Molecular diversity and relationships of fig associated nematodes from South Africa

Abstract

Nematodes of figs and fig wasps have received limited attention in Africa since their discovery in 1973. Sixteen of the 25 species of native South African figs were sampled for nematode associates using molecular barcoding with three loci (SSU, LSU D2-D3 and mtCOI) and fourteen (93%) were positive for at least one nematode species. Thirty-three putative species of nematodes were identified and classified according to the loci that were amplified and successfully sequenced. Fourteen putative nematode species were classified as Aphelenchoididae, of which nine were identified as Ficophagus from four species of Ficus from the section Galoglychia (i.e., five ex F. burkei including one shared with F. natalensis, one ex F. glumosa, one ex F. lutea, and one ex F. stuhlmannii) and one species ex F. sur from the section Sycomorus. In addition, there were four nematode species classified as Schistonchus s.s. from section Galoglychia figs (i.e., one ex F. burkei, two ex F. trichopoda, and one ex F. glumosa). There was also one species of Bursaphelenchus nematode recovered from F. sur from the section Sycomorus. Sixteen putative nematode species were classified as Diplogastridae, of which eight occurred in two clades of what is currently called Parasitodiplogaster with one (P. salicifoliae) being recovered from two Ficus species in the section Urostigma (F. salicifolia and F. ingens) and seven diplogastrids being associated with six species of Ficus from the section Galoglychia (i.e., two ex F. burkei including P. sycophilon, one ex F. stuhlmannii, one ex F. burtt-davyi, one ex F. trichopoda, one ex F. abutilifolia and one ex F. sansibarica). Three Acrostichus spp., a Teratodiplogaster and a Pristionchus species were recovered from F. sur and two Teratodiplogaster spp. and Pristionchus sycomori were recovered from F. sycomorus with both Ficus species belonging to the subgenus and section Sycomorus. The identities of the previously described T. martini and Parasitodiplogaster doliostoma (= Pristionchus sp. 35) are discussed. Lastly, there was a panagrolaimid identified from F. petersii.

Introduction

Martin et al. [1] first reported about the amazing morphological diversity of nematodes inside figs from six native species of Ficus from Zimbabwe in 1973 and invited participation from the global nematological community to help elucidate their identities and biology. Because non-native (introduced) figs were reported to be without fig wasp pollinators and did not have any nematode associates, a tight ecological and evolutionary relationship between nematodes, fig wasps, and figs was hypothesized [1]. Even though a few nematodes have been described from Africa, work elsewhere has shown that nematodes from two families, the Aphelencoididae and Diplogastridae, are frequently associated with figs [2]. In general, members of the Diplogastridae can have useful typological characters for species/generic separations whereas members of the Aphlenchoididae often do not [3, 4], but cryptic species are possible within both groups [5–7]. Furthermore, diverse phenotypes can be produced by a single genotype in some members of both families [8–11]. For instance, Susoy et al. [8] reported how a diverse array of up to 5 phenotypes can be manifested by a single new species of Pristionchus (i.e., P. sycomori Susoy, Kanzaki, Kruger, Ragsdale & Sommer in Susoy et al. and Pristionchus sp. 35) in sycones of F. sycomorus L. and F. sur Forsskål in South Africa, respectively.

While both nematode families have been described from a Ficus in the African section/subsection Galoglychia [12, 13], it is not clear if this pattern is common in Africa. Galoglychia and three of the other five sections of Ficus that occur in Africa, Sycidium, Sycomorus and Urostigma reach right down to South Africa [14]. Subsections of these three sections also occur in Asia, Southeast Asia and Australia where both nematode lineages have been found [14].

Poinar [12] described the first new species/genus of fig/fig wasp nematode from Africa in 1979, i.e., Parasitodiplogaster sycophilon Poinar (Diplogastridae) from the fig wasp, Elizabethiella stuckenbergi Grandi (Agaonidae) in figs (sycones) of Ficus burkei (Miq.) from Zimbabwe. Vovlas et al. [13] described Schistonchus africanus Vovlas, Troccori, van Noort & van den Berg (Aphelenchoididae) from figs of F. burkei and the fig wasp E. stuckenbergi from South Africa. Kanzaki et al. [15] described Teratodiplogaster martini Kanzaki, Giblin-Davis, Davies & Center (Diplogastridae) from Ficus sp. and Parasitodiplogaster doliostoma Kanzaki, Giblin-Davis, Davies & Center from F. sur from fixed materials provided from Martin et al.’s [1] original collections, and Jauharlina et al. [16] outlined the association dynamics between Parasitodiplogaster sp. and its fig wasp host E. baijnathi Wiebes and sycones of F. burtt-davyi Hutchinson from South Africa. More recently, Wöhr et al. [17, 18] re-described Parasitodiplogaster sycophilon from F. burkei and described Parasitodiplogaster salicifoliae Wöhr, Greeff, Kanzaki & Giblin-Davis from figs of F. salicifolia Vahl / F. ingens (Miq.) from South Africa, respectively. Except for the most recent work [8, 17, 18], all African fig/fig wasp nematode taxonomy was done solely with traditional morphology, which can be misleading with certain nematode groups and requires molecular sequence data for placement of these taxa into a modern phylogenetic framework.

Since adult or juvenile nematodes are phoretic/necromenic/or parasitic on the species-specific fig wasps and fossil evidence suggests that Parasitodiplogaster’s relationship is at least 19 million years old and they may well have co-diversified with the pollination mutualism [19] we can expect these two families to frequent figs from southern Africa. The paucity of information from African Ficus sections/subsections that occur on other continents and the phenotypic conservatism and occasional extreme trophic plasticity suggest a broad survey in Africa would be insightful. In the present study, we surveyed as many native figs as possible over a four-year period in South Africa to examine the identity and patterns of nematode associates of figs.

Materials and methods

Ethics statement

Specific permissions were not required for the nematodes collected for the present study. The trees sampled for nematode collection were either inside the University of Pretoria campus or places open to the public. Endangered or protected species were not involved with the present study.

Nematode collection

Fig syconia in various phases (phases B-E) were collected from 25 Ficus species from many locations across South Africa (Fig 1, Table 1, S1 Table in S2 File). In general, fig syconia manifest phenologically in five phases based upon the pollination mutualism and corresponding development of the fig and its fig wasp pollinators as described by Janzen [20]. Because nematodes are vectored into the developing fig only after the syconia becomes receptive at phase B (female), phase A (pre-receptive or pre-female) figs were not used. In most cases, the ideal phases for nematode collection were early to late interfloral (phase C) or male (phase D) figs which were always used, if available. However, because fruit availability can be unpredictable, we collected phase B and E if it was the only fruit available. An average of 50 syconia were collected per crop and never less than 30. Collected figs were cut into ca 1–2 mm thick pieces and soaked in water for 10–15 min. Nematodes recovered from the sliced figs were hand-picked, identified to their taxonomic group at genus or family level at low magnification under a dissecting microscope according to body and stomatal/stylet shapes, and 3–5 individuals for each morphotypes were placed into either 100% ethanol or nematode digestion buffer (NDB) [21, 22], but less than that if there were insufficient specimens recovered. The rest of the individuals were killed by heat and fixed in formalin and processed into glycerin [23] as morphological vouchers. In addition, some individuals isolated from F. sur and F. sycomorus were transferred to DESS [24] to eventually compare their typological characters and molecular phylogenetic status for single nematodes.

Fig 1. Map of southern Africa, country names in large font, city names in small font.

Each color dot represents a fig species sampled in the area, mostly associated with cities. The color legend defines the fig species that each color represents.

Table 1. Host, locality and recovered nematode species from dissected fig syconia.

Fig section: subsection	Ficus species	Collection localities	Nematode species a	Previously reported pollinator wasp species b
Galoglychia: Chlamydodorae	F. burkei	Abel Erasmus pass, Phiphidi, Nzhelele Valley, Louis Trichardt, Hartbeespoort Dam	Parasitodiplogaster sycophilon, Parasitodiplogaster n. sp. 6, Ficophagus n. spp. 1, 2, 5, 6, 7, Schistonchus n. sp. 4	Elisabethiella stuckenbergi
	F. natalensis	Ballito	Ficophagus n. sp. 7	Elisabethiella socotrensis
	F. burtt-davyi	Eastern Cape	Parasitodiplogaster n. sp. 2	Elisabethiella baijnathi
	F. petersii	Nelspruit	Unidentified panagrolaimidc	Alfonsiella binghami
	F. craterostoma	Pretoria	Morphological vouchers only, not genotyped d	Alfonsiella pipithensis
Galoglychia: Platyphyllae	F. stuhlmannii	Nelspruit	Parasitodiplogaster n. sp. 5, Ficophagus n. sp. 8	Alfonsiella binghami
	F. abutilifolia	Pretoria	Parasitodiplogaster n. sp. 3	Elisabethiella comptoni
	F. trichopoda	Ballito	Parasitodiplogaster n. sp. 1, Schistonchus n. spp. 1, 2	Elisabethiella bergi breviceps
	F. glumosa	Nelspruit, Pretoria	Schistonchus n. sp. 3	Elisabethiella glumosae
Galoglychia: Galoglychia	F. lutea	Ballito	Ficophagus n. sp. 9	Allotrizoon heterandromorphum
Galoglychia: Caulocarpae	F. sansibarica	Nelspruit	Parasitodiplogaster n. sp. 4	Courtella armata
Urostigma: Urostigma	F. ingens	Pretoria	Parasitodiplogaster salicifoliae	Platyscapa soraria
	F. salicifolia	Pretoria, Nelspruit	Parasitodiplogaster salicifoliae	Platyscapa awekei
	F. cordata	Augrabies Falls	Morphological vouchers only, not genotypedd	Platyscapa desertorum
Sycomorus: Sycomorus	F. sur	Ballito, Pretoria	Teratodiplogaster cf. martini, Acrostichus n. spp. 1, 2, 3 (and 4), Pristionchus sp. 35 ex Susoy et al. [8] (= cf. “Parasitodiplogaster” doliostoma ex Kanzaki et al. [15]), Ficophagus n. sp. 4	Ceratosolen capensis
	F. sycomorus	Pretoria, Nelspruit	Teratodiplogaster n. spp. 1, 2, Pristionchus sycomori	Ceratosolen arabicus

^a The numbers suggest the genotype code considered as different undescribed species based on the differences of molecular sequences.

^b Information from van Noort & Rasplus [14].

^c Not analyzed phylogenetically because only relatively short (ca 400 bps) fragment of D2-D3 LSU was obtained, and no close relative was found in GenBank, i.e., the closest sequences were Macrolaimus spp. with 80% of identity (see text).

^d Although multiple species of nematodes were found, molecular materials were not available, and only collection records are presented here.

Molecular profiles and phylogeny

The nematodes transferred to 100% ethanol or NDB were used for molecular samples to survey the diversity of nematode genotypes associated with different fig species from different locations, i.e., DNA was extracted from ethanol-fixed material using a QIAamp ® DNA Micro Kit (Qiagen, Germany) and NDB-treated samples were digested under 55°C for 20 min. Whereas the DESS-fixed materials were re-hydrated in a drop of sterile water, photo-documented (see below for methods), transferred to NDB and digested for use as template DNA.

We attempted to amplify and sequence three genetic loci, i.e., nearly full-length small subunit (SSU) and D2-D3 expansion segments of the large subunit (LSU) of ribosomal RNA genes and a partial region of the mitochondrial cytochrome oxidase subunit I (mtCOI) for all DNA template samples. Methodologies of Ye et al. [25] and Kanzaki and Futai [26] were employed for sequencing ribosomal RNAs and mtCOI. Briefly, amplification of SSU, LSU and mtCOI were attempted with primer sets, SSUF07 (5’—AAA GAT TAA GCC ATG CAT G—3’) and SSUnR (5’—TTA CGACTT TTG CCC GGT TC—3’), D2a (5’—ACA AGT ACC GTG AGG GAA AGT TG—3’) and D3b (5’—TCG GAA GGA ACC AGC TAC TA—3’), and COIF1 (5’—CCT ACT ATG ATT GGT GGT TTT GGT AAT—3’) and COIR2 (5’—GTA GCA GTA AAA TAA GCA CG—3’), respectively. Upon successful amplification, sequences were determined with Sanger sequencing using the purified amplicons. Sequences with ambiguous or poor quality electropherogram/chromatograms were omitted. The sequences were compared with those deposited in the GenBank database using BLASTN program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) for initial identification, and the generic status of all compared sequences were confirmed by their phylogenetic status. High quality sequences are summarized in Table 1 and S1 Table in S2 File.

Because two groups (= families), Aphelenchoididae and Diplogastridae were recognized, these families were phylogenetically analyzed separately for each locus using Bayesian Inference (BI) to determine their phylogenetic status within family (SSU and D2-D3) or other fig-associates (mtCOI). Compared sequences and their GenBank accession numbers are shown in Figs 2–7. The compared sequences were selected according to previous family-wide analyses, i.e., Kanzaki et al. [27] and Davies et al. [3] for Aphelenchoididae and Wöhr et al. [17], Zeng et al. [28] and Gonzalez et al. [29] for Diplogastridae.

Fig 2. Phylogenetic status of fig-associated species within the family Aphelenchoididae inferred from SSU under GTR + G model.

The parameters are: AIC = 56880.841, lnL = -28210.097, freqA = 0.26, freqC = 0.20, freqG = 0.27, freqT = 0.27, R(a) = 0.85, R(b) = 2.47, R(c) = 1.02, R(d) = 0.88, R(e) = 3.48, R(f) = 1.00, Pinva = n/a, Shape = 0.39. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Fig 7. Phylogenetic relationship of fig-associated Diplogastridae species inferred from mtCOI under GTR + G model.

The parameters are: AIC = 9938.549, lnL = -4858.882, freqA = 0.26, freqC = 0.12, freqG = 0.19, freqT = 0.43, R(a) = 0.44, R(b) = 3.03, R(c) = 1.72, R(d) = 0.68, R(e) = 3.24, R(f) = 1.00, Pinva = n/a, Shape = 0.32. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Compared sequences were aligned using the MAFFT program [30, 31] (https://mafft.cbrc.jp/alignment/server/index.html) with the default settings. The base-substitution models for each gene were determined using the MEGA 7 software [32] and the Akaike information criterion. Combined Bayesian analysis was performed using the MrBayes 3.2 software [33, 34]; four chains were run for 4 × 10⁶ generations, and Markov chains were sampled at intervals of 100 generations [35]. Two independent runs were performed, and after confirming convergence and discarding the first 2 × 10⁶ generations as ‘burn in’, the remaining topologies were used to generate a 50% majority-rule consensus tree.

Morphological observation

Several DESS-fixed materials were rehydrated and observed for their typological characters, particularly stomatal/stylet and pharyngeal morphologies and male and female tail characters which are usually used for diagnostic characters, prior to digestion. The materials were temporarily mounted on an agar pad, observed under a light microscope (Eclipse 80i, Nikon) with differential interference contrast (DIC) optics and photomicrographed using a microscope digital camera system (MC170 HD, Leica) connected to the microscope. The micrographs were edited using Photoshop Elements 3.0 (Adobe) for constructing micrographic figures (S1–S7 Figs in S1 File). Some formalin-fixed materials were also observed for identification of nematodes to the genus or family-level, and some typological characters were noted and illustrated using a camera-lucida drawing system (S8 Fig in S1 File).

Results

Sample collection

Fig fruits (syconia) were successfully collected from 16 out of 25 South African native Ficus species. Nematode DNA sequences were obtained from 14 fig species. However, because of shortages in the number of collected nematodes or material condition, only morphological vouchers were obtained from two of the fig species. Collection information and molecular identification (genotyping) are summarized in Table 1 and S1 and S2 Tables in S2 File.

Nematode identification

Nematodes were identified based on their typological characters and phylogenetic status.

Phylogenetically, three aphelenchoidid genera (Schistonchus, Ficophagus and Bursaphelenchus), four diplogastrid genera (Parasitodiplogaster, Teratodiplogaster, Pristionchus and Acrostichus), and an unidentified panagrolaimid species were recognized (Table 1, S1 Table in S2 File).

Three fig-associated aphelenchoidid genera previously considered Schistonchus s. l. (Schistonchus, Martininema and Ficophagus) are typologically similar to each other [3]. Therefore, the identification of these genera was confirmed using molecular phylogenetic analyses. The phylogenetic analyses of three loci suggested that the sequences belonging to subfamily Aphelenchoidinae are separated into four Schistonchus spp. and nine (or ten) Ficophagus spp. (Figs 2–4, Table 2).

Fig 4. Phylogenetic relationship of fig-associated Aphelenchoididae species inferred from mtCOI under GTR+G+I model.

The parameters are: AIC = 13195.267, lnL = -6530.39, freqA = 0.22, freqC = 0.12, freqG = 0.20, freqT = 0.46, R(a) = 0.07, R(b) = 2.45, R(c) = 1.17, R(d) = 0.49, R(e) = 2.12, R(f) = 1.00, Pinva = 0.44, Shape = 1.04. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Table 2. Number of nematode species collected in Ficus subsections.

Ficus section	Ficus subsection	Nematode group
		Aphelenchoididae			Diplogastridae				Panagrolaimidae
		Schistonchus s.s.	Bursaphelenchus	Ficophagus	Parasitodiplogaster	Teratodiplogaster	Pristionchus	Acrostichus
Galoglychia	Chlamydodorae	1	-	5	3	-	-	-	1
	Platyphyllae	3	-	2	3	-	-	-	-
	Galoglychia	-	-	1		-	-	-	-
	Calocarpe	-	-	-	1	-	-	-	-
Urostigma	Urostigma	-	-	-	1	-	-	-	-
Sycomorus	Sycomorus	-	1	1		3	2	3	-

These are all considered to be new nematode species and were isolated from various sections/subsections of figs in South Africa, namely subgenus Urostigma and section Galoglychia figs from three subsections (Chlamydodorae, Galoglychia and Platyphyllae) as well as the subgenus Sycomorus for Ficophagus spp. and subgenus Urostigma and section Galoglychia figs from two subsections (Chlamydodorae and Platyphyllae) for Schistonchus spp. However, their phylogenetic groupings were not clearly associated with host fig subsections when put into a global context (Figs 2–4).

Diplogastrid nematodes were molecularly separated (clustered) into eight Parasitodiplogaster spp. (P. sycophilon, P. salicifoliae, and six new species), three Teratodiplogaster spp. (T. cf. martini and two new species), two Pristionchus spp. (P. sycomori and Pristionchus sp. 35), and three new Acrostichus spp. (Figs 5–7).

Fig 5. Phylogenetic status of fig-associated species within the family Diplogastridae inferred from SSU under GTR + G + I model.

The parameters are: AIC = 50531.581, lnL = -25026.463, freqA = 0.25, freqC = 0.21, freqG = 0.27, freqT = 0.27, R(a) = 0.94, R(b) = 2.60, R(c) = 2.10, R(d) = 0.92, R(e) = 3.80, R(f) = 1.00, Pinva = 0.35, Shape = 0.53. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Parasitodiplogaster spp. were recovered from various figs in the subgenus Urostigma and phylogenetic groupings were in accordance with specific sections and subsections of host figs in South Africa, i.e., P. salicifoliae was isolated only from section Urostigma and subsection Urostigma figs whereas P. sycophilon and the other six new Parasitodiplogaster species were associated with section Galoglychia figs from three subsections (Caulocarpae, Chlamydodorae and Platyphyllae) (Figs 2–4). This contrasts with Pristionchus, Teratodiplogaster and Acrostichus which were found exclusively from figs in the subgenus and section Sycomorus and each was inferred as being monophyletic within each genus (Figs 2–4). Because we observed only one species of Parasitodiplogaster (n. sp. 2) associated with sycones of F. burtt-davyi from South Africa it is quite possible that it is conspecific with the species studied for its fig and fig wasp host dynamics by Jauharlina et al. [16].

The typological characters observed in DESS- and formalin-fixed materials are mostly in accordance with the phylogenetic status, i.e., the examined characters were basically identical to the generic characters or specific characters of nominal species, although some parts, e.g., male genital papillae, were sometimes not clear (S1–S8 Figs in S1 File).

In addition, a short fragment of LSU with ca 400 bp of an unidentified panagrolaimid species was recognized from F. petersii Warburg. However, further identification or phylogenetic analysis were not conducted for the nematode because the sequences closest to it were Macrolaimus spp. (Panagrolaimomorpha) with only 80% identity match.

Discussion

Taxonomic issues of fig-associated nematodes

Martin et al. [1] highlighted the potential for amazing diversity in the fig and fig wasp associated nematode fauna of Africa and the world with a survey of figs from Zimbabwe in southern Africa. The traditional typological/morphological approaches available at that time would have led to misinterpretations about this diversity and their life histories, especially in the subgenus Sycomorus clade figs where it appears that they observed the most diversity.

First, there was the issue of extremes in polyphenism of up to five morphotypes per genotype for a clade of, at that point, unknown subgenus Sycomorus fig associated Pristionchus species [8]. Within two Pristionchus spp. found in the present study, Pristionchus sp. 35 is considered conspecific to “Parasitodiplogaster” doliostoma, because although Pristionchus sp. 35 has not been formally described, it is phylogenetically close to P. borbonicus and P. sycomori, and the stomatal morphology of “P. doliostoma” is clearly similar to that of the type IV morph of these two Pristionchus spp. [8, 15], i.e., “P. doliostoma” is hypothesized as one morphotype of Pristionchus sp. 35. The species will need to be transferred to Pristionchus, but a formal taxonomic revision will be presented elsewhere. This polymorphism helps explain the misclassification of “Parasitodiplogaster” doliostoma by Kanzaki et al. [15] from original preserved specimens from Martin et. al [1]. Data presented herein and in Susoy et al. [8] for Pristionchus sp. 35 (= Parasitodiplogaster cf. doliostoma) and P. sycomori demonstrates that about half of the nematode species diversity Martin et al. [1] observed in each Sycomorus fig (i.e., F. sur and F. sycomorus) was probably due to a single species of Pristionchus that can manifest in morphotypes as divergent as five different diplogastrid genera, presumably to fill niches during the phenology of the developing syconium [8]. There is also the newly documented situation of extreme morphological divergence in Acrostichus ziaelasi Kanzaki, Liang, Chiu & Li from a tenebrionid beetle termitiphile [36] which converges in trophic morphology on another genus (Rhabditolaimus) which might help explain Martin et al.’s [1] observation of nematodes that looked like members of Cylindrocorpidae (= Rhabditolaimus) from Sycomorus figs. Given that A. ziaelasi is close to Acrostichus n. spp. 1 and 2 as well as three Asian Sycomorus fig clade associates (Figs 5 and 6) it is possible that some odd manifestation of trophic morphology in this genus of Sycomorus associated nematodes is yet to be observed. Parasitodiplogaster was reported from F. sycomorus in Susoy et al. [8] but was not recovered from Sycomorus figs in this study. Teratodiplogaster (i.e., T. cf. martini and Teratodiplogaster n. spp. 1 and 2) was delimited to Sycomorus figs and interestingly this clade was sister to the P. salicifoliae clade of nematodes from Urostigma/Urostigma figs (Fig 6).

Fig 6. Phylogenetic status of fig-associated species within the family Diplogastridae inferred from D2-D3 LSU under GTR + G + I model.

The parameters are: AIC = 55649.066, lnL = -27559.395, freqA = 0.20, freqC = 0.22, freqG = 0.33, freqT = 0.25, R(a) = 0.53, R(b) = 1.92, R(c) = 0.89, R(d) = 0.50, R(e) = 3.51, R(f) = 1.00, Pinva = 0.21, Shape = 0.94. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Second, and in contrast to the intraspecific morphological diversity in Pristionchus spp., fig and fig wasp associated aphelenchoidids show highly conserved (convergent) morphology which resulted for many years in the lumping of three disparate genera into Schistonchus sensu latu which have now been separated into Schistonchus s.s., Martininema and Ficophagus [3]. In addition, there is another aphelench lineage (Parasitaphelenchinae), i.e., a clade of Sycomorus fig associated Bursaphelenchus (e.g., Bursaphelenchus n. sp. 1 and B. sycophilus) (Figs 2 and 3, S2 Fig in S1 File) that morphologically converged on the mouth form (stylet) motif of Schistonchus s.l. This makes aphelenchoidid nematodes difficult to separate from typical Schistonchus s.l. especially in mixed populations of fig-associated nematodes [3, 27, 29]. For example, only one aphelenchoidid, S. africanus has been described from the studied region from F. burkei [13], and thus, one of the six aphelenchoidids (five Ficophagus and one Schistonchus spp.) recovered from F. burkei in this study could be conspecific to S. africanus. However, because of the typological similarity among fig-associated aphelenchoidids and the lack of detailed morphological and genetic information in the original description [9, 13], we could not resolve or further characterize S. africanus using current taxonomic standards. In addition, the number and quality of materials collected in the present study were not sufficient to characterize each genotype typologically. Therefore, the conspecificity and the phylogenetic status of S. africanus with any of the F. burkei isolates in this study remains unclear and will require a reverse taxonomy approach. In addition, a species of Bursaphelenchus which is closely related to B. sycophilus isolated from F. variegata in Japan (Figs 2 and 3) [27] was isolated from F. sur from Pretoria corroborating a subgeneric Sycomorus fig co-association for this fig-derived Bursaphelenchus clade. Susoy et al. [8] reported discovery of a Bursaphelenchus from F. sycomorus from Pretoria that will require further work for phylogenetic placement.

Fig 3. Phylogenetic status of fig-associated species within the family Aphelenchoididae inferred from D2-D3 LSU under GTR + G + I model.

The parameters are: AIC = 55193.907, lnL = -27357.223, freqA = 0.24, freqC = 0.19, freqG = 0.30, freqT = 0.27, R(a) = 0.45, R(b) = 2.61, R(c) = 0.92, R(d) = 0.64, R(e) = 3.31, R(f) = 1.00, Pinva = 0.11, Shape = 0.86. Posterior probability values exceeding 50% are given on appropriate clades. Fig-associated nematode species obtained in the present study and previous studies are written in red and blue, respectively. Section or subsection of host fig species are coded with color dots.

Lastly, there is the issue of mixed lineages and incomplete lineage sorting from movement of species of nematodes among the different pollinators or inquilines that might visit different Ficus species [37, 38]. Clearly, Martin et al. [1] made an extraordinary discovery about fig-associated nematodes in Africa, especially when it comes to Sycomorus clade figs, which is corroborated and further elucidated here with the aid of molecular inferences (Figs 2–7).

In addition to the two typical fig-associated families (Aphelenchoididae and Diplogastridae), an unidentified panagrolaimid was recognized in the present study. The precise phylogenetic status of the panagrolaimid is still unknown. This finding suggests that nematode colonization in fig syconia has occurred many times, more than we expected, and further diversity of fig associated nematodes will be discovered by more extensive collecting.

Host fig association

The phylogenetic relationships inferred for the fig-associated aphelenchoidid genera (Schistonchus and Ficophagus) recovered in this study were not clearly associated with geography or host fig subsections (Figs 2–4). The only true affiliation between Ficophagus and fig lineages appears to be between Central American delimited Pharmacosycea figs and their associated nematodes (both Ficophagus and Parasitodiplogaster), but in the South African figs, Ficophagus n. sp. 4 ex F. sur appeared consistently monophyletic with other Ficophagus spp. from Sycomorus clade figs from other continents in SSU and LSU inferences (Figs 2 and 3). Similarly, the discovery of Bursaphelenchus sp. 1 from F. sur from Pretoria and its monophyly with B. sycophilus from F. variegata from Japan [27] establishes a subgeneric Sycomorus fig co-association for a fig-adapted Bursaphelenchus clade. It also portends that the Bursaphelenchus reported from F. sycomorus by Susoy et al. [8] will be monophyletic with other Sycomorus clade fig associated Bursaphelenchus from Africa and Australasia when re-examined.

Poinar [12] described P. sycophilon as the first member of this genus from F. burkei (subsection Chlamydodorae) from Zimbabwe without molecular data. We can now place P. sycophilon with six new Parasitodiplogaster species that are all associated with Urostigma subgenus, section Galoglychia figs from three subsections (Caulocarpae, Chlamydodorae and Platyphyllae) which form an African delimited Parasitodiplogaster clade that is associated with an African delimited Ficus clade (Figs 5–7). In contrast, P. salicifoliae was isolated only from section Urostigma and subsection Urostigma figs from South Africa (F. salicifolia and F. ingens) but formed a monophyletic clade with other Parasitodiplogaster species from this clade of figs from Australia (i.e., P. australis Bartholomaeus, Davies, Ye, Kanzaki & Giblin-Davis) and China (i.e., P. religiosae Zeng, Zeng, Zhang, Ye, Cheng, Kanzaki & Giblin-Davis) (Figs 5 and 6).

Multiple lineages of a nematode genus were found in the same fig species, and these lineages included close relatives (a pair of sister species), in the present study. For example, F. burkei harbored two Parasitodiplogaster spp. (P. sycophilon and its close relative, Parasitodiplogaster n. sp. 6), and five Ficophagus spp. (Ficophagus n. spp. 1, 2, 5–7, where sp. 1 and sp. 2 are close to each other, and spp. 5–7 form a subclade), and F. sur had three or four closely related Acrostichus spp. (n. sp. 1–3 or 4), T. cf. martini, Pristionchus sp. 35 (= Parasitodiplogaster cf. doliostoma) and Ficophagus sp. 4 (Figs 2–7, Table 1, S1 Table in S2 File). Similar multiple colonization events and duplications of the lineages were found in previous studies, e.g., F. hispida L. harbors two Ficophagus spp. (F. centerae (Zeng, Giblin-Davis & Ye) and an undescribed Ficophagus species), three Martininema spp. (M. guangzhoensis (Zeng, Giblin-Davis & Ye), M. baculum (Davies, Bartholomaeus, Kanzaki, Ye & Giblin-Davis) and an undescribed Martininema species), and an undescribed Schistonchus sp. [39–41], and F. maxima Mill. has three Parasitodiplogaster spp. (P. maxinema Poinar & Herre, P. pharmaconema Kanzaki, Giblin-Davis, Ye, Herre & Center and an undescribed species which are all close to each other) and Ficophagus maxima Davies, Ye, Center, Kanzaki, Bartholomaeus, Herre, Esquivel & Giblin-Davis [42–44].

Multiple colonization events and the duplication of lineages are also well documented in wood and bark beetles and their associated nematodes. A species of bark beetle often harbors several different lineages of aphelenchoidids and diplogastrids [45, 46], and two closely related Bursaphelenchus spp. (B. luxuriosae Kanzaki & Futai and B. acaloleptae Kanzaki, Ekino, Maehara, Aikawa & Giblin-Davis) share the same host (Araliaceae trees) and carrier beetle (Acalolepta luxuriosa (Bates)) [47].

The wasp associations of the nematodes, e.g., host range, were not examined in the present study. These nematodes are carried by fig wasps, and thus, their fig host range is determined by the wasps. Several previous authors reported that host specificity of the fig wasps which is determined by chemical signals from the fig host can become less stringent. One species of wasp can be associated with multiple species of figs, and vice versa [37, 38, 48, 49]. In addition, the host switching, duplication of the lineages, and extinction and fill-in of the wasp lineages can drive the diversification of figs and their pollinating wasps [49, 50]. Because the nematodes are tightly associated with figs and fig wasp as the habitat/host and carrier/host, their diversification and host switching are likely to be affected by those of the carrier host. Further collections of figs, wasps and nematodes and specifying their host/carrier ranges, i.e., carrier wasp range of nematodes and host range of wasps, are necessary to understand their tripartite associations.

In the present study, we focused on the diversity of nematodes in relation to their host/habitat fig lineages but did not conduct extensive research involving the diversity and host range of the fig wasps. Further studies on biological and phylogenetic relationships among nematodes, fig wasps and fig trees will help reveal the mechanism(s) and give further insights into the evolutionary context of these tripartite relationships.

Our study has expanded and improved the clarity and resolution of Martin et al. [1] with a more extensive spatial/temporal snapshot of the fig-associated nematode fauna from South Africa with the aid of molecular technology. However, it still relied upon the luck of finding asynchronously fruiting fig trees in the proper phases with nematodes in good condition with different association rates with their fig wasp and/or fig hosts from a large area during a finite survey. Thus, there are potentially many more nematode species and interesting life history discoveries to be made to add upon what is reported here. There is also much reverse taxonomy needed to finish tying the genotypes reported in this study with well-defined morphotypes and biological/life history traits for South African species of fig associated nematodes as recently initiated by Wöhr et al. [17, 18] and Susoy et al. [8].

Supporting information

S1 File. Typological characters of several nematode species recovered from the fig materials.

Light micrographs or drawn are provided as supplementary figures.

(PDF)

S2 File. Detailed collection information and taxonomic status of Ficus species appeared in the present study.

(XLSX)

Data Availability

Data used in this study are included within the manuscript and Supporting Information files. The sequence data is in GenBank and the accession numbers for this data are included in Supporting Information Table 1 and Figs 2–7.

Funding Statement

This work was supported in part by the National Research Foundation of South Africa for financial support (grant 77256 to JMG) and Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (20H03026 to NK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript and therefore do not accept any liability in regard thereto.