From the Brazilian lowlands to the Andes: specialist fungus gnat pollination and self-incompatibility in two Malaxis species (Malaxidinae: Orchidaceae)

From the Brazilian lowlands to the Andes: specialist fungus gnat pollination and self-incompatibility in two Malaxis species (Malaxidinae: Orchidaceae) Fernando H. Calderon-Quispe1, Júlia M. Brandalise1, Emerson Mauricio Huaman2, Renan Pittella1, Rafael Becker1, Rodrigo B. Singer1 1 Programa de Pós-Graduação em Botânica, Departamento de Botânica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil 2 Escuela Profesional de Agronomía, Universidad Nacional de San Cristóbal de Huamanga, Ayacucho, Perú Corresponding author: Fernando H. Calderon-Quispe ([email protected]m) Academic editor: Marco Pellegrini♦ Received 6 July 2025♦ Accepted 29 September 2025♦ Published 8 December 2025 Abstract Background and aims – Malaxis is a cosmopolitan genus comprising approximately 300 species and is one of the most diverse within the subtribe Malaxidinae. However, to date, no detailed studies have addressed its reproductive biology in the Neotropics. This study aimed to document the floral traits, pollination mechanisms, breeding system, and fruiting success of two native Neotropical species. Material and methods – Plants of Malaxis parthoni were studied in Porto Alegre, southern Brazil (79 m a.s.l.), while individuals of M. excavata were examined at 3,500 m a.s.l. in Ayacucho, in the Peruvian Andes. To evaluate the breeding system, we tested for autonomous pollination and self-compatibility using flowers isolated from pollinators through bagging. Nectar production was assessed through qualitative tests to detect the presence of sugars in floral secretions. The pollination process, under natural conditions, was recorded in the field through video and photographs, and pollination efficiency and natural fruiting success were documented. Key results – Both Malaxis species possess nectar-producing flowers and were found to be pollinator-dependent and self-incompatible. Fungus gnats of the genus Mycomya (Mycetophilidae) acted as pollinators. In both species, pollinia were attached to the ventral part of the prothorax. In both species, a high percentage of flowers with pollinia removed was observed, indicating high pollination efficiency. On average, fruiting success was 11.24% in M. parthoni and 36.01% in M. excavata, the latter showing a statistically higher percentage. Conclusion – Our findings reveal that both species require cross-pollination to achieve fruit set, with fungus gnats acting as effective pollinators. The relatively high fruiting success compared to other congeners and self-incompatible orchids may result from a combination of factors, including the presence of floral rewards and high pollinator efficiency. This study provides the first comprehensive account of the reproductive biology of Neotropical Malaxis species. Keywords Atlantic Rainforest Biome, breeding system, gnat pollination, High Andes, Mycetophilidae, terrestrial orchids INTRODUCTION Pollination is one of the most crucial processes in plant reproduction as it is essential for maintaining plant populations and genetic diversity (Potts et al. 2010; Ratto et al. 2018; Feigs et al. 2022). In Orchidaceae Juss., more than 70% of the studied species are pollinator-dependent (Dressler 1981; Tremblay et al. 2005; Ackerman et al. 2023). This family exhibits significant morphological variation, primarily associated with adaptations that attract pollinators ranging from insects to birds (Tremblay et al. 2005; Barbosa et al. 2009; Calderon-Quispe and Plant Ecology and Evolution 158 (3): 476–492, 2025 https://doi.org/10.5091/plecevo.164210 Copyright Fernando H. Calderon-Quispe, Júlia M. Brandalise, Emerson Mauricio Huaman, Renan Pittella, Rafael Becker, Rodrigo B. Singer. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Plant Ecology and Evolution is published by Meise Botanic Garden and the Royal Botanical Society of Belgium. RESEARCH ARTICLE Plant Ecology and Evolution 158 (3): 476–492, 2025 477 Singer 2024). Pollination by dipterans (myophily) has been reported in the subfamilies Epidendroideae Lindl. ex Endl., Orchidoideae A.A.Eaton, and Cypripedioideae Lindl. ex Endl. (Han et al. 2022; Ackerman et al. 2023). However, there is a significant lack of research on fly pollination, mainly due to the general belief that dipterans play a minor role as pollinators (Mesler et al. 1980; Orford et al. 2015; Raguso 2020). Recent studies have shown that several orchid species exhibit a high degree of specialisation for pollination by flies (Blanco and Barboza 2005; Barbosa et al. 2009; Endara et al. 2010; Hayashi et al. 2025). In the Neotropics, species belonging to the subtribe Pleurothallidinae Lindl. ex G.Don are particularly well known for their strong association with dipteran-mediated pollination (Borba and Semir 2001; Endara et al. 2010; Bogarín et al. 2018). Several authors have proposed that dipterans also play a significant role as pollinators in the subtribe Malaxidinae Benth. & Hook.f. (Margońska et al. 2021; Jermakowicz et al. 2022; Ackerman et al. 2023; Margońska et al. 2025). However, available evidence on pollination biology in the subtribe remains limited. To date, pollination has only been reported for the following species within the subtribe. In Dienia ophrydis (J.Koenig) Seidenf., a species of Miridae Hahn, 1831 (order Hemiptera Linnaeus, 1758) was the sole visitor observed carrying pollinia and acting as a potential pollinator (Nuammee 2018). In Crepidium acuminatum (D.Don) Szlach., hoverflies (Syrphidae Latreille, 1802) and fungus gnats (Sciaridae Billberg, 1820) were identified as effective pollinators (Nuammee 2018), while in Oberonia japonica (Maxim.) Makino, gall midges (Cecidomyiidae) were confirmed as pollinators (Sunakawa et al. 2024). The fungus gnat Phronia digitata Hackman, 1970 (Mycetophilidae Newman, 1834) was collected and observed carrying pollinia of Hammarbya paludosa (L.) Kuntze, suggesting that this species could act as its pollinator (Reeves and Reeves 1984; Argue 2014). Similarly, in Liparis gigantea C.L.Tso (a genus closely related to Malaxis Sol. ex Sw.), pollination by a fungus gnat (Mycomya sp., Mycetophilidae) was recently reported (Qi et al. 2024). For Malaxis, pollination is likewise presumed to involve fungus gnats, although confirmed evidence exists only for the European Malaxis monophyllos (L.) Sw., where the fungus gnat Mycomya fimbriata (Meigen, 1818) (Mycetophilidae) was identified as pollinator (Claessens and Kleynen 2011). Fungus gnats are small dipterans principally grouped in the Mycetophilidae and Sciaridae families (Burdíková et al. 2024). Pollination mediated by this group of insects is apparently uncommon and remains poorly studied, likely due to the challenges of field observation. Their small body size and activity patterns, typically at dawn and dusk, with some species being nocturnal, make them particularly difficult to observe (Jakovlev 2012; Mochizuki and Kawakita 2018). Strategies of pollination by Mycetophilids involve models of food rewards (nectar) and mimicry, such as brood-site deception and sexual deception (Song et al. 2014). In orchids, there is a known model of brood-site deceptive pollination by fungus gnats widely reported in orchids of the genus Corybas Salisb. (Diurideae Endl. ex Butzin) (Kelly et al. 2013; Kuiter 2020; Han et al. 2022), and sexually deceptive pollination, also by fungus gnat, reported in species of the genus Lepanthes Sw. in the Neotropics (Blanco and Barboza 2005) or Pterostylis R.Br. in Australia (Phillips et al. 2014; Reiter et al. 2019; Hayashi et al. 2022). The breeding system within the subtribe Malaxidinae is also variable, with some species relying on crosspollination for fruit set, while others, such as species of Liparis Rich., are self-compatible and capable of rainassisted self-pollination. In the latter case, natural or artificial rain droplets rapidly dislodge the anther caps, often causing them to detach from the flowers. As a result, the pollinia frequently fell directly into the stigmatic cavity, leading to self-pollination (Catling 1980; Suetsugu 2019; Nuammee 2018). In the genus Malaxis, the breeding system remains poorly understood, as evidence is limited; although reports of autogamy exist for several Paleotropical species (Argue 2014). By contrast, Malaxis massonii (Ridl.) Kuntze (the only Neotropical species studied to date) is self-incompatible and pollinatordependent (Aragon and Ackerman 2001). Despite the genus Malaxis being one of the most diverse genera of the subtribe and being widely distributed, its reproductive biology is largely unexplored. Additionally, most studies within the subtribe, including preliminary observations in Malaxis, have focused on European and Asian species, with little attention given to Neotropical representatives, which include the High Andean species. This lack of information may result from the inconspicuous nature of these orchids, which typically produce small, greenish, ephemeral flowers and grow in sparse populations (Jermakowicz et al. 2022). To contribute to the understanding of Neotropical Malaxis, we investigated the reproductive biology of two terrestrial species: Malaxis parthoni C.Morren and M. excavata (Lindl.) Kuntze. Both are native and widely distributed throughout the Neotropics, with M. parthoni ranging from Mexico to northern Argentina (POWO 2025b), and M. excavata from Mexico to northwestern Argentina (POWO 2025a). In this study, M. parthoni was examined at low altitudes in southern Brazil, while M. excavata was studied in a High Andean environment of Peru. To address key knowledge gaps, we focused on three central questions: (1) What is the breeding system of each species? (2) Are pollinators involved in their reproduction, and if so, which taxa? (3) What is their reproductive performance under natural conditions? Thereafter, we proposed the following hypotheses: (1) Owing to preceding literature on Malaxidinae, Malaxis parthoni and M. excavata may be self-incompatible and depend on pollinators for pollination. (2) Diptera are expected to be as effective pollinators in both species, based on floral traits, suggestive of dipteran pollination. To test these hypotheses, we conducted field studies assessing breeding systems through controlled pollination Calderon-Quispe et al.: Specialist fungus gnat pollination of Malaxis species478 experiments, recorded floral visitors and pollination events, and quantified fruiting success under natural conditions. MATERIAL AND METHODS Study system and species description The subtribe Malaxidinae includes approximately 1250 species across 14 genera, with a predominantly tropical and subtropical distribution (Kolomeitseva et al. 2024; Zeng et al. 2024). Malaxis is one of the most diverse genera within the subtribe, comprising ca 300 species found worldwide, especially in tropical and subtropical regions (Chinchilla et al. 2022; Jermakowicz et al. 2022). Most species are terrestrial, bearing fibrous superficial roots and tunicate pseudobulbs. Plants typically have one or two leaves per sympodial unit and produce inflorescences as spikes, racemes, or corymbs. The flowers are small, non-resupinate, usually pale green, and lack accessory structures in their pollinia (i.e. naked pollinia) (Dressler 1981; Cameron 2005). In the Americas, a total of 143 Malaxis species have been documented and distributed from North America to Argentina (Ulloa Ulloa et al. 2017; Chinchilla et al. 2022). Mexico is recognised as the most diverse country for the genus, hosting 71 species. Brazil harbours 11 species (Santos and Smidt 2023), while eight species are recorded in Peru (Ulloa Ulloa et al. 2017). Here, we examined two Malaxis species. Malaxis parthoni (Fig. 1) and M. excavata (Fig. 2) are terrestrial orchids with comparable plant heights, ranging from 14.52 to 39.33 cm. Both species typically produce two leaves. In our study sites, M. parthoni displayed a highly dispersed distribution, with scarce and isolated individuals, whereas M. excavata occurred in dense aggregations. The leaves of M. parthoni are elliptic with acute apices (Fig. 1A), while those of M. excavata are elliptic-lanceolate, also ending in acute tips (Fig. 2A). The main difference between the two species lies in the shape and size of the labellum: in M. parthoni, the labellum is relatively flattened (Fig. 1F), whereas in M. excavata it is concave with a truncate apex (Fig. 2I). Study area Malaxis parthoni was investigated in southern Brazil, specifically in Morro Santana (Porto Alegre municipality; 30°04’01.59”S, 51°07’29.69”W; 79 m) and Morro São Pedro (Viamão municipality; 30°10’55.82”S, 51°05’55.59”W; 91 m), both located in the state of Rio Grande do Sul, near the southern limit of the Atlantic Rainforest Biome (Mata Atlântica) (Fig. 3A, C, E). The climate in this region is characterised by a mean annual temperature ranging from 18 to 20°C and an average annual precipitation of 1,300 to 1,500 mm, with no distinct dry season throughout the year (Overbeck et al. 2005). Malaxis excavata was studied in the Andean region of Ayacucho, Peru, at an elevation of 3,500 m (13°00’10.25”S, 74°08’46.84”W) (Fig. 3A, B, D). According to Holdridge’s life zone classification, this area corresponds to the Subtropical Montane Humid Forest, characterised by a mean annual temperature of 12.9°C and a maximum average total precipitation of 1,190 mm (INRENA 1995). Unlike the Brazilian sites, the Ayacucho region exhibits a clearly defined wet and dry season, with the wet season usually extending from late October to March and the dry season from April to early October. In this area, M. excavata grows within a monospecific forest dominated by Alnus acuminata Kunth (Betulaceae). Maps were developed using QGIS v.3.22.2. Elevation maps were obtained from WorldClim 2.1 (Fick and Hijmans 2017). Floral features Given that floral traits are considered key to understanding pollination processes, and floral attractants (Woodcock et al. 2014; Assis 2023), floral and inflorescence features including flower colour, scent, size, sexual organ position, reward type, and inflorescence morphology were described and measured from fresh anthesis-stage flowers of five individuals per species (6 to 10 flowers in M. parthoni and 6 to 8 flowers in M. excavata). As hypothesised, Diptera may serve as potential pollinators. In orchids pollinated by this group of insects, fragrance emission is commonly associated with pollinator attraction (Tan et al. 2002; Jermakowicz et al. 2022). To test this, the timing of fragrance release was recorded through hourly olfactory inspections of inflorescences, all conducted by the same observer (Calderon-Quispe and Singer 2024). Flower longevity was also recorded, as it determines the period during which flowers remain available to attract and receive visits (Primack 1985). To quantify longevity, flowers were isolated from pollinators using tulle bags (Calderon-Quispe and Singer 2024). In total, 20 flowers of M. parthoni (from 10 individuals) and 30 flowers of M. excavata (from 10 individuals) were monitored. During pollinator observations, we also noted the presence of a watery secretion on the labellum. However, the volume was minimal, hence a qualitative test for sugar content was performed to determine whether the secretion could be classified as nectar. Using a glucose monitoring system, Bioland G-245-3, a drop of distilled water was deposited on the labellum to wash the surface, and the resulting liquid was then absorbed using a reactive glucose test strip, allowing for qualitative assessment of sugar presence (n= 10 flowers per species) (Power et al. 2018). The voucher specimens of M. excavata were deposited in the Herbario Sur Peruano, Instituto Científico Michael Owen Dillon (HSP), and those of M. parthoni in the Herbário do Instituto de Ciências Naturais, Universidade Federal do Rio Grande do Sul (ICN). Pollinator observations Diurnal and nocturnal observations were conducted for both species. Diurnal observations took place between 06:00 and 18:00 h, while nocturnal observations were Plant Ecology and Evolution 158 (3): 476–492, 2025 479 Figure 1. Vegetative and floral characteristics of Malaxis parthoni. A. Habit. B. Upper view of the corymbiform inflorescence. C. Frontal view of a flower. D. Back view of a flower. E. Longitudinal section of a flower. F. Detached labellum and column. G. Cavity of the labellum with visible nectar. H. Naked pollinia. I. Lateral view of the labellum and column, showing the stigmatic surface (white arrowhead). Calderon-Quispe et al.: Specialist fungus gnat pollination of Malaxis species480 Figure 2. Vegetative and floral characteristics of Malaxis excavata. A. Habit. B. Upper view of the corymbiform inflorescence. C. Frontal view of the inflorescence. D. Frontal view of a flower. E. Back view of a flower. F. Naked pollinia. G. Column showing the stigmatic surface. H. Stigmatic surface with pollinia attached (white arrowhead). I. Labellum with visible nectar (blue ellipse). J. Developing fruit. Plant Ecology and Evolution 158 (3): 476–492, 2025 481 Figure 3. Study sites for pollinator observations and breeding system experiments. The map shows the country boundaries in South America (A), highlighting the Peruvian department of Ayacucho, where Malaxis excavata was studied (B), and the Brazilian state of Rio Grande do Sul, where M. parthoni was studied (C). Satellite imagery illustrates the study area in Peru (D), as well as the two in Brazil (E). carried out from 18:00 to 24:00 h. Observations of M. parthoni were conducted in June and July of 2023 and 2024, whereas observations of M. excavata were performed in January 2024. For each species, a total of 80 h per species was conducted over five days. Only insects that effectively removed and inserted pollinia were considered pollinators (Adams and Lawson 1993). Pollinators of M. parthoni were photographed and filmed using a Nikon D5300 camera and a Nikon AF-S VR 105-mm macro lens, while those of M. excavata were documented with a Sony DSCHX400V 215-mm camera. Video recordings were used to describe visitor behaviour. Fungus gnats (Mycomya Rondani, 1856) visiting the flowers were collected using an aspirator. Two individuals per species were sampled and preserved in 70% ethanol. The Mycomya species collected in Peru was deposited in the Museo Sur Peruano, Instituto Cientifico Michael Owen Dillon (MSP), while the voucher specimens collected in Brazil were deposited in the Museu de Ciências Naturais, Secretaria do Meio Ambiente e Infraestrutura (MCN), Porto Alegre, Brazil. Breeding system treatments Ten individuals were isolated from pollinators using tulle bags, through which air and light could pass, thereby minimising effects on the flowers and plants (Sagili et al. 2025). Four treatments, previously applied to Neotropical orchids following the methodologies of Calderon-Quispe and Singer (2024), Sanguinetti and Singer (2014), and Buzatto et al. (2022), were conducted on each individual: intact flowers, to test for autonomous autogamy (i.e. Calderon-Quispe et al.: Specialist fungus gnat pollination of Malaxis species482 Table 1. Floral measurements and longevity features of Malaxis parthoni and M. excavata. Data are presented as minimum– maximum values (mean ± standard error; sample size). Different uppercase letters indicate significant differences between species (Mann-Whitney U test, p < 0.05). Feature Malaxis parthoni Malaxis excavata Flowers per inflorescence 98–206 (136.90 ± 10.04; 10)A18–102 (44.38 ± 6.47; 15)B Flowers in simultaneous anthesis 12–20 (15.8 ± 0.93; 10)A11–38 (20.16 ± 1.31; 25)B Flower longevity (days) 8–21 (13.75 ± 0.91; 20)A14–17 (15.86 ± 0.19; 20)B Pedicel length (cm) 0.53–0.91 (0.75 ± 0.03; 10) 0.71–1.02 (0.90 ± 0.03; 8) Sepal length (mm) 2.36–3.16 (2.66 ± 0.06; 10) 3.45–3.66 (3.58 ± 0.02; 8) Sepal width (mm) 0.59–1.40 (1.03 ± 0.08; 10) 0.99–1.31 (1.24 ± 0.03; 8) Lateral petal length (mm) 1.59–2.59 (2.08 ± 0.09; 10) 2.23–3.45 (2.75 ± 0.16; 8) Lateral petal width (mm) 0.15–0.21 (0.18 ± 0.03; 10) 0.29–0.58 (0.38 ± 0.03; 8) Labellum length (mm) 2.14–2.43 (2.24 ± 0.03; 10) 2.57–2.91 (2.74 ± 0.04; 6) Labellum width (mm) 2.01–2.24 (2.14 ± 0.03; 10) 1.56–1.86 (1.71 ± 0.04; 6) Column length (mm) 0.63–0.80 (0.69 ± 0.03; 6) 0.71–1.12 (0.95 ± 0.06; 6) Column width (mm) 0.74–1.01 (0.86 ± 0.04; 6) 0.81–0.98 (0.90 ± 0.02; 6) Pollinia length (mm) 0.35–0.37 0.45–0.46 Pollinia width (mm) 0.20–0.24 0.23–0.26 Stigmatic surface length (mm) 0.32–0.33 0.44–0.46 Stigmatic surface width (mm) 0.70–0.72 0.89–0.91 Ovary length (mm) 1.87–2.06 (1.96 ± 0.03; 6) 2.76–3.13 (2.97 ± 0.05; 6) Ovary width (mm) 0.71–0.87 (0.79 ± 0.02; 6) 1.37–1.48 (1.42 ± 0.01; 6) Note: Statistical comparisons were performed only for the number of flowers per inflorescence, the number of flowers in anthesis, and flower longevity. Measurements without mean ± standard error and sample size are due to the very small size of the structures, which prevented us from performing additional measurements to obtain these data. whether flowers can set fruit without any pollinator intervention); emasculation, to test for apomixis (i.e. fruit development in the absence of pollen and consequently fecundation), which consists of removing the pollinia; manual self-pollination, to assess self-compatibility (i.e. the capacity to produce fruits and seeds from pollen of the same flower); and manual cross-pollination, to evaluate fruit set when pollen comes from a different individual. For M. parthoni, five flowers per treatment were applied to each of 10 individuals (50 flowers per treatment in total), whereas for M. excavata, only three flowers per treatment could be used per individual (30 flowers per treatment in total). No statistical comparisons were applied, since only one treatment produced fruits. Pollination efficiency and fruiting success Pollination efficiency was assessed using Nilsson’s male efficiency factor, calculated as the ratio of the percentage of pollinated flowers (flowers per inflorescence on which pollinia were deposited on the stigmatic surface) and pollen donor flowers (flowers per inflorescence from which pollinia were removed) (Nilsson et al. 1992; Buzatto et al. 2022). Accordingly, we recorded the number of pollinated flowers, and pollen donor flowers were evaluated in 10 individuals of Malaxis parthoni and 21 individuals of M. excavata. The number of pollinated flowers was statistically compared to the number of donor flowers using the non-parametric Wilcoxon signed-rank test, after assessing normality with the Shapiro-Wilk test (p < 0.05). In the same localities where pollinator observations were carried out, 10 individuals per species were collected just at the end of the flowering period (i.e. upon fruit maturation). Plants were monitored weekly to determine the fruit maturation. For each individual, fruit set success was calculated by dividing the number of fruits formed by the number of flowers per inflorescence (Castro et al. 2022; Calderon-Quispe and Singer 2024). Statistical comparisons of Nilsson’s male efficiency factor and fruiting success between species were performed using the non-parametric Mann-Whitney U test, after testing for normality with the Shapiro-Wilk test (p < 0.05). All statistical comparisons were done in RStudio v.2023.12.1.402 (R Core Team 2024), and the graph was generated using the R package ggplot2 v.3.5.2 (Wickham 2016). Plant Ecology and Evolution 158 (3): 476–492, 2025 483 RESULTS Flower and inflorescence features Both species produce corymbiform inflorescences (Figs 1B, 2B). Statistically, M. parthoni produces significantly more flowers than M. excavata (Mann-Whitney U test, p < 0.05), with floral counts ranging from 98 to 206 in M. parthoni, and from 18 to 102 in M. excavata (Table 1). Flowers are greenish, non-resupinate, and pedicellate. Pedicel lengths vary from 0.53 to 0.91 cm in M. parthoni, and from 0.71 to 1.02 cm in M. excavata (Table 1). Sepals are oblong-lanceolate in M. parthoni and ellipticoblong in M. excavata. The lateral petals are linear and convolute in both species. The labellum of M. parthoni is flattened, trilobate with an entire margin, an acute apex, and inconspicuous, nearly rounded lateral lobes. In contrast, the labellum of M. excavata is trilobate, truncate, and concave, featuring a longitudinal crest on its abaxial surface. Both species secrete drops of liquid containing sugars, as confirmed by a qualitative assay, which likely serves as a floral reward for pollinators. This secretion can therefore be regarded as nectar, released through two cavities at the base of the labellum (Figs 1G, 2I) and also from the fleshy anterior region of the middle lobe, which is more robust and visibly nectariferous in M. excavata (blue ellipse in Fig. 2I) compared to M. parthoni. The column in both species is dorso-ventrally compressed (Figs 1F, 2D) and bears two naked pollinia at the subapical dorsal position (Figs 1H, 2F). The stigma surface is bilobed and positioned apically on the column (Figs 1I, 2GH). The ovary is green and glabrous in both species (Figs 1D, 2E), as are the fruits (Fig. 2J). Overall, the flowers of M. excavata are larger than those of M. parthoni (Table 1). Flower phenology, longevity, and scent emission The flowering period of M. parthoni extends from June to August, while that of M. excavata occurs between December and February. Flowers are long-lived, ranging from 8 to 21 days in M. parthoni and from 14 to 17 days in M. excavata, with significantly longer floral duration in the latter species (Mann-Whitney U test, p < 0.05; Table 1). In both species, the flowers are considered long-lived. The floral scent of M. parthoni resembles that of fresh fish and was detected between 14:30 and 18:00 h, with peak intensity around 17:00 h. In contrast, M. excavata emits an acrid scent, detected between 16:00 and 20:00 h, peaking around 19:00 h. Pollinators and pollinator behaviour Fungus gnats of the genus Mycomya (Mycetophilidae) acted as pollinators of both species (Suppl. material 1 and 2). Individuals of a species provisionally designed as Mycomya sp.1 pollinated Malaxis parthoni, while those Table 2. Pollinator behaviour, pollination efficiency, and fruiting success in Malaxis parthoni and M. excavata. Data are presented as minimum–maximum values (mean ± standard deviation). Different lowercase letters indicate significant differences within species (Wilcoxon signed-rank test, p < 0.05), while different uppercase letters indicate significant differences between species (MannWhitney U test, p < 0.05). Pollination features Malaxis parthoni Malaxis excavata Pollinator Mycomya sp.1 (Mycetophilidae) Mycomya sp.2 (Mycetophilidae) Flowers visited (n) 2–23 (6.36 ± 5.90) 1–23 (5.6 ± 5.29) Time spent per flower (s) 2–22 (7.09 ± 5.32) 12–313 (73.62 ± 61.65) Time spent per inflorescence (s) 10–65 (36.07 ± 18.38) 87–1032 (363.55 ± 326.82) Flowers acting as donors (%) 27.77–100.00 (66.79 ± 23.85)a29.41–100.00 (84.65 ± 16.52)a Flowers pollinated (%) 11.11–41.67 (24.15 ± 10.95)b11.76–96.67 (57.14 ± 21.38)b Male efficiency (Nilsson index) 0.18–0.50 (0.35 ± 0.10)A0.30–1.00 (0.67 ± 0.20)B Fruiting success (%) 4.03–35.00 (11.24 ± 9.90)A5.00–85.71 (36.01 ± 20.02)B Table 3. Percentage of fruit set resulting from different breeding system treatments. Fruits were produced exclusively under crosspollination. Data are based on 10 individual plants used for each species of Malaxis. Numbers in parentheses represent the number of fruits obtained over the number of flowers used in each treatment. Treatments Malaxis parthoni Malaxis excavata Intact flowers 0% (0/50) 0% (0/30) Emasculation 0% (0/50) 0% (0/30) Self-pollination 0% (0/50) 0% (0/30) Cross-pollination 90.00% (45/50) 76.67% (23/30) Calderon-Quispe et al.: Specialist fungus gnat pollination of Malaxis species484 of another species of the same genus (Mycomya sp.2) pollinated Malaxis excavata. A total of 59 pollinator interactions were recorded for M. parthoni and 83 for M. excavata. In M. parthoni, pollinator activity occurred between 13:30 and 18:00 h, with a peak in visitation between 16:00 and 17:00 h (Fig. 4). During each visit, pollinators interacted with 2 to 23 flowers, spending between 2 and 22 seconds per flower, and between 10 and 65 seconds per inflorescence (Table 2). In M. excavata, interactions were recorded between 16:00 and 20:30 h, with a peak between 18:00 and 18:30 h (Fig. 4). Pollinators visited 1 to 23 flowers per visit, spending 12 to 313 seconds per flower and 87 to 1032 seconds per inflorescence (Table 2). Pollination process The pollination process was similar in both Malaxis species. Both exhibit corymbiform inflorescences (Figs 1B, 2B) that serve as a landing platform for pollinators, as the flowers are considerably smaller than the body size of the visiting insects (Figs 5A–D, 6A–C). Occasionally, more than two individuals of Mycomya were observed arriving at the inflorescences simultaneously (Figs 5B, 6A). Pollinators typically begin by feeding on nectar secreted along the margin of the labellum (Figs 5D, 6B). In M. excavata, this behaviour is more conspicuous due to a pronounced thickening along the labellar margin, where a greater volume of nectar is secreted (Suppl. material 2). Notably, pollinators of M. excavata were frequently observed feeding exclusively on this marginal nectar, without necessarily accessing the nectar accumulated in the cavities of the labellum. In contrast, M. parthoni has a more flattened labellum, making nectar secretion along the margin less prominent (Suppl. material 1). After feeding at the labellar margin, Mycomya individuals in both species may proceed to feed on nectar secreted by the cavities, where nectar accumulates in greater quantity. To access this nectar, the insects must insert their head into the inner part of the labellum (Figs 5C, 6C). It is at this point that the ventral part of the prothorax and the area behind the mouthparts come into contact with the pollinia that are located at the subapical dorsal position of the column (Figs 1H, 2F), which then adhere to these regions (Figs 5D–F, 6D–F). When the insect visits another flower and feeds on nectar from cavities of the labellum, the pollinia are deposited onto the stigma, which is located at the apex of the column. Breeding system, pollination efficiency, and fruiting success No fruit development was observed in bagged intact flowers or emasculated individuals of either Malaxis species, indicating that both are pollinator-dependent for fruit set. Likewise, no fruit was produced under manual self-pollination, suggesting that both species require cross-pollination to achieve successful reproduction (Table 3). In both species, the number of flowers acting as pollen donors was significantly higher than the number of pollinated flowers per inflorescence (Wilcoxon signedrank test; p < 0.05; Table 2). In M. excavata, the number of pollinated flowers tended to be higher than in M. parthoni (Table 2). Nilsson’s male efficiency index ranged from 0.18 to 0.50 in M. parthoni (Table 2), while in M. excavata it was notably higher, ranging from 0.30 to 1.00 (Table 2), indicating greater pollination efficiency in the latter species (Mann-Whitney U test, p < 0.05; Table 2). Fruiting success in M. parthoni was consistently low, ranging from 4.03% to 35.00% (Table 2). In contrast, M. excavata showed higher values, with fruiting success ranging from 5.00% to 85.71% (Table 2). Figure 4. Visits by Mycomya species to Malaxis parthoni (studied in southern Brazil) and M. excavata (studied in the Peruvian Andes) flowers, recorded at 30-minute intervals for a total of 80 hours of observation per species of Malaxis. Plant Ecology and Evolution 158 (3): 476–492, 2025 491 (Mycetophilidae) in southwestern Yunnan, China. 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Ecology and Evolution 14(4): e11295. https://doi. org/10.1002/ece3.11295 SUPPLEMENTARY MATERIALS Supplementary material 1 Pollination process of Malaxis parthoni. https://doi.org/10.5091/plecevo.164210.suppl1 Supplementary material 2 Pollination process of Malaxis excavata. https://doi.org/10.5091/plecevo.164210.suppl2