Papilionidae Research Articles

A test of the Grant-Stebbins pollinator-shift model of floral evolution.

Pollinators are thought to play a key role in driving incipient speciation within the angiosperms. However, the mechanisms underlying floral divergence in plants with generalist pollination systems, remains understudied. Brunsvigia gregaria displays significant geographical variation in floral traits and are visited by diverse pollinator communities. Because pollinators are often shared between populations, we investigated whether specific pollinators are responsible for driving floral divergence between them. Three distinct ecotypes were identified, each dominated by three different pollinators: bees, swallowtail butterflies, and long-proboscid flies. Across seven populations, we found a pattern of association between style length and the morphology of pollinators that visit the flowers most frequently and contact the reproductive parts most often. Furthermore, we found significant linear, quadratic and correlational selection on flower number, tepal length and style length within the butterfly- and bee-dominated populations. We also found partial evidence for divergent selection on these traits between experimental sites. Our findings suggest that a handful of key pollinators that vary in their importance have the potential to drive population-level divergence in floral traits, which may lead to pollination ecotype formation.

Wiped out by an earthquake? The 'extinct' Taiwanese swallowtail butterfly (Lepidoptera, Papilionidae) was morphologically and genetically distinct.

For the first time, we obtained for the first time a COI DNA barcode from museum specimens of the Old World swallowtail butterfly endemic to Taiwan, Papilio machaon ssp. sylvina, that has disappeared since the devastating Jiji earthquake in 1999 that shook Central Taiwan. We demonstrate that this population was not only phenotypically distinct, but also had a unique mitochondrial haplotype among all other Holarctic populations of P. machaon. The life history of P. m. sylvina from rearing experiments carried out in the 1990s is illustrated and discussed.

Puzzling androecium development in Bauhinia galpinii (Fabaceae) facilitates wing pollination by butterflies

Floral adaptation to pollinators often involves key modifications of floral development. We investigated floral development and its function for pollination in Bauhinia galpinii, a legume species native to eastern and southern Africa with a unique arrangement of stamens and staminodes. Flowers and floral buds at different stages were prepared for observation under Scanning Electron Microscopy (SEM), Light Microscopy (ML), and X-ray Computed Tomography (XCT). Pollinator observations were conducted in populations in southern Africa and floral advertising and reward traits were quantified. Five (rarely six) sepals emerge helically, followed by the petal primordia in unidirectional order. The carpel arises synchronously with the stamens and expands rapidly. Stamen initiation occurs unidirectionally, with three abaxial antesepalous stamens fully developing while other stamen primordia remain sterile and underdeveloped (staminodial). The number of adaxial staminode tips can fluctuate by branching, regardless of their original whorl. Concentric collateral bundles supply all the staminodes. Swallowtail butterflies transfer pollen through their fluttering wings that touch the abaxial anthers and stigma while feeding on nectar in the hypanthium located on the adaxial side. Bauhinia galpinii exhibits an androecium characterised by secondary, unstable branching of staminodes, which form adaptive structures suited to its pollinators. The variation in androecium structure may be attributed to heterochronic changes during development, marked by an early carpel initiation and its basal expansion leading to spatial restriction. This ontogenetic contingency, associated with selective pressures from pollinators, notably swallowtail butterflies, seems to play a role in selecting this complex and distinct floral architecture.

Reproductive ecology and egg parasitism of the Samoan swallowtail butterfly

AbstractWe investigated the reproductive ecology and effects of egg parasitism on the Samoan swallowtail butterfly (Papilio godeffroyi), which survives only on Tutuila Island, American Samoa, after having disappeared from the much larger islands of Upolu and Savai‘i in independent Samoa. During monthly surveys of its only known host plant, Micromelum minutum, across eight sites in 2013 and 2014, we collected eggs, eggshells, larvae, pupae, and pupal exuviae. Live specimens were reared under laboratory conditions to determine reproductive outcomes, developmental rates, and sex ratios, as well as parasitoid attack frequencies, brood sizes, and sex ratios. Sixty‐six of 448 (14.7%) eggs produced larvae, 47 of which became adults. The sex ratio was approximately even overall and within each developmental stage. Eggs were slightly larger on individual host trees and in host tree stands that yielded more eggs per unit of foliage, indicating that ovipositing females responded to some features of host trees and stands. Eggs hatching female or male larvae were similar in size, and the sexes developed at similar rates. A newly described species of parasitoid wasp, Ooencyrtus pitosina (Encyrtidae), emerged from 73.6% of 382 butterfly eggs that failed to hatch in the laboratory (62.7% of 448 eggs overall). Forty‐one other eggs contained dead parasitoid larvae. An additional, unidentified Ooencyrtus wasp species emerged from a single P. godeffroyi egg. No parasitoids were reared from P. godeffroyi larvae or pupae. Of 656 P. godeffroyi eggshells collected in the field and examined in the laboratory, 62.2% showed signs of having been parasitized by O. pitosina. There was no evidence that parasitism rates were density‐dependent. O. pitosina brood sizes ranged from 1 to 5, with the sex ratio skewed toward females (2.40 F:1.00 M). Larger parasitoid broods were associated with slightly larger host eggs, indicating that female wasps may adjust brood size according to host egg size or that fewer wasp larvae are able to complete development in smaller eggs. Techniques used to rear both P. godeffroyi and O. pitosina in the laboratory could be applied to a captive‐rear, wild‐release program, which may facilitate reestablishment of the species in Samoa.

Conservation imperatives for swallowtail butterflies (Lepidoptera: Papilionidae): a case study in the north bank landscape of river Brahmaputra, Bodoland Territorial Region, India

The decline of swallowtail butterflies in forest habitats, which was not a significant concern about two decades ago, has now garnered attention worldwide, leading to their designation as globally endangered by the International Union for Conservation of Nature. A recent study conducted in the north bank landscape (NBL) of river Brahmaputra, specifically under the Bodoland Territorial Region in India, has shed light on the remarkable diversity of swallowtail butterfly species in the area. The study documented a total of 35 species representing eight different genera. Notably, three species are endemic to northeastern India, and 12 enjoy federal protection. The findings suggest that the studied landscape plays a crucial role in supporting larval host plants and adult resources of swallowtail butterflies along with the other abiotic factors. These butterflies depend on 25 plant species from six families as essential food sources. Unfortunately, these host plants, valued for their traditional medicinal properties, are being overexploited. Urgent conservation measures are imperative to safeguard the habitats of swallowtail butterflies and other wildlife in the NBL under Bodoland Territorial Region, as they face significant threats from practices such as agriculture, illegal tree felling, forest fires, and cattle farming.

Identification and in vivo functional analysis of furanocoumarin-responsive cytochrome P450s in a Rutaceae-feeding Papilio butterfly.

The Order Lepidoptera contains nearly 160,000 described species and most of them are specialist herbivores that use restricted plant species as hosts. Speciation that originated from host shift is one of the important factors for the diversification of Lepidoptera. Because plants prepare secondary metabolites for defense against herbivores, with varying profiles of the components among different plant taxa, the specialist herbivores need to be adapted to the toxic substances unique to their host plants. Swallowtail butterflies of the genus Papilio consist of over 200 species. Approximately 80% of them utilize Rutaceae plants, and among the remaining species, a specific subgroup uses phylogenetically distant Apiaceae plants as larval hosts. Rutaceae and Apiaceae commonly contain toxic secondary metabolites, furanocoumarins, and molecular phylogenetic studies support the concept that Apiaceae feeders were derived from Rutaceae feeders. Molecular mechanisms underlying furanocoumarin tolerance in Papilio butterflies have been investigated almost exclusively in an Apiaceae feeder by an in vitro assay. In contrast, there is little information regarding the Rutaceae feeders. Here, we focused on a Rutaceae feeder, Papilio xuthus, and identified two furanocoumarin-responsive cytochrome P450-6B (CYP6B) genes, of which one was an ortholog of a furanocoumarin-metabolizing enzyme identified in the Apiaceae-feeding Papilio while the other was previously unreported. We further conducted in vivo functional analysis using the CRISPR/Cas9 system, revealing a contribution of these CYP6Bs to furanocoumarin tolerance of P. xuthus larvae. Our findings suggest that co-option of furanocoumarin-metabolizing CYP6B enzymes at least partially contributed to the host shift from Rutaceae to Apiaceae in Papilio butterflies.

The Role of Floral Morphology and Nectar Chemistry in Host Plant Selection by Indian Swallowtail Butterflies (Papilionidae)

The Role of Floral Morphology and Nectar Chemistry in Host Plant Selection by Indian Swallowtail Butterflies (Papilionidae)

Examining elevation and thermoregulatory trait differences of endemic tropical swallowtail butterflies to assess their vulnerability to climate change

Abstract Tropical montane insects are vulnerable to climate change if up‐hill range retractions reduce range sizes and increase extinction risk. Endemic species will be particularly at risk if they disproportionately occur at higher elevations, although threats may be ameliorated if they have morphological traits associated with improved thermoregulatory ability. We examine the vulnerability of endemic swallowtail butterflies on Sulawesi (Indonesia) to climate change by quantifying inter‐specific variation in traits associated with thermoregulation (body size and melanism) and whether species' traits are associated with the elevation of species' ranges. We collected data on size, melanism and elevation of 29 swallowtail butterfly species (Lepidoptera: Papilionidae) from 754 specimens at the Natural History Museum (United Kingdom) and the Zoologische Staatssammlung München (Germany). Endemic butterflies were less melanised (15 endemic species, mean = 81% melanised; 14 non‐endemic species = 89% melanised) but similar to non‐endemics in size (endemic, mean = 56 mm; non‐endemic = 53 mm) and elevation (endemic, mean = 352 m.a.s.l.; non‐endemic: 289 m). There was no evidence that species size or melanism patterns were related to elevation in either group. Few differences in elevation of endemic and non‐endemic species imply that endemic swallowtail butterflies are not disproportionately more threatened by climate‐driven mountaintop extirpation than non‐endemic species. Further work is needed to examine whether paler wings alter the ability of endemic species to thermoregulate and adapt to changing climates. In the absence of reductions in anthropogenic global greenhouse gas emissions, on‐going climate warming will continue to threaten tropical montane species.

A draft reference genome assembly of California Pipevine, Aristolochia californica Torr.

The California Pipevine, Aristolochia californica Torr., is the only endemic California species within the cosmopolitan birthwort family Aristolochiaceae. It occurs as an understory vine in riparian and chaparral areas and in forest edges and windrows. The geographic range of this plant species almost entirely overlaps with that of its major specialized herbivore, the California Pipevine Swallowtail Butterfly Battus philenor hirsuta. While this species pair is a useful, ecologically well-understood system to study co-evolution, until recently, genomic resources for both have been lacking. Here, we report a new, chromosome-level assembly of A. californica as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 531 scaffolds spanning 661 megabase (Mb) pairs, with a contig N50 of 6.53 Mb, a scaffold N50 of 42.2 Mb, and BUSCO complete score of 98%. In combination with the recently published B. philenor hirsuta reference genome assembly, the A. californica reference genome assembly will be a powerful tool for studying co-evolution in a rapidly changing California landscape.

Spectral variation and pigmentary basis of ornamental and mimetic wing colour patches of swallowtail butterflies

Abstract Colours and colour patterns are diverse traits that are often used as visual signals. To test ecological and evolutionary drivers of these visual signals, a clear understanding of their nature and variation is necessary. Here we characterize variation in wing coloration of mormon swallowtail butterflies (Papilio, subgenus Menelaides). These species exhibit two kinds of colour patches on black wings: creamy white/yellow/green patches that are presumably used as sexual ornaments, and pure white patches that are presumably used as mimetic signals. Using reflectance spectrophotometry we quantified spectral properties of black wing background and colour patches between sexes, wing surfaces, and mimicry status. We found that brightness and saturation of the black background were less variable across sexes, wing surfaces, and mimetic/nonmimetic status. However, colour contrast and saturation were higher on dorsal surfaces than on ventral surfaces, and colour contrast between the black background and colour patches was higher in males than in females. Colour patches in nonmimetic butterflies were brighter and more saturated across the whole colour spectrum compared to mimetic butterflies. Thus, our study characterizes colour variation in an extraordinary signal radiation on the wings of swallowtail butterflies, a model clade in ecology, evolution, and genetics. Our results generate numerous hypotheses about the ecological and sexual functions of these colour patches, which may guide further behavioural experiments. Finally, using liquid chromatography-mass spectrometry we identified the pigment papiliochrome-II to be the chemical basis of the presumed sexual ornaments in mormon swallowtails.

An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types

BackgroundThe frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system.ResultsThe components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut.ConclusionsThese findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Genomics of the relict species Baronia brevicornis sheds light on its demographic history and genome size evolution across swallowtail butterflies.

Relict species, like coelacanth, gingko, tuatara, are the remnants of formerly more ecologically and taxonomically diverse lineages. It raises the questions of why they are currently species-poor, have restrained ecology, and are often vulnerable to extinction. Estimating heterozygosity level and demographic history can guide our understanding of the evolutionary history and conservation status of relict species. However, few studies have focused on relict invertebrates compared to vertebrates. We sequenced the genome of Baronia brevicornis (Lepidoptera: Papilionidae), which is an endangered species, the sister species of all swallowtail butterflies, and is the oldest lineage of all extant butterflies. From a dried specimen, we were able to generate both long-read and short-read data and assembled a genome of 406 Mb for Baronia. We found a fairly high level of heterozygosity (0.58%) compared to other swallowtail butterflies, which contrasts with its endangered and relict status. Taking into account the high ratio of recombination over mutation, demographic analyses indicated a sharp decline of the effective population size initiated in the last million years. Moreover, the Baronia genome was used to study genome size variation in Papilionidae. Genome sizes are mostly explained by transposable elements activities, suggesting that large genomes appear to be a derived feature in swallowtail butterflies as transposable elements activity is recent and involves different transposable elements classes among species. This first Baronia genome provides a resource for assisting conservation in a flagship and relict insect species as well as for understanding swallowtail genome evolution.

Male behavior in a swallowtail butterfly (Battus philenor) ensures directional iridescent sexual signal is visible to females during courtship

Male behavior in a swallowtail butterfly (Battus philenor) ensures directional iridescent sexual signal is visible to females during courtship

Convergence in sympatric swallowtail butterflies reveals ecological interactions as a key driver of worldwide trait diversification

Ecological interactions can promote phenotypic diversification in sympatric species. While competition can enhance trait divergence, other ecological interactions may promote convergence in sympatric species. Within butterflies, evolutionary convergences in wing color patterns have been reported between distantly related species, especially in females of palatable species, where mimetic color patterns are promoted by predator communities shared with defended species living in sympatry. Wing color patterns are also often involved in species recognition in butterflies, and divergence in this trait has been reported in closely related species living in sympatry as a result of reproductive character displacement. Here, we investigate the effect of sympatry between species on the convergence vs. divergence of their wing color patterns in relation to phylogenetic distance, focusing on the iconic swallowtail butterflies (family Papilionidae). We developed an unsupervised machine learning-based method to estimate phenotypic distances among wing color patterns of 337 species, enabling us to finely quantify morphological diversity at the global scale among species and allowing us to compute pairwise phenotypic distances between sympatric and allopatric species pairs. We found phenotypic convergence in sympatry, stronger among distantly related species, while divergence was weaker and restricted to closely related males. The convergence was stronger among females than males, suggesting that differential selective pressures acting on the two sexes drove sexual dimorphism. Our results highlight the significant effect of ecological interactions driven by predation pressures on trait diversification in Papilionidae and provide evidence for the interaction between phylogenetic proximity and ecological interactions in sympatry, acting on macroevolutionary patterns of phenotypic diversification.

IMPLEMENTASI DIMENSI FRAKTAL BOX COUNTING DAN K-MEANS DALAM KLASIFIKASI JENIS KUPU-KUPU (LEPIDOPTERA) BERDASARKAN BENTUK SAYAP

Butterflies are insects that have aesthetic beauty and are also important assets in the field of nature protection. In nature, butterflies have a crucial role in maintaining ecosystems, especially in the process of pollination of flowering plants which contributes to the natural propagation of plants. Butterfly wings have an important role in identifying butterfly species based on size, shape and color. In mathematics, there is the concept of fractals which is a very suitable method for modeling natural phenomena and used as a tool to represent natural objects. In this study, a classification of butterfly species was carried out based on the shape of the butterfly's wings. A total of 100 butterfly images were used in this study, the first step was to convert the RGB images to grayscale and then segment the butterfly wings. The area is then used to identify butterfly wing type patterns using edge detection using the Canny method. Furthermore, by using the fractal box counting method, the resulting dimensional values are used in the clustering process. The results of the dimension values will be classified using the K-Means grouping method with five groups, namely Adonis Butterfly, Julia Butterfly, Brown Siproeta Butterfly, Metal Marks Butterfly, and African Giant Swallowtail Butterfly, showing an accuracy of 89%.

A draft reference genome assembly of the Pipevine Swallowtail butterfly, Battus philenor hirsuta.

The California Pipevine Swallowtail Butterfly, Battus philenor hirsuta, and its host plant, the California Pipevine or Dutchman's Pipe, Aristolochia californica Torr., are an important California endemic species pair. While this species pair is an ideal system to study co-evolution, genomic resources for both are lacking. Here, we report a new, chromosome-level assembly of B. philenor hirsuta as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 109 scaffolds spanning 443 mega base (Mb) pairs, with a contig N50 of 14.6 Mb, a scaffold N50 of 15.2 Mb, and BUSCO complete score of 98.9%. In combination with the forthcoming A. californica reference genome, the B. philenor hirsuta genome will be a powerful tool for documenting landscape genomic diversity and plant-insect co-evolution in a rapidly changing California landscape.

Reproductive response of the Samoan swallowtail butterfly to variability in host plant and habitat characteristics

AbstractThe Samoan swallowtail butterfly (Papilio godeffroyi) has become restricted to Tutuila Island, American Samoa. Factors driving its extirpation on other islands may be partly due to the availability and suitability of habitat, given the singular association we observed of P. godeffroyi with its host plant, Micromelum minutum. We expected that as a host plant specialist, P. godeffroyi might respond to variation in the physical traits and habitat context of M. minutum, which could help identify conservation opportunities throughout its present and former range. Because the ecology of P. godeffroyi is largely unknown, we conducted systematic surveys during 15 months in 2013 and 2014 on Tutuila to investigate its reproductive response to the physical and phenological variability of individual host trees and to the structural and geographical variability of the stands in which they occurred. We searched foliage for eggs, larvae, and pupae and monitored seasonal trends in herbivory and production of leaves, flowers, and fruits on 117 host trees in 8 rainforest stands, mostly within the National Park of American Samoa (NPSA), starting in March 2013 and ending in August 2014. We observed high variability in butterfly reproductive output between individual trees and stands, due largely to a strong negative relationship between leaf biomass (at both tree and stand levels) and oviposition density (number of eggs per foliar biomass) and frequency (number of eggs per survey). Distribution patterns of larvae and pupae were less distinct but generally followed trends in oviposition. Our findings indicate that the butterfly is reproducing widely across Tutuila and is likely to respond positively to management to increase the availability of small host trees in low‐density stands. Thus, the species is a good candidate for conservation management focusing on habitat.

De novo assembly of a chromosome-level reference genome of the ornamental butterfly Sericinus montelus based on nanopore sequencing and Hi-C analysis.

Sericinus montelus (Lepidoptera, Papilionidae, Parnassiinae) is a high-value ornamental swallowtail butterfly species widely distributed in Northern and Central China, Japan, Korea, and Russia. The larval stage of this species feeds exclusively on Aristolochia plants. The Aristolochia species is well known for its high levels of aristolochic acids (AAs), which have been found to be carcinogenic for numerous animals. The swallowtail butterfly is among the few that can feed on these toxic host plants. However, the genetic adaptation of S. montelus to confer new abilities for AA tolerance has not yet been well explored, largely due to the limited genomic resources of this species. This study aimed to present a chromosome-level reference genome for S. montelus using the Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. The final assembly was composed of 581.44Mb with an expected genome size of 619.27Mb. Further, 99.98% of the bases could be anchored onto 30 chromosomes. The N50 of contigs and scaffolds was 5.74 and 19.12Mb, respectively. Approximately 48.86% of the assembled genome was suggested to be repeat elements, and 13,720 protein-coding genes were predicted in the current assembly. The phylogenetic analysis indicated that S. montelus diverged from the common ancestor of swallowtails about 58.57-80.46 million years ago. Compared with related species, S. montelus showed a significant expansion of P450 gene family members, and positive selections on eloa, heatr1, and aph1a resulted in the AA tolerance for S. montelus larva. The de novo assembly of a high-quality reference genome for S. montelus provided a fundamental genomic tool for future research on evolution, genome genetics, and toxicology of the swallowtail butterflies.

Examining the female immigrant and minority representation in Shunji Iwai's “Swallowtail Butterfly”

Bu makale, Swallowtail Butterfly (Shunji Iwai, 1996) filmi bağlamında kadın mülteci ve azınlık temsilini çok-disiplinli bir açıdan incelemektedir. Filmin furusato veya dil bariyerleri gibi yerli/yabancı temasını ilgilendiren konularıyla başlayan makale, ardından filmin kadın hikayelerini feminist teori ile incelemektedir. Dolayısıyla bu makale, küreselleşmenin hızla yaşandığı günümüzde, çoklu açılardan filmin güncel bir perspektifle kapsamlı bir analizini sunmayı amaçlamaktadır.

Swallowtail butterflies and hawkmoths contribute equally to the pollination of Habenaria dentata (Orchidaceae)

Mixed pollination system with both diurnal and nocturnal visitors contributing to pollen transfer and seed set have been documented in many angiosperm species. The flowers of Habenaria species are usually green or white, with a long nectar secreting spur, and are usually interpreted as specialized for pollination by nocturnal moths. With a few exceptions, nocturnal moths and diurnal butterflies are both reported as pollinators of Habenaria species. However, the relative contributions made by moths versus butterflies to the reproductive success in the same orchid species has rarely been studied. We investigated the floral morphology, pollinators, pollination efficiency, and breeding system of Habenaria dentata in southern China. We showed that two species of diurnal swallowtail butterflies (Papilio helenus and Papilio memnon) and a species of crepuscular hawkmoth (Eupanacra mydon) were this orchid's effective pollinators. The pollinaria were attached to the eyes of these three insect species. Pollinator exclusion experiments showed that pollinarium removal and pollinium deposition rates on stigmas by butterflies (47.51% and 38.64%, respectively) were not significantly different from those made by hawkmoths (50.70% and 34.47%, respectively), indicating that diurnal and crepuscular visits contributed equally to reproductive success. As natural rates of insect-mediated pollination of our population of H. dentata resulted in fruit set rates of 93.33%, rejected the prospect that this population was pollinator limited. Hand pollination experiments indicated that H. dentata was self-compatible but without autonomous self-pollination. The proportion of well-developed seeds with large embryos in self-pollinated fruits was significantly lower than in cross-pollinated fruits, suggesting natural selection favors xenogamy. Our results highlighted the importance of both butterflies and hawkmoths contributing to the reproductive success of a rare orchid with a mixed pollination system.