Introduction
Ficus amplissima, a tree species of flowering plants, is a member of the family Moraceae, usually referred to as the fig or mulberry family. It is also known as the Indian bat tree or Indian bat fig. It is indigenous to Sri Lanka, the Maldives, central and southern peninsular India, and the Western Ghats of India, with a sizable distribution worldwide. F. amplissima is rarely found in the northern and eastern Indian states – Bihar, Gujarat, and Rajasthan, while it is widespread in southern states – Andhra Pradesh, Goa, Karnataka, Maharashtra, Tamil Nadu, Telangana, and Kerala (Wiebes Reference Wiebes1992; Chaudhary et al. Reference Chaudhary, Sudhakar, Kumar, Bajpai, Tiwari and Murthy2012). The species has medicinal properties, being used in Ayurveda, Siddha, and Unani medicine to treat diabetes. The tree’s bark has natural antidiabetic and antioxidant properties that lower blood sugar levels (Karuppusamy and Thangaraj Reference Karuppusamy and Thangaraj2013). This fig belongs to the subgenus Urostigma, section Urostigma, subsection Urostigma; monoecious, semi-deciduous tree species that is commonly found in Delhi and the northern parts of India. An insect species, Eupristina sp., is the sole pollinator of this fig species and a consistent carrier of nematodes (Wiebes Reference Wiebes1992). So far, no nematode species related to Schistonchus s.l. associated with Ficus amplissima have been identified or thoroughly studied.
The group Schistonchus sensu lato (s.l.) (Gasparrini Reference Gasparrini1864) Cobb, Reference Cobb1927 was reclassified into three distinct genera in 2015: Schistonchus, Ficophagus, and Martininema (Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015). As of now, the genus Ficophagus comprises 33 valid species. The genus Schistonchus alone comprises 15 species, while Martininema includes only four valid species (Waghmare et al. Reference Waghmare, Khan, Somvanshi, Islam, Sultana and Kundu2023; Kanzaki et al. Reference Kanzaki, Tanaka and Giblin-Davis2023; Zeng et al. Reference Zeng, Zhang, Giblin-Davis and Roberts2023a, Reference Zeng, Zhang, Giblin-Davis and Robertsb; Zeng et al. Reference Zeng, Zhang, Giblin-Davis and Roberts2024). The genus Ficophagus Davies & Bartholomaeus, Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 was separated from Schistonchus s.l. Cobb, Reference Cobb1927 (Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015). So far, seven Ficophagus species, Ficophagus antherobenghalensis, Ficophagus cuculloracemosus, Ficophagus flagelloracemosus, Ficophagus mucrobenghalensis, Ficophagus religiosus, Ficophagus glomerata, and Ficophagus virens have been described from India (Bajaj and Tomar Reference Bajaj and Tomar2014; Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015, Reference Davies, Ye, Kanzaki, Center, Bartholomaeus, Herre, Esquivel and Giblin-Davis2017; Gupta et al. Reference Gupta, Tahseen and Borges2021; Waghmare et al. Reference Waghmare, Somvanshi and Khan2022). All these descriptions are primarily based on morphological and morphometrical characteristics. A new species, Ficophagus glomerata Gupta, Tahseen and Borges, Reference Gupta, Tahseen and Borges2021, was described using morphological and molecular data from Ficus racemosa from Karnataka, India. Ficophagus virens (Bartholomaeus, Davies, Kanzaki, Ye and Giblin-Davis, Reference Bartholomaeus, Davies, Ye, Kanzaki and Giblin-Davis2009) Davies and Bartholomaeus, Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 and F. religiosus were further characterised from Ficus virens and F. religiosa, respectively, from Delhi, India (Waghmare et al. Reference Waghmare, Somvanshi and Khan2022; Waghmare et al. Reference Waghmare, Khan, Somvanshi, Islam, Sultana and Kundu2023). A recent survey of Ficus species on the Pusa Campus of ICAR-Indian Agricultural Research Institute (IARI), New Delhi, India, revealed a morphologically distinct and undescribed species of Ficophagus within the figs of Ficus amplissima. This newly discovered population is herein described as Ficophagus siddiqii n. sp. Molecular phylogenetic analysis provided a robust framework supporting the distinctiveness of F. siddiqii n. sp. Thus, the species is described by an integrative approach combining morphological and molecular data.
Materials and methods
Nematode material
Nematode specimens were collected from figs at developmental phases B and C (Giblin-Davis et al. Reference Giblin-Davis, Center, Nadel, Frank and Ramirez1995) of a Ficus amplissima tree located at ICAR-IARI, New Delhi, India (28°38′25.8″N, 77°10′6.1″E; elevation 189 m above mean sea level). Figs were sliced apart individually in a beaker filled with clear tap water. After an hour in the suspension, nematodes and accompanying wasps were visible. Live nematodes were handpicked into double-distilled water (ddH₂O) for subsequent DNA extraction, amplification, and sequencing. For morphological studies, specimens were collected and heat-killed using a hot-water bath (Hooper Reference Hooper and Southey1986), then fixed in Triethanolamine Formalin (TAF) solution (2 mL triethanolamine, 7 mL formalin, 91 mL distilled water). Fixed specimens were processed to anhydrous glycerin using the glycerol–ethanol dehydration method for morphometric analysis under light microscopy and subsequently transferred to 100% glycerin for permanent slide mounts (Seinhorst Reference Seinhorst1959).
Morphological and morphometrical study
Mature male and female specimens were handpicked under an Olympus stereozoom binocular microscope (SZ-9 Olympus, Japan). Specimens were processed by Seinhorst’s glycerol–ethanol method (1959), mounted in anhydrous glycerin on clean glass slides and sealed by the wax-ring procedure (De Maeseneer and d’Herde Reference de Maeseneer and D’Herde1963). The photomicrographs were taken with a compound microscope (Zeiss Axiocam M2m, Germany) equipped with differential interference contrast (DIC) lenses. de Manian (de Man Reference de Man1884) and other ratios (De Grisse Reference De Grisse1964) were determined based on the measurements taken on a compound microscope (Olympus BX50, Japan) fitted with an ocular micrometer and a drawing tube.
Molecular techniques
A single female nematode was put into a 0.2 mL microcentrifuge tube with 25 μL of sterile water for molecular characterisation. Equal parts of lysis buffer with the following ingredients were added: 0.2 M NaCl, 0.2 M Tris-HCl (pH 8.0), 1% (v/v) β-mercaptoethanol, and 2 μL proteinase K (stock: 10 mg/mL) (Holterman et al. Reference Holterman, van der Wurff, van den Elsen, van Megen, Bongers, Holovachov, Bakker and Helder2006). The lysis occurred in a thermocycler at 65°C for 2 h with periodic flicking, followed by a 10 min incubation at 95°C. Two μL of the lysate was used as a DNA template in the Polymerase Chain Reaction (PCR) reaction, while the remaining lysate was maintained at −20°C. Forward and reverse primer pairs were used in the PCR amplification; for D2–D3 expansion segment of the large subunit 28S rRNA gene (LSU) - D2A (5′-ACAAGTACCGTGAGGGAAAGT-3′) and D3B (5′-TGCGAAGGAACCAGCTACTA-3′) (Nunn Reference Nunn1992), for 18S rRNA gene (SSU) SSUF07 (5′-AAAGATTAAGCCATGCATG-3′) and SSUR26 (5′-CATTCTTGGCAAATGCTTTCG-3′) (Floyd et al. Reference Floyd, Abebe, Papert and Blaxter2002), for the COI gene, COI-F1 (5′-CCTACATGATTGGTGGTTTTGGTAATTG-3′) and COI-F2 (5′-GTAGCAGCAGTAAAATAAGCACG-3′) (Kanzaki and Futai Reference Kanzaki and Futai2002) and for ITS gene, 18S(F) (5′-TTGATTACGTCCCTGCCCTTT-3′) and 26S(R) (5′-TTTCACTCGCCGTTACTAAGG-3′) (Vrain et al. Reference Vrain, Wakarchuk, Lévesque and Hamilton1992). Two μL of lysed DNA was used to set up the PCR reactions in a 0.5 mL microcentrifuge tube containing 25 μL GoTaq® Green master mix (cataloguenumber M712, Promega, Madison, USA); 1 μL of each forward and reverse primer (10 μM); and nuclease-free water to a total volume of 50 μL. The thermal cycling programme included a denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 2 min. The last extension was carried out for 10 min at 72°C. According to the manufacturer’s protocol, PCR products were cleaned using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA). After confirming the band size on a 1.2% agarose gel electrophoresis of the PCR-amplified products, the purified PCR products were sent for outsourced Sanger sequencing (AgriGenome Labs Pvt. Ltd., Kerala, India). The raw sequences were manually compared with the chromatograms and modified to ensure quality. Molecular Evolutionary Genetics Analysis (MEGA) X was used for each marker gene to align and generate a consensus sequence (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018). A Basic Local Alignment Search Tool (BLAST) search was conducted using the recently acquired D2–D3 and SSU sequences to locate any sequences of additional Ficophagus species available in the National Center for Biotechnology Information (NCBI) GenBank. A dataset of sequences was then produced for phylogenetic analysis. The closely related NCBI GenBank sequences were individually aligned using ClustalW in MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018) with the default values for each marker. The most appropriate model to generate a phylogenetic tree was evaluated, and evolutionary history was inferred using MrBayes 3.2.6’s Bayesian inference (BI) method (Ronquist et al. Reference Ronquist, Teslenko, Van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The ideal nucleotide substitution model for the analyses was GTR + G + I, which includes a proportion of invariable sites, gamma-distributed rate variation across sites, and general time-reversible substitution. A random tree was used to start the analysis, which was carried out across 106 generations for each gene. The Markov chains were sampled every 1,000 generation intervals or when the standard deviation of split frequencies was less than 0.01. Burn-in samples were eliminated, and subsequent trees were used to produce a consensus tree that followed the 50% majority criterion.
Results
The Ficophagus population recovered from the Indian bat tree (Ficus amplissima) was described as Ficophagus siddiqii n. sp., and it was characterised based on morphology, morphometrics, and molecular data. All the morphological characters and their morphometrics of this new species from India are presented in Table 1.
Morphometrics of Ficophagus siddiqii n. sp. in Ficus amplissima from India. All measurements are in μm (except ratio) and in the form: mean ± s.d. (range). Specimens mounted in glycerin
Table 1. Long description
The table presents measurements in micrometers (mean plus or minus standard deviation and range) for Male (Holotype and Paratypes, n = 45) and Reproductive Female (Paratypes, n = 45) specimens.
Key measurements include:
* Body length (L): Male Holotype is 498, Paratypes average 494; Females average 446.7.
* De Man indices (a, b, c, c prime): Male c prime is 1.4; Female c prime is significantly higher at 4.0.
* V/T ratio: Male Holotype is 36.3; Female average is 67.5.
* Stylet length: Male Holotype is 23; Female average is 17.4.
* Tail length: Male Holotype is 19; Female average is 33.4.
* Spicule length: Male Holotype is 16, Paratypes average 15.4; not applicable for females.
* Ovary or Testis length: Male Holotype is 181; Female average is 313.3.
Other anatomical markers include anterior end to base metacorpus, pharyngeal gland, lip diameter and height, conus and shaft stylet, knob dimensions, S E pore, vulval position, P U S, and spermatheca dimensions.
Descriptions of the new species
Ficophagus siddiqii n. sp.
Ficophagus siddiqii n. sp. in lateral view. A: Entire reproductive female; B: Entire male; C: Anterior end of male; D: Female posterior vulva and ovary; E: Anterior end of female with secretory-excretory pore; F: Posterior end of female; G: Posterior end of male with spicule morphology and tail (scale bars: A–B = 25 μm; C–G = 20 μm).
Figure 1. Long description
A multi-panel scientific illustration containing seven panels labeled A through G.
Panel A, on the far right, shows a full reproductive female in lateral view, appearing as a long, slender, straight body with internal reproductive organs visible. A scale bar below indicates 25 micrometers.
Panel B, in the upper center, shows a full male in lateral view, characterized by a curved posterior end. A scale bar below indicates 25 micrometers.
Panel C, located above the center scale bar, provides a close-up of the male anterior end, showing the head structure and internal canal.
Panel D, at the top left, displays the female posterior vulva and ovary, showing a row of nucleated cells within the reproductive tract.
Panel E, on the far left, shows the anterior end of a female, highlighting the secretory-excretory pore and the bulbous structure of the esophagus.
Panel F, below panel D, shows the posterior end of a female, tapering to a sharp point.
Panel G, at the bottom center, provides a detailed view of the male posterior end, showing the curved tail and the hook-shaped spicule morphology used for mating.
A central scale bar for panels C through G indicates 20 micrometers.
Photomicrographs of Ficophagus siddiqii n. sp.: A: Anterior and posterior ends of reproductive females (arrowhead showing secretory-excretory pore, vulva position); B: Posterior end of reproductive female showing vulva and PUS (arrowhead pointing to terminal end of sac); C–D: Anterior end of female EP (arrowhead); E: Anterior and posterior ends of female showing vulva and tail shape (scale bars A–E = 10 μm).
Figure 2. Long description
A collection of five light microscopy panels labeled A through E, each containing a 10 mu m scale bar.
* Panel A: Shows three segments of the nematode. The central segment is the anterior end. An arrowhead on the left side of the central segment points to the secretory-excretory pore. An arrowhead on the right segment points to the vulva position.
* Panel B: A close-up of the posterior region. An arrowhead points to the vulva and the terminal end of the post-uterine sac, known as the P U S.
* Panel C and D: Two separate views of the anterior end. In panel D, an arrowhead points to the E P, or secretory-excretory pore, located just below the head region.
* Panel E: Displays three distinct sections. On the left is the anterior end. In the center, an arrowhead points to the vulva. On the right is the posterior end, showing a tapered, pointed tail shape.
Photomicrographs of Ficophagus siddiqii n. sp. A and C: Anterior ends of male showing EP (arrowheads) and head; B and D: Posterior ends of males showing by arrowheads rostrate spicules and caudal papillae (arrowheads) (scale bars A–D = 10 μm).
Figure 3. Long description
A four-panel set of light microscopy photos labeled A through D. Each panel includes a vertical scale bar representing 10 micrometers.
* Panel A (top-left) and Panel C (bottom-left) show the anterior ends of male nematodes. The bodies are elongated and cylindrical. An arrowhead in each panel points to the E P, or excretory pore, located slightly below the rounded head region.
* Panel B (top-right) and Panel D (bottom-right) show the posterior ends of male nematodes. The tails are strongly curved or coiled. Arrowheads point to the rostrate spicules, which are curved, sclerotized reproductive structures protruding near the tail.
* In Panel D, additional arrowheads highlight the caudal papillae, which appear as small sensory bumps along the ventral surface of the tail region.
Measurements
See Table 1.
Description
Male from figs (n = 45)
Body cylindrical, ventrally curved, spiral upon fixation. Head continuous, nearly 3/4 as high as wide at base. Cuticle with fine annulations. Lateral fields with four faint lateral lines at mid-body, the outermost being the most prominent. Secretory-excretory pore (SE pore) 11.9 (7.0–14) μm from the anterior end, close to the base of the lip region. Deirids and hemizonids not visible. Stylet 21.8 (18–24) μm long, weakly sclerotized with small, rounded stylet knobs. Length of conus and shaft 8.8 (3.0–12) μm and 13 (8.0–17) μm, respectively. Knobs height and diameter: 2.0 (2.0–2.0) μm and 2.5 (2.5–2.5) μm. Procorpus cylindrical, 2.5–2.6 stylet lengths long, ending in well-developed median pharyngeal bulb. Median pharyngeal bulb oval to oblong, 52.6 (49–58) μm in length from the anterior end. Pharyngo-intestinal junction just beneath the pharyngeal bulb. Nerve ring at the level of the pharyngo-intestinal junction. Basal pharyngeal lobe 108.4 (82–128) μm long, three pharyngeal gland nuclei, separate. Maximum body diameter 16.3 (14–18) μm. Testis outstretched or reflexed- 193.4 (108–208) μm in length from cloaca to anterior distal end of testis. Vas deferens barely offset, not muscular, distal end extending to cloaca. Testis located at 39.2% (21.2–45.5%) of total body length from the anterior end. Sperm amoeboid, cloacal body width 13.4 (11–17) μm. Spicules paired, slender, stout, rose-thorn-shaped and pointed distally. Capitulum with depression in the middle. Condylus relatively tall and rounded to truncate. Rostrum large, bluntly conical. Lamina dorsal line smoothly, symmetrically curved. Junction of rostrum and calomus smooth. Calomus not extending up to tip of lamina. Spicule tip finely rounded, without cucullus. Gubernaculum not observed. Three pairs of subventral caudal papillae arranged as one conspicuous adcloacal pair (P3) and two post-cloacal pairs (one near halfway between cloacal and tail terminus (P3a), the other pair (P4), 3–5 μm away near tail tip). Tail conoid, 1.1–1.6 anal body widths long, tip broadly rounded, without mucro. Bursa or bursal flap not observed.
Entomophilic female (from fig/wasp)
Observed but not collected for drawings, photomicrographs, or measurement.
Reproductive female from figs (n = 45)
Body cylindrical, almost straight, slightly ventrally curved upon being heat-killed. Anterior end slightly offset, continuous head, nearly 3/4 as high as wide at base. Cuticle with fine annulations, lip height and diameter 2.2 (2.0–3.0) μm to 4.7 (3.0–7.0) μm, respectively. Stylet 17.4 (16–19) μm long, strongly sclerotized stylet with small, rounded knobs. Length of conus and shaft 5.8 (4.0–8.0) μm and 11.6 (10–12) μm, respectively. Knobs height and diameter 1.4 (1–5) μm and 2.0 (2.0–2.0) μm. Lateral field with four faint lateral lines as in males. SE pore at 13.2 (12–16) μm from the anterior extremity, close to the base of the labial region. Deirids and hemizonids not visible. Procorpus, pharyngeal bulb, nerve ring, pharyngeal glands, and pharyngo-intestinal junction as for males. Median pharyngeal bulb 59.5 (55–66) μm in length from the anterior end; basal pharyngeal lobe 94 (89–99) μm long. Maximum body diameter 16.4 (14.5–18) μm. Vulva at 301.2 (297–307) μm from the anterior end, at 67.5% (64.6–75.1%) of the body. Vulval lips slightly protruding from its mouth and vulval flap present; vulval body width 14.8 (14–16) μm. Vagina slightly inclined anteriorly. Oviduct not well demarcated, crustaformeria not differentiated under light microscopy, uterus long and elongated. Ovary straight or reflexed, oocytes arranged in a single row. Spermatheca of round to oblong shape, 10–14 × 6.5–9.0 μm in size, containing amoeboid sperm. Post-uterine sac (PUS) 5–10 μm long from the vulva. Tail elongated conoid, rounded, 3.3–4.5 anal body widths long, tapering with spike-like tail terminus or pointed tail end.
Type host and locality
Male and reproductive females collected from figs of Ficus amplissima L. growing at ICAR-IARI, Pusa campus, New Delhi, India (28°38′25.8″N, 77°10′6.1″E; elevation 189 m above mean sea level).
Type material
Holotype male, one male, and one reproductive female paratype of Ficophagus siddiqii n. sp. are deposited in the National Nematode Collection of India (NNCI) – Division of Nematology, ICAR-Indian Agricultural Research Institute, New Delhi, while one male and one reproductive female paratype are deposited in the Zoological Survey of India, Kolkata, India.
Etymology
The species is named in remembrance of eminent nematode taxonomist Dr. Mohammad Rafiq Siddiqi.
Diagnosis and relationships
Ficophagus siddiqii n. sp. can be easily diagnosed by a cylindrical body, straight, slightly ventrally curved to spiral-shaped upon heat fixation/killed, a continuous head region with a faintly offset labial disc, stylet length of both male and female 21.8 (18–24) μm and 17.4 (16–19) μm, respectively and tail conoid, pointed or spike-like. Post-uterine sac length (PUS) 8.3 (5–10) μm, spermatheca round to oblong-shaped. Ficophagus siddiqii n. sp. is characterised by having the EP opening near the lips, a short-length PUS, rose-thorn-shaped spicules with a condylus relatively tall and rounded to truncate. Rostrum large, stout, bluntly conical, and three pairs of caudal papillae on the male tail. It is also distinguished by its biogeography and host plant, being collected from Ficus amplissima, and its status as a distinct species is corroborated by molecular data from sequencing of 28S D2–D3, 18S SSU, COI, and ITS (Figs. 4–7).
Bayesian inference analysis of Ficophagus siddiqii n. sp. using the D2–D3 expansion region of the LSU rDNA gene. The evolutionary history was inferred by Bayesian analysis using the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR + G + I). The branch length and scale bar are indicated.
Figure 4. Long description
A phylogenetic tree diagram with a root at the bottom left labeled A J 5 4 5 0 1 9 Pratylenchus thornei. The tree branches out to the right with bootstrap values indicated at the nodes.
* The uppermost and largest clade contains numerous species of Ficophagus, including F. altermacrophylla, F. benjamina, and F. virens.
* Near the bottom of the Ficophagus group, the species P V 4 3 5 4 9 2 Ficophagus siddiqii n. sp. is highlighted in bold text, branching off a node with a support value of 89.
* Below the Ficophagus clade is a distinct group containing Martininema species, such as M. baculum and M. guangzhouensis.
* The next clade down consists of Schistonchus species, including S. caprifici and S. superbus.
* A scale bar at the bottom center indicates a genetic distance of 0.5 substitutions per site.
In overall morphological appearance, Ficophagus siddiqii n. sp. is most similar to Ficophagus flagelloracemosus (Bajaj and Tomar Reference Bajaj and Tomar2014) from fig sycones of F. racemosa from India. Ficophagus siddiqii n. sp. is similar to F. flagelloracemosus in having females with a similar body size 446.7 (401–462) μm vs 475.9 (442–504) μm, c ratio 13.4 (11.5–14.9) vs 13.9 (12.8–16.8), distance of SE pore from anterior end 13.2 (12–16) μm vs 11.4 (11–13) μm, and tail length 33.4 (30–36) μm vs 34.3 (30–38) μm, and in the number and position of pairs of caudal papillae (three, with one pair adcloacal on the posterior lip, one at mid-tail and one near the tip) but it differs in a-ratio 27.3 (24.3–31.1) vs 26.9 (24.3–30.3), b ratio 4.7 (4.3–5.1) vs 9.0 (8.0–10.1), V 67.5 (64.6–75.1) vs 76.2 (74.7–78.2), c′ ratio 4.0 (3.3–4.5) vs 4.5 (3.4–5.2), stylet length 17.4 (16–19) μm vs 14 μm. In males, a few characters are similar with some deviations (body size 494 (431–512) μm vs 379.8 (343–446) μm, c′ value 1.4 (1.1–1.6) vs 1.3 (1.2–1.4)), but it differs in having a larger a ratio 29.8 (14–36.4) vs 27.3 (22.8–32.9), smaller ratio b 4.6 (3.9–5.9) vs 9.1 (8.1–11.2), larger c ratio 27.4 (24.5–34) vs 24.3 (21.1–27.5), spicule length 15.4 (14–17) μm vs 10.1 (9.0–11) μm and sperm (amoeboid vs flagellate). However, both can be distinguished by female body shape, tail shape and tail tip, and oesophageal lobe. Ficophagus siddiqii n. sp. having female body cylindrical, straight upon heat killing, but it is C-shaped in F. flagelloracemosus upon fixation; tail tip pointed to bluntly round vs narrowly rounded; tail with a small digitate process as in F. flagelloracemosus, but it is absent in Ficophagus siddiqii n. sp., long esophageal lobe vs short to medium. Based on morphometric characterisation, Ficophagus siddiqii n. sp. was also found to be similar to F. benjamina (Bartholomaeus, Davies, Ye, Kanzaki and Giblin-Davis, Reference Bartholomaeus, Davies, Ye, Kanzaki and Giblin-Davis2012) by having short PUS 8.3 (5.0–10) μm vs 8.0 (5.0–10) μm and body diameter 16.4 (14.5–18) μm vs 17 (14–22) μm in females but differs in most of the other morphometric characters. Ficophagus siddiqii n. sp. from India completely differs from other described Ficophagus species in its body shape as well as in length. However, females of both species (i.e., Ficophagus siddiqii n. sp. and F. benjamina) can be separated by spermatheca structure, ovary length, and tail tip. Ficophagus siddiqii n. sp. has a rounded to ovoid spermatheca, whereas no true spermatheca in F. benjamina, ovary not extending beyond metacorpus in any of specimens vs extending beyond metacorpus in few specimens, no tail mucro in females of any specimens vs mucronate tail in one of the specimens.
Ficophagus siddiqii n. sp. females are somewhat similar to F. africanus (Vovlas, Troccoli, van Noort and Van den Berg, Reference Vovlas, Troccoli, Van Noort and Den Berg1998) Davies and Bartholomaeus, Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 females in shape; however, it differs in larger body diam. 16.4 (14.5–18) vs 11.3 (10–13.7 μm), stylet length longer 17.4 (16–19) vs 14.6 (13.2–15.7 μm), and SE pore distance anterior from stylet knobs 13.2 (12–16) vs 18.1 (14.7–26.8 μm), a smaller body size 446.7 (401–462) vs 481 (429–559) μm, smaller a, b, c and c′ ratios (27.3 (24.3–31.1) vs 42.2 (33.6–52.2), 4.7 (4.3–5.1) vs 5.6 (5.2–6.1), 13.4 (11.5–14.9) vs 14.5 (9.9–18.5) and 4.0 (3.3–4.5) vs 5.0 (4.0–6.3), respectively), anterior end to metacorpus base length smaller 59.5 (55–66) vs 85 (75–98) μm, anterior end to pharyngeal gland base length smaller 94 (89–99) vs 144 (122–170) μm, smaller tail 33.4 (30–36) vs 33.6 (30.2–47) μm, shorter PUS (5.0–10) vs PUS one body width long (9.0–19) and also in males it differs by having a longer body size (L = 494 (431–512) vs 448 (340–538) μm, lower b ratio 4.6 (3.9–5.9) vs 6.1 (5.0–6.9), larger c ratio 27.4 (24.5–34) vs 16.9 (15–21.7), and lower c′ ratio 1.4 (1.1–1.6) vs 1.9 (1.6–2.1), anterior end to metacorpus base length smaller 52.6 (49–58) vs 74 (59–86) μm, anterior end to pharyngeal gland base length smaller 108.4 (82–128) vs 127 (102–151) μm, short tail 18.1 (15–20) vs 26.6 (21.8–30.4) μm), and short spicule 15.4 (14–17) vs 21 (17.8–23.5) μm and in having a P3, P3a, P4 arrangement of the caudal papillae however it is different arrangement P2, P3, P3a.
Molecular phylogenetic analyses based on D2–D3 (LSU), SSU, and COI sequences indicated that Ficophagus siddiqii n. sp. is closely related to six characterised species: F. altermacrophylla (Lloyd and Davies, Reference Lloyd and Davies1997) (AB535534) from Australia; F. altissimus (Zeng, Ye, Huang, Li and Giblin-Davis, Reference Zeng, Ye, Huang, Li and Giblin-Davis2013) (KC526930) from China; F. zealandicus (Zhao, Li, Davies and Ye, Reference Zhao, Li, Davies and Ye2015) (KM276663) from New Zealand; F. bullenei (Davies, Ye, Kanzaki, Center, Bartholomaeus, Herre, Esquivel and Giblin-Davis, Reference Davies, Ye, Kanzaki, Center, Bartholomaeus, Herre, Esquivel and Giblin-Davis2017) (KJ638395) from Central America; F. elizabethae (Davies, Bartholomaeus, Li, Zhao, Ye and Giblin-Davis, Reference Davies, Bartholomaeus, Li, Zhao, Ye and Giblin-Davis2020) (KY996314) from eastern Australia; and F. richardi (Davies, Bartholomaeus, Li, Zhao, Ye and Giblin-Davis, Reference Davies, Bartholomaeus, Li, Zhao, Ye and Giblin-Davis2020) (KJ638372) from eastern Australia. Additionally, molecular phylogenetic analyses revealed a close relationship with five uncharacterised Ficophagus taxa, including four from South Africa: Ficophagus sp. 7 NK-2021 (LC606054), Ficophagus sp. 8 NK-2021 (LC606114), Ficophagus sp. 8 NK-2021 (LC606113), and Ficophagus sp. 9 NK-2021 (LC606075), as well as Ficophagus sp. 5069 KD3 ex Ficus microcarpa (KM817192) from Singapore. Based on ITS sequences, Ficophagus siddiqii n. sp. formed a separate clade in the phylogenetic tree because there were not enough sequences of other Schistonchus s.l. group species to be adequately studied.
Ficophagus siddiqii n. sp. is sister to F. zealandicus and F. elizabethae based on phylogenetic inference from the SSU sequences. However, it differs from F. zealandicus by the absence of a gubernaculum, different spicule morphology (rostrum large, stout, bluntly conical vs distinct but low rostrum; condylus relatively tall and rounded to truncate vs condylus not prominent; broadly rounded tip vs tip offset, conoid with mucron), a lower a ratio 30.4 (23.9–36.4) vs 33.2 (30.5–37.6) in male, and a smaller spicule 15.4 (14–17) vs 16.8 (16–18) μm. In females, PUS is smaller 8.3 (5.0–10) vs 13.6 (10.0–16.0) and it also differs from F. elizabethae with different spicule morphology (rostrum large, stout, bluntly conical vs distinct but low narrow to broad rostrum; condylus relatively tall and rounded to truncate vs prominent, broad and bluntly rounded; broadly rounded tip vs tip offset, conoid, broadly rounded, usually mucronate), a lower a-ratio 30.4 (23.9–36.4) vs 34.6 (28.9–38.1) in male, and a smaller spicule 15.4 (14–17) vs 18 (17–19) μm.
In overall appearance, males of Ficophagus siddiqii n. sp. are close to those of Ficophagus richardi, F. altermacrophylla, F. bullenei, and F. altissimus based on D2–D3 of LSU and COI sequences. However, the collection sites of these nematodes are from fig hosts that are geographically different (Australia, USA, and China) and associated with different pollinator wasp species (Pleistodontes greenwoodi Grandi and P. xanthocephalus Lopez-Vaamonde, Dixon, Cook and Rasplus vs P. froggatti in Australia, Pegoscapus spp. in USA, Eupristina altissima in China, respectively).
Ficophagus siddiqii n. sp. differs from F. richardi in spicule form, having a small rostrum, high condylus vs more depressed one. Females of Ficophagus siddiqii n. sp. have a cylindrical, almost straight body vs ventrally C-shaped; lateral field with four faint lateral lines vs two lines; elongated tail, conoid and rounded, tapering with spike-like tail terminus or pointed tail end vs tip usually bluntly rounded, occasionally narrower, V-shaped hyaline area, mucron absent. In females, PUS is smaller, 8.3 (5.0–10) vs 13 (8.0–21).
Ficophagus siddiqii n. sp. differs from F. altermacrophylla in spicule shape (condylus depressed at middle and rostrum more prominent, stout in Ficophagus siddiqii n. sp. vs curved junction between rostrum and calomus, no cucullus), rostrum stout and broadly squared vs variable but not broadly squared, lacking gubernaculum vs with gubernaculum-like structure in males and the respective positions of the genital papillae (three pairs; one adcloacal, one posterior to mid tail and one near tail tip vs two or three pairs; one precloacal, one adcloacal and one posterior to mid-tail).
Ficophagus siddiqii n. sp. differs from F. bullenei males with high condylus vs low condylus, large rostrum, stout square vs small rostrum, lateral field with four faint lateral incisures vs three incisures, and different pollinator wasps associated: Eupristina sp. vs Pegoscapus gemellus.
Ficophagus siddiqii n. sp. differs from F. altissimus in having a reproductive female tail tip morphology (without mucron vs with mucron), spicule morphology and size (rose-thorn-shaped, without cucullus vs hook-shaped with a cucullus); in males, lateral field with four faint lateral lines vs three lines; spicule tip without vs with cucullus; spicule appears sturdier vs slender; high condyles vs small condyles. In females, having body cylindrical, almost straight, slightly ventrally curved vs ventrally arcuate or C-shaped; tail elongated conoid, rounded, tapering with spike-like tail terminus or pointed tail end vs conoid, tail tip rounded with a mucron.
In having a rose-thorn-shaped spicule with a ventrally hooked dorsal limb, Ficophagus siddiqii n. sp. differs from all described species of Ficophagus except F. altissimus. Having a P3, P3a, P4 arrangement of the caudal papillae, it is similar to all described species of Ficophagus, except F. altermacrophylla and F. centerae, which have an arrangement of P2, P3 and P3a.
Molecular phylogenetic relationships
Partial SSU, LSU D2–D3 expansion segments, COI, and ITS were sequenced to infer molecular phylogenetic placement of Ficophagus siddiqii n. sp. among other Schistonchus s.l. species. The LSU, SSU, COI, and ITS sizes were 419 bp, 782 bp, 636 bp, and 841 bp, respectively. The nematode sequences were deposited in the GenBank database under the accession numbers listed in Figures 4–7. The tree inferred based on Bayesian analysis from D2–D3 of LSU (Fig. 4) using Pratylenchus thornei Sher & Allen, 1953 as an outgroup suggested that: (i) three distinct monophyletic clades, i.e., Ficophagus clade, Martininema clade and Schistonchus clade, and species/populations within the Ficophagus clade, Martininema clade and Schistonchus clade are divided into somewhat highly-supported clades with 67%, 98% and 100% posterior probability (pp), respectively; (ii) Ficophagus siddiqii n. sp. and other species of Ficophagus are clustered in a medium-supported (67% pp) monophyletic clade, although the new species is in an unsupported clade close to species/populations of Ficophagus sp. 9 NK-2021, Ficophagus sp. 7 NK-2021, F. zealandicus, F. richardi, F. altermacrophylla, and F. elizabethae; (iii) nine selected species/populations of Martininema are in another monophyletic clade with 98% pp; and (iv) nine selected species/populations of Schistonchus are in another monophyletic clade with 100% pp.
The tree inferred from SSU (Fig. 5) using Parasitodiplogaster salicifoliae as the outgroup suggested that: (i) Ficophagus siddiqii n. sp. and the selected total of 40 species/populations are distinctly clustered into three highly-supported (88%, 83% and 100% pp, respectively) monophyletic clades, i.e., Ficophagus clade, Martininema clade and Schistonchus clade, and ii) Ficophagus siddiqii n. sp. is in a monophyletic clade with Ficophagus sp. 5069 KD3 Ficus microcarpa, F. elizabethae, F. zealandicus with high support (100% pp) and is sister to Ficophagus sp. 5069 KD3 Ficus microcarpa.
Bayesian inference analysis of Ficophagus siddiqii n. sp. using the 18S rDNA (SSU) marker and the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR + G + I). The branch length and scale bar are indicated.
Figure 5. Long description
The phylogenetic tree begins at a root on the far left labeled K R 7 8 0 0 6 8 Hirshmanniella mucronata. Moving right, the tree splits into several major clades.
At the top, a large cluster includes various Ficophagus species such as F. parensis, F. virens, and F. bullenei.
In the middle section, Ficophagus siddiqii n. sp. (labeled P V 4 2 2 6 1 2) is positioned within a clade that includes F. zealandicus, F. elizabethae, and F. auriculae. This specific branch is highlighted in bold text.
Below this, another major clade contains F. yoponensis and F. maxima, followed by a group of Martininema species including M. guangzhouensis and M. semicordatae.
At the bottom right, a distinct clade features Schistonchus species, including S. caprifici and S. superbus.
Numerical values representing bootstrap support are placed at each node, ranging from 51 to 100. A scale bar at the bottom center indicates a genetic distance of 0.04.
The tree inferred from COI (Fig. 6) using Hirschmanniella mucronata (Das, Reference Das1960) Luc and Goodey, 1964 as the outgroup suggested that: (i) four genera, Schistonchus, Aphelenchoides, Ficophagus, and Martininema, are clustered into a single monophyletic clade with 91% pp; (ii) the ten selected species/populations of Bursaphelenchus Fuchs, 1937 are in another monophyletic clade with 100% pp; (iii) Ficophagus siddiqii n. sp. (new species) is in an unsupported clade close to species/populations of F. bullenei and F. altissimus; and (iv) the mitochondrial COI-based Bayesian phylogenetic analysis could not resolve the phylogenetic position of Ficophagus siddiqii n. sp.
Bayesian inference analysis of Ficophagus siddiqii n. sp. using the mtCOI marker using the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR + G + I). The branch length and scale bar are indicated.
Figure 6. Long description
The phylogenetic tree branches from a root on the left toward terminal nodes on the right.
* The top major clade consists of Schistonchus caprifici with multiple accessions including E U 2 8 7 6 1 7 and O K 0 6 7 4 0 4, followed by Schistonchus sp. 4 N K 2 0 2 1 and a cluster of Aphelenchoides paradalianensis.
* The middle section contains the genus Ficophagus. This includes clusters of Ficophagus fleckeri, Ficophagus centerae, Ficophagus costaricana, and Ficophagus sp. 8 N K 2 0 2 1.
* Positioned centrally within this group is the target species P V 4 2 2 7 9 8 Ficophagus siddiqii n. sp., shown in bold text on a distinct branch.
* Below the target species are Ficophagus bullenei and Schistonchus hirtus.
* The lower section of the tree features clades for Martininema baculum, Aphelenchoides xylocopae, and a large group of Bursaphelenchus species including B. xylophilus, B. mucronatus, B. doui, and B. microcarpae.
* The tree is rooted at the bottom by Parasitodiplogaster salicifoliae L C 6 0 6 1 2 4.
* Numerical bootstrap values are provided at the nodes, such as 1 0 0, 9 1, and 5 6, indicating branch support.
* A scale bar at the bottom represents 0.6 units of genetic distance.
The tree inferred from ITS (Fig. 7): only two Schistonchus s.l. species were available in NCBI data, such as Ficophagus sp. 5069 KD3 ex Ficus microcarpa (KM817192) and Martininema guangzhouensis 5068 KD1 ex Ficus hispida (KM817189), so for a more detailed comparison, Ficophagus population/species sequences were not available in NCBI data for our obtained sequences of Ficophagus siddiqii n. sp., so we were unable to determine the phylogenetic position of the current species.
Bayesian inference analysis of Ficophagus siddiqii n. sp. using the ITS marker and the General Time Reversible substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites (GTR + G + I). The branch length and scale bar are indicated.
Figure 7. Long description
The phylogenetic tree begins at a root on the far left, branching rightward. A major node with a bootstrap value of 100 separates the outgroup, L N 8 2 7 6 2 4 Teratorhabditis synpapillata at the bottom, from the main ingroup.
The main ingroup splits into two primary clusters. The upper cluster is further divided into several sub-clades. At the top, a clade with bootstrap values of 100 and 97 includes two specimens of Bursaphelenchus abruptus (A M 4 0 0 2 4 4 and A B 0 6 7 7 5 6). Below this, a clade with a value of 99 contains Aphelenchoides subtenuis (K Y 6 9 5 1 2 9) and Aphelenchoides primadentus (M F 9 9 1 9 6 2).
Directly below these is the target species, P V 4 5 4 5 1 4 Ficophagus siddiqii n. sp., highlighted in bold.
Continuing down the upper cluster, a clade with values of 71 and 95 contains three specimens of Aphelenchoides bicaudatus (M F 6 6 9 5 1 1, M K 7 7 5 0 2 6, and H Q 2 5 9 0 6 4). This is followed by two specimens of Aphelenchoides sp. (E U 2 8 7 6 0 1 and E U 1 8 6 0 7 1) at a node of 70 and 99. Three individual branches follow: Bursaphelenchus minutus (L R 7 4 6 5 4 2), Aphelenchoides rotundicaudatus (M K 3 0 5 9 0 4), and Aphelenchoides arachidis (K C 7 7 1 2 7 8).
The lower cluster, marked by a node of 100, contains four species: Martininema guangzhouensis 5 0 6 8 K D 1 ex Ficus hispida (K M 8 1 7 1 8 9), Aphelenchoides varicaudatus (H Q 2 8 3 3 5 1), Aphelenchoides fragariae (M K 2 9 2 1 2 3), and Ficophagus sp. 5 0 6 9 K D 3 ex Ficus microcarpa (K M 8 1 7 1 9 2).
A scale bar at the bottom left indicates a genetic distance of 0.7.
Discussion
This study confirms the discovery of a novel aphelenchoidid nematode, Ficophagus siddiqii n. sp., associated with the figs of Ficus amplissima (Indian bat tree) at ICAR-IARI, Pusa Campus, New Delhi, India. Using an integrative taxonomic approach combining morphological and molecular analyses, we provide the first global record of a Schistonchus s.l.-related species from F. amplissima.
Although fig-nematode associations play an important role in Ficus-pollinator mutualisms, no Schistonchus s.l. species has previously been reported from F. amplissima. Furthermore, many Indian Ficophagus species remain poorly characterised because they rely primarily on subtle morphological traits without molecular support. F. siddiqii n. sp. is distinguished by a moderately slender body, with the greatest body diameter (16–18 μm) occurring at mid-body length, a weakly offset lip region, a straight, robust stylet with small rounded or sloping knobs, and a conoid tail with a finely to broadly rounded terminus lacking a mucro. Females possess a monodelphic-prodelphic reproductive system with a post-uterine sac about 0.2 vulval body diameter long, whereas males have rose-thorn-shaped spicules, a tall rounded or truncate condylus, a blunt conical rostrum, and three pairs of caudal papillae. These characters distinguish the species from closely related taxa, including F. virens (Bartholomaeus, Davies, Ye, Kanzaki and Giblin-Davis, Reference Bartholomaeus, Davies, Ye, Kanzaki and Giblin-Davis2009) Davies and Bartholomaeus 2015 in Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 , F. glomerata (Gupta, Tahseen and Borges, Reference Gupta, Tahseen and Borges2021), F. centerae (Zeng, Giblin-Davis and Ye, Reference Zeng, Giblin-Davis and Ye2007) Davies and Bartholomaeus, Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 in Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015, and F. microcarpus (Zeng, Ye, Giblin-Davis, Li, Zhang and Du, Reference Zeng, Ye, Giblin-Davis, Li, Zhang and Du2011) Davies and Bartholomaeus, Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015 in Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015, which differ in spicule morphology, capitulum structure, tail shape, and papillae arrangement. Four distinct lateral field incisures provide an additional diagnostic character.
The species is further characterised by an exceptionally slender cylindrical female body (14.5–18.0 μm wide), narrower than all described congeners except F. bullenei (9–12 μm). Despite this similarity, F. siddiqii n. sp. differs from F. bullenei in both sexes by its longer, more robust stylet with distinct basal knobs, a longer post-uterine sac, a more anterior vulva, a conoid to finely pointed female tail, and differences in spicule length, curvature, capitulum shape, gubernaculum structure, and ventral genital papillae. Its diagnostic distinction is further supported by unique b and c ratios that fall outside the known range of F. bullenei and other Ficophagus species.
Phylogenetic analyses based on SSU, D2–D3, COI, and ITS sequences consistently recovered F. siddiqii n. sp. as a distinct lineage within the Ficophagus clade. The SSU dataset recovered a monophyletic group with Ficophagus sp. 5069 KD3 from F. microcarpa (Singapore), F. zealandicus (New Zealand), and F. elizabethae (Australia), with strong support in the SSU phylogeny (100% posterior probability) and moderate support in the COI (57%) and ITS (82%) datasets. These results suggest a shared evolutionary ancestry among these taxa, followed by independent evolutionary divergence. In contrast, the D2–D3 tree showed only moderate support (67% posterior probability), likely reflecting the relatively short sequence length (419 bp), and grouped the new species with F. zealandicus, F. elizabethae, F. richardi, F. altermacrophylla, and two unidentified African Ficophagus species (Ficophagus sp. 9 NK-2021 and Ficophagus sp. 7 NK-2021).
The phylogenetic analyses based on molecular inference (Figs. 4 and 5) support the three major lineages recognised within Schistonchus s.l., consistent with previous studies (Davies et al. Reference Davies, Ye, Kanzaki, Bartholomaeus, Zeng and Giblin-Davis2015; Zeng et al. Reference Zeng, Huang, Zhang, Zeng, Shi, Zhang, Peng, Ye and Giblin-Davis2019). F. siddiqii n. sp. is clearly nested within the Ficophagus lineage. Beyond its molecular placement, the new species is distinguished from its closest relatives by host specificity, biogeography, and pollinator associations. F. siddiqii n. sp. is exclusively associated with F. amplissima in India (section Urostigma, subsection Urostigma) and is linked with Eupristina wasps. In contrast, F. zealandicus is associated with F. macrophylla in New Zealand (section Malvanthera, subsection Malvantherae), while F. elizabethae occurs on multiple Australian hosts, including F. rubiginosa and F. obliqua (section Malvanthera, subsection Platypodeae) and F. macrophylla, with both species being associated with Pleistodontes wasps.
Together, the distinctive morphology, unique morphometrics, ecological specialisation, and nucleotide differences across all four molecular markers firmly establish F. siddiqii n. sp. as a novel species with no evidence of recent gene flow with other described congeners, suggesting long-term co-evolution with F. amplissima.
In conclusion, F. siddiqii n. sp. represents the first Schistonchus s.l.-related species described from F. amplissima and expands our understanding of Ficophagus diversity. This study demonstrates the value of integrative taxonomy in resolving fig-nematode diversity and highlights the need for broader surveys of Ficus-associated nematodes across tropical ecosystems.
Acknowledgements
The authors thank the Post Graduate School, ICAR –Indian Agricultural Research Institute, New Delhi and the Division of Nematology for supporting this study.
Financial support
No financial support.
Competing interests
The authors declare no competing interests.
Ethical standard
The authors affirm that all procedures involved in this study complied with the relevant national and institutional ethical guidelines for the collection, handling, care, and dissection of animals.
