Six new species of Asphondylia (Diptera: Cecidomyiidae) damaging flower buds and fruit of Australian Acacia (Mimosaceae)

Authors


  • Unpublished for the purposes of zoological nomenclature (Art. 8.2, ICZN)

Abstract

Six species of gall midge are described from Australian acacias. Asphondylia bursicola Kolesik sp.n. and A. occidentalis Kolesik sp.n. form galls on fruit; A. germinis Kolesik sp.n., A. pilogerminis Kolesik sp.n. and A. glabrigerminis Kolesik sp.n. induce severe deformation of flower buds; and A. acaciae Kolesik sp.n. causes galls on both fruit and flower buds. Galled flower buds do not produce flowers, and galled fruit produce no or undeveloped seeds. Host ranges of the new species comprise between two and eight acacia hosts. Larval, pupal and male morphology, together with phylogenetic analyses of a 410-bp fragment of the mitochondrial cytochrome b gene, were used to characterize the new species. For A. bursicola, A. germinis, A. pilogerminis, A. glabrigerminis and A. acaciae, the intraspecific divergence values were between 0.2 and 3.4%, and the interspecific divergence values ranged between 5.1 and 10.5%. For A. occidentalis, the only species with geographical distribution confined solely to Western Australia, the intraspecific divergence was between 6.6 and 10.3%, and the interspecific difference from the other five new species was between 9.3 and 13.9%. In contrast to Dasineura spp. from Acacia, for which the morphology was more informative in species recognition than the cytochrome b sequence, in Asphondylia spp. treated here the partial cytochrome b sequence data provided better species recognition than did the morphology. Several of the new Asphondylia have potential as biological control agents in ecosystems in which Australian acacias are invasive and their sexual reproduction needs to be restricted. A list of Australian acacias whose reproductive organs are destroyed by known gall midges, all belonging to Dasineura and Asphondylia, is provided.

Introduction

Australian acacias are utilized worldwide as garden ornamentals, and in some countries are cultivated for agro-forestry purposes, fodder production, land reclamation and production of edible seeds (Vercoe, 1989; Thomson et al., 1994; Doran & Turnbull, 1997; Maslin et al., 1998; Turnbull et al., 1998). Inevitably, as a consequence of widespread redistribution, some Australian acacias have naturalized beyond areas of intentional cultivation, causing considerable economic and environmental harm, and are consequently subject to control programs. In South Africa, 13 species of Australian acacias became targets of biological control (Henderson, 2001), and currently there is a range of phytophagous guilds being utilized or tested to weaken their fitness and seed production (Dennill et al., 1999; Adair, 2004), including gall midges Dasineura dielsi Skuse used against Acacia cyclops (Adair, 2005) and D. rubiformis Kolesik recently introduced to control A. mearnsii (Adair, 2004; Impson et al., 2008). Acacia is Australia's largest plant genus, with close to 1000 described species (Maslin, 2001) incorporating a diverse range of morphological and ecological adaptations that enable species to occupy an extraordinary range of habitats. Accordingly, the phytophagous fauna associated with Acacia appears to be similarly diverse (New, 1984) and includes a rich assemblage of gall-forming Cecidomyiidae that utilizes the reproductive organs of the host plant (Adair, 2004; Kolesik et al., 2005). Dasineura dominates the Cecidomyiidae fauna of Australian acacias and frequently co-occurs with Asphondylia, often on the same tree, inflorescence or even the same flower head. Asphondylia is a large cosmopolitan genus with almost 300 species worldwide (Gagné, 2004), including 15 from Australia (Kolesik & Veenstra-Quah, 2008). Until now, no Asphondylia had been described from Australian Acacia. This paper is a second taxonomic report from a survey of potential biological control agents of Australian acacias and describes six new species of Asphondylia that feed on flower buds or fruit and prevent seed production.

Materials and methods

Collection and preparation of galls and insects

Between 1998 and 2008, 141 Australian Acacia species were surveyed for cecidomyiid galls on flowers and fruit at 502 field sites in Western Australia, South Australia, Victoria, Tasmania, New South Wales, Australian Capital Territory and Queensland. If cecidomyiid galls were located, the plant organs affected, gall condition and stage of insect development were recorded, and insect voucher specimens were collected. At selected sites, representative samples of fresh, mature galls were used to annotate gall size, shape, colour, external and internal morphology, and number of individual flower galls forming gall clusters on flower heads. The position and colour of immature cecidomyiid stages (larvae, pupae) within galls were recorded, and representative samples were either preserved in 70% ethanol or reared to adults for mounting and identification. Newly emerged adults were collected and preserved in 70% ethanol. Pupal skins were collected and stored dry in gelatine capsules. Permanent microscope slides were prepared for type series and other material. For each accession, five or six specimens each of larvae, pupae (or pupal skins), females and males were macerated in 15–20% KOH (larvae and pupae were perforated laterally with a thin needle to improve maceration), washed in 20% acetic acid, 70% and then 99% ethanol, cleared in Histoclear or clove oil, and mounted in Canada balsam on glass slides under round glass coverslips 10 mm in diameter. Whole larvae and pupae were mounted dorsoventrally. Adults were dissected into four pieces, with the particular body parts mounted separately: wings and head frontally, thorax laterally, abdomen dorsoventrally or for some of the female specimens laterally. The type series were deposited in the South Australian Museum, Adelaide (SAMA) and the Australian National Insect Collection, Canberra (ANIC).

Morphology

Insect morphology was investigated with bright-field and phase-contrast microscopy. Length measurements were made with a digital imaging system and refer to type series unless stated otherwise. Drawings were made with the aid of a microscope drawing tube. Terminology of adults and pupae follows Gagné (1981) and that of larvae follows Gagné (1989). States of seven larval, three pupal and one male morphological characters were used to delineate differences between the six new species (Table 1). Seven morphological characters were informative about phylogenetic relationships. Binary coding was employed for the morphological characters that were treated as unordered multistate characters. Phylogenetic analyses were executed in paup*4 version beta 10 (Swofford, 2002) using maximum parsimony (MP). Trees were generated using the heuristic search option with tree-bisection-reconnection (TBR) branch swapping and stepwise addition using 1000 random sequence addition replicates. Nodal support was assessed from MP analysis of 10 000 non-parametric bootstrap replicates (full heuristic search; ‘as is' stepwise addition of taxa). Trees were rooted using Asphondylia mcneilli Kolesik & Veenstra-Quah, an Australian species that induces flower gall on Sclerolaena diacantha (Nees) Benth. (Chenopodiaceae), as an outgroup.

Table 1. Matrix of morphological characters for Asphondylia spp.
CharacterL1L2L3L4L5L6L7P1P2P3M1
  1. Characters in larva (L), pupa (P) and male (M).

  2. L1 Antennae short (0) or long (1).

  3. L2 Antennae tapering at distal half (0) or tapering at full length (1).

  4. L3 Number of lateral papillae 3 (0), 4 (1) or 5 (2).

  5. L4 Anterior third of spatula narrow (0) or wide (1).

  6. L5 Spatula with incision between inner and outer teeth shallower (0) or deeper (1) than incision between inner teeth.

  7. L6 Spatula with inner height of inner teeth larger than (0), as large as (1) or smaller that (2) inner height of outer teeth.

  8. L7 Corniform papillae of terminal segment small (0) or large (1).

  9. P1 Antennal horns pointed (0), tapered (1) or obtuse (2).

  10. P2 Distance between anterior lower horns and posterior lower horn longer (0) or shorter (1) than between anterior lower horns.

  11. P3 Inner edges of antennal horns smooth (0) or serrated (1).

  12. M1 Gonostyle in posterior view 1.5× (1) or less than 1.5× (0) longer than wide.

A. acaciae 00010011000
A. germinis 00010012000
A. pilogerminis 11110001000
A. bursicola 11100010000
A. glabrigerminis 11110101000
A. occidentalis 11111200111
A. mcneilli 11210111210

DNA extraction, amplification, sequencing and phylogenetic analysis

A 410-bp fragment of the mitochondrial cytochrome b gene was amplified, sequenced and aligned for representative accessions of the six Asphondylia species described in this study. Total DNA was extracted from immature stages or adults using a standard phenol-chloroform extraction protocol (Sambrook et al., 1989). For very small specimens for which phenol-chloroform extraction was considered unfeasible, the entire pupa or larva was added to the polymerase chain reaction (PCR) reaction tube. DNA extraction, amplification and sequencing followed the detailed procedures described in Kolesik et al. (2005). Contarinia loti (AY017535) and Dasinuera rubiformis (AY278712) were designated as outgroups to the Asphondylia ingroup species. Sequences have been deposited in GenBank, and the accession numbers with associated field data are given in ‘DNA analysis'. Field data for sequences of accessions that were examined morphologically are given in ‘Types’ or ‘Other material examined’. MP analyses of morphological and DNA sequences were performed in paup*4 version beta 10. Trees were generated using the heuristic search option with TBR branch swapping and stepwise addition using 1000 random sequence addition replicates. Maximum likelihood (ML) analysis was performed in phyml version 2.4.5 (Guindon & Gascuel, 2003) with the AIC-selected GTR +Γ model of sequence evolution (Yang, 1994). To evaluate nodal support, 1000 non-parametric bootstrap replicates were evaluated in paup*4 (MP) and phyml (ML). In this study, nodes with bootstrap values < 50% are considered as not supported, bootstrap values between 50 and 70% as weakly supported, and bootstrap values > 70% as strongly supported.

Key to immature stages of Asphondylia inducing flower bud and fruit galls on Australian Acacia

Until now, no asphondylias had been described from Australian acacias. In addition to the six new species included in the key we know of several undescribed Asphondylia species attacking Australian acacias, all causing damage to the reproductive organs of the host plant (Adair et al., 2000). First instar Asphondylia larvae, not identified to species level, were found to cause flower bud galls on Acacia nilotica (L.) Willd. ex Del., A. hockii De Wild., A. reficiens Wawra and A. tortilis (Forsk.) Hayne in Kenya (Gagné & Marohasy, 1993). These larvae have three lateral papillae on each side and thus would key to couplet 2 containing Asphondylia acaciae and A. germinis. Although until now no Asphondylia had been described from Acacia flowers or fruit worldwide, two species are known to feed on leaves of non-Australian acacias: Asphondylia trichocecidarum Mani causes hairy spherical galls on Acacia leucophloea Willd. in India (Mani, 1934), and Asphondylia napiformis Gagné induces turnip-shaped galls on Acacia mellifera (Vahl) Benth. in Kenya (Gagné & Marohasy, 1993). While the larva of A. trichocecidarum is not known, the description of A. napiformis is based solely on larvae that have three setose lateral papillae on each side and would key to couplet 2 in the key given below. The shape of the spatula in A. napiformis differs from that in A. acaciaesp.n. and A. germinissp.n. The key is based on larvae and pupae. For additional morphological characters distinguishing the six new species see Table 1.

  • 1Larva with three lateral papillae per side (Fig. 3P).…… 2
  • Larva with four lateral papillae per side (Fig. 4G)…… 3
  • 2Pupal antennal horns acute (Fig. 3M)…A. acaciaesp.n.
  • Pupal antennal horns blunt (Fig. 4D)…A. germinissp.n.
  • 3Anterior part of larval spatula narrow (Fig. 4K), pupal antennal horns pointed and smooth (Fig. 4L).……………………………………..A. bursicolasp.n.
  • Anterior part of larval spatula wide (Fig. 4G), pupal antennal horns tapered and smooth (Figs 4H, P) or serrated mesally (Fig. 5H).……………………………… 4
  • 4Pupa with antennal horns serrated mesally (Fig. 5H) and anterior lower horns closer to each other than to posterior lower horn (Fig. 5F), larval spatula with incision between inner and outer teeth deeper than incision between inner teeth (Fig. 5C).…………………A. occidentalissp.n.
  • Pupal antennal horns smooth (Fig. 4H, P) and anterior lower horns closer to posterior lower horn than to each other (Fig. 3L), larval spatula with incision between inner and outer teeth shallower than incision between inner teeth (Fig. 4O) ...................................................5
  • 5Larval spatula with inner height of inner teeth larger than inner height of outer teeth (Fig. 4G).……………… . …………………………….. A. pilogerminissp.n.
  • Laval spatula with inner height of inner teeth as large as inner height of outer teeth.……. A. glabrigerminissp.n.
Figure 3.

Asphondylia acaciae from Acacia retinoides. A–G, male; H–J, female; K–N, pupa; O–R, larva. A, Head; B, wing; C, gonostyle posteriorly; D, last three flagellomeres; E, genitalia dorsally; F, first tarsomere; G, last six flagellomeres; H, end of abdomen laterally; I, end of ovipositor laterally; J, end of abdomen dorsally; K, last abdominal segment dorsally; L, head ventrally; M, antennal horns ventrally; N, prothoracic spiracle; O, head; P, sternal spatula with adjacent papillae; R, terminal segment dorsally.

Figure 4.

A–D, Asphondylia germinis from Acacia xanthina; E–H, A. pilogerminis from Acacia baileyana; I–L, A. bursicola from Acacia mearnsii; M–P; A. glabrigerminis from Acacia mearnsii. A, larval head; B, larval terminal segment dorsally; C, larval sternal spatula with adjacent papillae; D, pupal antennal horns ventrally; E, larval head; F, larval terminal segment dorsally; G, larval sternal spatula with adjacent papillae; H, pupal antennal horns ventrally; I, larval head; J, larval terminal segment dorsally; K, larval sternal spatula with adjacent papillae; L, pupal antennal horns ventrally; M, larval head; N, larval terminal segment dorsally; O, larval sternal spatula with adjacent papillae; P, pupal antennal horns ventrally.

Figure 5.

Asphondylia occidentalis from Acacia pentadenia. A, larval head; B, larval terminal segment dorsally; C, larval sternal spatula with adjacent papillae; D, male gonostyle posteriorly; E, male gonostyle dorsally; F, pupal head ventrally; G, pupal prothoracic spiracle; H, pupal antennal horns ventrally.

Descriptions of species

Genus Asphondylia Loew

Asphondylia (as subgenus of Cecidomyia Meigen) Loew, 1850: 21 and 37 Citation list after 1850 in Gagné (2004), Veenstra-Quah et al. (2007) and Kolesik & Veenstra-Quah (2008)

Type species. Cecidomyia sarothamniLoew, 1850

Diagnosis.

Asphondylia contains species with the first tarsomere bearing a ventrodistal spine, the ovipositor with a pair of large basal lobes, female flagellomeres 9–12 progressively shorter, the gonocoxite with a ventro-apical lobe and a dorsally situated gonostyle that is about as wide as long and bears 2 basally merged teeth.

Asphondylia is a cosmopolitan genus currently comprising nearly 300 species. Fifteen species have been described from Australia previously, with 12 of them attributed to known host plants–A. dodonaeae feeds on Dodonaea viscosa (Sapindaceae), A. inflata on Halosarcia pergranulata, A. ericiformis on H. indica, A. floriformis and A. sarcocorniae on Sarcocornia quinqueflora, A. vesicaria on Atriplex vesicaria, A. mcneilli on Scleroleana diacantha, A. tonsura on Enchylaena tomentosa (Chenopodiaceae), A. anthocercidis on Anthocercis littorea, A. paucidentata on Solanum aviculare and S. lineafolium, A. obscura on S. chenopodioides and S. physalifolim, A. sturtiana on S. sturtianum (Solanceae) (Kolesik, 1995, 1997; Kolesik et al., 1997, 2000; Veenstra-Quah et al., 2007; Kolesik & Veenstra-Quah; 2008).

Characters shared by all six species

Larva.

Head capsule strongly sclerotized, with no postero-lateral extensions. Spatula with four anterior teeth, shaft broadened at both mid-length and base, depending on species, with three or four lateral papillae per side, surrounded anteriorly and laterally by extensive sclerotized area. Six terminal papillae comprising one corniform pair and two setose pairs, position and number of papillae varying among specimens in some species. Small sclerotized area surrounds corniform and one pair of setose papillae in some species. Anus positioned terminally. Colour of the last instar orange.

Pupa.

Antennal horns prominent. One upper and three lower frontal horns. One setose and one asetose papilla to either side of lower frontal horns. One setose and one asetose lateral facial papilla. Prothoracic spiracle long, tapered, finely serrated distally, bent at half length, trachea reaching one-third total length. Abdominal dorsal spines simple. Colour: fresh pupa with non-sclerotized parts orange, sclerotized parts brown; mature pupa brown.

Male.

Head. Antenna: scape broadest distally, length around 1.5 × breadth at distal end, around 2 × length pedicel; pedicel wider than long; flagellomeres 12 in number, first flagellomere about 4 × length of scape; flagellomeres evenly cylindrical; circumfila dense, anastomosing, equally spread along segments. Eye facets close together, hexagonoid, eye bridge 9 to 11 facets long. Labella hemispherical. Palpus 3 segmented, first segment shortest, third slightly longer than second.

Thorax. Wing with C vein ending near wing apex, R1 vein ending slightly anteriorly to wing mid-length. Claws simple, as long as empodia. Ventro-distal spine on the first tarsomere long, bent at right angle at distal half.

Genitalia. Gonocoxite with ventro-apical lobe; gonostyle longer than wide in posterior view, apical teeth unequal in size; aedeagus elongate and narrow distally, as long as cerci; hypoproct with incision one-third height; cerci triangular.

Colour. Eyes, setae and scales black; sclerotized body parts brown; non-sclerotized parts of thorax and abdomen orange.

Female.

Head. Flagellomeres progressively shortened. Circumfila sparse, comprising two longitudinal bands connected by two transverse bands.

Abdomen.

Abdominal sternite 7 about 2 × longer than sternite 6. Depending on species, ovipositor needle 1.4–1.7 × longer than sternite 7. Basal lobes hemispherical in dorso-ventral view, with shallow incision.

Otherwise as in male.

Asphondylia acaciae Kolesik sp.n.

(Figs 1A–D, 2E, 3)

Figure 1.

Galls of Asphondylia on Acacia spp. A, Asphondylia acaciae galled buds of Acacia melanoyxlon; B, Asphondylia acaciae galled buds of Acacia verticillata; C, Asphondylia acaciae galled buds of Acacia longifolia; D, Asphondylia acaciae galled fruit of Acacia longifolia; E, Asphondylia bursicola galled fruit of Acacia mearnsii; F, Asphondylia glabrigerminis galled buds of Acacia mearnsii with protruding pupal exuviae; G, longitudinal section of Asphondylia glabrigerminis galled bud of Acacia irrorata showing pupa in development chamber; H, longitudinal section of Asphondylia glabrigerminis galled bud of Acacia mearnsii showing compacted melanised fungal hyphae and pycnidia developing in lower right corner; I, Asphondylia occidentalis galled fruit of Acacia littorea (right hand side of photo); J, Asphondylia occidentalis galled fruit of Acacia pentadenia; K, Asphondylia pilogerminis galled buds of Acacia parvipinnula with pupal exuviae protruding from two galls; L, Asphondylia pilogerminis galled buds of Acacia mearnsii.

Figure 2.

Galls of Asphondylia on Acacia spp., external and internal morphology. A, Asphondylia seminis galled fruit on Acacia mearnsii, B, longitudinal section of seed gall of Asphondylia seminis on Acacia mearnsii; C, Asphondylia pilogerminis galled bud on Acacia mearnsii, left gall with emergence hole; D Asphondylia glabrigerminis galled bud on Acacia mearnsii; E, Asphondylia acaciae galled bud on Acacia verticillata; F, dissected bud gall of Asphondylia glabrigerminis on Acacia mearnsii; note melanized hyphal layer in left gall and larva amongst enlarged stamens at bottom of right gall.

Types.

Victoria, Australia. Holotype male, Gunamatta (38° 25′S, 144°52′E), ex fruit gall on Acacia retinoides collected 21.X.1998 (RJA2578), 29-002949 (SAMA). Paras: two males, three females, two pupae, two larvae (SAMA, 29-002950 to 29-002958), two males, two females, one pupa, two larvae (ANIC), collected with holotype.

Other material examined.

RJA2635: New South Wales, Australia, Myall Lakes (32°30′S, 152°18′E), ex bud gall on Acacia floribunda collected 29.ix.1998, one male, one pupa. RJA2730: New South Wales, Australia, Tenterfield (29°62783′S, 152°1115′E), ex bud gall on Acacia floribunda collected 24.iv.1999, one female, one pupal skin. RJA2650: Victoria, Australia, Lilydale (37°45′S, 145°22′E), ex bud gall on Acacia verticillata collected 17.ix.1998, ten males, five females, five pupae, ten larvae. RJA2765: Victoria, Australia, Malmsbury (37°10′S, 144°21′E), ex bud gall on Acacia melanoxylon collected 2.ix.1999, five larvae. RJA2772: New South Wales, Australia, Sydney Royal National Park (34°06365′S, 151°0586′E), ex bud gall on Acacia ulicifolia collected 27.ix.1999, five males, five females, six pupal skins. RJA2649: Victoria, Australia, Bittern (38°58148′S, 145°3032′E), ex fruit gall on Acacia longifolia subsp. sophorae collected 24.i.1998, five males, three females, three pupal skins. RJA2896: Victoria, Australia, Frankston (38°20177′S, 145°2795′E), ex fruit gall on Acacia longifolia subsp. sophorae collected 28.i.1999, one male, two females, six pupae, six larvae. RJA2666: Victoria, Australia, Bittern (38°58148′S, 145°3032′E), ex bud gall on Acacia paradoxa collected 6.iii.1999, six pupae, six pupal skins, six larvae. RJA2656: Victoria, Australia, Elphinstone (37°06′S, 144°20′E), ex bud gall on Acacia paradoxa collected 2.ix.1998, four males, five pupae, six larvae. RJA2615: Western Australia, Australia, Perth (32°07′S, 115°45′E), ex fruit gall on Acacia cyclops collected 31.x.1998, one pupal skin. RJA2771: Western Australia, Australia, Yanchep NP (31°54488′S, 115°6841′E), ex fruit gall on Acacia cyclops collected 26.vii.1999, two larvae.

DNA analysis.

RJA3193 (GenBank accession number AY277743): New South Wales, Australia, Uki (28°48735′S, 153°2179′E), ex bud gall on Acacia floribunda collected 9.iv. 2004. RJA 2578 (GenBank accession number AY277768), RJA2650 (GenBank accession number AY277757), RJA2765 (GenBank accession number AY277762), RJA2772 (GenBank accession number GQ1217766), RJA2649 (GenBank accession number AY277763), RJA2666 (GenBank accession number AY277759).

Description.

Larva (Fig. 3O–R). Length 2.53 mm (2.05–3.34 mm). Antennae short, tapering at distal half. Sternal spatula with anterior third wide, incision between inner and outer teeth shallower than incision between inner teeth, inner height of inner teeth longer than inner height of outer teeth. Lateral papillae 3 per side. Corniform papillae on terminal segment as long as adjacent setae. Pupa (Fig. 3K–N). Length 3.09 mm (2.91–3.35 mm). Antennal horns tapered, inner edges smooth. Distance between anterior lower facial horns and posterior lower facial horn longer than distance between anterior lower horns. Female (Fig. 3G–J). Wing length 3.19 mm (3.14–3.30 mm), width 1.23 mm (1.16–1.30 mm). Ovipositor needle 1.5 × (1.3–1.7 ×) longer than sternite 7. Male (Fig. 3A–F). Wing length 2.92 mm (2.40–3.14 mm), width 1.17 mm (1.00–1.30 mm). Gonostyle 1.2 × longer than wide (Fig. 3C).

Etymology.

The name is derived from the generic name of the host plants. Common name: ‘bud and fruit galler’.

Biology and distribution (Figs 1A–D, 2E).

Larvae of A. acaciae induce galls on flower buds and fruit of several species of Acacia from the sections Phyllodineae, Pluirnerves and Juliflorae in southern Australia. Bud galls develop from buds within a compound flower head. Mature bud galls are small (mean length 2.2 mm, mean width 2.9 mm, n = 15) and vary in shape from oblong-elliptic, obovate to oblate. The exterior surface is glabrous to sparsely pubescent and usually with a nipple-like mucro on the apex. Galls contain single larvae that pupate within the spherical chamber. The pupa emerges from the gall and the exuvium remains lodged in the gall wall for a short period following adult's emergence. The other type of gall induced by A. acaciae, the fruit gall, occurs on immature pods where larvae are intimately associated with developing ovules. Early instar larvae feed on the inner wall of the locule before moving to ovules where they are associated with a fungal mantle of the symbiont Botrysphaeria dothidea (Adair et al., 2009). The larvae prevent the ovule from development, and the mature larva is generally found within the partial remains of the ovule. Pupation occurs within the locule, and adults emerge through the wall of green mature fruit. Infected locules become slightly enlarged and distorted, but often can be difficult to distinguish superficially from normal fruit. Fruit with A. acaciae generally will dehisce normally to the point of the first locule containing a larva; thereafter dehiscence is prevented. Asphondylia acaciae is multivoltine and may alternate between buds and fruit of suitable host species. Although gall densities can be high on occasional individual hosts, most populations are sparse to rare, probably as a result of parasitoid activity.

Host plants.

Acacia retinoides, A. floribunda, A. verticillata, A. melanoxylon, A. ulicifolia, A. longifolia subsp. sophorae, A. paradoxa, A. cyclops. Possible hosts (see details in ‘Collections not ascertained to species'): A. truncata, A. rubida.

Asphondylia bursicola Kolesik sp.n.

(Figs 1E, 4I–L)

Types.

Victoria, Australia. Holotype male, Bundoora (37° 71828′S, 145°0465′E), ex fruit gall on Acacia mearnsii collected 16.x.1998 (RJA2597), 29-002959 (SAMA). Paras: two males, three females, three pupae, three larvae (SAMA, 29-002960 to 29-002970), two males, two females, two pupae, two larvae (ANIC), collected with holotype.

Other material examined.

RJA2575: Australian Capital Territory, Australia, Canberra (35°27994′S, 149°11095′E), ex fruit gall on Acacia mearnsii collected 9.xi.1998, three males, five females, four pupae, six pupal skins, two larvae. RJA2775: New South Wales, Australia, Mittagong (34°43803′S, 150°4701′E), ex fruit gall on Acacia deanei collected 29.ix.1999, three males, five females, four pupae, six pupal skins. RJA2664: New South Wales, Australia, Braidwood (35°32064′S, 149°8765′E), ex fruit gall on Acacia decurrens collected 20.xii.1998, five males, five females, five pupae, one larva. RJA2870: New South Wales, Australia, Sydney Royal National Park (34°06365′S, 151°0587′E), ex fruit gall on Acacia irrorata collected 27.ix.1999, four pupal skins.

DNA analysis.

RJA2597 (GenBank accession number AY277752), RJA2775 (GenBank accession number AY277767). RJA3171 (GenBank accession number AY277753): New South Wales, Australia, Holbrook (35°93412′S, 147°1349′E), ex fruit gall on Acacia decurrens collected 13.xii.2000. RJA3005 (GenBank accession number AY277755): New South Wales, Australia, Bateman's Bay (35°741′S, 150°1641′E), ex fruit gall on Acacia irrorata collected 9.vii.2000.

Description.

Larva (Figs 4I–K). Length 3.44 mm (3.30–3.73 mm). Antennae long, tapering at full length. Sternal spatula with anterior third narrow, incision between inner and outer teeth shallower than incision between inner teeth, inner height of inner teeth longer than inner height of outer teeth. Lateral papillae 4 per side. Corniform papillae on terminal segment as long as adjacent setae. Pupa (Fig. 4L). Length 4.48 mm (4.42–4.60 mm). Antennal horns pointed, inner edges smooth. Distance between anterior lower facial horns and posterior lower facial horn longer than distance between anterior lower horns. Female. Wing length 4.24 mm (4.18–4.30 mm), width 1.47 mm (1.42–1.53 mm). Ovipositor needle 1.7 × (1.5–1.7×) longer than sternite 7. Male. Wing length 3.67 mm (3.49–3.84 mm), width 1.29 mm (1.23–1.35 mm). Gonostyle 1.2 × longer than wide (Fig. 3C).

Etymology.

The name combines ‘bursa’, Latin for bag or pod, and ‘cola’, Latin for dweller, referring to the fact that larvae develop within fruit pods of the host plant. Common name: ‘bipinnate pod galler’.

Biology and distribution (Fig. 1E).

Asphondylia bursicola is restricted to eastern Australia and feeds on acacias in the section Botrycephalae (bipinnate acacias), particularly those close to Acacia mearnsii. Larvae develop in locules as described for fruit galls of A. acacia, and gall symptoms are also similar to those of fruit galls of A. acaciae. Adult A. bursicola emerge from mid-spring to mid-summer, when fruit is fully developed but still green. On most hosts, the next season's fruit is not present at the time of adult emergence, and it is unclear at what developmental stage this insect survives the inter-fruiting period. Asphondylia bursicola can be locally abundant, but is mostly occasional to rare on host trees. The westernmost occurrence of A. bursicola detected in this study was in the Grampians Mountains, Victoria, close to the westernmost natural occurrence of A. mearnsii. Unlike Dasineura rubiformis (Adair, 2004), A. bursicola is not present in Western Australia, where A. mearnsii, the insect's main host, is planted as shade and windbreak trees in the southwest region.

Host plants.

Acacia mearnsii, A. deanei, A. decurrens, A. irrorata.

Asphondylia germinis Kolesik sp.n.

(Fig. 4A–D)

Types.

Western Australia. Holotype pupa, Iloway (30° 47823′S, 115°1034′E), ex bud gall on Acacia xanthina collected 22.vii.1999 (RJA 2769), 29-002971 (SAMA). Paras: two pupae, three larvae (SAMA, 29-002972 to 29-002976), two pupae, two larvae (ANIC), collected with holotype.

DNA analysis.

RJA2769 (GenBank accession number AY277761). RJA3213 (GenBank accession number AY277746): Western Australia, Australia, Albany (35°09888′S, 117°9676′E), ex bud gall on Acacia littorea collected 19.iv.2002. RJA2614 (GenBank accession number AY277760): Western Australia, Australia, Maragret River (33°96683′S, 115°0172′E), ex bud gall on Acacia divergens collected 01.xi.1998. RJA3192 (GenBank accession number AY277764): New South Wales, Australia, Marulan (34°73712′S, 149°8342′E), ex bud gall on Acacia genistifolia collected 08.iv.2002. RJA3085 (GenBank accession number AY277747): Western Australia, Australia, Dwellingup (32°68372′S, 116°0391′E), ex bud gall on Acacia urophylla collected 19.vi.2001.

Description.

Larva (Fig. 4A–C). Length 2.95 mm (2.79–3.37 mm). Antennae short, tapering at distal half. Sternal spatula with anterior third wide, incision between inner and outer teeth shallower than incision between inner teeth, inner height of inner teeth longer than inner height of outer teeth. Lateral papillae 3 per side. Corniform papillae on terminal segment as long as adjacent setae. Pupa (Fig. 4D). Length 2.60 mm (2.35–2.91 mm). Antennal horns obtuse, inner edges smooth. Distance between anterior lower facial horns and posterior lower facial horn longer than distance between anterior lower horns. Female. Not known. Male. Not known. Microtrichia-covered part of gonostyle 1.2× longer than wide.

Etymology.

The name is derived from ‘germen’, Latin for bud, and refers to the host plant organ, the flower bud, malformed by the new species. Common name: ‘bud galler’.

Biology and distribution.

Asphondylia germinis galls flower buds of Phyllodineae acacias and is widely distributed in southwest Western Australia, but with a disjunct occurrence on Acacia genistifolia in eastern Australia. It is likely that additional hosts in eastern Australia will be confirmed for A. germinis with more collecting. Larvae of A. germinis form galls in a similar manner to as described for bud galls of A. acaciae. Galls range in shape from elliptic to oblong to obovate. The apex is acute to broadly obtuse with a nipple-like mucro. The external gall surface is glabrous to densely pubescent or hirsute. Adult emergence occurs mostly from April to July.

Host plants.

Acacia xanthina, A. littorea, A. divergens, A. genistifolia, A. urophylla. Possible hosts (see details in ‘Collections not ascertained to species'): A. truncata, A. rubida.

Asphondylia glabrigerminis Kolesik sp.n.

(Figs 1F–H, 2D, 2F, 4M–P)

Types.

Victoria, Australia. Holotype pupa, Bundoora (37° 73278′S, 145°0465′E), ex glabrous bud gall on Acacia mearnsii collected 10.viii.1998 (RJA2620), 29-002977 (SAMA). Paras: one female, two pupae, five larvae (SAMA, 29-002978 to 29-002985), one pupa, five larvae (ANIC) collected with holotype.

Other material examined.

RJA2637: New South Wales, Australia, Braidwood (35°32406′S, 145°8763′E), ex glabrous bud gall on Acacia deanei collected 24.ix.1998, four males, five females, six pupal skins. RJA3008: New South Wales, Australia, Braidwood (35°32406′S, 145°8763′E), ex glabrous bud gall on Acacia deanei collected 09.vii.2000, five larvae. RJA2745: Victoria, Australia, Molesworth (37°16608′S, 145°54592′E), ex glabrous bud gall on Acacia mearnsii collected 10.ix.1998, five females, five pupal skins. RJA2781: New South Wales, Australia, Mittagong (34°45367′S, 150° 4346′E), ex glabrous bud gall on Acacia mearnsii collected 29.ix.1999, five males, five females, six pupal skins.

DNA analysis.

RJA2637 (GenBank accession number AY277749), RJA3008 (GenBank accession number AY277756). RJA3016 (GenBank accession number AY277758): New South Wales, Australia, Casula (33°95327′S, 150°878′E), ex glabrous bud gall on Acacia mearnsii collected 12.vii.2000.

Description.

Larva (Fig. 4M–O). Length 2.55 mm (2.21–2.91 mm). Antennae long, tapering at full length. Sternal spatula with anterior third wide, incision between inner and outer teeth of equal depth, inner height of inner teeth as long as inner height of outer teeth. Lateral papillae 4 per side. Corniform papillae on terminal segment shorter than adjacent setae. Pupa (Fig. 4P). Length 4.19 mm (4.05–4.42 mm). Antennal horns tapered, inner edges smooth. Distance between anterior lower facial horns and posterior lower facial horn longer than distance between anterior lower horns. Female. RJA2637–wing length 3.63 mm (3.49–3.86 mm), width 1.32 mm (1.28–1.33 mm). Ovipositor needle 1.5 × (1.4–1.6×) longer than sternite 7. Male. RJA2637–wing length 3.20 mm (3.07–3.37 mm), width 1.20 mm (1.14–1.26 mm). Gonostyle 1.2 × longer than wide (Fig. 3C).

Etymology.

The name is derived from ‘glabra’ and ‘germen’, Latin for hairless and bud, respectively, and refers to the gall appearance. Common name: ‘glabrous bud galler’.

Biology and distribution (Figs 1F–H, 2D, 2F).

Asphondylia glabrigerminis is restricted to Botrycephaleae acacias in eastern Australia. Larvae develop in flower buds and form globose galls that contract abruptly into a distinct nipple-like mucro on the apex. Slight lateral lobes are apparent in some specimens. Galls are green and mostly wholly glabrous, although occasionally sparsely pubescent with appressed white hairs. Galls are small (mean length 3.2 mm, mean width 3.1 mm, n = 36) and usually develop in clusters of up to 28 galls per flower head. Asphondylia glabrigerminis is univoltine and adults emerge from September to October. The main host species, Acacia mearnsii and A. deanei, are in bud at the time of adult emergence. Adults rest on foliage during the day and swarm around the canopy of the host in the late afternoon. Oviposition occurs on immature to well-developed buds. The fungal gall symbiont Botryosphaeria dothidea forms pycnidia in the gall wall in some seasons, and conidia are present at the time of adult emergence (Adair et al., 2009). Asphondylia glabrigerminis often co-occurs with A. pilogerminis on the same host tree, inflorescence or flower head. Abundant populations of A. glabrigerminis occur sporadically, but usually only persist for up to three successive seasons before declining as a result of parasitoid activity.

Host plants.

Acacia mearnsii, A. deanei.

Asphondylia occidentalis Kolesik sp.n.

(Figs 1I–J, 5)

Types.

Western Australia. Holotype male, Denmark-Walpole (34°92708′S, 116°57739′E), ex fruit gall on Acacia pentadenia collected 14.ix.2007 (RJA3404), 29-002986 (SAMA). Paras: two females, two pupal skins (SAMA, 29-002987 to 29-002990), one male, one female, two pupal skins (ANIC), collected with holotype.

Other material examined.

RJA3087: Western Australia, Australia, Shannon-Walpole (34°60028′S, 116°4375′E), ex fruit gall on Acacia pentadenia collected 23.vi.2001, five larvae (SAMA 29-02987 to 29-02990). RJA2768: Western Australia, Albany (35°09888′S, 117°9676′E), ex fruit gall on Acacia littorea collected 29.vii.1999, five larvae. RJA3407: Western Australia, Albany (35°07973′S, 117°87542′E), ex fruit gall on Acacia littorea collected 15.ix.2007, two males, three females, five pupal skins.

DNA analysis.

RJA3087 (GenBank accession number AY277750). RJA3209 (GenBank accession number AY277754): Western Australia, Australia, Kalbarri (27°69953′S, 114°175′E), ex fruit gall on Acacia rostellifera collected 20.iv.2002. RJA3210 (GenBank accession number AY277751): Western Australia, Australia, ‘The Loop’ (27°55672′S, 114°4347′E), ex fruit gall on Acacia ramulosa collected 20.iv.2002.

Description.

Larva (Fig. 5A–C). RJA3087–length 2.65 mm (2.33–3.35 mm). Antennae long, tapering at full length. Sternal spatula with anterior third wide, incision between inner and outer teeth deeper than incision between inner teeth, inner height of inner teeth shorter than inner height of outer teeth. Lateral papillae 4 per side. Corniform papillae on terminal segment shorter than adjacent setae. Pupa (Fig. 5F–H). RJA3087–length 4.72 mm (4.58–4.84 mm). Antennal horns pointed, inner edges serrated. Distance between anterior lower facial horns and posterior lower facial horn shorter than distance between anterior lower horns. Female. Wing length 4.00 mm (3.84–4.12 mm), width 1.38 mm (1.35–1.40 mm). Ovipositor needle 1.6 × (1.5–1.6×) longer than sternite 7. Male (Fig. 5D, E). Wing length 4.56 mm (4.42–4.70 mm), width 1.66 mm (1.58–1.74 mm). Gonostyle 1.5 × longer than wide (Fig. 3C).

Etymology.

‘Occidentalis’ is Latin for western and refers to the Australian distribution of the new species. Common name: ‘western pod galler’.

Remarks.

The high values of intraspecific divergence between the three A. occidentalis accessions indicate a barrier to gene flow between the populations from which the accessions were collected. As the three localities are in close proximity it is unlikely that the divergence is a result of geographic isolation. The fact that all pairwise divergence values for all three A. occidentalis accession are fairly high and that they do not cluster with any other sequence type but form a monophyletic group in the phylogenetic analysis indicates that the cytochrome b sequencing data accurately reflect the underlying diversity in this clade, possibly as a result of the existence of several related cryptic species.

Biology and distribution (Fig. 1I–J).

Asphondylia occidentalis is restricted to Western Australia, where it forms fruit galls on acacias in the sections Pulchellae and Phyllodineae. Gall symptoms are similar to those described for fruit galls of A. acaciae. The phenology of A. occidentalis is not well known, but adults emerge from galls in spring.

Host plants.

Acacia pentadenia, A. rostellifera, A. ramulosa, A. littorea.

Asphondylia pilogerminis Kolesik sp.n.

(Figs 1K–L, 2C, 4E–H)

Types.

Victoria, Australia. Holotype male, Pearcedale (38° 18147′S, 145°2188′E), ex pubescent bud gall on Acacia baileyana collected 17.viii.1998 (RJA2639), 29-002991 (SAMA). Paras: one female, four pupae, four larvae (SAMA, 29-002992 to 29-002300), three pupae, four larvae (ANIC), collected with holotype.

Other material examined.

RJA2782: New South Wales, Australia, Mittagong (34°45367′S, 150°4346′E), ex pubescent bud gall on Acacia mearnsii collected 29.ix.1999, three males, five females, six pupal skins. RJA2640: New South Wales, Australia, Doughboy (35°24858′S, 149°6697′E), ex pubescent bud gall on Acacia mearnsii collected 24.ix.1998, ten males, ten females, five pupae. RJA2641: Australian Capital Territory, Australia, Canberra (35°22211′S, 149°1765′E), ex pubescent bud gall on Acacia baileyana collected 22.ix.1998, five males, five females, five pupal skins. RJA2634: New South Wales, Australia, Braidwood (35°42811′S, 149°6989′E), ex pubescent bud gall on Acacia dealbata collected 22.ix.1998, five males, five females, seven pupal skins. RJA2653: Victoria, Australia, Bittern (38°3451′S, 145°1719′E), ex pubescent bud gall on Acacia dealbata collected 13.ix.1998, six males, three females, five pupae, ten larvae. RJA2654: Victoria, Australia, Bendigo (36°90769′S, 144°2187′E), ex pubescent bud gall on Acacia dealbata collected 3.ix.1998, six pupae, five larvae.

DNA analysis.

RJA2782 (GenBank accession number AY277744), RJA2641 (GenBank accession number AY277748). RJA3199 (GenBank accession number AY277765): New South Wales, Australia, Casino (28°86335′S, 152°7858′E), ex pubescent bud gall on Acacia irrorata collected 9.iv.2002. RJA3115 (GenBank accession number AY277745): Victoria, Australia, Stawell (37°36525′S, 143°16993′E), ex pubescent bud gall on Acacia baileyana collected 21.ix.2001. RJA3116 (GenBank accession number AY277766): Australia, Stawell (37°3652′S, 143°16993′E), ex pubescent bud gall on Acacia baileyana collected 21.ix.2001.

Description.

Larva (Fig. 4E–G). Length 2.96 mm (2.72–3.33 mm). Antennae long, tapering at full length. Sternal spatula with anterior third wide, incision between inner and outer teeth shallower than incision between inner teeth, inner height of inner teeth longer than inner height of outer teeth. Lateral papillae 4 per side. Corniform papillae on terminal segment shorter than adjacent setae. Pupa (Fig. 4H). Length 3.80 mm (3.53–4.07 mm). Antennal horns tapered, inner edges smooth. Distance between anterior lower facial horns and posterior lower facial horn longer than distance between anterior lower horns. Female. RJA2782–wing length 3.98 mm (3.63–4.14 mm), width 1.49 mm (1.40–1.56 mm). Ovipositor needle 1.4 × (1.3–1.4 ×) longer than sternite 7. Male. RJA2782–wing length 3.80 mm (3.60–4.07 mm), width 1.47 mm (1.42–1.51 mm). Gonostyle 1.2 × longer than wide.

Etymology.

The name is derived from ‘pilosus’ and ‘germen’, Latin for hairy and bud, respectively, and refers to the gall appearance. Common name: ‘pubescent bud galler’.

Biology and distribution (Figs 1K–L, 2C).

Asphondylia pilogerminis forms bud galls on Botrycephaleae acacias in eastern Australia. Galls are small (mean length 4.6 mm, mean width 2.5 mm, n = 36) oblong-elliptic to orbiculate. The exterior gall surface is typically hoary pubescent to densely pubescent with erect to antrorse white hairs. Asphondylia pilogerminis is multivoltine and may alternate between host species as a means of bridging the interbud period. It regularly co-occurs with A. glabrigerminis on Acacia mearnsii, but the galls of the two midges can be readily distinguished in the field by differences in the gall indumentum.

Host plants.

Acacia baileyana, A. mearnsii, A. irrorata, A. dealbata.

Collections not ascertained to species

The following four collections could not be ascertained to species and belong to either Asphondylia acaciae or A. germinis. RJA2618: Western Australia, Yalgorup (32°91231′S, 115°6959′E), ex bud gall on Acacia truncata collected 31.x.1998, three pupal skins. RJA2766: Western Australia, Cervantes (30°47823′S, 115°1034′E), ex bud gall on Acacia truncata collected 21.vii.1999, one pupal skin, five larvae. RJA2767: Western Australia, Cervantes (30°05′S, 115°1034′E), ex bud gall on Acacia truncata collected 21.vii.1999, one pupa. RJA2888: Western Australia, Nambung NP (30°56142′S, 115°1039′E), ex bud gall on Acacia truncata collected 21.vii.1999, six larvae. RJA2760: Australian Capital Territory, Canberra (35°22211′S, 149°1764′E), ex bud gall on Acacia rubida collected 25.vii.1998, five larvae.

Phylogeny

Morphology-based phylogeny

Of the 11 morphological characters recorded (Table 1), only seven were parsimony-informative, which is reflected in the generally unresolved tree topology recovered from parsimony analysis of these characters (Fig. 6). There was strong support for a sister-taxon relationship between A. acaciae and A. germinis, and some support for the basal position of A. occidentalis, but species relationships were otherwise unresolved.

Figure 6.

Strict consensus of four trees recovered from maximum parsimony analysis of morphological characters. Asphondylia mcneilli was used as the outgroup. The numbers above the branches represent bootstrap values from 10 000 bootstrap replicates. Excluding parsimony-uninformative characters, total tree length = 12, consistency index = 0.83, and homoplasy index = 0.67. The scale bar indicates the number of changes along a branch.

Cytochrome b-based phylogeny

Parsimony analysis of the partial cytochrome b sequences recovered 12 equally parsimonious trees, the strict consensus of which was well resolved and identical to the phyml maximum likelihood (ML) tree with respect to the species relationships among the six Asphondylia species (Fig. 7). All accessions identified to species level based on morphological and ecological traits as well as on life history formed monophyletic species groups based on DNA sequence analysis, regardless of the method of phylogenetic analyses used. Both ML and MP analyses recovered the sister-taxon relationship between A. acaciae and A. germinis, congruent with the morphology-based topology. MP and ML analyses also recovered a sister-taxon relationship between A. glabrigerminis and A. bursicola, although strong support for this node was lacking. MP and ML analyses recovered A. occidentalis as the most basal taxon with strong support. Uncorrected interspecifc divergence values between all Asphondylia species ranged between 9.3 and 13.9%, whereas intraspecific divergence values between all Asphondylia species except A. occidentalis ranged between 0.2 and 3.4% (Table 2). Interestingly, intraspecific divergence values within A. occidentalis ranged from 6.6 to 10.3%, indicating a high level of population substructuring, perhaps owing to the existence of cryptic species.

Figure 7.

Cladogram of 1 of 12 maximum-parsimony trees recovered from a heuristic search of cytochrome b sequence data. Tree length = 355, consistency index = 0.69, retention index = 0.79. The numbers represent maximum-parsimony bootstrap values > 50% followed by maximum-likelihood bootstrap values > 50%. GBG, glabrous bud gall; PBG, pubescent bud gall. The scale bar indicates the number of nucleotide changes along a branch.

Table 2. Nucleotide differences (below diagonal/asterisks) and uncorrected sequence divergences (above) for Asphondylia spp.
  1234567891011121314151617181920212223242526272829
  1. Accession numbers are indicated in parentheses.

1 A. acaciae (RJA3193) * 1.0%0.7%0.5%0.7%1.5%1.5%6.3%7.1%7.3%6.6%6.1%8.8%8.5%8.5%8.5%8.5%10.0%10.0%10.0%9.5%9.0%9.3%9.3%13.4%13.7%11.7%25.6%26.0%
2 A. acaciae (RJA2650)4 * 1.2%1.0%1.2%2.0%1.5%6.8%7.6%7.8%7.1%6.6%8.3%8.0%8.0%8.0%8.0%9.8%9.8%9.8%9.3%8.5%8.8%8.8%12.9%13.2%11.2%25.1%26.0%
3 A. acaciae (RJA2765)35 * 0.7%0.7%1.7%1.7%6.6%7.3%7.6%6.8%6.3%9.0%8.8%8.8%8.8%8.8%10.0%9.8%10.0%9.8%9.0%9.3%9.5%13.7%13.9%11.5%25.7%26.3%
4 A. acaciae (RJA2772)243 * 0.2%1.0%1.0%5.9%6.6%6.9%6.1%5.6%8.4%8.1%8.1%8.1%8.1%9.6%9.6%9.6%9.1%8.6%8.8%8.8%13.3%13.5%11.5%25.7%25.6%
5 A. acaciae (RJA2649)3531 * 0.7%0.7%5.6%6.4%6.6%5.9%5.4%8.1%8.3%8.3%7.8%7.8%9.3%9.3%9.3%8.8%8.3%8.6%8.6%13.0%13.3%11.3%25.7%25.4%
6 A. acaciae (RJA2578)68743 * 1.5%5.9%7.1%6.8%6.1%5.6%7.8%8.0%8.0%7.6%7.6%8.5%8.6%9.0%8.5%8.0%8.3%8.3%12.7%13.0%11.5%24.9%25.8%
7 A. acaciae (RJA2666)667436 * 5.4%6.1%6.3%5.6%5.1%7.3%7.6%7.6%7.1%7.1%8.5%8.6%8.5%8.0%7.6%7.8%7.8%12.2%12.5%10.5%24.9%25.1%
8 A. germinis (RJA3213)26282724232422 * 2.7%2.0%1.5%1.5%7.1%7.1%7.3%7.3%7.1%9.5%10.0%10.0%9.5%8.3%8.8%8.8%11.5%11.2%10.0%24.6%25.7%
9 A. germinis (RJA2614)2931302726292511 * 2.0%2.4%2.9%6.8%6.8%7.1%8.0%7.8%10.0%10.0%10.0%9.5%8.8%9.3%8.8%12.0%11.5%9.5%24.4%26.0%
10 A. germinis (RJA3192)3032312827282688 * 2.2%2.2%7.6%7.6%7.3%8.3%8.0%10.0%10.0%10.5%10.0%9.0%9.5%9.5%11.7%12.2%10.7%25.1%26.8%
11 A. germinis (RJA3085)272928252425236109 * 1.0%6.3%6.6%7.6%6.6%6.6%9.5%10.0%10.0%9.5%8.5%9.0%8.5%12.0%11.5%9.3%24.1%25.4%
12 A. germinis (RJA2769)2527262322232161294 * 6.1%6.3%6.8%6.3%6.3%9.0%9.5%9.5%9.0%7.8%8.3%8.3%12.0%12.0%9.8%24.6%25.5%
13 A. pilogerminis (RJA2782)363437343332302928312625 * 1.0%2.2%2.2%2.2%8.3%8.8%8.3%7.8%7.1%7.3%6.8%12.5%12.0%10.2%23.30%25.4%
14 A. pilogerminis (RJA3199)3533363334333129283127264 * 2.2%2.7%2.2%8.3%8.8%8.3%7.8%6.8%7.1%6.6%12.2%11.7%10.2%23.20%25.2%
15 A. pilogerminis (RJA3116)35333633343331302930312899 * 3.4%3.2%8.5%8.6%8.5%8.0%7.6%7.8%7.8%12.0%12.0%11.2%23.9%27.0%
16 A. pilogerminis (RJA3115)35333633323129303334272691114 * 0.5%8.3%8.8%8.3%7.8%6.8%7.1%7.1%12.5%12.5%10.7%24.0%25.7%
17 A. pilogerminis (RJA2641)35333633323129293233272699132 * 8.0%8.5%8.0%7.6%6.3%6.6%6.6%12.2%12.2%10.7%23.9%25.8%
18 A. bursicola (RJA2597)4140413938353539414139373434353433 * 0.5%0.5%0.5%7.8%8.0%8.5%13.9%13.7%12.2%25.9%28.1%
19 A. bursicola (RJA2775)41404039383535414141413936363536352 * 1.0%1.0%7.8%8.1%8.6%13.7%13.5%12.0%26.0%28.8%
20 A. bursicola (RJA3171)414041393837354141434139343435343324 * 0.5%7.8%8.0%8.5%13.4%13.2%11.7%26.1%28.1%
21 A. bursicola (RJA3005)3938403736353339394139373232333231242 * 7.8%8.0%8.0%13.4%13.2%11.7%25.6%27.8%
22 A. glabrigerminis (RJA2637)373537353433313436373532292831282632323232 * 0.5%1.0%11.7%10.8%11.5%25.4%25.8%
23 A. glabrigerminis (RJA3008)3836383635343236383937343029322927333333332 * 1.0%11.7%11.2%12.0%25.4%26.3%
24 A. glabrigerminis (RJA3016)38363936353432363639353428273229273535353344 * 11.7%10.8%11.5%24.6%25.8%
25 A. occidentalis (RJA3087)555356545352504749484949515049515057565555484848 * 6.6%10.3%23.8%26.2%
26 A. occidentalis (RJA3209)56545755545351464750474949484951505655545444464427 * 7.6%24.3%26.4%
27 A. occidentalis (RJA3210)4846474746474341394438404242464444504948484749474231 * 23.9%26.4%
28 Dasineura rubiformis (RJA2609)1061031071061061021021011001039910196959899981041061071071051041019810098 * 27.2%
29 Contarinia lotii 97979895949694969710095959594101969610510710510496989698999992 *

Combined phylogeny

The sequence and morphological data were combined and analysed by MP, as there were no strongly supported nodes in one dataset that were in conflict with strongly supported nodes in the other dataset. The sister-taxon relationship between A. acaciae and A. germinis was strongly supported as expected, given the presence of this node in both morphological and DNA-based topologies (Fig. 8). A clade comprising A. pilogerminis, A. glabrigerminis, and A. bursicola, present in the cytochrome b topology but not in the morphology-based phylogeny, was weakly supported, indicating that both data types provide some signal for this relationship. The combined topology was largely similar to the cytochrome b phylogeny, reflecting the greater number of parsimony-informative characters contributed by the cytochrome b dataset than by the morphological dataset.

Figure 8.

Unrooted cladogram of the strict consensus of the two best parsimony trees recovered from a heuristic search of combined molecular and morphological character data. Tree length = 140, consistency index = 0.84, retention index = 0.44. The numbers at the nodes represent maximum parsimony bootstrap values > 50%. The scale bar indicates the number of changes along a branch.

List of Australian Acacia species attacked by gall midges

The inventory (Table 3) contains 38 Australian Acacia spp. damaged by Asphondylia as described in this paper or by Dasineura as described in Kolesik et al. (2005). In addition to these, several other Australian acacias were found in this study to be attacked by Asphondylia and Dasineura but were unidentified to species level (Adair et al. 2000).

Table 3. List of Australian Acacia spp. attacked by gall midges.
HostGaller midgeCommon nameOrgan utilized by larvae
Acacia aneura F. Muell. ex Benth Dasineura glauca Grey fluted gallerOpen flowers
Acacia baileyana F. Muell. Dasineura pilifera Hairy inflated gallerOpen flowers
Acacia baileyana Asphondylia pilogerminis Pubescent bud gallerFlower buds
Acacia cyclops A. Cunn.ex G. Don Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia cyclops Dasineura dielsi Small fluted gallerOpen flowers
Acacia dealbata Link Dasineura pilifera Hairy inflated gallerOpen flowers
Acacia dealbata Asphondylia pilogerminis Pubescent bud gallerFlower buds
Acacia deanei (R.T.Baker) M.B. Welch Asphondylia glabrigerminis Glabrous bud gallerFlower buds
Acacia deanei Dasineura glomerata Common flower gallerOpen flowers
Acacia deanei Asphondylia bursicola Bipinnate pod gallerFruit
Acacia decurrens Willd. Dasineura pilifera Hairy inflated gallerOpen flowers
Acacia decurrens Asphondylia bursicola Bipinnate pod gallerFruit
Acacia divergens Benth. Asphondylia germinis Bud gallerFlower buds
Acacia elata A. Cunn. ex Benth. Dasineura glomerata Common flower gallerOpen flowers
Acacia floribunda (Vent.) Willd. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia genistifolia Link Asphondylia germinis Bud gallerFlower buds
Acacia hakeoides A.Cunn. ex Benth. Dasineura glomerata Common flower gallerOpen flowers
Acacia implexa Benth. Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia irrorata Sieber ex Spreng. Dasineura fistulosa Elongate fluted gallerOpen flowers
Acacia irrorata Asphondylia bursicola Bipinnate pod gallerFruit
Acacia irrorata Asphondylia pilogerminis Pubescent bud gallerFlower buds
Acacia littorea Maslin Asphondylia germinis Bud gallerFlower buds
Acacia littorea Asphondylia occidentalis Western pod gallerFruit
Acacia longifola (Andrews) Willd. Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia mearnsii De Wild. Dasineura fistulosa Elongate fluted gallerOpen flowers
Acacia mearnsii Asphondylia glabrigerminis Glabrous bud gallerFlower buds
Acacia mearnsii Dasineura glomerata Common flower gallerOpen flowers
Acacia mearnsii Asphondylia bursicola Bipinnate pod gallerFruit
Acacia mearnsii Asphondylia pilogerminis Pubescent bud gallerFlower buds
Acacia mearnsii Dasineura rubiformis Tiny flower gallerOpen flowers
Acacia maidenii F.Muell. Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia melanoxylon R. Br. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia melanoxylon Dasineura furcata Forked fluted gallerOpen flowers
Acacia melanoxylon Dasineura glomerata Common flower gallerOpen flowers
Acacia oldfieldii F. Muell. Dasineura oldfieldii Ram's horn flower gallerOpen flowers
Acacia omalophylla Cunn. ex Benth. Dasineura glauca Grey fluted gallerOpen flowers
Acacia oshanesii F. Muell. & Maiden Dasineura oshanesii Small inflated gallerOpen flowers
Acacia paradoxa DC. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia pendula A.Cunn. ex G. Don Dasineura glauca Grey fluted gallerOpen flowers
Acacia pentadenia Lindley Asphondylia occidentalis Western pod gallerFruit
Acacia pycnantha Benth. Dasineura glomerata Common flower gallerOpen flowers
Acacia ramulosa W. Fitzg. Dasineura glauca Grey fluted gallerOpen flowers
Acacia ramulosa Asphondylia occidentalis Western pod gallerFruit
Acacia retinoides Schltdl. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia retinoides Dasineura glomerata Common flower gallerOpen flowers
Acacia rostellifera Benth. Asphondylia occidentalis Western pod gallerFruit
Acacia saligna (Labill.) H.L. Wendl. Dasineura sulcata Groove gallerOpen flowers
Acacia schinoides Benth. Dasineura glomerata Common flower gallerOpen flowers
Acacia longifolia subsp. sophorae (Labill.) Benth. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia longifolia subsp. sophorae Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia sophorae x oxycedrus Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia stricta (Andrews) Willd. Dasineura acaciaelongifoliae Obtuse fluted gallerOpen flowers
Acacia ulicifolia (Salisb.)Court Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia urophylla Benth. in J.Lindley Asphondylia germinis Bud gallerFlower buds
Acacia verticillata (L’Her.) Willd. Asphondylia acaciae Bud and pod gallerFlower buds, fruit
Acacia xanthina Benth. Asphondylia germinis Bud gallerFlower buds

Discussion

Acacia has a large Australasian distribution and includes close to 1000 species of trees and shrubs, which were geologically recently segregated from far less numerous American, African and Asian taxa (Maslin et al., 2003). Australian acacias are divided into seven sections and all except the Lycopodiifoliae are known to host gall-forming cecidomyiids (Adair, 2004, 2005). Despite the remarkable diversity of acacias present in Australia, only three cecidomyiid genera have so far been recorded–Dasineura (Kolesik et al., 2005), Asphondylia and an undescribed genus of the tribe Cecidomyiini (Adair, 2007b). Dasineura and Asphondylia account for all but one species and are confined to the reproductive organs of their hosts. The undescribed genus contains a single species that forms spherical galls on the leaf pinnules of some Botrycephalae acacias, including A. dealbata and A. deanei.

Asphondylia is a worldwide genus that occurs on a broad range of host families, with the bulk of the species recorded from Asteraceae, Fabaceae and Chenopodiaceae (Gagné, 2004). Previously only two species of Asphondylia had been formally described from Acacia: A. trichocecidarum from India (Mani, 1934) and A. napiformis from Kenya (Gagné & Marohasy, 1993). Eight other species from Asphondyliini genera, Aposchizomyia and Schizomyia, attack African and Indian species of Acacia, but the majority of described species from Acacia belong to genera in the tribes Cecidomyiini, Dasineurini, Alycaulini and the Lestodiplosini (Gagné, 2004; Kolesik et al., 2005).

Three general life-history patterns are apparent in Australian Asphondylia feeding on Acacia: (1) flower bud galling (A. germinis, A. glabrigerminis, A. pilogerminis), (2) fruit galling with larvae living within loculi of developing pods (A. bursicola, A. occidentalis), and (3) bud and fruit galling, where larval development takes place alternatively in buds and fruit of a single host species or in buds and fruit of different acacia hosts (A. acaciae). All species pupate within the galls.

Unlike Dasineura galls on Australian acacias (Kolesik et al., 2005), the morphology of Asphondylia galls is rather uniform and provides only a few informative characters to assist with species identification and phylogenetic analyses. Similar to other Cecidomyiidae, Asphondylia spp. from Australian acacias experience frequent attacks from ecto- and endoparasitic Hymenoptera (Adair & Neser, 2006), often at remarkably high levels. However, parasitoid pressure on Australian Asphondylia appears to have had a limited influence on the phylogenic diversification of gall morphology. It is possible that the radiation of gall morphology of Asphondylia galls has been constrained by the absence of fungal symbionts. Alternatively, Australian Asphondylia may be an evolutionary young component of the fauna yet to develop distinctive morphological features, or Asphondylia galls on acacias are under little ecological pressure to evolve modifications to their present structures.

Despite the similarity of insect and gall morphology among Asphondylia feeding on Acacia, phylogenetic analysis of DNA sequence data, together with the morphology of larvae and pupae, life-history traits, and ecological traits, allows reliable identification of species in this group. Phylogenetic analyses of the cytochrome b sequence data in this study resulted in generally well-resolved topologies, indicating its utility as a diagnostic marker for delineating species boundaries in Cecidomyiidae. However, nodal support for deeper nodes in the phylogeny was limited. Inclusion of sequence data from a more slowly evolving mitochondrial gene, such as cytochrome oxidase I (see Uechi et al., 2004; Veenstra-Quah et al., 2007; Kolesik & Veenstra-Quah, 2008 for use of this gene in the taxonomy of Asphondylia), in addition to sequence data from a nuclear gene such as the internal transcribed spacer gene, will probably lead to greater support for deeper nodes.

Asphondylia have a potential role in the biological control of Australian acacias in regions where these plants cause ecological problems, for example in Southern Africa, India, southern Europe, New Zealand and some parts of Australia (Adair, 2007a). The Asphondylia species described here develop on either buds or fruit but not on vegetative organs, and most are confined to a narrow range of related host species, which makes them optimal control agents aimed at reducing sexual reproduction but not at host-plant destruction. Galls of Asphondylia from Australian acacias are smaller than or of the same biomass as non-galled organs and therefore are unlikely to create significant resource sinks, avoiding concerns in relation to possible negative impacts on the growth performance of host plants. In South Africa, the reduction of seed output is a primary objective of the biological control program for invasive Australian Acacia species that are utilized as silvicultural crops or harvested for domestic purposes (Adair, 2002, 2004), and therefore it is conceivable that some of the new species described here will join Dasineura dielsi and D. rubiformis as control agents of Acacia mearnsii and other Australian acacias in South Africa.

Acknowledgements

We thank the South African Department of Water Affairs and Forestry for funding (to RJA) through the Working Water Programme to undertake surveys and collect gall-forming Cecidomyiidae in southern Australia. The taxonomic part of the project was funded by the Australian Research Council (grant to PK). We thank Jana Kolesik (University of Adelaide) for preparing permanent microscopic slides, Brooke Adair for helping with ink drawings of galls, Bruce Maslin (Western Australian Herbarium) for identifying numerous acacia specimens, and Raymond J. Gagné (USDA, Washington, DC) for commenting on an early draft of the manuscript.

Ancillary