D3 region topology.

The root-lesion nematode, P. zeae was found in rice from Upala, Alajuela, and sugarcane from Cañas, Guanacaste. Previously, an identification of this nematode with morphological techniques in four regions of Costa Rica: Atlantic, North Pacific (Guanacaste), Central Pacific, and Southeast was made (^{López et al., 1987}). The results from this research could have implications for nematode management because both crops are common in Guanacaste. Therefore, the presence of this crops permits the reproduction and survival of the nematode species and might reduce the positive impact of the sugar cane/rice rotation system (^{Nzogela et al., 2020}; ^{Santos et al., 2012}). P. zeae has been associated with significant yield reduction in sugarcane and rice crops (^{Namu et al., 2018}; ^{Nzogela et al., 2020}; ^{Singh et al., 2020}). The genetic heterogeneity shown within the P. zeae populations from Costa Rica is consistent with previous reports (^{Carta et al., 2001}; ^{Mwamula et al., 2020}; ^{Subbotin et al., 2008}), and supports that P. zeae could be a species complex. In other taxa, cryptic species have been proposed within P. parazeae, P. coffeae, and P. hippeastri (^{De Luca et al., 2010}; ²⁰¹²; ^{Inserra et al., 2007}; ^{Palomares-Rius et al., 2014}; ^{Wang et al., 2015}).

The D3 segment did not provide a good resolution for the identification of the Pratylenchus species associated with sugarcane in Grecia, Alajuela. Even though Blast Search identified the sample as P. hippeastri with 100 % similarity, the phylogenetic analysis placed this population within a clade formed by P. hippeastri, P. floridensis, and P. parafloridensis. The last two species are considered cryptic species within the P. hippeastri group. The use of a larger fragment as the D2-D3 region for the characterization of Pratylenchus species, due to the D2 segment variability was recomend by ^{Subbotin et al. (2008)}. The D3 segment allowed the identification of the root-lesion nematode. P. hippeastri was confirmed in the central region of Costa Rica in strawberry (^{Brenes-Campos et al., 2022}).

P. pseudocoffeae was associated with aster plants in Puriscal. Previously, this nematode was found in chrysanthemum from Heredia (^{Zamora-Araya et al., 2016}). Worldwide, P. pseudocoffeae has been documented in ornamental plants as chrysanthemum, artemisia, aster, and grasses (^{Inserra et al., 1998}; ^{Uesugi et al., 2012}). Pathogenicity assays should be performed to evaluate the host susceptibility of P. pseudocoffeae in aster and determine the level of damage.

The Pratylenchus sample extracted from banana was grouped in a large clade (with a poor resolution, BPP=64) with species considered members of the P. coffeae species complex, such as P. speijeri and P. coffeae. These two species are considered cryptic species (^{De Luca et al., 2012}; ^{Palomares-Rius et al., 2014}), and their separation as different species is unreliable using morphological and morphometric methods (^{De Luca et al., 2012}). Pratylenchus speijeri was described and isolated from Musa in Ghana (^{De Luca et al., 2012}) and weeds (Rottboellia cochinchinensis, Panicum maximum, Acalypha ciliata, Sida acuta, Brachiaria deflex, and Feuria aestuans) on previously planted fields with banana in the same country (^{Brentu et al., 2013}). In Costa Rica, there is little information related to the Pratylenchus species affecting the banana crop. The presence of P. coffeae and other Pratylenchus spp. have been reported but no molecular characterization was provided (^{Fernández Solano & Quesada Solís, 2013}).

In the present study, P. gutierrezi was found in two locations affecting coffee, Orosí, Cartago, and Tarrazú, San José. The root-lesion nematode P. gutierrezi was initially reported on coffee in San Antonio, Cinco Esquinas, Alajuela (^{Morgan Golden et al., 1992}; ^{Zamora Araya et al., 2016}), then found on yampee (Dioscorea trifida) in the south region of Costa Rica (^{Humphreys-Pereira et al., 2017}). As the nematode was identified in two new locations, caution must be taken with the movement of infected seedling material from one locality to another, to avoid further nematode dissemination.

The root-lesion nematode, P. penetrans was the species most frequently found in this study, associated with four (onion, strawberry, gypsophila, and lily) out of 12 crops, collected from the provinces of Cartago and Heredia. Pratylenchus penetrans has a wide host range (^{Bélair et al., 2007}; ^{Castillo & Vovlas, 2007}), and previous reports from other countries showed the synergistic relationship between this species and pathogenic fungi and bacteria (^{Figueiredo et al., 2021}; ^{LaMondia, 2003}). Therefore, studies to determine the damage caused by Pratylenchus species to different crops in Costa Rica are essential, as well as the identification of other hosts to establish accurate management strategies against Pratylenchus plant-parasitic nematode.

Despite the low resolution of the phylogenetic analysis with the Pratylenchus samples obtained from leather-leaf fern (Birrisito, Cartago) and potato (San Juan de Chicua, Cartago), the blast search for both samples resulted in the highest similarity values with P. bolivianus (100 % identity). Two root-lesion nematodes species had been identified on leather-leaf fern in Costa Rica, P. penetrans in Coris, Cartago (^{López & Salazar, 1990}), and P. bolivianus in San Isidro, Heredia (^{Zamora-Araya et al., 2016}). Further studies on nematode surveys in these crops and molecular species identification should be performed to determine this species distribution in Cartago and in other parts of the country.

Some sequences of Pratylenchus spp. collected from potato and onion were grouped in a clade with sequences of P. crenatus. A query in the GenBank showed the highest similarity to sequences of P. crenatus (100 % identity). This nematode has been reported on potatoes in several countries such as Belgium (^{Pelsmaeker & Coomans, 1987}), United States (^{Florini et al., 1987}; ^{Wheeler et al., 1994}), Canada (^{Kimpinski & Smith, 1988}; ^{Olthof et al., 1982}), and in onions from New Zealand (^{Knight et al., 1997}). Crop rotation between potato and onion is common in the northern area of Cartago, which might be favoring an increase in the nematode population density. The characterization of this nematode population using more regions of the genome and morphology, pathogenicity assays, and the screening for tolerant varieties should be a priority since both crops are essential for the economy of northern Cartago.

The nematode P. brachyurus was found associated with black pepper from Muelle, San Carlos, Alajuela. No previous reports were found of this nematode on black pepper in Costa Rica. This species was identified using morphological methods in pineapple from Pital, San Carlos (^{López & Salazar, 1990}). Later, ^{Humphreys-Pereira et al. (2017)} using the D3 region identified P. brachyurus associated with yellow yam (Los Chiles, Alajuela), yampee (Los Chiles, Alajuela and Sabalito, Coto Brus), and pineapple (Upala, Alajuela). These results could indicate that this species could have a wide distribution in Alajuela province. P. brachyurus is an important specie in yield reduction in different crops in South Africa as maize and cassava (^{Coyne et al., 2018}). Therefore, further pathogenicity analysis in the country should be done.

The results from this research increase knowledge on the status of Pratylenchus species in the country. It is important to extend sampling to other provinces of the country and in different plant hosts, as well as to explore other ecological conditions. This information is valuable for the establishment of alternative control strategies.

Conclusions

A total of nine Pratylenchus species were identified in 12 crops using the D3 region of the 28S gene. These results reflect the great diversity of Pratylenchus in the country and establish a starting point for future research on the management of these nematodes.

In the Chorotega region of Costa Rica, the species P. zeae was found associated with sugarcane and rice.

In the Atlantic region, a species from the P. coffeae complex group and highly similar to P. speijeri was identified on banana, whereas in the Northern region of the country, P. brachyurus was found for the first time in black pepper.

In the Central region, P. pseudocoffeae was found associated for the first time with aster, P. gutierrezi in coffee, a species highly similar with P. crenatus in onion and potato, P. penetrans in onion, strawberry, gypsophila, and lily, P. bolivianus with leather-leaf fern and potato, and a species from the P. hippeastri complex group in sugarcane. The species P. zeae, P. penetrans, and P. gutierrezi appear to have a wide geographic distribution in the country.

The morphological, morphometric, and the amplification of more regions of the genome could help to have more robust results and it is suggested for further investigations.