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Biological Journal of the Linnean Society, 2014, 111, 699–718. With 4 figures
REVIEW ARTICLE
An emerging example of tritrophic coevolution between
flies (Diptera: Fergusoninidae) and nematodes
(Nematoda: Neotylenchidae) on Myrtaceae host plants
LEIGH A. NELSON1, KERRIE A. DAVIES2*, SONJA J. SCHEFFER3, GARY S. TAYLOR4,
MATTHEW F. PURCELL5, ROBIN M. GIBLIN-DAVIS6, ANDREW H. THORNHILL7 and
DAVID K. YEATES1
1
CSIRO Ecosystem Sciences, Clunies Ross Street, Acton, ACT 2601, Australia
Australian Centre for Evolutionary Biology and Biodiversity, and School of Agriculture, Food and
Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia
3
Systematic Entomology Lab, USDA-ARS, 10300 Baltimore Av., Beltsville, MD 20705, USA
4
Australian Centre for Evolutionary Biology and Biodiversity, and School of Earth and
Environmental Sciences, The University of Adelaide, North Terrace, Adelaide, SA 5005, Australia
5
CSIRO Ecosystem Sciences/USDA ARS Australian Biological Control Laboratory, GPO Box 2583,
Brisbane, Qld 4001, Australia
6
Fort Lauderdale Research and Education Center, University of Florida, IFAS, 3205 College Av.,
Fort Lauderdale, FL 33314, USA
7
Australian Tropical Herbarium, James Cook University, Cairns, Qld 4870, Australia
2
Received 9 September 2013; revised 14 November 2013; accepted for publication 14 November 2013
A unique obligate mutualism occurs between species of Fergusonina Malloch flies (Diptera: Fergusoninidae) and
nematodes of the genus Fergusobia Currie (Nematoda: Neotylenchidae). These mutualists together form different
types of galls on Myrtaceae, mainly in Australia. The galling association is species-specific, and each mutualism in
turn displays host specificity. This tritrophic system represents a compelling arena to test hypotheses about
coevolution between the host plants, parasitic nematodes and the fergusoninid flies, and the evolution of these
intimate mutualisms. We have a basic knowledge of the interactions between the host plant, fly and nematode in this
system, but a more sophisticated understanding will require a much more intensive and coordinated research effort.
Summaries of the known Fergusonina/Fergusobia species associations and gall type terminology are presented. This
paper identifies the key advantages of the system and questions to be addressed, and proposes a number of
predictions about the evolutionary dynamics of the system given our understanding of the biology of the mutualists.
Future research will profitably focus on (1) gall cecidogenesis and phenology, (2) the interaction between the fly larva
and the nematode in the gall, and between the adult female fly and the parasitic nematode, (3) the means by which
the fly and nematode life cycles are coordinated, (4) a targeted search of groups in the plant family Myrtaceae that
have not yet been identified as gall hosts, and (5) establishment and comparison of the phylogenetic relationships of
the host plants, fly species and nematodes. Recently derived phylogenies and divergence time estimation studies of
the Diptera and the Myrtaceae show that the fly family Fergusoninidae is less than half the age of the Myrtaceae,
discounting the hypothesis of cospeciation and coradiation of the fly/nematode mutualism and the plants at the
broadest levels. However, cospeciation may have occurred at shallower levels in the phylogeny, following the
establishment of the fly/nematode mutualism on the Myrtaceae. © 2014 The Linnean Society of London, Biological
Journal of the Linnean Society, 2014, 111, 699–718.
ADDITIONAL KEYWORDS: cospeciation – COI – morphology – tritrophic interaction – tritrophic
specialization.
*Corresponding author. E-mail: [email protected]
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
699
700
L. A. NELSON ET AL.
INTRODUCTION
Nematode
Species of Fergusonina Malloch flies (Diptera:
Fergusoninidae) form unique species-specific associations with nematodes of the genus Fergusobia Currie
(Nematoda: Neotylenchidae), in the only known case
of obligate mutualism between nematodes and insects
(Giblin-Davis, 1993). First reported by Morgan (1933),
the Fergusonina/Fergusobia mutualists together
form galls on plants of the family Myrtaceae, mainly
in Australia, although some are from India, New
Guinea, New Zealand and The Philippines (Harris,
1982; Siddiqi, 1986, 1994; Taylor et al., 2007). Galls
have been recorded predominantly from Eucalyptus
species, although Angophora, Corymbia, Leptospermum, Melaleuca, Metrosideros and Syzygium
are also hosts (Currie, 1937; Tonnoir, 1937; Harris,
1982; Siddiqi, 1986, 1994; Giblin-Davis et al., 2004b;
Taylor, 2004; Taylor et al., 2007; Taylor & Davies,
2008; Davies et al., 2010b; K. A. Davies et al., unpubl.
data).
Galls comprise one or more separate chambers or
locules, each containing an individual fly larva and
associated nematodes (Giblin-Davis et al., 2004a).
The fly larvae feed on the cells lining the wall of their
associated locule.
The life cycles of the flies and their associated
nematodes are summarized in Figure 1. Fergusobia
nematodes apparently induce galls on host plants
via pharyngeal gland secretions produced during
feeding on host cells (Currie, 1937; Giblin-Davis et al.,
2001b). Fergusonina flies facilitate transport to new
host plants and provide nutrition to their parasitic
nematodes (e.g. Currie, 1937; Fisher & Nickle, 1968;
Giblin-Davis et al., 2001b; Taylor, Head & Davies,
2005; Taylor & Davies, 2008). The system is therefore
described as a mutualism. The Fergusobia nematodes
of the amphimictic generation mate within the
locules, and fertilized females enter the haemocoel of
third-instar female Fergusonina larvae (Fig. 1) by
an unknown mechanism. The parasitic female nematode has a highly modified, absorptive epidermis
(Giblin-Davis et al., 2001a). It lays eggs within the
fly haemocoel, giving rise to juvenile nematodes
which are deposited by the fly, together with its
eggs, into meristematic plant tissues (Currie, 1937;
Giblin-Davis et al., 2001b).
The origin of the fly/nematode mutualism is
unclear. Flies are known to be parasitized by various
tylenchid nematodes, e.g. Howardula and Parasitylenchus (Poinar, Jaenike & Shoemaker, 1998).
Fergusobia is the only known tylenchid nematode
that has both insect- and plant-parasitic generations (Siddiqi, 2000). However, tylenchid nematodes
with insect- and fungal-parasitic generations are
known (Siddiqi, 2000). Fergusobia nematodes repre-
Parasitic ♀
IN FLY
Fly
Eggs
Eggs
Juveniles
Oviposition
IN GALL
Juveniles
Eggs
Parthenogenetic ♀
1st
Amphimictic ♀+♂
Fertilised
preparasitic ♀
2nd
Nematodes
into ♀ larvae
Larval
instars
3rd
Pupae
Figure 1. Life cycles of Fergusonina flies and Fergusobia
nematodes. Female flies deposit eggs and juvenile nematodes into meristematic plant tissue. The phytophagous
nematodes deposited by the fly develop into parthenogenetic females, which lay eggs in the gall. These eggs develop
into phytophagous male and female nematodes, which
mate. By the time the fly eggs hatch, adult male nematodes
are present in the gall; females develop later. Fertilized
pre-parasitic female nematodes enter third-instar female
fly larvae and become entomoparasitic. There are three fly
larval instars. During pupation (or following emergence of
the fly), female parasitic nematodes lay fertilized eggs into
fly haemolymph. Juvenile nematodes hatch and move to fly
oviducts, awaiting oviposition.
sent a large and potentially ancient radiation.
Molecular evidence suggests a single origin of the
Fergusonina/Fergusobia mutualism (Ye et al., 2007;
Davies et al., 2010b). Possible scenarios for the
evolution of its parasitism were discussed by
Giblin-Davis et al. (2003) and Taylor et al. (2005), and
further phylogenetic studies using suitable molecular
markers could be used to understand the evolutionary
history of this unique relationship. Unfortunately, no
molecular phylogeny is available of the suborder
Hexatylina, to which Fergusobia belongs, but would
greatly enhance understanding of its evolutionary
relationships.
Much of the research on the Fergusonina/
Fergusobia mutualism over the last two decades
stemmed from its application as a potential biocontrol
agent of Melaleuca quinquenervia, a serious weed in
Florida, USA (Goolsby, Makinson & Purcell, 2000;
Davies, Makinson & Purcell, 2001; Giblin-Davis
et al., 2001b; Scheffer et al., 2004, 2013; Taylor, 2004;
Center et al., 2011; Pratt et al., 2013). More recent
research has taken advantage of these detailed
studies and has addressed the broader evolutionary
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
and coevolutionary aspects of this tritrophic mutualistic system (Ye et al., 2007; Davies et al., 2010b).
Further collecting has also extended the plant host
range (Davies et al., 2010b), and molecular genetic
techniques have been used to further elucidate the
biology of the system (Scheffer et al., 2004, 2013).
Synthesizing this information, we make some predictions about coevolutionary patterns and processes in
the system.
GALL
TYPES
Specific pairs of Fergusonina flies and Fergusobia
nematodes cause different gall types on different
plant tissues. There are five primary gall types based
on the location of the gall, and each of these types is
divided into unilocular versus multilocular forms. In
addition, flower bud gallers may attack the stigma,
stamen or ovary of the host plant. The most commonly collected galls, because they are easily seen,
are large multilocular galls developing from terminal
leaf buds. The more cryptic unilocular galls, occupying positions such as small axial buds, may be underrepresented in collections. In an effort to standardize
and normalize the terminologies used to delineate
gall types in the literature, a summary has been
prepared (Table 1), and common gall forms are illustrated in Figure 2.
Several gall types were defined by Currie (1937),
and re-examined by Taylor et al. (2005). Since then,
histological study has revealed that differences in gall
types are determined by the placement and timing
of oviposition by Fergusonina flies (Giblin-Davis
et al., 2004a). Only a few Fergusonina/Fergusobia
pairs have been found galling more than one tissue.
Examples are Fergusonina lockharti, which produces
701
both multilocular axial and terminal leaf bud galls
on Eucalyptus camaldulensis (Taylor & Davies,
2010), and F. turneri, which produces galls on both
shoots and flower buds on Melaleuca quinquenervia
(Goolsby, Makinson & Purcell, 2000). Other separate
Fergusonina/Fergusobia species have been collected
from the stigma, stamen and ovary of flower bud galls
(Giblin-Davis et al., 2004a).
Molecular phylogenies so far indicate that while
gall type is conserved among some closely related
Fergusobia clades, gall types have evolved more than
once (Ye et al., 2007; Davies et al., 2010b). The kinds
and frequencies of transitions between gall types
should be assessed when a phylogeny of the flies is
available that includes reliable information on host
species and gall type.
FLY
AND NEMATODE DIVERSITY
The specificity of the mutualism to different host
species, and host tissue types, implies that there may
be many hundreds of Fergusonina/Fergusobia species
pairs across the Myrtaceae. Allopatric mutualists
are also known in different parts of the range of
widespread host species such as E. pauciflora and
E. camaldulensis (Fisher & Nickle, 1968; Taylor &
Davies, 2010; Nelson, Scheffer & Yeates, 2011a, b;
Davies et al., 2012a). This is a significant case of
cryptic diversity in the Australasian entomological
and nematological fauna (Austin et al., 2004; Raven &
Yeates, 2007; Hodda & Nobbs, 2008).
Gaining insight into the Fergusonina/Fergusobia
system involves the combined analysis of all three
trophic levels involved. However, obtaining information on all three components, namely the identification and characterization of each species in the
Table 1. A summary of gall types induced by the Fergusonina/Fergusobia mutualism on Myrtaceae
Organ
Location
Locule type
Leaf
Petiole (A)
Unilocular
Multilocular
Unilocular
Multilocular
Unilocular
Multilocular
Multilocular
Unilocular
Flat (B)
Shoot bud
Flower
bud (G)
Axillary (C)
Terminal (D)
Stigma
Stamen (primordial
tissue)
Ovary
Gall form
Schematic of common gall locations
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
702
L. A. NELSON ET AL.
Figure 2. Representative Fergusonina/Fergusobia gall forms. A, leaf ‘pea’ galls (unilocular) from E. pauciflora; B, shoot
bud gall from M. dealbata; C, axial leaf bud gall from E. camaldulensis; D, flat leaf gall from E. leucoxylon; E, flower bud
gall from E. microcarpa; F, ‘leafy’ leaf bud gall from E. aromaphloia; G, terminal leaf bud gall from E. obliqua. All galls
except A are multilocular. Note exit holes in C and G. Scale bars = 1 cm.
tritrophic plant/nematode/fly interaction, poses distinct challenges. To date, records have been collected
for about 200 tritrophic associations (Tables 2 and 3;
G. S. Taylor and K. A. Davies, unpubl. data).
Description of a Fergusobia nematode is usually
undertaken only when all taxonomically informative
stages of the life cycle have been obtained. Nematodes
are only found in locules when eggs or the fly larvae
are present. Obtaining the taxonomically important
life stages for the Fergusonina flies (larvae, pupae
and adult) is easier than for the Fergusobia nematodes. The fly larvae and pupae remain in the galls
for longer, and provided that third-stage larvae are
present, adult flies can usually be reared from the
galls. The cuticular dorsal shield is unique among
Diptera larvae. It varies between species, from comprising a few raised spicules (being almost absent) to
transverse rows of raised, sclerotized spicules to large
plates with ridges or comb-like processes (Currie,
1937; Taylor et al., 2005). Large multilocular galls can
be partially dissected to remove larvae and/or pupae,
leaving the remainder of the gall intact for emergence
of adult specimens.
Often a Fergusonina/Fergusobia gall is found on a
myrtaceous host plant, but insufficient material is
available for positive identification of the plant, fly or
nematode species (Davies et al., 2010b). Of the 38
described Fergusonina species (Currie, 1937; Fisher &
Nickle, 1968; Siddiqi, 1986, 1994; Davies & Lloyd,
1996; Davies & Giblin-Davis, 2004; Taylor, 2004;
Taylor et al., 2007; Taylor & Davies, 2008, 2010;
Nelson, Scheffer & Yeates, 2011a, b; Davies et al.,
2012a, b; Purcell et al., 2013), only 14 have complete
records for corresponding myrtaceous host, gall type,
fly larval dorsal shield morphology and a named
Fergusobia nematode (Table 2). However, information
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
Flower bud
‘leaf galls’
C. maculata (Malloch, 1932; Morgan,
1933)
E. melanophloia (Tonnoir, 1937)
E. crebra (poss.) (Tonnoir, 1937)
E. odorata (Currie, 1937)
E. hemiphloia (Currie, 1937)
M. viridiflora (Taylor, 2004)
E. bridgesiana (Currie, 1937; Tonnoir, 1937)
E. amygdalina (Tonnoir, 1937)
Euc. spp. (Tonnoir, 1937)
M. leucadendra (Taylor, 2004)
E. macrorryncha (Tonnoir, 1937)
Eucalyptus spp.
E. pauciflora species-group
C. maculata (Malloch, 1932; Morgan,
1933; Currie, 1937)
E. meliodora (Tonnoir, 1937)
Euc. spp. (Currie, 1937)
E. camaldulensis (Taylor et al., 1996)
E. amygdalina (Tonnoir, 1937)
C. ptychocarpa (Taylor & Davies,
2008)
M. nervosa (Taylor, 2004)
biseta Malloch
1932
brimblecombi
Tonnoir 1937
burrowsi Taylor
2004
carteri Tonnoir
1937
centeri Taylor
2004
curriei Tonnoir
1937
davidsoni
Tonnoir 1937
daviesae Nelson
& Yeates 2011
eucalypti
Malloch 1932
evansi Tonnoir
1927
flavicornis
Malloch 1925
frenchi Tonnoir
1937
giblindavisi
Taylor 2008
goolsbyi Taylor
2004
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
Rosette gall at base of axial shoot
buds, = basal galls of axial shoot
buds
Flower bud
‘leaf galls’
Terminal leaf bud
Terminal leaf bud
Unknown
Leaf bud
Nodular, terminal or axial shoot bud;
shiny
‘leaf galls’
leafy leaf bud
Nodular, terminal or axial shoot bud
Flower-bud
Flower-bud
Unknown
Unknown
atricornis
Malloch 1925
Gall type
Myrtaceous host
Fergusonina sp.
North coastal Qld
Mareeba (Qld)
Eastern Australia
Brisbane (Qld)
Cairns (Qld)
Emerald (Vic)
WA, SA, Vic. & NSW
Canberra (ACT)
Adelaide (SA)
Bodalla & Batemans Bay
(NSW)
Follows high-elevation snow
gum distribution
Adelaide (SA)
Canberra (ACT)
Cairns (Qld)
Cardwell (Qld)
Canberra (ACT) &
Southern tablelands (NSW)
Emerald (Vic)
Adelaide (SA)
North coastal Qld
Canberra (ACT) & Qld
Qld
SA
Vic
Bodalla (NSW)
Sydney (NSW)
Canberra (ACT)
Distribution
3 bands – 2 of them confluent
(blended into 1)
Raised sclerotized spicules
on the 2nd and 3rd thoracic segments
Unknown
Plate with 3, mostly 4 and rarely 5
anterior-projecting prongs similar
to F. lockharti
Two sclerotized plates
4 sclerotized plates, with first and
second, and third and fourth,
confluent to form 2 almond-shaped
areas
9 separate transverse sclerotized
bands
Unknown
Approx. 6 separate transverse
sclerotized bands
5 broad black sclerotized plates; first
anterior 2 confluent
2 patches of heavily sclerotized
cuticle
5 broad black sclerotized plates
2 hooks with scoop-like projections
rising from base
Unknown
Unknown
Dorsal shield
Fb. species 1 (Davies &
Giblin-Davis, 2004)
ptychocarpae Davies 2008
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
leucadendrae Davies &
Giblin-Davis 2004
tumifaciens Currie 1937 (on
E. bridgesiana)
viridiflorae Davies &
Giblin-Davis 2004
Unknown
Unknown
Unknown
Fergusobia sp.
Table 2. Summary of described Fergusonina flies and Fergusobia nematodes, their myrtaceous host, gall type, distribution based on collection records and larval
dorsal shield type
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
703
C. maculata (Malloch, 1932; Morgan,
1933)
E. rudis (Tonnoir, 1937)
E. camaldulensis (Taylor & Davies, 2010)
M. nervosa (Taylor, 2004)
Metrosideros excelsa (Taylor et al.,
2007)
Unknown
E. hemiphloia (Tonnoir, 1937)
E. gomphocephala (Tonnoir, 1937)
E. macrorryncha (Tonnoir, 1937)
E. pauciflora species-group
(Nelson & Yeates, 2011)
E. amygdalina (Tonnoir, 1937)
M. nervosa (Taylor, 2004)
M. nervosa (Taylor, 2004)
Unknown
Syzygium cumini (Siddiqi, 1986)
E. pauciflora species-group
C. abbreviata (Taylor & Davies, 2008;
Davies et al., 2010a)
E. dalrympleana (Nelson & Yeates,
2011)
E. blakelyi (Tonnoir, 1937)
E. camaldulensis (Tonnoir, 1937)
E. tereticornis (Currie, 1937)
lockharti
Tonnoir 1937
makinsoni
Taylor 2004
metrosiderosae
Taylor 2007
microcera
Malloch 1924
morgani Tonnoir
1937
newmani
Tonnoir 1937
nicholsoni
Tonnoir 1937
omlandi Nelson
& Yeates 2011
pescotti Tonnoir
1937
purcelli Taylor
2004
schefferae Taylor
2004
scutellata
Malloch 1925
syzygii Harris
1982
taylori Nelson &
Yeates 2011
thomasi Taylor
2008
thornhilli
Nelson &
Yeates 2011
tillyardi Tonnoir
1937
E. polyanthemos (Currie, 1937)
greavesi Currie
1937
gurneyi Malloch
1932
Myrtaceous host
Fergusonina sp.
Table 2. Continued
Flower bud
Terminal leaf bud
Flower bud
Terminal leaf bud
Axillary bud
Unknown
Nodular, terminal or axial shoot bud
galls with fine dense pubescence
Nodular, terminal or axial shoot bud
galls with fine pubescence
‘leaf gall’
Terminal leaf bud
Flower bud
Small pea-like unilocular galls on
young stems and leaf buds
Flower bud
Unknown
Unilocular shoot bud gall
Terminal or axial shoot bud galls;
densely convoluted finely pubescent
‘leaf gall’
Terminal leaf
Axial shoot bud
Flower bud
‘stem-tip’
Gall type
Canberra
Naracoorte (SA)
Vic. (Currie, 1937)
Abercrombie River (NSW)
Kimberley Region (WA)
Follows high-elevation snow
gum distribution
India
Sydney (NSW)
Heavily sclerotized, with raised
spicules and 7–8 teeth
Only known from pupa: 2 patches
heavily sclerotized cuticle
Unknown
9 separate transverse sclerotized
bands
Several patches of sclerotized cuticle
with patterns of raised ridges and
spicules
Unknown
5 narrow black sclerotized plates
5 broad black sclerotized plates
Coastal north Qld
Coastal north Qld
Unknown
7 (sometimes 8) separate transverse
sclerotized bands
Heavily sclerotized with plates
lacking teeth
Heavily sclerotized with two or three
teeth
Unknown
Unknown
Small with
about 20 sparse raised sclerotized spicules
7 broad black sclerotized plates
Black sclerotized plate with 3–5
anterior projecting teeth with
raised sclerotized spicules
Unknown
4 chitinous plates in two pairs
Dorsal shield
Emerald (Vic)
Follows low-elevation snow
gum distribution
Canberra (ACT)
Clare (SA)
Perth (WA)
Vic
Sydney (NSW) (Malloch,
1932)
New Zealand
Northern Qld
South QLD, VIC, SA and WA.
Mundaring (WA)
Batemans Bay (NSW)
Canberra (ACT)
Distribution
curriei on E. camaldulensis)
(Fisher & Nickle, 1968)
Unknown
Unknown
Unknown
jambophila Siddiqi 1986
Unknown
nervosae Davies &
Giblin-Davis 2004
cajuputiae Davies &
Giblin-Davis 2004
Unknown
Unknown
juliae Davies 2012
gomphocephalae Davies
2012a
Unknown
Unknown
pohutakawa Davies 2007
dealbatae Davies &
Giblin-Davis 2004
brittenae Davies 2010 (on
E. camaldulensis)
Unknown
Unknown
Fergusobia sp.
704
L. A. NELSON ET AL.
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
E. camaldulensis (Davies et al.,
2012a)
Unknown
Axial bud ‘stem’ galls
Flower bud
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
E. delegatensis (Davies et al., 2013a)
E. diversifolia (Davies et al., 2013a)
Angophora floribunda (Davies et al.,
2013a)
E. tereticornis (Davies et al., 2013a)
Angophora nr. woodsiana (Davies
et al., 2013a)
Unknown
Unknown
Unknown
Unknown
Unknown
*AS1, AS2 = abdominal segments 1, 2.
**TS1, TS2 = thoracic segments 1, 2.
E. porosa (Davies et al., 2013b)
E. cosmophylla (Davies et al., 2013a)
Unknown
Unknown
Large leaf bud gall
Large leaf bud gall
Large leaf bud gall
Large leaf bud gall
Large leaf bud gall
Large leaf bud gall
Flat leaf gall
Unilocular leaf ‘pea’ galls
E. fibrosa (Davies et al., 2012b)
Unknown
Stigma gall
Flat leaf gall
E. fasciculosae (Davies et al., 2012b)
Unknown
Flower bud
Corymbia sp. (Davies et al., 2012a)
E. eugenioides (Davies et al., 2012b)
Unknown
n/a
E. microcarpa (Davies et al., 2013b)
Found in soil (Siddiqi, 1986)
Unknown
Flower bud
Unknown
E. deglupta (Siddiqi, 1994; Davies
et al., 2010b)
Unknown
Flower bud
Flat leaf
Terminal and axial bud ‘stem’ galls
Terminal leaf bud
Terminal or axial shoot bud and
flower bud
Unknown
E. leucoxylon (Davies & Lloyd, 1996)
E. deglupta (Siddiqi, 1994; Davies
et al., 2010b)
C. tessellaris (Siddiqi, 1986; Davies
et al., 2010a)
Unknown
Unknown
E. baxteri species complex
williamsensis
Nelson &
Yeates 2011
Unknown
M. quinquenervia (Davies & Giblin-Davis, 2004)
M. fluviatilis (Davies & Giblin-Davis, 2004)
turneri Taylor
2004
Pimpama (QLD)
Sydney (NSW)
Mudgee (NSW)
Meningie (SA)
Ben Lomond (TAS)
Mylor (SA)
Strathalbyn (SA)
Adelaide (SA)
Woodburn (NSW)
Adelaide (SA)
Shute Harbour (QLD)
Goolwa (SA)
Canberra (ACT)
India
Philippines
Bulolo (PNG)
Adelaide (SA)
Coastal Qld
Grampians (Vic)
Coastal Qld
North coastal NSW
Absent
Heavily sclerotized with two or three
teeth
Absent
Eight or 9 broad transverse rows of
sclerotized raised spicules
Seven or 8 broad transverse rows of
sclerotized raised spicules
3 confluent cuticular plates with 6 to
9 short forwardly projecting teeth
2 patches heavily sclerotized cuticle
2 patches heavily sclerotized cuticle
Absent
Bars of spicules
3 confluent cuticular plates, with 2
forwardly projecting teeth and a
ridge on posterior margin of AS2
Cuticular plate of AS1 confluent with
short broad plate on AS2 and a
heavily sclerotized plate on TS1; 2
rows of 8–10 short teeth on AS1
3 confluent patches of sclerotized
cuticle
Unknown
Unknown
Unknown
2 patches heavily sclerotized cuticle
Shield restricted to a broad area of
weakly sclerotized spicules along
anterior margin TS2
8 separate transverse sclerotized
bands
6 narrow black sclerotized plates
pimpamensis
Davies 2013
minimus
Lisnawita 2013
floribundae
Davies 2013
diversifoliae
Davies 2013
delegatensae
Davies 2013
cosmophyllae
Davies 2013
porosae Davies 2013
microcarpae
Davies 2013
rileyi Davies 2012
camaldulensae Davies 2012
morrisae Davies 2012
fasciculosae Davies 2012
eugenioidae Davies 2012
indica (Jairajpuri 1962)
Siddiqi 1986
philippinensis Siddiqi 1994
brevicauda Siddiqi 1994
fisheri Davies & Lloyd 1996
magna Siddiqi 1984 sensu
Davies 2010
Unknown
quinquenerviae Davies &
Giblin-Davis 2004
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
705
706
L. A. NELSON ET AL.
Table 3. A list of Fergusonina eucalypt hosts sorted by the taxonomic treatment of Brooker, Slee & Connors (2006)
Species
Subgenus
Angophora costata
Angophora floribunda
Angophora subvelutina
Corymbia trachyphloia
Corymbia papuana
Corymbia tessellaris
Corymbia torelliana
Corymbia citriodora
Corymbia maculata
Corymbia abbreviata
Corymbia gummifera
Corymbia intermedia
Corymbia ptychocarpa
Eucalyptus acmenoides
Eucalyptus amygdalina
Eucalyptus coccifera
Eucalyptus elata
Eucalyptus nitida
Eucalyptus tenuiramis
Eucalyptus baxteri
Eucalyptus eugenioides
Eucalyptus ligustrina
Eucalyptus macrorhyncha
Eucalyptus delegatensis
Eucalyptus haemastoma
Eucalyptus pauciflora
Eucalyptus racemosa
Eucalyptus sieberi
Eucalyptus obliqua
Eucalyptus stricta
Eucalyptus planchoniana
Eucalyptus marginata
Eucalyptus diversifolia
Eucalyptus stellulata
Eucalyptus olsenii
Eucalyptus cloeziana
Eucalyptus coolabah
Eucalyptus pruinosa
Eucalyptus albens
Eucalyptus intertexta
Eucalyptus largiflorens
Eucalyptus microcarpa
Eucalyptus moluccana
Eucalyptus odorata
Eucalyptus polybractea
Eucalyptus populnea
Eucalyptus porosa
Eucalyptus crebra
Eucalyptus fibrosa
Eucalyptus melanophloia
Eucalyptus siderophloia
Eucalyptus baueriana
Eucalyptus fasciculosa
Eucalyptus polyanthemos
Eucalyptus leucoxylon
Eucalyptus melliodora
Eucalyptus sideroxylon
Eucalyptus yalatensis
Eucalyptus zopherophloia
Angophora
Angophora
Angophora
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Corymbia
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Idiogenes
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Section
Subsection
Apteria
Blakearia (ghost gums)
Blakearia (ghost gums)
Cadagaria
Politaria (spotted gums)
Politaria (spotted gums)
Rufaria (red bloodwoods)
Rufaria (red bloodwoods)
Rufaria (red bloodwoods)
Rufaria (red bloodwoods)
Amentum (white mahoganies)
Aromatica (peppermints)
Aromatica (peppermints)
Aromatica (peppermints)
Aromatica (peppermints)
Aromatica (peppermints)
Capillulus (stringy barks)
Capillulus (stringy barks)
Capillulus (stringy barks)
Capillulus (stringy barks)
Cineraceae
Cineraceae
Cineraceae
Cineraceae
Cineraceae
Eucalyptus (the ashes)
Eucalyptus (the ashes)
Insolitae
Longistylus
Longistylus
Longitudinales
Nebulosa
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Adnataria
Bisectae
Bisectae
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
(the
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
boxes
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
and
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
ironbarks)
Series
Arboreae
Frutices
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Apicales
Terminales
Terminales
Terminales
Terminales
Terminales
Terminales
Destitutae (pith glands absent)
Glandulosae (pith glands present)
Aquilonares
Aquilonares
Buxeales
Buxeales
Buxeales
Buxeales
Buxeales
Buxeales
Buxeales
Buxeales
Buxeales
Siderophloiae
Siderophloiae
Siderophloiae
Siderophloiae
Heterophloiae
Heterophloiae
Heterophloiae
Melliodorae
Melliodorae
Melliodorae
Subulatae
Accedentes
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
707
Table 3. Continued
Species
Subgenus
Section
Subsection
Series
Eucalyptus platypus
Eucalyptus loxophleba
Eucalyptus
gomphocephala
Eucalyptus lesouefii
Eucalyptus blakelyi
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Bisectae
Bisectae
Bolites
Glandulosae (pith glands present)
Glandulosae (pith glands present)
Erectae
Loxophlebae
Symphyomyrtus
Symphyomyrtus
Eucalyptus dealbata
Symphyomyrtus
Eucalyptus tereticornis
Symphyomyrtus
Eucalyptus lockyeri
Symphyomyrtus
Eucalyptus camaldulensis
Symphyomyrtus
Eucalyptus rudis
Symphyomyrtus
Eucalyptus cupularis
Symphyomyrtus
Dumaria
Exsertaria
gums)
Exsertaria
gums)
Exsertaria
gums)
Exsertaria
gums)
Exsertaria
gums)
Exsertaria
gums)
Exsertaria
gums)
Eucalyptus cosmophylla
Eucalyptus robusta
Symphyomyrtus
Symphyomyrtus
Incognitae
Latoangulatae
Eucalyptus interstans
Symphyomyrtus
Eucalyptus
parramattensis
Eucalyptus bridgesiana
Eucalyptus globulus
Eucalyptus johnstonii
Eucalyptus dalrympleana
Eucalyptus viminalis
Eucalyptus aromaphloia
Eucalyptus nicholii
Eucalyptus aggregata
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Liberivalvae (red
from the ovary
Liberivalvae (red
from the ovary
Maidenaria
Maidenaria
Maidenaria
Maidenaria
Maidenaria
Maidenaria
Maidenaria
Maidenaria
Eucalyptus ovata
Symphyomyrtus
Maidenaria
Triangulares
Eucalyptus
Eucalyptus
Eucalyptus
Eucalyptus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Maidenaria
Platysperma
Platysperma
Sejunctae
Triangulares
mannifera
brevifolia
confluens
cladocalyx
(red gums and white
Rufispermae
Erythroxylon
(red gums and white
Erythroxylon
(red gums and white
Erythroxylon
(red gums and white
Phaeoxylon
(red gums and white
Rostratae
(red gums and white
Singulares
(red gums and white
Subexsertae
(white
gums)
Annulares
(red
mahoganies)
gums; disc free
roof)
gums; disc free
roof)
on gall type, host plant species and dorsal shield
morphology of the third-instar fly larva provides considerable information on which Fergusonina/
Fergusobia pair is present within a given gall (Davies
et al., 2010b).
PLANT
HOST DIVERSITY
A summary of plant host records for the Fergusonina/
Fergusobia mutualism on Myrtaceae was given by
Davies et al. (2010b), and is updated here (Table 3).
Euryotae
Euryotae
Euryotae
Euryotae
Euryotae
Triangulares
Triangulares
Triangulares
Bridgesianae
Globulares
Semiunicolores
Viminales
Viminales
Acaciiformes
Acaciiformes
Foveolatae
(swamp
gums)
Foveolatae
(swamp
gums)
Microcarpae
Confirmed Fergusonina/Fergusobia galls have been
reported from more than 80 species of eucalypts
(Angophora, Corymbia and Eucalyptus), 12 Melaleuca,
one Metrosideros, two Leptospermum and two
Syzygium species. These plant taxa represent only five
(Melaleuceae, Eucalypteae, Syzygieae, Leptospermeae
and Metrosidereae) of the 17 Myrtaceae tribes in
Wilson’s (2011) classification.
Figure 3, adapted from Thornhill et al. (2012),
shows the relationships between these five tribes, and
the divergence times between them. A number of very
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
708
L. A. NELSON ET AL.
Figure 3. Chronogram of the tribes of Myrtaceae adjusted from Thornhill et al. (2012). Shaded tribes highlight
Fergusonina hosts and show that no sister tribes in Myrtaceae have been found to host the flies. The figures next to each
tribe name indicate how many species have been recorded as hosts versus the number of species recognized in the tribe.
*Tribes that do not have any representative taxa in Australia.
diverse tribes of Myrtaceae are not yet recorded as
hosts of the Fergusonina/Fergusobia mutualism and
it is likely that only a fraction of the diversity of the
association is known. For example, only two of 632
species of Syzygium have been recorded with galls,
and a systematic survey of the genus would probably
reveal many more host species. Syzygium is an important component of Old World tropical rainforest
flora (Biffin et al., 2006), and its species richness and
lineage diversity are centred in the Australasian
region. New Guinea has about 200 species of
Syzygium, Malaya 200 species and Borneo 150
species (Biffin et al., 2006) but Australia and the
South Pacific are relatively species-poor. Fergusobia
jambophila is recorded as galling shoot buds on
S. cumini in India (Siddiqi, 2000). However, no
fergusoninid flies were found in a survey of fruits
collected from S. paniculatum or S. australe in New
South Wales (Juniper & Britton, 2010).
Furthermore, Leptospermeae and its sister tribe
Chamelaucieae occur over much of Australia, yet only
two Leptospermum species are recorded as hosts.
Other Myrtaceae tribes such as Backhousieae,
Tristanieae, Kanieae, Syncarpieae, Lophostemoneae
and Xanthostemoneae could also be hosts. These
tribes occur in tropical or wet forests of Australasia.
If galls occur on any of these rainforest tribes they
may provide important insights into the evolutionary
development of the mutualism.
The Fergusonina/Fergusobia mutualism has most
commonly been found on Angophora, Corymbia and
Eucalyptus, including the two largest sub-genera of
the last named. There are some eucalypt groups,
however, with few or no species recorded as hosts
(see Fig. 3 and Table 3). In particular, none of the
22 species of the Eucalyptus sub-genus Eudesmia
has been recorded as a host and only four of the
250 species in the Eucalyptus sub-genus Symphyo-
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
myrtus section Bisectae. Some unidentified physiological characteristics (e.g. semiochemicals, leaf
toughness) of these clades may prevent colonization
by Fergusonina/Fergusobia. On the other hand,
some eucalypt groups seem to favour the mutualism,
such as the Eucalyptus sub-genus Symphyomyrtus
sections Exsertaria (red gums), Adnataria (boxes and
ironbarks) and Maidenaria, and the sub-genus Eucalyptus section Cineraceae (peppermints) (Table 3).
GALL
PHENOLOGY
Mature galls of most well-studied Fergusonina/
Fergusobia mutualisms generally appear to occur
annually, often during the cooler winter and spring
months, which in southern Australia may also be
wetter. In a two-year study of F. turneri on
M. quinquenvervia in northern New South Wales and
south-eastern Queensland, Australia, populations
were found to follow an annual cycle correlated with
temperature and bud density, but not rainfall
(Goolsby et al., 2000). Gall numbers were highest in
the cooler winter months of August and September,
when new bud growth is abundant. In South Australia, the phenology of E. camaldulensis was
observed over a 2-year period, and the densities of
three gall forms developing on that host was also
highly seasonal (Head, 2008). Greatest density of
growing points, axial leaf buds (galled by Fergusonina
sp. with Fb. camaldulensae) and flower buds (galled
by F. tillyardi with Fb. curriei) occurred in mid-winter
to spring (July to September). In contrast, most terminal bud galls (F. lockharti with Fb. brittenae) were
found from mid-spring to summer (October to February) (Head, 2008). The occurrence of Fergusonina
galls on high-elevation snow gums was also seasonal,
with mature galls occurring in spring (September to
November) (Nelson et al., 2011b). The spatial distribution of galls within a host tree is affected by the
distribution of suitable meristematic tissues (growing points, axial leaf and flower buds). Different
Fergusonina species differ in their preferred location
on host trees. In a study of galling species on
E. camaldulensis in South Australia, most terminal
leaf bud galls occurred on the northern and eastern
quadrants of trees, while axial leaf bud galls and
flower bud galls occurred more in western and southern quadrants (Head, 2008).
The galls of some Fergusonina/Fergusobia mutualisms appear to be less seasonal and/or less abundant
than others. Obviously, species may vary in life histories and abundances because of any number of
ecological and evolutionary factors, but some variation could be due to climactic aseasonality and/or
extremes caused either directly or indirectly through
effects on host plants. In addition, the prevalence
709
and seasonality of species-specific (or gall-specific)
parasitoids (generally Hymenoptera) and inquilines
(generally Lepidoptera) attacking the fergusoninids
and their galls could influence species abundances as
well as lead to natural selection on seasonality
(Currie, 1937; Taylor, Austin & Davies, 1996).
CECIDOGENESIS
The process of gall formation, or cecidogenesis, is a
neoplastic outgrowth of plant tissue as a defensive
mechanism following herbivory (Schick & Dahlsten,
2003). It involves complex interactions between
plants and causative organisms, with highly specific
reciprocal adaptations between plant host and gallinducer (e.g. Stone & Schönrogge, 2003; Raman,
2011). The chemical interactions between gallforming insects and their host plants are poorly
understood (Raman, 2010). Gall-forming insects
commonly show preference for undifferentiated
meristematic plant tissue as oviposition sites (e.g.
Mani, 1964; Fritz et al., 1987; Price, Fernandes &
Waring, 1987; Raman, 2010). Little is known about
how Fergusonina flies select an oviposition site.
Their oviposition behaviour may be influenced by a
combination of visual and olfactory cues including
semiochemicals and plant hormones (auxins, e.g.
indole-acetic acid), which are particularly prevalent
in meristematic tissue (Raman, 2010). Oviposition
preference in the shoot fly (Atherigona soccata) in
seedling sorghum is positively correlated with nitrogen content of the growing tip (Ogwaro & Kokwaro,
1981). Most Fergusonina/Fergusobia galls occur
on young foliage, presumably with higher nitrogen
content (Larsson & Ohmart, 1988; Edwards &
Wanjura, 1991).
Experimental injection of juvenile Fergusobia
nematodes into shoot buds (Giblin-Davis et al., 2001b)
led to some gall and nematode development. Thus,
the nematode appears to induce gall development, but
the fly larva is responsible for the internal structure
of the gall. Nematodes failed to develop in vitro on
callus culture (Head, 2008). Given their strong stylet,
it is most likely that Fergusobia nematodes feed on
plant material (Giblin-Davis et al., 2001b, 2004a),
although Currie (1937) suggested that they may also
feed on excretions of the co-occurring fly larvae.
In recent years, considerable efforts have been
made to understand the process of cecidogenesis
in gall-forming pest nematodes. All plant-parasitic
nematode genomes examined to date (e.g. Bert et al.,
2008; Bird, Williamson & Abad, 2009; Dieterich &
Sommer, 2009; van Megen et al., 2009; Kikuchi et al.,
2011) have genes coding for secreted enzymes that
degrade cell walls, which may have originated from
horizontally transferred bacterial or fungal genes
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
710
L. A. NELSON ET AL.
(Smant et al., 1998; Blaxter, 2007; Davis, Hussey &
Baum, 2009; Kikuchi et al., 2011). Understanding the
process of cecidogenesis in pest nematodes such as
the cyst and root-knot nematodes will provide
a molecular model against which gall-formation in
Fergusonina/Fergusobia can be examined and compared. Like Fergusobia, these pest nematodes are
tylenchids. However, phylogenetic analyses suggest
that gall-formation has evolved independently on
more than one occasion within the tylenchids (Bert
et al., 2008; van Megen et al., 2009), and differing
mechanisms of cecidogenesis may occur.
The role of the dorsal shield of the larval
fergusoninids in cecidogenesis is unknown. It is probably used as a mechanical scraper within gall locules
(Taylor, 2004), expanding the locule, possibly determining internal gall architecture, and/or producing
pelletized food material for the fly larvae and/or
nematodes (Giblin-Davis et al., 2004b). Plant material
is often seen wedged under the hooks of larvae possessing them.
COORDINATION
OF FLY/NEMATODE LIFE CYCLES
Clearly, the life cycles of the fly and the nematode are
closely coordinated (Fig. 1). The cues which regulate
this coordination and its evolution are unknown, but
may be hormonal. For example, in the beetle parasite
Contortylenchus brevicomi, fourth-stage juvenile
nematodes were stimulated to moult while within the
body cavity of the host larva (Gibb & Fisher, 1986,
1989), but ecdysis was inhibited by high concentrations of juvenile hormone in the host, and nematodes
moulted once they left the larva. Fergusobia nematodes parasitic in the pupae of the flies may be
stimulated to moult by the high concentrations of
ecdysteroid known to be present in dipteran puparia
(Walker & Denlinger, 1980), which could be tested in
vitro.
Between one and 50 juvenile nematodes were associated with Fergusonina eggs deposited in flower buds
(Giblin-Davis et al., 2004a). Apparently, nematodes
are deposited passively with eggs during oviposition
(Currie, 1937) and it is not clear whether the fly is
able to regulate the number of nematodes deposited
with each egg.
Nematodes occur only in female larvae, pupae and
flies (Davies et al., 2001; Giblin-Davis et al., 2001b;
Scheffer et al., 2013). Sex pheromones may be recognized in the selection of female Fergusonina larvae
and pupae. Nematodes select for female hosts in other
systems, including the tylenchid Sphaerularia bombi
which only invades queen bumblebees (Poinar &
Van Der Laan, 1972) and the fig-wasp–nematode
tritrophic interactions in Ficus sycones (Krishnan
et al., 2010).
How Fergusobia nematodes enter third-instar
Fergusonina larvae is unknown. Common routes for
nematode invasion of insect larvae include penetration through the cuticle or via spiracles, mouth or
anus (e.g. Triggiani & Poinar, 1976; Georgis & Hague,
1981). Nematode entry is not likely via the highly
sclerotized, convoluted spiracles of Fergusonina
larvae. The nematode suborder Hexatylina, which
includes Fergusobia, contains nematodes parasitizing
insects and other invertebrates. Those seen penetrating host insects have enlarged pharyngeal glands and
apparently use secretions from them to weaken the
host cuticle, and stylet thrusts to cut a hole for entry
(Welch, 1959; Poinar & Doncaster, 1965; Bedding,
1972; Poinar et al., 1993). The reduced stylet and
smaller pharyngeal glands of pre-parasitic Fergusobia
(Davies & Giblin-Davis, 2004) does not suggest penetration of the Fergusonina larvae via the cuticle.
They may enter via the larval anus.
A female nematode developing with a male fly larva
in a unilocular gall will be unable to develop into a
parasitic female and reproduce. However, it is unclear
if nematodes within a multilocular gall in locules
with male larvae are at a reproductive dead-end. It
is likely that nematodes can move between locules,
especially given observations of coalescing locules
(Giblin-Davis et al., 2004a) at later stages in the gall
life cycle. Because adult male flies do not have nematodes, there can be no sexual transfer of nematodes
between fly sexes (Scheffer et al., 2013).
Between one and 15 parasitic female nematodes
develop per female larva or fly (Currie, 1937; Davies
et al., 2001; Giblin-Davis et al., 2001b; Pratt et al.,
2013). It is not known if or how the number of
nematodes per female is regulated to prevent
overexploitation of the host. Hundreds to thousands
of infective juvenile nematodes and nematode eggs
occur in the haemocoel of the abdomens of adult
female flies, but the juveniles have cuticles suggesting
that they do not absorb nutrients from the fly. Variation in nematode densities per female fly could mean
that species interdependency is context-dependent
(Pratt et al., 2013), i.e. that low nematode numbers
could result in low or irregular transference rates
during oviposition and limit gall development and
hence fly performance, and that high densities may
negatively influence survival or fitness of the host fly.
This will be difficult to test.
The mechanism underlying evasion or suppression
of the fly immune system by the nematodes is
not known, but is a further indication of complex
coevolutionary interactions between Fergusonina and
Fergusobia. The models developed to explain immune
suppression in parasitoid wasp/host interactions may
well prove useful in studies of how Fergusobia parasitic females and juveniles in the haemocoel of
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
Fergusonina flies escape or evade the fly immunological system (Schmidt & Theopold, 2004; Schmidt et al.,
2005; Schmidt, 2007). A major difference, however, is
that in the case of the Fergusonina/Fergusobia
immune interactions, it is in the best interest of the
fly to facilitate immune suppression in some way,
while in the case of a parasitized insect it is not. An
alternative model would be that of the immune
evasion system present in some filarial nematodes
(Maizels et al., 2001).
HOST-PLANT INTERACTIONS WITH
FERGUSONINA/FERGUSOBIA
It is well known that plant chemistry may influence
the ecology and speciation of phytophagous insects,
and both inter- and intraspecific variation can be
important (e.g. Jolivet, 1998). Recent work shows that
susceptibility to herbivory can vary even within a
single Eucalyptus tree (Padovan et al., 2013). About
20–25% of insects associated with eucalypts are herbivores (Majer, Recher & Keals, 1999), and many of
these have high degrees of specificity for particular
subgenera and groups of species, e.g. gall-forming
eriococcid scale insects (Cook, 2001; Cook & Gullen,
2004). Fergusonina/Fergusobia mutualisms are a
further example, with each usually specific to a single
host species. However, very little is known about the
constraints that determine this specificity.
The Myrtaceae are well known as sources of essential oils and other plant secondary compounds
(Lassak & McCarthy, 1983; Brophy & Doran, 1996;
Wheeler, 2007). Such compounds typically provide
defence against generalist insect and other herbivores
by being distasteful and/or toxic. However, specialist
insects adapted to tolerate the secondary compounds
of their host plants may in fact use the presence of
these compounds for host location and recognition
(Jolivet, 1998). Although tissues of host plants may
contain high levels of noxious compounds, galls containing herbivorous insects commonly have modified
levels of plant secondary as well as nutritive compounds within their tissues, particularly in the modified cells lining the gall upon which the gallers
feed (Hartley, 1998). Many of the host plants of the
Fergusonina/Fergusobia mutualism are known to
contain high levels of secondary compounds. For
example, in the following known hosts, leaves of E.
loxophleba contain sideroxylonal (Foley & Lassak,
2004); those of the Eucalyptus subgenus Symphyomyrtus contain formylated phloroglucinal compounds
(Eschler et al., 2000); E. cladocalyx, E. leucoxylon,
E. polyanthemos, E. viminalis, E. diversifolia and
E. ovata contain the glycoside prunasin (Gleadow
et al., 2008); and M. quinquenervia has the terpenoids
E-nerolidol and viridiflorol (Giblin-Davis et al., 2005).
711
In fact, in Fergusonina/Fergusobia galls, oil glands
are usually external to the individual gall locules
(Giblin-Davis et al., 2004b), suggesting that the
mutualists are able to avoid oil gland involvement or
integration during cecidogenesis. Thus, the secondary
compounds are more likely to have a role as attractants for the flies and to function in host selection than
to deter gall development.
The only direct evidence that we have of resistance
to the Fergusonina/Fergusobia mutualism comes
from a field observation (Giblin-Davis et al., 2001b). In
M. quinquenervia, hypersensitive responses to oviposition by F. turneri followed large numbers of ovipositions in one bud. Thus, plants may react to oviposition
by the fly/nematode mutualism, but whether this is a
specific response to Fergusonina oviposition or a generalized wound response is not known.
Leaf ‘toughness’ (resistance to fracture per unit
fracture area) (Ohmart & Edwards, 1991; Steinbauer,
Clarke & Madden, 1998) may function in defence
against some gall formers. As eucalypt foliage ages,
leaf ‘toughness’ increases and nitrogen content
declines (Larsson & Ohmart, 1988; Edwards &
Wanjura, 1991). However, as Fergusonina/Fergusobia
mutualists are usually found associated with young
foliage or flower buds (Currie, 1937; Goolsby,
Makinson & Purcell, 2000; Taylor & Davies, 2010) and
fly larvae and nematodes feed within galls, ‘toughness’
may not have a role in defence in this system.
Because many gall-forming nematodes are economically important plant pathogens, plant resistance to them has been extensively studied (e.g.
Williamson & Kumar, 2006). Sequencing of the
genomes of some of these, followed by mapping, will
lead to identification of genes involved in virulence
and pathogenicity. This knowledge could be used
in studies of host selection by the Fergusonina/
Fergusobia mutualists.
FLY–NEMATODE
COSPECIATION
Cospeciation (co-cladogenesis) between closely interacting organisms has been studied extensively over
the past several decades, resulting in the common
view that cospeciation is most likely in cases of vertical (rather than horizontal) transmission of the
interaction (Moran & Baumann, 2000; Page, 2003;
Hosokawa et al., 2006). The Fergusonina/Fergusobia
mutualism appears to be an exceptional candidate for
cospeciation given the apparent strict vertical transmission of nematodes between fly generations (Davies
et al., 2010b). However, recent molecular evidence
suggests that large multilocular galls can have multiple conspecific fly foundresses, potentially allowing
for horizontal transfer of nematodes between offspring of different females (Purcell, 2012). Switching
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
712
L. A. NELSON ET AL.
of nematode lineages between conspecific fly lineages
could occur frequently in the many Fergusonina/
Fergusobia mutualisms that form large multilocular
galls (Giblin-Davis et al., 2004b; Ye et al., 2007; Taylor
& Davies, 2010; Davies et al., 2010b; Purcell, 2012).
Potential departure from the theory of strict vertical
transmission of nematodes has evolutionary implications for the Fergusonina/Fergusobia mutualism and
adds complexity to the evolutionary dynamics. Occasional host-switches by the nematode may also help
explain anomalies observed in comparative analyses
of molecular datasets (Ye et al., 2007; Davies et al.,
2010b).
Mating between nematodes associated with the
offspring of different fly foundresses, even when
conspecific, could result in increased genetic diversity
in the nematodes of multilocular galls as compared
with nematodes in unilocular galls, as Fergusobia
species forming unilocular galls from a single fly
female would never have an opportunity for moving
between fly lineages (Taylor & Davies, 2010). Having
reduced genetic variation could influence the evolutionary potential of nematode species for adaptation
to environmental challenges as well as for host shifts
to new plant species. It should be possible to compare
the genetic variability of nematodes from multilocular
galls with that of nematodes from unilocular galls.
SPECIATION
AND EVOLUTION OF HOST-SELECTION
Host range may affect diversification rates of ecologically specialized gall-forming insects (Hardy & Cook,
2010). In the case of the Fergusonina/Fergusobia
mutualisms, most are highly specialized and found
only on one or a few closely related host plant species
(Davies et al., 2010b; Nelson et al., 2011a), suggesting
that speciation events typically occur in conjunction
with a shift to a new host plant species or tissue type.
Because the female Fergusonina fly chooses new
oviposition sites for galling, it is the behaviour and
host plant/tissue specificity of the flies that provide
the initial micro-evolutionary variation upon which
speciation processes can act. The ability of the nematodes both to initiate and to function within the gall
is critical and a major source of selection on the
oviposition choices made by the female flies. A female
fly that oviposits onto a new plant species or plant
tissue type in which the nematodes cannot function
normally will have lower fitness than the flies choosing their normal host.
Little work has been undertaken on host choice for
the fly/nematode mutualism. In the only work published to date, Wright et al. (2013) made a series
of no-choice oviposition and development tests to
assess host use by the F. turneri/F. quinquenerviae
mutualism in Florida. Oviposition and gall develop-
ment was assessed on eight species of Myrtaceae
native to Florida, eight species phylogenetically
related to its natural host M. quinquenervia and five
non-myrtaceous species. Female flies did not probe
any non-myrtaceous species, but did probe 11 of 16
tested Myrtaceae. Fly eggs and nematodes were
deposited in four of these. However, galls developed
and matured only on M. quinquenervia. Thus, the
mutualism has a narrow range of host choices, and
evidence that the fly/nematode mutualism is highly
host-specific, with one or at the most two host plant
species, is supported.
There are many circumstances that could lead to
fly oviposition on a non-host plant species or tissue,
most notably a lack of suitable oviposition sites (e.g.
Wright et al., 2013). The extreme and often unpredictable weather patterns common in Australia may
greatly influence the timing and general availability
of oviposition sites present on any particular host
plant species. In the absence of suitable sites on a
certain host plant species or tissue, a female may
‘dump’ her eggs on whatever non-host is available
(e.g. Kostal, 1993; Messina, Morrey & Mendenhall,
2007; Wright et al., 2013). This may provide an initial
impetus for host shifting onto new host plant taxa or
tissue types. Oviposition sites on non-traditional host
plants or tissue types would lead to strong selection
on larval and nematode performance to incorporate
the plant as a new host. In turn, this could lead
to population divergence and speciation under a
wide variety of genetic models and scenarios, e.g.
sympatric, parapatric and ecological speciation
(Tilmon, 2008; Butlin, Bridle & Schluter, 2009).
One of the key questions about the Fergusonina/
Fergusobia mutualism is whether phylogenies of the
nematode–fly associations are congruent with the
radiation of their host plants. The family Myrtaceae
comprises 17 tribes, 130–150 genera and nearly 6000
species. It is widespread in the southern hemisphere
with centres of diversity in Australia, South-East Asia
and South America (Wilson, 2011). The most recent
estimated crown age of the family is the Late Cretaceous (∼85 Mya) (Thornhill et al., 2012), and most
tribes had split from each other by the Oligocene (see
Fig. 4). Of the five tribes that have been found to
be hosts of the Fergusonina/Fergusobia mutualism,
none are sister groups, and divergence times between
these host tribes date back to the Late Cretaceous,
Palaeocene and Eocene. Recent dating of the fly
radiations suggests that Fergusoninidae diverged
from its sister family Asteiidae around 42 Mya
(Wiegmann et al., 2011), and a crown age for the
family is unknown but is younger than 42 Mya. Thus,
the Fergusoninidae did not cospeciate with the
Myrtaceae tribes on which they occur and evolved
when most tribes of Myrtaceae had already diversi-
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TRITROPHIC COEVOLUTION BETWEEN FLIES AND NEMATODES
713
Figure 4. Dendrogram of the eucalypts based on A. H. Thornhill et al. (unpubl. data) showing how the major subgenera
and sections are related to each other and where the concentration of Fergusonina host species occurs in the phylogeny.
fied. The dating of the Fergusonina phylogeny should
provide us with a clearer timeframe of when the
group radiated around Australia. While the coevolution of Fergusonina on Myrtaceae at the broadest
level is precluded because of the different geological
ages of the groups, coevolution within smaller subgroups of flies and plants that have diversified in
more recent times (e.g., in the last 10–15 Mya) is still
possible.
Similarity in the molecular sequences and morphology of the dorsal shields of the Fergusonina flies and
their associated Fergusobia nematodes, respectively
from flat leaf galls on host species from Eucalyptus
section Adnataria and terminal shoot bud galls on host
species from section Exsertaria, suggests cospeciation
(Davies et al., 2010b, 2013a, b). Dated phylogenies of
these host Eucalyptus groups are needed to confirm
this potential radiation with host species.
Clearly, the development of a Fergusonina/
Fergusobia gall depends on the close biological relationship between the mutualists themselves that
has evolved over millions of years, and their ability
to initiate and produce galls on a particular host
Myrtaceae species and tissue. Speciation and diversification in the Fergusonina/Fergusobia mutualism
probably involve some combination of cospeciation
with the host plants, speciation tracking host plant
phylogeny, and speciation via host-shifts and changes
to the tissue type selected for galling. Determining
the relative importance of these and their associated
mechanisms will require a multidisciplinary approach
including molecular systematics, phytochemistry,
population genetics, behaviour and evolutionary
ecology. Our understanding of speciation and diversification in the Fergusonina/Fergusobia/host plant
system is still in its earliest stages.
CONCLUSIONS
While understanding of the Fergusonina/Fergusobia
association is limited, its life cycle is established, and
it is known that flies and nematodes have a complex
and obligatory mutualism, each of which is generally
host-specific. At present, little is known of fly behaviour, host choice, the process of cecidogenesis, factors
determining host resistance or the selection pressures
operating on the mutualism. However, the genetic
tools needed to analyse many of these are now available, and models against which hypotheses can be
tested are becoming increasingly available.
Plant-feeding insects comprise an extraordinary
proportion of extant biodiversity (Strong, Lawton &
Southwood, 1984; Mitter, Farrell & Wiegmann, 1988;
Schoonhoven, van Loon & Marcel, 2005), and provide
important and compelling examples of evolution.
Given the complex and obligate nature of the
Fergusonina/Fergusobia mutualism, and their relationships with their myrtaceous hosts, the development of robust, dated, phylogenies for all three
trophic levels will provide a unique study system for
cospeciation and coevolution. A robust phylogeny
of both flies and nematodes at deep levels is necessary
for co-phylogenetic comparisons, but is only available for the flies. Such phylogenies will also allow
© 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 699–718
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L. A. NELSON ET AL.
us to further understand the evolutionary and
coevolutionary history of host-use, gall-formation and
factors such as development of the dorsal shield.
The apparent vertical transmission of Fergusobia
across Fergusonina generations led to the prediction
of congruent phylogenies between Fergusonina and
Fergusobia (Giblin-Davis et al., 2004b; Ye et al., 2007;
Davies et al., 2010b). However, the two phylogenetic
studies of the flies and nematodes published to date
(Scheffer et al., 2004; Ye et al., 2007) did not show
strict congruence when compared ad hoc. Genetic
evidence that multilocular galls may have multiple
foundresses possibly allowing for lateral transmission
of nematodes provides at least one mechanism for a
departure from strict cospeciation, and others are no
doubt awaiting discovery. We now predict that strict
co-phylogeny between the fly and nematodes will be
found more often in clades of unilocular gallers rather
than multilocular gallers where nematodes can probably move between locules.
Recent studies providing divergence time estimates
for both the Myrtaceae and the Diptera show that
the fly family Fergusoninidae is much younger than
the Myrtaceae, thus rejecting the hypothesis of
cospeciation of the flies and the plants at deep levels.
We expect that tritrophic co-phylogeny between the
Fergusoninidae, their mutualist nematodes and their
myrtaceous host plants will most likely be found at
low taxonomic levels in the Myrtaceae, such as within
clades of host Eucalyptus or Corymbia species that
have been diversifying more recently than the 42 Mya
crown age of Fergusoninidae.
ACKNOWLEDGEMENTS
Special thanks to Ted and Barb Center, Paul Pratt,
Greg Wheeler, Scott Blackwood, Phil Tipping, Susan
Wright, Weimin Ye and Dorota Porazinska among
many others at the USDA IPRL (Invasive Plant
Research Lab) and the University of Florida, IFAS
FLREC in Fort Lauderdale, Florida, for their help and
support during studies of this amazing group of flies
and nematodes for deployment as a biological control
agent against M. quinquenervia in the United States.
Some of the research summarized in this article was
supported by USDA Special Grants in Tropical and
Subtropical Agriculture (CRSR-99-34135-8478 and
CRSR-03-34135-14078) to R.G.D.; and by Australian
Biological Resources Studies grants to K.A.D.
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