Fergusobia morrisae, Davies, Kerrie, Giblin-Davis, Robin, Ye, Weimin, Taylor, Gary & Thomas, Kelley, 2012

Davies, Kerrie, Giblin-Davis, Robin, Ye, Weimin, Taylor, Gary & Thomas, Kelley, 2012, Nematodes from galls on Myrtaceae. III. Fergusobia from flower bud and stigma galls on Eucalyptus, with descriptions of four new species, Zootaxa 3532, pp. 1-36 : 20-35

Description of Fergusobia morrisae n. sp. Davies

( Figs 6 , 7 J, 8N)

Measurements. See Table 5 .

Material examined. The description presented here is based on measurements of 19 parthenogenetic females, 19 males and 11 pre-parasitic infective females from flower bud galls on Eucalyptus fibrosa subsp. fibrosa F. Muell. , associated with an unknown species of Fergusonina ; from Shute Bay Lookout, Shute Bay, Queensland, Australia (20°18´S 148°47´E), collected by Kerrie Davies, 20.v.2002.

Holotype. Parthenogenetic female, with a paratype male and infective female, on a slide deposited at the Australian National Insect Collection ( ANIC), Canberra, Australian Capital Territory, Australia.

Paratypes. Vouchers deposited at the Waite Insect and Nematode Collection ( WINC), the University of Adelaide, Adelaide, South Australia (slides number 004328); and the Queensland Museum, Brisbane, Queensland, Australia.

Description. Parthenogenetic female. Body C-shaped when heat-relaxed, dorsally curved with ventral side convex and most curvature in posterior third of body; smaller than amphimictic pre-parasitic female and males; body narrowing behind vulva. Cuticle with obscure annules ~1 µm spacing, weak longitudinal striae apparent when viewed with light microscope; lateral fields not seen.

Cephalic region ~70% diameter of body at anterior end, offset, unstriated, 2–3 µm long; with rounded outline in lateral view and circum-oral area raised. Stylet 7–8 µm long, with conus 40–50% of total length, basal knobs ~2 µm diameter at base, round.

Orifice of dorsal oesophageal gland 1–2 µm posterior to stylet knobs. Anterior fusiform part of digestive tract diameter 52–62% of body diameter (n = 5), length 2.4–3.1 times diameter (n = 5); lumen of tract broadening at ~50% length of dorsal oesophageal gland. Oesophageal glands large to enormous, diameter 56–75% of body diameter (n = 5), extending over intestine, distance from head to end of glands 53 (30–63) % of total body length. Secretory/excretory pore with prominent duct, opening anterior to oesophageal gland nucleus, at about 30% length from anterior of gland; secretory/excretory cell ovoid ~2 µm long, displacing gland dorsally. Hemizonid extending over two to three annules, one annule anterior to secretory/excretory pore.

Reproductive tract variable in length, extending part-way along dorsal oesophageal gland; usually straight but sometimes with one flexure (in 4 of 19 specimens); oviduct with oocytes not in rows; uterus containing no eggs or occasionally one egg (in 3 of 19 specimens); vulva with protruding lips, flat or forming a depressed slit. Tail conoid, length 1.4–2.3 times anal body diameter, rounded tip.

Infective pre-parasitic female. Infecting mature larval stage of Fergusonina sp. or pupa. Body arcuate or Jshaped when heat-relaxed, with most curvature posterior to vulva; maximum body diameter at mid-body with head and tail regions similar in diameter; tapering slightly posterior to vulva. Cuticle with obscure annulations, longitudinal striae not seen; lateral fields not seen. Large nuclei present in epidermis posterior to vulva, and in intestinal wall.

Cephalic area occupying 80–90% of body diameter at anterior end, continuous; circum-oral area flat; stylet slender, 6–10 µm long, weakly sclerotised with tiny round basal knobs 1–2 µm diameter; conus 40–50% of total length.

Orifice of dorsal oesophageal gland 1–2 µm posterior to stylet knobs; oesophageal glands diameter 38 (29–44) % of body diameter (n = 5), extending over intestine, distance from head to end of glands on average 18 (14–25)% of total body length. Anterior fusiform part of digestive tract diameter 35–55% of body diameter, length 1.9–3.3 times diameter (n = 5).

Secretory/excretory pore opening 59–85 µm posterior to anterior body end, opposite oesophageal gland nucleus; with prominent duct. Secretory/excretory cell similar to that of parthenogenetic female, ~5 µm long. Hemizonid not seen.

Uterus 60–70% of body length in uninseminated females, packed with sperm in inseminated females; vagina perpendicular to body axis; reproductive tract extending to nerve ring, with large cap cell; hypertrophied in some specimens. Vulval lips raised. Tail broad, length 0.7–2 times diameter at anus, tip bluntly rounded or almost hemispherical.

Male. Body arcuate, C- or J-shaped when heat-relaxed, tail region more or less curved ventrally. Cuticle with obscure annules ~1 µm wide, weak longitudinal striae apparent when viewed with light microscope; lateral fields not seen.

Cephalic region offset, 2–3 µm long, circum-oral area flat or slightly raised, with lightly sclerotised framework; stylet 7–10 µm long, conus 40–50% of total length; stylet knobs round, ~2 µm diameter.

Anterior fusiform part of digestive tract diameter 60–75% of body diameter, length 2.3 (2–2.7) times diameter (n = 5). Oesophageal glands diameter 62 (50–71) % of body diameter (n = 5), extending over intestine, distance from head to end of glands on average 28 (24–32) % of total body length. Lumen of intestinal tract broadening posterior to oesophageal gland.

Secretory/excretory pore opening on area of raised cuticle, opposite or posterior to oesophageal gland nucleus; duct obscure; secretory/excretory cell not seen. Hemizonid extending over two or three annules, 2 – 4 annules anterior to secretory/excretory pore.

Reproductive tract with single testis, variable in length, extending to distal end of dorsal oesophageal gland or part-way along it; straight or very occasionally reflexed (1 of 19 specimens); testis, seminal vesicle and vas deferens not clearly differentiated. Bursa smooth; prominent or obscure; arising just posterior to secretory/ excretory pore, 75 – 83% along length of body as measured from tail tip. Spicules paired, angular near middle, robust, heavily sclerotised; manubrium not offset, with similar diameter to shaft; blade with slight convex curvature on proximal edge, bluntly rounded tip; opening terminal. Inconspicuous muscles associated with cloaca. Tail curved, ventrally concave, length 1.6–2.4 times diameter at cloaca, with rounded tip.

Diagnosis and relationships. Fergusobia morrisae n. sp. is morphologically characterized by the combination of a C-shaped parthenogenetic female with a narrowly conoid tail, an arcuate or J-shaped infective female with most body curvature behind the vulva and a short tail with an almost hemispherical tip, and arcuate or J-shaped males with strongly sclerotised, angular spicules and long peloderan bursa.

Morphologically, Fergusobia morrisae n. sp. is similar to F. eugenioidae n. sp. and F. juliae n. sp.

In shape, the parthenogenetic female of F. morrisae n. sp. (open C to C-shape, with most curvature in posterior third of body) is similar to F. eugenioidae n. sp. It differs in shape from F. curriei , F. f i s h e r i, F. n e r v o s a e, F. quinquenerviae (more open C-shape); from F. camaldulensae , F. fasciculosae n. sp., F. pohutukawa , and F. viridiflorae (arcuate to open C-shape); and from F. rileyi (almost straight to arcuate). They are shorter (262–347 µm, mean 291 µm) than F. i n d i c a (525–626 µm), F. magna (418–780 µm), and F. tumifaciens (415 µm), and generally shorter than F. ptychocarpae (320–461 µm, mean 390 µm) and F. brittenae (328–461 µm; mean 389 µm). The raised circum-oral area separates F. morrisae n. sp. from F. brevicauda , F. dealbatae , F. leucadendrae , and F. philippinensis , in which it is not raised. The stylet (7–8 µm) is shorter than in F. eugenioidae n. sp. (9–11 µm). The cuticle does not swell on fixation, differentiating it from F. jambophila , in which it does swell. The hemizonid is 1 annule anterior to the secretory/excretory pore in F. m o r r i s a e n. sp., but immediately anterior in F. ptychocarpae , 11–12 annules anterior in F. cajuputiae , and 8 or 9 annules anterior in F. brittenae . The parthenogenetic female of F. morrisae n. sp. generally has a longer stylet (7–8 µm) than F. juliae n. sp. (5–7 µm).

The infective female of F. morrisae n. sp. is similar in shape to F. eugenioidae n. sp., F. juliae n. sp., and F ptychocarpae (strongly curved in posterior region). It differs in shape from F. m a g n a, F. brittenae , F. dealbatae , F. fisheri , F. nervosae , F. quinquenerviae , and F. viridiflorae (arcuate); from F. curriei and F. f a s c i c u l o s a e n. sp. (open C-shape); and from F. rileyi (almost straight). Infective females are longer (322–395 µm) than those of F. cajuputiae (239–309 µm) and F. leucadendrae (227–291 µm). The cephalic region in F. morrisae n. sp. is continuous and flat, separating it from F. camaldulensae and F. eugenioidae n. sp. in which the circum-oral area is raised; and from F. ptychocarpae in which the cephalic region is flat but distinctly offset. The hemispherical tail tip, and strongly curved body posterior to the vulva differs from that of F. brevicauda and F. camaldulensae , (straighter); and F. philippinensis (truncate tip). The general form of infective females of F. juliae n. sp. is similar to that of F. morrisae n. sp., but a cuticular plate is present around the vulva in the former, and absent in the latter.

In shape (arcuate to J-shape), the males of F. morrisae n. sp. differ only from those of F. jambophila (almost straight). In length (347–413 µm), they are smaller than males of F. magna (446–588 µm); and larger than those of F. fasciculosae n. sp. (274–336 µm), F. nervosae (277–312 µm), and F. quinquenerviae (256–329 µm). The ratio ‘a’ (8.5–10.7) of F. morrisae n. sp. males is smaller than in F. juliae n. sp. (11.1–15.3). Stylet length (7–10 µm) is smaller than in F. r i l e y i (11–13 µm). Tail shape (arcuate, with bluntly rounded tip) differs from that of F. ptychocarpae and F. viridiflorae (more slender), from F. brittenae (shorter and broader); and from F. philippinensis (with truncate tip). In length (30–45 µm), the tail is shorter than in F. pohutukawa (50–61 µm). Spicule length (18–23 µm) is larger than in F. leucadendrae (14–17 µm). In having a bursa arising near the secretory/excretory pore, males of F. morrisae n. sp. differ from F. brevicauda , F. camaldulensae , F. curriei , F. dealbatae , F. eugenioidae n. sp., F. fisheri , F. juliae n. sp., and F. tumifaciens , all of which have a shorter bursa. The hemizonid is further anterior to the secretory/excretory pore in F. morrisae n. sp. than in F. cajuputiae (respectively, 2–4 annules anterior vs at the level of the pore).

The status of F. morrisae n. sp. as a new species is supported by its sequence divergence based upon phylogenetic analyses of sequences of D2/D3 and COI ( Figs 1 , 2 ). From phylogenetic analyses ( Fig. 1 ) based on sequences of D2/D3, F. morrisae n. sp. is genetically closest to Fergusobia sp. collected from FBGs (voucher 1) on E. microcarpa , F. juliae n. sp. and F. fasciculosae n. sp. The blast search of the 861 bp sequenced from D2/D3 revealed the highest matches as vouchers 1 (MSp 22), 2 (MSp 27), 63 (MSp 9), 65 ( F. fasciculosae n. sp.), and 70 (MSp 21), with 96–97% identity, 24–37 bp differences, and 9–14 gaps. The blast search of the 618 bp sequenced from COI revealed the highest matches as vouchers 24, 444, 452 and 465 (all F. m a g n a), with 95–96 identity, 24–30 bp differences and no gaps.

Etymology. Named for Krystalynne Morris, Hubbard Center for Genome Studies, University of New Hampshire, in recognition of her contributions to work on nematode phylogeny.

morphospecies, see Davies et al. 2012).

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Host plant WINC Body Tail shape Shape of tail Uterus Spicule Fergusonina Reference Species Species number shape tip sp.(where

known) E. fibrosa F. morrisae 0 0 4328 C Conoid Bluntly Not

n. sp. rounded extensile Infective

females

...... continued on the next page Host plant WINC Body Tail shape Shape of tail Uterus Spicule Fergusonina Reference

Species Species number shape tip sp.(where known)

Nematode morphology. Habitus drawings of whole nematodes collected from FBGs are presented in Fig. 7 and drawings of the forms of spicules of the males are shown in Fig. 8 . Brief notes on the morphology of Fergusobia nematodes recovered from FBGs on Corymbia and Eucalyptus spp. are presented in Table 6 .

Collections of Fergusobia nematodes and their associated fly larvae from flower bud galls on Corymbia and Eucalyptus are presented in Table 1. No nematode material was available from FBGs collected or recorded from C. maculata by Morgan (1933), collected from C. abbreviata by Taylor & Davies (2008), collected from E. coolabah by K.A. Davies (unpub. data), recorded from E. crebra , E. melanophloia , E. odorata or E. hemiphloia by Currie (1937), and collected from Eucalyptus sp. (Indooroopilly) by G.S. Taylor (unpub. data).

A range of species and morphospecies ( Fig. 7 , Table 6 ) of Fergusobia nematodes have been collected from FBGs, and morphological form is not related to particular host plant groups, suggesting that the nematodes developing in particular plant groups come from more than one lineage. Parthenogenetic females have body shapes varying from arcuate to a tight C-shape, but shape is not correlated with host plant groups. The uterus is extensile in one morphospecies collected from E. baxteri (subgenus Eucalyptus ) and in F. brevicauda from E. deglupta (subgenus Minutifructus) ( Siddiqi 1994), but is not extensile in other species or morphospecies from FBG. Infective females vary similarly in general body shape, without an apparent pattern. Males also vary in body shape. Most morphospecies have angular spicules ( Fig. 8 ), but the size of the angle varies considerably, and one (from E. deglupta ) has sub-arcuate spicules. Similarly, the ratio of the length of the manubrium and shaft to the length of the “foot” differs among morphospecies, but is not correlated with host plant groups. All males collected from FBGs on hosts from the subgenus Symphyomyrtus have a relatively short bursa (from 15–60% of body length), but Fergusobia morphospecies with both the long and short bursa were collected from hosts of the subgenus Eucalyptus . Sequencing data also suggests variability is not correlated with particular host plant groups ( Figs 1 , 2 ). There is thus strong evidence suggesting that the nematodes developing in particular host plant groups come from more than one lineage, and that host-switching occurred widely or that homoplasy is common.

Gall forms. The histology of some Fergusobia / Fergusonina FBGs was described by Giblin-Davis et al. (2004a). FBGs are club-shaped and larger than uninfested flower buds ( Currie 1937, Giblin-Davis et al. 2004a, Taylor et al. 2005). Representative FBGs are shown in Fig. 9 . Each unique Fergusobia / Fergusonina mutualism appears to induce formation of one particular gall form (Davies & Giblin-Davis 2004; Davies et al. 2010a). It is therefore of interest to compare the gall forms induced by nematode/fly mutualisms thought to be related, from two groups of genetically close host plants.

On E. macrophylla and E. obliqua , both from subgenus Eucalyptus , locules originate in a proliferation of anther cells, are membrane-bound, and are each attached to the floret wall by an anther filament. Hypertrophied cells line the lumens of the rounded locules. Filaments in infested buds are 2–3 times the diameter of those of uninfested buds. In E. macrophylla galls, the stigma is not apparent, but it develops in similar galls on E. obliqua . In FBGs on E. microcarpa (Subgenus Symphyomyrtus ), locules similarly originate in a proliferation of anther cells, and the operculum is not released ( Giblin-Davis et al. 2004a, Taylor et al. 2005). Giblin-Davis et al. (2004a) also examined FBGs from E. camaldulensis (subgenus Symphyomyrtus ), but unfortunately no specific gall material was kept as vouchers for the samples sequenced here. In these, galled tissue appeared to be a proliferation of the disc and tissues either from the base of the stigma or from the stamens. The operculum was lost in more mature galls. Ovaries appeared similar to those of uninfested buds. Anthers were not seen, but remains of filaments were present. Locules were irregular in shape. Style galls from E. fasciculosa are inconspicuous, containing 1–3 locules, and indicated by a slightly swollen style after shedding of the operculum and dehiscence of stamens ( Taylor et al. 2005).

Thus, histological studies suggest that the tissue (anther, stigma or stamens) galled within flower buds varies with the particular mutualisms. While only anther tissue was galled in flower buds examined from hosts of the subgenus Eucalyptus , the number of studies is too limited to allow conclusions on whether this is characteristic of galls on hosts from that subgenus.

Shield forms. Because the Fergusobia / Fergusonina mutualism is specific, the morphology of the dorsal shield of the third instar fly larvae and pupae ( Taylor et al. 2005) can be used as a guide in identifying the phylogenetic group to which an associated nematode belongs ( Davies et al. 2010a). Brief notes on the morphology of the dorsal shield of Fergusonina fly larvae associated with respective Fergusobia nematodes collected from various FBGs are presented in Table 7 . The dorsal shields are illustrated in Fig. 10 .

...... continued on the next page Plant host sp. Fly sp. Gall WINC Site/s Shield form Referenc

form number e

E. Fn. tillyardi FBG 0 0 3304 Adelaide, SA Shield formed from plate on AS 1 †

camaldulensis confluent with a short broad plate on anterior margin of AS 2 and a short broad weakly sclerotised transverse band on posterior margin of AS 2; 7–8 long

anteriorly projecting teeth arise from posterior margin of AS 1; middle teeth are broad, long and blade-like and outer teeth are narrower and more sharply tipped

E. Fn. tillyardi FBG ACT, VIC, SA Dorsal plate, with a rake formed of Currie

camaldulensis , 5–7 hooks (teeth) 1937

blakelyi ,

tereticornis

E. coolabah FBG 0 0 4949 Anna Creek Shield formed from plate on AS 1 † Station, SA confluent with a short broad plate on anterior margin of AS 2 and a short broad transverse band on posterior margin of AS 2; 2 short anteriorly projecting teeth arise from posterior margin of AS 1

E. fasciculosa STG 0 0 3017 Nr. Goolwa, SA Shield formed from plate on AS 1 † confluent with a short broad plate on anterior margin of AS 2 and a short broad heavily sclerotised plate on TS3; 8–10 short sharp anteriorly projecting teeth arise from posterior margin of AS 1; second row of shorter teeth arise from posterior margin of AS 1

E. fibrosa FBG 0 0 4201 Shute Harbour, Shield comprises 3 confluent † QLD cuticular plates, with 2 forwardly projecting teeth arising at middle of second plate and a posterior ridge developed into a

backwardly-directed projection

E. largiflorens FBG 0 63831 Tooleybuc, NSW Shield comprises 3 confluent † cuticular plates, with 5 forwardly projecting teeth arising at middle of second plate

E.? melliodora FBG 0 0 3401 Forbes, NSW †

E. microcarpa FBG 0 0 4878 Horsham, VIC Shield formed from plate on AS 1 † confluent with a short broad plate on anterior margin of AS 2 and a strongly sclerotised broad plate on TS3; 2 short anteriorly projecting teeth arise from posterior margin of AS 1

...... continued on the next page Abbreviations: FBG flower bud gall; AS abdominal segment; STG stigma gall; TS thoracic segment; † Taylor unpub. obs.

Fly larvae from FBGs on Corymbia have shields comprising a few bands of sclerotised spicules. Larvae from FBGs from subgenus Eucalyptus have shields comprising plain sclerotised plates or dots which are elaborated in some to become hinged dots/plates ( Currie 1937; G.S. Taylor unpub. data). In larvae collected from FBGs on subgenus Symphyomyrtus , all shields have the ‘plates with teeth’ form. Various degrees of elaboration are seen, being greatest in Fn. tillyardi from E. camaldulensis and Fergusonina spp. from E. fasciculosa and E. fibrosa respectively ( Currie 1937; G.S. Taylor unpub. data). Thus, it appears that fergusoninid phylogeny is more closely correlated with host plant group than that of the nematodes.

Fly larvae with shields of the ‘plates with teeth’ form developing in FBGs have been collected only from subgenus Symphyomyrtus sections Adnataria and Exsertaria. These two sections are genetically distinct ( Steane et al. 2002), but commonly have overlapping distributions ( Brooker & Kleinig 1999; 2001). This suggests that host switching could have occurred within the section, or that homoplasy is common.

Fly larvae with shields of the ‘plates with teeth’ form were collected from FBGs and also from terminal leaf bud galls and some small galls, again only from hosts of the subgenus Symphyomyrtus , from six sections (including Adnataria and Exsertaria) ( Davies et al. 2010a, K.A. Davies unpub. data). Shields comprising plain plates have been collected from hosts of the subgenus Symphyomyrtus as well from Eucalyptus , but not from FBGs ( Davies et al. 2010a).

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Host plant distributions and biogeography of Fergusobia . Information on the taxonomy and natural distribution of plants from which Fergusobia / Fergusonina galls have been collected can be used to predict Fergusobia distribution and biogeography, and as a guide for future collections. In addition, knowledge of the genetic relationships of the host plants can be used to test hypotheses about co-speciation of plant and nematode/fly mutualisms. In Table 8 , brief notes are presented on the distribution and taxonomic status of the various Corymbia and Eucalyptus spp. from which the Fergusobia / Fergusonina FBGs were collected ( Brooker & Kleinig 1999, 2004). Given these host distributions, and the wide range of sites from which the Fergusobia / Fergusonina mutualism has been collected, it is clear that FBGs occur widely in Australasia and the Philippines. However, at present it is not possible to determine where they developed.

Papua New Guinea was separated from the Australian mainland by the flooding of Torres Strait after the last glacial period, ca 8000 years ago. However, before that time a land bridge to Papua New Guinea was present more often than not ( Dodson 1994). This would have allowed colonization and re-colonization of Papua New Guinea by plants and animals from Australia, and may have enabled distribution of the Fergusobia / Fergusonina mutualism both north from Australia and south from Asia.

It is not clear if E. deglupta , a host of the fly/nematode mutualism ( Siddiqi 1994) found outside Australia in tropical south-east Asia, is a relic of early myrtaceous development, or an instance of long-distance dispersal. It occurs in New Guinea, New Britain, the Indonesian islands of Sulawesi and Ceram, and in Mindanao in the southern Philippines ( Brooker & Kleinig 2004).The phylogeography of Philippine fauna and flora is complex ( Jones & Kennedy (2008). While there is a high level of endemism, several colonization routes have been suggested, including a route from Sulawesi. Such colonization events were apparently followed by island-by-island differentiation within the Philippines ( Jones & Kennedy 2008). Eucalyptus deglupta could have reached Mindanao via Sulawesi, as proposed by Jones & Kennedy (2008). Siddiqi’s finding of F. brevicauda and F. philippinensis , morphologically different species both developing on E. deglupta , suggests that the two species are genetically distant. A host switch could have occurred within the Philippines, and/or the ancestors of these Fergusobia species reached the Philippines independently.

Relationships. In previous work, the molecular analyses of D2/D3 from isolates of F. eugenioidae n. sp., F. fasciculosae n. sp., F. juliae n. sp., and F. morrisae n. sp. ( Fig. 1 ; Ye et al. 2007, Davies et al. 2010a) indicated that flower bud gallers occurred in several different clades and were associated with fly larvae having several forms of dorsal shield. These results are confirmed here. Similar groupings of F. eugenioidae n. sp., F. fasciculosae n. sp., F. juliae n. sp., albeit with different positions in the tree, were inferred with sequences of MtCOI, but F. morrisae n. sp. appeared to be basal ( Fig. 2 ).

In our analyses, clades of Fergusobia nematodes are associated with fergusoninid flies having similar shield forms. In the D2/D3 phylogenetic tree ( Fig. 1 ), FBGers ( F. curriei ) from E. camaldulensis grouped with terminal leaf bud gallers ( F. brittenae ) from the same host plant species, and both were associated with fly larvae with similar shield forms ( Davies et al. 2010a). Fergusobia ptychocarpae grouped with Fergusobia (MSpp 33, 83 and 2) from four different gall forms all associated with flies lacking or with shields reduced to a few spicules, from Angophora , Corymbia and Eucalyptus host species. Fergusobia fasciculosae n. sp. from E. fasciculosa grouped with pea gallers (MSpp 28 and 9) and all were associated with fly larvae having shields with two spots of sclerotised cuticle. These formed a larger group with F. morrisae n. sp. and F. juliae n. sp., associated with fly larvae having shields comprising plates with or without teeth.

From the COI phylogenetic tree ( Fig. 2 ), sequences from Fergusobia (Msp 27) from E. obliqua appear to be anomalous. This was discussed in Davies et al. (2010a). With COI, F. morrisae n. sp. is sister to Msp 22 from FBG on E. microcarpa ; F. curriei remains sister to F. brittenae ; F. eugenioidae n. sp. is sister to F. juliae n. sp. in a clade with fergusobids from FBGsFBGs from E. obliqua (MSp 27); and F. fasciculosae n. sp. groups with pea gallers as in the D2/D3 tree. Fergusobia juliae n. sp. appears to be a species complex, suggesting possible genetic drift between the geographically widely separated collection sites in the ACT and SA.

Figures

FIGURE 1. The Bayesian consensus tree inferred from D 2 / D 3 under TVM + I + G model (lnL = 4001.6121; freqA = 0.2903; freqC = 0.1443; freqG = 0.2394; freqT = 0.3259; R (a) = 0.8475; R (b) = 3.4236; R (c) = 1.9889; R (d) = 0.455; R (e) = 3.4236; R (f) = 1; Pinva = 0.522; Shape = 0.5933). Posterior probability values exceeding 50 % are given on appropriate clades. (Tree labels comprise nematode species, location (Australia state code), gall type, host plant species and GenBank accession number.

FIGURE 2. The Bayesian consensus tree inferred from COI under GTR + I + G model (lnL = 3999.9661; freqA = 0.2104; freqC = 0.1017; freqG = 0.1948; freqT = 0.4931; R (a) = 1.2258; R (b) = 7.2392; R (c) = 1.5064; R (d) = 0.0698; R (e) = 4.1806; R (f) = 1; Pinva = 0.5476; Shape = 1.5992). Posterior probability values exceeding 50 % are given on appropriate clades.

FIGURE 6. Fergusobia morrisae n. sp. (all in lateral view): A, Entire infective female; B, Head of infective female; C, Tails of infective females; D, Entire parthenogenetic female; E, Head of parthenogenetic female; F, Tails of parthenogenetic females; G, Entire male; H, Head of male; I, Tails of males. Scale bars: A, D, G = 50 μm. B, E, H = 5 μm. Tails not drawn to scale. Line drawings indicate ranges of habitus of the various stages.

FIGURE 7. Habitus drawings of Fergusobia spp. (most undescribed) collected from flower bud galls (P and Q redrawn from Siddiqi 1994): A, F. ptychocarpae from C. ptychocarpa; B, F. eugeniodae n. sp. from E. eugenioides; C, F. j u l i a e n. sp. from E. macrorrhyncha; D, MSp 27 from E. obliqua; E, MSp 65 from E. obliqua; F, MSp 64 from E. pauciflora; G, F. tumifaciens from E. bridgesiana (formerly stuartiana); H, MSp 54 from E. tereticornis; I, F. fasciculosae n. sp. from E. fasciculosa; J, F. morrisae n. sp. from E. fibrosa; K, MSp 71 from E. largiflorens; L, MSp 72 from E. melliodora; M, Msp 22 from E. microcarpa; N, Msp 75 from E. populnea; O, Msp 62 from E. pruinosa; P, F. brevicauda from E deglupta; Q, F. philippinensis from E. deglupta. Scale bars = 50 μm. MSp numbers as in Davies et al. (2012).

FIGURE 8. Spicules from Fergusobia spp. collected from flower bud galls (K – L redrawn from Siddiqi 1994): A, F. eugeniodae n. sp. from E. eugenioides; B, F. juliae n. sp. from E. macrorrhyncha; C, MSp 65 from E. obliqua; D, MSp 27 from E. obliqua; E, F.? curriei from E. camaldulensis; F, F.? curriei from E. camaldulensis; G, F. fasciculosae n. sp. from E. fasciculosae; H, MSp 71 from E. largiflorens; MSp 75 from E. populnea; J, MSp 62 from E. pruinosa; K, F. brevicauda from E. deglupta; L, F. philippinensis from E. deglupta; M, from E. pauciflora; N, F. m o r r i s a e n. sp. from E. fibrosa; and O, MSp 54 from E. tereticornis. Scale bars = 5 µm. MSp numbers as in Davies et al. (2012).

FIGURE 9. Dried flower bud galls (arrowed): A, from E. eugenioides associated with F. eugenioidae n. sp.; B, from E. camaldulensis associated with F.? curriei; and C, from E. obliqua associated with MSp 65. Scale bars = 5 mm.

FIGURE 10. Shield forms from L 3 fergusoninid larvae from flower bud galls (A – C redrawn from Currie (1937); D, E = automontage images, original): A, Fn. nicholsoni from flower bud galls on E. macrorrhyncha; B, Larva (Currie’s Sp. 5) from flower bud galls on E. pauciflora; C, Fn. tillyardi (newly moulted) from flower bud gall on E. camaldulensis; D, larva from style gall on E. fasciculosa; E, larva from flower bud gall on E. fibrosa. Scale bars = 50 µm.

Tables

TABLE 5. Measurements (µm) of Fergusobia morrisae n. sp. from E. fibrosa (mean ± S. D. (range )).

	Holotype Parthenogenetic females	Males	Infective females
n	1 19	19	11
Length	274 291±22.2 (262–347)	384±23.7 (347–413)	365±21.9 (322–395)
a	7.8 8.3±0.6 (7.4–9.3)	9.6±0.9 (8.5–10.7)	10.0±0.8 (8.5–11.1)
b’	1.6 1.9±0.4 (1.6–3.3)	3.6±0.4 (3.1–4.2)	5.7±1.1 (4.0–6.9)
c	9.9 12.5±2.2 (9.9–15.8)	9.9±1.0 (8.8–11.3)	14.3±2.7 (11.2–21.1)
c’	1.9 1.8±0.3 (1.3–2.3)	2.1±0.2 (1.6–2.4)	1.4±0.4 (0.7–2.0)
V %	82.4 83.2±1.8 (79.1–86.7)		76.3±2.2 (73.1–79.5)
T %		69.7±7.8 (53.5–79.7)
Body diameter	35 35±2.6 (33–45)	40±4.3 (32–52)	36±2.4 (33–43)
Stylet length	7 7.5±0.3 (7–8.0)	7.9±1.2 (7–10)	7.7±1.6 (6–10)

TABLE 6. Comparative morphology of species of Fergusobia from flower bud galls on Corymbia and Eucalyptus. (MSp

Host plant Species	Species	WINC number	Body shape	Tail shape	Shape of tail tip	Uterus	Spicule	Fergusonina sp.(where known)	Reference
C. ptychocarpa	F. ptychocarpae	0 0 3268 0 0 4334 0 0 4795	C	Slender, conoid, curved, about 2x as long as wide	Bluntly rounded	Not extensile		giblindavisi	Taylor & Davies 2007
E. obliqua	MSp A	0 0 3470	Open C or C	Short, slender	Narrowly rounded	Not extensile			Davies et al. 2010a
E. obliqua	MSp B	003096-7	Open C or C	Short, broad, sub- cylindroid	Bluntly rounded	Not extensile			Davies et al. 2010a
E. baxteri		0 0 4581	arcuate		Bluntly to broadly rounded	extensile
E. pauciflora		0 63854	C	slender	Bluntly rounded	Not extensile
E. camaldulensis	F. curriei	0 63739	Open C	Short, sub- triangular	Sub-pointed	Not extensile		tillyardi	Fisher & Nickle 1968; Tonnoir 1937
E. tereticornis		0 63851	Open C	Short, sub- triangular	Bluntly rounded	Not extensile
E. largiflorens		0 63831	C	Slender, sub- cylindroid	Bluntly rounded	Not extensile
E.? melliodora		0 0 3401	C	Short, broad	Bluntly to broadly rounded	Not extensile
E. microcarpa		0 0 4878	C		Bluntly rounded	Not extensile
E. populnea		0 0 3087	C		Bluntly rounded	Not extensile
E. pruinosa		0 0 3445	C		Bluntly rounded	Not extensile
E. deglupta	F. philippinensis		C	Slender,	Bluntly rounded	Not extensile			Siddiqi 1994
E. deglupta	F. brevicauda		C	Short, broad	Broadly rounded	Extensil e			Siddiqi 1994
E. eugenioides	F. eugenioidae n. sp.	0 0 4312	Arcuate, open C or C	conoid	Bluntly rounded	Not extensile
E. fasciculosa	F. fasciculosae n. sp.	0 0 4498		curved	Broadly rounded to sub- hemispherical	Not extensile
E. macrorrhyncha	F. juliae n. sp.	0 0 4310 0 0 4516	Arcuate to C	Conoid to sub-triangular	Bluntly rounded	Not extensile		nicholsoni	Currie 1937

TABLE 7. Form of the dorsal shield on Fergusonina larvae and pupae found associated with Fergusobia nematodes from flower galls on Corymbia and Eucalyptus spp.

Plant host sp.	Fly sp.	Gall form	WINC number	Site/s	Shield form	Referenc e
Corymbia
C. abbreviata	Fn. thomasi	FBG	0 0 3443	Kimberley Region, WA	N/A	Taylor & Davies 2008
C. maculata	Fn. gurneyi	FBG		Bateman’s Bay, NSW	N/A	Malloch 1932
C. maculata	Fn. biseta	FBG		Bodalla and Bateman’s Bay, NSW	N/A	Malloch, 1932
C. maculata	Fn. eucalypti	FBG		Bateman’s Bay, NSW	4 sclerotised plates, with first and second, and third and fourth, confluent to form 2 almondshaped areas	Currie 1937
C. ptychocarpa	Fn. giblindavisi	FBG	0 0 4333	Various, coastal QLD	Series of raised sclerotised spicules on TS 2 and 3	Taylor & Davies 2008
Eucalyptus subgenus Eucalyptus E. baxteri FBG 0 0 4581 Grampians, VIC Plain shield extending over 3 segments, fused; no teeth †	E. baxteri		FBG	0 0 4581	Grampians, VIC	Plain shield extending over 3 segments, fused; no teeth	†
E. baxteri		FBG	0 0 4581	Grampians, VIC	Plain shield extending over 3 segments, fused; no teeth	†
E. eugenioides		FBG	0 0 4221	Canberra, ACT	3 confluent dots of sclerotised cuticle	†
E. macrorrhyncha	Fn. nicholsoni	FBG	0 0 4516	Canberra, ACT; Clare, SA	Short broad sclerotised plate on posterior margin of TS3 confluent with a broader plate on AS1, and a narrower plate on anterior margin of AS2	Currie 1937; †
E. obliqua		FBG	0 0 3094, 0 0 3096	Stirling, SA; Fleurieu Peninsular, SA	Short broad sclerotised plate on posterior margin of TS3 confluent with a broader plate on AS1, and a narrower plate on anterior margin of AS2	†
E. obliqua		FBG	0 0 3470		N/A
E. pauciflora	Fergusonina n. sp. 5	FBG	0 63798, 0 63854	Mt. Kosciusko, NSW; Canberra, ACT	Weakly sclerotised rectangular plate on TS3 forming concave plate on posterior margin; hinged to a chitinous hump on anterior margin of AS1, covered with protuberances; a second concave plate at posterior margin of AS2 hinged with convex plate on anterior margin of AS3	Currie 1937; †
Eucalyptus subgenus Symphyomyrtus

TABLE 8. Nomenclature and distribution of Corymbia and Eucalyptus known to be hosts for Fergusobia / Fergusonina flower bud gallers.

Host sp.	Common names and remarks	Geographic range
Corymbia
Section Septentrionales
C. abbreviata	Scraggy bloodwood	Eastern Kimberley, WA; and adjacent parts of NT
C. maculata	Spotted gum	Coastal plains and hills of NSW (and inland to Albury/Wodonga), with a disjunct population near Orbost in VIC
C. ptychocarpa	Swamp bloodwood. Widely grown as an ornamental; fly/nematode mutualism not recorded from within native range of host; known only from Brisbane and Cairns (Taylor & Davies 2008)	Scattered in the Kimberley Region of WA, northern NT and far northwest QLD, associated with creeks and damp sites
Eucalyptus subgenus Eucalyptus
Section Capillulus
Eucalyptus baxteri	Brown stringybark	Mount Lofty Range (SA) south-eastwards to VIC; also in sandy ‘desert’ country of SA and VIC
E. eugenioides	Thin leaved stringybark	Coastal NSW and adjacent ranges and tablelands, extending into south-east QLD
E. macrorrhyncha	Red stringybark	Common, widespread in Great Dividing Range of NSW and VIC, with disjunct population near Clare, SA
Section Cineraceae
E. pauciflora	Snow gum	Highest alpine parts of the Great Dividing range in NSW and VIC, disjunct populations in south-east of SA and north-east of TAS
Section Eucalyptus
E. obliqua	Messmate stringybark	South-eastern Australia, coastal ranges and tablelands
Eucalyptus subgenus Symphyomyrtus
Section Exsertaria
E. blakelyi	Red gum	Northern and southern tablelands of NSW, extending into adjacent VIC
E. camaldulensis	River red gum. The most widespread eucalypt in Australia	Rivers, creeks and valleys of south-eastern Australia and WA
E. tereticornis	Forest red gum	Coast and coastal ranges of NSW from Bateman’s Bay north through QLD to New Guinea
Section Adnataria
E. crebra	Ironbark	North west slopes and plains of NSW, north through eastern QLD to the southern part of Cape York Peninsula