# A New Interactive Web-Based Polytomous Key for Species Identification of Pin Nematodes of the GenusParatylenchus Micoletzky, 1922 (Nematoda: Paratylenchinae) with the Use of Ribosomal and Mitochondrial Genes

Authors: Juan E. Palomares-Rius; Guillermo León-Ropero; Ilenia Clavero-Camacho; Antonio Archidona-Yuste; Carolina Cantalapiedra-Navarrete; Pablo Castillo
Journal: Journal of Zoological Systematics and Evolutionary Research
 (2022)

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## Abstract

Pin nematodes of the genusParatylenchus comprise 140 species. This group of nematodes is characterized by a quite homogeneous morphology and cosmopolitan distribution and is prevalent in cultivated and natural soil ecosystems. The present study describes the first interactive and illustrated web-assisted polytomous identification key for the genus Paratylenchus. The updated Paratylenchus species polytomous key was based on a wide list of 24 diagnostic characters generated for the 140 species comprising this genus. Here we developed a web-assisted method to achieve an easy and accurate Paratylenchus species characterization that will greatly improve the identification of these plant-parasitic nematodes for many diagnostic laboratories and researchers. However, this identification needs to be completed with the use of molecular markers available for the species due to the existence of species complexes studied in former researches. This idea is pointed in the polytomous key in the specific species complexes up-to-know. In some cases, the presence in the soil as survival stage of few individuals in the fourth-stage juvenile (J4) required the use of molecular markers for species identification. We suggest the use of at least a fragment of mitochondrial COI gene for species identification or the combination of nuclear D2-D3 regions of the 28S rRNA and the COI to complement each marker. However, for some species complexes, the use of the D2-D3 regions alone has not enough resolution to separate the putative species inside the species complex. Web-based polytomous key was constructed using the free software Xper3, for computers and mobile devices (smartphones, tablets, and pocket PCS).


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## Body

## 1. Introduction

Pin nematodes of the genusParatylenchus Micoletzky, 1922 [1] are obligate plant ectoparasitic nematodes of small body length (≤600 μm) with wide variable stylet length range (10-120 μm), extensively distributed worldwide in different natural environments and crops [2–5]. Tarjan [6] reviewed the genus Paratylenchus for the first time and proposed the first dichotomic key for 16 nominal species identification and based on 7 main diagnostic characters. During the last decades, there were numerous taxonomical discussions debating about the validity of the genera Gracilacus, Paratylenchoides, and Gracilpaurus, based on stylet length, heavy sclerotization in the lip region, or the presence of tubercles or punctations on the cuticle of mature females [7–9]. However, recent integrative taxonomic studies including ribosomal and mitochondrial genes questioned the monophyly of Tylenchulidae and do not support the validity of these genera that were considered synonyms of Paratylenchus [3–5, 10]. In any case, the Paratylenchus species description increased exponentially to 140 nominal species in the last 61 years (Table S1, 58 of them molecularly characterized), including the confirmation of examples of cryptic diversity [3–5, 10]. Although several diagnostic keys have been published during this period [2, 8, 11–14], given the large number of Paratylenchus species and their high morphological and morphometric similarity, the species identification of these nematodes is often a very difficult task. Ghaderi et al. [2, 14] pragmatically divided the genus into eleven groups based on the three most stable characters, such as stylet length, number of lines in the lateral field, and the presence/absence of vulval flaps in females. Within each group, species can be identified using dichotomic keys based on presence/absence of males, shape of the spermatheca, tail terminus, etc., following a hierarchical system. In addition, Ghaderi et al. [14] proposed a diagnostic compendium based on seven items: pragmatical group, lip region from lateral view, males and their stylets, spermatheca shape, fourth-juvenile stylet, cuticle annulation, and tail terminus shape. Although these keys can help in the morphometrical identification of Paratylenchus spp., it still lacks a useful, wider, holistic, and accurate key using a wide list of morphological and morphometrical traits, included in a comprehensive web-based polytomous key which may help in the identification of known and new species within this genus, as well as the potential separation of species complexes differing in a few traits. Over the last two decades, the advance of new web-based techniques represents an excellent opportunity to generate useful species key tools for helping in nematode identification [15]. These tools, such as Xper3, allow the integration of large amounts of quantitative data, as well as accurate and precise descriptions using a wider set of qualitative and quantitative characters that can be applied simultaneously with no restrictions to the specimen to be identified [15, 16]. They are available in every browser at no charge [15, 16]. In Xper3, species identification is achieved in several steps by gradual elimination of species names that do not meet the selection criteria at each step of the process.Additionally to the use of morphological data for species identification, molecular-based approaches integrated with morphology and morphometric data have revealed an exponential increase in the number of cryptic species in the phylum Nematoda over recent decades [17–19]. In plant-parasitic nematodes, this phenomenon of cryptic speciation can be explained on the basis of genetic mutations and/or ecological adaptations to geographical location or host range [20, 21]. More specifically, pin nematodes are characterized by a conserved morphology that has led to the use of DNA barcoding with different fragments of nuclear and mitochondrial DNA to clarify different species groups [3–5, 10]. Several studies demonstrated the existence of cryptic diversity within the genus Paratylenchus and highlighted the need for correct and accurate species identification in food security and pest management strategies [3–5, 10]. The knowledge of intraspecific and interspecific sequence variability of the 28S rRNA and the mitochondrial cytochrome c oxidase subunit I (COI) genes within Paratylenchus is important to detect species misidentifications/incorrect identifications deposited in GenBank (https://www.ncbi.nlm.nih.gov/) or cryptic speciation [3–5, 10]. However, morphological data of nematode individuals prior to DNA extraction significantly improve species delimitation accuracy in comparison to simply molecular taxonomy [19]. An emblematic example of high cryptic species diversity within Paratylenchus comprises P. straeleni-complex species, distinguishing 4-9 putative species [3–5, 10], and one new species recently described from southern Spain, viz., P. parastraeleni [4], which 33 years before was identified as P. straeleni only based on morphometry and morphology [22]. Similarly, Van den Berg et al. [10] and Singh et al. [3] studied numerous Paratylenchus populations revealing several species complexes with few morphological differences in the main diagnostic characters, but clearly differing in molecular markers. Some examples of these species’ complexes include P. aquaticus complex (P. humilis, P. pandus, P. triincisus, and P. variatus), P. hamatus complex (Paratylenchus sp. 2 and Paratylenchus sp. CaD), and Paratylenchus nanus complex (P. projectus, P. neoprojectus, and P. neoamblycephalus). Another excellent example includes P. microdorus-complex species, extensively reported in Spain in cultivated and natural environments [23–26]. Recently, analyses based on integrative taxonomy identified different species with the basic morphology of P. microdorus, but molecularly well separated, including P. recisus, P. variabilis, P. veruculatus, and P. zurgenerus [4, 5, 27]. Probably, these potential misidentifications can also be referred to the numerous records of P. microdorus in other countries, such as Bulgaria, Germany, Hungary, Poland, and Romania [2], which need further investigation. Species belonging to the genus Paratylenchus display a particular resting stage which accumulates in soil under adverse environmental conditions [2, 4]. This state is nonfeeding, molting to adults after stimulation by host-plant roots, and may provide some useful data for species identification [2]. Usually the resting stage is fourth-stage juvenile (J4), but third-stage (J3) appears in other species, recognized by granular body contents and presence/absence of stylet [2]. This survival strategy makes an identification based on only morphological and morphometrical characters difficult. Thus, the establishment of clear and unequivocal molecular markers for species identification is even more necessary. The identification of Paratylenchus spp. became more complicated with the usual presence of several species in the same soil sample. Using an integrative taxonomy, we detected even four species within a soil sample and the coexistence of almost identical species sharing the same niche and the same host in our recent studies in cultivated Prunus spp. and natural environments in Spain [4, 5]. For these reasons, it is important to include molecular markers and more than one individual in the integrative taxonomy of this genus.Therefore, the objectives of this research were as follows: (1) to facilitateParatylenchus species identification, developing an open access web-assisted, interactive, and illustrated polytomous key based on a wide list of diagnostic and morphometric traits (freely accessible at https://nemabioli.ias.csic.es/paratylenchus/index.html), and (2) to evaluate and review the usefulness of D2-D3 regions of the 28S rRNA gene and the COI gene fragment for Paratylenchus species identification for some groups of species complexes within this genus.


## 2. Materials and Methods

### 2.1. Database of Paratylenchus Species and Diagnostic Characters

For constructing theParatylenchus database, we considered only the type population for each Paratylenchus nominal species according to the latest monograph of the genus published by Ghaderi et al. [2], as well as other Paratylenchus species populations based only on integrative taxonomical identification [3–5, 10, 28]. This is because of the great cryptic diversity detected in the recent studies of pin nematodes [3–5, 10, 28], which may alter the real morphometric delimitations for each species. For that, we analysed all main diagnostic Paratylenchus characters, comprising 11 numerical and 13 categorical characters (Table 1 and Figure 1). Selection for quantitative character division in subgroups within each diagnostic numerical character was based on K-means clustering method [29]. All data analyses were performed with the R software, version 4.1.0 [30]. The library NbClust v. 2.0.4 [31] was used to perform the clustering using Euclidean distances. We tested from 2 to 6 clusters. The best number of clusters was selected testing all indexes to optimize the K-means cluster number for one variable each time based on the NbClust library. The best number of groups was obtained as the number with the major quantity of accepted indexes. Once the best number of clusters was obtained, we calculated it using the library factoextra v. 1.0.7. using Euclidean distances. Range of the clusters for each character was checked for incongruences and limits included in the figures (Figure 2). All figures were created using the ggplot2 package version 3.3.3 [32]. All database characters could be obtained upon reasonable request to the authors.Table 1
Morphometric and morphological characters used to distinguishParatylenchus spp. in the web-assisted polytomous key. Some characters are ranked in the program (shown in the table), and the rest are ranked to 3.
(A) Female stylet length (rank 5)A1: <23.5 μmA2: 23.5-40.0 μmA3: 40.1-69.5 μmA4: >69.5 μm(B) Excretory pore levelB1: at median bulb level or anteriorB2: at isthmus levelB3: at basal bulb level or posterior(C) Number of lines in the lateral field (rank 4)C1: 2 linesC2: 3 linesC3: 4 linesC4: indistinct lines(D) Advulval flap (rank 4)D1: presentD2: absent(E) Lip region shape in lateral viewE1: conoidE2: roundedE3: disc-shapeE4: truncate, concave, anteriorly flattenedE5: cap-like structure(F) Submedian lobes in lateral viewF1: without submedian lobesF2: with small submedian lobesF3: with prominent submedian lobes(G) Vulva-anus distanceG1: ≤32.75 μmG2: 32.76-47.50 μmG3: >47.50 μm(H) Spermatheca shapeH1: rounded to sphericalH2: elongated, ovalH3: not developed, inconspicuous(I)Femalestyletlenght/L×100I1: ≤12.20%I2: 12.21-21.8%I3: >21.8%(J) MaleJ1: unknownJ2: present(K) Male tail shapeK1: conoidK2: rounded, cylindricalK3: filiform terminusK4: unknown(L) Male styletL1: unknownL2: without styletL3: with stylet(M) c’ ratioM1: ≤2.25M2: 2.26-2.85M3: 2.86-3.35M4: 3.36-3.95M5: >3.95(N) b ratioN1: ≤2.85N2: 2.86-3.65N3: 3.66-4.25N4: >4.25(O) Body length (L)O1: ≤253 μmO2: 253-303 μmO3: 304-356 μmO4: 357-426 μmO5: >426 μm(P) Spicules’ lengthP1: ≤14.25 μmP2: 14.26-16.75 μmP3: 16.76-19.75 μmP4: 19.76-23.0 μmP5: >23.0 μm(Q) Female tail terminus shapeQ1: acute or pointedQ2: subacute to finely roundedQ3: bluntly roundedQ4: digitate, lobed, or indentedQ5: serrated(R) V ratio (%)R1: ≤76.0%R2: 76.1-81.0%R3: >81.0%(S) Female tail lengthS1: ≤26.4 μmS2: >26.4 μm(T) J4 styletT1: absentT2: presentT3: unknown(U) c ratioU1: ≤13.25U2: 13.26-17.25U3: >17.25(V) Female tail shapeV1: conoidV2: cylindricalV3: subcylindrical, elongate(W) Cuticle annulationW1: normal annulation, ca. 1 μmW2: cuticle with punctationsW3: cuticle annuli crenate(X) a ratioX1: ≤21.75X2: >21.7Figure 1
Morphological categorical characters and subgroups used in the web-assistedParatylenchus species polytomous key.Figure 2
K-means of morphometric numerical characters ofParatylenchus species for subgroup separation.


### 2.2. Xper3 Software Polytomous Key

The web-based polytomous key was constructed using the free softwareXper3 version 1.4.0 (https://www.xper3.fr), it is compatible for computers and mobile devices (smartphones, tablets, and pocket PCS), and it is freely accessible at https://nemabioli.ias.csic.es/paratylenchus/index.html. This software allows for the selection of any character (morphological or morphometrical) within a set of descriptors for the identification of Paratylenchus. Descriptors were ranked with the built-in ranking system of Xper3 (rank 1: little important and 5: highly important) according to their distinct nature and relative simplicity to study. Measurements and indexes for all species were read in as quantitative data with fixed ranges. Completeness of the database was checked using the inbuilt tools for analysis of the data. The tool “Checkbase” is an automatic search for various errors in the database, for instance, items with identical descriptions, undescribed or inapplicable descriptors, and descriptors described as “unknown.” A direct link to these errors provides a handy means for quick and easy revision. Xper3 lists all Paratylenchus species including all descriptors which can be edited and weighted. A view of the complete description matrix with the possibility to search for undescribed items and the revision of these is of great help to make sure that each species is completely described. We ranked descriptors because some characters showed greater stability inside the same species and also less prone to create artefacts during nematode fixation prior measurements, such as female stylet length, number or lines in lateral fields, and the presence or absence of advulval flaps. Moreover, these characters were recommended by other authors to create groups [14]. This is also a great way to simplify and speed up the identification process, allowing quick and reliable results, for instance, rank 5 for female stylet length as a descriptor highly important, rank 4 for the number of lines in lateral field and advulval flap, and rank 3 for the rest of morphometric and morphological characters (Table 1). In addition, the morphological and morphometric characters of the male (viz., male tail shape, male stylet, and spicule length) were conditioned to the presence of males (Table 1). Finally, in Xper3, it is possible to select various species or groups for comparison of all characters. Although users can decide the starting character to initiating the Paratylenchus identification, the authors suggest to follow the order and weighted proposed. In any case, the section “History” provides a helpful means to look up the identification process and correct potential mismatches without resetting the complete selection. In the case that the aided key leads to several species, the resulting identification will be based on the individual comparison of the target species with original descriptions and the help of molecular markers.Graphical picture/s available in an illustrated window will appear for categorical characters (viz., advulval flap, excretory pore level, lip region shape, submedian lobes, female and male tail shape, female tail terminus, and cuticle annulation) when the user clicks on these web characters; then, the user enters the corresponding appropriate state for his/her sample data.The present interactive and illustrated web-based polytomous key has been successfully tested on popular browsers in Windows, Linux, and MacOS.


### 2.3. Sequence Variability Analyses of D2-D3 Regions and the COI Fragment ofParatylenchus Species

A total of 340 and 292 sequences assigned to the genusParatylenchus were retrieved from GenBank and used to calculate the intraspecific and interspecific sequence variability of D2-D3 regions and the COI gene fragment, respectively. For sequence selection from NCBI, we have taken into consideration all the annotated misidentifications indicated by recent papers based on integrative taxonomical approaches [3–5, 10]. These molecular markers are commonly used in the molecular identification of species in this genus. Internal transcribed spacer (ITS) region and partial 18S rRNA are also used, but the first one has less sequences deposited in GenBank in comparison to D2-D3 regions, and partial 18S rRNA has a lower resolution for species identification than the other most frequently used markers [3]. For interspecific sequence variability, one consensus sequence was obtained from Paratylenchus each species, resulting an alignment of 69 and 57 consensus sequences of 575 and 320 bp from D2-D3 regions and the COI fragment, respectively. Sequence alignment was manually edited using BioEdit [33] in order to improve the default multialignment. Then, pairwise identity expressed in percentage among taxa was computed using Sequence Demarcation Tool version 1.2 (SDT v1.2) with MAFFT alignment options and adjustment for missing data [34]. On the other hand, for the intraspecific sequence variability, one dataset from each species with more than one available sequence (Tables S2-S3) was created and aligned using MAFFT v. 7.450 using the FFT-NS-2 algorithm [35]. Then, intraspecific sequence variability was calculated for both molecular markers in the number of nucleotide differences using BioEdit [33].


## 2.1. Database of Paratylenchus Species and Diagnostic Characters

For constructing theParatylenchus database, we considered only the type population for each Paratylenchus nominal species according to the latest monograph of the genus published by Ghaderi et al. [2], as well as other Paratylenchus species populations based only on integrative taxonomical identification [3–5, 10, 28]. This is because of the great cryptic diversity detected in the recent studies of pin nematodes [3–5, 10, 28], which may alter the real morphometric delimitations for each species. For that, we analysed all main diagnostic Paratylenchus characters, comprising 11 numerical and 13 categorical characters (Table 1 and Figure 1). Selection for quantitative character division in subgroups within each diagnostic numerical character was based on K-means clustering method [29]. All data analyses were performed with the R software, version 4.1.0 [30]. The library NbClust v. 2.0.4 [31] was used to perform the clustering using Euclidean distances. We tested from 2 to 6 clusters. The best number of clusters was selected testing all indexes to optimize the K-means cluster number for one variable each time based on the NbClust library. The best number of groups was obtained as the number with the major quantity of accepted indexes. Once the best number of clusters was obtained, we calculated it using the library factoextra v. 1.0.7. using Euclidean distances. Range of the clusters for each character was checked for incongruences and limits included in the figures (Figure 2). All figures were created using the ggplot2 package version 3.3.3 [32]. All database characters could be obtained upon reasonable request to the authors.Table 1
Morphometric and morphological characters used to distinguishParatylenchus spp. in the web-assisted polytomous key. Some characters are ranked in the program (shown in the table), and the rest are ranked to 3.
(A) Female stylet length (rank 5)A1: <23.5 μmA2: 23.5-40.0 μmA3: 40.1-69.5 μmA4: >69.5 μm(B) Excretory pore levelB1: at median bulb level or anteriorB2: at isthmus levelB3: at basal bulb level or posterior(C) Number of lines in the lateral field (rank 4)C1: 2 linesC2: 3 linesC3: 4 linesC4: indistinct lines(D) Advulval flap (rank 4)D1: presentD2: absent(E) Lip region shape in lateral viewE1: conoidE2: roundedE3: disc-shapeE4: truncate, concave, anteriorly flattenedE5: cap-like structure(F) Submedian lobes in lateral viewF1: without submedian lobesF2: with small submedian lobesF3: with prominent submedian lobes(G) Vulva-anus distanceG1: ≤32.75 μmG2: 32.76-47.50 μmG3: >47.50 μm(H) Spermatheca shapeH1: rounded to sphericalH2: elongated, ovalH3: not developed, inconspicuous(I)Femalestyletlenght/L×100I1: ≤12.20%I2: 12.21-21.8%I3: >21.8%(J) MaleJ1: unknownJ2: present(K) Male tail shapeK1: conoidK2: rounded, cylindricalK3: filiform terminusK4: unknown(L) Male styletL1: unknownL2: without styletL3: with stylet(M) c’ ratioM1: ≤2.25M2: 2.26-2.85M3: 2.86-3.35M4: 3.36-3.95M5: >3.95(N) b ratioN1: ≤2.85N2: 2.86-3.65N3: 3.66-4.25N4: >4.25(O) Body length (L)O1: ≤253 μmO2: 253-303 μmO3: 304-356 μmO4: 357-426 μmO5: >426 μm(P) Spicules’ lengthP1: ≤14.25 μmP2: 14.26-16.75 μmP3: 16.76-19.75 μmP4: 19.76-23.0 μmP5: >23.0 μm(Q) Female tail terminus shapeQ1: acute or pointedQ2: subacute to finely roundedQ3: bluntly roundedQ4: digitate, lobed, or indentedQ5: serrated(R) V ratio (%)R1: ≤76.0%R2: 76.1-81.0%R3: >81.0%(S) Female tail lengthS1: ≤26.4 μmS2: >26.4 μm(T) J4 styletT1: absentT2: presentT3: unknown(U) c ratioU1: ≤13.25U2: 13.26-17.25U3: >17.25(V) Female tail shapeV1: conoidV2: cylindricalV3: subcylindrical, elongate(W) Cuticle annulationW1: normal annulation, ca. 1 μmW2: cuticle with punctationsW3: cuticle annuli crenate(X) a ratioX1: ≤21.75X2: >21.7Figure 1
Morphological categorical characters and subgroups used in the web-assistedParatylenchus species polytomous key.Figure 2
K-means of morphometric numerical characters ofParatylenchus species for subgroup separation.


## 2.2. Xper3 Software Polytomous Key

The web-based polytomous key was constructed using the free softwareXper3 version 1.4.0 (https://www.xper3.fr), it is compatible for computers and mobile devices (smartphones, tablets, and pocket PCS), and it is freely accessible at https://nemabioli.ias.csic.es/paratylenchus/index.html. This software allows for the selection of any character (morphological or morphometrical) within a set of descriptors for the identification of Paratylenchus. Descriptors were ranked with the built-in ranking system of Xper3 (rank 1: little important and 5: highly important) according to their distinct nature and relative simplicity to study. Measurements and indexes for all species were read in as quantitative data with fixed ranges. Completeness of the database was checked using the inbuilt tools for analysis of the data. The tool “Checkbase” is an automatic search for various errors in the database, for instance, items with identical descriptions, undescribed or inapplicable descriptors, and descriptors described as “unknown.” A direct link to these errors provides a handy means for quick and easy revision. Xper3 lists all Paratylenchus species including all descriptors which can be edited and weighted. A view of the complete description matrix with the possibility to search for undescribed items and the revision of these is of great help to make sure that each species is completely described. We ranked descriptors because some characters showed greater stability inside the same species and also less prone to create artefacts during nematode fixation prior measurements, such as female stylet length, number or lines in lateral fields, and the presence or absence of advulval flaps. Moreover, these characters were recommended by other authors to create groups [14]. This is also a great way to simplify and speed up the identification process, allowing quick and reliable results, for instance, rank 5 for female stylet length as a descriptor highly important, rank 4 for the number of lines in lateral field and advulval flap, and rank 3 for the rest of morphometric and morphological characters (Table 1). In addition, the morphological and morphometric characters of the male (viz., male tail shape, male stylet, and spicule length) were conditioned to the presence of males (Table 1). Finally, in Xper3, it is possible to select various species or groups for comparison of all characters. Although users can decide the starting character to initiating the Paratylenchus identification, the authors suggest to follow the order and weighted proposed. In any case, the section “History” provides a helpful means to look up the identification process and correct potential mismatches without resetting the complete selection. In the case that the aided key leads to several species, the resulting identification will be based on the individual comparison of the target species with original descriptions and the help of molecular markers.Graphical picture/s available in an illustrated window will appear for categorical characters (viz., advulval flap, excretory pore level, lip region shape, submedian lobes, female and male tail shape, female tail terminus, and cuticle annulation) when the user clicks on these web characters; then, the user enters the corresponding appropriate state for his/her sample data.The present interactive and illustrated web-based polytomous key has been successfully tested on popular browsers in Windows, Linux, and MacOS.


## 2.3. Sequence Variability Analyses of D2-D3 Regions and the COI Fragment ofParatylenchus Species

A total of 340 and 292 sequences assigned to the genusParatylenchus were retrieved from GenBank and used to calculate the intraspecific and interspecific sequence variability of D2-D3 regions and the COI gene fragment, respectively. For sequence selection from NCBI, we have taken into consideration all the annotated misidentifications indicated by recent papers based on integrative taxonomical approaches [3–5, 10]. These molecular markers are commonly used in the molecular identification of species in this genus. Internal transcribed spacer (ITS) region and partial 18S rRNA are also used, but the first one has less sequences deposited in GenBank in comparison to D2-D3 regions, and partial 18S rRNA has a lower resolution for species identification than the other most frequently used markers [3]. For interspecific sequence variability, one consensus sequence was obtained from Paratylenchus each species, resulting an alignment of 69 and 57 consensus sequences of 575 and 320 bp from D2-D3 regions and the COI fragment, respectively. Sequence alignment was manually edited using BioEdit [33] in order to improve the default multialignment. Then, pairwise identity expressed in percentage among taxa was computed using Sequence Demarcation Tool version 1.2 (SDT v1.2) with MAFFT alignment options and adjustment for missing data [34]. On the other hand, for the intraspecific sequence variability, one dataset from each species with more than one available sequence (Tables S2-S3) was created and aligned using MAFFT v. 7.450 using the FFT-NS-2 algorithm [35]. Then, intraspecific sequence variability was calculated for both molecular markers in the number of nucleotide differences using BioEdit [33].


## 3. Results

### 3.1. Interactive Web-Based Polytomous Key of Paratylenchus spp.

The interactive and illustrated web-basedParatylenchus polytomous key (version 1, April 2022, 140 species) is hosted and freely available on the server of the Spanish National Research Council (CSIC), Madrid, Spain at https://nemabioli.ias.csic.es/paratylenchus/index.html, and it is freely accessible to any user.Quantitative character division in subgroups within each diagnostic numerical character was based on K-means clustering method (Figure2). All data analyses were performed testing from 2 to 6 clusters, and the best number of groups was obtained as the number with the major number of indexes, checking for incongruences and limits included in the figures (Figure 2). The best subgroups for these characters comprised 5 subgroups for female body length (L), c’ ratio, and spicule length; 4 subgroups for female stylet length and b ratio; 3 subgroups for percentage of female stylet length/L, vulva-anus distance, V ratio, and c ratio; and 2 subgroups for female tail length and a ratio (Figure 2).The introduction of sample data inXper3 implies that the software will exclude every taxon which does not fit the set of states and give a list of the remaining possible species names. After sample data introduction, the interactive key enables the simultaneous comparison of all characters of the unknown Paratylenchus population with the data of Paratylenchus-database species and provides and displays the identified species if all characters are coincident with those of species in the database. Alternatively, the web application will provide a list of species that are the most similar to the user’s nematode sample. In addition, the identified species or the relevant species group provides a link to NCBI accessions in case they have any available molecular markers for species confirmation. Authors recommend verifying species identification and potential grouping with other species using molecular markers if the species selected have available sequence data or species-specific PCR primers. This key will be updated if new species are described or any taxonomical change is provided for any of the 140 nominal valid species. Some of these species’ complexes will be studied in more detail in the next point, but in this interactive polytomous key species, we included the original descriptions as well as those reports based on molecular markers, but we did not include species not described formally. However, the topotype and the measured populations are included in the key (see the example of P. straeleni with topotype population, Belgium and USA, respectively).


### 3.2. Sequence Variability of D2-D3 Regions and the COI Fragment Genes within Paratylenchus spp. and Species Complexes

Intraspecific and interspecific sequence variability within the genusParatylenchus was analysed based on nuclear and mitochondrial DNA (D2-D3 regions of 28S rRNA and the COI fragment). The intraspecific sequence identity of D2-D3 regions detected in the 67 studied Paratylenchus species ranged from 100 to 97% (from 0 to 28 different nucleotides per 575 nucleotide positions), while for the 63 Paratylenchus species included in the COI fragment analyses, these values ranged from 100 to 95% (from 0 to 31 different nucleotides per 320 nucleotide positions). Interestingly, the amount of intraspecific variability was found to be highly variable, and the majority of species with more than one sequence available in GenBank showed low or none intraspecific variability (Tables S2-S3). For example, for both markers, D2-D3 regions and the COI fragment, no variability was detected for some species, such as P. amundseni, P. elachistus, P. holdemani, P. tateae, P. variabilis, or P. verus, while other species, such as P. enigmaticus, P. goodeyi, P. straeleni, and P. veruculatus, presented highly diverse haplotypes. These species have been previously studied, and some of them were identified as a complex of cryptic species by Singh et al. [3] and Clavero-Camacho et al. [5]. Therefore, these species are difficult to be separated morphologically. The study of these complexes is shown in Table 2.Table 2
Species complexes included in the polytomous key with molecular markers needed for putative species separation. Many of these putative species are not formally described as a new species. Species separation data were obtained from Singh et al. [3] and Clavero-Camacho et al. [4, 5].
SpeciesD2-D3 regionsCOIn. speciesa% similaritySeq. GenBankbSuitabilitycn. speciesa% similaritySeq. GenBankbSuitabilitycP. aquaticus273.2-98.54 (4)Yes281.12 (2)YesP. enigmaticus299.8-10017 (2)No397.1-1007 (4)YesP. goodeyi198.3-10032 (9)No794.3-10019 (15)YesP. hamatus/P. sp. 2/P. sp. CaD/P. tenuicaudatus494.4-10049 (15)Yes493.7-10017 (4)YesP. sheri1∗99.6-1006 (3)No294.3-1009 (3)YesP. straeleni/P. parastraeleni693.0-10014 (9)No1091.1-10029 (14)YesP. veruculatus297.4-10017 (7)No393.7-10013 (6)YesaBased on species separation methods [3] or similarity cut-off (approx. 98% for D2-D3 regions and 97.5% for the COI fragment). bNumber of different haplotypes in brackets. cSuitability to separate species inside the species-complex taking the number of species with COI marker for the D2D3 regions. ∗This marker cannot separate P. sheri from P. israelensis and P. neoamblycephalus.Multiple alignments of D2-D3 regions and the COI fragment consensus sequences betweenParatylenchus spp. showed identities ranging from 68% to 99% and 70% to 98%, respectively (Figure 3). The nucleotide differences among Paratylenchus species ranged from 2 to 188 nucleotides for D2-D3 region and from 7 to 88 nucleotides for COI from an alignment of 575 and 320 bp length, respectively. The pairwise similarity observed between Paratylenchus species was, in almost all cases, below 95% (Figure 3), except for some species complexes, such as P. pandatus, P. macrodorus, and P. wuae, which showed similarity values of 98% for the D2-D3 regions; however, for the COI fragment, this value was of 94% (Figure 3). These three species comprise a species complex with similar morphology in several diagnostic characters including stylet length, excretory pore level, lateral field lines, and female tail shape, but clearly differing in others, such as advulval flap (absent in P. pandatus vs. present), lip region shape (conoid in P. wuae vs. rounded), submedian lobes (prominent in P. wuae vs. small), spermatheca (rounded in P. wuae vs. elongated-oval), and shorter vulva-anus distance in P. wuae vs. large in P. macrodorus and P. pandatus [2, 36].Figure 3
Colour-coded pairwise nucleotide sequence identity matrix ofParatylenchus spp. of (a) D2-D3 regions and the (b) COI fragment sequences from GenBank using SDT v1.2. Each coloured cell represents a percentage identity score between two sequences (one indicated horizontally to the left and the other vertically at the bottom).
(a)(b)Almost identical pairwise identity was found forP. sheri, P. israelensis, and P. neoamblycephalus. The D2-D3 region sequences of these species were 98% similar among them, but the similarities among the COI fragment sequences were 93-94%. However, these three species, although sharing some similar morphology in several diagnostic characters including stylet length, lateral field lines, presence of vulval flaps, female tail shape, and tail terminus, clearly differed in others, such as spermatheca (rounded in P. sheri vs. elongated-oval), submedian lobes (prominent in P. israelensis vs. small and absent in P. sheri and P. neoamblycephalus, respectively), and shorter vulva-anus distance in P. sheri vs. large in P. israelensis and P. neoamblycephalus [2, 5].The species complex comprisingP. colinus, P. aciculus, P. aculentus, P. audriellus, and P. paralatescens showed a 99% similarity in D2-D3 regions, but unfortunately, only the COI fragment sequences of P. aciculus and P. aculentus were available in GenBank, with 90% similarity between them. This case has previously been studied by Singh et al. [3] and Munawar et al. [37], who distinguished P. colinus, P. aciculus, P. aculentus, and P. paralatescens according to some morphological characters, including stylet length (larger in P. paralatescens), excretory pore position (at the isthmus level in P. audriellus and P. colinus), lateral field lines (four in P. audriellus vs. three), advulval flaps (present in P. audriellus vs. absent), vulva-anus distance (larger in P. aciculus and P. paralatescens vs. shorter), and spermatheca (elongate-oval in P. aciculus and P. paralatescens vs. rounded) [3, 5, 38].Finally, similarity values from 97 to 99% were found for the D2-D3 regions amongP. neoprojectus, P. projectus, P. coronatus, Paratylenchus sp. C SAS 2019, and Paratylenchus sp. 4 (Figure 3). However, Paratylenchus sp. 4 and P. projectus can be separated on the basis of COI sequences (91% similar to each other). Sequence data on 28S rRNA and ITS from P. neoprojectus and P. projectus suggest that both species can be synonymized, since minor morphological differences can be detected between them [3, 37]; however, additional data on the COI for P. neoprojectus are needed for confirming this new status. Likewise, D2-D3 from P. coronatus is highly similar to P. neoprojectus (98.3% similarity), and the detailed comparison of morphometrics from both species suggests that both can be synonymized [37, 39]. Unfortunately, both species have no available COI sequences for confirmation.


## 3.1. Interactive Web-Based Polytomous Key of Paratylenchus spp.

The interactive and illustrated web-basedParatylenchus polytomous key (version 1, April 2022, 140 species) is hosted and freely available on the server of the Spanish National Research Council (CSIC), Madrid, Spain at https://nemabioli.ias.csic.es/paratylenchus/index.html, and it is freely accessible to any user.Quantitative character division in subgroups within each diagnostic numerical character was based on K-means clustering method (Figure2). All data analyses were performed testing from 2 to 6 clusters, and the best number of groups was obtained as the number with the major number of indexes, checking for incongruences and limits included in the figures (Figure 2). The best subgroups for these characters comprised 5 subgroups for female body length (L), c’ ratio, and spicule length; 4 subgroups for female stylet length and b ratio; 3 subgroups for percentage of female stylet length/L, vulva-anus distance, V ratio, and c ratio; and 2 subgroups for female tail length and a ratio (Figure 2).The introduction of sample data inXper3 implies that the software will exclude every taxon which does not fit the set of states and give a list of the remaining possible species names. After sample data introduction, the interactive key enables the simultaneous comparison of all characters of the unknown Paratylenchus population with the data of Paratylenchus-database species and provides and displays the identified species if all characters are coincident with those of species in the database. Alternatively, the web application will provide a list of species that are the most similar to the user’s nematode sample. In addition, the identified species or the relevant species group provides a link to NCBI accessions in case they have any available molecular markers for species confirmation. Authors recommend verifying species identification and potential grouping with other species using molecular markers if the species selected have available sequence data or species-specific PCR primers. This key will be updated if new species are described or any taxonomical change is provided for any of the 140 nominal valid species. Some of these species’ complexes will be studied in more detail in the next point, but in this interactive polytomous key species, we included the original descriptions as well as those reports based on molecular markers, but we did not include species not described formally. However, the topotype and the measured populations are included in the key (see the example of P. straeleni with topotype population, Belgium and USA, respectively).


## 3.2. Sequence Variability of D2-D3 Regions and the COI Fragment Genes within Paratylenchus spp. and Species Complexes

Intraspecific and interspecific sequence variability within the genusParatylenchus was analysed based on nuclear and mitochondrial DNA (D2-D3 regions of 28S rRNA and the COI fragment). The intraspecific sequence identity of D2-D3 regions detected in the 67 studied Paratylenchus species ranged from 100 to 97% (from 0 to 28 different nucleotides per 575 nucleotide positions), while for the 63 Paratylenchus species included in the COI fragment analyses, these values ranged from 100 to 95% (from 0 to 31 different nucleotides per 320 nucleotide positions). Interestingly, the amount of intraspecific variability was found to be highly variable, and the majority of species with more than one sequence available in GenBank showed low or none intraspecific variability (Tables S2-S3). For example, for both markers, D2-D3 regions and the COI fragment, no variability was detected for some species, such as P. amundseni, P. elachistus, P. holdemani, P. tateae, P. variabilis, or P. verus, while other species, such as P. enigmaticus, P. goodeyi, P. straeleni, and P. veruculatus, presented highly diverse haplotypes. These species have been previously studied, and some of them were identified as a complex of cryptic species by Singh et al. [3] and Clavero-Camacho et al. [5]. Therefore, these species are difficult to be separated morphologically. The study of these complexes is shown in Table 2.Table 2
Species complexes included in the polytomous key with molecular markers needed for putative species separation. Many of these putative species are not formally described as a new species. Species separation data were obtained from Singh et al. [3] and Clavero-Camacho et al. [4, 5].
SpeciesD2-D3 regionsCOIn. speciesa% similaritySeq. GenBankbSuitabilitycn. speciesa% similaritySeq. GenBankbSuitabilitycP. aquaticus273.2-98.54 (4)Yes281.12 (2)YesP. enigmaticus299.8-10017 (2)No397.1-1007 (4)YesP. goodeyi198.3-10032 (9)No794.3-10019 (15)YesP. hamatus/P. sp. 2/P. sp. CaD/P. tenuicaudatus494.4-10049 (15)Yes493.7-10017 (4)YesP. sheri1∗99.6-1006 (3)No294.3-1009 (3)YesP. straeleni/P. parastraeleni693.0-10014 (9)No1091.1-10029 (14)YesP. veruculatus297.4-10017 (7)No393.7-10013 (6)YesaBased on species separation methods [3] or similarity cut-off (approx. 98% for D2-D3 regions and 97.5% for the COI fragment). bNumber of different haplotypes in brackets. cSuitability to separate species inside the species-complex taking the number of species with COI marker for the D2D3 regions. ∗This marker cannot separate P. sheri from P. israelensis and P. neoamblycephalus.Multiple alignments of D2-D3 regions and the COI fragment consensus sequences betweenParatylenchus spp. showed identities ranging from 68% to 99% and 70% to 98%, respectively (Figure 3). The nucleotide differences among Paratylenchus species ranged from 2 to 188 nucleotides for D2-D3 region and from 7 to 88 nucleotides for COI from an alignment of 575 and 320 bp length, respectively. The pairwise similarity observed between Paratylenchus species was, in almost all cases, below 95% (Figure 3), except for some species complexes, such as P. pandatus, P. macrodorus, and P. wuae, which showed similarity values of 98% for the D2-D3 regions; however, for the COI fragment, this value was of 94% (Figure 3). These three species comprise a species complex with similar morphology in several diagnostic characters including stylet length, excretory pore level, lateral field lines, and female tail shape, but clearly differing in others, such as advulval flap (absent in P. pandatus vs. present), lip region shape (conoid in P. wuae vs. rounded), submedian lobes (prominent in P. wuae vs. small), spermatheca (rounded in P. wuae vs. elongated-oval), and shorter vulva-anus distance in P. wuae vs. large in P. macrodorus and P. pandatus [2, 36].Figure 3
Colour-coded pairwise nucleotide sequence identity matrix ofParatylenchus spp. of (a) D2-D3 regions and the (b) COI fragment sequences from GenBank using SDT v1.2. Each coloured cell represents a percentage identity score between two sequences (one indicated horizontally to the left and the other vertically at the bottom).
(a)(b)Almost identical pairwise identity was found forP. sheri, P. israelensis, and P. neoamblycephalus. The D2-D3 region sequences of these species were 98% similar among them, but the similarities among the COI fragment sequences were 93-94%. However, these three species, although sharing some similar morphology in several diagnostic characters including stylet length, lateral field lines, presence of vulval flaps, female tail shape, and tail terminus, clearly differed in others, such as spermatheca (rounded in P. sheri vs. elongated-oval), submedian lobes (prominent in P. israelensis vs. small and absent in P. sheri and P. neoamblycephalus, respectively), and shorter vulva-anus distance in P. sheri vs. large in P. israelensis and P. neoamblycephalus [2, 5].The species complex comprisingP. colinus, P. aciculus, P. aculentus, P. audriellus, and P. paralatescens showed a 99% similarity in D2-D3 regions, but unfortunately, only the COI fragment sequences of P. aciculus and P. aculentus were available in GenBank, with 90% similarity between them. This case has previously been studied by Singh et al. [3] and Munawar et al. [37], who distinguished P. colinus, P. aciculus, P. aculentus, and P. paralatescens according to some morphological characters, including stylet length (larger in P. paralatescens), excretory pore position (at the isthmus level in P. audriellus and P. colinus), lateral field lines (four in P. audriellus vs. three), advulval flaps (present in P. audriellus vs. absent), vulva-anus distance (larger in P. aciculus and P. paralatescens vs. shorter), and spermatheca (elongate-oval in P. aciculus and P. paralatescens vs. rounded) [3, 5, 38].Finally, similarity values from 97 to 99% were found for the D2-D3 regions amongP. neoprojectus, P. projectus, P. coronatus, Paratylenchus sp. C SAS 2019, and Paratylenchus sp. 4 (Figure 3). However, Paratylenchus sp. 4 and P. projectus can be separated on the basis of COI sequences (91% similar to each other). Sequence data on 28S rRNA and ITS from P. neoprojectus and P. projectus suggest that both species can be synonymized, since minor morphological differences can be detected between them [3, 37]; however, additional data on the COI for P. neoprojectus are needed for confirming this new status. Likewise, D2-D3 from P. coronatus is highly similar to P. neoprojectus (98.3% similarity), and the detailed comparison of morphometrics from both species suggests that both can be synonymized [37, 39]. Unfortunately, both species have no available COI sequences for confirmation.


## 4. Discussion

MorphologicalParatylenchus species identification is particularly complex because of the large number of species within the genus (140), the great phenotypic plasticity (including remarkable cryptic diversity), and the limited availability of molecular markers for many species. Then, the use of available software, such as Xper3, allows to analyse large amounts of descriptive data facilitating the identification process. Morphological identification of Paratylenchus species can be reduced in time and steps with respect to dichotomous keys. As an example, the identification of Paratylenchus ciccaronei and P. neoamblycephalus in morpho-group 3 (stylet<40 μm, 4 lines in lateral fields and advulval flaps present) by Ghaderi et al. [2] requires 49 steps, whereas with our interactive web-based key, only 8 and 6 steps are needed, respectively. This means an optimization of Paratylenchus spp. identification, as well as a potential comparison with closer related species. The free access of any user to the polytomous key assures that everybody accessing to the link will be working with the latest version, since any change or updating of the database will not affect the permanent URL. Because the polytomous key and database are deposited at a public institutional research organization (CSIC) linked to a nematological research group with young researchers, we assure the long-term storage of these data. Although some morphometric characters and indexes showed a narrow range of measurements and ratios (viz., styletlength/bodylength×100, V, vulva-anus distance, a, c, and tail length), we maintain the separation in subgroups estimated by K-means method (Figure 2), since it can help in the separation of some species groups or cryptic species (i.e., P. microdorus complex). The new web-based tool can also help to new species identification. Still, because of the great phenotypic plasticity of these nematodes, the Xper3 results must be corroborated with the original description or redescriptions of the species, as well as multilocus molecular markers, to find the correct species. Van den Berg et al. [10] proposed the synonymy of P. pandus, P. triincisus, and P. variatus with P. aquaticus; however, the species comparison under the Xper3 polytomous key separates these species by the corresponding matrix codes (Table S4). We have confirmed that application of the new Xper3 polytomous key is decisive and efficient in the morphological-morphometrical separation of Paratylenchus species comprising species closely related morphologically to P. aquaticus [10]. Consequently, since no molecular characterization of these proposed synonymized species is provided, we maintain a conservative proposal considering all of them as valid species until molecular data can confirm if these few differences can be attributable to intraspecific variation or to the real cryptic species.Several recently published studies have contributed to unravelling the species delineation within the genusParatylenchus, resolving many misidentifications, describing new species, and assigning molecular data to known species. This is important because the amount of sequences deposited in GenBank has greatly increased in recent years [3–5, 37]. The use of both D2-D3 regions and the COI fragment sequences appears most promising for species identification of Paratylenchus genus, being the COI more effective to separate putative species [3–5, 37]. The sequences of D2-D3 regions showed the resolution necessary to separate the majority of Paratylenchus species. However, some species-complex groups remain unresolved with D2-D3 regions (showing 99% similarity), viz., P. wuae-P. pandatus-P. macrodorus, P. colinus-P. aciculus-P. aculentus-P. audriellus-P. paralatescens, and P. sheri-P. israelensis-P. neoamblycephalus, requiring the COI fragment sequences for its correct identification and even the analyses of the morphological data. On the contrary, a high intraspecific variability within this marker was found in some other species, such as P. straeleni, P. enigmaticus, or P. veruculatus. These may suggest intraspecific genetic diversity or even cryptic species that need to be clarified; however, further studies are still needed to determine whether or not this high genetic diversity within the genus Paratylenchus is linked to the intrinsic characteristics of this taxonomic group.


## 5. Conclusions

The new web-based application to identifyParatylenchus species allows users to easily use the developed polytomous key for this genus, as well as an efficient tool for characterizing unknown and still undescribed species. In addition, the compatibility of this web application with any operation system and other mobile devices (smartphones, tablets, and pocket PCS) gives a great versatility, facilitating its use for any user irrespective of access to complicated electronic devices. This interactive and illustrated polytomous key will be very useful for nematologists and specialist-diagnosticians to identify Paratylenchus species in agricultural, forest, and natural environments. Even so, this key should be combined with nuclear and mitochondrial molecular markers in a multilocus analysis. We propose D2-D3 regions of 28S rRNA gene and the COI gene fragment as molecular markers due to their sequence variability and availability in public databases. In spite of that, some complex-species groups need further studies for confirming the specific status.



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*Source: 6216266-2022-07-04.xml*