Phylogeny and taxonomy of a new clade of Australian Heliozelidae in the genus Prophylactis Meyrick, 1897 (Lepidoptera, Adeloidea) pollinating Boronia (Rutaceae: Sapindales)

Douglas J. Hilton; D. Andy Young; Liz Milla; Mengjie Jin; Stephen Wilcox; Qike Wang; Verena C. Wimmer; Jinny Chang; Henning Kallies; Andie Hall; Marina Watowich; Carly A. Busch; Jordan Wilcox; Aileen Swarbrick; Marlene Walter; Don Sands; Davina Paterson; David C. Lees; Marco F. Duretto; Adnan Moussalli; Mike Halsey; Axel Kallies

doi:10.3897/asp.83.e130334

Abstract

Heliozelidae are a group of small monotrysian moths with a near world-wide distribution. While the Heliozelidae fauna of the Palaearctic and Nearctic is relatively well known, few studies have examined Heliozelidae in other regions of the world. If known, described species are leaf miners as larvae; however, the early biology of species outside of the Northern Hemisphere is poorly understood. Here, we describe a group of heliozelid moths that are specialised pollinators of the iconic plant genus Boronia Sm. (Rutaceae) from the south of Western Australia. Females of this group are characterised by the presence of a pollen-collecting structure on the dorsal side of the abdomen that is unique among known Lepidoptera. We propose that these moths are involved in a brood pollination mutualism with their species-specific host plant, where females lay eggs into and pollinate Boronia flowers, and larvae consume developing seeds. Molecular phylogenetic analyses using seven mitochondrial protein coding genes recovered a monophyletic group of pollinator species that belong to a larger group of Rutaceae-associated Australian Heliozelidae. The remainder of this group lack this pollen-collecting structure, providing insights into the evolution of pollination relationships. We resurrect the genus name Prophylactis Meyrick, 1897 stat. rev. and describe 15 new species based on a combination of morphological and molecular characters and host plant information: Prophylactis albiflorallax Hilton, Young & Kallies sp. nov., P. binbin Hilton, Young & Kallies sp. nov., P. clavatallax Hilton, Young & Kallies sp. nov., P. crassifoliallax Hilton, Young & Kallies sp. nov., P. crenulatallax Hilton, Young & Kallies sp. nov., P. gracilipax Hilton, Young & Kallies sp. nov., P. heterophyllax Hilton, Young & Kallies sp. nov., P jasperae Hilton, Young, Milla & Kallies sp. nov., P. megastigmallax Hilton, Young, Halsey, Milla & Kallies sp. nov., P. molloyax Hilton, Young & Kallies sp. nov., P. octandrallax Hilton, Young, Milla & Kallies sp. nov., P. pulchellax Hilton, Young & Kallies sp. nov., P. purdieanallax Hilton, Young & Kallies sp. nov., P. strictallax Hilton, Young, Halsey & Kallies sp. nov., and P. tetrandrallax Hilton, Young, Milla & Kallies sp. nov.

Keywords

Pollination, Mutualism, New Species, Molecular phylogeny, Pseliastis, Hoplophanes

1. Introduction

As their name suggests, Heliozelidae are a family of day-flying moths. Globally, there are 128 described species in 12 genera (E van Nieukerken and DJ Hilton, unpublished checklist). While most knowledge is centred on the Northern Hemisphere fauna, a robust molecular phylogeny for the family suggested its origin and major radiation in Australia, with many new genera and hundreds of species requiring description (Milla et al. 2018, 2020).

Heliozelidae larvae are commonly considered to be leaf-miners, which excise an elliptical case from the leaf being mined inside of which they pupate (Common 1990). This trait has resulted in the common name for the family - Shield Bearing Moths. Based on the data presented here, however, and supported by additional unpublished results, it is now evident that multiple lineages of Australian heliozelid moths do not conform to this leaf-mining lifestyle and therefore the common name may be of limited relevance.

The unusual nature of the heliozelid moths described in this study was first noticed when we collected female specimens bearing a distinct cleft-like structure at the tip of their abdomen to which pollen was almost invariably found to be attached. Field observations showed that female heliozelid moths belonging to this group lay their eggs into Boronia Sm. (Rutaceae) flowers, and after hatching, the larvae consume developing seeds. Using molecular phylogenetic analyses, we show that these heliozelid species form a monophyletic clade within the genus Prophylactis Meyrick, 1897, which we redescribe and resurrect from synonymy with Hoplophanes Meyrick, 1897. We also discuss evidence suggesting that the relationship between the pollinator heliozelid species and their Boronia hosts is a previously unrecognised brood pollination mutualism, which could shed light on the evolution of specialised pollination relationships.

The interaction between insects and flowers is thought to be a key contributor to the Cretaceous radiation of angiosperms (Regal 1977; Mulcahy 1979; Grimaldi 1999; Van der Niet et al. 2014; Kawahara et al. 2019). In most cases, these relationships are promiscuous with plant species pollinated by a range of insects and these insects visiting many species of plants. Many insects obtain food, in the form of pollen or nectar, in exchange for the pollination service (Landry 2010). More rarely, however, plants provide breeding sites for the insects as a reward for pollination. These relationships are known as brood pollination mutualisms (Sakai 2002) and are highly specific and sometimes mutually obligate, with both the plant and the insect dependent on each other for reproductive success (Kato and Kawakita 2017).

Brood pollination mutualisms serve as vital study systems for evolution of cooperation and for coevolutionary biology (Hembry and Althoff 2016). The best known and most widely studied relationships involve figs (Ficus L., Moraceae), which are inhabited and pollinated by fig wasps (Agaoninae, Hymenoptera) (Herre et al. 2008; Janzen 1979; Weiblen 2002; Wiebes 1979) and yuccas (Yucca L. and Hesperoyucca (Engelm.) Baker, Agavaceae), which are exclusively pollinated by yucca moths (Tegeticula Zeller, 1873 and Parategeticula Davis, 1967, Prodoxidae) (Davis 1967; Riley 1892). In recent years, additional brood pollination mutualisms have been described, for example between leafflower trees (Breynia J.R.Forst. & G.Forst., Flueggea Willd., Glochidion J.R.Forst. & G.Forst. and Phyllanthus L.; Phyllanthacae) and their pollinating moths (Epicephala Meyrick, 1880; Gracillariidae) (Chheang et al. 2022) or the Globeflower (Trollius europaeus L.) and its pollinating flies (Chiastocheta Pokorny, 1889; Anthomyiidae) (Pellmyr 1989). Overall, however, brood pollination mutualisms are relatively rare.

Boronia is a genus of Rutaceae with 129 named species in Australia and four species in New Caledonia (Duretto et al. 2023). Many genera of Australian Rutaceae, including Boronia, produce relatively simple flowers with four or five petals, uniformly sized fertile anthers and a simple stigma (Duretto et al. 2013). In contrast, a strongly supported monophyletic clade of eighteen species of Boronia section Boronia found in south-western Australia produce flowers with a remarkably diverse range of morphology (Duretto et al. 2023). This clade is sub-divided into three series, Heterandrae, Persistens and Variabiles, and in addition to the well-known and commercially cultivated Boronia megastigma Nees ex Bartl., it also includes the threatened species Boronia clavata P.G.Weston (Duretto 2013, 2019, 2023).

In this study, we describe the discovery, taxonomy and phylogeny of 15 new species of Heliozelidae, which we found to be pollinators of B. megastigma and 12 related Boronia species of the B. megastigma clade.

2. Materials and Methods

2.1. Material

Lepidoptera morphological terminology follows Common (1990) and Van Nieukerken and Eiseman (2021). Alar expanses are the widest distance between the outer margins of both forewings of spread specimens. Measurements are given as means +/- standard deviations. Botanical nomenclature follows the International Plant Names Index (IPNI, accessed in October 2024). Plant specimens were identified in the field by our co-authors DAY, DS, MFD and MH. Boronia nomenclature and phylogeny were based on relevant publications including Duretto et al. (2013, 2019, 2023).

Label data of holotype and lectotype specimens are quoted in verbatim, with line breaks indicated by semicolons. Wing and genitalia slides were essentially produced according to protocols outlined in Common (1990). Label data of paratypes and additional specimens are generally given in a standardised form showing the essential information of collecting locality, date, and collector. Longitude and latitude measurements, where available, are given as decimal degrees. Records of plant locations since 1990 were obtained from The Australasian Virtual Herbarium (https://avh.chah.org.au) and The Atlas of Living Australia (https://www.ala.org.au). Plant and moth records were then used in a custom application (https://cadegraaf.shinyapps.io/mothMapv4) to create the distribution map.

Material considered in the study was collected in Australia under a range of permits valid in the various States and Territories. All the material relevant to the description of new species was collected in Western Australia under permits SF004955, SF005627, SF008181, ES001883, CE000962 and CE001510. Adult moths were swept or directly captured from the host plants during the daytime.

In accordance with the conditions of research permits, holotypes and some paratypes are deposited in the Western Australian Museum, Perth (WAM). Other paratypes are deposited in Australian National Insect Collection, Canberra (ANIC); Natural History Museum, London, UK (NMHUK) and the collections of Doug Hilton (CDH), Axel Kallies (CAK), Melbourne and D’Estrees Entomology (DESS), Kangaroo Island.

2.2. Morphological characterisation

The morphological characters of all the studied species were imaged using both light microscopy and scanning electron microscopy (SEM). The dorsal views of the pinned male and female specimens were imaged using a Zeiss SteREO Discovery V8 stereoscope coupled with a Nikon camera (Figs 2, 4, 7H, 8–11), while male and female genitalia slides were photographed using Zeiss LSM 880 or 980 microscopes equipped with a 25×08 multi-immersion lens (Figs 14, 15, S1). Wings and genitalia of the moths were first treated in 10% KOH solution at 90°C for 5-10 min to reveal the structure, then embedded in Euparal, imaged under the microscope with variable focal distance, and stacked using Helicon Focus (Helicon Soft Ltd., Kharkiv, Ukraine) to increase the depth of view (Figs 7A, 7D, 14, 15, S1).

We additionally obtained SEM images of the head, thorax, and abdomen of all the species (Figs 6, 12). Pinned specimens were mounted on aluminium stubs, placed in a glass desiccator for 48 hours to dry thoroughly before being coated with gold in a sputter coater for 45 seconds (estimated gold thickness of 5 nm) and imaged on a Philips XL-30 Field Emission ESEM (Philips Corp., Eindhoven, Netherlands) with an acceleration voltage of 5 kV, captured using a secondary electron detector (SE), and a scan speed of 40 S per frame. The images were processed on Windows 11 platform using Adobe Photoshop CS8 (Adobe Systems, Inc., San Jose, CA, USA).

2.3. DNA extraction, library building and sequencing

2.3.1. Genomic DNA Extraction

Legs or abdomens from 102 adult moth specimens were used to generate genomic DNA for next generation sequencing. DNA was extracted from moths individually and prepared according to the Macherey-Nagel NucleoSpin® Tissue XS protocol. The integrity and quantity of the individual DNA samples were assessed on the Agilent TapeStation 4200 using the Genomic tape and reagents. Abdomens were retained to allow genitalia to be examined.

2.3.2. Library Preparation and Genome Sequencing

An input of 100 ng of genomic DNA was prepared and indexed for Illumina sequencing using the TruSeq DNA sample Prep Kit (Illumina) as per manufacturer’s instruction. The library was quantified using the Agilent TapeStation and the Qubit™ RNA assay kit for Qubit 2.0® Fluorometer (Life technologies). The indexed libraries were then prepared and diluted to 750 pM for paired end (2×150 base) sequencing on a NextSeq2000 instrument using the P1 300 cycle kit using v3 chemistry (Illumina) as per manufacturer’s instructions. The base calling and quality scoring were determined using Real-Time Analysis on board software v2.4.6, while the fastq file generation and de-multiplexing utilised bcl2fastq conversion software v2.15.0.4 (RRID:SCR_015058).

2.3.3. Sequencing of Historic Specimens

DNA extraction and sequencing for the type specimen of P. argochalca (NHMUK013697112) was conducted at the Natural History Museum (NHMUK, formerly BMNH), following the ancient DNA protocol from Rohland et al. (2018) with modifications to work at smaller volumes on a Kingfisher Flex robot (Hall et al. 2023). Tissues (e.g. a single leg) were lysed overnight at 56°C in 90 µl of lysis buffer C (Korlević et al. 2021). Lysate was transferred to deep well plates with 900 µl of binding buffer (Dabney et al. 2013). For each sample, 10 µl of silica bead suspension (prepared following Rohland et al. 2018) was added to each sample well, containing lysate and binding buffer, and the plate loaded onto the Kingfisher to complete the extraction process. DNA extracts were quantified using a Qubit fluorimeter and the Qubit HS dsDNA assay kit (ThermoFisher Scientific). Libraries were prepared with unique dual indexing for each specimen following Kapp et al. (2021), with the following modification: post-PCR, samples were diluted with 1.1× EBT and cleaned with 0.75× SPRI bead solution (Cytiva Sera-Mag SpeedBeads™ Carboxyl Magnetic Beads, Fisher Science) as described in Rohland and Reich (2012). Libraries were pooled with other uniquely indexed museum specimens and sequenced on a NovaSeq S4 2×150 bp lane (Novogene), generating 66 million reads (33 million PE reads) for this sample.

2.4. Phylogenetic relationship reconstruction

To aid alpha taxonomic inference we utilised whole genome next generation sequencing data generated as part of our ongoing research of Australian Heliozelidae. We assembled mitogenomes using the program MitoZ (version 3.6, Meng et al. 2019). The final dataset incorporated 65 samples representative of the 15 pollinator species described herein and additional 34 samples of Prophylactis. Representatives from the genus Pseliastis Meyrick, 1897 (Table S2) were used as outgroup based on Milla et al. (2020). The MitoZ pipeline first uses Fastp (version 0.21, Chen et al. 2018) for adapter and quality trimming of raw sequence data, followed by de novo assembly using MEGAHIT (version 11.0, D. Li et al. 2015). Assembled contigs containing mitochondrial protein coding sequences were then identified using Hidden Markov Model profiles derived for a curated set of Arthropods using HMMER (version 3.1b2, http://hmmer.org). Resulting contigs were annotated and collated across all samples producing individual FASTA files per gene. Final alignments were produced using MAFFT (v7.490, Katoh and Standley 2013) with default settings. We focussed on seven protein coding genes (COI, COII, ATP6, CYTB, ND1, ND4, ND5) for which taxa representation was near complete (see Table S2 for NCBI Accession Numbers) and estimated a maximum likelihood phylogeny using IQ-TREE version 1.6.12 (Chernomor et al. 2016; Minh et al. 2020). We treated each gene as a separate partition, identifying best BIC model for each partition using ModelTest (Kalyaanamoorthy et al. 2017), and assessed nodal support using non-parametric bootstrap and SH-like approximate likelihood ratio test (SH-aLRT) (Guindon et al. 2010).

2.5. Etymology

For all but two of the species described here, we use all or part of the host-plant’s specific name along with all or part of the suffix “-allax” (ἀλλάξ = reciprocal), which is derived from the verb ἀλλάσσω, a transitive word meaning “change”, “exchange for”, or “alternate”. We have taken this approach to create relatively euphonious specific names for the pollinator species. All these species names should be treated as nouns in apposition meaning “the one that is reciprocal to the host plant concerned”.

2.6. Abbreviations

ANIC—Australian National Insect Collection, Canberra, Australia; MVM—Museum Victoria, Melbourne. CAK—personal collection of Axel Kallies, Melbourne, Australia; CDH—personal collection of Douglas J. Hilton, Bend of Islands, Australia; GP—genitalia preparation; NP—National Park; NR—Nature Reserve; SF—State Forest; FR—Flora Reserve; CP—Conservation Park.

3. Results

3.1. Pollination of Boronia by Heliozelidae

For decades, Western Australian Boronia species of the B. megastigma species group, unusual for their floral morphology including cup-shaped flowers and specialised anthers (Fig. 1), have been speculated to have specialist pollinators, likely to be moths (Quick 1963; MacTavish and Menary 1997; Plummer et al. 1999). Remarkably, however, no “Boronia moths” have been collected and deposited in an institutional collection, photographed or described. To address this issue and to uncover the identity of these elusive Boronia pollinators, for the last 10 years we systematically searched Australian Boronia species during their flowering period. To date, we have examined 65 of the 129 described Boronia species, including all 18 species in the B. megastigma group and representatives of all other major clades of Boronia (Table S1).

Figure 1.

Boronia sp. and their associated Prophylactis sp. moths in the field. A, B Boronia megastigma; C B. megastigma and its associated pollinator moth P. megastigmallax sp. nov.; D B. heterophylla and its associated pollinator moth P. heterophyllax sp. nov.; E B. tetrandra and its associated moth P. tetrandrallax sp. nov.

Across Australia, we collected >200 species of Heliozelidae moths visiting Rutaceae flowers. Around half of these species were found to belong to Pseliastis, a genus formerly known from just three species and only known from Tasmania (Meyrick 1897). Pseliastis species are usually characterised by the presence of a fascia across the forewing and occasionally other markings such as spots (Fig. 2A, B). In addition to Boronia, Pseliastis moths were also found on other Rutaceae genera including Cyanothamnus Lindl., Phebalium Vent., Zieria Sm., Correa Andrews, Philotheca Rudge, Asterolasia F.Muell. and Eriostemon Sm. (our unpublished data). A second group of Heliozelidae moths found on Rutaceae flowers differed from Pseliastis by their plain forewings, usually light brown with a metallic silver or gold sheen and the absence of any defined wing markings (Fig. 2C, D). Such moths were particularly abundant on Boronia species in south-western Australia, but they were also found in south and eastern Australia. While most of these species were observed on Boronia, some were also found on other Rutaceae such as Cyanothamnus and Zieria. Closer examination revealed that these moths fell into two morphotypes, those in which the female moths exhibit a simple abdomen and those in which the dorsal tip was invaginated to create a unique, complex and previously unrecognised structure to which pollen was invariably attached (Fig. 3). While moths lacking a pollen-collecting structure were found across a wide range of Boronia and other Rutaceae species across Australia, those with a pollen-collecting structure were found only in south-western Western Australia and were exclusively associated with the species of Boronia megastigma clade (senso Duretto et al. 2023).

Figure 2.

Heliozelidae species, dorsal view. A, B Pseliastis sp. Grampians National Park, VIC; C, D Prophylactis megastigmallax sp. nov.; Paratype, MMP005414 (C); Paratype, MMP005415 (D). Scale bars = 1 mm. A,C: male; B,D: female.

Figure 3.

Photo of a freshly caught female of P. crenulatallax sp. nov. with pollen attached to the distal end of dorsal side of the abdomen.

Previous molecular studies revealed a well-supported monophyletic branch for these plain Rutaceae-associated moths (Milla et al. 2018, 2020), which we noticed to have a general similarity with Prophylactis argochalca Meyrick, 1897 (Fig. 4A, B), the type species of the genus Prophylactis, currently considered a synonym of Hoplophanes (Fig. 4E, F). Our morphological observations, and molecular studies confirmed this association, and placed P. argochalca at an early diverging lineage within this branch (Fig. 5; Table S2). Species with abdominal pollen-collecting structures formed a monophyletic group embedded inside this branch suggesting that all these species could be considered to belong to a single genus, Prophylactis.

Figure 4.

Heliozelidae species, dorsal view. A, B Prophylactis argochalca; Paralectotype, NHMUK 013697110 (A); Lectotype, NHMUK 013697111 (B); C, D Prophylactis sp., non-pollinator group species; E, F Hoplophanes sp.; Scale bars = 1 mm. A, C, E: male; B, D, F: female.

Figure 5.

Phylogeny of Prophylactis species obtained from seven mitochondrial protein-coding genes. Nodal support values, non-parametric bootstrap followed by SH-like approximate likelihood ratio test, are only displayed for poorly resolved relationships (bootstrap <75%). Insert Top left: Topology of Boronia phylogeny (Duretto et al. 2023).

3.2. The taxonomic status and phylogeny of the genus Prophylactis

Prophylactis was described by Meyrick (1897) and initially contained the type species, P. argochalca, and two other species, P. aglaodora Meyrick, 1897 and P. chalcopetala Meyrick, 1897. Later, Meyrick (1922) added a fourth species, P. memoranda Meyrick, 1922. Subsequently, Nielsen (1996) synonymized Prophylactis with Hoplophanes, albeit without presenting any evidence for this action. Based on the examination of the syntypes of P. argochalca and more than 10,000 specimens of Heliozelidae from across Australia, it became clear that Prophylactis is a genus distinct from Hoplophanes. Morphologically, Prophylactis species differ from Hoplophanes and other Australian genera of Heliozelidae based on their head shape and scaling, eye-colour, wing venation, forewing pattern and bionomics. The results of our morphological studies were supported by molecular data (Milla et al. 2018, 2020), which showed that species of Prophylactis are clearly separated from Hoplophanes and other heliozelid genera, including Pseliastis. Both Prophylactis and Pseliastis are associated exclusively with Rutaceae, while Hoplophanes closely associates with and, where the biology is known, feeds on Ericaceae (our unpublished data). Based on morphological, molecular and biological evidence, we therefore resurrect the genus Prophylactis stat. rev.

To gain more detailed insight into the phylogeny of Prophylactis, we sequenced the genomes of a total of 103 specimens, including species with and without a pollen-collecting structure. Importantly, we also sequenced a syntype of P. argochalca, which we here designate as the lectotype. Phylogenetic analysis showed multiple, well supported lineages in Prophylactis, with the pollen-bearing species forming a single, well supported, monophyletic group, while species lacking the pollen-collecting structures formed several distinct lineages (Fig. 5; Table S2). Notably, in our previous studies, we referred to some of these groups as putative genera, i.e. “genus 1”, “genus 2”, “genus 7”, “genus 11” and “genus 15” (Milla et al. 2018, 2020). Following a parsimonious approach, however, we refrain from erecting new genera and consider all species, including the pollinator species group, as belonging to Prophylactis. Within the pollinator species group, there are four well supported clades (Fig. 5; Table S2). Clade 1 contains seven species, but their relationships remain poorly resolved except for the branch (P. gracilipax sp. nov. + P. pulchellax sp. nov.). Within clade 2, P. albiflorallax sp. nov. and P. octandrallax sp. nov. are recovered as sister taxa that group with P. crenulatallax sp. nov. Clade 3 contains three species with a basal split of P. crassifoliallax sp. nov. to the other two. Clade 4 groups P. jasperae sp. nov. and P. binbin sp. nov. together with good support.

3.3. Characterization of the Pollinator Species Group

Pollen-bearing species described in this study form a monophyletic group within Prophylactis, which we term the ‘Pollinator Species Group’. Morphologically, this group is characterised by the females having abdominal modifications forming unique, species-specific pollen-collecting structures (Figs 6A, B, 12, S1). These structures are formed from an invagination of tergites VII and VIII to create a cleft with interior spines, setae and/or scales that trap pollen. The structures of the pollen-collecting clefts are species-specific and morphologically diverse. Some species show relatively simple and compact structures, formed by lateral ridges interspaced by a central plate, all of which are covered by simple short setae to which pollen adheres (e.g. P. megastigmallax sp. nov., Figs 12A, S1A), others have long, Venus Flytrap-like structures covered with very long setae that capture and trap pollen (e.g. P. gracilipax sp. nov., Figs 12F, S1F). Yet other species bear a medial ridge that bifurcates the cleft (e.g. P. purdieanallax sp. nov., Figs 12K, S1K). The tip of the abdomen has long setae and spines (Fig. 6A, B), with the oviscapt being highly sculpted with overlapping plates and teeth. In contrast to the highly modified abdomens of the females in the pollinator group, the abdomens of non-pollinator females are simple and unremarkable (Fig. 6H). Males of the pollinator group are more difficult to distinguish from other Prophylactis, although the presence of a brush of elongate, androconial scales on the hindwing (Fig. 7H) in most species, serves to distinguish them from non-pollinator Prophylactis species in Western Australia.

Figure 6.

SEM photos of Prophylactis species. A, B P. megastigmallax sp. nov.; female abdomen, dorsal view showing the pollen-collecting structure with pollen attached; C P. heterophyllax sp. nov., head, lateral view; D-F P. megastigmallax sp. nov., head, ventral view (D); eye, showing intra-ommatidial setae (E); antennal flagellomeres near the distal end (F); G P. crenulatallax sp. nov.; male abdominal tergites; H Prophylactis sp., non-pollinator group species, female abdomen, dorsal view. Scale bars = 100 µm.

Figure 7.

Wing photos and interpretative illustrations of Heliozelidae species. A-D Prophylactis megastigmallax sp. nov., Paratype male, MMP005306 (A, B); Paratype female, MMP005307 (C, D); E Prophylactis sp., non-pollinator group species, male; F Hoplophanes tritocosma Meyrick, 1897, male; G Pseliastis sp., male; H P. molloyax, Paratype male, MMP005420, blue arrows indicating the androconial brush. Scale bars = 1 mm.

As outlined in the descriptions below, many species of Prophylactis look similar and can be best distinguished by their different hostplants and molecular barcodes. Pollinator species belonging to Clade 1 share the following characters: males with androconial brushes on hindwings, female pollen-collecting structure V-shaped or Y-shaped, length at most twice of width, and lateral plates not expanded towards apex. Clade 2 contains three species that all share a rather slender pollen-collecting structure in the females. Males of P. albiflorallax sp. nov. and P. octandrallax sp. nov. have androconial brushes on their hindwing, while this is absent in P. crenulatallax sp. nov. Clade 3 also contains three species P. purdieanallax sp. nov., P. tetrandrallax sp. nov. and P. crassifoliallax sp. nov. Females have long pollen-collecting structures which include a longitudinal ridge. Males lack the androconial brush. Clade 4, containing P. jasperae sp. nov. and P. binbin sp. nov., is sister to the other species in the pollinator group. Females have simple linear pollen-collecting structures while as for Clade 3, males lack the androconial brush.

3.4. Biology of pollinating Prophylactis

Based on collective evidence, all pollinator group species are pollinators of Boronia species in the Boronia megastigma clade (senso Duretto et al. 2023). In the field, they can be routinely observed flying over or perched on their hostplant. While females were invariably observed on the hostplant itself, often in their flowers, males could also be seen on plants closely adjacent to their host. Females were observed to oviposit directly into flowers. Based on observations of P. megastigmallax sp. nov., larvae feed within the developing seeds. Mature larvae fall to the ground, becoming prepupal in a firm, sub-surface cocoon and eclose the following year. Adults emerge in winter, spring or early summer, coinciding with the flowering period of their hostplants. While some Boronia species flower over an extended period of weeks, the pollinating species of Prophylactis have a more discreet emergence period early in the flowering cycle. Adults appear to be exclusively diurnal and have not been collected at light. They generally begin flying two to four hours after sunrise and in fine weather have a flight window of 6 to 7 hours. Moths have also been collected on cold, wet and windy days, though they are less active during these conditions. Some species can occur in moderate to high density swarms of thousands and perhaps tens of thousands of individuals, especially early in the flowering season within high density colonies of the host Boronia.

Although moths typically stay close to their host plants, there is evidence that they move between different Boronia colonies, even when these are commercially planted within their natural range but spatially distant from existing wild populations. In one instance, pollinator moths were noted around B. megastigma within 24 hours of introduction of the plants to a nursery near Nannup. The nearest wild plants from which the moths could have travelled were at least 800 m away, and the nearest significant stands were approximately 2 km distant.

Most species of the Boronia megastigma clade (senso Duretto et al. 2023) are associated with a unique species of moth pollinators. Two Boronia species, however, were found to be associated with two pollinators: Boronia crassifolia Bartl. was associated with P. binbin sp. nov. and P. crassifoliallax sp. nov., and Boronia octandra P.G.Wilson with P. jasperae sp. nov. and P. octandrallax sp. nov. Despite extensive searching, no pollinating moth has yet been found associated with Boronia crassipes Bartl., Boronia virgata P.G.Wilson, Boronia oxyantha Turcz., Boronia capitata Benth. or Boronia nematophylla F.Muell. Notably, many of the Boronia species linked to pollinators were also associated with one or several non-pollinator Prophylactis species.

4. Discussion

In this study, we describe 15 new species of Heliozelidae that are characterised by females bearing highly specialised and previously unrecognised pollen-collecting structures on the dorsal tip of the abdomen. Furthermore, we describe a new putative brood pollination mutualism involving these new species and their Boronia host plants (Rutaceae). Based on a combination of morphologic, molecular and host plant information, we show that these species belong to the genus Prophylactis, which we resurrect from synonymy.

The results of this study are based on more than 10 years of dedicated field and laboratory work. During this period, we collected several hundred heliozelid species, approximately 200 of which belonged to a clade tightly associated with Rutaceae. Most of these moths belonged to two genera: Pseliastis and Prophylactis. The latter included a group of species characterised by a unique pollen-collecting structure on the dorsal tip of the abdomen of female moths. Molecular phylogenetic analyses showed that the species bearing this pollen-collecting structure formed a monophyletic group nested among Prophylactis that lacked this structure. While the majority of Prophylactis species had neither a pollen-collecting structure nor pollen attached to their bodies, pollen was found to be almost invariably attached to the pollen-collecting structure in all 15 species described in this paper. Given that their larvae consume seeds, pollination by the moths would benefit their reproductive success by ensuring their hostplants would set seeds, thus increasing the likelihood that larvae have sufficient food once they hatch. The plant could also benefit from a species-specific pollinator by conserving pollen resources. Thus, a mutually beneficial relationship between Prophylactis and their Boronia hosts can be envisaged. The obligate nature of at least some of the relationships between Prophylactis moth pollinators and their hostplants has been suspected for decades; however, this needs to be explicitly tested for each species and will be the subject of future publications (Milla et al. in preparation).

The sacrifice of a minority of the seeds produced because of larval predation, might appear to be a negative aspect of this association for the plant-partner. If this was the case, then the highly specific nature of the pollination provided by the moths could be seen as compensation for this sacrifice. Some evidence shows that a large shed-seed load may, in some cases, significantly lower germination rates (e.g. Tielbörger and Prasse 2009). The predation of a seed fraction at larval stage by the insect-partner may therefore potentially prove to be a net benefit to the plant-partner in the relationship outlined here.

The pollinator moths described in this study form a monophyletic group, suggesting that the ability to pollinate using the novel pollination structure has evolved only once within the genus. Our current phylogenetic analysis based on mitochondrial protein coding genes was not sufficient to reconstruct all phylogenetic relationships within the pollinator group. Indeed, the limited phylogenetic signal could not resolve the short internal tree branches for the crown group, Clade 1, suggesting that speciation events occurred closely spaced in time. Within Clade 2, a relatively long distance was recovered between P. crenulatallax sp. nov. and (P. albiflorallax sp. nov. + P. octandrallax sp. nov.). This observation is consistent with the finding that their hostplants belong to different series (Duretto et al. 2023). While Boronia crenulata Sm. was classified as belonging to series Variabiles, Boronia albiflora R.Br. ex Benth and Boronia octandra were placed in series Persistens. Species within Clades 3 and 4 were also associated with Boronia belonging to series Persistens; however, apomorphic characters for these clades are elusive (Duretto et al. 2023). We are currently working to better resolve the phylogenetic relationships between moth species to explore the potential co-evolution of the heliozelid pollinators and their Boronia host species.

Many Boronia species with a pollinating Prophylactis species are also hosts for non-pollinator Prophylactis and Pseliastis species. This may suggest that the relationship between pollinators and their hosts may be exploited by non-pollinator group moths. Additional studies, however, are needed to assess the complex relationships between moth species and their hostplants. Better understanding these relationships may also aid conservation of some of these Boronia species. For example, conservation efforts for B. clavata, a species restricted to two disjunct sites along rivers and creeks and listed as threatened, need to account for the impact of environmental stresses such as bushfire and insecticide use in adjacent agricultural land on the pollinating moth. Additionally, consideration should be given to listing both the pollinating moths and its non-pollinating counterpart for protection, given both are dependent on their threatened hostplant for survival.

Overall, our study has uncovered a new group of heliozelid moths characterised by the presence of a unique pollen-collecting structure on females and linked to the pollination of their Boronia hostplants.

5. Taxonomy

5.1. Prophylactis Meyrick, 1897, stat. rev.

Prophylactis Meyrick, 1897: 408. Type species: Prophylactis argochalca Meyrick, 1897, by original designation.

Description.

Cream-white, grey-beige, or metallic beige with metallic gold, silver and sometimes violet sheen. Wingspan 4 to 9 mm. — Head: Uniformly cream, yellow, beige or light brown coloured, lighter ventrally; clothed with appressed lamellar scales (Fig. 6C). Roughly triangular in lateral view, hemispherical in dorsal and ventral view (Fig. 6D). Anterior edge of head capsule arched, with indents adjacent to eyes and below anterior tentorial pits, which are prominent. Scales on the occiput spathulate with indented apex, forming a collar at the boundary with the prothorax (Fig. 6C). Scales on the frons large and partially covering the mouthparts (Fig. 6C, D), while on the vertex larger and more spathulate. Labrum small and triangular; without pilifers. Mandibles vestigial or absent. Maxilla consisting of an ovoid “basal piece” (Kristensen 1999), likely the fused cardio and stipes, on which are small four-segmented palps and a well-developed proboscis that is unscaled and much longer than labial palps but sometimes coiled and obscured by scales. Labium bearing porrect three-segmented labial palps. Segments cylindrical with length ratio approximately 2:1:2, and the terminal segment with notch at apex. Labial palps surface covered with dense scales, cream on the base segment, while brownish on the apical two. Eyes red (Fig. 1D) extending to the ventral margin of the head capsule and past the lateral and posterior margins (Fig. 6D); intra-ommatidial setae present anteroventrally (Fig. 6E). Antennae approximately two-thirds the length of forewing; scapes and pedicels cylindrical, similar in length and twice as long as flagellomeres; 25 to 35 cylindrical flagellomeres with two annuli containing 8 sets of 5 overlapping scales in the basal ring and 8 sets of 4 to 7 overlapping scales in the distal ring (Fig. 6F). — Thorax: Dorsal surface of prothorax and mesothorax uniformly cream, yellow, beige or light brown with a metallic sheen; similar colour as the head and the basal margin of the costa on the forewings; dorsal surface of metathorax darker and similar in colour to dorsal surface of abdomen; all segments lighter laterally and ventrally. — Legs: Legs brown with metallic sheen dorsally, cream ventrally; spur formula 0-2-4; protibia with large epiphysis; cream tibial brush present on hind legs of both males and females (Fig. 10G, H). — Wings: Forewings lanceolate. Hindwings falcate with distal half of costa concave. Dorsal surface of forewings ranging in colour from cream, grey-beige, to beige with silver or gold sheen, sometimes with faint blue shine and some species with scattered lighter and darker scales; dorsum without spots or fasciae, except for a highly distinctive undescribed species from Western Australia. Ventral surface generally light brown or brown; fringe of similar colour as dorsal surface. Dorsal surface of hindwings generally slightly darker than forewing, beige or light brown with less silver or gold sheen than forewing; ventral surface grey to grey-brown, lighter than ventral surface of forewing, and patches black, cream or yellow androconial scales present in some species (P. molloyax sp. nov., P. clavatallax sp. nov. and P. binbin sp. nov.) and an androconial brush found more widely (Fig. 7H); fringe same colour as dorsal surface. In some species, males with 15-20 flattened androconial scales (similar in length to the frenulum) forming a brush and originating from the dorsal surface of hindwings just below the costal margin distal of the frenulum for half the length of the wings (Fig. 7H). — Wing coupling: In males, frenula composed of a single bristle of approximately 1/3 length of hindwing (Fig. 7A, B, E, F); while approximately 15-20 pseudofrenular bristles present along the costa, each about 2/3 the length of the frenula. Retinacula formed from a series of scales and hooks running above Sc from base to midway along the costal margin. In female, frenula absent but with 15 to 20 pseudofrenular bristles. — Wing venation: Forewings (Sc, Rs1 to Rs4, M, CuA1 and 1A+2A) and hindwings (Sc+R1, Rs, M1, M2, M3, CuA1, CuA2 and 1A+2A) with eight terminal veins (Fig. 7A-E). Venation similar throughout the genus with minor differences on forewings between and within species in: (i) strength of cross veins, resulting in cell being closed (Fig. 7B) or open (Fig. 7D) and (ii) position of cross veins being either before (Fig. 7B) or after the Rs2 branch. — Abdomen: Grey-brown to brown dorsally, sometimes metallic beige; cream ventrally. In both males and females, tergites covered with small deeply serrated scales, becoming less numerous on posterior tergites, especially tergites VII and VIII (in males). On each tergite, scales larger but less serrated and appear lighter toward the posterior margin (Fig. 6G). Sternum (S) IIa butterfly-shaped with a central line of stronger sclerotization. SIIp U-shaped with thin apodemes extending anterior of SIIa. SIII to SVI in female rectangular, more heavily sclerotized than tergites. Tergite (T) I trapezoidal with narrower anterior margin; surface membranous but with strong marginal sclerotization along the anterior edge at point of articulation with metathorax, lateral edges, and lateral parts of posterior margin. TII trapezoidal, wider anteriorly with stronger sclerotization of the anterior part of the lateral margin. TIII to TVI rectangular to roughly square. — Abdomen males: Scales projecting from posterior margin of tergite VIII partly covering the valvae (Fig. 6G). — Abdomen females: Unmodified (Fig. 6H) or with TVII and TVIII modified to form pollen-collecting structures (Figs 6A, B, 12, S1). — Female genitalia: (Fig. 13). SVIII weakly truncate at terminal end. Tip of oviscapt sharply pointed; anterior and posterior apophyses usually subequal in length, a long interapodemal process present between anterior apophyses. In some species, anterior apophyses weakly bent inward near apical 1/3. In some species, the tip of posterior apophyses with oval sclerotised plate, the plate surface covered with short, dense, strong spines. — Male genitalia: (Figs 14, 15). Vinculum (SIX) long and wide, forming a half-cone, approximately 2/3 of total length of genitalia. Tegumen (TIX) short, truncate or slightly trapezoid, uncus and gnathos differ between species. Valva almost triangular, distally narrowed with blunt terminal ends, with stalked pectinifer at apical 1/2-1/3 of inner margin, pecten covered with dense blunt comb-like sensilla. Transtilla elongate arch-like, median area usually with broad projection, subapical processes long. Juxta present, arrow shape to spathulate. Phallus simple, usually straight and very long, basal margin smooth, apex usually forming a sharp hook or spine. Phallocrypt covered with very dense, sclerotised, short but sharp spines, usually with one or two subapical spines.

Diagnosis.

Species in the genus Prophylactis can be readily distinguished from other genera of Australian Heliozelidae by their plain forewings, that lack any fasciae or spots (Figs 2C, D, 4A-D, 8–11), and their red eyes. Hoplophanes Meyrick 1897, from which we have resurrected Prophylactis from synonymy, and Pseliastis Meyrick 1987, which like Prophylactis are found associated with Rutaceae, have black not red eyes. Pseliastis almost invariably have a fascia at the midpoint of the forewing and often additional medial and distal bands. Hoplophanes often, but not always, have forewings with fasciae. Wing venation is also useful in distinguishing the three genera. Prophylactis species (Fig. 7A-E) can be separated from Hoplophanes (Fig. 7F) by the forewing, which lacks R1 and one branch of CuA, and the hindwing which has Rs stalked to M near the apical area and Rs-M cross veins absent. Prophylactis species can be separated from Pseliastis (Fig. 7G) by hindwing CuA, which always has two branches, but only one in Pseliastis.

Remarks.

The appearance of Prophylactis moths depends on the direction and nature of the light. Thus, live moths in their natural environment (Fig. 1C-E) appear quite different to set specimens under artificial light (Figs 8–11). Furthermore, the colour depends on the wear of specimen, with fresh specimens appearing lighter, while worn specimens due to the loss of the superficial scales appear darker. The descriptions here are for set moths in good condition.

Figure 8.

Prophylactis species, dorsal view. A, B P. megastigmallax Hilton, Young, Halsey, Milla & Kallies sp. nov.; Paratype, MMP005414 (A); Holotype, MMP005413 (B); C, D P. strictallax Hilton, Young, Halsey & Kallies sp. nov.; Paratype, MMP005406 (C); Holotype, MMP005405 (D); E, F P. heterophyllax Hilton, Young & Kallies sp. nov.; Paratype, MMP005417 (E); Holotype, MMP005416 (F); G, H P. molloyax Hilton, Young & Kallies sp. nov.; Paratype, MMP005420 (G); Holotype, MMP005419 (H). Scale bars = 1 mm. A, C, E, G: male; B, D, F, H: female.

Figure 9.

Prophylactis species, dorsal view. A, B P. clavatallax Hilton, Young & Kallies sp. nov.; Holotype, MMP005390 (A); Paratype, MMP005391 (B); C-D P. gracilipax Hilton, Young & Kallies sp. nov.; Paratype, MMP005401 (C); Holotype, MMP005400 (D); E, F P. pulchellax Hilton, Young & Kallies sp. nov.; Paratype, MMP005408 (E); Holotype, MMP005407 (F); G, H P. crenulatallax Hilton, Young & Kallies sp. nov.; Paratype, MMP005403 (G); Holotype, MMP005402 (H). Scale bars = 1 mm. A,C,E,G: males; B,D,F,H: females.

Figure 10.

Prophylactis species, dorsal view. A, B P. albiflorallax Hilton, Young & Kallies sp. nov.; Holotype, MMP005387 (B); C, D P. octandrallax Hilton, Young, Milla & Kallies sp. nov.; Paratype, MMP005389 (C); E, F P. purdieanallax Hilton, Young & Kallies sp. nov.; MMP005399 (E); Holotype, MMP005394 (F); G, H P. tetrandrallax Hilton, Young, Milla & Kallies sp. nov.; Paratype, MMP005393 (G); Holotype, MMP005392 (H). Scale bars = 1 mm. A, C, E, G: males; B, D, F, H: females.

Figure 11.

Prophylactis species, dorsal view. A, B P. crassifoliallax Hilton, Young & Kallies sp. nov.; Paratype, MMP5425 (A) Holotype, MMP004318 (B); C, D P. jasperae Hilton, Young, Milla & Kallies sp. nov.; Holotype, MMP005388 (D); E, F P. binbin Hilton, Young & Kallies sp. nov.; Paratype, MMP005423 (E); Holotype, MMP005422 (F). Scale bars = 1 mm. A, C, E: males; B, D, F: females.

Life history.

Pollinator and non-pollinator group species of Prophylactis appear to be highly hostplant specific, being associated with a single species of Boronia, or in the case of some undescribed non-pollinator group Prophylactis species, associated with a single species of Cyanothamnus or Zieria. Females were observed to oviposit directly into flowers. P. megastigmallax sp. nov. places its eggs individually at the base of the reduced antipetalous anther on the ovary surface. Larvae emerge from the ovary base and eat their way into the seed they are feeding on basally through the early seed pod. Examination of seedheads containing more advanced larvae showed they usually consume only a single seed per larva. On achieving maturity in late spring or early summer, the larvae were observed emerging from an ovoid hole at the base of the seed within which they had developed. The larvae then dropped onto the soil where they crawled briefly, before burying themselves and becoming prepupal in a firm, sub-surface cocoon. Similar observations were made on a different non-pollinator species of Prophylactis, which develops in the seedheads of Zieria arborescens Sims.

Distribution.

Prophylactis species, like their hostplants, are widely distributed across Australia, being found in Western Australia from Kalbarri in the north to Cape Arid in the south, across South Australia and on the east coast from near Charters Towers in Queensland, throughout New South Wales, Victoria and Tasmania. With additional collecting efforts it is almost certain that the distribution will expand. The Boronia-pollinating species of Prophylactis, described below, are found exclusively in southern Western Australia where their hostplants grow (Figs S2B-F, S3, S4); however, B. crassifolia is found close to the border with South Australia and both the plant and its associated moths may be found in that state (Duretto et al. 2013).

Composition of the genus.

At present we include the following species in the genus: P. argochalca (the type species), P. aglaodora comb. rev., P. chalcopetala comb. rev., P. memoranda comb. rev. and the 15 species described below. While P. aglaodora and P. chalcopetala appear to be typical non- pollinator group species of the genus likely to be associated with Boronia, P. memoranda does not appear to be closely related to other species in the genus. Its precise position will be subject of a future revision of the family.