Bursaphelenchus penai n. sp. (Tylenchomorpha ... · Aphelenchoididae), a phoretic associate of ambrosia beetles (Coleoptera: Scolytinae) from avocado in Florida Natsumi KANZAKI1,2,∗,

Nematology 16 (2014) 683-693 brill.com/nemy

Bursaphelenchus penai n. sp. (Tylenchomorpha:Aphelenchoididae), a phoretic associate of ambrosia beetles

(Coleoptera: Scolytinae) from avocado in Florida

Natsumi KANZAKI 1,2,∗, Robin M. GIBLIN-DAVIS 1, Daniel CARRILLO 3,Rita DUNCAN 3 and Rafael GONZALEZ 1

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

2 Forest Pathology Laboratory, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan3 Tropical Research and Education Center, University of Florida/IFAS, 18905 SW 280 Street, Homestead, FL 33301, USA

Received: 4 January 2014; revised: 19 March 2014Accepted for publication: 19 March 2014; available online: 7 May 2014

Summary – During an experimental host-plant survey for the invasive redbay ambrosia beetle, Xyleborus glabratus, in Homestead,Florida, it and three native species of ambrosia beetle, Xyleborus affinis, X. volvulus and Xyleborinus gracilis were found to be associatedwith a Bursaphelenchus species. This nematode species, isolated from ambrosia beetles from the tribe Xyleborini, was cultured onMonilinia fructicola or Botryotinia fuckeliana for further study and was determined to be new to science and a putative sister species toB. kiyoharai because of two apomorphic characters in males, viz., possession of a tail spike vs the typical bursal flap, and the apparentabsence of the P1 ventral single papilla, both typically plesiomorphic characters for the genus. Additionally, B. kiyoharai is associatedwith X. serriatus suggesting that the host and microbiome associations that are shared between these two species, both of whichare carried by ambrosia beetles, may have ecological and biological significance in their evolution and lineage radiation. Molecularphylogenetic analyses of the near-full-length small subunit (SSU: 18S) and the D2/D3 expansion segments of the large subunit (LSU:28S) confirmed that B. penai n. sp. is very closely related to B. kiyoharai which is a member of the B. fungivorus clade that includesB. thailandae and B. willibaldi. Bursaphelenchus penai n. sp. and B. kiyoharai both share very similar overall spicule morphology withthe B. fungivorus clade. The new species is described and can be typologically differentiated from B. kiyoharai by the position of thehemizonid and excretory pore and its different geographical and host associations.

Keywords – description, insect associate, molecular, morphology, morphometrics, phoresy, phylogeny, secondary character loss,taxonomy, Xyleborini.

The redbay ambrosia beetle, Xyleborus glabratus Eich-hoff (Coleoptera: Curculionidae: Scolytinae), togetherwith its associated and highly pathogenic fungal mutual-ist, Raffaelea lauricola T.C. Harr., Fraedrich & Agayeva,is an invasive symbiotic complex in the south-easternUSA where it has caused extensive damage to mature red-bay, Persea borbonia (L.), since soon after its initial de-tection in coastal Georgia in 2002 (Fraedrich et al., 2008;Harrington et al., 2008). It has spread to southern Floridaand threatens the avocado (Persea americana Mill.) in-dustry as well as a variety of native plants in the lau-rel family (Lauraceae). The complex is native to SouthEast Asia and is thought to have been introduced into the

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

USA via wood packing or shipping materials. This beetle-fungus symbiosis established in host plants that were al-ready inhabited by a complex of ambrosia beetles. Forinstance, 14 species of ambrosia beetles are found asso-ciated with avocado wood in different parts of Florida(Carrillo et al., 2012). Scolytid beetles have symbiotic as-sociations with a number of microorganisms, especiallywith symbiotic fungi, but also with mites and nematodes(Cardoza et al., 2008). Recent research revealed an un-precedented case of lateral transfer of R. lauricola amongnative and exotic ambrosia beetles inhabiting avocadosand other lauraceous trees (Carrillo et al., 2014). It istherefore possible that other symbiotic associations could

© Koninklijke Brill NV, Leiden, 2014 DOI:10.1163/15685411-00002797

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have resulted with the establishment of the X. glabratus-R. lauricola complex. The oak-hickory ambrosia beetle,Xyleborinus gracilis (Eichhoff), is one of a number of na-tive species of ambrosia beetles in southern Florida andthe Caribbean, including Xyleborus affinis Eichhoff andX. volvulus (Fabricius), which have a wide host range onstressed, dead or dying trees (Atkinson & Peck, 1994).Ambrosia beetles were collected and identified from in-fested trees in natural areas with known Xyleborus glabra-tus attacks during an experimental survey evaluating thepropensity of attack of X. glabratus and native or natu-ralised ambrosia beetles on young and healthy, or stressed,potted redbay or avocado (Simmonds) trees. Nematodeswere sometimes observed in the galleries or on adult bee-tles from galleries of these trees in Homestead, Florida. Inone case, we collected adult Xyleborinus gracilis from ayoung infested avocado tree and cultured a nematode thatwas identified as a new species nearest to the recently de-scribed B. kiyoharai from Japan (Kanzaki et al., 2011).Subsequently, we successfully cultured this same nema-tode species from adult Xyleborus affinis, X. glabratus andX. volvulus. It is described here as B. penai n. sp.

Materials and methods

NEMATODE ISOLATION AND MORPHOLOGICALOBSERVATION

Xyleborinus gracilis adults were collected from theirgalleries in an infested avocado tree where nematodescould be seen on 1 June 2013. They were collected anddissected alive onto 1.5% water agar media and incubatedat room temperature for 2 weeks before examining fornematodes. Nematodes that successfully multiplied weretransferred to a PDA agar that had been previously inoc-ulated with Botryotinia fuckeliana (de Bary) or Moniliniafructicola (Winter) Honey. Adult nematodes from 10-day-old cultures were heat-killed and fixed in TAF, processedwith a glycerin-ethanol series using modified Seinhorst’smethods (Minagawa & Mizukubo, 1994) and mountedin glycerin according to the methods of Maeseneer &d’Herde (described in Hooper, 1986). Morphological ob-servations with a microscope were conducted using liv-ing materials obtained from 1- to 2-week-old cultures andglycerin-mounted permanent slides.

Subsequently, three other ambrosia beetle species, Xyle-borus affinis, X. glabratus and X. volvulus, were collectedfrom dead avocado trees in Dade County, Florida, be-tween December 2013 and January 2014. The beetles

were dissected on 1.5% water agar media as describedabove.

MOLECULAR CHARACTERISATION AND PHYLOGENY

A DNA sample from B. penai n. sp. was collected andprepared as described by Kikuchi et al. (2009) and Tanakaet al. (2012). The DNA base sequences of partial riboso-mal DNA (ca 1.6 kb near-full-length small subunit (SSU)and 0.7 kb D2/D3 expansion segment of large subunit(D2/D3 LSU)) were determined for B. penai n. sp. fol-lowing Kanzaki & Futai (2002) and Ye et al. (2007). Thedetermined molecular sequences were used in a BlastNhomology search (available online at http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) to establish theclosely related species group, and the near-full-lengthSSU was compared with other Bursaphelenchus species.The species compared in these analyses (operational tax-onomic unit: OTU) were selected as Clade 2 of the genusaccording to the homology search results and the specieslist in Kanzaki & Tanaka (2013).

The molecular phylogenetic status of the new specieswas determined using a Maximum Likelihood analysis us-ing an online version of PhyML 3.0 (Guindon et al., 2010:available online at www.atgc-montpellier.fr/phyml/). Thecompared sequences were aligned using the MAFFT pro-gram (Katoh et al., 2002; available online at: http://align.bmr.kyushu-u.ac.jp/mafft/software/) and the base substi-tution model was determined as GTR + I + G usingMODELTEST version 3.7 (Posada & Crandall, 1998)under the AIC model selection criterion. The Akaike-supported model, log likelihood (ln L), Akaike infor-mation criterion values, proportion of invariable sites,gamma distribution shape parameters, and substitutionrates were used in the phylogenetic analyses. The pa-rameters for analysis were calculated as follows. Log-likelihood: −4317.86465; Gamma shape parameter:0.454; Proportion of invariant: 0.631; nucleotide fre-quencies: f(A) = 0.26180, f(C) = 0.18809, f(G) =0.26074, f(T) = 0.28937. GTR relative rate parameters:A/C = 1.01810, A/G = 2.72285, A/T = 1.76504, C/G =0.32127, C/T = 5.93148, G/T = 1.00000. The tree topolo-gies obtained from the analysis was evaluated with 1000bootstrap pseudoreplications.

The identities of additional materials, i.e., the culturednematodes isolated from Xyleborus affinis, X. glabratusand X. volvulus were confirmed by the molecular se-quence of partial SSU which was previously employed asa ‘barcode’ sequence by Powers et al. (2009).

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Results

Bursaphelenchus penai* n. sp.(Figs 1-7)

MEASUREMENTS

See Table 1.

DESCRIPTION

General

Small- to medium-sized species, i.e., 602-913 μm and757-1009 μm in length for males and females, respec-tively. Body cylindrical, ventrally arcuate, or straightwhen killed by heat treatment. Male tail weakly orstrongly recurved ventrally at times, causing it to over-lap with body. Cuticle thin, annulated, lateral field withfour incisures. Head distinctly offset from body, sepa-rated by a clear constriction, lip region in lateral viewsquarish-round, ca twice as broad as high. Stylet in twoparts, a short cone ca one-third of total stylet lengthand a shaft with clear basal swellings. Procorpus cylin-drical, ca 2.5-3.0 stylet lengths long, ending in a well-developed metacorpus. Dorsal pharyngeal gland orificeopening into lumen of metacorpus mid-way between an-terior end of metacorpal valve and anterior end of meta-corpus. Pharyngo-intestinal junction immediately poste-rior to metacorpus. Dorsal pharyngeal gland overlappingintestine dorsally, ca 4-6 metacorpal lengths long. Nervering surrounding pharyngeal glands and intestine at ca onestylet length posterior to pharyngo-intestinal junction. Ex-cretory pore ca 2-3 stylet lengths posterior to nerve ring.Hemizonid located near excretory pore, i.e., relative posi-tion to excretory pore varying between slightly anterior topore to ca one stylet length posterior to, indistinct in fixedmaterials (but a little clearer in live materials), observedonly in a few individuals.

Male

Gonad on ventral and/or right of intestine, outstretched,anterior end reflexed in some individuals, sperm amoe-boid. Tail region weakly to strongly arcuate. Spicules

* The species is named in honour of Professor Jorge Peñafor his many contributions to the study of natural enemiesand symbionts of pest insects in tropical fruit and vegetableagroecosystems before retiring from the University of Florida-IFAS, Tropical Research and Education Center.

paired, separate, wide in middle region with clear dor-sal and ventral limbs. Capitulum (condylus + rostrum)smoothly depressed in middle region. Condylus long, dis-tinctive, squared. Rostrum distinctive, rounded, finger-like. Dorsal limb conspicuous, ending ca one-fifth ofspicule length from distal end of spicule. Ventral limbconspicuous, straight, weakly tapering to a blunt end, be-ginning immediately posterior to capitulum. Thin, trian-gular, membrane-like cuticle connecting dorsal and ven-tral limbs. Cucullus and gubernaculum absent. Distalspike-like projection occupying ca 50-60% of total taillength (cloacal slit to tail spike tip). Bursal flap absent.Six (three pairs) of conspicuous genital papillae present,first subventral pair (P2) located immediately anterior tocloacal slit, second subventral pair (P3) located ca 70%of cloaca-spike root length from cloacal slit, third ventralpair (P4) midway between P3 and root of spike. No singleprecloacal papilla (P1) observed, probably vestigial.

Female

Reproductive tract on right of intestine, comprisingovary, oviduct, spermatheca, crustaformeria, uterus,vagina + vulva and post-uterine sac. Ovary single, an-teriorly outstretched, anterior end reflexed in some indi-viduals. Oocytes present in multiple (2-5) rows in mostparts of ovary and a single well-developed oocyte some-times located at posterior end of ovary. Oviduct tube-like, constructed of large oval-shaped cells connectingovary and crustaformeria, sometimes occupied by well-developed oocyte. Spermatheca (receptaculum seminis)constructed of rounded cells, present as a branch over-lapping oviduct, i.e., branching out to left from anteriorend of crustaformeria, slightly irregular oval shape, some-times filled with well-developed sperm. Crustaformerianot conspicuous, formed of small, rounded cells. Uterusshort with thick wall, sometimes containing a developingegg and several sperm. Vagina slightly inclined anteri-orly, a pair of three-celled structures in uterus nearest tovagina (uterus/post-uterine sac junction). Vulva a simpleslit in ventral view, without a flap in lateral and ventralviews. Post-uterine sac ca 2-4 vulval body diam. long, ex-tending for ca 50% of vulva to anus distance, sometimesfilled with sperm; sperm stored in post-uterine sac appar-ently larger and more transparent compared with those inspermatheca. Anus a small, dome-shaped slit in ventralview. Tail long, ca ten times longer than anal body diam.(ABD), straight or weakly recurved ventrally when killedby heat, cylindrical, smoothly tapering to a pointed tail tip(= filiform).

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Fig. 1. Bursaphelenchus penai n. sp. A: Adult female in the left lateral view. The reproductive system actually behind (i.e., to the right)of the intestine, but is drawn from this side to show the relative position of spermatheca; B: Adult male in the right lateral view; C:Anterior part of adult female in right lateral view (h = hemizonid; ep = excretory pore); D: Female reproductive tract in left lateralview (ov = ovary; od = oviduct; sp = spermatheca; cr = crustaformeria; ut = uterus; pus = post-uterine sac; v = vulval opening); E:Female tail in right lateral view; F: Female anal opening in ventral view; G: Male tail in ventral view (P + number = genital papillae);H: Male spicule in right lateral view; I: Male tail in left lateral view (P + number = genital papillae).

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Fig. 2. Anterior part of adult female of Bursaphelenchus penain. sp. Excretory pore encircled at corresponding position (neckregion is a little twisted to show ventral view) and hemizonidindicated with an arrow.

TYPE HOST, INSECT CARRIER AND LOCALITY

The type materials were obtained from cultured nema-todes. The culture was started from a population of B.

Fig. 3. Left lateral view of female Bursaphelenchus penai n. sp.vulval region. The cuticular pronged structure is indicated byarrowheads.

penai n. sp. which was isolated from adults of an am-brosia beetle, Xyleborinus gracilis, collected on 1 June2013 from beetle galleries bored into a dead avocado tree(Persea americana) in Homestead, FL, USA.

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Fig. 4. Ventral view of female Bursaphelenchus penai n. sp.vulval region in different focal planes. A: Vulval opening; B:Vaginal region; C: Vagina-uterus junction showing cuticularpronged structure (arrowhead).

TYPE MATERIAL

The type material was obtained from 10-day-old cul-tures. Holotype, nine paratype males and ten paratype fe-males deposited in the United States Department of Agri-culture Nematode Collection (USDANC), Beltsville, MD,USA; and ten paratype males and ten paratype females,deposited in the Forest Pathology Laboratory collection,Forestry and Forest Products Research Institute (FFPRI),Tsukuba, Japan. In addition to the type material, mass-fixed material in formalin-glycerin or processed to de-hydrated glycerin was deposited at Fort Lauderdale Re-search and Education Center, University of Florida/IFAS.

DIAGNOSIS AND RELATIONSHIPS

Bursaphelenchus penai n. sp. is characterised by thefollowing: four-lined lateral field, position of the hem-izonid (inconsistent in relation to excretory pore), malespicule with conspicuous dorsal and ventral limbs, num-ber and arrangement of male genital papillae (three pairsof genital papillae arranged anteriorly as precloacal sub-ventral P2, subventral postcloacal P3, and ventral P4 atthe root of the tail spike), P4 comprising paired papillae –not ‘glandpapillae’ sensu Ryss et al. (2005), and missingP1 ventral single papilla which is present in most speciesin the genus (e.g., Kanzaki et al., 2011), male tail withoutbursal flap but with a spike-like projection, female vulvawithout flap and tail very long and tapering with pointedtip.

The new species is typologically closest to B. kiyoharai,i.e., these two species share unique tail characters of malesand females. Both species possess a male tail spike instead

Fig. 5. Left lateral view of female Bursaphelenchus penai n. sp.tail. Tail tip indicated by arrowhead.

of a bursal flap, and an unusual female tail, which isextremely long for the genus. Bursaphelenchus penai n.sp. and B. kiyoharai are typologically almost identical andcan only be distinguished from one another by the positionof the hemizonid, which is located around the excretory

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Fig. 6. Left lateral view of male Bursaphelenchus penai n.sp. tail in different focal planes. A: Mid-body (co = cloacalopening; P + number = genital papillae); B: Intermediatebetween mid-body and surface (P + number = genital papillae);C: Superficial view (P + number = genital papillae).

Fig. 7. Ventral view of male Bursaphelenchus penai n. sp. tailin different focal planes. A: Mid-body showing ventral view ofspicule; B; Intermediate between mid-body and surface showingventral view of spicule tip (P + number = genital papillae); C:Superficial view (co = cloacal opening; P + number = genitalpapillae); D: Tail spike (P + number = genital papillae).

pore (slightly anterior to excretory pore to ca one styletlength posterior to excretory pore) vs consistently locatedimmediately posterior to excretory pore (Kanzaki et al.,2011). Based on morphometric values, B. penai n. sp.is distinguished from B. kiyoharai by a slightly more

Table 1. Morphometrics of Bursaphelenchus penai n. sp. Allmeasurements are in μm and in the form: mean ± s.d. (range).Character Male Female

Holotype Paratypes Paratypes

n – 19 20L 632 733 ± 98 874 ± 73

(602-913) (757-1009)a 31.1 33.3 ± 3.5 36.0 ± 3.3

(27.3-37.8) (30.3-41.3)b 10.0 11.1 ± 1.2 13.0 ± 0.9

(9.6-13.6) (11.7-15.2)c 11.3 11.7 ± 1.4 6.6 ± 0.5

(9.0-14.3) (5.5-7.5)c′ 4.6 4.8 ± 0.4 10.5 ± 1.0

(4.2-5.5) (9.0-12.2)M 36.4 35.0 ± 2.0 34.6 ± 1.6

(31.8-39.1) (32.0-36.4)T or V 61.1 61.3 ± 6.0 66.1 ± 0.9

(44.7-69.9) (63.3-67.7)Max. body diam. 20.3 22.2 ± 3.5 24.4 ± 1.9

(18.0-30.0) (20.9-27.3)Lip diam. 6.4 6.9 ± 0.4 7.3 ± 0.4

(6.4-7.5) (7.0-8.1)Lip height 2.9 3.5 ± 0.4 3.6 ± 0.3

(2.9-4.1) (2.9-4.1)Stylet conus 4.6 4.8 ± 0.5 4.9 ± 0.3

(4.0-5.8) (4.6-5.2)Stylet length 12.7 13.8 ± 0.9 14.2 ± 0.7

(12.2-15.7) (12.8-15.7)Median bulb length 14.5 16.1 ± 0.8 17.0 ± 1.1

(14.5-17.4) (14.5-18.6)Median bulb diam. 10.4 12.0 ± 1.1 12.5 ± 0.7

(10.4-14.5) (11.6-13.9)Median bulb 1.39 1.35 ± 0.1 1.35 ± 0.1

length/diam. (1.20-1.61) (1.25-1.48)Excretory pore from 95 98 ± 6.4 105 ± 6.2

anterior end (87-111) (95-117)Excretory pore from 33 34 ± 4.9 39 ± 5.0

posterior end of (26-44) (31-47)median bulb

Hemizonid from 104 99 ± 7.5 105 ± 5.5anterior end (88-117) (96-115)

Hemizonid from 9.3p 2.2p ± 3.5 0.1p ± 2.6excretory pore1) (3.5a-9.3p) (6.4a-4.1p)

Spicule length2) 12.6 14.2 ± 0.9 –(12.6-15.9)

posteriorly located excretory pore (87-111 μm for malesand 95-117 μm for females vs 77-93 μm for males and85-94 μm for females) (Kanzaki et al., 2011).

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Table 1. (Continued.)

Character Male Female

Holotype Paratypes Paratypes

Spicule length3) 14.5 16.1 ± 1.0 –(14.5-17.4)

Gonad length4) 386 448 ± 71 169 ± 17.6(330-605) (132-193)

Post-uterine sac length – – 67 ± 20.5(36-132)

Post-uterine sac/vulva – – 40.7 ± 10.7to anus (%) (19.7-69.3)

Tail length 56 63 ± 5.1 132 ± 11(54-73) (113-155)

Anal or cloacal 12.2 13.1 ± 1.0 12.7 ± 0.8body diam. (11.6-15.1) (11.6-13.9)

1) The characters ‘a’ and ‘p’ mean ‘anterior to’ and ‘posterior to’excretory pore, respectively.2) Curved along arc from bottom of capitulum depression todistal end.3) Condylus tip to distal end measured in a straight line.4) Length from cloacal or vulval opening to anterior end ofgonad.

MOLECULAR PROFILES AND PHYLOGENETIC STATUS

The molecular sequences of near-full-length of smallsubunit and D2/D3 expansion segments of ribosomalRNA were determined for B. penai n. sp. and weredeposited in the GenBank database with the accessionnumbers, AB901293 and AB901292.

The molecular phylogenetic analysis based upon near-full-length SSU revealed that B. penai n. sp. is close toB. kiyoharai, B. thailandae Braasch & Braasch-Bidasak,2002 and B. parathailandae Gu, Wang & Chen, 2012(Fig. 8). However, the new species is readily distinguishedfrom B. thailandae and B. parathailandae by the male tailpossessing a tail spike vs a bursal flap and female tail longand filiform vs long conical (Braasch & Braasch-Bidasak,2002; Gu et al., 2012).

Molecularly, the new species is differentiated from B.kiyoharai, B. thailandae and B. parathailandae by morethan 15 bp of substitutions and 3-7 bp of gaps (insertionsor deletions) in ca 1.6 kb of near-full-length SSU andmore than 35 bp of substitutions in ca 0.7 kb of D2/D3LSU. This difference level is clearly higher than thedifferences among B. xylophilus (Steiner & Buhrer, 1934)Nickle, 1970 and its close relatives (see Kanzaki et al.,

Fig. 8. Phylogenetic relationship among Bursaphelenchus spp. belonging to Clade 2 of the genus inferred from near full length SSUsequences. Phylogenetic relationship analysed with Maximum Likelihood analysis under GTR + I + G model. Bootstrap supportshigher than 50% are indicated in the figure. Bursaphelenchus abruptus (basal to the genus) and B. clavicauda (Clade 1 of the genus),served as outgroup species.

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2012) and is consistent for species level differences in thegenus (Ye et al., 2007).

BIOLOGICAL CHARACTER

Bursaphelenchus penai n. sp. is a mycophagous nema-tode that can be easily maintained on M. fructicola orB. fuckeliana at room temperature with continuous fort-nightly to monthly subculturing.

In addition to the type carrier insect, the new speciesis also associated with three other ambrosia beetles:Xyleborus affinis, X. glabratus and X. volvulus, collectedbetween December 2013 and January 2014 from deadavocado trees in Dade County, Florida. The SSU barcodesequences of these materials were identical to that of thetype strain.

Discussion

MORPHOLOGY OF FEMALE REPRODUCTIVE TRACT

During the morphological observation a characteris-tic structure was confirmed in the female gonad. Thenew species has a spermatheca as an anteriorly ex-tended branch originating from the anterior end of thecrustaformeria, this branch laterally overlapping theoviduct. This character has been confirmed (published)in three parasitaphelenchids, i.e., Sheraphelenchus sucusKanzaki & Tanaka, 2013, B. niphades Tanaka, Tanaka,Akiba, Aikawa, Maehara, Takeuchi & Kanzaki, 2014, B.xylophilus and some other parasitaphelenchids (Hasegawaet al., 2004; Kanzaki & Tanaka, 2013; Tanaka et al., 2014;Kanzaki, unpubl. obs.). Further, this character has beenconfirmed in several other parasitaphelenchids, i.e., Bur-saphelenchus spp., including B. kiyoharai, Ruehmaphe-lenchus spp. and Parasitaphelenchus sp. (Kanzaki, un-publ. obs.). However, the character has not been re-ported in the other subfamilies of the Aphelenchoididaeand some species in the other subfamilies, e.g., Aphe-lenchoides spp. (Bert et al., 2008) and Peraphelenchusorientalis Kanzaki, Tanaka, Ikeda, Taki, Sugiura & Mat-sumoto, 2013 (Kanzaki et al., 2013a) and Schistonchusspp. (Davies et al., 2010), have been confirmed to lack abranch in the female gonad.

Parasitaphelenchinae is a relatively derived group in theAphelenchoididae (e.g., Kanzaki & Giblin-Davis, 2012;Kanzaki & Tanaka, 2013). Thus, the character of thespermatheca forming a branch (Fig. 1D) is consideredto be a derived (apomorphic) character in the subfamily

Parasitaphelenchinae (Hasegawa et al., 2004; Kanzaki &Tanaka, 2013; Tanaka et al., 2014). Observation of thecharacter on more species and a family/subfamily-widecomparison may reveal the origin of the character andclarify the systematic definition of the subfamily andgenera.

PHORETIC ASSOCIATION WITH AMBROSIA BEETLES

The nematodes belonging to the Parasitaphelenchi-nae (Bursaphelenchus, Parasitaphelenchus, Ruehmaphe-lenchus and Sheraphelenchus) are associated with manydifferent groups of insects, especially the bark beetles(summarised by Poinar, 1975; Hunt, 1993, 2008). How-ever, only a few species (Ruehmaphelenchus spp., B. kiy-oharai: Kanzaki et al., 2011, 2013b) are known as asso-ciates of ambrosia beetles. Interestingly, B. penai n. sp.,a close relative of B. kiyoharai, is also associated withan ambrosia beetle, Xyleborinus gracilis, and some otherambrosia beetles belonging to Xyleborini. Thus, the am-brosia beetle association could be regarded as a diagnos-tic biological character of the clade including B. kiyoharaiand B. penai n. sp.

Most parasitaphelenchids have been described from thetemperate to the subarctic zones of the northern hemi-sphere (e.g., Ryss et al., 2005) where the bark beetle faunais divergent. By contrast, the ambrosia beetles are morediverse and abundant in warmer regions (warm temper-ate to tropical areas) (Hayashi et al., 1984). More surveysin tropical regions may reveal a much greater diversity ofambrosia beetle-associated parasitaphelenchids.

Different ambrosia beetle genera from different geo-graphical regions may provide isolation to gene flow thatmay help establish or maintain species boundaries for as-sociated species of nematodes. Allopatry that was presentin regional populations of ambrosia beetles and their sym-bionts in the late 1800s-1900s has probably been com-promised by the globalisation of trade between countriesand the frequent movement of wood and wood-packingmaterials with their corresponding xylem-inhabiting am-brosia beetles and associated fungal, bacterial, nematodeand mite symbionts. This can create confusion about theorigins of newly discovered symbionts. In the presentcase, as early as 2004 at a site in Hilton Head Island,South Carolina, which was exhibiting redbay tree die-off,an exotic (Xyleborus glabratus) and two native ambrosiabeetles (Xyleborinus gracilis and Ambrosiodmus obliquusLeConte) were isolated together from the branches ofmany dead or dying redbay trees (Fraedrich et al., 2008).This questions the centre of origin of B. penai n. sp. Is it a

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natural associate of Xyleborinus gracilis, or is it an intro-duced associate of Xyleborus glabratus that has ‘jumpedship’ to native ambrosia beetles? In addition to Xylebori-nus gracilis, B. penai n. sp. is also associated with Xyle-borus affinis, X. glabratus and X. volvulus from avocadofrom Dade Co., Florida. Further survey work is neededto assess the nematode fauna from native ambrosia bee-tles and/or X. glabratus in areas where it has invaded andnot invaded in the USA for comparisons with the nema-tode fauna of X. glabratus in its native range in South EastAsia (Giblin-Davis et al., 2013).

Acknowledgements

This study was supported in part by a Grant-in-Aidfor Scientific Research, #24658147 from The Ministryof Education, Culture, Sports, Science and Technology,Japan to senior author and by Florida Department ofAgriculture and Consumer Services and Florida AvocadoCommittee grants to Dr Jorge E. Peña. The authorssincerely thank Ami Akasaka, FFPRI for assistance inmolecular sequencing, and Noriko Shimoda for preparingmounted materials.

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