https://orcid.org/0000-0001-8752-1674
Figures
Abstract
The nematode genus Bursaphelenchus is a highly divergent group. This genus mainly consists of mycophagous entomophilic species, but some species have specialized as obligate or facultative plant parasites, facultative insect parasites, or exhibit feeding dimorphism (phenotypic plasticity) leading to mycophagous and predatory forms. In the present study, a new Bursaphelenchus species, B. suri n. sp. was isolated from fresh syconia (figs) of Ficus sur and is described and illustrated based on its typological characters and molecular phylogenetic status. The new species is characterized by its highly derived feeding structures found in obligate plant parasites, lip possessing a labial disc and a long and thick stylet with a long conus and extremely well-developed basal swellings. In addition, slender body of both sexes is characteristic of the species. The new species is phylogenetically and typologically closely related to B. sycophilus, i.e., these two species share the characteristic feeding structures and form a well-supported clade within the B. fungivorus group in the genus. Biologically, these two species are both isolated from fresh figs of the section Sycomorus. However, the new species differs from B. sycophilus by the length of the female post-uterine sac and the shape of the male spicule, i.e., the new species has a long post-uterine sac and spicule condylus without dorsal recurvature. Thus, the new species is the second obligate fig parasite of the genus, and the evolutionary relationship between the B. suri n. sp. and B. sycophilus clade and section Sycomorus figs is hypothesized as an example of adaptive radiation with more species to be discovered.
Citation: Kanzaki N, Kruger MS, Greeff JM, Giblin-Davis RM (2022) Bursaphelenchus suri n. sp.: A second Bursaphelenchus syconial parasite of figs supports adaptive radiation among section Sycomorus figs. PLoS ONE 17(4): e0265339. https://doi.org/10.1371/journal.pone.0265339
Editor: Ebrahim Shokoohi, University of Limpopo, SOUTH AFRICA
Received: August 10, 2021; Accepted: February 20, 2022; Published: April 6, 2022
Copyright: © 2022 Kanzaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported in part by the National Research Foundation of South Africa for financial support (grant 77256 to J.M.G.) and Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (20H03026 to N.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Therefore, any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and therefore funders do not accept any liability in regard thereto.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The specific pollination system of figs (Ficus spp.) and fig wasps has been studied as a model system for co-evolution and diversification [1–4]. There, several other invertebrates, e.g., mites and nematodes are involved as phoretic associates and parasites of the wasps, plant (syconia tissue) parasites, microbe feeders and predators [5–9]. For example, the nematode genus Parasitodiplogaster Poinar has been examined as a case study of host-parasite virulence evolution [5] and Pristionchus Kries was revealed as a radiation of nematodes that manifest extreme trophic diversity through divergent developmental phenotypes in section Sycomorus figs [10]. After the early reports of fig/fig wasp-associations with nematodes [11, 12], more than ten genera of nematodes have been reported to have specialized associations with the fig pollination system [9–11, 13–19].
The nematode superfamily Aphelenchoidoidea is a divergent group in terms of feeding life history traits and insect (invertebrate) associations. The superfamily is derived from soil-dwelling fungal feeders, and currently contains fungal feeders, plant parasites, insect parasites, and predators [20–22]. Within this superfamily, there are at least four obligate fig/fig wasp associated lineages, where the nematodes are carried by fig wasps, and feed on internal syconia tissues of figs [17, 19]. In addition, all four lineages are phylogenetically separable from each other and are sisters to corresponding mycophagous lineages [17, 19], suggesting that these four clades occurred independently from mycophagous lineages. Interestingly, the ingestive (stomatal) structure of all four lineages are similar to each other in being specialized for plant-parasitism, i.e., a morphological and functional convergence has occurred [17, 19], and these lineages could be an exciting system to study adaptive radiation and the origins of nematode plant parasitism.
In previous studies, Kanzaki et al. [17] and Kruger et al. [23] reported that two species of the genus Bursaphelenchus Fuchs are associated with figs, i.e., B. sycophilus from F. variegata (Blume) in Japan [17] and an undescribed Bursaphelenchus sp. from F. sur Forssk. in South Africa [23]. The purpose of this study was to describe and illustrate Bursaphelenchus suri n. sp. to elucidate its close relationship with B. sycophilus from Japan, which it shares specialized morphology and biology within Sycomorus figs.
Materials and methods
Ethics statement
Specific permissions were not required for the nematodes collected for the present study. The fields (trees) used for nematode collection were on the grounds of the University of Pretoria campus. Endangered or protected species were not involved with the present study.
Nematode collection
The procedures for collecting specimens were provided in the previous study [23]. In short, various-phased syconia of F. sur were collected from trees planted on the grounds of the campus of the University of Pretoria, South Africa (GPS: 25°45’20” S, 28°13’40”E, 1370 m a.s.l.) in Nov. 2015, cut into small pieces in distilled water, and nematodes were hand-picked from the water with a stainless-steel insect pin under a dissecting microscope.
Collected nematodes were morphologically studied for genus or family-level identification. Some specimens were heat-killed at 55°C for one min. and fixed in TAF (2.0% triethanolamine, 7.0% formalin, 91% distilled water) for morphological vouchers, while the others were fixed in DESS [24] for molecular profiles.
Morphological observation
The TAF-fixed materials were processed to glycerin and mounted according to the modified Seinhorst method [25] and the Maeseneer and d’Herde method [26], respectively. The mounted materials were observed under a light microscope (Eclipse 80i, Nikon). Morphological drawings and morphometric analyses were conducted using a drawing tube attached to the microscope. Photomicrographs were taken using a digital camera system (MC170 HD, Leica). All drawings and micrographs were edited to construct figures using Photoshop 2020 (Adobe).
Molecular profiles and phylogeny
The molecular sequences of ca 1.7 kb of the small subunit (SSU) and ca 0.7 kb of the D2-D3 expansion segments of the large subunit (D2-D3 LSU) ribosomal RNA genes and ca 0.6 kb of mitochondrial cytochrome oxidase subunit I (mtDNA) were determined and deposited to the GenBank database in the previous study [23]. Briefly, DESS-fixed nematodes were rehydrated, observed under the microscope for typological identification, and individually transferred to nematode digestion buffer [27, 28]. The nematodes were digested at 55°C for 30 min. and used for PCR template to determine the sequences according to the methodologies of Ye et al. [29] and Kanzaki and Futai [30].
For the phylogenetic analysis, compared sequences were selected according to the results of a BLAST homology search and previous studies [31, 32], i.e., because the BLAST search suggested that the new species of nematode was closest to B. sycophilus Kanzaki, Tanaka, Giblin-Davis & Davies, and belonged to the fungivorus group of the genus, those nematode species belonging to the fungivorus group and several other Bursaphelenchus, Parasitaphelenchus Fuchs, Ruehmaphelenchus Goodey and Sheraphelenchus Nickle species belonging to other parasitaphelenchid clades were used for the Bayesian phylogenetic analyses based on SSU and D2-D3 LSU.
The compared sequences were aligned separately using MAFFT (available online at http://mafft.cbrc.jp/alignment/server/) [33, 34], and the base substitution model was determined using MEGA7 [35] under the Akaike information criterion (AIC) for model selection. Bayesian analysis was performed separately to confirm the tree topology of each gene using MrBayes 3.2 [36, 37]; four chains were run for 4 × 106 generations. Markov chains were sampled at intervals of 100 generations [38]. Two independent runs were performed, and, after confirming the convergence of runs and discarding the first 2 × 106 generations as burn-in, the remaining topologies were used to generate a 50% majority-rule consensus tree.
Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:011FAF51-3B7C-4D4C-9228-CF0DC12B164D. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS.
Results
Phylogenetic status
Bursaphelenchus suri n. sp. belongs to subgroup 1 of the fungivorus group sensu Kanzaki et al. [31], and is phylogenetically closest to B. sycophilus, the other fig-associated species of the genus (Figs 1 and 2). These two species formed a maximally supported clade within the group.
For model selection, AIC = 19854.013; lnL = -9829.881. Analytical parameters are: freqA = 0.26, freqC = 0.19, freqG = 0.27, freqT = 0.28; R(a) = 1.11, R(b) = 2.70, R(c) = 1.46, R(d) = 0.54, R(e) = 4.66, R(f) = 1.00; Pinva = 0.35; Shape = 0.31. Posterior probability values exceeding 50% are given on appropriate clades. The type locality of each species marked with an asterisk suggests that the material was isolated from wooden packing material imported from that country.
For model selection, AIC = 24371.263; lnL = -12088.342. Analytical parameters are: freqA = 0.21, freqC = 0.19, freqG = 0.32, freqT = 0.28; R(a) = 0.17, R(b) = 1.68, R(c) = 0.53, R(d) = 0.34, R(e) = 3.05, R(f) = 1.00; Pinva = 0.32; Shape = 0.88. Posterior probability values exceeding 50% are given on appropriate clades. The type locality of each species marked with an asterisk suggests that the material was isolated from wooden packing material imported from that country.
Taxonomic description
Bursaphelenchus suri Kanzaki, Kruger, Greeff & Giblin-Davis n. sp. urn:lsid:zoobank.org:act:7A4B50E1-451A-4A73-9034-0106BD144756 (Figs 3–5).
A: Female; B: Male; C: Lip and stylet region of male; D: Body surface of male showing lateral field and annulations; E: Head to pharyngeal region of male; F: Posterior part of female gonad; G: Female tail; H: Male tail; I: Male tail tip; J, K: Male spicule. All subfigures are in the right lateral view except for H (ventral view).
A: Lip and stylet region in two different focal planes (ld = labial disc; cn = stylet conus; sh = stylet shaft; bs = basal swelling); B: Pharyngeal part in two different focal planes (p-i = pharyngo-intestinal junction; hz = hemizonid; nr = nerve ring; ep = secretory-excretory pore); C: Anterior end of testes; D: Male tail in four different focal planes (P + number = genital papillae; bu = bursal flap). All subfigures are in right lateral view.
A: Ovary to vulval region in five different focal planes (ov = ovary; od = oviduct; sp = spermatheca; ct = connective tissue); B: Vulval region in three different focal planes (vo = vulval opening; cr = crustaformeria; ut = uterus); C: Post-uterine sac; D: Anal region (ao = anal opening); E: Tail tip. All subfigures are in right lateral view.
Description.
According to the journal’s requirement, a short description of the comparative diagnostic characters is given here. The typical morphologies for the genus and species group appear in the short description are provided in the previous species descriptions [16, 31, 32]. A detailed typological description is given in S1 Text.
Adult. Relatively large and slender species of the genus. Body shape and cuticle structures are typical to the genus, with four-lined lateral field, but the structure is vague, and internal lines are difficult to observe. Lip with six equal-sized sectors, roundish rectangular to triangular in lateral view. A labial disc present at the anterior end, and its edge appears as two short projections in lateral view. Stylet with narrow lumen separated into a long conus and a shaft with clear, very well-developed and somewhat tear-drop-shaped basal swellings. Procorpus with clear procorpal tube, about two stylet lengths long, metacorpus (= median bulb) well-developed, and pharyngo-intestinal junction structures are typical to the genus. Dorsal pharyngeal gland ca 7–10 metacorpal lengths long. Nerve ring at about one stylet length (about 1.5 metacorpal lengths) posterior to metacorpus. Hemizonid about 1.5 stylet length (about 2 metacorpal lengths) posterior to metacorpus. Secretory-excretory pore at immediately posterior or almost same level of hemizonid.
Male. Body and gonadal structure are typical to the genus. Anterior end of testis outstretched (8 out of 9 type specimens) or reflexed (1 out of 9). Spermatocytes arranged in multiple (3–5) rows. Spicules typical for the fungivorus group of the genus. Gubernaculum absent. Tail smoothly tapering in anterior 2/3, and distal 1/3 narrowing abruptly. Bursal flap conspicuous, an oval shape with a triangular projection at the posterior end. Three pairs of genital papillae present, but ventral precloacal papilla (P1) which is present in most other parasitaphelenchid nematodes not observed in light microscope, possibly vestigial.
Female. Structure and position of reproductive tract typical to the genus. A pair of three-celled structures usually found in the genus was not confirmed, possibly because of material condition, but this region was somewhat sclerotized. Vulva without any flap apparatus. Post-uterine sac long, 6.3–9.7 vulval body diam. long, extending for almost half or more (47–73%) of vulva to anus distance. Tail slender, 5.6–10.7 anal body diam. long, elongate conoid in shape, smoothly tapering to pointed terminus.
Morphometrics.
Morphometric values are summarized in Table 1.
Type material.
Holotype male (Collection accession number: 51322), eight paratype males (51323–51330), and ten paratype females (51331–51340) deposited in the National Collection of Nematodes (NCN) at the Nematology Unit, Biosystematics, ARC-Plant Health and Protection, Roodeplaat, Pretoria.
Type habitat and locality.
The type material was obtained from a syconium of Ficus sur growing on the campus of the University of Pretoria, Pretoria, South Africa (GPS: 25°45’20” S, 28°13’40”E, 1370 m a.s.l.) in November 2015.
Diagnosis and relationship.
In addition to generic characters, Bursaphelenchus suri n. sp. is characterized by its lip structure, possessing a labial disc; long stylet with long conus occupying more than half of the total stylet length and large basal swellings; four-lined inconspicuous lateral field, male spicule with slightly dorsally truncate and roundish-squared condylus, dorsal and ventral limbs and membrane-like part between the limbs; bursal flap with a conspicuous projection; female gonad without clearly branching spermatheca; and relatively long female tail.
Based on its lip and stylet morphology and biology, i.e., association with a section Sycomorus fig, B. suri n. sp. is obviously close to B. sycophilus, i.e., forming a well-supported clade with the other fig-syconia-parasite. The phylogenetic status within the group corroborates their typological and biological similarities (Figs 1–5) [17, 23]. However, the new species can be distinguished from B. sycophilus by typological differences in adult morphology, namely the post-uterine sac length in females (6–10 times vulval body diameter, extending for almost half or more (47–73%) of the vulva to anus distance in B. suri n. sp. vs 3–5 times vulval body diameter, extending less than half (40–48%) of the vulva to anus distance in B. sycophilus) and condylus shape in male spicules (slightly dorsally truncate and roundish-squared in B. suri n. sp. vs strongly dorsally arcuate in B. sycophilus).
Bursaphelenchus maxbassiensis (Massey) Baujard also shares a modification of lip structure and stylet with long conus and large basal swellings with the two fig-parasites [16, 17, 39, 40]. However, B. suri n. sp. is distinguished from B. maxbassiensis by its lip shape, roundish square in lateral view with a labial disc vs laterally expanded to form an umbrella-like shape, and the labial disc was not observed; position of secretory-excretory pore, posterior vs anterior to metacorpus; male spicule morphology, with slightly dorsally truncate and roundish-squared condylus and relatively clear dorsal and ventral limbs vs dorsally recurved and pointed condylus and without clear ventral limb; male bursal flap shape, with pointed vs rounded tip; female tail shape long vs short conoid [16, 39].
Discussion
Biological characters of the B. fungivorus group
The new species and its closest relative, B. sycophilus belong to subgroup 1 of the fungivorus group of the genus [31]. Although most Bursaphelenchus spp. are generally associated with wood-boring beetles, particularly bark beetles that inhabit the above ground parts of dead trees [20, 41–43], the fungivorus group contains several soil-inhabiting species (B. hunti (Steiner) Giblin & Kaya, B. fungivorus Franklin & Hooper, B. gonzalezi Loof, B. seani Giblin & Kaya and B. rockyi Wang, Fang, Maria, Gu & Ge) [44–48], and their insect associations are rather variable compared with other intrageneric groups, e.g., soil-dwelling bees (B. seani) [47], a stag beetle (B. tadamiensis Kanzaki, Taki, Masuya & Okabe) [49], ambrosia beetles (B. kiyoharai Kanzaki, Maehara, Aikawa, Masuya & Giblin-Davis and B. penai Kanzaki, Giblin-Davis, Carrillo, Duncan & Gonzalez) [50, 51], and fig wasps (B. sycophilus and B. suri n. sp.) [17, 23]. In addition to the loss of the bursal flap in B. kiyoharai and B. penai, B. kiyoharai, B. penai, B. sycophilus and B. suri n. sp. have also lost (or possess vestigial) a P1 genital papilla in males [50, 51]. Considering the central phylogenetic placement within the genus [42, 43], the loss of a bursal flap and P1 papilla among typical Bursaphelenchus species (with a plesiomorphic P1 papilla and bursal flap) supports some genetic plasticity in the genus and the group.
Phylogenetically, the two fig-associates were sister to a clade containing B. braaschae Gu & Wang, B. tadamiensis and B. willibaldi Schönfeld, Braasch & Burgermeister (Fig 1). Within these three species, B. tadamiensis has been isolated from a stag beetle collected in Fukushima, Japan, i.e., the species inhabits the decaying wood of a broad-leaved tree [49], but the biological characters of the other two species are unknown because they were isolated from wood packing materials [52, 53]. Currently, we do not have a clear understanding of the evolutionary history of fig-associated Bursaphelenchus, e.g., how their niche and feeding preferences evolved. Although it seems likely that it involved an early introduction of a fungivorus group shared ancestor into a sycone of an ancestor fig of section Sycomorus by another insect vector with a host switch to fig wasps and a subsequent adaptive radiation involving the evolution from mycophagy to plant-parasitism. The genetic distances within the clade of fig-associates (fungivorus subgroup 1) and the nearest relatives of fungivorus subgroups 2 and 3 have relatively long branch lengths (Fig 1). This is somewhat reminiscent of another clade currently placed within a large Bursaphelenchus clade based upon molecular phylogenetic analysis. Ruehmaphelenchus appears to be associated and radiating chiefly with ambrosia beetles [54]. Thus, further survey work is justified for the 14 species of figs in the subsection Sycomorus and six species of the subsection Neomorphe in the subgenus and section of Sycomorus [55] (https://www.figweb.org/Ficus/Classification_of_figs/index.htm) which could reveal more new species and a clearer pattern of the evolutionary history and adaptive radiation of the fig-associated members of subgroup 1 of the fungivorus group. This was foreshadowed by Susoy et al. [10] in their paper reporting on the section Sycomorus radiation of at least seven species of Pristionchus where they also recovered Bursaphelenchus spp. from F. sycomorus L. and F. mauritiana Lam., but not F. racemosa L. (see Suppl. Table 3 in Susoy et al., [10]). In addition, during the sequencing of dauers from the pollinating fig wasp Ceratosolen coecus (Coquerel) emerging from ripe figs of Ficus mauritiana, no Bursaphelenchus sp. were recovered questioning if the fig wasp pollinator is the insect carrier of the fungivorus group radiation [10]. Kanzaki et al. [17] hypothesized that because the host-specific fig wasp, Ceratosolen appendiculatus (Mayr) was the only insect found in the syconium of F. variegata from which type materials of B. sycophilus were obtained, it was the likely carrier (or host) insect of the nematode. Using this logic, the likely carrier host for B. suri n. sp. would be Ceratosolen capensis Grandi since it is the primary pollinator of F. sur in South Africa, but fig wasps were not collected from B. suri n. sp. positive figs to sequence dauers for confirmation of the carrier association in the current study. Additionally, Kanzaki et al. [31] included B. maxbassiensis as a member of subgroup 1 of the fungivorus group because of similarities of its stylet morphology to B. sycophilus, but without molecular confirmation, this bark beetle associate may represent an example of convergence, which begs for re-isolation, sequencing, and further study. Further efforts to collect close relatives of fig-associated Bursaphelenchus followed by detailed biological and genetic analyses will help elucidate the evolutionary history of these highly derived species.
Some morphological characteristics of the new species and B. sycophilus.
In addition to its fig association, B. suri n. sp. shares several important characters with B. sycophilus. Particularly, the presence of a labial disc, long conus and extremely well-developed basal swellings of the stylet, and slender body are highly characteristic.
Within the superfamily Aphelenchoidoidea, the labial disc has been confirmed in several Ruehmaphelenchus and Aphelenchoides Fischer species (fungal feeders) [56, 57], Anomyctus xenurus Allen (hypothesized to be a predator) [58, 59], and some species of the fig-associated genera, Schistonchus Cobb, Ficophagus Davies & Bartholomaeus and Martininema Davies & Bartholomaeus [19], but not in regular fungal feeders [20, 22]. The lip structure and stylet morphology are tightly related to their feeding habits, and the structures are hypothesized to be adapted to plant parasitism, where the nematodes penetrate host epidermal cells, which are physically harder than fungal hyphae. However, because the fig-associated species have not been cultured successfully, the usage of their lips and stylets have not been observed. Further culturing attempts are necessary for these species and also for the confirmation of plant parasitism within Ruehmaphelenchus spp., which has a typical fungal feeders’ stylet.
The slender body may be an adaptation for B. suri n. sp. and B. sycophilus to their habitat inside section Sycomorus figs. In the family Diplogastridae, which also contains several fig-associated lineages, several aquatic lineages, e.g., the aquatic group of Allodiplogaster Paramonov & Sobolev, have slender bodies [60]. Interestingly, Teratodiplogaster Kanzaki, Giblin-Davis, Davies, Ye, Center & Thomas and the fig-associated clade of Pristionchus sharing Sycomorus figs with B. sycophilus and B. suri n. sp. also have long and slender bodies, while most of the other non-Sycomorus fig-associates e.g., Parasitodiplogaster and Caenorhabditis Osche have rather stout bodies [61–63]. Considering their host/habitat fig species, the figs of section Sycomorus are filled with liquid, while others are not [55]. In fact, these lineages are crawling (swimming) in the cavity of figs filled with liquid during the interfloral phase [10, 62]. Similar to the tendency in Diplogastridae, where swimmers have long and slender bodies, B. suri n. sp. and B. sycophilus are considered to inhabit the cavity of the figs filled by the liquid, feeding on the internal surface tissue of figs. Contrastingly, the Ficophagus sp. sharing the same fig with B. suri n. sp. has a stouter body [23], and it is assumed to be a more sedentary species inhabiting the fig tissue. Thus, although both species are plant parasites and feed on fig tissue, their niches may be segregated within the fig.
Remarks on future studies.
The section Sycomorus fig-associated Pristionchus spp. manifest feeding polymorphism to occupy a variety of niches according to the age of the figs [10]. Examination of the syconial ecology of section Sycomorus figs with different nematode faunal components could help elucidate how different species utilize and compete for different temporal and spatial resources. Such specialization could explain how the clade-specific radiations of certain nematode groups, such as Pristionchus, Teratodiplogaster, and Bursaphelenchus was triggered.
The tripartite relationship among fig, fig wasp, and nematodes is an intriguing system for the study of evolutionary biology. In addition, microbes and other invertebrates, e.g., mites, are involved in the system. However, most studies have been conducted as field surveys because of difficulties in the establishment of cultured materials for experimental research. To examine the system in more detail, laboratory strains will be a big advance. Of the animal interactants, nematodes are most predisposed to being cultured in the laboratory, e.g., Caenorhabditis inopinata can be cultured, and utilized as a satellite model system [9]. Further attempts at culturing fig-associated nematodes, including Bursaphelenchus species, should be undertaken to allow for more extensive studies.
Supporting information
S1 Text. Typological description of Bursaphelenchus suri n. sp. in traditional telegraphic style.
https://doi.org/10.1371/journal.pone.0265339.s001
(DOCX)
Acknowledgments
The authors sincerely thank Dr. Kerrie A. Davies, University of Adelaide, for her mentorship of M.S.K. concerning nematode taxonomy, morphology, and scientific illustration and her friendship and collaboration on many nematode adventures with N.K. and R.G-D. The authors are grateful to the University of Pretoria for a Postgraduate Study Abroad Bursary and international student registration fee award that allowed M.S.K. to learn from her co-authors in person.
References
- 1. Molbo D, Machado CA, Sevenster JG, Keller L, Herre EA. Cryptic species of fig-pollinating wasps: Implications for the evolution of the fig–wasp mutualism, sex allocation, and precision of adaptation. Proc Nat Acad Sci USA. 2003; 100:5867–5872. pmid:12714682
- 2. Machado CA, Robbins N, Gilbert MTP, Herre EA. Critical review of host specificity and its coevolutionary implications in the fig/fig-wasp mutualism. Proc Nat Acad Sci USA. 2005; 102:6558–6565. pmid:15851680
- 3. Herre EA, Jandér KC, Machado CA. Evolutionary ecology of figs and their associates: recent progress and outstanding puzzles. Ann Rev Ecol Evol Syst. 2008; 39:439–458.
- 4. Wang G, Zhang X, Herre EA, McKey D, Machado CA, et al. Genomic evidence of prevalent hybridization throughout the evolutionary history of the fig-wasp pollination mutualism. Nat Comm. 2021; 12:718. pmid:33531484
- 5. Herre EA. Population structure and the evolution of virulence in nematode parasites of fig wasps. Science 1993; 259:1442–1445. pmid:17801279
- 6. Herre EA. Factors affecting the evolution of virulence: nematode parasites of fig wasps as a case study. Parasitology 1995; 111:S179–S191.
- 7. Krishnan A, Muralidharan S, Sharma L, Borges RM. A hitchhiker’s guide to a crowded syconium: how do fig nematodes find the right ride? Funct Ecol. 2010; 24:741–749.
- 8. Jauharlina J, Lindquist EE, Quinnell RJ, Robertson HG, Compton SG. Fig wasps as vectors of mites and nematodes. Afr Entomol. 2012; 20:101–110.
- 9. Kanzaki N, Tsai IJ, Tanaka R, Hunt VL, Liu D, et al. Biology and genome of a newly discovered sibling species of Caenorhabditis elegans. Nat Comm. 2018; 9: 3216. pmid:30097582
- 10. Susoy V, Herrmann M, Kanzaki N, Kruger M, Nguyen CN, Rödelsperger C, et al. Large-scale diversification without genetic isolation in nematode symbionts of figs. Sci Adv. 2016; 2:e1501031. pmid:26824073
- 11. Gasparrini G. Sulla maturazione e la qualita dei fichi dei contorni di Napoli. Atti della Accademia Pontaniana 1864; 9:99–118.
- 12. Martin GC, Owen AM, Way JI. Nematodes, figs and wasps. J Nematol. 1973; 5:77. pmid:19319308
- 13. Cobb NA. Note on a new nema, Aphelenchus retusus, with a proposed division of Aphelenchus into three subgenera. J Parasitol. 1927; 14:57–58.
- 14. Poinar GO. Parasitodiplogaster sycophilon gen. n., sp. n. (Diplogasteridae: Nematoda), a parasite of Elisabethiella stuckenbergi Grandi (Agaonidae: Hymenoptera) in Rhodesia. Proc K Ned Akad Wet C. 1979; 82:375–381.
- 15. Vovlas N, Troccoli A, Noort SV, Van den Berg E. Schistonchus africanus n.sp. (Aphelenchida: Aphelenchoidiodae) associated with Ficus thonningii (Moraceae) and its pollinator wasp Elisabethiella stuckenbergi (Chalcidoidea: Agaonidae). J Nematol. 1998; 30:404–410. pmid:19274232
- 16. Kanzaki N, Giblin-Davis RM, Center BJ. Redescription of four North American Bursaphelenchus species from Massey’s type material. Nematology 2009; 11:129–150.
- 17. Kanzaki N, Tanaka R, Giblin-Davis RM, Davies K. New plant-parasitic nematode from the mostly mycophagous genus Bursaphelenchus discovered inside figs in Japan. PLoS ONE 2014; 9:e99241. pmid:24940595
- 18. Davies KA, Ye W, Giblin-Davis RM, Thomas WK. Ficotylus congestae gen. n., sp. n. (Anguinata), from Ficus congesta (Moraceae) sycones in Australia. Nematology 2009; 11:63–75.
- 19. Davies KA, Bartholomaeus F, Kanzaki N, Ye W, Giblin-Davis RM. A review of the taxonomy, phylogeny, distribution and co-evolution of Schistonchus (Nematoda: Aphelenchoididae) and proposal of Ficophagus n. gen. and Martininema n. gen. Nematology 2015; 17:761–829.
- 20.
Hunt DJ. Aphelenchida, Longidoridae and Trichodoridae: their systematics and bionomics. Wallingford, CABI Publishing; 1993.
- 21. Hunt DJ. A checklist of the Aphelenchoidea (Nematoda: Tylenchina). J Nematode Morph Syst. 2008; 10(2007):99–135.
- 22.
Kanzaki N, Giblin-Davis RM. Aphelenchoidea. In: Manzanilla-López RH, Marba´n-Mendoza N, editors. Practical plant nematology. Jalisco: Colegio de Post-graduados and Mundi-Prensa, Biblioteca Básicade Agricultura; 2012. 161–208.
- 23. Kruger MS, Kanzaki N, Giblin-Davis RM, Greef JM. Molecular diversity and relationships of fig associated nematodes from South Africa. PLoS ONE 2021; 16: e0255451. pmid:34375357
- 24. Yoder M, De Ley IT, King IW, Mundo-Ocampo M, Mann J, Blaxter M, et al. DESS: a versatile solution for preserving morphology and extractable DNA of nematodes. Nematology 2006; 8:367–376.
- 25. Minagawa N, Mizukubo T. A simplified procedure of transferring nematodes to glycerol for permanent mounts. Jpn J Nematol. 1994; 24:75.
- 26.
Hooper DJ. Handling, fixing, staining and mounting nematodes. In: Southey JF. Editor, Laboratory methods for work with plant and soil nematodes. London: Her Majesty’s Stationery Office; 1986. pp. 59–80.
- 27. Kikuchi T, Aikawa T, Oeda Y, Karim N, Kanzaki N. A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification (LAMP). Phytopathology 2009; 99:1365–1369. pmid:19900002
- 28. Tanaka R, Kikuchi T, Aikawa T, Kanzaki N. Simple and quick methods for nematode DNA preparation. Appl Entomol Zool. 2012; 47:291–294.
- 29. Ye W, Giblin-Davis RM, Braasch H, Morris K, Thomas WK. Phylogenetic relationships among Bursaphelenchus species (Nematoda: Parasitaphelenchidae) inferred from nuclear ribosomal and mitochondrial DNA sequence data. Mol Phylogenet Evol. 2007; 43:1185–1197. pmid:17433722
- 30. Kanzaki N, Futai K. A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within xylophilus group. Nematology 2002; 4:35–41.
- 31. Kanzaki N, Aikawa T, Maehara N, Thu PQ. Bursaphelenchus kesiyae n. sp. (Nematoda: Aphelenchoididae), isolated from dead wood of Pinus kesiya Royle ex Gordon (Pinaceae) from Vietnam with proposal of new subgroups in the B. fungivorus group. Nematology 2016; 18:133–146.
- 32. Kanzaki N, Ekino T, Ide T, Masuya H, Degawa Y. Three new species of parasitaphelenchids, Parasitaphelenchus frontalis n. sp., P. costati n. sp., and Bursaphelenchus hirsutae n. sp. (Nematoda: Aphelenchoididae), isolated from bark beetles from Japan. Nematology 2018; 20:957–1005.
- 33. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002; 30:3059–3066. pmid:12136088
- 34. Kuraku S, Zmasek CM, Nishimura O, Katoh K. aLeaves facilitates on-demand exploration of metazoan gene family trees on MAFFT sequence alignment server with enhanced interactivity. Nucleic Acids Res. 2013; 41:W22–28. pmid:23677614
- 35. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870–1874. pmid:27004904
- 36. Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001; 17:1754–1755. pmid:11524383
- 37. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61:539–542. pmid:22357727
- 38. Larget B, Simon DL. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Mol Biol Evol. 1999; 16:750–759.
- 39. Massey CL. Omemeea maxbassiensis n. gen., n. sp. (Nematoda: Aphelenchoididae) from galleries of the bark beetle Lepersinus californicus Sw. (Coleoptera: Scolytidae) in North Dakota. J Nematol. 1971; 3:289–291. pmid:19322384
- 40.
Massey CL. Biology and taxonomy of nematode parasites and associates of bark beetles in the United States. Agriculture Handbook No. 446. Washington DC: USDA, Forest Service; 1974.
- 41. Ryss A, Vieira P, Mota MM, Kulinich O. A synopsis of the genus Bursaphelenchus Fuchs, 1937 (Aphelenchida: Parasitaphelenchidae) with keys to species. Nematology 2005; 7:393–458.
- 42. Ryss AY, Subbotin SA. Coevolution of wood-inhabiting nematodes of the genus Bursaphelenchus Fuchs, 1937 with their insect vectors and plant hosts. Zhurnal Obshchei Biologii 2017; 78:32–61. pmid:30024676
- 43. Kanzaki N, Giblin-Davis RM. Diversity and plant pathogenicity of Bursaphelenchus and related nematodes in relation to their vector bionomics. Cur For Rep. 2018; 4:85–100.
- 44. Steiner G. Opuscula miscellanea nematologica, II. Proc Helminthol Soc Wash. 1935; 2:104–110.
- 45. Franklin MT, Hooper DJ. Bursaphelenchus fungivorus n. sp. (Nematoda: Aphelenchoidea) from rotting gardenia buds infected with Botrytis cinerea Pers. ex Fr. Nematologica 1962; 8:136–142.
- 46. Loof PAA. Free-living and plant parasitic nematodes from Venezuela. Nematologica 1964; 10:201–300.
- 47. Giblin R.M. & Kaya H.K. (1983). Bursaphelenchus seani n. sp. (Nematoda: Aphelenchoididae) a phoretic associate of Anthophora bomboides stanfordiana Cockerell, 1904 (Hymenoptera: Anthophoridae). Rev Nématol. 1983 6:39–50.
- 48. Wang X, Fang Y, Maria M, Gu J, Ge J. Description of Bursaphelenchus rockyi n. sp. (Nematoda: Aphelenchoididae) in peat moss from Russia. Nematology 2019; 21:253–265.
- 49. Kanzaki N, Taki H, Masuya H, Okabe K. Bursaphelenchus tadamiensis n. sp. (Nematoda: Aphelenchoididae), isolated from a stag beetle, Dorcus striatipennis (Coleoptera: Lucanidae), from Japan. Nematology 2012; 14: 223–233.
- 50. Kanzaki N, Maehara N, Aikawa T, Masuya H, Giblin-Davis RM. Description of Bursaphelenchus kiyoharai n. sp. (Tylenchina: Aphelenchoididae) with remarks on the taxonomic framework of the Parasitaphelenchinae Rühm, 1956 and Aphelenchoidinae Fuchs, 1937 Nematology 2011; 13:787–804.
- 51. Kanzaki N, Giblin-Davis RM, Carrillo D, Duncan R, Gonzalez R. Bursaphelenchus penai n. sp. (Tylenchomorpha: Aphelenchoididae), a phoretic associate of ambrosia beetles (Coleoptera: Scolytinae) from avocado in Florida. Nematology 2014; 16: 683–693.
- 52. Schönfeld U, Braasch H, Burgermeister W. Bursaphelenchus spp. (Nematoda: Parasitaphelenchidae) in wood chips from sawmills in Brandenburg and description of Bursaphelenchus willibaldi sp. n. Russ J Nematol. 2006; 14:119–126.
- 53. Gu J, Wang J. Description of Bursaphelenchus braaschae sp. n. (Nematoda: Aphelenchoididae) found in dunnage from Thailand. Russ J Nematol. 2010; 18:59–68.
- 54. Kanzaki N, Giblin-Davis RM, Gonzalez R, Duncan R, Carrillo D. Description of Ruehmaphelenchus juliae n. sp. (Tylenchina: Aphelenchoididae) isolated from an ambrosia beetle, Xylosandrus crassiusculus (Motschulsky), from South Florida. Nematology 2015; 17:639–653.
- 55.
van Noort S, Rasplus J-Y. Figweb: figs and fig wasps of the world. 2021 [cited 23 May 2021]. Available from: https://www.figweb.org/Figs_and_fig_wasps/index.htm.
- 56. Kanzaki N. Description of Ruehmaphelenchus fujiensis n. sp. (Tylenchomorpha: Aphelenchoididae) isolated from dead wood of Quercus crispula from Yamanashi, Japan. Nematology 2021; 23:in press.
- 57. Hooper DJ, Ibrahim SK. Aphelenchoides nechaleos n. sp. and A. paranechaleos n. sp. (Nematoda: Aphelenchoididae) from rice plants. Fundam Appl Nematol. 1994; 17:153–160.
- 58. Allen MW. 1940. Anomyctus xenurus, a new genus and species of Tylenchoidea (Nematoda). Proc Helminthol Soc Wash. 1940; 7:96–98.
- 59. Hooper DJ, Cooke DA. Some observations on Anomyctus xenurus Allen, 1940 Nematologica 1967; 13:320–321.
- 60. Kanzaki N, Ragsdale EJ, Giblin-Davis RM. Revision of the paraphyletic genus Koerneria Meyl, 1960 and resurrection of two other genera of Diplogastridae (Nematoda). Zookeys 2014; 442:17–30. pmid:25349487
- 61. Poinar Jr GO, Herre EA. Speciation and adaptive radiation in the fig wasp nematode Parasitodiplogaster (Diplogasteridae: Rhabditida) in Panama. Rev Nématol. 1991; 14:361–374.
- 62. Kanzaki N, Giblin-Davis RM, Davies K, Ye W, Center BJ, et al. Teratodiplogaster fignewmani gen. nov., sp. nov. (Nematoda: Diplogastridae) from the syconia of Ficus racemosa in Australia. Zool Sci. 2009; 26:569–578. pmid:19719410
- 63. Zeng Y, Giblin-Davis RM, Ye W. Two new species of Schistonchus (Nematoda: Aphelenchoididae) associated with Ficus hispida in China. Nematology 2007; 9:169–187.