Pollinator rarity as a threat to a plant with a specialized pollination system
RYAN D. PHILLIPS
1,2,3*, ROD PEAKALL
1, BRYONY A. RETTER
2,3, KIRKE MONTGOMERY
2, MYLES H. M. MENZ
4, BELINDA J. DAVIS
2,3, CHRISTINE HAYES
1, GRAHAM R. BROWN
5,6, NIGEL D. SWARTS
7,8and KINGSLEY W. DIXON
2,91Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia
2Kings Park and Botanic Garden, The Botanic Gardens and Parks Authority, Fraser Avenue, West Perth, WA 6005, Australia
3School of Plant Biology, The University of Western Australia, Crawley, WA 6009, Australia
4Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland
5Museum and Art Gallery of Northern Territory, GPO Box 4646, Darwin, NT 0801, Australia
6Research institute for Environment and Livelihoods, Charles Darwin University, Darwin, NT 0909, Australia
7Royal Tasmanian Botanic Gardens, Queens Domain, Hobart, TAS 7000, Australia
8Tasmanian Institute of Agriculture, University of Tasmania, Private Bag 98, Hobart, TAS 7000, Australia
9Department of Agriculture and Environment, Curtin University, WA 6102, Australia
An increasing diversity of highly specialized pollination systems are being discovered, many of which are likely to be vulnerable to anthropogenic landscape modification. Here, we investigate if a specialized pollination system limits the persistence of Caladenia huegelii(Orchidaceae), an endangered species pollinated by sexual deception of thynnine wasps. Once locally common in part of its geographical range,C. huegeliiis now largely restricted to small habitat remnants in urban areas. Pollinator surveys coupled with DNA barcoding detected a single pollinator taxon, a small form ofMacrothynnus insignis. Phylogenetic analysis revealed that smallM. insignisfrom within the range ofC. huegelii are strongly divergent from other wasp populations, suggesting that some reproductive isolation may exist. Although common in intact landscapes outside the range of C. huegelli, small M. insignis individuals were recorded at only 4% of sites in suitableC. huegelii habitat. Accordingly, reproductive success in C. huegeliiwas low compared with related Caladeniaspp., with 33–60% of populations failing to set fruit in any given year. As such, populations are likely to now persist primarily through individual plant longevity rather than reproduction. Due to the low reproductive success ofC. huegelii, ongoing human intervention will almost certainly be needed to sustain the species. Future research will need to focus on optimizing hand pollination to maintain reproduction and high seed fitness. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 511–525.
ADDITIONAL KEYWORDS: habitat fragmentation – orchid – sexual deception.
INTRODUCTION
Although pollination by multiple animal species pro- vides greater reproductive assurance, most animal-
pollinated plants are specialized on a subset of the available pollinator community (Fenster et al., 2004;
Rosas-Guerrero et al., 2014). Further, there is increasing recognition that there is a wide range of specialized pollination systems, with plants being reliant on just one or a few species of pollinators
*Corresponding author. E-mail: Ryan.Phillips@anu.edu.au
511 Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-ctuy3kdcag0q3
(Johnson, 2010). In these systems, specialization can be mediated through a diversity of processes includ- ing exaggerated morphology for which only one polli- nator species can reach the nectar (Nilsson et al., 1987), unusual rewards of interest to a small portion of the pollinator community (Houston et al., 1993;
Pauw & Bond, 2011) or chemical attractants that exploit sensory biases of a particular pollinator (Schiestlet al., 2003; Brodmannet al., 2008; Bohman et al., 2014). As a consequence of relying on just a few pollinator species for sexual reproduction, low density or narrow geographical range of the pollinator can contribute to rarity of the plant (Phillips et al., 2014a). Further, should a pollinator species undergo a decline following anthropogenic landscape modifica- tion, plant populations are also likely to decline (Anderson et al., 2011; Pauw & Hawkins, 2011;
Geerts & Pauw, 2012). In extreme scenarios, local extinctions of pollinators can leave plant populations entirely reliant on clonal reproduction. This situation is exemplified by the oil-producing orchids pollinated by the beeRediviva peringueyi, with local extinction of the bees leading to local extinctions of the orchids, and the less clonal species suffering extinction more rapidly (Pauw & Hawkins, 2011).
Orchidaceae are characterized by a high incidence of specialized pollination strategies, with many orchid species reliant on only one or few pollinator species (Tremblay, 1992; Schiestl & Schluter, 2009).
Of these strategies, one of the most diverse and geographically widespread is pollination by sexual deception (Gaskett, 2011; Phillips et al., 2014b). In this pollination strategy, long-range attraction of pol- linators occurs through mimicry of the sex phero- mone of female insects, with pollination achieved by the male during attempted courtship or copulation with the flower (Schiestl et al., 1999, 2003; Bohman et al., 2014). As a by-product of mimicking the spe- cific sex pheromones of insects, sexually deceptive orchids often have only a single known pollinator species (Paulus & Gack, 1990; Phillips et al., 2009b;
Peakallet al., 2010; Gaskett, 2011). However, studies investigating populations of sexually deceptive orchids over a broad geographical range are increas- ingly revealing geographical variation in pollinator species (Breitkopf et al., 2013; Menz et al., 2015b;
Phillips et al., 2015). Furthermore, DNA barcoding has demonstrated that some groups of sexually deceived pollinators contain morphologically cryptic taxa (Griffithset al., 2011; Menzet al., 2015b), poten- tially obscuring the exact number of species actually involved in pollination.
Due to their high pollinator specificity, lineages of sexually deceptive orchids may show higher levels of intrinsic rarity than other pollination strategies, a prediction that has been supported by a regional
analysis in south-western Australia (Phillips et al., 2011). Further, a detailed investigation of correlates of rarity in the sexually deceptive genus Drakaea Lindl. suggested that rarity was most likely caused by the absence of pollinators from otherwise suitable orchid habitat, rather than low levels of reproductive success within existing populations (Phillips et al., 2014a). However, meta-analyses have shown that in other genera sexually deceptive species tend to have lower levels of fruit set than related orchids with more generalized pollination strategies (Phillips et al., 2009b; Gaskett, 2011), suggesting low fruit set could cause rarity in some sexually deceptive species.
Caladenia R.Br. is a diverse genus of terrestrial orchids (>375 taxa) with centres of diversity in south- western and south-eastern Australia (Phillips et al., 2009a), two regions with extensive habitat removal.
Although many Caladenia taxa are pollinated by nectar-foraging insects (Faast et al., 2009; Phillips et al., 2009b), and a small portion are self-pollinating (Jones, 2006),>100 species representing three subgen- era are believed to be pollinated by sexual deception of thynnine wasps (Stoutamire, 1983; Bower, 2001;
Phillips et al., 2009b). Caladenia contains >70 taxa that are currently federally listed as threatened or endangered (Department of the Environment, 2015), over half of which are believed to be pollinated via sexual deception. This trend raises the question of whether pollination by sexual deception is contribut- ing to rarity either through intrinsic rarity of some wasp species or vulnerability of specialized pollination systems to landscape modification. Given the diversity of the genus, understanding causes of rarity in sexu- ally deceptiveCaladeniamay yield useful information for the management of a large number of threatened taxa.
Caladenia huegeliiRchb.f. is a rare species endemic to south-western Australia (Hoffman & Brown, 2011) (Fig. 1). It is currently known only from the Swan Coastal Plain between Perth and Busselton (Fig. 2), preferring areas of banksia woodland on the Bassen- dean dune system (Hoffman & Brown, 2011). Once locally common in at least part of its range (Hopper &
Brown, 2001),C. huegeliihas undergone an extensive decline associated with the large scale habitat destruction for agriculture, housing and light industrial areas, which has continued in recent decades (Brown, Thomson-Dans & Marchant, 1998;
Department of Environment and Conservation, 2008).
Of the 48 known populations, no plants were recorded at 21 of them during the last major surveys (Department of Environment and Conservation, 2008). Further, 17 of the surviving populations con- tained less than five flowering plants (Department of Environment and Conservation, 2008). Due to the small and isolated nature of the remaining popula-
tions,C. huegeliihas been listed as endangered under the Australian federal EPBC Act.
Although habitat clearance has been the major driver of species decline in C. huegelii, surviving
populations may be limited through interspecific interactions, particularly in a now heavily frag- mented landscape. Investigations into the mycorrhi- zal ecology ofC. huegeliihave shown that, although it Figure 1. Macrothynnus insignis s.l.(A), the pollinator of the sexually deceptive orchidCaladenia huegelii(B). Panel (A) illustrates specimens of the large and small forms ofM. insignis s.l.Only the small form is responsible for pollination of C. huegelii. Photographs (A) by Belinda Davis, and (B) by Ryan Phillips.
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Perth Kwinana
Yallingup Busselton
Northcliffe Broke Inlet
Albany
C. huegelii C. thinicola C. huegelii fruit set Place names
Figure 2. Geographical range of Caladenia huegelii and C. thinicola based on records in the Western Australian Herbarium, theC. huegeliirecovery plan and personal observations. Marked in red are sites where fruit set forC. huegelii was recorded.
relies on a single species of mycorrhizal fungus for germination and annual growth (Swartset al., 2010), field germination rates are comparable to co-occurring commonCaladeniaspp. (Swartset al., 2010) and the fungus is geographically widespread (Davis et al., 2015). Alternatively, relatively little is known about the pollination biology of the species. Like most members of Caladenia subgenus Calonema (Benth.) Hopper & A.P.Br., pollinator exclusion experiments have shown that C. huegeliiis reliant on a vector for pollination and is self-compatible (Retter, 2009).
However, a single season survey by See (2006) indi- cated that the availability of pollinators could be limiting reproductive success of this species. Prelimi- nary observations revealed that on rare occasions C. huegelii attracts a large form of the thynnine wasp Macrothynnus insignis with behaviour indica- tive of sexual deception, in which the wasp approaches the flower with the sustained zig-zag flight characteristic of its behaviour when tracking a sex pheromone plume (e.g. Peakall, 1990; Phillips et al., 2013). However, this wasp is too large to be a pollinator and, even when locally common, only rarely exhibits attraction to the flower. A small form of M. insignis pollinates the putative sister species, Caladenia thinicolaHopper & A.P.Br. (Phillips et al., 2009b), raising the possibility that this wasp or a close relative could also be involved in the pollination of C. huegelii. The morphological study of Brown (1995) did not reveal any morphological difference between the small and large forms of M. insignis s.l. other than a disjunction in size (Fig. 1; total length of small form: mean = 19.6 ± 0.5 mm (s.e.);
range = 15.2–23.9 mm (n= 17); total length of large form: mean = 31.2 ± 0.5 mm; range = 27.2–34.3 mm (n= 15); R.D. Phillips, unpublished data), but the apparent differences in response to the orchids sug- gests that they may represent different taxa.
The overall objective of this study was to determine if a specialized pollination system could limit persis- tence ofC. huegelii. Specifically, we sought to answer the following questions:
(1) Which taxon is responsible for the pollination of C. huegelii?
(2) What is the current abundance of the pollinator?
(3) What is the geographical range of the pollinator relative to the orchid?
(4) Is C. huegelii characterized by low reproductive success compared to related species?
To help resolve the taxonomy of the putative polli- nators, we take advantage of DNA barcoding, which has proved to be highly informative for detecting morphologically cryptic species of thynnine wasps (Griffiths et al., 2011; Menzet al., 2015b).
MATERIALS AND METHODS POLLINATOR SURVEYS AND OBSERVATIONS
Observations of pollinator behaviour were under- taken using the pollinator baiting method, in which picked orchid flowers are moved to a new part of the landscape to renew the pollinator response (Stoutamire, 1974; Peakall, 1990). Bait flowers were picked at the base of the stem and kept fresh in vials of water for up to 2 weeks in a portable refrigerator atc. 4 °C. Although observations in otherCaladenia spp. suggest there is no detectable decrease in polli- nator attraction over this period, most bait flowers were used in experiments within 4 days of being picked. In all baiting trials, two flowers ofC. huegelii were used. ForC. huegelii, flowers were sourced from populations represented by the voucher specimens PERTH07501080 and PERTH07439938 in the Western Australian Herbarium (locations withheld as a requirement of permits to study rare flora). Polli- nator observations were undertaken in sunny weather when ambient temperatures exceeded 18 °C.
We recorded if the responding pollinator exhibited the zig-zag flight associated with tracking a sex pheromone plume, if they attempted copulation with the flower and if they came into contact with the column (see Phillipset al., 2013). Where possible, before leaving the flower, floral visitors were netted for identification and subsequent genetic analysis.
Due to the rapid attraction of pollinators to experi- mentally presented bait flowers, this method can also be used as an effective tool to survey the abundance and distribution of sexually deceived pollinators (Phillipset al., 2014a). Pollinator surveys were under- taken at 92 sites across the full geographical range of C. huegelii. Sites were chosen based on soil type and broad vegetation community matching that of C. huegelii populations (Fig. 3; see Department of Environment and Conservation, 2008 for more precise details of habitat). Due to the extensive habitat clear- ing in this region, our survey was able to cover most large bushland remnants on publically accessible land. Surveys were undertaken progressively through September and October of 2008, 2011 and 2012.
Baiting was conducted in a 20 m × 40 m rectangle with bait positions at each corner of the rectangle and mid-way along its two long sides, giving a total of six baiting positions at each site. Each position was baited for 2 min. It has previously been shown for other orchids pollinated by sexual deception of thyn- nine wasps that this survey design yields 90% con- gruence in presence/absence of the pollinator across years (Phillipset al., 2014a). Nonetheless, this design was repeated across 2 years at 22 sites and three years at 17 sites.
DISTRIBUTION OF THE POLLINATOR –MUSEUM RECORDS AND BAITING WITH OTHER ORCHIDS In light of evidence in 2011 that the small form of M. insignisis a pollinator ofC. huegelii (see results), we took two complementary approaches to resolve the distribution of this taxon beyond the range ofC. hue- gelli. One of us (GRB) searched the collections of the Western Australian Museum, the Western Australian Department of Agriculture and the Australian National Insect Collection for records of this wasp.
Secondly, following the same survey design as outlined above, baiting was undertaken with orchids known to attractM. insignis, either as a pollinator or a ‘minor responder’, defined as wasps that are sexually attracted to the flower, but exhibit inappropriate size or behaviour to achieve pollination (sensu Bower, 1996). The small form of M. insignis pollinates C. thinicola(Phillipset al., 2009b), but it is also occa- sionally attracted to C. attingens Hopper & A.P.Br.
subsp. attingens and C. granitora Hopper & A.P.Br., but has not been observed to actually alight on the flower in either of these species (Phillipset al., 2009b) (RD Phillipset al., unpublished data). We undertook baiting withC. thinicola(32 sites),C. attingenssubsp.
attingens(60 sites) andC. granitora(five sites) within the natural distribution of these orchids (Fig. 3;
voucher populations: C. attingens subsp. attingens:
PERTH08604347, RDP0229, RDP0281; RDP0286;
C. granitora: PERTH005536294; C. thinicola:
PERTH08604347; PERTH08604460, RDP0276, RDP0299). At multiple sites, representatives of the visiting wasps were collected for identification and DNA barcoding.
DNABARCODING – IDENTIFICATION AND DISTRIBUTION OF THE POLLINATOR
Due to recent discoveries of morphologically cryptic species in some genera of thynnine wasps (Griffiths et al., 2011; Menzet al., 2015b), genetic analysis was undertaken to help resolve species boundaries within M. insignis s.l. Specimens of M. insignis collected from all orchid species used in the baiting studies were used for genetic analysis (Supplementary Table S1). These specimens extended along the coast from Kwinana in the north to Albany in the south- east, a distance of c. 520 km (Fig. 4). In addition, specimens were included that were collected by oppor- tunistic sweep-netting of patrolling males or catching mating pairs while feeding on nectar plants. In total, 20 and 19 individuals, respectively, of the small and large forms ofM. insignis s.l. were sequenced.
DNA barcoding using the mitochondrial DNA CO1 locus was used to investigate species boundaries in M. insignis s.l. This locus has previously been useful for resolving species-level differences in thynnine wasps (Griffithset al., 2011; Menzet al., 2015b). DNA extraction followed the methods of Griffiths et al.
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Baiting with C. huegelii Baiting with other species M. insignis present
Figure 3. Sites where the small form ofMacrothynnus insignishas been recorded and sites where surveys for it were undertaken using orchids as bait flowers.
(2011). Initially, the primer set of Folmeret al. (1994), Hebertet al. (2004) and Hajibabaeiet al. (2006) was used for DNA amplification. However, this set of primers failed to readily amplify DNA of M. insignis s.l. Following failed attempts to develop primers for this locus, we instead used the Simon et al. (1994) primer set. These primers amplify a region of CO1 immediately adjacent to the region amplified by the Folmer primers, in the 3′ direction. This set of primers successfully amplified DNA for all individuals ofM. insignis, except those that responded toC. hue- gelii. For these individuals we developed a final set of primers (MthynCO1B.1F GATTYTTTGGWCAYCCA- GAAG; MthynCO1B.1R AATTTTTTAAAGTAT- GATGGT), which successfully amplified these specimens for the Folmer et al. (1994) region. To confirm that the CO1 region used by Simon et al.
(1994) yields comparable results to the Folmer et al.
(1994) region, we sequenced between four and 28 individuals of severalZaspilothynnusspp., using the same individuals as Menzet al. (2015b).Zaspilothyn- nus was chosen as the genus for comparison as phy- logenetic evidence provides strong support that Macrothynnus is nested within Zaspilothynnus (RD Phillips et al., unpublished data).
A multiple sequence alignment was performed in Geneious v7.1.4 (Kearse et al., 2012). Sequence data
were analysed using maximum likelihood and Bayes- ian analyses. The maximum likelihood analysis was run in PHYML (Guindon & Gascuel, 2003) via a plug-in in Geneious v7.1.4 (Kearseet al., 2012), with 1000 bootstrap replicates using the GTR + G model of nucleotide substitution. The Bayesian analysis was run in Mr Bayes 3.2.2 (Huelsenbeck & Ronquist, 2001) through the CIPRES portal (Miller, Pfeiffer &
Schwartz, 2011). Two parallel runs of the analysis were run for 2 million generations (first 5% discarded as burn in, Markov chain sampled every 1000 gen- erations with four chains), using the GTR + G model.
However, the analysis ceased afterc.860 000 genera- tions as the standard deviation of the split frequen- cies reached<0.01. We quantified the maximum level of sequence divergence within putative species using Geneious. To confirm that the Simon et al. (1994) region of CO1 yielded comparable results to the Folmer et al. (1994) CO1 region previously used for barcoding studies of thynnine wasps, we calculated the correlation between the level of sequence diver- gence within species for both regions of the CO1 locus using JMP v9.0.0 (SAS Institute Inc., 2010).
REPRODUCTIVE SUCCESS
Hand pollination experiments have shown that fruit- ing of C. huegelii is pollen- rather than resource-
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C. huegelii
M. insignisscp clade M. insignissouth clade
Figure 4. Records of the two clades of the small form of Macrothynnus insignis relative to the geographical range of Caladenia huegelii. Distributional data forC. huegeliicome from the Western Australian Herbarium and theC. huegelii recovery plan.
limited, with no difference in the proportion of seeds containing embryos between self-pollination and out- crossing treatments (Retter, 2009). Although the number of seeds produced byC. huegeliihas not been quantified, the closely relatedC. arenicola Hopper &
A.P.Br. produces c. 30 000 seeds per capsule (Batty, 2001). Fruit set was assessed at a total of ten popula- tions in 2008, 2011 and 2012. Data collection focused on urban remnants in the south-eastern suburbs of Perth, Western Australia, the core range ofC. huegelii (Fig. 2). Fruit set was recorded in these populations in 2008 and 2012. In 2011, fruit set was recorded in some of these suburban sites plus three sites from the southern part of the geographical range ofC. huegelii.
The ability to collect data was limited in some sites by low flowering in some years and permission to access study sites. Percentage fruit set was calculated as the number of fully formed fruits divided by the number of flowers in the population, thoughC. huegeliiis primar- ily single-flowered (Hopper & Brown, 2001). For each site, the percentage fruit set was averaged across years to give a mean for that site. The average percentage fruit set across populations of C. huegelii was com- pared with data available in the literature for other members ofCaladeniasubgenusCalonema(Peakall &
Beattie, 1996; Phillips et al., 2009b, 2013). As a measure of the actual reproductive output per popula- tion, the number of fruits formed in each year was used to calculate a mean for each population and an overall mean across populations.
RESULTS
POLLINATOR SURVEYS AND OBSERVATIONS Pollinator observations across the entire geographical range ofC. huegeliirevealed that only the small form of M. insignis was both attracted to C. huegelii and exhibited the necessary behaviour for pollen removal and deposition. During the present study, there were no instances of the large form of M. insignis being attracted to C. huegelii. Despite an extensive multi- year survey, only six M. insignis individuals were attracted toC. huegelii, none of which carried pollinia.
All of the responding wasps displayed the character- istic zig-zag flight associated with tracking a sex pheromone plume. Three out of the six responding wasps crawled into the position needed for pollination, with at least two attempting copulation with the flower. Pollinators were observed at only four of the 92 sites surveyed (4.3%), with only one of these sites supporting a known population ofC. huegelii. At each of these sites they were only detected in 1 of the 2 years in which they were surveyed. By comparison, when baiting withC. thinicola outside the range ofC. hue- gelii, the small form ofM. insigniswas recorded at 13 of the 32 sites surveyed (40.6%).
DISTRIBUTION OF THE POLLINATOR BASED ON MUSEUM RECORDS
Searches of museum collections revealed that prior to this study the small form of M. insignis was known from only three specimens, collected from Kwinana, Yallingup and Northcliffe (Fig. 3). Of these locations, Kwinana is broadly within the natural range of C. huegelii and the other locations are within the range of C. thinicola. In addition to wasps collected within the core range of C. huegeliiand C. thinicola, baiting with orchids yielded specimens of the small form ofM. insignisfrom Albany (C. granitora), North- cliffe (C. attingens subsp. attingens) and Broke Inlet (C. thinicola) (Fig. 4).
DNABARCODING – IDENTIFICATION AND DISTRIBUTION OF THE POLLINATOR
Comparison with the data of Menzet al. (2015b) dem- onstrated that sequence divergence within species is strongly correlated for the two regions of the CO1 sequence locus (R= 0.97), thereby confirming the utility of the Simon et al. (1994) region for thynnine wasps. Phylogenetic analysis resolved three strongly supported clades within M. insignis s.l. (Fig. 5). The large form ofM. insigniswas sister to the two clades of the small form of M. insignis. One of these clades contained only wasps from the Swan Coastal Plain (SCP clade), including all three sequenced pollinators of C. huegelii. The other clade contained samples ranging from Yallingup to Albany (South clade), including wasps attracted toC. thinicola,C. granitora and C. attingens subsp. attingens. Maximum sequence divergence within each of these clades was low (0% in the SCP clade; 1.2% in the South clade), but comparable to that seen in some other species of thynnine wasp (Table 1). The minimum level of sequence divergence between the large form of M. insignis and the other two clades was c. 10%, which is comparable with levels of interspecific variation in other genera of thynnine wasps. The minimum sequence divergence between the SCP clade and the South clade was 4.7%, which is less than that seen within the widespreadZ. nigripes, but greater than in other thynnine species.
REPRODUCTIVE SUCCESS
Averaged across all populations, fruit set for C. hue- gelii was 3.8 ± 1.1 (S.E.)% (Table 2), which is lower than that observed in other sexually deceptive members ofCaladeniasubgenusCalonema(range for other species: 9.9–20.5%; Table 3). Out of the ten populations, fruit set was observed in seven, with 33–60% of populations failing to set fruit in any given year. Of the seven populations in which fruit set data
was recorded in multiple years, five populations expe- rienced at least one year with no fruit set. Overall, there was on average 1.0 ± 0.3 fruits produced per population per year.
DISCUSSION
POLLINATOR SPECIES OF C. huegelii
Baiting at over 90 sites across the geographical range of C. huegelii yielded a single taxon of sexually
deceived pollinator, the small form of M. insignis, despite the presence of diverse thynnine wasp com- munities in these habitats (Menzet al., 2015a). This apparently high specificity matches other sexually deceptive orchids, which are typically pollinated by one or few insect species (Phillips et al., 2009b;
Gaskett, 2011). Although males of the large form of M. insignis were observed patrolling for females at>15 survey sites, none of them was attracted to the bait flowers, confirming that this taxon is not a pol- Figure 5. Phylogenetic analysis of the mtDNA CO1 locus (‘Simon’ region) for Macrothynnus insignis s.l.and related species in the genusZaspilothynnus. One of the clades ofM. insignis is the pollinator ofCaladenia huegelii. SCP clade:
wasps collected from the Swan Coastal Plain; South clade: wasps collected from the southern part of the range of M. insignis. Numbers at nodes above the line are bootstrap values from a maximum likelihood analysis, numbers below are posterior probabilities from a Bayesian analysis. Numbers in parentheses indicate the number of identical sequences.
linator ofC. huegelii. Furthermore, the degree of CO1 divergence between the small and large forms of M. insignis (10%) falls well outside typical intraspe- cific variation (most species<2%; Table 1), leading us to conclude that these sympatric forms represent dif- ferent species.
Baiting for pollinators with other sexually decep- tive Caladenia spp. revealed that the small form of M. insignisoccurs over a wide area beyond the range ofC. huegelii. Indeed, in some of the southern parts of its distribution it appears to be locally common in areas with large amounts of intact habitat. However, populations from the SCP clade and southwards along the coast from Yallingup (South clade) form two diver- gent clades. While the level of genetic divergence between these clades (4.7%) is comparable with that seen in the widespread Zaspilothynnus nigripes, it exceeds the level of divergence seen in other closely
related members of Zaspilothynnus. Although levels of divergence within the clades are low across mod- erate distances (SCP = 0% over 90 km; South 1.2%
over 300 km), the 4.7% sequence divergence occurs over a distance of only 50 km between clades. This higher than expected genetic divergence raises the possibility that a level of reproductive isolation may exist between these two clades and that M. insignis s.l. could represent a complex of up to three species.
Biogeographic evidence supports the plausibility of this situation, as the SCP is characterized by high levels of endemism in both plants and animals (Hopper & Gioia, 2004; Cogger, 2014), often with considerable changes in community composition with adjoining regions.
Further resolution of the species boundaries within M. insignis s.l. could be important for understand- ing distributional limits in C. huegelii. Critically, Table 1. Percentage sequence divergence in clades ofMacrothynnus insignis s.l.and closely related members of the genus Zaspilothynnus. The Folmeret al. (1994) region is that typically used for DNA barcoding studies. The Simonet al. (1994) region is immediately adjacent to the Folmeret al. (1994) region but in the 3′direction. ‘Small’ and ‘large’ refers to two morphologically distinct forms ofM. insignis. X: data could not be obtained due to consistent failure to amplify DNA.
N= number of wasp individuals sequenced
Taxon % divergence ‘Folmer’ CO1 % divergence ‘Simon’ CO1
Macrothynnus insignis– large X 0.5 (N= 19)
Macrothynnus insignis– small – SCP clade X 0 (N= 4)
Macrothynnus insignis– small – South clade X 1.2 (N= 16)
Macrothynnus insignis –small – both clades X 4.7 (N= 20)
Zaspilothynnus dilatatus 0.6 (N= 4) 0.5 (N= 5)
Zaspilothynnus gilesi(red-legged) 2.3 (N= 7) 1.5 (N= 7)
Zaspilothynnus gilesi(black-legged) 0.7 (N= 12) 0.6 (N= 10)
Zaspilothynnus nigripes 5.8 (N= 28) 5.4 (N= 28)
Zaspilothynnussp. A 1.5 (N= 6) 0.5 (N= 4)
Zaspilothynnussp. B 2.6 (N= 10) 1.5 (N= 10)
Zaspilothynnus rugicollis 0 (N= 7) 0 (N= 5)
Zaspilothynnus seductor 1.1 (N= 11) 0.7 (N= 9)
Table 2. Percentage fruit set in populations ofCaladenia huegelii. Numbers in parentheses are the number of flowering plants in that year. IF = insufficient flowering for reproductive success to be scored. (–) population was not surveyed
2008 2011 2012 Average
Wandi 0 (7) IF IF 0
Fraser S 0 (12) 0 (9) 0 (14) 0
Dennis B 14.28 (7) IF 0 (5) 7.14
Ken Hurst L 0 (18) 7.1 (28) 2.94 (34) 3.35
Ken Hurst S 0 (10) – 7.69 (13) 3.85
Caladenia 9.09 (11) – 3.37 (27) 6.4
Fraser L 11.76 (33) – 4.28 (70) 8.02
Shirley Balla – 0 (8) – 0
Kooljenerrup – 9.1 (22) – 9.10
Ruabon NR – 0 (4) IF 0
experimental opportunities provided by sexually deceptive pollination systems may enable tests of whether the SCP and South clades are likely to exhibit reproductive isolation via differences in sex pheromones. Experimental presentation of calling females is one plausible method for conducting choice tests with male thynnines (Alcock, 1981). Similarly, choice tests, in which both pollinator clades are exposed toC. huegeliiandC. thinicola(as per Bower, 1996), could provide evidence of differences in sex pheromones should the two orchid species differ in floral odour. Testing pollinator responses to the floral odour of C. huegelii and C. thinicola with gas chromatography-electroantennographic detection (GC-EAD) would provide a test of whether the two clades of pollinator detect the same components of the floral odour. Although this technique has been used as a starting point to determine sex pheromone systems in other thynnine wasps, synthesis of compounds and experimental validation of sexual attraction in the field are needed to confirm that compounds detected in GC-EAD function in attraction (Schiestl et al., 2003; Peakall et al., 2010; Bohman et al., 2014).
However, due to the large number of experimental animals needed, larger populations of M. insignis from the SCP clade will need to be found before these approaches will become feasible in this system.
POLLINATOR RARITY AND LOW REPRODUCTIVE SUCCESS IN C. huegelii
Despite intensive survey effort, the small form of M. insignis was only recorded from four sites within
the geographical range of C. huegelii, with only one containing a known population ofC. huegelii. Due to the low number of thynnine wasp specimens collected from the SCP in previous decades, it is impossible to judge whether the small form ofM. insignisis natu- rally rare in this region or has declined through habitat clearance and fragmentation of the remaining vegetation. However, at least some decline seems likely to be based on the extensive declines, and in some cases local extinction, documented for many vertebrate species on the SCP following extensive habitat clearance (Storr & Johnstone, 1988; How &
Dell, 2000; Davis, Gole & Roberts, 2013).
The current scarcity of the small form ofM. insignis on the SCP suggests that an absence of pollinators from areas of suitable habitat forC. huegeliicould be contributing to population decline in this orchid. This prediction is supported by the low levels of reproduc- tive success in C. huegeliicompared to related sexu- ally deceptive orchids (Table 3), with 33–60% of populations failing to set fruit in any given year.
Comparison with the closely relatedC. thinicola pro- vides further support for this argument. Caladenia thinicolais pollinated exclusively by the small form of M. insignis, but is allopatric toC. huegelii. Within the range of C. thinicola, the small form of M. insignis was recorded at 40.6% of sites (versus 4% inC. hue- gelii), with much higher fruit set inC. thinicolathan in C. huegelii (20.5% for C. thinicola vs. 3.8% for C. huegelii).
Low levels of fruit set in C. huegelii, despite the apparent absence of a thynnine wasp pollinator the study populations, raises the question of how these Table 3. Percentage fruit set in members ofCaladeniasubgenusCalonema, listed by pollination strategy. Species have only been included if data is available for three or more populations. Numbers are the percentage fruit set ± standard error with the number of populations measured in parentheses
Taxon Fruit set (%) Reference
Sexual deception
Caladenia arenicola 9.9 ± 3.1 (4) (Phillipset al., 2009b)
Caladenia attingenssubsp. attingens 19.9 ± 7.3 (12) (Phillipset al., 2009b)
Caladenia ferruguinea 14.4 ± 9.9 (3) (Phillipset al., 2009b)
Caladenia huegelii 3.8 ± 1.1 (10) Present study
Caladenia pectinata 12.3 ± 5.6 (8) (Phillipset al., 2013)
Caladenia tentaculata 14.7 ± 8.6 (9) (Peakall & Beattie, 1996)
Caladenia thinicola 20.5 ± 3.1 (5) (Phillipset al., 2009b)
Food deception
Caladenia longicaudasubsp. eminens 42.6 ± 9.2 (4) (Phillipset al., 2009b)
Caladenia serotina 50.2 ± 15.7 (3) (Phillipset al., 2009b)
Caladenia speciosa 40.0 ± 6.9 (3) (Phillipset al., 2009b)
Nectar producing
Caladenia rigida 33.7 ± 3.1 (8) (Bickerton, 1997; Phillipset al., 2009b)
Unresolved strategy
Caladenia behrii 14.9 ± 1.7 (3) (Petit & Dickson, 2005)
pollination events are achieved. One possibility is that the small form ofM. insignisdoes occur in these remnants at low abundance, but our surveys did not detect them. Alternatively, pollination may occur in the absence of a sexually deceived pollinator by occa- sional food deception events. Although to the human eye, C. huegelii has the dull red labellum character- istic of many sexually deceptive Caladenia (Phillips et al., 2009b), it also has large, cream coloured peri- anth parts that may occasionally attract nectar- foraging insects. This possibility is supported by an observation of pollination of the sexually deceptive Caladenia behrii Schltdl. by a food-foraging fly (Dickson & Petit, 2006). On rare occasions, thynnine wasp individuals belonging to species that typically do not alight on or attempt copulation with orchids can achieve pollination (Peakall et al., 2010).
Although such events may contribute to pollination of C. huegelii, observations in other sexually deceptive orchids suggest that they are likely to be rare and therefore difficult to observe (Phillips et al., 2009b;
Peakall et al., 2010; Phillips, 2010). Should C. hue- gelii have the capacity to attract pollinators by either of these mechanisms, it may confer some resil- ience in the absence of its primary sexually deceived pollinator.
Given the recent fragmentation of the landscape and the evidence for long life spans in at leastCalad- enia subgenus Calonema (25+ years, Swarts et al., 2009), several of these populations ofC. huegeliimay be below the threshold for persistence but have not yet suffered local extinction, a situation that has been termed an ‘extinction debt’ (Tilmanet al., 1994). This phenomenon has been shown for both plant and pol- linator communities, but the response to fragmenta- tion may be more rapid for pollinators (Sang et al., 2010; Pauw & Bond, 2011; Pauw & Hawkins, 2011) due to their short generation times (Kuussaariet al., 2009). Because of the consistently low fruit set and the rarity of the pollinating thynnine wasp, detailed empirical data on population growth rates are needed to assess the potential for long-term population per- sistence and the predicted time until extinction (e.g.
Kuussaari et al., 2009). Although some groups of Caladeniashow extensive clonal reproduction via the proximal production of daughter tubers (Dixon &
Tremblay, 2009), clonality in wild plants is believed to be rare or absent inC. huegelii and related species, suggesting that persistence of populations is more likely to be related to longevity than clonality.
PROSPECTS FOR THE MANAGEMENT OF C. huegelii Given the decline of the species as a whole (Brown et al., 1998; Department of Environment and Conservation, 2008) and the low reproductive success
of the remaining populations, future management of C. huegelii may require direct intervention through hand pollination, supplemental planting or the estab- lishment of new populations. Although supplemen- tary hand pollination has been shown to increase population sizes in Caladenia hastata (Nicholls) Rupp (N. Reiter, pers. comm.), there are limited data available on the costs to the individual of repeated fruiting for this group of orchids (but see Coates &
Duncan, 2009). Given the small and isolated nature of many populations of C. huegelii (Department of Environment and Conservation, 2008), which may already exhibit some inbreeding depression, we would strongly recommend consideration of the possible benefits of ‘genetic rescue’ by hand pollination between populations (Frankham, 2015). Species such as C. huegelii, which are not autogamous, have consistent habitat preferences (Department of Environment and Conservation, 2008) and have populations that were recently connected (Swarts et al., 2009), are highly likely to show increased fitness following pollen transfer between populations, with minimal risk of outbreeding depression (Frankham et al., 2011; Frankham, 2015).
Bolstering or establishing populations by the plant- ing of seedlings is also likely to be feasible for this species. Techniques for the cultivation and planting of Caladenia are well refined, with multiple successful reintroductions being conducted in south-eastern Aus- tralia (Wright et al., 2009). Critically, pollinator surveys provide an important method for establishing candidate sites for the establishment of new popula- tions. Alternatively, although reintroduction of polli- nating insects has been undertaken on other continents (e.g. http://www.bumblebeereintroduction .org/), reintroductions of thynnine wasps are likely to be challenging. Thynnine wasps have a complex life cycle in which females parasitize underground scarab beetle larvae (Ridsdill-Smith, 1970) and adults are reliant on either nectar or sugary secretions from other insects as a food source (Brown & Phillips, 2014). Given that the specific requirements are known for few, if any, thynnine species, it is unlikely that pollinator reintroductions or management of habitat specifically for pollinators will be achieved in the foreseeable future without a concerted research effort to understand the biology of these species better.
IMPLICATIONS FOR FLORA THAT REQUIRE POLLINATION MANAGEMENT
To manage cases successfully in which the pollinator is virtually absent, some critical information will be needed before implementing hand pollination.
For example, as some plant species show a cost of
reproduction (Primack & Hall, 1990; Obeso, 2002), there will be an optimum interval between artificial reproductive events. Similarly, there may be an optimal distance range of pollen transfer (within populations, between nearby populations, between distant regions) that maximizes fitness or adaptive potential. Ideally, the success of crosses should be evaluated with germination experiments in the field, as the effects of inbreeding may increase at later life history stages (e.g. Smithson, 2005). Further, when undertaking crosses between plants it will be neces- sary to avoid artificial selection (e.g. favouring larger flowered individuals). We are unaware of any cases involving management to sustain populations in the absence of pollinators. However, given the declines recorded in many groups of pollinators (Potts et al., 2010), we predict that these are going to become increasingly important topics for the conservation of rare plant species (Menz et al., 2011).
ACKNOWLEDGEMENTS
Funding was provided by Jandakot Airport Holdings and an Australian Research Council Linkage grant (LP110100408) to RP and KWD, a grant from the Australian Orchid Foundation to RDP, and grants from the Holsworth Wildlife Research Endowment to RDP and MHMM. During parts of this project RDP and MHMM were supported by Australian Postgradu- ate Awards. Thank you to Keith Smith, Alyssa Wein- stein and Jeff Hardwick for assistance with fieldwork.
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