Nematology 17 (2015) 639-653 brill.com/nemy

Description of Ruehmaphelenchus juliae n. sp. (Tylenchina:Aphelenchoididae) isolated from an ambrosia beetle, Xylosandrus

crassiusculus (Motschulsky), from South Florida

Natsumi KANZAKI 1,∗, Robin M. GIBLIN-DAVIS 1, Rafael GONZALEZ 1,Rita DUNCAN 2 and Daniel CARRILLO 2

1 Fort Lauderdale Research and Education Center, University of Florida/IFAS, 3205 College Avenue,Davie, FL 33314-7799, USA

2 Tropical Research and Education Center, University of Florida/IFAS, 18905 SW 280 Street, Homestead, FL 33301, USA

Received: 3 March 2015; revised: 27 March 2015Accepted for publication: 27 March 2015; available online: 7 May 2015

Summary – During a survey of nematode associates of ambrosia beetles from dead and dying red bay and avocado trees affected by thelaurel wilt epidemic in southern Florida, a Ruehmaphelenchus species was isolated from the non-native ambrosia beetle, Xylosandruscrassiusculus. The new species is characterised by its possession of an oral disc at the stoma opening, three lines in the lateral field,male spicule with clear dorsal and ventral limbs connected by elongated triangular cuticle, thin membrane-like tissue and cuticularbridge-like structure, conical tail with pointed tip of males and conical tail with digitate mucro of females. The new species is verysimilar to four previously described species: R. asiaticus, R. digitulus, R. thailandae and R. sirisus, and can be distinguished only bysome minor morphological differences in male tail characters, i.e., spicule morphology, position of genital papillae and tail tip shape,and morphometric values. However, the new species is phylogenetically unique, i.e., it is the basal taxon of the Ruehmaphelenchusclade and close to Bursaphelenchus spp. Ruehmaphelenchus juliae n. sp. is therefore proposed based on its morphological diagnosticcharacters and molecular sequences of near-full-length of SSU, internal transcribed spacer region, D1, D2 and D3 expansion segmentsof LSU ribosomal RNA and partial mitochondrial COI genes.

Keywords – Coleoptera, Curculionidae, laurel wilt disease, molecular, morphology, morphometrics, Persea americana, Perseaborbonia, phylogeny, Raffaelea lauricola, Scolytinae, taxonomy, USA, Xyleborini.

Previous surveys of ambrosia beetles from dead and dy-ing red bay Persea borbonia (L.) and avocado P. ameri-cana (Miller) trees affected by the laurel wilt disease insouthern Florida, USA (Carrillo et al., 2012) suggestedthat nematodes associated with introduced and invasiveambrosia beetle species, e.g., Xyleborus glabratus Eich-hoff, might be a source of arriving nematodes that couldbe passed among populations of native ambrosia beetlespecies (Giblin-Davis et al., 2013; Kanzaki et al., 2014).Alternatively, nematodes that had long been associatedwith native species of ambrosia beetles could be mov-ing onto arriving species of ambrosia beetles and creat-ing the opportunity for new interactions. We have contin-ued surveying introduced and native ambrosia beetles fortheir nematode associates and discovered an undescribedRuehmaphelenchus species from the introduced granulate

∗ Corresponding author, e-mail: natsumikanzaki@hotmail.com

ambrosia beetle Xylosandrus crassiusculus (Motschul-sky) (Coleoptera: Curculionidae: Scolytinae: Xyleborini)in southern Florida.

Based upon molecular phylogenetic inferences, Rueh-maphelenchus Goodey, 1963 is currently a derived cladewith a relatively long branch (for small subunit (SSU)and long subunit (LSU) ribosomal RNA genes) withinthe assemblage of species and two other genera (Para-sitaphelenchus Fuchs, 1930 and Sheraphelenchus Nickle,1970) currently comprising Bursaphelenchus Fuchs, 1937(Goodey, 1963; Hunt, 1993, 2008). Kanzaki et al. (2011)suggested that Ruehmaphelenchus should probably be ajunior synonym of Bursaphelenchus until further molec-ular and morphological work is done to help resolvethe classification of Bursaphelenchus. Ruehmaphelenchusis sometimes included in the Aphelenchoidinae Skar-

© Koninklijke Brill NV, Leiden, 2015 DOI 10.1163/15685411-00002896

N. Kanzaki et al.

bilovich, 1947 clade by phylogenetic analyses based onsequences of internal transcribed spacer (ITS) regions ofthe rRNA genes (Braasch et al., 2006; Gu & Wang, 2010,2012). Additional work is needed to resolve the classifi-cation status of the genus.

In the present study, the newly discovered nematode isdescribed and figured as R. juliae n. sp., and its molecularphylogenetic status is determined based upon near-full-length of SSU, internal transcribed spacer region (ITS1and 2), D1, D2 and D3 expansion segments of LSUribosomal RNA and partial mitochondrial COI genes.

Materials and methods

NEMATODE ISOLATION AND MORPHOLOGICAL

OBSERVATION

Dead branches from ambrosia beetle-infested red bayor avocado ‘Simmonds’ trees affected by the laurel wiltpathogen (Raffaelea lauricola Harrington, Fraedrich &Aghayeva) from natural areas in and around Homestead,FL, USA, were placed for several weeks in vented rear-ing containers in the laboratory to allow for the emer-gence of adults for subsequent beetle dissections andnematode rearing attempts during 2014. Beetles wereidentified, rinsed externally and dissected alive onto 1.5%water agar media in groups of 5-10. The resulting plateswere incubated at room temperature (ca 20-25°C) for 2-3 weeks. Nematodes that successfully propagated on theplates were transferred to a LGPDA (19.5 g potato dex-trose agar, 30.8 g glycerin, 500 ml dH2O with 1 ml of25% lactic acid aseptically added as the agar cooled) thathad been previously inoculated with Monilinia fructicola(Winter) Honey, and kept as a laboratory strain with aculture code CBAY2. Morphological observations includ-ing drawings, micrographs and morphometrics were madewith a microscope using living material that was obtainedfrom 1-week-old cultures. To examine the morphomet-ric range of the species, adult nematodes from 2-week-old cultures on 2.0% malt extract agar (MEA: malt ex-tract: 20 g; agar: 20 g and brought to 1 l with distilledwater) previously inoculated with Botryotinia fuckeliana(de Bary) (= Botrytis cinerea Pers.) were also examined.The type materials were obtained from 2-week-old cul-ture on MEA-B. fuckeliana, heat-killed and fixed in TAF(triethanolamine:formalin:distilled water = 2:7:91), pro-cessed with a glycerin-ethanol series using Seinhorst’smethod, and mounted in glycerin according to the meth-ods of Maeseneer and d’Herde (described in Hooper,1986).

MOLECULAR CHARACTERISATION AND PHYLOGENY

A DNA sample from R. juliae n. sp. was collected andprepared as described by Kikuchi et al. (2009) and Tanakaet al. (2012). About 3.6 kb of DNA base sequences ofpartial ribosomal DNA including near-full-length of 18S,internal transcribed spacers 1 and 2, 5.8S and D1 throughD3 expansion segments of 28S (near-full SSU, ITS andD1-D2-D3 LSU) and ca 0.7 kb of partial sequence ofmitochondrial cytochrome oxidase subunit I (mtCOI)were determined for R. juliae n. sp. following Kanzaki &Futai (2002) and Ye et al. (2007).

Determined sequences were compared to sequencesfrom other Ruehmaphelenchus and Bursaphelenchusspecies that had been deposited in the GenBank databasewith accession numbers LC031813 (ribosomal RNAgenes) and LC031814 (mtCOI). The species that were in-cluded in these analyses were selected, and the outgroupspecies were determined as Aphelenchoides stammeri(Körner, 1954) and B. abruptus Giblin-Davis, Mundo-Ocampo, Baldwin, Norden & Batra, 1993 according tothe phylogenetic analyses conducted by Kanzaki et al.(2014, 2015). The GenBank accession numbers of com-pared sequences are provided in Table 1, and the un-aligned sequences are provided separately (SSU and D2-D3) as FASTA files (Supplementary Texts 1, 2 in the on-line edition of this journal, which can be accessed viahttp://booksandjournals.brillonline.com/content/journals/15685411).

The molecular phylogenetic status of the new specieswas determined by Bayesian analysis based on near-fullSSU and D2-D3 LSU. First, sequences from differentspecies were aligned using the MAFFT program (Katohet al., 2002; web version available at: http://align.bmr.kyushu-u.ac.jp/mafft/software/) and the base-substitutionmodels for each gene were determined using MOD-ELTEST version 3.7 (Posada & Crandall, 1998) underAkaike information criterion (AIC). For both genes, themodel was selected as GTR + I + G, which has the small-est AIC value among 56 models compared. Bayesian anal-yses were performed to confirm the tree topology based oncombined sequence (= SSU + D2-D3) using MrBayes3.2 (Huelsenbeck & Ronquist, 2001); four chains wererun for 4 × 106 generations, and Markov chains weresampled at intervals of 100 generations (Larget & Simon,1999). Two independent runs were performed and, afterconfirming convergence of runs and discarding the first2 × 106 generations as ‘burn-in’, remaining topologieswere used to generate a 50% majority-rule consensus tree.

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Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Table 1. Parasitaphelenchid species used for the phylogenetic analyses.

Genus Species SSU D2-D3 Intrageneric clade

Aphelenchoides stammeri AB368535 AM396582 OutgroupBursaphelenchus abruptus AY508010 AY508073 OutgroupRuehmaphelenchus asiaticus – AM269475 I (Ruehmaphelenchus)Ruehmaphelenchus digitulus JN377732 JN377730 I (Ruehmaphelenchus)Ruehmaphelenchus formosanus AB808718 AB808719 I (Ruehmaphelenchus)Ruehmaphelenchus juliae n. sp. LC031813 LC031813 I (Ruehmaphelenchus)Ruehmaphelenchus sp. NK202 AB368534 AB597984 I (Ruehmaphelenchus)Bursaphelenchus abietinus AY508011 AY508074 IBursaphelenchus anamurius – FJ643489 IBursaphelenchus antoniae AM279709 AM279710 IBursaphelenchus borealis AY508012 AY508075 IBursaphelenchus chengi – EU107359 IBursaphelenchus clavicauda AB299221 AB299222 IBursaphelenchus “corneolus” HQ407406 JQ765870 IBursaphelenchus eggersi AY508013 AY508078 IBursaphelenchus eremus – AM396568 IBursaphelenchus gerberae AY508024 AY508092 IBursaphelenchus hellenicus AY508017 AY508083 IBursaphelenchus hildegardae AM397013 AM396569 IBursaphelenchus hofmanni AY508018 AY508084 IBursaphelenchus hylobianum AY508019 AY508085 IBursaphelenchus mazandaranense JN153102 JN153103 IBursaphelenchus niphades AB849469 AB849479 IBursaphelenchus osumiana AB918706 AB918707 IBursaphelenchus paracorneolus AY508027 AY508095 IBursaphelenchus paraparvispicularis GQ421483 GQ429010 IBursaphelenchus parvispicularis AB218829 AB368537 IBursaphelenchus pinasteri AM397016 AM396574 IBursaphelenchus poligraphi AY508028 AY508096 IBursaphelenchus rainulfi AM397017 EU295498 IBursaphelenchus rufipennis AB368529 AB368530 IBursaphelenchus sakishimanus LC027461 LC027462 IBursaphelenchus sexdentati AY508031 AY508101 IBursaphelenchus sinensis AB232162 EU752257 IBursaphelenchus tusciae AY508033 AY508104 IBursaphelenchus vallesianus AM397020 AM396578 IBursaphelenchus yongensis AM397023 AM396581 IBursaphelenchus sp. AF037369 – IBursaphelenchus sp. JH2004 AY284650 – IBursaphelenchus sp. JH2013 KF164829 KF164833 IBursaphelenchus arthuri AM397010 AM396564 IIBursaphelenchus arthuroides HQ599188 HQ599190 IIBursaphelenchus braaschae GQ845409 GQ845408 IIBursaphelenchus cocophilus AY509153 AY508076 IIBursaphelenchus fungivorus AY508016 AY508082 IIBursaphelenchus kiyoharai AB597253 AB597254 IIBursaphelenchus parathailandae JN377724 JN377722 IIBursaphelenchus penai AB901293 AB901292 IIBursaphelenchus platzeri AY508026 AY508094 IIBursaphelenchus seani AY508029 AY508097 IIBursaphelenchus sycophilus AB901291 – II

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Table 1. (Continued.)

Genus Species SSU D2/D3 Intrageneric clade

Bursaphelenchus tadamiensis AB635398 AB635398 IIBursaphelenchus thailandae AM397019 AM396577 IIBursaphelenchus willibaldi AM397021 AM396579 IIBursaphelenchus sp. JH2013B KF314804 KF314807 IIBursaphelenchus africanus JF317266 HM623784 IIIBursaphelenchus anatolius AY508025 AY508093 IIIBursaphelenchus kevini AY753531 AY753532 IIIBursaphelenchus debrae – EF488813 IIIBursaphelenchus conicaudatus AB067757 AB299227 IIIBursaphelenchus doui NK217 AB299224 AB299226 IIIBursaphelenchus fagi – JX683686 IIIBursaphelenchus firmae AB650015 AB650014 IIIBursaphelenchus fraudulentus AY508015 AY508079 IIIBursaphelenchus koreanus JX154585 – IIIBursaphelenchus luxuriosae AB097864 AB299228 IIIBursaphelenchus macromucronatus – EU256382 IIIBursaphelenchus masseyi – JQ287495 IIIBursaphelenchus mucronatus AY508023 AY508086 IIIBursaphelenchus obeche – EU159108 IIIBursaphelenchus okinawaensis AB358983 AB358982 IIIBursaphelenchus paraburgeri HQ727727 HQ727726 IIIBursaphelenchus paraluxuriosae JF966206 JF966204 IIIBursaphelenchus populi HQ699855 FJ998281 IIIBursaphelenchus singaporensis AM397018 AM396576 IIIBursaphelenchus tokyoensis AB430445 AB430446 IIIBursaphelenchus trypophloei – FJ998283 IIIBursaphelenchus xylophilus AM397022 AY508105 IIIBursaphelenchus sp. JG2011D JN377728 JN377726 IIIBursaphelenchus sp. JW2012 JX154584 – IIIBursaphelenchus sp. P3851 JF317267 JF317269 IIIBursaphelenchus sp. PL72 – KF772174 IIIBursaphelenchus sp. SAM2 – FJ661083 IIIBursaphelenchus sp. SAM7 – FJ661084 IIIBursaphelenchus sp. TM2013 – KF736975 IIIBursaphelenchus sp. ZLR KJ653442 KJ653443 IIISheraphelenchus sucus AB808720 AB808721 III (Sheraphelenchus)

∗ Subclade numbers for Parasitaphelenchinae were given following Kanzaki et al. (2011).

Results

Ruehmaphelenchus juliae* n. sp.(Figs 1-5)

MEASUREMENTS

See Table 2.

* Named in honour of Dr Julia I. Giblin, daughter of the secondauthor.

DESCRIPTION

Adults

Mid- to large-sized species: 737-1034 μm and 866-1291 μm in length for males and females, respectively.Body cylindrical, ventrally arcuate when killed by gentleheat. Male tail strongly recurved ventrally. Cuticle thin,finely annulated with a lateral field possessing three in-cisures. Lip region distinctly offset from body, separatedfrom body by a weak constriction and with six equal-

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Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Fig. 1. Ruehmaphelenchus juliae n. sp. A: Female; B: Male.

sized lip sectors forming hexagonal shape in en face view,squarish-rounded, and ca 3-4 times as broad as tall in lat-eral view. An oral (labial) disc observed at anterior endof lip region and ca one-third of lip in diam. Stylet sep-arated into two parts: a short conus occupying ca one-third to two-fifths of total stylet length, and a shaft witha clear basal swelling, but not forming clear basal knobs.Procorpus cylindrical, ca 2.5-3.0 stylet lengths long, con-nected to a well developed muscular metacorpus (medianbulb). Dorsal pharyngeal gland orifice opening into lu-men of metacorpus midway between anterior end of meta-corpal valve and anterior end of metacorpus. Pharyngo-intestinal junction immediately posterior to metacorpus.Dorsal pharyngeal gland ca 2.5-3.0 metacorpal lengthslong, overlapping intestine dorsally. Nerve ring surround-ing pharyngeal glands and intestine, situated immediatelyposterior to pharyngo-intestinal junction. Excretory porelocated ca 1.5-2.0 stylet lengths posterior to metacorpus.Hemizonid located ca 1.5-2.0 stylet lengths posterior toexcretory pore.

Male

Gonad outstretched or with reflexed anterior part insome individuals. Vas deferens composed of large flat-tened cells, separated from other parts of gonad. Posteriorend of testis and intestine fusing to form a narrow tube atlevel of spicule. Sperm amoeboid; spermatocytes arrangedin multiple (2-5) rows in anterior two-thirds of testis andwell developed sperm tightly packed in remainder. Tailregion clearly arcuate ventrally. Spicules paired, separate,possessing clear dorsal and ventral limbs that are almostparallel to each other at middle of spicule. Dorsal limbca 1.8 times as long as ventral limb, both weakly ven-trally arcuate and smoothly narrowing to bluntly pointeddistal end. Anterior end of dorsal limb forming roundedcondylus. Rostrum absent, i.e., anterior end of ventrallimb rounded, not forming a clear rostrum. Dorsal andventral limbs connected by three different tissues: ablade-like cuticle; a thin, triangular, membrane-like struc-ture; and limb-like cuticularised tissue. First tissue type(= membrane-like lamina) connecting whole parts of dor-sal and ventral limbs with its distal end forming a roundedtip to spicule, second type connected along entire lengthof ventral limb and anterior part of dorsal limb, narrow-ing anteriorly to form an elongated triangle, third partconnected to distal end of ventral limb and at a pointtwo-thirds from anterior end of dorsal limb. Small notch-like separation (cleavage) present at distal tip. Cucullusand gubernaculum absent. Tail short, conical in lateralview, broad in ventral view, possessing pointed tip. Seven

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Fig. 2. Ruehmaphelenchus juliae n. sp. A: Anterior region; B: Lip region and stylet; C: En face view showing oral disc; D: Body surfacestructure; E: Right lateral view of female gonad; F: Right lateral view of vulval region; G: Ventral view of female anus and rectum; H:Right lateral view of female tail; I: Left lateral view of male tail; J: Ventral view of male tail; K: Left lateral view of spicule; L: Ventralview of spicule; M: Early-stage egg; N: Late-stage egg.

papilla-like (i.e., not glandular) genital papillae (a singleventral papilla and three subventral pairs) present: singleventral papilla (P1) ca one cloacal body diam. (CBD) an-terior to cloacal opening (CO); first subventral pair (P2)slightly anterior to CO, which is dome-shaped slit in ven-tral view; second subventral pair (P3) one CBD posteriorto CO; third subventral pair (P4) one-third CBD posteriorto P3, slightly more ventrally located compared with P3.P1 slightly smaller than P2-P4; P2-P4 more or less samein size.

Female

Reproductive tract including an ovary, oviduct, sper-matheca, crustaformeria, uterus, vagina + vulva, and post-uterine sac. Ovary single, anteriorly outstretched, anteriorend reflexed in some individuals. Oocytes present in mul-tiple (2-5) rows in anterior half of ovary and well de-veloped oocytes arranged in a single row in remainder.Oviduct tube-like, horizontal wrinkle-like pattern some-times appearing on its surface, constructed of oval-shapedcells, connecting ovary and crustaformeria, sometimes oc-

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Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Fig. 3. Ruehmaphelenchus juliae n. sp. A: Anterior region (ep =excretory pore; h = hemizonid); B-D: Lip and stylet in differentfocal planes (od = oral disc). This figure is published in colourin the online edition of this journal, which can be accessedvia http://booksandjournals.brillonline.com/content/journals/15685411.

cupied by well developed oocyte. Spermatheca (receptac-ulum seminis) present between oviduct and crustaforme-ria, slightly irregular oval shape, sometimes filled withwell developed sperm. Crustaformeria not conspicuous,formed by relatively large, rounded cells. Uterus shortwith thick wall, sometimes with an early-stage egg whichhas many verrucae-like small expansions on its surface.Vagina perpendicular to body surface or slightly inclined

anteriorly and uterine wall nearest to vagina (uterus/post-uterine sac junction) thickened. Vulva a simple slit in ven-tral view, lacking any vulval flap apparatus in lateral andventral views. Post-uterine sac well developed, ca 2.5 vul-val body diam. long, occupying ca half of vulva-anus dis-tance, sometimes filled with sperm. Rectum present, caone anal body diam. (ABD) long; intestine-rectum junc-tion constricted by sphincter muscle. Anus a small dome-shaped slit in ventral view. Tail ca three ABD long, coni-cal, smoothly tapering to a pointed or unclearly mucronatetip, i.e., conical or digitate mucro sometimes present.

TYPE HOST, CARRIER INSECT AND LOCALITY

Ruehmaphelenchus juliae n. sp. (culture code CBAY2)was isolated from the bodies of Xylosandrus crassiusculusthat emerged from caged dead branches from a red baytree from a levy south of Tamiami trail, FL, USA (GPScode: 25°44′10.9′′N, 80°29′51.3′′W) on 13 March 2014.

TYPE MATERIAL

Type materials were obtained from 2-week-old cul-tures. Holotype male, nine paratype males and ten pa-ratype females deposited in the United States Depart-ment of Agriculture Nematode Collection (USDANC),Beltsville, Maryland, USA, with collection ID numbers T-687t (holotype male), T-6519p-T6527p (paratype males)and T-6528p-T6537p (paratype females); five paratypemales and five paratype females deposited in the For-est Pathology Laboratory collection, Forestry and For-est Products Research Institute (FFPRI), Tsukuba, Japan,with collection ID numbers Ruehmaphelenchus juliaePM01-05 (paratype males) and PF01-05 (paratype fe-males). In addition to the type materials, mass-fixed ma-terials fixed in TAF and those processed into dehydratedglycerin were deposited at Fort Lauderdale Research andEducation Center, University of Florida/IFAS, Fort Laud-erdale, FL, USA (FLREC) and FFPRI.

DIAGNOSIS AND RELATIONSHIPS

Ruehmaphelenchus juliae n. sp. is characterised by thepresence of an oral disc, lateral field with three lines,spicule morphology (clear dorsal and ventral limbs con-nected by the three different structures described above),male tail conical with pointed tip and female tail conicalwith digitate mucro. In addition to the adult morpholo-gical characters, presence of verrucae-like expansions onthe surface of early-stage eggs has not been reported inany of the other known species in the genus.

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Fig. 4. Ruehmaphelenchus juliae n. sp. A-C: Right lateral view of male tail in different focal planes; D-F: Left lateral view of spicule(triangular piece is indicated with an arrowhead in E; and bridge connecting dorsal and ventral limb and notch-like separation at distalend of spicule are indicated with arrowheads in F); G-K: Ventral view of male tail in different focal planes. Genital papillae and cloacalopening are labelled P1, P2, P3, P4 and CO. This figure is published in colour in the online edition of this journal, which can be accessedvia http://booksandjournals.brillonline.com/content/journals/15685411.

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Fig. 5. Ruehmaphelenchus juliae n. sp. A-C: Right lateral view of female vulval region in different focal planes; D-F: Ventral viewof female vulval region in different focal planes; G, H: Left lateral view of female tail in different focal planes; I-L: Early-stage eggin different focal planes; M, N: Late-stage egg in different focal planes. This figure is published in colour in the online edition of thisjournal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

Currently, Ruehmaphelenchus contains seven species:R. martini (Rühm, 1955) Goodey, 1963; R. ipidicola(Rühm, 1956) Kanzaki, Li, Lan & Giblin-Davis, 2014;R. asiaticus Braasch, Gu, Burgermeister, Brandstetter& Metge, 2006; R. thailandae Gu & Wang, 2010; R.digitulus Gu & Wang, 2012; R. sirisus Bajaj, 2012; and R.formosanus Kanzaki, Masuya, Taki, Okabe & Chen, 2013.In addition to these seven species, an undescribed species,Ruehmaphelenchus sp. NK202, which is molecularlyseparable from several nominal species, has been reported(Kanzaki et al., 2013).

Ruehmaphelenchus juliae n. sp. is morphologicallysimilar to R. asiaticus, R. digitulus and R. thailandae, eachsharing three lines in the lateral field, the conspicuousventral and dorsal limbs of the male spicule, short andconical female tail with pointed or unclearly mucronatedtip, and the absence of a flap apparatus at the vulva.The new species may also be typologically similar to R.sirisus, i.e., although the number of lateral lines is notdescribed, the other characters overlap between these twospecies.

Based upon the original descriptions, R. juliae n. sp.is distinguished from R. asiaticus by spicule morphology,i.e., new species has triangular membrane-like structureson both sides (shown in Figs 2L; 4E), which has not beendescribed for R. asiaticus, and male tail tip morphology,i.e., distal cone-shaped part of new species is clearlylonger than that of R. asiaticus (Braasch et al., 2006);from R. thailandae by its spicule morphology presencevs absence of the triangular membrane-like structures,female tail morphology, absence vs presence of longmucron, and the arrangement of the male genital papillae(ratio between P2-P3/P3-P4 is ca 3-6 vs >10; calculatedfrom drawings and photographs or morphometric tablesprovided in Gu & Wang, 2010); from R. digitulus by itsspicule morphology presence vs absence of the triangularmembrane-like structures, and male tail tip morphology,i.e., distal cone-shaped part of new species is clearlylonger than that of R. digitulus (Gu & Wang, 2012); andfrom R. sirisus by its spicule morphology presence vsabsence of the triangular membrane-like structures (Bajaj,2012).

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Table 2. Morphometrics of Ruehmaphelenchus juliae n. sp. All measurements are in μm and in the form: mean ± s.d. (range).

Character Permanent slides Temporary water mounts

Male Female Male Female

Holotype Paratypes Paratypes

n – 14 15 15 15L 892 859 ± 93 1005 ± 92 944 ± 53 1129 ± 77

(737-1036) (866-1201) (886-1034) (1000-1291)a 33.9 33.0 ± 2.0 29.2 ± 1.7 25.7 ± 1.9 23.0 ± 2.0

(28.8-35.7) (26.1-32.1) (23.3-30.5) (19.4-27.6)b 12.5 11.9 ± 1.1 13.7 ± 1.2 12.9 ± 0.5 14.9 ± 1.1

(10.0-13.9) (11.9-15.9) (12.2-13.8) (12.7-16.5)c 36.8 35.5 ± 2.8 23.4 ± 3.1 34.4 ± 3.2 25.5 ± 2.1

(31.3-41.1) (20.2-33.0) (29.8-40.2) (20.1-28.5)c′ 1.7 1.8 ± 0.1 3.3 ± 0.3 1.8 ± 0.2 2.4 ± 0.2

(1.6-2.1) (2.7-3.9) (1.4-2.0) (2.0-2.7)T or V 45.1 46.3 ± 4.9 82.5 ± 0.8 82.4 ± 4.2 82.4 ± 0.8

(36.8-55.1) (81.6-84.6) (75.9-89.5) (81.5-84.4)M 37.9 38.6 ± 1.9 37.8 ± 2.0 37.6 ± 1.6 38.3 ± 2.0

(35.5-41.4) (34.4-41.2) (34.3-40.0) (34.5-41.4)Lip diam. 8.8 8.7 ± 0.4 10.2 ± 0.6 9.7 ± 0.3 11.3 ± 0.4

(7.7-9.3) (8.8-10.8) (9.3-10.5) (10.9-12.0)Lip height 4.1 4.1 ± 0.3 4.4 ± 0.3 4.1 ± 0.3 4.2 ± 0.4

(3.6-4.6) (3.6-4.6) (3.8-4.6) (3.4-5.1)Lip diam.:height ratio 2.1 2.2 ± 0.1 2.4 ± 0.2 2.4 ± 0.2 2.7 ± 0.2

(2.0-2.4) (2.1-2.6) (2.0-2.8) (2.2-3.2)Stylet conus 5.7 5.9 ± 0.3 6.3 ± 0.5 5.7 ± 0.4 6.3 ± 0.5

(5.7-6.2) (5.7-7.2) (5.1-6.3) (5.7-6.9)Stylet length 14.9 15.3 ± 0.6 16.7 ± 0.8 15.2 ± 0.7 16.4 ± 0.6

(13.9-16.0) (15.4-18.6) (14.7-17.3) (15.4-17.1)Max. body diam. 26 26 ± 3.1 35 ± 4.3 37 ± 2.3 49 ± 4.3

(21-33) (28-42) (31-41) (40-57)Vulval body diam. – – 26 ± 2.3 – 34 ± 2.2

(24-30) (31-40)Median bulb diam. 17.0 16.2 ± 1.0 19.2 ± 1.2 19.4 ± 1.1 21.7 ± 1.4

(13.9-18.0) (17.0-21.1) (17.1-21.4) (18.6-24.3)Median bulb length 22.7 21.8 ± 1.3 24.2 ± 1.7 21.3 ± 1.0 23.5 ± 1.9

(19.6-24.7) (22.2-28.4) (20.0-22.9) (18.6-25.7)Median bulb length: diam. ratio 1.3 1.3 ± 0.1 1.3 ± 0.1 1.1 ± 0.1 1.1 ± 0.1

(1.2-1.4) (1.2-1.4) (1.0-1.2) (1.0-1.2)Nerve ring from anterior end 76 78 ± 3.3 81 ± 2.4 79 ± 2.3 84 ± 2.6

(72-85) (76-84) (74-83) (80-89)Nerve ring from median bulb1) 5.1 6.2 ± 1.5 7.2 ± 1.4 5.4 ± 1.3 7.8 ± 2.2

(4.2-9.3) (4.2-9.3) (2.9-7.1) (5.7-14.3)Excretory pore from anterior end 97 96 ± 6.4 100 ± 2.4 102 ± 11.1 106 ± 10.1

(80-103) (85-113) (86-123) (86-119)Excretory pore from median bulb1) 26 24 ± 6.3 26 ± 7.9 28 ± 11.2 30 ± 9.3

(8.5-32) (12-41) (11-51) (11-43)Hemizonid from anterior end 121 117 ± 8.0 122 ± 5.8 130 ± 6.8 140 ± 15.7

(104-132) (114-136) (116-143) (116-176)Hemizonid from median bulb1) 50 45 ± 7.0 49 ± 6.3 57 ± 6.0 64 ± 14.8

(34-56) (42-67) (46-69) (41-99)

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Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Table 2. (Continued.)

Character Permanent slides Temporary water mounts

Male Female Male Female

Holotype Paratypes Paratypes

Gonad length2) 402 394 ± 49 433 ± 76 777 ± 58 757 ± 86(325-482) (348-608) (684-885) (577-869)

Cloacal/anal body diam. 13.9 13.4 ± 1.2 13.3 ± 0.7 15.8 ± 0.7 18.9 ± 1.6(11.9-15.5) (11.9-14.4) (14.7-17.3) (16.0-21.7)

Tail length 24 24 ± 2.2 43 ± 2.5 28 ± 2.4 44 ± 2.8(21-28) (36-47) (22-31) (41-50)

Spicule length (line)3) 21.1 20.7 ± 1.3 – 21.7 ± 1.0 –(19.1-23.7) (19.8-23.2)

Spicule length (curve)4) 17.0 16.4 ± 1.1 – 17.5 ± 1.4 –(14.4-18.6) (16.0-21.1)

Post-uterine sac length – – 76 ± 9.4 – 83 ± 7.8(63-102) (70-96)

Vulva-anus distance – – 132 ± 13 – 154 ± 16.1(113-153) (131-187)

Post-uterine sac/vulva-anus distance (%) – – 57.8 ± 3.6 – 54.3 ± 5.5(53.6-66.3) (43.4-64.8)

1) Distance from posterior end of median bulb.2) Length from cloacal or vulval opening to anterior end of gonad.3) Condylus tip to distal end measured in a straight line.4) Length curved along arc from capitulum depression to distal end.

Morphometric data were also used to distinguish R.juliae n. sp. from R. asiaticus, R. thailandae, R. digitulusand R. sirisus. The new species is distinguished from R.asiaticus by female b = 11.9-16.5 vs 9.0-11.9 (Braasch etal., 2006); from R. thailandae by female body length =866-1291 vs 504-620 μm, female b = 11.9-16.5 vs 8.4-9.8, V = 81.5-84.6 vs 78.9-80.5, female body diam. =28-57 vs 19.2-22.6 μm, male and female stylet length =15.4-18.6 vs 12.4-14.4 μm, male and female median bulblength = 18.6-28.4 vs 15.0-17.0 μm, male and femalemedian bulb diam. = 13.9-18.0 vs 6.9-8.2 μm, male andfemale excretory pore position = 80-123 vs 60-75 μmfrom anterior end, male spicule length = 19.1-23.7 μm(chord) and 14.4-21.1 μm (arc) vs 12.0-13.3 μm (chord)and 9.9-10.3 μm (arc), female post-uterine sac length =63-102 vs 51.7-56.6 μm, female anal body diam. = 11.9-21.7 vs 8.8-9.6 μm, and female tail length = 36-50 vs<33 μm (Gu & Wang, 2010); from R. digitulus by malec = 29.8-41.1 vs 39.4-54.4 and male c′ = 1.4-2.1 vs1.1-1.5, although they are slightly overlapping (Gu &Wang, 2012); and from R. sirisus by male c′ = 1.4-2.1 vs1.1-1.3, stylet length of males and females = 13.9-18.6vs 11-13 μm, female body length = 866-1291 vs 460-

632 μm, female b = 11.9-16.5 vs 8.2-10.3, median bulb infemales = 17.0-24.3 vs 9-15 μm in diam., and 18.6-28.4vs 13-20 μm in length, female excretory pore position =85-119 vs 60-71 μm from anterior end, and post-uterinesac length = 63-102 vs 43-55 μm (Bajaj, 2012).

MOLECULAR PROFILES AND PHYLOGENETIC STATUS

OF NEW SPECIES

Based on molecular phylogenetic analyses inferredfrom near-full-length SSU and D2-D3 LSU sequences,the new species formed a well supported clade with theother Ruehmaphelenchus species, including Ruehmaphe-lenchus sp. NK202, which was isolated from Xyleborusganshoensis Murayama from Okinawa, Japan (Kanzaki etal., 2013), and was the basal species of the clade (Fig. 6).The near-full-length SSU and/or D2-D3 LSU sequencesare available for R. asiaticus, R. digitulus, R. formosanus,Ruehmaphelenchus sp. NK202 and R. juliae n. sp. withall five species being clearly distinguishable with unusu-ally large levels of interspecific sequence divergence. Fur-ther, the ITS (ITS1, ITS2 and 5.8S ribosomal RNA) se-quence of R. thailandae is available in GenBank (acces-

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N. Kanzaki et al.

sion number GQ911597) and is clearly different fromthe ITS regions of R. juliae n. sp. (supporting data notshown).

Discussion

BIONOMICS OF Ruehmaphelenchus juliae N. SP.

The carrier insect of R. juliae n. sp. is a small (femalesare ca 2 mm long and males ca 1.5 mm) beetle, X. cras-siusculus, which was probably originally native to tropicaland subtropical Asia long before being widely introducedaround the world and then into South Carolina, USA, inthe 1970s (Atkinson et al., 2012). It is considered a seri-ous pest of a wide range of agricultural and horticulturaltrees (Frank & Sadof, 2011). Its ambrosial fungus is con-sidered to be a species of Ambrosiella, which are gener-ally not considered to be pathogenic (Dute et al., 2002).However, there is the possibility of transmission of wilt-inducing fungi by this and other ambrosia beetles (Davis& Dute, 1997; Carrillo et al., 2014).

Currently, some of the nominal Ruehmaphelenchusspp. have been described from packaging wood or thebark of dead trees (Braasch et al., 2006; Gu & Wang,2010, 2012; Bajaj, 2012), and their natural bionomicsare unknown. However, the other species are known tobe associated with ambrosia or bark beetles: R. martiniwas found under the elytra of Anisandrus dispar Ferr. asdauer juveniles (Rühm, 1955); R. ipidicola is associatedwith Dryocoetes spp. which are associated with relativelyold dead trees (Rühm, 1956); R. formosanus is associ-ated with Euwallacea fornicatus (Eichhoff); Ruehmaphe-lenchus sp. NK202 was isolated from X. ganshoen-sis (Kanzaki et al., 2013); and another undescribedRuehmaphelenchus species is associated with Platypusquercivorus (Murayama) (Kanzaki et al., 2013). Con-sidering the insect association of these latter-mentionedRuehmaphelenchus species, the genus appears to bemostly associated and radiating with certain lineages ofambrosia beetles and bark beetles.

ADDITIONAL REMARKS ON MORPHOLOGICAL

CHARACTERS

In the present study, several characteristic morpholo-gical traits were found in the new species. First, a clearoral disc was confirmed at the anterior end of the lipregion. A similar character has been described, drawn

and/or photographed for R. asiaticus, R. thailandae, R.digitulus and R. sirisus (Braasch et al., 2006; Gu & Wang,2010, 2012; Bajaj, 2012), and was confirmed in R. for-mosanus (Kanzaki, unpubl.). Considering the phyloge-netic relationships among Ruehmaphelenchus spp., thecharacter has been confirmed from the most basal speciesthrough to the most derived species. Thus, although thestructure has not been described for R. martini and R.ipidicola (Rühm, 1955, 1956), this could likely be a com-mon morphological character in the genus. Second, char-acteristic verrucae-like formations on the egg shell, whichhave not been described for the other Ruehmaphelenchusspecies (Rühm, 1955, 1956; Braasch et al., 2006; Gu &Wang, 2010, 2012; Bajaj, 2012; Kanzaki et al., 2013),were observed in R. juliae n. sp. Comparing the develop-mental stages of the eggs, the structure seems to be foundonly on the early- to middle-stage eggs, developed eggs,which contain the juvenile, sometimes lacking these ex-pansions. Egg surface structure is not usually used for tax-onomic characterisation of nematodes, and the characteris not usually examined, especially in descriptions basedon materials collected from the field. Therefore, we do notknow whether this character is specific to R. juliae n. sp.or is a common character of the clade. More cultured ma-terials and detailed developmental studies will reveal therelevance of this character for taxonomic studies.

By contrast, spike-like projections on the lateral regionof the spicule, which were found in R. formosanus andRuehmaphelenchus sp. NK202 and hypothesised to benerve endings (Kanzaki et al., 2013), were not found inR. juliae n. sp. Comparing the phylogenetic relationshipsamong these species, i.e., R. juliae n. sp. is basal to thegenus, and the other two are more derived, the charactermay have developed only in a certain group (lineage) ofthe genus.

Based on the morphological characters including mor-phometric values, R. juliae n. sp., R. asiaticus and R.digitulus are very similar to each other, i.e., molecularprofiles are almost the only characters that distinguishthese species (Braasch et al., 2006; Gu & Wang, 2012).However, despite their morphological similarity, they areclearly phylogenetically different with significantly longbranches (branches were so long that they would not fitFig. 6 without shortening). It could be interesting to com-pare Bursaphelenchus spp., R. juliae n. sp. and more de-rived Ruehmaphelenchus at the genome or transcriptomelevel.

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Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Fig. 6. Molecular phylogenetic relationships among Ruehmaphelenchus, Sheraphelenchus and Bursaphelenchus species. Bayesian treeinferred from near-full-length of SSU and D2-D3 LSU under GTR + I + G model. lnL = 11 878.3496; freqA = 0.2674; freqC =0.1694; freqG = 0.2535; freqT = 0.3097; R(a) = 1.4002; R(b) = 2.8221; R(c) = 1.3437; R(d) = 0.7125; R(e) = 5.5059; R(f) = 1;Pinvar = 0.4416; Shape = 0.4562 for near-full-SSU; lnL = 17 794.2188; freqA = 0.1815; freqC = 0.17; freqG = 0.3234; freqT =0.3251; R(a) = 0.558; R(b) = 2.8917; R(c) = 1.0505; R(d) = 0.6494; R(e) = 4.8359; R(f) = 1; Pinvar = 0.2929; Shape = 0.8046 forD2-D3 LSU. Posterior probability values exceeding 50% are given on appropriate clades.

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AMBROSIA BEETLES AND THE ASSOCIATION WITH

NEMATODES

Rühm (1955, 1956) reported that dauer juveniles of R.martini and R. ipidicola are found from under the elytraand body segments of their carrier beetles. However,the insect-associated stage of the nematodes and thenematode-harbouring organ of the other interactions havenot been clarified so far as the nematodes were isolatedfrom the dissected insect body kept on media plates (e.g.,Kanzaki et al., 2013). In the present study, similar to thecase of R. formosanus, the dauer juvenile of R. juliae n.sp. was not recognised during the dissection, probablybecause the number of associated nematodes was low.Additional survey work and careful beetle dissections arenecessary to elucidate the insect association of the newspecies.

Xylosandrus crassiusculus is a member of the Xylo-borini, a tribe that uses haplodiploidy and inbreeding asthe sex determination and mating system (Kirkendall,1983). Haploid males live in the galleries and mate withtheir siblings. Pre-dispersal mating within the family oforigin and monogynous initiation of galleries allow sin-gle females to establish successful populations in non-native locations and leads to founder effects and geneticbottlenecking. Members of the Xyloborini practise fun-gal agriculture, but unlike analogous attine ants and cer-tain fungal-feeding termites that provision their nests withwood and maintain fungal gardens, they simply build theirgalleries into the cellulose substrate (Biedermann, 2007).Larval and adult beetle grazing in the galleries may en-courage longer maintenance of the ‘crop’ fungi withoutdetrimental successional changes to other non-nutritive ortoxic fungi (Biedermann, 2007). These life history detailshave led to an apparently rapid speciation rate amongstthe Xyloborini and could have important ramifications fornematodes associates of such beetles. For example, dothe long branches between Ruehmaphelenchus species se-quenced thus far represent rapid speciation events? If so,then what is different between ambrosia beetle-associatedRuehmaphelenchus and the ‘Bursaphelenchus kiyoharai’clade of ambrosia-associated mycophagous nematodes?Do these ambrosia beetle-associated nematodes conferany population benefits or costs to the beetle hosts? Is itpossible that nematode feeding and defecation could helpwith recycling and maintenance of ‘crop’ fungi under cer-tain conditions?

Acknowledgement

We thank Ms Mayrine Silva for assistance with dis-sections, culturing and sequencing attempts during thisproject.

References

Atkinson, T.H., Foltz, J.L., Wilkinson, R.C. & Mizell,R.F. (2012). Xylosandrus crassiusculus (Motschulsky) (In-secta: Coleoptera: Curculionidae: Scolytinae). Featured crea-tures. Fact sheet. University of Florida, available onlineat: http://entnemdept.ufl.edu/creatures/trees/asian_ambrosia_beetle.htm.

Bajaj, H.K. (2012). Ruehmaphelenchus sirisus sp. n. and Al-biziaphelenchus arthrorostrus gen. n., sp. n. (Aphelenchoi-didae: Aphelenchida) from bark of Albizia lebbeck (L.) Benthinfected with bark borers. Indian Journal of Nematology 42,118-124.

Biedermann, P.H.W. (2007). Social behaviour in sib matingfungus farmers: intra- and interspecific cooperation in am-brosia beetles. Masterarbeit der Philosophisch-naturwissen-schaftlichen Fakultät der Universität Bern, Bern, Switzer-land.

Braasch, H., Gu, J., Burgermeister, W., Brandstetter, M. &Metge, K. (2006). Description of Ruehmaphelenchus asiati-cus sp. n. (Nematoda: Aphelenchoididae) and remarks on thegenus Ruehmaphelenchus. Journal of Nematode Morphologyand Systematics 9, 39-43.

Carrillo, D., Duncan, R.E. & Peña, J.E. (2012). Ambrosia beetles(Curculionidae: Scolytinae) that breed in avocado wood inFlorida. Florida Entomologist 95, 573-579.

Carrillo, D., Duncan, R.E., Ploetz, J.N., Campbell, A.F., Ploetz,R.C. & Peña, J.E. (2014). Lateral transfer of a phy-topathogenic symbiont among native and exotic ambrosiabeetles. Plant Pathology 63, 54-62.

Davis, M.A. & Dute, R.R. (1997). Fungal associates of theAsian ambrosia beetle, Xylosandrus crassiusculus. SouthernNursery Association Research Conference 42, 106-112.

Dute, R.R., Miller, M.E., Davis, M.A., Woods, F.M. & McLean,K.S. (2002). Effects of ambrosia beetle attack on Cerciscanadensis. IAWA Journal 23, 143-160.

Frank, S.D. & Sadof, C.S. (2011). Reducing insecticide volumeand nontarget effects of ambrosia beetle management innurseries. Journal of Economic Entomology 104, 1960-1968.

Fuchs, A.G. (1930). Neue an Borken- und Rüsselkäfer gebun-dene Nematoden, halbparasitische und Wohnungseinmieter.Zoologische Jahrbücher, Abteilung für Systematik, Ökologieund Geographie der Tiere 59, 505-646.

Fuchs, A.G. (1937). Neue arasitische und halbparasitische Ne-matoden bei Borkenkäfern und einige andere Nematoden.1 Teil. Zoologische Jahrbücher, Abteilung für Systematik,Öekologie und Geographie der Tiere 70, 291-380.

652 Nematology

Ruehmaphelenchus juliae n. sp. from an ambrosia beetle

Giblin-Davis, R.M., Mundo-Ocampo, M., Baldwin, J.G., Nor-den, B.B. & Batra, S.W.T. (1993). Description of Bursaphe-lenchus abruptus n. sp. (Nemata: Aphelenchoididae), an as-sociate of a digger bee. Journal of Nematology 25, 161-172.

Giblin-Davis, R.M., Kanzaki, N. & Davies, K.A. (2013). Nema-todes that ride insects: unforeseen consequences of arrivingspecies. Florida Entomologist 96, 770-780.

Goodey, J.B. (1963). Soil and freshwater nematodes, 2nd edi-tion. London, UK, Methuen.

Gu, J. & Wang, J. (2010). Description of Ruehmaphelenchusthailandae n. sp. (Nematoda: Aphelenchoididae) found indunnage from Thailand. Nematology 12, 869-876.

Gu, J. & Wang, J. (2012). Description of Ruehmaphelenchusdigitulus sp. n. (Nematoda: Aphelenchoididae) found inpackaging wood from Taiwan. Nematology 14, 489-498.

Hooper, D.J. (1986). Handling, fixing, staining and mountingnematodes. In: Southey, J.F. (Ed.). Laboratory methods forwork with plant and soil nematodes. London, UK, HerMajesty’s Stationery Office, pp. 59-80.

Huelsenbeck, J.P. & Ronquist, F. (2001). MR BAYES: Bayesianinference of phylogenetic trees. Bioinformatics 17, 1754-1755.

Hunt, D.J. (1993). Aphelenchida, Longidoridae and Trichodori-dae: their systematics and bionomics. Wallingford, UK, CABInternational.

Hunt, D.J. (2008). A checklist of the Aphelenchoidea (Nema-toda: Tylenchina). Journal of Nematode Morphology and Sys-tematics 10(2007), 99-135.

Kanzaki, N. & Futai, K. (2002). A PCR primer set for determina-tion of phylogenetic relationships of Bursaphelenchus specieswithin xylophilus group. Nematology 4, 35-41.

Kanzaki, N., Maehara, N., Aikawa, T., Masuya, H. & Giblin-Davis, R.M. (2011). Description of Bursaphelenchus kiy-oharai n. sp. (Tylenchina: Aphelenchoididae) with remarkson the taxonomic framework of the ParasitaphelenchinaeRühm, 1956 and Aphelenchoidinae Fuchs, 1937. Nematology13, 787-804.

Kanzaki, N., Taki, H., Masuya, H., Okabe, K. & Chen, C.-Y.(2013). Description of Ruehmaphelenchus formosanus n. sp.(Tylenchina: Aphelenchoididae) isolated from Euwallaceafornicates from Taiwan. Nematology 15, 895-906.

Kanzaki, N., Li, H.-F., Lan, Y.-C. & Giblin-Davis, R.M. (2014).Description of two Pseudaphelenchus species (Tylenchomor-pha: Aphelenchoididae) associated with Asian termites andproposal of new subfamily Tylaphelenchinae n. subfam. Ne-matology 16, 963-978.

Kanzaki, N., Okabe, K. & Kobori, Y. (2015). Bursaphelen-chus sakishimanus n. sp. (Tylenchomorpha: Aphelenchoi-didae) isolated from a stag beetle, Dorcus titanus sakishi-manus Nomura (Lucanidae), on Ishigaki Island in Japan. Ne-matology, in press (NEM 2887).

Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002). MAFFT:a novel method for rapid multiple sequence alignment basedon fast Fourier transform. Nucleic Acids Research 30, 3059-3066.

Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N. & Kanzaki, N.(2009). A rapid and precise diagnostic method for detectingthe pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification (LAMP). Phytopathology99, 1365-1369.

Kirkendall, L.R. (1983). The evolution of mating systemsin bark and ambrosia beetles (Coleoptera: Scolytidae andPlatypodidae). Zoological Journal of the Linnean Society 77,293-352.

Körner, H. (1954). Die Nematodenfauna des vergehendenHolzes und ihre Beziehungen zu den Insekten. ZoologischeJahrbücher (Systematik) 82, 245-353.

Larget, B. & Simon, D.L. (1999). Markov chain Monte Carloalgorithms for the Bayesian analysis of phylogenetic trees.Molecular Biology and Evolution 16, 750-759.

Nickle, W.R. (1970). A taxonomic review of the genera ofthe Aphelenchoidea (Fuchs, 1937) Thorne, 1949 (Nematoda:Tylenchida). Journal of Nematology 2, 375-392.

Posada, D. & Crandall, K.A. (1998). MODELTEST: testing themodel of DNA substitution. Bioinformatics 14, 817-818.

Rühm, W. (1955). Über einige an holzbrütende Ipiden gebun-dene Nematodenarten. Zoologischer Anzeiger 155, 70-83.

Rühm, W. (1956). Die Nematoden der Ipiden. ParasitologischeSchriftenReihe 6, 1-435.

Skarbilovich, T.S. (1947). [Revision of the systematics of thenematode family Anguillulinidae Baylis and Daubney, 1926.]Doklady Akademy Nauk SSR 57, 307-308.

Tanaka, R., Kikuchi, T., Aikawa, T. & Kanzaki, N. (2012).Simple and quick methods for nematode DNA preparation.Applied Entomology and Zoology 47, 291-294.

Ye, W., Giblin-Davis, R.M., Braasch, H., Morris, K. & Thomas,W.K. (2007). Phylogenetic relationships among Bursaphelen-chus species (Nematoda: Parasitaphelenchidae) inferred fromnuclear ribosomal and mitochondrial DNA sequence data.Molecular Phylogenetics and Evolution 43, 1185-1197.

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