Multi-locus phylogenetic analyses uncover species boundaries and reveal the occurrence of two new entomopathogenic nematode species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp.

Heterorhabditis nematodes used in this study were collected by us during different nematode collection campaigns carried out in Rwanda, Mexico, and India, or were collected by different collaborators at different locations around the world (Table S1) (Bai et al., 2013; Bhat et al., 2021b; Bruno et al., 2020; Carrera, 2015; Fallet et al., 2020; Mukuka et al., 2010; Rana et al., 2020; Yan et al., 2016).

One representative nematode isolate of each new species, MEX-39 and Rw14_N-C4a, was selected for detailed morphological and morphometrical characterization. First- and second-generation adult nematodes were obtained by dissecting infected G. mellonella larvae in Ringer’s solution. Infective juveniles (IJs) were collected after their emergence from G. mellonella larvae in White traps (White, 1927). Nematodes were killed with water at 60°C, then fixed in triethanolamine formalin (7  ml formalin, 2  ml triethanolamine, 91 ml ddH₂O), then dehydrated by the Seinhorst’s method, and finally transferred to glycerine (Bhat et al., 2019b; Courtney et al., 1955; Seinhorst, 1959, 1962). Nematodes were mounted in small drops of glycerine on permanent glass slides with extra layers of paraffin wax to prevent the flattening of the nematodes (Bhat et al., 2021a). Morphological measurements were taken using the Nikon DS-L1 software built in a phase contrast microscope (Nikon Eclipse 50i). Between 20 and 25 specimens at each developmental stage were measured. Light microscopy photographs were taken using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with differential interference contrast optics (DIC) and a Nikon Digital Sight DS–U1 camera. For the scanning electron microscopy (SEM), nematode specimens preserved in glycerine were processed as described by Abolafia (2015). For this, the nematodes were re–hydrated in distilled water, dehydrated in ethanol-acetone, critical-point dried with liquid carbon dioxide, mounted on SEM stubs with copper tape and coated with gold in a sputter coater. Specimens were observed and microphotographs were captured using a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany). All micrographs were processed using Adobe® Photoshop® CS. The obtained morphometrical characters were compared with those published in previous studies describing all the species of the genus, independently of their current status (valid, species inquirendae, synonym, etc) (Abd-Elgawad and Ameen, 2005; Agüera de Doucet and Doucet, 1986; Andaló et al., 2006; Bhat et al., 2019a; Bhat et al., 2021b; Edgington et al., 2011; Gardner et al., 1994; Hunt and Nguyen, 2016; Kajol et al., 2020; Kakulia and Mikaia, 1997; Khan et al., 1976; Liu, 1994; Liu and Berry, 1996; Li et al., 2012; Malan et al., 2008; Malan et al., 2014; Maneesakorn et al., 2015; Nguyen et al., 2004; Nguyen et al., 2006; Nguyen et al., 2008; Pereira, 1937; Phan et al., 2003; Plichta et al., 2009; Poinar and Veremchuk, 1970; Poinar, 1976; Poinar et al., 1987; Poinar et al., 1992; Rana et al., 2020; Sagun et al., 2015; Shahina et al., 2017; Shamseldean et al., 1996; Stock, 1993; Stock et al., 1996; Stock, 1997; Stock et al., 2002; Stock et al., 2009; Turco, 1970; Vanlalhlimpuia et al., 2018; Wouts, 1979).

Self-crossing and cross-hybridization experiments were carried out on lipid agar plates as described by Dix et al. (1992). Heterorhabditis ruandica n. sp. Rw14_N-C4a and H. zacatecana n. sp. MEX-39 were self-crossed, hybridized with each other and with H. bacteriophora CH21 (Rana et al., 2020). For this, one second–generation male and one second–generation virgin female were placed on lipid agar plates (35 mm diam.) and incubated at 27°C. Ten independent plates per crossing type were set. Progeny production was observed daily for a period of five consecutive days. Experiments were repeated three times under the same conditions.

Genomic DNA from about 10 to 20 thousand nematodes was extracted using the genomic DNA isolation kit following manufacturer’s instructions (Norgen Biotek Corp., Thorold, Ontario, Canada). The following genes/genomic regions were amplified by polymerase chain reaction (PCR): the D2–D3 expansion segments of the 28S rRNA, the internal transcribed spacer (ITS) region of the rRNA, the cytochrome c oxidase I (COI), the thin filament (F-actin)-associated protein (unc-87), and the calmodulin 1 (cmd-1). Primers used were selected based on previous publications (Dhakal et al., 2020; Joyce et al., 1994b; Regeai et al., 2009; Subbotin et al., 2006) (Table S2). PCR reactions consisted of 1 µL of genomic DNA, 12.5 µL of EmeraldAmp GT PCR Master Mix (Takara Bio, Shiga, Japan), 0.5 µL of both forward and reverse primers at 10 mM and 10.5 µL of dH₂O. The PCR reaction was performed using a thermocycler (Mastercycler nexus gradient, Eppendorf, Germany) with the following settings: (i) for ITS and D2–D3, 1 cycle of 1 min at 98°C followed by 35 cycles of 10 sec at 98°C, 30 sec at 50°C, 1 min 30 sec at 72°C, and by a single final elongation step at 72°C for 10 min; (ii) for cmd-1 and unc-87, 1 cycle of 1 min at 98°C followed by 40 cycles of 10 sec at 98°C, 30 sec at 50°C, 30 sec at 72°C, and by a single final elongation step at 72°C for 10 min; (iii) for COI, 1 cycle of 1 min at 98°C followed by 40 cycles of 10 sec at 98°C, 30 sec at 40°C, 30 sec at 72°C, and by a single final elongation step at 72°C for 10 min. PCR was followed by electrophoresis (45 min, 100 V) of 5 μl of PCR products in a 1% TBA (Tris–boric acid–EDTA) buffered agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, California, USA). PCR products were purified using the FastGene Gel/PCR extraction kit (Nippon Genetics Co., Japan) and sequenced using reverse and forward primers by Sanger sequencing (Microsynth AG, Balgach, Switzerland). Obtained sequences were manually curated and trimmed and deposited in the NCBI under the accession numbers given in Table S3. Sequences of the following nematode strains were obtained in this study: Heterorhabditis ruandica n. sp. (isolates Rw18_M-Hr1a, Rw18_M-Hr1b, and Rw14_N-C4a), H. zacatecana n. sp. (isolates MEX-39, MEX-40, and MEX-41), H. bacteriophora (isolates DE2, DE6, PT1, IT6, EN01, and TT01); H. georgiana Hbb, H. beicherriana H06, H. indica CH7, and H. atacamensis MEX-20. To complete this data set and to obtain genomic sequences of nematodes that belong to all the validly described species of the genus Heterorhabditis, we searched the database of the National Center for Biotechnology Information (NCBI) by the Basic Local Alignment Search Tool (BLAST) using the accession numbers of the sequences obtained previously (Dhakal et al., 2020) (Table S3). Resulting sequences were used to reconstruct phylogenetic relationships by the Maximum Likelihood method based on the following nucleotide substitution models: Hasegawa–Kishino–Yano (HKY + I) (cmd-1), Tamura–Nei (TN93 + G + I) (COI), Kimura 2-parameter (K2 + G) (D2–D3 and ITS), and Tamura 3-parameter (T92) (unc-87). To select the best substitution model, best-fit nucleotide substitution model analyses were carried out in MEGA 7 (Hasegawa et al., 1985; Kimura, 1980; Kumar et al., 2016; Nei and Kumar, 2000). Sequences were aligned with MUSCLE (v3.8.31) (Edgar, 2004). The trees with the highest log likelihood are shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. In some cases, a discrete Gamma distribution (+G) was used to model evolutionary rate differences among sites and the rate variation model allowed for some sites to be evolutionarily (+I). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Graphical representation and edition of the phylogenetic tree were performed with Interactive Tree of Life (v3.5.1) (Chevenet et al., 2006; Letunic and Bork, 2016).

The Photorhabdus entomopathogenic bacteria associated with H. ruandica n. sp. Rw14_N-C4a and H. zacatecana n. sp. MEX-39 nematodes were isolated as described by Machado et al. (2019), (2021b). Briefly, Galleria mellonella larvae (Lepidoptera: Pyralidae) were exposed to 100 nematode infective juveniles. Three to four days later, insect cadavers were surface–sterilized and cut open with a blade. Bacteria-digested internal organs were spread onto LB agar plates and incubated at 28°C for 24 to 48 h. Photorhabdus-like colonies were sub-cultured until monocultures were obtained. A single primary form colony was then selected and used for further experiments. Bacteria primary forms were determined by examining colony characteristics and by examining pigments uptake on NBTA plates (LB agar plates supplemented with 25 mg l^–1 bromothymol blue and 4 mg l^–1 triphenyl-2,3,5-tetrazolium chloride). The strains were further sub-cultured and maintained on LB agar plates at 28°C. To establish their taxonomic identities, we reconstructed phylogenetic relationships based on whole genome sequences of the isolated bacteria and all the different species/subspecies of the genus Photorhabdus (Machado et al., 2021a, b). To obtain genomic sequences, genomic DNA was extracted and purified using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Switzerland) following manufacturer’s instructions. The resulting DNA was used for library preparation using the TruSeq DNA PCR-Free LT Library Prep (FC-121-3003) kit. Indexed libraries were then pooled at equimolar concentrations and sequenced (2 × 150 bp) on an Illumina HiSeq 3000 instrument. Genomes were assembled using the Bactopia pipeline (Petit and Read, 2020). Briefly, the raw Illumina reads were quality trimmed using Trimmomatic 0.39 (Bolger et al., 2014). The resulting reads were assembled with SPAdes 3.14.1 (k-mer sizes of 31, 51, 71, 91, and 111 bp) (Bankevich et al., 2012). Scaffolds with a mean read-depth smaller than 20% of the median read-depth of the longer scaffolds (≥5,000 bp) as well as scaffolds that were shorter than 200 bp were removed. The final assemblies were polished using Pilon 1.22 (Walker et al., 2014). Genome sequences were deposited in the National Centre for Biotechnology Information. Accession numbers are listed in Table S4. Phylogenetic relationships were reconstructed based on the assembled genomes and the genome sequences of all validly published species of the genus (Machado et al., 2021a, b). For this, core genome alignments were created using Roary 3.6.2 (Page et al., 2015). Using this alignment, a maximum likelihood tree was constructed using Fasttree 2.1.10 based on the Jukes-Cantor + CAT nucleotide evolution model (Price et al., 2009, 2010). These analyses were carried out in Galaxy (Afgan et al., 2018). Whole genome sequence similarities were calculated by the digital DNA-DNA hybridization (dDDH) method using the recommended formula 2 of the genome-to-genome distance calculator (GGDC) web service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Auch et al., 2010a, 2010b; Meier-Kolthoff et al., 2013, 2014).

Figures 1–4, Tables 1 and 3–6.

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Body 0.65 to 0.86 mm long, C-shaped after fixation. Cuticle almost smooth, with transversal striae poorly developed. Lateral field not visible. Lip region with six lips developed but not fused bearing six acute labial papillae at oral margin and four rounded cephalic papillae at the base of lips. Oral opening almost rounded with thick margins. Amphidial apertures pore-like, ovoid and located posterior to lateral labial papillae. Stoma rhabditoid type, 1.2 to 2.3 times the lip region width, with short cheilostom with poorly refringent rounded cheilorhabdia, short gymnostom with refringent bar-like rhabdia, and long stegostom surrounded by the pharyngeal collar and bearing bar-like pro-mesorhabdia and small poorly refringent meta-telorhabdia. Pharynx poorly developed with robust corpus without differentiated metacorpus, short and slightly narrow isthmus and pyriform bulb with poorly visible valvular apparatus. Nerve ring encircling the isthmus at 58 to 75% of neck length, just anterior to basal bulb. Excretory pore located at basal bulb level, located at 61 to 97% of neck length. Cardia poorly developed, surrounded by intestinal tissue. Intestine without differentiations. Cardiac anterior end with thin walls. Genital system monorchic, laterally reflexed. Spicules well-developed, separate, with small angular manubrium, calamus poorly developed, and robust lamina with acute tip, scarcely prominent dorsal hump and poorly developed ventral velum. Gubernaculum with manubrium straight and slightly ventrally curved corpus, 40 to 50% of spicule length. Tail conoid with acute tip, ventrally curved posteriorly, flanked by the bursa. Bursa peloderan, with nine pairs of genital papillae (1 + 2/3 + 3), one of them probably the phasmid: three pairs pre-cloacal (GP1–GP3) and six pairs post-cloacal being three pairs at mid tail length (GP4–GP6) and three pairs (GP7–GP9) terminal; GP1 and GP2 more spaced, GP2 and GP3 closely spaced (Figs. 1–4).

Body 2.91 to 4.12 mm long, arcuate with general morphology similar to male, having labial papillae very acute and prominent. Nerve ring encircling the isthmus at 56 to 78% of neck length. Excretory pore located at or posterior to basal bulb, located at 67 to 103% of neck length. Genital reproductive system didelphic-amphidelphic with ovaries well developed, reflexed, oviducts and uteri not well visible, vagina very short and vulva small having transverse slit opening. Rectum slender, 0.8 to 1.3 times longer than the anal body diameter. Anus with prominent lips. Tail conoid with acute tip lacking mucro, having cellular part simple at its junction with the hyaline part. Phasmids inconspicuous (Figs. 1–4).

Body similar to, but usually smaller than hermaphroditic females, 1.13–1.61  mm long. Rectum very long, almost twice longer than the anal body diameter. Anus with posterior lip very prominent. Tail conoid with acute tip lacking mucro, having cellular part bifurcated at its junction with the hyaline part (Figs. 1–4).

Body 0.5 to 0.6 mm long, with habitus slightly ventral curved after fixation. Cuticle with transversal striae at anterior end, with both transversal and longitudinal striae at neck region and only with longitudinal striae at rest of body. Lip region lacking differentiate lips, bearing six labial papillae and cephalic papillae not visible. Amphidial apertures very reduced. Oral opening closed, having triradial symmetry. Stoma tubular, about twice the lip region wide. Pharynx slender, with long and narrow corpus, very narrow isthmus and pyriform basal bulb. Nerve ring surrounding the isthmus. Excretory pore at or just posterior to basal bulb. Cardia reduced, surrounded by intestinal tissue. Reproductive system absent. Rectum poorly visible. Anus closed. Tail conoid elongate with acute tip without mucro. Terminal hyaline part 37 to 54% of tail length (Figs. 1–4).

Body 0.47 to 0.56 mm long, with habitus almost straight after fixation. Cuticle with only transversal striae. Lip region lacking differentiate lips, and labial and cephalic papillae not visible. Oral opening rounded, closed, bearing a large, very refringent dorsal tooth. Amphidial apertures very prominent. Stoma tubular, slightly longer than the lip region wide. Pharynx, nerve ring and excretory pore location similar to the sheathed stage. Cardia reduced, surrounded by intestinal tissue. Rectum poorly visible. Anus closed. Tail conoid with very acute tip without mucro. Terminal hyaline part absent (Figs. 1–4).

Heterorhabditis ruandica n. sp. is characterized by having hermaphrodite females 2.91 to 4.12  mm long, amphimictic females 1.13 to 1.61  mm long, males 0.65 to 0.86  mm long, and IJs 0.50 to 0.59  mm long. Cuticle with poorly visible annuli in adults, with longitudinal crests in IJ2 and with well-developed annuli in IJ3. Lip region with six low lips having thin and acute lipplets in adults. Lips are poorly developed in IJ2 and bearing a large refringent dorsal tooth in IJ3. Stoma reduced in adults and tubular in IJ. Pharynx robust and short in adults and narrow and slender in IJ. Female reproductive system didelphic–amphidelphic. Anal body diameter in hermaphrodites 29 to 51 µm long, in amphimictic females 18 to 34 µm long, and in males 15 to 25 µm long. Tail short and conoid with acute terminus at cellular part in hermaphrodite females (63-98 µm long, c  =  34-51, c′  =  1.7-2.6) and slightly bifurcated in amphimictic females (62-88 µm long, c  =  16-24, c′  =  1.9-3.6). Tail conoid-elongate in IJ2 (49-64  µm long, c  =  8.0-12, c′  =   3.1-6.2) and IJ3 (22-39  µm long, c = 7.6-8.6, c′  =  3.4-5.8). Male reproductive system monorchic, with spicules 34 to 50  µm long having reduced manubrium 15 to 21  µm long, bursa peloderan bearing nine pairs of genital papillae (1 + 2/3 + 3).

Heterorhabditis ruandica n. sp. is morphologically similar to H. egyptii, H. bacteriophora, H. georgiana, and H. beicherriana, and can be distinguished from these species mainly by adult and infective juvenile characters (Tables 3–6). Heterorhabditis ruandica n. sp. can be distinguished from H. egyptii by the distance from the anterior end to the nerve ring in IJs (52-64 vs. 78-100  µm), the presence of a cephalic tooth in IJs (large vs. apparently small or absent). Additionally, hermaphroditic females of these two species differ in size (2.91-4.12 vs. 2.10-3.10), body diameter (209-274 vs. 107-164  µm), and in the distance from the anterior end to the excretory pore (106-153 vs. 154-205). Amphimictic female of H. ruandica n. sp. and H. egyptii differ in the size of their tails (62-88 vs. 56-78  µm).

Heterorhabditis ruandica n. sp. IJs can be distinguished from the IJs of H. bacteriophora by the distance between the anterior end and the excretory pore (67-90 vs. 87-110  µm) and the distance between the anterior end and the nerve ring (52-64 vs. 72-93  µm), and by the tail length (49-65 vs. 83-112  µm). The males of Heterorhabditis ruandica n. sp. can be distinguished from the males of H. bacteriophora by the distance from the anterior end to the excretory pore (61-109 vs. 114-130  µm) and by the lower D% value (61-97 vs. 117). Hermaphroditic and amphimictic females also show various morphometric differences (Tables 3–6).

Heterorhabditis ruandica n. sp. can be distinguished from H. beicherriana by the size of IJs (496-591 vs. 566-687  µm), the distance between the anterior end and the excretory pore (67-90 vs. 100-122  µm) and between the anterior end and the nerve ring (52-64 vs. 85-106  µm), and by neck (103-131 vs. 118-146  µm) and tail lengths of IJs (49-65 vs. 86-111  µm). The body length of H. ruandica n. sp. males is shorter than the body length of H. beicherriana males (652-863 vs 889-1192  µm). Males can also be distinguished by body diameter (40-51 vs. 51-73  µm), and by the distance between the anterior end and the excretory pore (61-109 vs. 130-157  µm) and between the anterior end and the nerve ring (56-74 vs. 81-108  µm), by neck (84-117 vs. 116-143  µm), tail (21-29 vs. 32-45  µm) and gubernaculum (15-21 vs. 22-27  µm) lengths, and by the D% value (61-97 vs. 102-120). Several other morphometric differences were also observed in hermaphroditic and amphimictic females (Tables 3–6).

Heterorhabditis ruandica n. sp. can be distinguished from H. georgiana by the anterior end to the excretory pore (67-90 vs. 97-113  µm) and the anterior end to the nerve ring (52-64 vs. 74-94  µm) distances, and by the tail length (49-65 vs. 86-108  µm) of IJs. The males can be distinguished by the anterior end to the excretory pore (61-109 vs. 101-145  µm) and the anterior end to the nerve ring (56-74 vs. 72-93  µm) distances, and by tail (21-29 vs. 29-41  µm) and gubernaculum (15-21 vs. 20-28  µm) length, and by D% values (61-97 vs. 100-122). Several other morphometric characters of hermaphroditic and amphimictic females differ between these two species (Tables 3–6).

The type hosts are unknown as the nematodes of this genus can be hosted by different insect species and were isolated from soil samples by the Galleria baiting technique (Bedding and Akhurst, 1975; White, 1927). Nematode strains H. ruandica n. sp. Rw18_M-Hr1a and Rw18_M-Hr1b were collected in the district of Karongi, Western province of the Republic of Rwanda (Decimal degrees coordinates: -2.131500, 29.325467) in a moist habitat along a river bench covered with sweet potato plants. Heterorhabditis ruandica n. sp. Rw14_N-C4a nematodes were collected in a ploughed cropland on terraces in a hilly area near Kanyirandori village, Tare sector, Nyamagabe district, Southern province of the Republic of Rwanda (Decimal degrees coordinates: -2.500000, 29.483333).

Rw14_N-C4a nematodes are the type material for Heterorhabditis ruandica n. sp. Holotype male, and 15 paratype hermaphrodites, males and amphimictic females and 15 third stage juveniles were deposited in the National Nematode Collection of India, IARI, New Delhi, India. Additional specimens were deposited at the nematode collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Spain, under the following slide numbers: Rwa001-01 to -12 (25 hermaphrodite females and 6 juveniles), Rwa002-01 to -05 (8 amphimictic females and 9 males), and Rwa003-01 to -02 (8 juveniles). Nematode cultures are maintained in the Institute of Biology, University of Neuchatel, Switzerland and in the Rwanda Agriculture and Animal Resource Development Board, Rubona, Rwanda.

The specific name refers to the country, the Republic of Rwanda (Africa), where the type material, Heterorhabditis ruandica n. sp. Rw14_N-C4a nematodes, used to phenotypically characterize the species, were collected.

Figures 5–8, Tables 2 and 3–6

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Body 0.81 to 0.91  mm long, J-shaped after heat killing and body arcuate posteriorly. Cuticle almost smooth, with transversal striae poorly developed. Lateral field not visible. Lip region with six lips poorly developed bearing six acute labial papillae at oral margin and four rounded cephalic papillae at base of lips. Oral opening almost rounded with thick margin. Amphidial apertures pore-like, ovoid and located posterior to lateral labial papillae. Stoma rhabditoid type, 0.9 to 1.6 times the lip region width, with short cheilostom with poorly refringent rounded cheilorhabdia, short gymnostom with refringent bar-like rhabdia, and long stegostom surrounded by the pharyngeal collar and bearing bar-like pro-mesorhabdia and small poorly refringent meta-telorhabdia. Pharynx poorly developed with robust corpus without differentiated metacorpus, short and slightly narrow isthmus and robust pyriform bulb with poorly visible valvular apparatus. Nerve ring encircling the isthmus at 61% to 96% of neck length, just anterior to basal bulb. Excretory pore located at or posterior to the basal bulb, located at 78% to 134% of neck length. Cardia poorly developed, surrounding by intestinal tissue. Intestine without differentiations. Genital system monorchic, laterally reflexed. Spicules well developed, separate, with more or less rounded manubrium, calamus poorly developed, and thinner and slender lamina with acute tip, scarcely prominent dorsal hump and poorly developed ventral velum. Gubernaculum with manubrium slightly ventral curved and straight corpus, 40% to 60% of spicule length. Tail conoid with acute tip, ventrally curved posteriorly, flanked by the bursa. Bursa peloderan, with nine pairs of genital papillae (1 + 2/3 + 3), one of them probably the phasmid: three pairs pre-cloacal (GP1–GP3) and six pairs post-cloacal being three pairs at mid tail length (GP4–GP6) and three pairs (GP7–GP9) terminal; GP1 and GP2 more spaced, GP2 and GP3 closely spaced (Figs. 5–8).

Body 4.41 to 6.18  mm long, arcuate with general morphology similar to male, having labial papillae more acute and prominent. Genital reproductive system didelphic–amphidelphic with ovaries well developed, reflexed, oviducts and uteri not well visible, vagina very short and vulva small having transverse slit opening. Rectum slender, about 1.5 times longer than the anal body diameter. Anus with prominent lips. Tail conoid with acute tip lacking mucro, having cellular part simple at its junction with the hyaline part. Phasmids inconspicuous (Figs. 5–8).

Body similar to, but usually smaller than hermaphrodites, 1.95 to 2.80  mm long. Rectum very long, about twice longer than the anal body diameter. Anus with posterior lip more prominent. Tail conoid with acute tip lacking mucro, having cellular part simple at its junction with the hyaline part (Figs. 5–8).

Body 0.49–0.58  mm long, with habitus slightly ventral curved after fixation. Cuticle with transversal striae at anterior end, with both transversal and longitudinal striae at neck region and only with longitudinal striae at rest of body. Lip region lacking differentiate lips, bearing six labial papillae and cephalic papillae not visible. Amphidial apertures very reduced. Oral opening closed, having triradial symmetry. Stoma tubular, about twice the lip region wide. Pharynx slender, with long and narrow corpus, very narrow isthmus and pyriform basal bulb. Nerve ring surrounding the isthmus. Excretory pore at or just posterior to basal bulb. Cardia reduced, surrounded by intestinal tissue. Reproductive system absent. Rectum poorly visible. Anus closed. Tail conoid elongate with acute tip without mucro. Terminal hyaline part 31% to 56% of tail length (Figs. 5–8).

Body 0.47 to 0.55  mm long, with habitus slightly ventral curved after fixation. Cuticle with only transversal striae. Lip region lacking differentiate lips, and labial and cephalic papillae not visible. Oral opening rounded, closed, bearing a small dorsal tooth. Amphidial apertures very prominent. Stoma tubular, slightly longer than the lip region wide. Pharynx, nerve ring and excretory pore location similar to the sheathed stage. Cardia reduced, surrounded by intestinal tissue. Rectum poorly visible. Anus closed. Tail conoid with acute tip without mucro. Terminal hyaline part absent (Figs. 5–8).

Heterorhabditis zacatecana n. sp. is characterized by having hermaphrodite females 4.41 to 6.18  mm long, amphimictic females 1.9 to 2.7  mm long, males 0.81 to 0.91  mm long, and IJs 0.49 to 0.57  mm long. Cuticle with poorly visible annuli in adults, with longitudinal crests in IJ2 and with well-developed annuli in IJ3. Lip region with six low lips having thick and acute lipplets in adults. Lips are poorly developed in IJ2 and bearing a small refringent dorsal tooth in IJ3. Stoma reduced in adults and tubular in IJs. Pharynx robust and short in adults, and narrow and slender in IJs. Female reproductive system didelphic–amphidelphic. Anal body diameter in hermaphrodites 34 to 58  µm long, in amphimictic females 31 to 41 µm long, and in males 13 to 22  µm long. Tail short and conoid with acute terminus at cellular part in hermaphrodite females (63-87  µm long, c  =  52-90, c′  =  1.2-2.4). and in amphimictic females (45-75  µm long, c  =  31-63, c′  =  1.3-2.0). Tail conoid-elongate in IJ2 (52-63  µm long, c = 7.9–9.8, c′  =  4.0-6.5) and in IJ3 (25-34  µm long, c = 8.2-10.5, c′  =  4.3-6.7). Male reproductive system monorchic, with spicules 38 to 55  µm long having conoid manubrium 15 to 25  µm long, bursa peloderan bearing nine pairs of genital papillae (1 + 2/3 + 3).

Heterorhabditis zacatecana n. sp. is morphologically similar to H. ruandica n. sp., H. amazonensis, H. bacteriophora, H. georgiana and H. beicherriana, and can be distinguished from these species mainly by adults and infective juvenile characters (Tables 3–6). Heterorhabditis zacatecana n. sp. can be distinguished from H. ruandica n. sp., one of the morphologically most similar species, by the shape of the male spicule (slender vs. robust) and the manubrium size (large vs. small), the size of hermaphrodites (4.41-6.18 vs. 2.91-4.12  mm), the hermaphrodite neck length (174-231 vs. 134-159  µm), and the hermaphrodite c ratio (52-90 vs. 34-51). The size of amphimictic females (1.95-2.80 vs. 1.13-1.61  µm), the shape of the tail tip (acute and longer vs. with mucro), the type of cellular–hyaline junction part (simple vs. bifurcated), the body diameter (160-228 vs. 68-83  µm), the a (11-15 vs. 15-20), b (16-21 vs. 9-14), and c ratios (31-63 vs. 16-24) and the anal body diameter (31-41 vs. 18-34  µm) differ also between H. zacatecana n. sp. and H. ruandica n. sp. IJs anterior ends also differ between these two species (small vs. large), and the presence of a cephalic tooth (small or absent vs. refringent and large).

Morphologically, the IJs of H. zacatecana n. sp. can be distinguished from the IJs of H. amazonensis by their size (493-578 vs. 567-612  µm), the distance from the anterior end to the nerve ring (59-72 vs. 76-93  µm), the neck length (96-124 vs. 107-132  µm), the tail length (52-63 vs. 98-115 µm), the a (19-24 vs. 24-29), c (8.2-10.5 vs. 5.1-6.1), and c′ (4.3-6.7 vs. ca. 7.3  µm) ratios and the E% (128-184 vs. 89-109). Moreover, hermaphroditic females differ in body size (4.41-6.12 vs. 3.52-5.59  mm), tail length (62-87 vs. 104-154  µm) and anal body diameter (34-58 vs. 59-85  µm). Amphimictic females of these two species differ also in body size (1.95-2.80 vs. 1.28-2.07  µm), tail length (45-75 vs. 25-38  µm), and body diameter (160-228 vs. 70-122). Male sizes differ between H. zacatecana n. sp. and H. amazonensis (0.81-0.91 vs. 0.69 vs. 0.83  mm) and body diameter (41-56 vs. 36-43  µm).

Heterorhabditis zacatecana n. sp. IJ can be distinguished from H. bacteriophora by the distance from the anterior end to the nerve ring (59-72 vs. 72-93  µm), and the tail length (52-63 vs. 83-112  µm). In the case of males, they differ in the distance from the excretory pore to the anterior end (77-109 vs. 114-130  µm) and in body diameter (41-56 vs. 38-46  µm). Several morphometric differences were also observed in hermaphrodites and amphimictic females (Tables 2–6).

Heterorhabditis zacatecana n. sp. IJs can be distinguished from H. beicherriana IJs by the distance from anterior end to the excretory pore (72-99 vs. 100-122  µm) and the distance from the anterior end to the nerve ring (59-72 vs. 85-106  µm), the tail length (52-63 vs. 86-111), values of a (19-24 vs. 24-29), c´ (4.3-6.7 vs. 6.0-7.4), and c (8.2-10.5 vs. 5.9-6.8) ratios, and the E% value (128-184 vs. 103-121). Males can be differentiated by differences in neck (71-108 vs. 116-143  µm) and tail (21-33 vs. 32-35  µm) lengths, the distance from the anterior end to the excretory pore (77-109 vs, 130-157  µm) and from the anterior end to the nerve ring (60-78 vs. 81-100  µm). Several morphometric differences were also observed in hermaphrodites and amphimictic females of these two species (Tables 2–6).

Heterorhabditis zacatecana n. sp. IJs can be distinguished from H. georgiana IJs by differences in body diameter (23-27 vs. 17-26  µm), tail length (52-63 vs. 86-108  µm), and anterior end to excretory pore (72-99 vs. 97-113  µm) and anterior end to nerve ring distances (59-72 vs. 74-94  µm). The a, b and c ratios, E% and D% of IJs differ also in these two species. The males of these two species differ in anterior end to excretory pore (77-109 vs. 101-145  µm) and anterior end to nerve ring distances (60-78 vs. 72-93  µm), and neck (71-108 vs. 100-122  µm) and tail (21-33 vs. 29-41  µm) lengths. Several morphometric characters of hermaphroditic and amphimictic females differ between these two species (Tables 2–6).

The type hosts are unknown as the nematodes of this genus can be hosted by different insect species and were isolated from soil samples by the Galleria baiting technique (Bedding and Akhurst, 1975; White, 1927). Heterorhabditis zacatecana n. sp. MEX-39 and MEX-40 nematodes were collected in maize fields in Villanueva (Zacatecas, Mexico; decimal degrees coordinates: 22.161371, -102.887940), and Heterorhabditis zacatecana n. sp. MEX-41 nematodes were collected in maize fields in Apaseo el Alto (Guanajuato, Mexico; decimal degrees coordinates: 20.470774, -100.59571).

MEX-39 nematodes are the type material for Heterorhabditis zacatecana n. sp. Holotype male, 15 paratype and 15 third stage juveniles were deposited in the National Nematode Collection of India, IARI, New Delhi. Additional specimens were deposited in the nematode collection of the Department of Animal Biology, Plant Biology and Ecology of the University of Jaén, Spain, under the following slide numbers: Mex001-01 to -03 (6 hermaphrodite females), Mex002-01 to -04 (8 amphimictic females and 3 males), and Mex003-01 to -04 (14 juveniles). Nematode cultures are maintained in the Institute of Biology, University of Neuchatel, Switzerland.

The specific name refers to the Mexican state, Zacatecas, where the type material, Heterorhabditis zacatecana n. sp. MEX-39 nematodes, used to phenotypically characterize the species were collected.

No progeny was observed when males and females of H. ruandica n. sp. Rw14_N-C4a and of H. zacatecana n. sp. MEX-39 were left to interact. No progeny was observed when males and females of H. ruandica n. sp. Rw14_N-C4a and of H. bacteriophora CH21 were left to interact. No progeny was observed when males and females of H. zacatecana n. sp. MEX-39 and of H. bacteriophora CH21 were left to interact. When males and females of H. ruandica n. sp. Rw14_N-C4a were crossed, fertile progeny was observed. When males and females of H. zacatecana n. sp. MEX-39 were crossed, fertile progeny was observed. When males and females of H. bacteriophora CH21 were crossed, fertile progeny was observed. Similarly, H. zacatecana n. sp. MEX-39 and H. zacatecana n. sp. MEX-40 nematodes produced fertile progeny, and H. ruandica n. sp. Rw18_M-Hr1a and H. ruandica n. sp. Rw14_N-C4a nematodes produced fertile progeny. These results provide further support for the heterospecific status of the Rwandan and the Mexican nematode populations.

Phylogenetic reconstructions based on nuclear and mitochondrial genes (ITS, D2–D3, COI, umc-87, and cmd–1), either individually or concatenated, confirm that the nematodes of the genus Heterorhabditis are grouped into three major clades: the “Megidis-group”, the “Indica-group” and the “Bacteriophora-group”, which is consistent with previous studies (Dhakal et al., 2020) (Fig. 9, Fig. S1). The clade of the “Bacteriophora-group” is, in turn, separated into five subclades. Three of them are composed of already described species: H. beicherriana, H. georgiana, and H. bacteriophora, and two of them are composed of two new, undescribed species, which we named here H. zacatecana n. sp., and H. ruandica n. sp. (Fig. 9, Fig. S1). Clearer phylogenetic separations within the species of the clade of the “Bacteriophora–group” were observed when phylogenies were reconstructed based on COI, ITS, or on concatenated sequences of COI, ITS, and D2–D3 (Fig. 9, Fig. S1). Closer inspection at the ITS, D2–D3 and COI sequences reveals unambiguous genetic differences between the nematodes of the “Bacteriophora–group” (Fig. 10). Sequence similarity scores and nucleotide difference counts show a closer relationship between H. bacteriophora, H. ruandica n. sp., and H. zacatecana n. sp. nematodes (Fig. 11 and Figs. S2-S6). Heterorhabditis ruandica n. sp. and H. bacteriophora share 99.1% and differ in 6 nucleotide positions in the ITS sequences flanked by primers TW81 and AB28, share 99.8% and differ in 1 nucleotide position in the D2–D3 sequences flanked by primers D2A and D3B, and share 94.1 to 94.7% and differ in 18 to 19 nucleotide positions in the COI sequences flanked by primers HCF and HCR (Fig. 11 and Figs. S2-S6). Heterorhabditis zacatecana n. sp. and H. bacteriophora share 99.4% and differ in 4 nucleotide positions in the ITS sequences flanked by primers TW81 and AB28, share 99.8% and differ in 1 nucleotide position in the D2–D3 sequences flanked by primers D2A and D3B, and share 94.1 to 94.4% and differ in 19 to 20 nucleotide positions in the COI sequences flanked by primers HCF and HCR (Fig. 11 and Figs. S2-S6). Heterorhabditis ruandica n. sp. and H. zacatecana share 99.7% and differ in 2 nucleotide positions in the ITS sequences flanked by primers TW81 and AB28, share 100% and differ in no nucleotide position in the D2–D3 sequences flanked by primers D2A and D3B, and share 97.6% to 98.2% and differ in 6–8 nucleotide positions in the COI sequences flanked by primers HCF and HCR (Fig. 11 and Figs. S2-S6). Noteworthy, we observed almost no intraspecific variations within the nematodes of the “Bacteriophora-group” at different genetic loci (Figs. 10, 11, and Figs. S2–S6). However, the sequences of the COI gene show very interesting signatures of population–specific polymorphism (Figs. 10D-F, 11). Specifically, Heterorhabditis ruandica n. sp. Rw18_M-Hr1a and Rw18_M-Hr1b nematodes that were collected in the same western Rwandan region differ from the Heterorhabditis ruandica n. sp. Rw14_N-C4a nematodes collected in a southern Rwandan region in a transitional nucleotide change (g.1212A > G) (Fig. 10D). Moreover, H. zacatecana n. sp. MEX-39 and MEX-40 nematodes collected in north-central Mexico and H. zacatecana n. sp. MEX-41 nematodes collected in central Mexico differ in three transitional nucleotide changes (g.1257T > C, g.1324T > C, and g.1464A > G) (Fig. 10D-F). Hence, due to its highly conserved species–specific polymorphism, and the consistent population-specific polymorphic patterns, the COI gene emerges as an important phylogenetic marker also for the genus Heterorhabditis, in a similar manner as it is for many other taxonomic groups (Hebert et al., 2003; Pentinsaari et al., 2016).

Figure 9:

Figure 10:

Figure 11:

In a recent study, Dhakal et al. (2020) studied several hundreds of ITS sequences of Heterorhabitis nematodes and recognized that nematodes identified as H. bacteriophora are represented by at least three haplotypes, some of which, the authors hypothesized, could represent new species. We contrasted their results and the sequences they used with the sequences we generated and found out that H. bacteriophora DE2, DE6, EN01, HP88, IT6, and PT1 nematodes represent Dhakal’s haplotype 1 (Figs. S7 and S8). Dhakal’s haplotype 2 is actually a mixture of two haplotypes: one represented by H. zacatecana n. sp. MEX-39, MEX-40, MEX-41, and by strains N2 and MK with identical ITS sequences; and one represented by strains UP2A2, 267, 269, 270, 271, 275, and 276 with identical ITS sequences. Strains MEX-39, MEX-40, MEX-41, N2 and MK differ in a transitional nucleotide change (g.2049A > G) with strains UP2A2, 267, 269, 270, 271, 275, and 276. Hence, strains N2, MK are likely H. zacatecana, and strains UP2A2, 267, 269, 270, 271, 275, and 276 might represent a new species. However, full characterization is needed to prove this hypothesis. Dhakal’s haplotype 3, represented by NGPS20, among others isolates, might also represent a new species, but again full characterization is needed to prove this hypothesis. In addition, our analyses reveal what we call a fourth haplotype, to follow Dhakal’s system, which is represented by H. ruandica Rw18_M-Hr1a, Rw18_M-Hr1b, and Rw14_N-C4a nematodes (Figs. S7 and S8). Phylogenetic reconstructions show a clear phylogenetic separation between all these haplotypes (Fig. S8). Hence, some of the haplotypes described by Dhakal et al. (2020) represent new species, closely related to H. bacteriophora, and some others likely represent new species, which highlights the power of statistical parsimony network analyses to uncover undescribed species of the genus Heterorhabditis, and supporting previous hypothesis regarding the taxonomic status of these nematode isolates (Bruno et al., 2020; Dhakal et al., 2020; Fallet et al., 2020).

Up to now, the bacterial genus Photorhabdus Boemare, Akhurst and Mourtant 1993 contains 27 taxa, including species and subspecies (Machado et al., 2021b). Phylogenetic relationship reconstructions based on whole genome sequences show that the bacterial symbionts isolated from H. zacatecana n. sp. MEX-39 and H. ruandica n. sp. Rw14_N-C4a nematodes, named here as MEX-39 and RW14-46, respectively, show high similarity with two of the already described Photorhabdus species: Photorhabdus kleinii and P. laumondii subsp. laumondii, respectively (Fig. 12). Photorhabdus kleinii MEX-39 shares 87–88% digital DNA–DNA hybridization (dDDH) with other members of the same species, while P. laumondii subsp. laumondii RW14-46 shares 89% digital DNA–DNA hybridization (dDDH) with other members of the same species, (Fig. S9).

Figure 12:

We revised the original publications of all synonymized species and based on their morphology and molecular data (when available), we reinforce the synonymized status of most of them (Khan et al., 1976; Wouts, 1979; Stock, 1993; Gardner et al., 1994; Liu, 1994; Stock et al., 1996; Plichta et al., 2009; Stock et al., 2009; Maneesakorn et al., 2015; Hunt and Nguyen, 2016; Shahina et al., 2017; Dhakal et al., 2020). However, the original description of H. bacteriophora provided by Poinar (1976) shows males with very anterior GP1 while in its synonymized species H. heliothidis (Khan, Brooks & Hirschmann, 1976) Poinar, Thomas & Hess, 1977 (=Chromonema heliothidis Khan et al., 1976) the GP1 appears more posterior (Khan et al., 1976; Poinar, 1976). Hence, it is likely that both species are not conspecific. Therefore, we declare H. heliothidis (Khan, Brooks & Hirschmann, 1976) Poinar, Thomas & Hess, 1977 as species inquirenda. Heterorhabditis hoptha and H. poinari were poorly described (Turco, 1970; Kakulia and Mikaia, 1997). Original descriptions lack differentiated description of all diagnostic characters of adult and larval stages. According to this, both species should remain in the list of species inquirendae. Heterorhabditis egyptii and H. hambletoni were described showing all diagnostic characters of adults and larvae stages. According to this, both species are considered valid herein (Pereira, 1937; Abd-Elgawad and Ameen, 2005). The lack of molecular data, however, impairs their inclusion in future phylogenetic studies. Nevertheless, new species description should contrast morphological characters with these species. An updated dichotomous key to identify the species of the genus Heterorhabditis is provided (Fig. 13, Tables 3-6).

Figure 13:

Considering the results of this study and the analyses of all the literature that describes new species of the genus Heterorhabditis, the updated list of the species of the genus, including their status, is as follows.