Heterorhabditis caligo n. sp. (Rhabditida: Heterorhabditidae): A New Entomopathogenic Nematode from Pichilemu Sand Dunes, Chile

San-Blas, Ernesto; Morales-Montero, Patricia; Bastidas, Brynelly; Půža, Vladimir; Machado, Ricardo A. R.

doi:10.2478/jofnem-2025-0045

Figures & Tables

Light microscopy photographs of the hermaphrodite and amphimictic female of Heterorhabditis caligo n. sp. (A) anterior region of hermaphrodite, (B) anterior region of the amphimictic female, (C) detail of the head showing the labial papillae of hermaphrodites, (D) detail of the head showing the labial papillae of the amphimictic female, (E) detail of the vulva of the hermaphrodite, (F,G) tail variations of the hermaphrodites, (H) tail of the amphimictic female. Scale bars: A–D and F–H = 20 μm; E = 10 μm.

Drawings of the hermaphrodite of Heterorhabditis caligo n. sp. (A) anterior region, (B) head region, (C) vulva; (D–F) Tail variations of the hermaphrodites, Scale bars: A–B and D–F = 20 μm; C = 10 μm.

Light microscopy photographs of males of H. caligo n. sp. (A) anterior part of the male (B) male in toto; (C,D) Lateral and ventral view of papillary arrangement gubernaculum of first-generation male in ventral view; (E,F) tail showing spicula and gubernaculum shapes. Scale bars: A = 20 μm; B = 100 μm; C–F = 10 μm.

Drawings of male and IJs of Heterorhabditis caligo n. sp. (A) anterior region of male; (B) head; (C) anterior region of IJ; (D) posterior portion of male in lateral view, showing spicules, gubernaculum and papillae arrangement; (E) posterior portion of male in ventral view, showing spicules, gubernaculum and papillae arrangement; (F) posterior portion of IJ showing amphids. Scale bars: A–B = 20 μm; C–F = 10 μm. IJs, infective juveniles.

Light microscopy photographs of IJs of Heterorhabditis caligo n. sp. (A) anterior part showing bacterial cells in the intestine (arrow), (B) detail of the head showing the dorsal tooth, (C) genital primordium, (D) basal bulb showing subventral gland (arrow), (E) tessellate pattern of the cuticle, (F) tail showing phasmid (arrow). Scale bars = 10 μm. IJs, infective juveniles.

Approximately-maximum-likelihood phylogenetic trees reconstructed from concatenated sequences of orthogroups of different Heterorhabditis species. A total of 4,600 single-copy orthogroups, comprising 1,846,787 amino acid positions, were analyzed. Numbers at the nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. Heterorhabditis mexicana from the indica clade and H. marelatus from the megidis clade could not be included in the analyses due to lack of laboratory cultures.

Maximum-likelihood phylogenetic tree reconstructed from concatenated sequences of the following protein-coding genes: cytochrome c oxidase, cytochrome b, and NADH dehydrogenase of the mitochondrial genomes of different Heterorhabditis species. A total of 9,494 nucleotide positions were analyzed. The genes were concatenated in the following order: cob, cox-1, cox-2, cox-3, nad-1, nad-2, nad-3, nad-4, nad-4l, nad-5, and nad-6. Accession numbers of the concatenated sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. Heterorhabditis mexicana from the indica clade and H. marelatus from the megidis clade could not be included in the analyses due to lack of laboratory cultures.

Maximum-likelihood phylogenetic tree reconstructed from the whole ribosomal RNA operons of different Heterorhabditis species. A total of 5,927 nucleotide positions were analyzed. Accession numbers of the sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. Heterorhabditis mexicana from the indica clade and H. marelatus from the megidis clade could not be included in the analyses due to lack of laboratory cultures.

Maximum aphylogenetic tree reconstructed from the concatenated sequences of the ITS region of the rRNA gene and the cytochrome c oxidase subunit I (cox-1) gene of different Heterorhabditis species. A total of 1,482 nucleotide positions were analyzed. The ITS sequences of H. marelatus and H. mexicana were obtained from the NCBI using the accession numbers AY321479 and EF043444, respectively. The ITS sequences of all the other isolates were extracted from whole ribosomal RNA operons. These sequences were then trimmed to obtain sequences that cover the region flanked by the commonly used primers TW81 and AW28. The cox-1 sequences of H. marelatus and H. mexicana were obtained from the NCBI using the accession numbers EF043419 and EF043422, respectively. The sequences of all the other isolates were extracted from whole mitochondrial genomes. These sequences were then trimmed to obtain sequences that cover the region flanked by the commonly used primers HCF and HCR. Accession numbers of the nucleotide sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. ITS, internal transcribed spacer; NCBI, National Center for Biotechnology Information.

Maximum-likelihood phylogenetic tree reconstructed from the nucleotide sequences of the calmodulin 1 (cmd-1) gene. A total of 738 nucleotide positions were analyzed. Accession numbers of the nucleotide sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. Trees were rooted at the midpoint.

Maximum-likelihood phylogenetic tree reconstructed from the nucleotide sequences of the thin filament F-actin-associated protein (unc-87) gene. A total of 465 nucleotide positions were analyzed. Accession numbers of the nucleotide sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. Trees were rooted at the midpoint.

Phylogenetic reconstruction based on core genome sequences of Photorhabdus type strains with validly published names. A total of 3,526,609 nucleotide positions (3,455 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Bar represents 0.05 nucleotide substitutions per sequence position. Accession numbers of the genome sequences used for the reconstruction are shown in Table S4 in the Supplementary Material.

Maximum-likelihood phylogenetic tree reconstructed from the sequences of the ITS region of the rRNA gene of different Heterorhabditis species. A total of 1075 nucleotide positions were analyzed. The sequences of H. marelatus and H. mexicana were obtained from the NCBI using the accession numbers AY321479 and EF043444, respectively. The sequences of all the other isolates were extracted from whole ribosomal RNA operons. These sequences were then trimmed to obtain sequences that cover the region flanked by the commonly used primers TW81 and AW28. Accession numbers of the nucleotide sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at the nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position. ITS, internal transcribed spacer.

Maximum-likelihood phylogenetic tree reconstructed from the sequences of the cytochrome c oxidase subunit I (cox-1) gene of different Heterorhabditis species. A total of 401 nucleotide positions were analyzed. The sequences of H. marelatus and H. mexicana were obtained from the NCBI using the accession numbers EF043419 and EF043422, respectively. The sequences of all the other isolates were extracted from whole mitochondrial genomes. These sequences were then trimmed to obtain sequences that cover the region flanked by the commonly used primers HCF and HCR. Accession numbers of the nucleotide sequences used for the analyses are shown in Table S3 in the Supplementary Material. Numbers at the nodes represent bootstrap values based on 500 replications. Bars represent average nucleotide substitutions per sequence position.

Features and assemble statistics of the nematode genomes generated and/or used in this study_

Species	Strain designation(s)	L50	N50	L90	N90	Genome size (bp)	GC content (%)
H. amazonensis	APURE	185	94744	692	21806	65101680	33.72
H. americana	S10	432	48365	1815	6421	72782968	33.07
H. americana	S8	635	33153	2886	3735	72234711	33.27
H. atacamensis	31873	212	82969	940	12449	67224907	32.98
	33031	218	81903	981	11470	67409700	33.09
	Brecon	585	35417	2251	6475	69889708	32.85
	EN01	593	35153	2635	4057	71959660	33.02
	IR2	422	50708	1521	10601	70203201	32.90
	m13e	787	27231	3030	4697	72585993	33.08
H. bacteriophora	MG618b	639	33083	2791	4049	71868631	33.02
	NL	424	49858	1597	9515	71077392	33.14
	S12	594	35368	2637	4102	71872306	33.00
	SGI-170	415	50445	1801	5833	73250772	33.13
	Px-SPH	396	53254	1778	5674	73984962	33.22
	Z1	383	54630	1655	6479	73293000	33.13
H. baujardi	PUC-He-TD1	166	102718	632	23255	64662943	33.73
	PUC-He-TD2	210	86004	814	15671	66175497	33.60
	Cherry	387	53208	1595	7903	71960759	32.97
H. beicherriana	CN4	740	28317	3011	4301	70797395	32.83
	H06	586	35927	2488	4871	70742037	32.82
	M6	378	55114	1600	7533	72276565	33.06

H. caligo n. sp.	UOH-032	216	90000	991	11663	72194284	33.78

	H4	410	52252	1770	6413	73080526	33.16
H. casmirica	HM	412	51774	1771	6469	73036779	33.15
H. casmirica	HP1	496	40787	1808	9330	70153441	32.94
H. downesi	P2, pur2	582	33455	2189	6602	66295249	33.33
H. downesi	P3, pur3	344	53355	1310	11663	65144250	33.18
H. floridensis	332	195	92043	712	21459	64561023	33.74
	K22	190	94213	711	21075	64790060	33.79
	Hbb	617	34453	2713	4209	71543875	32.94
H. georgiana	Kesha	432	49201	1880	5965	72925328	33.06
	Point Reyes	425	49428	1803	6471	72527224	33.03
	AB, HeM	225	79854	917	12606	65680349	33.80
	Bartow	247	73385	1017	10433	66304658	33.86
H. indica	IARI, IARI-EPN-Hms1	73	234422	298	60148	67546827	32.34
H. indica	LN2	244	74414	1018	10332	66427391	33.91
	Rebecca	223	81088	916	11868	65936969	33.83
H. megidis	UK211	234	77932	1025	11317	66720551	33.23
H. noenieputensis	SF669	272	66412	1186	7769	66740923	34.02
H. ruandica	Rw18_M-Hr1a	456	46969	2072	4705	73994590	32.80
H. ruandica	Rw18_M-Hr1b	451	48699	2058	5143	74807992	33.81
H. safricana	SF281	181	92924	847	12279	66942788	33.06
H. taysearae	Benin	235	78313	928	12299	66991707	34.04
H. zacatecana	MEX-39	450	46782	1866	6629	72489781	33.01
H. zacatecana	MEX-41	611	34263	2558	4886	71022245	32.85
H. zealandica	Blue, SF41	283	66513	1280	8325	67614449	33.11
H. zealandica	Green, MJ2C	216	83151	979	10895	67060566	33.09
Heterorhabditis sp.	CRI-LC	410	50459	1639	7686	72017000	32.97

National Center for Biotechnology Information (NCBI) databank accession numbers of the nematode sequences used for phylogenetic reconstructions_

Species	Strain designation(s)	Whole rRNA Operon	cob	Partial	Complete
Species	Strain designation(s)	Whole rRNA Operon	cob	*cox-1*	*cox-1*	*cox-2*	*cox-3*	*nad-1*	*nad-2*	*nad-3*	*nad-4*	*nad-4I*	*nad-5*	*nad-6*	*unc-87*	*cmd-1*
H. amazonensis	APURE	PQ345856	PQ428464	PQ368640	PQ341126	PQ428511	PQ428558	PQ428605	PQ428652	PQ428699	PQ367523	PQ428746	PQ428793	PQ428840	PQ367570	PQ367617
H. americana	S10	PQ345891	PQ428499	PQ368684	PQ341161	PQ428546	PQ428593	PQ428640	PQ428687	PQ428734	PQ367558	PQ428781	PQ428828	PQ428875	PQ367605	PQ367652
	S8	PQ345893	PQ428501	PQ368683	PQ341163	PQ428548	PQ428595	PQ428642	PQ428689	PQ428736	PQ367560	PQ428783	PQ428830	PQ428877	PQ367607	PQ367654
H. atacamensis	31873	PQ345852	PQ428460	PQ368641	PQ341122	PQ428507	PQ428554	PQ428601	PQ428648	PQ428695	PQ367519	PQ428742	PQ428789	PQ428836	PQ367566	PQ367613
	33031	PQ345853	PQ428461	PQ368642	PQ341123	PQ428508	PQ428555	PQ428602	PQ428649	PQ428696	PQ367520	PQ428743	PQ428790	PQ428837	PQ367567	PQ367614
	Brecon	PQ345860	PQ428468	PQ368643	PQ341130	PQ428515	PQ428562	PQ428609	PQ428656	PQ428703	PQ367527	PQ428750	PQ428797	PQ428844	PQ367574	PQ367621
	EN01	PQ345864	PQ428472	PQ368644	PQ341134	PQ428519	PQ428566	PQ428613	PQ428660	PQ428707	PQ367531	PQ428754	PQ428801	PQ428848	PQ367578	PQ367625
	IR2	PQ345872	PQ428480	PQ368645	PQ341142	PQ428527	PQ428574	PQ428621	PQ428668	PQ428715	PQ367539	PQ428762	PQ428809	PQ428856	PQ367586	PQ367633
H. bacteriophora	m13e, TT01	PQ345876	PQ428484	PQ368646	PQ341146	PQ428531	PQ428578	PQ428625	PQ428672	PQ428719	PQ367543	PQ428766	PQ428813	PQ428860	PQ367590	PQ367637
	MG618b	PQ345882	PQ428490	PQ368647	PQ341152	PQ428537	PQ428584	PQ428631	PQ428678	PQ428725	PQ367549	PQ428772	PQ428819	PQ428866	PQ367596	PQ367643
	NL	PQ345883	PQ428491	PQ368648	PQ341153	PQ428538	PQ428585	PQ428632	PQ428679	PQ428726	PQ367550	PQ428773	PQ428820	PQ428867	PQ367597	PQ367644
	S12	PQ345892	PQ428500	PQ368650	PQ341162	PQ428547	PQ428594	PQ428641	PQ428688	PQ428735	PQ367559	PQ428782	PQ428829	PQ428876	PQ367606	PQ367653
	SGI-170, SGI170	PQ345896	PQ428504	PQ368651	PQ341166	PQ428551	PQ428598	PQ428645	PQ428692	PQ428739	PQ367563	PQ428786	PQ428833	PQ428880	PQ367610	PQ367657
	Px-SPH, PxSPH	PQ345889	PQ428497	PQ368649	PQ341159	PQ428544	PQ428591	PQ428638	PQ428685	PQ428732	PQ367556	PQ428779	PQ428826	PQ428873	PQ367603	PQ367650
	Z1	PQ345898	PQ428506	PQ368652	PQ341168	PQ428553	PQ428600	PQ428647	PQ428694	PQ428741	PQ367565	PQ428788	PQ428835	PQ428882	PQ367612	PQ367659
H. baujardi	PUC-He-TD1	PQ345887	PQ428495	PQ368653	PQ341157	PQ428542	PQ428589	PQ428636	PQ428683	PQ428730	PQ367554	PQ428777	PQ428824	PQ428871	PQ367601	PQ367648
	PUC-He-TD2	PQ345888	PQ428496	PQ368654	PQ341158	PQ428543	PQ428590	PQ428637	PQ428684	PQ428731	PQ367555	PQ428778	PQ428825	PQ428872	PQ367602	PQ367649
	Cherry	PQ345861	PQ428469	PQ368655	PQ341131	PQ428516	PQ428563	PQ428610	PQ428657	PQ428704	PQ367528	PQ428751	PQ428798	PQ428845	PQ367575	PQ367622
H. beicherriana	CN4	PQ345862	PQ428470	PQ368656	PQ341132	PQ428517	PQ428564	PQ428611	PQ428658	PQ428705	PQ367529	PQ428752	PQ428799	PQ428846	PQ367576	PQ367623
	H06	PQ345866	PQ428474	PQ368657	PQ341136	PQ428521	PQ428568	PQ428615	PQ428662	PQ428709	PQ367533	PQ428756	PQ428803	PQ428850	PQ367580	PQ367627
	M6	PQ345877	PQ428485	PQ368658	PQ341147	PQ428532	PQ428579	PQ428626	PQ428673	PQ428720	PQ367544	PQ428767	PQ428814	PQ428861	PQ367591	PQ367638
H. caligo n. sp.	UOH-032	PV871102	PV873291	PV873292	PV873292	PV873293	PV873294	PV873295	PV873296	PV873297	PV873298	PV873300	PV873299	PV873301	PV892898	PV892895
	H4	PQ345867	PQ428475	PQ368659	PQ341137	PQ428522	PQ428569	PQ428616	PQ428663	PQ428710	PQ367534	PQ428757	PQ428804	PQ428851	PQ367581	PQ367628
H. casmirica	HM	PQ345869	PQ428477	PQ368660	PQ341139	PQ428524	PQ428571	PQ428618	PQ428665	PQ428712	PQ367536	PQ428759	PQ428806	PQ428853	PQ367583	PQ367630
	HP1	PQ345870	PQ428478	PQ368661	PQ341140	PQ428525	PQ428572	PQ428619	PQ428666	PQ428713	PQ367537	PQ428760	PQ428807	PQ428854	PQ367584	PQ367631
H. downesi	P2, pur2	PQ345884	PQ428492	PQ368662	PQ341154	PQ428539	PQ428586	PQ428633	PQ428680	PQ428727	PQ367551	PQ428774	PQ428821	PQ428868	PQ367598	PQ367645
	P3, pur3	PQ345885	PQ428493	PQ368663	PQ341155	PQ428540	PQ428587	PQ428634	PQ428681	PQ428728	PQ367552	PQ428775	PQ428822	PQ428869	PQ367599	PQ367646
H. floridensis	332	PQ345854	PQ428462	PQ368664	PQ341124	PQ428509	PQ428556	PQ428603	PQ428650	PQ428697	PQ367521	PQ428744	PQ428791	PQ428838	PQ367568	PQ367615
	K22	PQ345873	PQ428481	PQ368665	PQ341143	PQ428528	PQ428575	PQ428622	PQ428669	PQ428716	PQ367540	PQ428763	PQ428810	PQ428857	PQ367587	PQ367634
	Hbb	PQ345868	PQ428476	PQ368668	PQ341138	PQ428523	PQ428570	PQ428617	PQ428664	PQ428711	PQ367535	PQ428758	PQ428805	PQ428852	PQ367582	PQ367629
H. georgiana	Kesha	PQ345874	PQ428482	PQ368666	PQ341144	PQ428529	PQ428576	PQ428623	PQ428670	PQ428717	PQ367541	PQ428764	PQ428811	PQ428858	PQ367588	PQ367635
	Point Reyes	PQ345886	PQ428494	PQ368667	PQ341156	PQ428541	PQ428588	PQ428635	PQ428682	PQ428729	PQ367553	PQ428776	PQ428823	PQ428870	PQ367600	PQ367647
	AB, HeM	PQ345855	PQ428463	PQ368669	PQ341125	PQ428510	PQ428557	PQ428604	PQ428651	PQ428698	PQ367522	PQ428745	PQ428792	PQ428839	PQ367569	PQ367616
H. indica	Bartow	PQ345857	PQ428465	PQ368670	PQ341127	PQ428512	PQ428559	PQ428606	PQ428653	PQ428700	PQ367524	PQ428747	PQ428794	PQ428841	PQ367571	PQ367618
	IARI, IARI-EPN-Hms1	PQ345871	PQ428479	PQ368671	PQ341141	PQ428526	PQ428573	PQ428620	PQ428667	PQ428714	PQ367538	PQ428761	PQ428808	PQ428855	PQ367585	PQ367632
	LN2	PQ345875	PQ428483	PQ368672	PQ341145	PQ428530	PQ428577	PQ428624	PQ428671	PQ428718	PQ367542	PQ428765	PQ428812	PQ428859	PQ367589	PQ367636
	Rebecca	PQ345890	PQ428498	PQ368673	PQ341160	PQ428545	PQ428592	PQ428639	PQ428686	PQ428733	PQ367557	PQ428780	PQ428827	PQ428874	PQ367604	PQ367651
H. megidis	UK211	PQ345897	PQ428505	PQ368674	PQ341167	PQ428552	PQ428599	PQ428646	PQ428693	PQ428740	PQ367564	PQ428787	PQ428834	PQ428881	PQ367611	PQ367658
H. noenieputensis	SF669	PQ345895	PQ428503	PQ368675	PQ341165	PQ428550	PQ428597	PQ428644	PQ428691	PQ428738	PQ367562	PQ428785	PQ428832	PQ428879	PQ367609	PQ367656
H. ruandica	Rw18_M-Hr1a, Ma	PQ345878	PQ428486	PQ368676	PQ341148	PQ428533	PQ428580	PQ428627	PQ428674	PQ428721	PQ367545	PQ428768	PQ428815	PQ428862	PQ367592	PQ367639
	Rw18_M-Hr1b, Mb	PQ345879	PQ428487	PQ368677	PQ341149	PQ428534	PQ428581	PQ428628	PQ428675	PQ428722	PQ367546	PQ428769	PQ428816	PQ428863	PQ367593	PQ367640
H. safricana	SF281	PQ345894	PQ428502	PQ368678	PQ341164	PQ428549	PQ428596	PQ428643	PQ428690	PQ428737	PQ367561	PQ428784	PQ428831	PQ428878	PQ367608	PQ367655
H. taysearae	Benin	PQ345858	PQ428466	PQ368685	PQ341128	PQ428513	PQ428560	PQ428607	PQ428654	PQ428701	PQ367525	PQ428748	PQ428795	PQ428842	PQ367572	PQ367619
H. zacatecana	MEX-39, MEX39	PQ345880	PQ428488	PQ368686	PQ341150	PQ428535	PQ428582	PQ428629	PQ428676	PQ428723	PQ367547	PQ428770	PQ428817	PQ428864	PQ367594	PQ367641
	MEX-41, MEX41	PQ345881	PQ428489	PQ368679	PQ341151	PQ428536	PQ428583	PQ428630	PQ428677	PQ428724	PQ367548	PQ428771	PQ428818	PQ428865	PQ367595	PQ367642
H. zealandica	Blue, SF41	PQ345859	PQ428467	PQ368681	PQ341129	PQ428514	PQ428561	PQ428608	PQ428655	PQ428702	PQ367526	PQ428749	PQ428796	PQ428843	PQ367573	PQ367620
	Green, MJ2C	PQ345865	PQ428473	PQ368680	PQ341135	PQ428520	PQ428567	PQ428614	PQ428661	PQ428708	PQ367532	PQ428755	PQ428802	PQ428849	PQ367579	PQ367626
Heterorhabditis sp.	CRI-LC, CRILC	PQ345863	PQ428471	PQ368682	PQ341133	PQ428518	PQ428565	PQ428612	PQ428659	PQ428706	PQ367530	PQ428753	PQ428800	PQ428847	PQ367577	PQ367624

Features and annotation statistics of the nematode genomes generated and/or used in this study_

Species	Strain designation(s)	BUSCO Genome assembly	Number of proteins	BUSCO proteins
H. amazonensis	APURE	83	11725	82
H. americana	S10	80	20149	81
H. americana	S8	80	18366	80
H. atacamensis	31873	82	13256	75
	33031	82	13239	81
	Brecon	81	15525	71
	EN01	80	19242	75
	IR2	81	19233	77
	m13e	80	19084	77
H. bacteriophora	MG618b	80	19127	81
	NL	80	15490	81
	S12	80	19200	81
	SGI-170	81	21150	83
	Px-SPH	81	24383	76
	Z1	81	21949	75
H. baujardi	PUC-He-TD1	83	11561	76
	PUC-He-TD2	83	11648	74
	Cherry	82	20528	80
H. beicherriana	CN4	80	13778	77
	H06	82	17382	81
	M6	82	25282	72

H. caligo n. sp.	UOH-032	82	16223	80

H. casmirica	H4	81	18294	72
	HM	80	18376	76
	HP1	80	15124	71
H. downesi	P2, pur2	80	12235	83
H. downesi	P3, pur3	81	12257	83
H. floridensis	332	83	11541	82
	K22	83	11587	76
	Hbb	80	19156	81
H. georgiana	Kesha	80	23309	76
	Point Reyes	81	20357	82
	AB, HeM	82	12823	82
	Bartow	82	13059	85
H. indica	IARI, IARI-EPN-Hms1	84	11844	81
	LN2	82	13088	80
	Rebecca	83	12933	81
H. megidis	UK211	82	13224	80
H. noenieputensis	SF669	82	13418	75
H. ruandica	Rw18_M-Hr1a	80	18441	81
H. ruandica	Rw18_M-Hr1b	81	18274	79
H. safricana	SF281	82	12947	75
H. taysearae	Benin	83	20647	70
H. zacatecana	MEX-39	80	24958	76
H. zacatecana	MEX-41	81	24402	81
H. zealandica	Blue, SF41	82	21057	81
H. zealandica	Green, MJ2C	83	20417	82
Heterorhabditis sp.	CRI-LC	81	19815	81

Morphometrics of Heterorhabditis caligo n_ sp_

Character	Males		Hermaphrodites Paratypes	Females Paratypes	IJs Paratypes

	Holotype	Paratypes
n	-	20	20	20	20
L	967	1,018 ± 80 (864–1,174)	3,616 ± 602 (2,855–4,950)	2,674 ± 302 (2,002–3,103)	669 ± 43 (568–723)
a	20.1	19 ± 1.08 (16.9–20.8)	14 ± 1.85 (11–18)	15 ± 0.83 (13–17)	27 ± 1.3 (23.7–29.1)
b	8.3	9 ± 0.53 (7.7–9.5)	16 ± 1.36 (13.5–19.19)	16 ± 1.5 (13.1–19.6)	5 ± 0.3 (4.2–5.2)
c	32.2	31 ± 3.45 (24–38.9)	40 ± 7.3 (27.7–55.2)	2 ± 1.8 (2.4–0.13)	6.8 ± 0.5 (5.4–7.7)
c′	1.3	1 ± 0.18 (1.1–1.8)	1.7 ± 0.33 (1.2–2.3)	15 ± 2.2 (11.8–19.6)	6 ± 0.4 (5.6–7.2)
V			46 ± 2.8 (40.3–50)	52 ± 2.47 (45.6–56.8)
Max. body diam.	48	54 ± 4.1 (45–59)	265 ± 39 (187–336)	183 ± 20.2 (133–210)	25 ± 1.5 (22–28)
Excretory pore	124	131 ± 7.7 (118–146)	239 ± 37.9 (180–310)	193 ± 16.5 (169–224)	119 ± 6.3 (105–128)
Nerve ring	71	74 ± 6.4 (63–92)	156 ± 29 (122–216)	103 ± 15 (77–141)	107 ± 4.2 (95–114)
Pharynx (ES)	117	118 ± 4.2 (112–126)	224 ± 36.6 (175–294)	173 ± 13.7 (148–197)	141 ± 4 (135–150)
Hemizonoid					113 ± 4.0 (106–117)
Testis reflection	128	133 ± 29.1 (85–245)
Tail length	30	33 ± 4 (25–41)	92 ± 11.9 (71–110)	83 ± 7.3 (74–99)	98 ± 9.7 (84–129)
Tail length without sheath					71 ± 5.1 (60–79)
Anal body diam.	24	23 ± 2.1 (21–27)	55 ± 10.6 (40–75)	42 ± 2.82 (38–50)	16 ± 0.8 (15–18)
Spicule length	51	50 ± 3 (41–52)
Gubernaculum length	21	21 ± 1.1 (18–23)
D%	106	112 ± 6.7 (102–129)			84 ± 4.3 (75–91)
E%	413	403 ± 52.3 (367–535)			121 ± 4 (94–135)
SW%	213	214 ± 24.4 (152–248)
GS%	41	42 ± 3 (35–49)

Pairwise comparison of digital DNA–DNA Hybridization (dDDH) scores (%) of P_ tasmaniensis UOH-032 and all the Photorhabdus type strains with validly published names_ Accession numbers of gene sequences used are shown in Table S4_

Strain	dDDH (%)
P. aballayi sp. nov. APURE^T	30.7
P. aegyptia BA1^T	31.5
P. africana CRI-LC^T	32.3
P. akhurstii subsp. akhurstii DSM 15138^T	31.2
P. akhurstii subsp. bharatensis H3^T	31.3
P. antumapuensis UCH-936^T	32.7
P. asymbiotica DSM 15149^T	30.7
P. australis subsp. australis DSM 17609^T	30.5
P. australis subsp. thailandensis PB68.1^T	30.8
P. bodei LJ24-63^T	32.6
P. caribbeanensis HG29^T	30.8
P. cinerea DSM 19724^T	31.7
P. hainanensis DSM 22397^T	31.1
P. heterorhabditis subsp. aluminescens Q614^T	32.7
P. heterorhabditis subsp. heterorhabditis SF41^T	31.7
P. hindustanensis H1^T	31.1
P. kayaii DSM 15194^T	32.6
P. khanii subsp. guanajuatensis MEX20-17^T	48.1
P. khanii subsp. khanii DSM 3369^T	47.6
P. kleinii DSM 23513^T	32
P. laumondii subsp. clarkei BOJ-47^T	32.1
P. laumondii subsp. laumondii TT01^T	31.9
P. luminescens subsp. luminescens ATCC 29999^T	30.8
P. luminescens subsp. mexicana MEX47-22^T	30.7
P. luminescens subsp. venezuelensis JAR^T	30.7
P. namnaonensis PB45.5^T	31.1
P. noenieputensis DSM 25462^T	31.1
P. stackebrandtii DSM 23271^T	47.1
P. tasmaniensis DSM 22387^T	85.3
P. temperata DSM 14550^T	51.1
P. thracensis DSM 15199^T	50.8
P. viridis Green^T	49

National Center for Biotechnology Information (NCBI) accession numbers of the Photorhabdus sequences used in this study_ Sequences generated in this study are shown in bold_

Strain	Genome
P. aballayi APURE^T	JAPFCD01
P. africana CRI-LC^T	JAXBVE01
P. aegyptia BA1^T	JFGV01
P. akhurstii subsp. akhurstii DSM 15138^T	RCWE01
P. akhurstii subsp. bharatensis H3^T	PUWU01
P. antumapuensis UCH-936^T	JAHZMK01
P. australis subsp. thailandensis PB68.1^T	LOMY01
P. australis subsp. australis DSM 17609^T	JONO01
P. asymbiotica ATCC 43949^T	RBLJ01
P. bodei LJ24-63^T	NSCM01
P. caribbeanensis DSM 22391^T	RCWB01
P. cinerea DSM 19724^T	PUJW01
P. hainanensis DSM 22397^T	RCWD01
P. heterorhabditis subsp. aluminescens Q614^T	JABBCS01
P. heterorhabditis subsp. heterorhabditis SF41^T	RCWA01
P. hindustanensis H1^T	PUWT01
P. kayaii DSM 15194^T	JAJAFZ01
P. khanii subsp. khanii DSM 3369^T	AYSJ01
P. khanii subsp. guanajuatensis MEX20-17^T	PUJY01
P. kleinii DSM 23513^T	JAJAFY01
P. laumondii subsp. clarkei BOJ-47^T	NSCI01
P. laumondii subsp. laumondii TT01^T	WSFH01
P. luminescens subsp. luminescens ATCC 29999^T	FMWJ01
P. luminescens subsp. mexicana MEX47-22^T	PUJX01
P. luminescens subsp. venezuelensis JAR^T	JAPFFZ01
P. namnaonensis PB45.5^T	LOIC01
P. noenieputensis DSM 25462^T	RCWC01
P. stackebrandtii DSM 23271^T	PUJV01
P. tasmaniensis DSM 22387^T	PUJU01
P. tasmaniensis UOH-32	XXXXX
P. temperata DSM 14550^T	JAJAFX01
P. thracensis DSM 15199^T	CP011104
P. viridis Green^T	JBEJZY01

Heterorhabditis caligo n. sp. (Rhabditida: Heterorhabditidae): A New Entomopathogenic Nematode from Pichilemu Sand Dunes, Chile

Figures & Tables

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Figure S1:

Figure S2:

Features and assemble statistics of the nematode genomes generated and/or used in this study_

National Center for Biotechnology Information (NCBI) databank accession numbers of the nematode sequences used for phylogenetic reconstructions_

Features and annotation statistics of the nematode genomes generated and/or used in this study_

Morphometrics of Heterorhabditis caligo n_ sp_

Pairwise comparison of digital DNA–DNA Hybridization (dDDH) scores (%) of P_ tasmaniensis UOH-032 and all the Photorhabdus type strains with validly published names_ Accession numbers of gene sequences used are shown in Table S4_

National Center for Biotechnology Information (NCBI) accession numbers of the Photorhabdus sequences used in this study_ Sequences generated in this study are shown in bold_

Paradigm

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