Aphelenchoidid Nematodes Associated with Two Dominant Ficus

Nematology 19 (2017) 323-331 brill.com/nemy

Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia ∗ Rina SRIWATI 1,YukoTAKEUCHI-KANEKO 2,J.JAUHARLINA 1 and Natsumi KANZAKI 3, 1 Department of Plant Protection, Faculty of Agriculture, Syiah Kuala University, Darussalam Banda Aceh 23111, Indonesia 2 Laboratory of Terrestrial Microbial Ecology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan 3 Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan Received: 20 November 2016; revised: 18 January 2017 Accepted for publication: 18 January 2017; available online: 22 February 2017

Summary – Aphelenchoidid nematodes associated with the syconia of two dominant fig species, Ficus hispida and F. racemosa,were surveyed in Banda Aceh, Indonesia. Nematodes were isolated from sycones and pollinating wasps of these two fig species from four localities in the area, and identified based on the molecular sequences of two genetic loci, D2-D3 expansion segments of large subunit ribosomal RNA (D2-D3 LSU) and mitochondrial cytochrome oxidase subunit I (mtCOI). Molecular sequences of D2-D3 LSU and mtCOI were successfully determined for 44 and 19 individual nematodes, respectively, and these sequences were separated into four clades, i.e., types A-D of D2-D3 LSU and types I-IV of mtCOI. Phylogenetic analysis of the DNA sequences deposited in the GenBank database showed that the DNA sequences corresponded to three species, namely, Martininema baculum (type B/II), Ficophagus fleckeri (types A/I, D/IV) and F .cf.centerae (type C/III). Within these species, F. fl e cke r i was separated into two clades as suggested in previous studies and thus it may possibly reflect the existence of two different taxa, F. fl e cke r i and a cryptic species. The Indonesian F .cf.centerae was monophyletic with, but clearly separated from, the Chinese population of F. centerae and thus the Indonesian population is potentially an undescribed species. Overall, the species composition of fig-associated aphelenchoidids in the Aceh region seemed intermediate between continental Chinese and Australian species. However, further material collections followed by detailed morphological analyses are necessary to characterise or describe these fig-associated aphelenchoidids in Indonesia. Keywords – Aphelenchoididae, biogeography, diversity, Ficophagus cf. centerae, Ficophagus fleckeri, Ficus hispida, Ficus racemosa, figs, Martininema baculum, molecular, plant-parasitic nematode, syconia.

Nematodes associated with figs (syconia of Ficus spp.) + Teratodiplogaster Kanzaki, Giblin-Davis, Davies, Ye, are now becoming an interesting model system for evolu- Center & Thomas, 2009, Pristionchus Kreis, 1932, Acros- tionary biology of parasitism/pathogenicity (Herre, 1993), tichus Rahm, 1928 and Rhabditolaimus Fuchs, 1914, de- phenotypic plasticity (e.g., Susoy et al., 2016), feeding rived from diplogastrid bacterial feeders (e.g., Kanzaki et habit (e.g., Giblin-Davis et al., 2003; Kanzaki et al., al., 2016; Susoy et al., 2016), and Caenorhabditis Osche, 2014a) and other topics. So far, at least ten lineages 1952 derived from a rhabditid bacterial feeder (Kanzaki of nematodes are known to be associated with figs and et al., 2014b). In addition, several genera have been re- fig wasps: namely, Bursaphelenchus Fuchs, 1937, Schis- ported from India (e.g., Lingaiah et al., 2012). However, tonchus Cobb, 1927, Martininema Davies & Bartholo- we cannot discuss the Indian species in detail due to a lack maeus in Davies et al., 2015 and Ficophagus Davies of detailed description, molecular barcoding information & Bartholomaeus in Davies et al., 2015, derived from and type material. aphelenchoidid fungal feeders (Kanzaki et al., 2014a; In the Asia-Oceania (West Pacific) region, fig- Davies et al., 2015), Ficotylus Davies, Ye, Giblin-Davis associated nematode surveys and species descriptions & Thomas, 2009 derived from a tylenchid plant parasite have been conducted in Australia (Bartholomaeus et al., (Davies et al., 2009), Parasitodiplogaster Poinar, 1979 2009, 2012; Davies et al., 2009, 2010, 2013a, b, 2015;

∗ Corresponding author, e-mail: [email protected]

Kanzaki et al., 2009), New Zealand (Zhao et al., 2015), according to the methods of Kanzaki & Futai (2002) and Vietnam (Susoy et al., 2016), China (Zeng et al., 2007, Ye et al. (2007). 2010, 2011, 2013a, b, c), India (Kumari & Reddy, 1984; The determined sequences were compared with the Reddy & Rao, 1986; Anand, 2002, 2005; Bajaj & Tomar, other fig-associated aphelenchoidids with maximum like- 2014) and Japan (Kanzaki et al., 2014b, c). Indonesia is lihood phylogenetic analyses. The sequences for compar- surrounded by the countries where the fig-associated ne- ison with present data were downloaded from the Gen- matode studies have been conducted and is a contact point Bank database (http://www.genome.jp/dbget-bin/www_ of the East and South Asian and Oceania regions. There- bfind?genbank-today). The phylogenetic analyses were fore, it is important to examine the fig-associated nema- performed in PhyML (Guindon et al., 2010: http://www. todes there as basic information for future biogeograph- atgc-montpellier.fr/phyml/) using the D2-D3 LSU and mt- ical studies. However, at present almost no information COI sequence data separately. Sequence alignments were about fig-associated nematodes has been reported from performed using the online version of MAFFT (Katoh et this region. al., 2002: http://mafft.cbrc.jp/alignment/server/). The nu- In the present study, nematodes associated with two cleotide substitution model was selected automatically in dominant Ficus L. species, F. racemosa L. and Ficus the online PhyML and the tree topology was evaluated hispida L., were examined at Aceh, Indonesia, based on with 1000 bootstrap pseudoreplicates. molecular barcoding focusing on aphelenchoidid plant parasites. Results

Materials and methods Adults and propagative juveniles were isolated from fig syconia, while the nematodes isolated from the wasps NEMATODE COLLECTION AND MATERIAL seemed to be young adult females. However, because PREPARATION of the poor condition of the fixed material, probably because the temperature for heat-killing was not sta- Syconia were collected from Ficus racemosa and F. ble, detailed morphological analysis was not conducted hispida growing in natural forest around Banda Aceh, and the nematodes were identified using molecular se- Indonesia in July 2013 (Table 1). Collected figs were quences. Although the species-specific morphological dissected in distilled water or kept at room temperature for characters were not clearly observed, the photomicro- fig wasp development. The emerged fig wasps were then graphs of DESS-fixed materials are available upon re- dissected for nematode isolation. Collected nematodes quest. were either heat-killed at 60¡C for 1 min and fixed in The D2-D3 LSU and mtCOI were successfully se- TAF (7.0% formalin, 2.0% triethanolamine, 91% distilled quenced for 44 and 19 individuals, respectively and the water) for morphological vouchers for future taxonomic sequences were divided into four genotypes for both loci. studies, or stored in DESS (Yoder et al., 2006) for Furthermore, D2-D3 LSU type B was separated into seven molecular material. subtypes, and mtCOI types I, II and III were separated into three, six and three subtypes, respectively (Fig. 1; Ta- MOLECULAR IDENTIFICATION ble 1). However, these variations could be derived from sequencing error or unclear (heterologous) bases often DESS-fixed nematodes were hand-picked from the found in those loci of fig-associated aphelenchoidids (e.g., reagent using a flame-sterilised stainless steel entomolog- Davies et al., 2015). The determined sequences were de- ical pin, washed in a drop of sterile water and observed posited in the GenBank database with the accession num- under a light microscope for casual morphotyping. Then, bers LC208749-LC208772 (Figs 1, 2). the nematodes were individually transferred to nematode Based upon phylogenetic analyses, four genotypes digestion buffer (Kikuchi et al., 2009; Tanaka et al., 2012) were tentatively identified as Martininema baculum and individually digested at 60¡C for 30 min. The nema- (Davies, Bartholomaeus, Kanzaki, Ye & Giblin-Davis, tode lysate served as PCR templates, and their amplicons 2013) Davies & Bartholomaeus in Davies et al., 2015 of D2-D3 expansion segments of large subunit ribosomal (D2-D3 type B; mtCOI type II); Ficophagus cf. cen- RNA (D2-D3 LSU) and partial fragment of mitochondrial terae (D2-D3 type C; mtCOI type III) and F. fleckeri cytochrome oxidase subunit I (mtCOI) were sequenced (Davies, Bartholomaeus, Kanzaki, Ye & Giblin-Davis,

324 Nematology Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia , , centerae centerae cf. cf. Martininema baculum Martininema baculum Ficophagus fleckeri Ficophagus fleckeri Martininema baculum Martininema baculum Ficophagus Martininema baculum Ficophagus Ð mtCOI genotype Nematode identification B6, B7, C D2-D3 genotype Fig wasp B1, B2, B6, B7 IIb, IIc Source of nematode R1 Syconia Ð Ð Ð H3 Syconia B3, B4, B5, CR4 IIa, IIb, IIIa, IIIb,R2 IIIc SyconiaH1 Syconia A A, D Syconia B1, B3, B4, B6 Ia, Ð Ib, If, Ic, IV Id, Ie R0 Syconia Ð Ð H4H5 Syconia Syconia B1, B2, B3, B4, B1 IIIc R3 Syconia Ð Ð Ð tree code Ficus racemosa Ficus hispida Ficus racemosa Ficus hispida Ficus racemosa Ficus racemosa E) E) E) E) 43 33 32 35 N, 95¡19 N, 95¡14 N, 95¡15 N, 95¡15 53 39 46 56 Summary of collection locality, tree species, individual tree code, source of nematode and nematode genotype. Lampeunerut (5¡30 Table 1. Locality (GPS)Leupung 1 (5¡20 Tree species Individual Leupung 2 (5¡22 Leupung 3 (5¡22

Vol. 19(3), 2017 325 R. Sriwati et al.

Fig. 1. Molecular phylogenetic relationships among ﬁg-associated aphelenchoidid species together with their associated Ficus sp. host. The maximum likelihood tree was inferred from D2-D3 expansion segments of the large subunit of ribosomal RNA. The substitution model was selected automatically as GTR + I + G(lnL=−11 584.95; AIC = 23 571.89; Pinvar = 0.176; Shape = 0.951). Bootstrap support exceeding 50% is shown for appropriate clades. This ﬁgure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

326 Nematology Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia

Fig. 2. Molecular phylogenetic relationship among ﬁg-associated aphelenchoidid species together with their associated Ficus sp. host. The maximum likelihood tree was inferred from partial code of mitochondrial cytochrome oxidase subunit I. The substitution model was selected automatically as GTR + I + G(lnL=−4507.82; AIC = 9145.64; Pinvar = 0.482; Shape = 1.244). Bootstrap support exceeding 50% is shown for appropriate clades. This ﬁgure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

2013) Davies & Bartholomaeus in Davies et al., 2015 fig-associated aphelenchoidids from Indonesia. The ne- (D2-D3 types A, D; mtCOI types I, IV). Because mtCOI matodes isolated from fig syconia were adults and prop- sequences of these three species have not been deposited agative juveniles; those from wasps appeared to be young in the GenBank database, molecular identification was adult females. This may suggest that the transmission of conducted based upon the D2-D3 LSU. Within these three these fig-associated aphelenchs is different from most of species, F. fleckeri was separated into two subclades with the other aphelenchs where the nematodes are carried by relatively high boot strap support (Figs 1, 2), as suggested previously by Davies et al. (2015). Also, F .cf.centerae insects as dauer juveniles. However, we consider several was relatively clearly separated from F. centerae (Zeng, more case studies will be necessary to confirm the gener- Giblin-Davis & Ye, 2007) Davies & Bartholomaeus in ality of this phenomenon. Davies et al., 2015 from China (DQ912928) (Fig. 1). Martininema baculum was originally described from Ficus racemosa and F. h i s p i d a from Australia as Schis- tonchus baculum (see Davies et al., 2013a). In the D2-D3 Discussion LSU analysis, the Australian (AB535541) and Indonesian In the present study, four genotypes of aphelenchoi- isolates formed a monophyletic clade, and the variation dids were isolated from two Ficus species, i.e., M. bac- between two populations was very small. Further, the host ulum and F .cf.centerae are associated with Ficus hisp- fig species (F. h i s p i d a ) is consistent between these popula- ida, and two genotypes of F. fleckeri are associated with tions. Thus, the Indonesian population is clearly conspe- Ficus racemosa. This is the first formal report of those cific to the Australian population and the wide distribu-

Vol. 19(3), 2017 327 R. Sriwati et al. tion of M. baculum, i.e., Northern Australia to the north- Davis & Ye, 2007) Davies & Bartholomaeus in Davies western part of Sumatra Island, was confirmed. et al., 2015 and Ficophagus centerae in China (Kumari & On the other hand, sequences of Ficophagus cf. cen- Reddy, 1984; Giblin-Davis et al., 2004; Zeng et al., 2007; terae are distinct from those of the Chinese population of Davies et al., 2010, 2013a, 2015). F. centerae, with a relatively clear branch. This may be Banda Aceh, Indonesia, the collection site in the because sequence lengths determined in the present study present study, is geographically located at the intersection were not long enough to characterise the species (ca 260 of Australia, India and Southern China (Fig. 3). Consider- bp of the D3 region) and the difference of available se- ing the biogeographical boundaries, Banda Aceh and Aus- quences might have affected the phylogenetic analysis. tralia, which share M. baculum and Ficophagus fleckeri, Ficophagus centerae was originally described from Fi- are located on opposite (west and east) sides of Wallace’s cus hispida in Guangzhou, South China, by Zeng et al. line. On the other hand, Banda Aceh and China, which (2007). More field surveys followed by molecular analy- are on the same side (west) of the line seem to have differ- sis are necessary to confirm the distribution and species ent genotypes of Ficophagus (F. centerae and F .cf.cen- status of F .cf.centerae. terae) from each other. The biogeographical boundaries Ficophagus fleckeri was described from Ficus race- were hypothesised based on terrestrial animal fauna (e.g., mosa and F. h i s p i d a from several different localities in Holt et al., 2013). However, the distribution of figs and fig- Northern Australia (Davies et al., 2013a). The host tree associated nematodes seems determined by different fac- species in the present study (Ficus racemosa) is consis- tors from one another, and from those of larger terrestrial tent with the original description. Interestingly, the species animals. For example, the host Ficus trees, which have a was separated into two clades with high bootstrap or pos- wide distribution range crossing the Wallace line (Harri- terior probability support in the present and previous stud- son et al., 2012; Pothasin et al., 2014), can be distributed ies (Davies et al., 2015), i.e., the separation was con- by birds (see Lambert & Marshall, 1991), whereas the ne- firmed in both Australian and Indonesian populations. matodes are distributed by wasps that are not expected to Based upon the branch length disparity between these fly long distances (see Giblin-Davis et al., 2003; Davies two clades, and inter- and intraspecific variation of other et al., 2015), i.e., the distribution and speciation of nema- species in D2-D3 LSU analysis, these two clades could todes could be more local, whilst that of the host figs has be different species. In the original description, molecu- become wider than that of the terrestrial animals (= bio- lar data were not provided for the type population (Davies geographical boundaries). Similarly, another fig species, et al., 2013a) and thus we cannot determine which of the Ficus variegata (Blume), is distributed from subtropical two clades contains the type population. Repeated field Japan to Northern Australia (Harrison et al., 2012), and is surveys followed by detailed morphological and molecu- associated with several genera of nematodes, Teratodiplo- lar observation are necessary to confirm the species status gaster, Ficophagus and Bursaphelenchus. However, the of these two clades. nematodes species between Australia and Japan seem to Ficus racemosa and F. h i s p i d a have very wide distribu- be different. This fig species harbours F. cassowaryi and tion range, i.e., both are found in the South China-India- an unidentified Teratodiplogaster sp. in Australia (Davies Northern Australia region (Harrison et al., 2012), and et al., 2013a; Kanzaki et al., 2014c), and B. sycophilus have been reported to be associated with many aphelen- Kanzaki, Tanaka, Giblin-Davis & Davies, 2014, T. varie- choidids: Ficus racemosa is associated with M. baculum, gatae Kanzaki, Woodruff & Tanaka, 2014, and an uniden- Ficophagus fleckeri and S. cassowaryi Davies, Bartholo- tified Ficophagus sp. in Ishigaki, Japan, the species not maeus, Kanzaki, Ye & Giblin-Davis, 2013 in Australia, appearing to overlap one another, i.e.,noBursaphelen- and with S. osmani Anand, 2002, S. racemosa Reddy & chus has been isolated from Australian Ficus variegata, Rao, 1986, S. cuculloracemosus Bajaj & Tomar, 2014, and Japanese and Australian Teratodiplogaster and Fi- S. flagelloracemosus Bajaj & Tomar, 2014, and S. mu- cophagus seem molecularly and/or morphologically dif- croracemosus Bajaj & Tomar, 2014 in India (Kumari & ferent from each other (Davies et al., 2013a; Kanzaki et Reddy, 1984; Reddy & Rao, 1986; Bajaj & Tomar, 2014); al., 2014a, c; Kanzaki, unpubl. obs.). whilst Ficus hispida is associated with M. baculum, Fi- Many fig-associated nematodes have been described cophagus fleckeri, an undescribed Ficophagus sp. M5, and from those areas and thus field surveys of Indonesian fig- S. hispida Kumari & Reddy, 1984 in Australia, plus S. associated nematodes should yield biogeographically in- hispida in India, and M. guangzouensis (Zeng, Giblin- teresting information, as indicated by the limited study

328 Nematology Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia

Fig. 3. Geographical position of collection area, Banda Aceh and surrounding areas in relation to Wallace’s line. Nematode species found in the present study and their close relatives are noted beneath their type localities, and the areas mentioned in the present study are also noted. herein. To understand the biodiversity and biogeography Anand, R.L. (2005). Association of a nematode Ceratosolenus (= factors and relationship between distribution and spe- racemosa gen. n., sp. n. (Cylindrocorporidae: Rhabditida ciation) of those nematode species, more field collections Goodey, 1939) a wasp (Ceratosolen sp. n.) and fig Ficus and molecular analyses, including carrier fig wasps, are racemosa. Uttar Pradesh Journal of Zoology 25, 59-62. necessary. Bajaj, H.K. & Tomar, V.V.S. (2014). Description of new and known species of Schistonchus Cobb, 1927 (Aphelenchida: Aphelenchoididae) from syconia of Ficus trees growing at Acknowledgements Hisar, Haryana. Indian Journal of Nematology 44, 193-211. Bartholomaeus, F., Davies, K., Kanzaki, N., Ye, W. & Giblin- Davis, R.M. (2009). Schistonchus virens n. sp. (Aphelenchoi- The authors sincerely thank Dr Kerrie A. Davies, Uni- didae) and Parasitodiplogaster australis n. sp. (Diplogastri- versity of Adelaide, for her valuable comments on the dae), from Ficus virens (Moraceae) in Australia. Nematology early version of the manuscript and editorial corrections, 11, 583-601. and Ms Noriko Shimoda and Ms Atsuko Matsumoto, FF- Bartholomaeus, F., Davies, K.A., Ye, W. & Giblin-Davis, R.M. PRI, for their technical assistance in molecular profiling. (2012). Schistonchus (Aphelenchoididae) from Ficus benjam- This study was supported, in part, by Grants-in-Aid for ina in Australia, with description of S. benjamina sp. n. Ne- Scientific Research (B), Nos 26292083, 26292178 and matology 14, 1005-1013. 15H04514 and Grant-in-Aid for Challenging Exploratory Cobb, N.A. (1927). Note on a new nema, Aphelenchus retusus, Research, No. 15K14896 from the Japan Society for the with a proposed division of Aphelenchus into three subgenera. Promotion of Science, Japan, and Scheme for Academic Journal of Parasitology 14, 57-58. Davies, K.A., Ye, W., Giblin-Davis, R.M. & Thomas, W.K. and Mobility Exchanges (SAME) programmes by The (2009). Ficotylus congestae gen. n., sp. n. (Anguinata), from Ministry of Research, Technology and Higher Education, Ficus congesta (Moraceae) sycones in Australia. Nematology Indonesia, No. 79/E4.2/PP/2013. 11, 63-75. Davies, K.A., Bartholomaeus, F., Ye, W., Kanzaki, N. & Giblin- Davis, R.M. (2010). Schistonchus (Aphelenchoididae) from References Ficus (Moraceae) in Australia, with description of S. aculeate n. sp. Nematology 12, 935-958. Anand, R.L. (2002). Schistonchus osmani n. sp. ex Ficus Davies, K.A., Bartholomaeus, F., Kanzaki, N., Ye, W. & Giblin- racemosa. Uttar Pradesh Journal of Zoology 22, 281-283. Davis, R.M. (2013a). Three new species of Schistonchus

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330 Nematology Aphelenchoidid nematodes associated with two dominant Ficus species in Aceh, Indonesia

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