Aphelenchoides fujianensis, Zhuo, Kan, Cui, Ruqiang, Ye, Weimin, Luo, Mei, Wang, Honghong, Hu, Xuenan & Liao, Jinling, 2010

Zhuo, Kan, Cui, Ruqiang, Ye, Weimin, Luo, Mei, Wang, Honghong, Hu, Xuenan & Liao, Jinling, 2010, Morphological and molecular characterization of Aphelenchoides fujianensis n. sp. (Nematoda: Aphelenchoididae) from Pinus massoniana in China, Zootaxa 2509, pp. 39-52 : 41-50

Aphelenchoides fujianensis n. sp.

Figs. 1–3

Measurements. See Table 2.

Material examined. Type-material: Holotype male, 20 paratype males and 20 paratype females are deposited in the USDA Nematode Collection, Beltsville, Maryland; two paratype females in the University of California Nematode Collection, Riverside, California; two paratype females in the Canadian National Nematode Collection, Ottawa, Canada and two paratype females at CABI Bioscience, UK Centre, Surrey, UK. Other voucher specimens and cultures are available at the Plant Nematode Research Laboratory, College of Resources and Environmental Sciences, South China Agricultural University, Guangzhou, China.

Description. Male. Body slender, posterior region curved ventrally when heat-relaxed; annules fine. Lateral fields with four incisures in mid-body. Lip region rounded in lateral view, unstriated, slightly offset. Stylet 12.5–14 μm long, with small basal swellings, stylet cone comprising ca. 45% of total stylet. Procorpus cylindrical, ca. four stylet lengths. Metacorpus oval, with conspicuous valve situated centrally. Nerve ring posterior to metacorpus. Excretory pore anterior to or level with nerve ring, posterior to metacorpus. Pharyngo-intestinal junction immediately posterior to metacorpus. Pharyngeal glands overlapping intestine dorsally for ca. 3–4 body diameters. Testis single, cells in single row, anteriorly outstretched. Spicules smoothly curved, rosethorn-shaped, apex small, rounded. Rostrum short, pointed. Gubernaculum absent. Tail conoid, terminus with a star-shaped mucro with two to three pointed processes. Three pairs of subventral caudal papillae observed: one pair located just anterior to cloacal aperture, the second slightly behind middle of tail, and the third subterminal. Bursa absent.

Female. About equal number as males. Body slender and ventrally curved when heat-relaxed. Anterior region and cuticle similar to male. Monodelphic, ovary outstretched, occupying 40–60% of body length, anterior end about two third of the distance from head to vulva reaching to pharyngeal glands, oocytes in one or two rows in posterior half of the ovary. Oviduct connecting ovary and spermatheca. Spermatheca oval, filled with sperms. Crustaformeria ovate-oblong, posterior to spermatheca, visible in some individuals. Uterus with thick wall, posterior to crustaformeria. Vagina inclined anteriorly at ca. 45° to body axis, both anterior and posterior vulval lips slightly protruding, vulval flap absent. Postvulval uterine sac elongate, 68–110 μm long, extending 32–44% of vulva-anus distance, ca. 3–4 vulval body widths or 5–8 anal body widths long, sperms usually present. Tail conoid, terminus with a star-shaped mucro with three pointed processes.

Diagnosis and relationships. Aphelenchoides fujianensis n. sp. is characterised by four lateral incisures in the lateral field, elongate postvulval uterine sac (extending ca. 32–44% of vulva-anus distance), usually filled with sperms, large spicules (24–30 μm), three pairs of caudal papillae, star-shaped mucro and relatively long body length (653–843 μm in the male and 803–941 μm in the female).

Aphelenchoides fujianensis n. sp. belongs to the Group 3 category of Aphelenchoides species sensu Shahina (1996), i.e., a group of species with tetramucronate spine or star-shaped terminus of the tail. The identification codes of OEPP/EPPO (2004) for A. fujianensis n. sp. are: A1-B2-C1-D1/3-E1-F1/2. To date, 21 known species in the Group 3 category of Aphelenchoides species sensu Shahina (1996) are described. Species comparisons in the Group 3 are compiled in Table 3 updated from Shahina (1996) and OEPP/EPPO (2004). The new species, on the basis of four lateral lines, star-shaped tail terminus, and conoid, slightly ventrally curved tail, appears morphologically most similar to A. besseyi , A. goodeyi Siddiqi & Franklin, 1967 , A. asteromucronatus Eroshenko, 1967 , A. siddiqii Fortuner, 1970 and A. silvester Andrassy, 1958 .

Aphelenchoides fujianensis n. sp. differs from A. besseyi by the size and shape of the male spicule (24–30 μm vs. 18–21 μm; short condylus vs. condylus absent), longer stylet (12.5–14 μm vs. 10–12.5 μm), longer female tail (46–58 μm vs 36–42 μm), longer postvulval uterine sac (5–8 anal body widths vs 2.5–3.5 anal body widths, extending for 32–44% vs. less than 33% of vulva-anus distance). It differs from A. goodeyi by the longer female body (803–941 μm vs. 460–610 μm), longer stylet (12.5–14 μm vs. 11.5–12.5 μm), position of the excretory pore (anterior to or level with nerve ring vs. posterior to or level with hind edge of nerve ring), longer gonad (anterior end reaching about two-thirds the distance from head to vulva, ending close to the pharyngeal glands vs. anterior end reaching one third to a half the distance from head to vulva), longer female tail (46–58 μm vs. 33–42 μm), mucro number (2–3 vs. 4), larger female b1 ratio [total body length divided by the distance from the anterior end to the base of the median bulb ( Siddiqi & Franklin, 1967)] (11–13 vs. 8–10 and the presence of males and of sperms in the postvulval uterine sac (both species were cultured on agar with fungi). From A. asteromucronatus , the new species differs by having a longer female body (803–941 μm vs. 390–530 μm), higher female c ratio (15.1–18.2 vs. 10.9–14.5), longer stylet (12.5–14 μm vs. 9 μm), longer postvulval uterine sac (3–4 vulval body widths vs. 1 vulval body width) and the presence of males. From A. siddiqii , by longer female body (803–941 μm vs. 370–700 μm), longer stylet (12.5–14 μm vs. 11–12.5 μm), longer postvulval uterine sac (3–4 vulval body widths vs. 0.5–1 vulval body width), longer female tail (46–58 μm vs. 26 μm) and longer spicule (24–30 μm vs. 16–17 μm). From A. silvester , by the head offset status (slightly offset from body vs. distinctly offset from body), longer female body (803–941 μm vs. 480–560 μm), longer stylet (12.5–14 μm vs. 9.5–10 μm), smaller female a ratio (31.5–36.3 vs. 37–38), higher female b ratio (9.8–13 vs. 8–9.7) and the presence of males.

Type-locality and habitat: Type specimens were obtained from a two-week-old culture on Pestalotia sp. The culture was initiated from one female and one male of A. fujianensis n. sp., which were collected in September 2008 by the first two authors from the wood of dead pine tree ( Pinus massoniana ), in Xiapu County, Fujian Province (latitude N 26.9 º, longitude E 120º), China.

Etymology: The specific epithet is derived from Fujian Province, where the new species was originally collected.

TABLE 2 Morphometrics of Aphelenchoides fujianensis n. sp. [Measurements are in μm and in the format: mean ± SD (range)] Male Female

Molecular characterizations and phylogenetic relationships. For molecular analysis, the 1552 bp nearfull-length 18S rDNA and the 710 bp partial mitochondrial COI genes were sequenced. A Blastn search of A. fujianensis n. sp. on the 18S rDNA revealed the highest match with A. besseyi (GenBank accession number AY508035 ). The identities of the two sequences are 97% (1528/1563) with 13 insertions/deletions (0.8%). The second highest match is A. ritzemabosi (DQ9901554) with 93% identifies (1454/1550) and 22 insertions/ deletions (0.9%). A Blastn search of A. fujianensis n. sp. on the mtCOI revealed the highest match with some Bursaphelenchus species, but the identities are less than 88%. Comparing with A. besseyi ( EU983281 ), A. fujianensis n. sp. has 102 variable sites with 83% identities (520/624) and no gaps indicating much higher variations in mtCOI than 18S. However, there is only 1 amino acid difference out of 197 amino acids between these two species which implied almost all the variations are on the synonymous sites for this protein coding gene and the tight phylogenetic relationships between the two species.

Fig. 4 represents a phylogenic tree based on 18S rDNA from a multiple alignment of 1625 total characters. This dataset has 851 constant characters (52.4%). The average nucleotide composition is as follows: 25.5% A, 19.5% C, 26.1% G and 28.9% T. According to previous molecular phylogenetic analysis ( Meldal et al., 2007, Zhao et al, 2008, Kanzaki et al., 2009), there is no significantly supported natural outgroup resolved for aphelenchoidoids. For the purpose of this study, a non aphelenchoidoid species Ditylenchus dipsaci was chosen as an outgroup taxon to root the tree. This tree inferred two main clades. Aphelenchoides fujianensis n. sp. is in a 100% supported monophyletic clade with other Aphelenchoides and Laimaphelenchus species with teramucronate spine or star-shaped terminus, but most closest to A. besseyi . Bursaphelenchus is placed in a separate clade far from A. fujianensis n. sp.

Fig. 5 represents a phylogenetic tree based on mtCOI from a multiple alignment of 591 total characters. This dataset has 321 constant characters (54.3%). The average nucleotide composition is as follows: 24.1% A, 11.9% C, 19.4% G and 44.5% T. This gene is A-T rich (68.6%) with a high proportion of Ts. Using Schistonchus caprifici as an outgroup taxon, this tree inferred two main clades. Aphelenchoides fujianensis n. sp. is in a 93% supported monophyletic clade with two Bursaphelenchus species. This group is in a 100% supported monophyletic clade with four other Aphelenchoides species including A. besseyi . The same dataset was translated into protein sequences and a Neighbor-joining tree was generated in Fig. 6 . This tree revealed A. fujianensis n. sp. is close to A. besseyi and another unidentified Aphelenchoides species. Interestingly, the tree inferred from protein sequences well separated A. fujianensis n. sp. with Bursaphelenchus species which are in the same clade inferred from DNA sequences ( Fig. 5 ).

Figures

FIGURE 1. Aphelenchoides fujianensis n. sp. A: Entire female; B: Entire male; C: Female anterior region; D: Vulval region; E: Lateral field; F: Female tail; H – J: Female tail tips; G: Male tail; K – N: Male tail tips. (All scale bars are 10 µm)

FIGURE 3. Scanning Electron Microscopy (SEM) observation of Aphelenchoides fujianensis n. sp. A: Female head. B: Female tail. C – D: Male tail.

FIGURE 4. The 10001 st Bayesian tree inferred from 18 S under GTR + I + G model (lnL = 13396.8457; freqA = 0.2545; freqC = 0.195; freqG = 0.2611; freqT = 0.2894; R (a) = 1.0777; R (b) = 2.5109; R (c) = 1.1882; R (d) = 0.7844; R (e) = 4.1621; R (f) = 1; Pinva = 0.2802; Shape = 0.5391). Posterior probability values exceeding 50 % are given on appropriate clades.

FIGURE 5. The 10001 st Bayesian tree inferred from COI under TVM + I + G model (lnL = 4576.3374; freqA = 0.2744; freqC = 0.0652; freqG = 0.1429; freqT = 0.5175; R (a) = 0.5338; R (b) = 40.2214; R (c) = 1.1291; R (d) = 9.7083; R (e) = 40.2214; R (f) = 1; Pinva = 0.2936; Shape = 0.1891). Posterior probability values exceeding 50 % are given on appropriate clades.

FIGURE 6. Neighbor-Joining tree inferred from COI encoded protein sequences.