Meloidogyne marylandi (Fig. 5) Measurements Measurements of J2 are presented in Table 2. Description Second-stage juvenile (J2) Fourteen individuals were measured and observed. Their bodies were vermiform, almost straight, and tapering at both extremities, but tapered more posteriorly. The head was not offset, and was observed with labial disk, while the lip region was without annulation. The stylet was delicate (11.5 μm to 13.4 μm long). The cone was straight, narrow, and sharply pointed; the shaft was observed to become slightly wider posteriorly, with a small rounded knob. The cuticular annulation was fine and distinct. The lateral field was prominent, with four incisures. The excretory pore was three to four annuli anterior to the hemizonid. The rectum was inflated, while the phasmids were small and indistinct. The tail tapered gradually, and was 49.5 μm to 54.0 μm long. The hyaline tail terminus was 11.2 (9.5–13.5) μm long, and the terminus was bluntly rounded. Males and females were not found. Identification Means and ranges of all the measurements of J2 (Table 2) of the Japanese population of M. marylandi overlapped with those of other Japanese, Israeli, and USA populations of the nematode. The morphological characteristics of J2 (hyaline) tail shape, hemizonid position, and female stylet knob shape were in agreement with the original and additional descriptions (Jepson and Golden, 1987; Araki, 1992; Oka et al., 2003), except that the tail length was slightly shorter. aAraki, 1992. bOka et al., 2003. cJepson and Golden, 1987. Molecular profiles and phylogenetic status The partial 18S region, full length ITS, 28S D2- D3 region, and the cytochrome oxidase subunit II (CO II) sequences of M. paramali n. sp. were deposited in GenBank with the accession numbers MW342769-MW342772, MW342760-MW342768, MW342773-MW342775, and ON430646-ON430649, respectively. The full length ITS and 28S D2-D3 region sequences of M. marylandi were deposited in GenBank with the accession numbers ON453657- ON453659 and ON453660-ON453661. The 18S phylogenetic tree demonstrated that M. paramali n. sp. clustered with M. mali as an independent clade and close to the basal branch of Meloidogyne with high support value (posterior probability = 100). The intraspecific sequence divergence value of the four acquired 18S sequences of M. paramali n. sp. is 0.23% to 1.45% (5– 22/1,724 bp). M. paramali n. sp. shares the smallest sequence divergence value of 2.58% to 2.64% (41– 45/1707 bp) with M. mali. The ITS phylogenetic tree demonstrated the new species clusters with M. mali and M. vitis as an independent clade with fully supported value (posterior probability = 100). The new species showed a sister phylogenetic relationship with M. mali with low supported value (posterior probability = 60). The intraspecific sequence divergence value of the nine acquired M. paramali n. sp. ITS sequences is 0.15% to 7.35% (1–48/653 bp). The new species also shares the smallest sequence divergence value of 11.86% to 18.80% (81/683–129/686 bp) and 17.85% to 19.70% (108/605–120/609 bp) with M. mali and M. vitis, respectively. The 28S D2-D3 phylogenetic tree also displayed a similar topology with the ITS tree. The new species clustered with M. mali, an unidentified species Meloidogyne sp. GKL-2016, and M. vitis as a clade with fully supported value (posterior probability = 100). However, the new species is sistered to the clade that is comprised of M. mali, M. vitis, and Meloidogyne sp. GKL-2016. The analysis of the three acquired sequences of M. paramali n. sp. showed the intraspecific sequence divergence value of 3.24% to 6.35% (25–49/772 bp). M. paramali n. sp. shares the smallest sequence divergence value of 5.96% to 9.46% (46/772–74/782 bp) with M. mali. Further, the new species shared the sequence divergence value of 8.42% to 10.49% (65–71/772 bp) and 8.17% to 9.33% (63/771–72/772 bp) with Meloidogyne sp. GKL-2016 and M. vitis, respectively. In the COII phylogenetic tree, M. paramali n. sp. clustered with M. mali and M. vitis into a fully supported clade (posterior probability = 100). However, the phylogenetic relationship between these three species has remained unresolved. The analysis of the four acquired M. paramali n. sp. intergenic regions between CO II and 16S rDNA sequences showed the intraspecific sequence divergence value of 0.14% to 0.44% (1/687–3/688 bp). M. paramali n. sp. shares the sequence divergence values of 13.19% to 13.37% (72–73/546 bp) and 18.92% to 19.22% (102/539– 104/541 bp) with M. mali and M. vitis, respectively. This molecular phylogenetic analysis supports the status of M. paramali n. sp. as a new species. The sequence analysis of ribosomal rDNA 28S and ITS regions showed a 98% to 100% sequence similarity between the M. marylandi population from Japan and the registered M. marylandi, but it was quite different from other similar species. Therefore, the nematode was identified as M. marylandi.

Published as part of Gu, Jianfeng, Fang, Yiwu, Ma, Xinxin, Shao, Baolin & Zhuo, Kan, 2023, Meloidogyne paramali n. sp. (Nematoda: Meloidogyninae) and First Report of M. marylandi in maple and yacca tree from Japan, pp. 1-18 in Journal of Nematology 55 (1) on pages 8-15, DOI: 10.2478/jofnem-2022-0036, http://zenodo.org/record/12175678