Aroonrat Kidyoo
email: aroonratm@hotmail.com

Manit Kidyoo

Paweena Ekkaphan

Rumsaïs Blatrix

Doyle Mckey

Magali Proffit

Specialized pollination by cecidomyiid flies and associated floral traits in Vincetoxicum sangyojarniae (Apocynaceae, Asclepiadoideae)

Keywords: Autonomous self-pollination, Diptera, mushroom-alcohol, nectar-rewarding flower, Nematocera, odour mimicry, Pollination Importance Index (PII), sucrose/hexose ratio

Vincetoxicum sangyojarniae combines nectar reward with food deception to attract its pollinators and ensures successful pollination through reciprocal mechanical fit between plant reproductive organs and insect structures.

only suitable pollinators can extract the pollinaria, a prerequisite for successful pollination.

INTRODUCTION

A topic of long-standing interest in pollination biology is the degree of specialization in plantpollinator interactions, which for the plant may range from obligatory specialists (flowers interacting only with one pollinator) to facultative generalists (flowers interacting with a broad range of pollinators) [START_REF] Johnson | Generalization versus specialization in plant pollination systems[END_REF][START_REF] Ollerton | Multiple meanings and modes: on the many ways to be a generalist flower[END_REF][START_REF] Armbruster | The specialization continuum in pollination systems: diversity of concepts and implications for ecology, evolution and conservation[END_REF].

Reproductive success of flowering plants depends on the attraction and use of their pollinators.

Floral traits responsible for pollinator attraction are thus expected to be adaptive responses to the pressures consistently exerted by pollinating agents [START_REF] Faegri | The Principles of Pollination Ecology[END_REF][START_REF] Fenster | Pollination syndromes and floral specialization[END_REF][START_REF] Schiestl | Pollinator-mediated evolution of floral signals[END_REF][START_REF] Armbruster | The specialization continuum in pollination systems: diversity of concepts and implications for ecology, evolution and conservation[END_REF]. Specialization in pollination between flowers and pollinators can be 'functional', i.e., in cases where pollinators may belong to different taxonomic groups but interact with the flower in the same way owing to similarity in their morphology and/or behaviour and thus exert similar selective pressures on floral traits (e,g., long-tongued nectar-foraging flies and bees) or 'ecological', i.e., interacting with a limited number of pollinator species of a particular taxonomic group. Both types of specialization are usually associated with 'phenotypic specialization' of flowers, i.e., highly modified floral traits that appear to have evolved in response to selection for specialization, making the flowers potentially accessible to only a limited set of visitors [START_REF] Ollerton | Multiple meanings and modes: on the many ways to be a generalist flower[END_REF][START_REF] Armbruster | The specialization continuum in pollination systems: diversity of concepts and implications for ecology, evolution and conservation[END_REF].

Overall, specialization is generally achieved through various floral specializations that target the legitimate pollinators, i.e., advertisements such as shape, size, colour and odours, that are associated with floral rewards to pollinators such as nectar, pollen, brood sites and shelter [START_REF] Faegri | The Principles of Pollination Ecology[END_REF] and mechanical fits, that are often morphological adaptations that only allow correctly proportioned visitors to access the plant's rewards or reproductive organs [START_REF] Martos | Chemical and morphological filters in a specialized floral mimicry system[END_REF][START_REF] Shuttleworth | Entering through the narrow gate: A morphological filter explains specialized pollination of a carrion-scented stapeliad[END_REF]. However, some flowers are deceptive, attracting their pollinators with false signals, often mimicry of odours, shape and colours of food or sexual partner, that are coupled with absence of rewards [START_REF] Dafni | Mimicry and deception in pollination[END_REF][START_REF] Endress | Diversity and Evolutionary Biology of Tropical Flowers[END_REF].

Flowers of Asclepiadoideae (Apocynaceae) are noteworthy for their complex floral morphology [START_REF] Endress | Apocynaceae[END_REF]. One of the most prominent of its structures involved in the flower-pollinator interaction is the corona, which can be of corolline or staminal origin [START_REF] Liede | A descriptive system for corona analysis in Asclepiadaceae and Periplocaceae[END_REF]) and consists of either fleshy staminal lobes or of a ring of connate staminal and thinner interstaminal parts [START_REF] Endress | Apocynaceae[END_REF]. Corona parts may function to mechanically lead a pollinator's body part to the guide rails (structures that are delimited by lateral margins of two adjacent anthers), which in turn play a key role in trapping a body part of the pollinator and guiding it into a position in which pollinarium removal or pollinium insertion takes place [START_REF] Kunze | Structure and function in asclepiad pollination[END_REF]Wyatt & Broyles 1994). In addition, for species in which interstaminal parts are present and basally fused to staminal parts, the corona serves as a nectar reservoir [START_REF] Liede | A descriptive system for corona analysis in Asclepiadaceae and Periplocaceae[END_REF]. The degree of fusion of staminal and interstaminal parts (either completely fused or with gaps between them) affects the level of access to nectar granted to different visitors [START_REF] Ollerton | Corona structure in Cynanchum: linking morphology to function[END_REF][START_REF] Shuttleworth | Entering through the narrow gate: A morphological filter explains specialized pollination of a carrion-scented stapeliad[END_REF].

The highly modified floral morphology of asclepiads has frequently been interpreted as a trend towards specialized plant-pollinator interactions. However, phenotypic specialization of flowers is not always associated with a high degree of pollinator specialization [START_REF] Johnson | Generalization versus specialization in plant pollination systems[END_REF][START_REF] Armbruster | The specialization continuum in pollination systems: diversity of concepts and implications for ecology, evolution and conservation[END_REF]). Generalist pollination systems can occur in plant taxa with highly modified flowers (e.g., [START_REF] Vogel | A survey of the function of the lethal kettle traps of Arisaema (Araceae), with records of pollinating fungus gnats from Nepal[END_REF][START_REF] Johnson | Generalized pollination, floral scent chemistry, and a possible case of hybridization in the African orchid Disa fragrans[END_REF]. For Asclepiadoideae, generalized pollination has been well outlined in Asclepias spp. [START_REF] Ollerton | Multiple meanings and modes: on the many ways to be a generalist flower[END_REF]. Some flowers, e.g. Orbea lutea subsp. lutea, appear to be generalist, attracting various taxonomic groups of flies, but can be further specialized through mechanical filters that exclude fly species that are not legitimate pollinators from reaching the reproductive parts [START_REF] Shuttleworth | Entering through the narrow gate: A morphological filter explains specialized pollination of a carrion-scented stapeliad[END_REF].

Likewise, Vincetoxicum (Asclepiadeae, Tylophorinae) s.l. [START_REF] Liede-Schumann | Vincetoxicum and Tylophora (Apocynaceae: Asclepiadoideae: Asclepiadeae)-two sides of the same medal: Independent shifts from tropical to temperate habitats[END_REF]) is characterized by small disk-shaped flowers with a very short corolla tube and widely spreading lobes, and a staminal corona made up of five fleshy staminal lobes, mostly completely separate or sometimes partially connected by much thinner interstaminal parts [START_REF] Forster | A taxonomic revision of Cynanchum L. (Asclepiadaceae: Asclepiadoideae) in Australia[END_REF][START_REF] Liede-Schumann | Vincetoxicum and Tylophora (Apocynaceae: Asclepiadoideae: Asclepiadeae)-two sides of the same medal: Independent shifts from tropical to temperate habitats[END_REF][START_REF] Kidyoo | Vincetoxicum siamicum sp. nov. (Asclepiadeae, Asclepiadoideae, Apocynaceae), a rheophyte from northeastern Thailand and preliminary observation on its in situ pollen germination[END_REF][START_REF] Kidyoo | Vincetoxicum siamicum sp. nov. (Asclepiadeae, Asclepiadoideae, Apocynaceae), a rheophyte from northeastern Thailand and preliminary observation on its in situ pollen germination[END_REF][START_REF] Thaithong | Handbook of Asclepiads of Thailand[END_REF]. In such 'open' flowers, the presence of a corona is thought to offset the lack of a narrow tubular corolla, which in many species functions to exclude non-pollinating visitors from reaching the centre of the flower where the rewards and the reproductive organs are found [START_REF] Endress | Diversity and Evolutionary Biology of Tropical Flowers[END_REF]. The arrangement and the varying degree of fusion of the corona parts may play a crucial role in selecting for the legitimate pollinators, whilst allowing the corolla to maintain its primary function of pollinator attraction [START_REF] Ollerton | Corona structure in Cynanchum: linking morphology to function[END_REF]Yamashiro et al. 2008) Most Vincetoxicum species studied to date are pollinated by insects belonging to five orders, i.e., Diptera, Lepidoptera, Coleoptera, Hymenoptera and Blattodea, with dipterans as the most frequent pollinators [START_REF] Lumer | The reproductive biology of Vincetoxicum nigrum (L.) Moench (Asclepiadaceae), a Mediterranean weed in New York State[END_REF]Yamashiro et al. 2008;Xiong et al. 2020). Floral visitation can occur during the day, e.g., in V. nigrum [START_REF] Lumer | The reproductive biology of Vincetoxicum nigrum (L.) Moench (Asclepiadaceae), a Mediterranean weed in New York State[END_REF] and V. tanakae, or primarily at night, e.g., in V. aristolochioides, V. floribundum and V. hainanense, or during both day and night, e.g., in V. nipponicum (Yamashiro et al. 2008;Xiong et al. 2020). [START_REF] Lumer | The reproductive biology of Vincetoxicum nigrum (L.) Moench (Asclepiadaceae), a Mediterranean weed in New York State[END_REF] reported several families of large flies as pollinators of V. nigrum. Among these, the most important are the blowflies (Calliphoridae) and flesh flies (Sarcophagidae). Yamashiro et al. (2008) recognized several functional groups of pollinators (fly, sawfly, moth, bee, wasp and beetle) in their study of 21 Vincetoxicum spp., and revealed that although certain Vincetoxicum species observed, e.g., V. acuminatum, have generalized insect pollination, most species are specialized to members of a single taxonomic group. Xiong et al. (2020) unveiled specialized cockroach pollination in V. hainanense. Moreover, several species of the genus have been found to be capable of autonomous self-pollination, setting fruits by self-fertilization without pollinator visits [START_REF] Lumer | The reproductive biology of Vincetoxicum nigrum (L.) Moench (Asclepiadaceae), a Mediterranean weed in New York State[END_REF][START_REF] Leimu | Variation in the mating system of Vincetoxicum hirundinaria (Asclepiadaceae) in peripheral island populations[END_REF]). Compared to floral morphology, breeding systems, and pollinator fauna, which have received relatively more attention, much less is known about the chemical composition of floral scents of Vincetoxicum, which, so far, has been documented only in V. hirundinaria [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF].

During fieldwork in eastern Thailand, we had the opportunity to investigate pollination ecology of the climbing species Vincetoxicum sangyojarniae [START_REF] Kidyoo | Molecular phylogenetic and morphological analyses support recognition of a new species of Vincetoxicum (Apocynaceae, Asclepiadoideae) from eastern Thailand[END_REF], found in a protected area within the Rajamangala University of Technology Isan Surin campus, Surin Province. Individuals of this species have also been cultivated in an open garden in Bangkok Province, central Thailand, outside the plant's native range, and comparative studies in the two localities were conducted. We address the following four points: (1) Are plants capable of setting fruit by autonomous self-pollination? (2) What floral visitors are legitimate pollinators in the native range at Surin, and in the cultivation site in Bangkok, and how specialized/generalized is the plant's pollination system? (3) Does the flower display particular characteristics involved in specialization on pollination by a single major taxonomic group of insects? (4) What compounds characterize floral scents of this species, and how are floral scents related to pollinator attraction?

MATERIALS AND METHODS

Study sites and species

Vincetoxicum sangyojarniae is a slender twiner recently described from Thailand [START_REF] Kidyoo | Molecular phylogenetic and morphological analyses support recognition of a new species of Vincetoxicum (Apocynaceae, Asclepiadoideae) from eastern Thailand[END_REF]). It has ovate to oblong-lanceolate leaves, branched cymes with one to four zigzag rachises, and 20-many-flowered. Its flower is wheel-shaped, 6-6.5 mm in diameter and reddishpurple in colour. Within flower, at the base of the stamens, five fleshy, broadly ovate, reddishbrown staminal corona lobes are inserted. Voucher specimens were deposited at BCU and BKF (A. Kidyoo 80: BKF, BCU; A. Kidyoo 81: BCU).

Research was carried out in the preserved area within Rajamangala University of Technology Isan Surin campus, Surin Province, eastern Thailand (14°50'52.4" N, 103°28'52.6" E, 150 m a.s.l.) where 20 to 30 individuals of Vincetoxicum sangyojarniae naturally grow in an open, disturbed area of about 50 x 30 m. Additional fieldwork was also conducted on five individual plants 6 months after being grown from cuttings in a private garden in Bangkok (13°42'10.0" N, 100°24'14.7" E, 5 m a.s.l.). The plants are perennial twiners. Each produces a single stem that branches and usually climbs from less than 1 m to 20 m above the ground. Branches of adjacent individuals are often entangled with one another. To avoid pseudoreplication, careful attention was paid to choose plants whose stems could be traced back to their point of origin from the ground. Furthermore, for cultivation in the garden at Bangkok, each cutting was collected from a distinct individual plant.

Floral biology and flower visitors

Plants were observed during three consecutive flowering seasons from October 2021 to November 2022. On the first day of anthesis, floral biology, insect species, numbers, and behaviour of floral visitors, and temporal patterns of visitation were recorded continuously with NV-GS75 digital cameras (Panasonic, Matsushita Electric Industrial Co., Ltd., Japan) on 43 and 26 inflorescences in the Surin site (8-30 November 2021) and the Bangkok site (5-11 January 2022), respectively. When possible, floral visitors were captured on sticky traps installed near the inflorescences, and by using an insect net or a hand-made aspirator. Each visitor captured or observed on video was checked for attached pollinaria. Additional observations on nectar secretion and floral scent emission were made in parallel with video recordings. In the first few days of study, only a few ants, which did not behave as pollinators, were found sporadically visiting flowers in the daytime. Thereafter, the study was focused on nocturnal visitation, beginning at 16:00, before the anthesis of the first flower of each inflorescence, and extending to 09:00 the next morning (3 hours after sunrise) to check if there were diurnal visitors. Visitation rate was computed over time. During this same time period of video recording, light intensity, temperature and relative humidity were recorded hourly using data loggers (iButton DS1923, Maxim Integrated, San Jose, CA, USA; HOBO Pendant Temp/Relative Light Two Channel and HOBO U23-001A Temperature/Relative Humidity Data Logger, Onset Computer Corporation, Bourne, MA, USA).

Flower visitors were classified into taxonomic groups and morphospecies based on their morphological characters. Moreover, flies were additionally identified to family or genus level based on morphology using the identification keys in [START_REF] Marshall | Flies-The Natural History and Diversity of Diptera[END_REF] and Kirk-Spriggs and Sinclair (2017). Owing to the large number of undescribed tropical dipteran taxa and the lack of up-to-date taxonomic literature, identification to generic (except for one morphospecies of ceratopogonid fly) and specific levels was not possible. Cockroach (Blattodea) samples were identified to generic level. Specimens are kept in the collections of A. Kidyoo at Chulalongkorn University, Bangkok, Thailand.

To observe the flower microstructure and the attachment of pollinaria on the proboscis of flies, a part of the flower and fly samples was processed under a scanning electron microscope (SEM).

Flower and insect samples were dehydrated in a graded ethanol series, then subjected to freezedrying using t-butyl alcohol following the method of [START_REF] Inou | A new drying method of biological specimens for scanning electron microscopy: the t-butyl alcohol freeze-drying method[END_REF], sputter-coated with gold and examined using a JEOL JCM-7000 scanning electron microscope (JEOL Ltd., Tokyo, Japan), at an accelerating voltage of 10 kV.

To check whether V. sangyojarniae is capable of setting fruit by autonomous self-pollination, a flower-bagging experiment was performed to assess the plant's abilities to self-fertilize in the absence of pollinators. Five inflorescences, each originating from a distinct individual plant and bearing 20 to 30 pre-anthesis flowers, were bagged (mesh size 0.5 x 0.65 mm) to prevent their receiving visitors for the duration of flowering. At the end of the experiment, the number of fruits initiated was counted.

Relative pollination importance of different insects

To evaluate the relative pollination importance of each morphospecies of flower visitors, a pollination importance index (PII) [START_REF] Johnson | Pollination and breeding systems of selected wildflowers in a southern African grassland community[END_REF][START_REF] Mochizuki | Pollination by fungus gnats and associated floral characteristics in five families of the Japanese flora[END_REF] was calculated based on four factors: 1) relative abundance (A); 2) pollen-carrying capacity (PCC), the mean number of pollen grains carried per individual; 3) host fidelity (F), the average proportion of the total pollen load containing host pollen; and (4) pollination efficiency (PE), the probability that the behaviour of a given visitor results in pollination. The plant species studied here packages its pollen grains into pollinia that pair together to form a pollinarium, which is transported as a whole by a pollinator. Also, a single pollinium is generally sufficient to fertilize all ovules within a flower. Thus, values for PCC assigned to an individual visitor can be simplified to either 0 (pollinaria absent) or 1 (with at least one pollinarium). No other pollinariaproducing plants at flowering stage were present in the surrounding area during the period of study (three congeners, i.e., Vincetoxicum indicum, V. flexuosum and V. potamophilum, were found growing at 10 m to 100 m from the studied species, but their flowering had either already ended or began later). Like other asclepiads, pollination occurs when the insect appendage is first trapped between the guide rails, which, in turn, guide the appendage into the groove of the corpusculum. The corpusculum is then pulled out and drags the rest of the pollinarium (pollinarium removal). When the animal visits another flower of the same species, one or both of the two pollinia may be transferred, or more precisely, be inserted into a guide rail and left within the stigmatic chamber where the pollen grains can germinate (pollinium insertion) [START_REF] Kunze | Structure and function in asclepiad pollination[END_REF][START_REF] Endress | Diversity and Evolutionary Biology of Tropical Flowers[END_REF]. Thus, although visitors might carry single pollen grains from any other flowers they may have visited, these pollen grains would not reach the concave stigmatic surface hidden behind the guide rails, and therefore were considered to have no effect on pollination success of the plant under consideration. Consequently, values for F assigned to an individual visitor are either 0 (pollinaria absent) or 1 (with at least one pollinarium). Similarly, values assigned to an individual visitor for PE are either 0 (for a visitor that did not contact the guide rails) or 1 (for a visitor that contacted the guide rails). The pollination importance value (PIV) of each morphospecies of flower visitor was obtained by multiplying all four values (PIV = A x PCC x F x PE) and, in turn, PII was calculated as the proportional contribution of PIV of a given morphospecies of flower visitors to total PIV of all visitors observed (PII = PIV/PIV).

Floral scents

Volatile organic compounds (VOCs) emitted by flowers were collected in situ on the first day of anthesis from the inflorescences during the period of peak insect visitation (between 18.00 and 24:00) by the dynamic headspace technique [START_REF] Proffit | Can chemical signals, responsible for mutualistic partner encounter, promote the specific exploitation of nursery pollination mutualisms? The case of figs and fig wasps[END_REF][START_REF] Auttama | Flowering phenology and trap pollination of the rare endemic plant Ceropegia thaithongiae in montane forest of northern Thailand[END_REF]. As the odour of the flowers was very faint and hardly detectable by the human nose, each sample was extracted from multiple inflorescences (each from a distinct plant individual for the Surin site): two samples at the Surin site, including 30 and 20 blooming flowers from two and three inflorescences, respectively, and one sample at the Bangkok site, including 65 blooming flowers from five inflorescences. Inflorescences were enclosed in bags made of polyethylene terephtalate (Nalophan, Kalle Nalo, Wiesbaden, Germany). Pure air, i.e., ambient air passed through two glass tubes filled with activated charcoal (Supelco, Bellefonte, PA, USA), was conducted into the bag at a flow rate of 400 ml/min using exclusively Teflon tubing. The air containing floral volatiles was pulled from the other side of the bag at a flow rate of 300 ml/min through an adsorbent tube made of borosilicate glass and filled with 20mg of Porapak Q embedded in borosilicate glass wool (ARS, Inc., Gainesville, FL, USA). Airflow was maintained through the bags by two membrane pumps (NMP850KNDC; KNF, Switzerland) that blew and pulled and were connected to the flow meters by which the flow rates were controlled. Extractions lasted for 3 hours. All sampling of floral volatiles was conducted under the same conditions, with a blank simultaneously collected from an empty bag for each extraction.

Trapped volatiles were eluted with 150 l of hexane (hexane, Optima for HPLC and GC, H303-4, Fisher Scientific, Leicestershire, UK), and 10 l of 0.003% toluene (toluene HPLC grade, Fisher Scientific, Leicestershire, UK) solution in hexane was added as an internal standard. Therefore, each sample vial contained 0.00019% v/v toluene before injection. The eluants were stored at -40 °C until analysis. The volatile extracts were subsequently analyzed by an Agilent 7890B (Agilent Technologies, Wilmington, Delaware, USA) gas chromatograph fitted with a HP-5MS column (length 30 m, inner diameter 0.25 mm, film thickness 0.25 mm), coupled to an Agilent 7000C triple quadrupole mass spectrometer (using MS Scan mode). Ultrahigh-purity helium was used as carrier gas at a flow rate of 1.2 ml/min. The column temperature was initially held at 50 °C for 3 min, then increased at a rate of 3 °C/min to 100 °C, 2.7 °C/min to 140 °C, 2.4 °C/min to 180 °C, and 10 °C/min to 250 °C, followed by an isothermal period of 5 min. The total run time was 63 min. The detector temperature was 250 °C. The injector was heated to 230 °C and was on split mode with a split ratio of 5:1, and the injection volume was 1.5 L. The conditions for electron impact ionization were an ion energy of 70 eV and the mass range scanned was 33-400 m/z. Data processing was carried out using MassHunter Qualitative Analysis Software (version B.06.00, Agilent Technologies, Inc. 2012). The retention times of a series of n-alkanes (standard solution of alkanes, 49452-U, Sigma Aldrich®, Munich, Germany) were used for converting retention times to retention indices. Identity of VOCs was determined by comparing the mass spectra with those in the NIST database (NIST 2011 library), and confirmed by comparing the calculated retention indices (relative to the n-alkanes) with those reported in [START_REF] Adams | Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry[END_REF] and other published literature (see Table 2).

Floral nectar collection and analysis of sugars

Inflorescences with fully developed flower buds of Vincetoxicum sangyojarniae grown at the Bangkok site were covered with a nylon mesh bag (mesh size 0.5 × 0.65 mm) to exclude any visitors. We collected nectar samples from 30 flowers, distributed in five plants. To check for potential diel patterns of nectar production within flowers, for each half of flowers (n = 15), nectar samples were collected either during the peak hour in pollinator visitation at night (i.e., 00:00 -00:30) or in the next morning before the flowers closed (after anthesis at dusk, the flower closed on the next morning, i.e., 06:00 -07:00), allowing the nectar to accumulate for the whole functional period of the flower. Due to the fact that the flowers of V. sangyojarniae produce a small volume of highly viscous nectar, the available nectar cannot be extracted from the flower with a microcapillary tube, and in turn, the nectar volume and percentage of sugars cannot be assessed in the field. Therefore, we used the washing technique adapted from [START_REF] Morrant | Field methods for sampling and storing nectar from flowers with low nectar volumes[END_REF] to extract the nectar for sugar content analysis by High Performance Liquid Chromatography (HPLC). To ensure that the solution would contain sugar concentration detectable by HPLC, we had to pool the nectar from five flowers to constitute one sample. For each sample, sets of five flowers were one by one removed from the inflorescence. Each flower was held by its pedicel using a forceps, then agitated in 0.5 ml of distilled water (within a 1.5 ml microcentrifuge tube), taking care not to let the cut surface sink in the water to avoid contamination from cell sap leakage from the cut surface. The microcentrifuge tubes were stored in a freezer at -20C until analysis. We thus obtained three samples for night-time and three for day-time sampling.

Prior to HPLC analysis, the solutions were filtered using a 0.22-mm nylon membrane into 11.6 x 32 mm, 2-mL short thread vials, sealed with 9-mm PP short thread cap PTFE/silicone liners. Nectar sugar content was analyzed by injecting 50 μL of fi L of filtered solution using a SIL-40 XR autosampler (Shimadzu, Japan) and chromatographed using a Rezex RPM-Monosaccharide Pb+ Column, 300 x 7.8 mm (Phenomenex, Torrance, CA, USA) heated to 60 ºC to separate the sugars. Type I Water (Pall Life Sciences, Port Washington, NY, USA) was used as mobile phase at a flow rate of 0.6 mL min-1. We also injected 50 μL of fi L of each standard sugar solution, i.e., glucose, fructose and sucrose, at 50 ppm and 100 ppm (in distilled water). Detection was achieved using an evaporative light scattering detector (ELSD-LTIII, Shimadzu, Japan) heated to 100 ºC with nitrogen at a flow rate of 2 mL min-1. Data acquisition and handling were done using LabSolutions software from Shimadzu.

Sugars were identified by comparison with the retention times of external standard peaks. The concentration of each sugar was determined by comparing the areas of the samples to those of the known standard peaks in the chromatogram. We converted the concentration (mg L -1 ) to sugar mass (mg) per flower by multiplying by 0.0005 because 0.5 mL of distilled water was used to wash nectar from flowers (following [START_REF] Morrant | Field methods for sampling and storing nectar from flowers with low nectar volumes[END_REF]., then divided by five (the number of flowers from which nectar was extracted for each sample). We examined whether sugar mass per flower differed between the nectar samples collected at night and those collected in the next morning. The pooling of samples from individual flowers, made necessary by the very small volumes of nectar secreted, resulted in sample sizes too small (n = 3 for each treatment) to enable statistical comparison of sugar mass collected at night and during the day. However, the observed differences were very large (see section 3.4 below).

RESULTS

Floral biology and flower visitors

At both study sites, opening of flowers began after 17:00 when the light intensity drastically decreased, and peak blooming was achieved by midnight. In full bloom, the corolla lobes were fully spread out. The flowers are disk-shaped with open access to the reproductive organs at the centre (Fig. 1). The mean number ( SE) of blooming flowers per inflorescence observed on each day was 7.2  0.5 (n = 43 inflorescences) at the Surin site and 9.3  0.6 (n = 26 inflorescences) at the Bangkok site. Anthesis of a single flower often lasted two or rarely three days. The flowers closed during the day and re-opened at night. Soon after opening of the flowers, nectar was secreted, and accumulation was clearly visible in the shallow cup-shaped corolla tube around the gynostegium (Figs. 1B,2A). After anthesis, the corolla lobes closed up, wilted, and abscised.

During 18-h observation periods on each inflorescence based on video recordings and direct observation, at the Surin site (n = 43 inflorescences), we recorded 95 insect individuals of 13 morphospecies belonging to five orders, Diptera, Blattodea, Hymenoptera, Lepidoptera and Orthoptera, whereas at the Bangkok site (n = 26 inflorescences), 108 insect individuals of only three morphospecies belonging to the order Diptera were registered (Table 1). Diptera, which represented the largest group of flower visitors, were categorized into five taxonomic groups: cecidomyiid fly, ceratopogonid fly, psychodid fly, other Nematocera and culicid mosquito. Flies of two families, Cecidomyiidae and Ceratopogonidae (genus Forcipomyia) (Fig. 3A-D), dominated the visitor assemblage at the Surin site, representing 31% and 6% of all individuals observed, respectively. Only one morphospecies of Cecidomyiidae was detected at the Surin site (Fig. 3A-B), and was the main floral visitor (Table 1). Likewise, another morphospecies of cecidomyiid fly (Fig. 3C) was almost the only flower visitor at the Bangkok site, representing 97% of all individuals observed. Other insects that frequently visited flowers at the Surin site, but to a much lesser extent than cecidomyiid and ceratopogonid flies, were cockroach nymphs, ants, and mosquitos (Table 1, Fig. S1). Of all flowers observed during the study period, only about 0.4% set fruit at the Surin site, and no fruit set was found at the Bangkok site.

The results of the flower-bagging experiment showed that in all bagged inflorescences, no flowers set fruit, indicating that self-fertilization did not occur in the absence of pollinator visits.

Relative pollination importance

Remarkably, all flower visitors were observed purposefully feeding on nectar. No visitors exhibited mating or oviposition behaviours. Whilst foraging for nectar, all major visitors, i.e. cecidomyiid and ceratopogonid flies (Videos 1 & 2), cockroaches (Videos 3 & 4) and ants, contacted both the anthers and guide rails (between which a pollinium must be inserted if pollination is to occur). However, apart from a single individual cockroach nymph, only cecidomyiid and ceratopogonid flies were found to carry pollinaria (Table 1, Fig. 3B, Fig. S1, Video 1). Also, Cecidomyiidae were observed at times inserting the pollinia into the guide rails (Table 1, Fig. S1, Video 2). They carried pollinaria on one (or both) maxillary palpus (palpi) (Fig. 4B). The SEM images (Fig. 4) clearly showed that the dorsal side of the second segment (each palpus has three segments) of the maxillary palpus (52-58 microns in length, 17-21 microns in diameter) was locked in the corpuscular groove (72-77 micron in length, 26-30 micron in diameter at the basal entrance) (Figs. 2F,4C). Note that the upper end of the groove fitted firmly to the constricted joint of the first and second segments of the maxillary palpus (Fig. 4D). Examination of the flowers pollinated by cecidomyiid flies revealed several pollinia lodged in the stigmatic chambers (Fig. 2A,D,G). The pollinium (101-129 micron in equatorial diameter and 140-152 micron in polar diameter) was slipped into the entrance (129-138 micron in diameter) at the lower part of the guide rail and drawn farther upwards to the large opening below the style-head (the topmost part of the style, see 'sh' in Fig. 2A) where the pollen tubes grew from the pollinium through the lower surface of the style-head downwards the styles (Fig. 2G). Usually, the translator arm broke off, leaving a single inserted pollinium in each chamber (the pollinium was thus completely hidden behind the anther wings, see Fig. 2C-D). Sometimes, the flies detached themselves from the corpusculum by leaving a part of the maxillary palpus inside the corpuscular groove (Fig. 2C), therefore achieving only a single fertilization with a whole pollinarium.

As confirmed by the pollination importance index (PII) scores (Table 1), ranging from 0.8 (Surin site) to 1 (Bangkok site), cecidomyiid flies were the most important pollinators of Vincetoxicum sangyojarniae. They visited flowers between 17:00 and 05:00 the following morning (Fig. 5). At the Surin site, visits of pollinators began at 17:00, just before the disappearance of the last sunlight, at average temperature of 27 C and relative humidity of 65 %, then peaked at 18:00 when light intensity became zero with average temperature of 26 C and relative humidity of 70% (Fig. 5). At the Bangkok site, visits of pollinators began later, at 18:00, when it was almost completely dark, the temperature decreased to 30C and the relative humidity increased to more than 50%. The visits then peaked during the period from 22:00 to midnight when the temperature declined to about 25-26C and the relative humidity increased to 71-73% (Fig. 5). Pollinator activities ceased before sunrise at both study sites. It appears that in both sites, flower visitation began when the night fell, and peak visit occurred when temperatures were relatively cool (26 C) and humidity was high (70-73%). This finding explains the difference in peak time of visits over the night observed between the two sites (Fig. 5).

Floral scents

Flowers of Vincetoxicum sangyojarniae have a very faint grassy, astringent, sweet odour with a slight note reminiscent of rubber, that is hardly detectable to the human nose. Chemical analysis showed that the floral scent bouquet was composed of 22 VOCs (Table 2), most of which are aliphatic and monoterpenoid compounds. (E)--ocimene, 1-octen-3-ol, (E)-4,8-dimethyl-1,3,7nonatriene (E-DMNT) and N-(3-methylbutyl)acetamide were the major components, each with relative amount more than 5%.

Floral nectar collection and analysis of sugars

The only sugar component detected in all nectar samples extracted from the flowers of V. sangyojarniae was sucrose. The other two sugars investigated, i.e., glucose and fructose, were not detected. Among the remaining peaks (i.e., components other than the three most common sugars) in the chromatogram, none was consistently found across samples. The mean sugar mass (mg) per flower (mean  SE) for the nectar collected at night and in the next morning were 0.0115  0.0015 and 0.0061  0.001, respectively. Although statistical comparison is impossible given our small sample sizes (n = 3 for each case), the almost two-fold difference in the mean values and the low dispersion of the values (Fig. S2) suggest that the apparent difference is real and biologically meaningful.

DISCUSSION

In the present study we characterized the floral biology and the degree of pollinator specialization of V. sangyojarniae. We also analysed floral traits that target the legitimate pollinators in two localities (in natural habitat and in cultivation outside the plant's native range).

Specialization for cecidomyiid fly pollination and associated floral morphological traits

The is likely the main reward sought by all of its nocturnal insect visitors (but see below the discussion about floral volatiles). Despite the diversity of visitors observed at the Surin site, most of the major visitors other than cecidomyiid and ceratopogonid flies, i.e. cockroaches, mosquitoes and ants, were nectar robbers that fed on nectar (cockroaches, in particular) without providing any pollination services. Only flies of the family Cecidomyiidae were observed at high frequency to remove pollinaria from the anthers, to carry pollinaria when arriving at the flowers, and to insert pollinia into the guide rails. They are thus effective pollinators at both study sites.

Flies of the family Ceratopogonidae (genus Forcipomyia) also visited the flowers and removed pollinaria, although much less frequently than did cecidomyiids. Ceratopogonidae can thus be regarded as potential alternative pollinators. Surprisingly, nymphs and adults of the cockroach Blattella bisignata (Ectobiidae) were reported in China to be specialized pollinators of V. hainanense, carrying pollinaria on their legs and mouthparts (Xiong et al. 2020). In our study, the cockroach Margattea sp. (nymphs in particular) frequently visited flowers at the Surin site, and whilst probing for nectar, their maxillary and labial palps, labium and labrum, as well as their legs, were very frequently in contact with the plant's floral reproductive organs. However, with the exception of one individual, no pollinaria were found attached to their body parts (Table 1). The flowers of V. hainanense measure about 22 mm in diameter (Xiong et al. 2020). This flower size is about one and a half (adults) to three (nymphs) times larger than the length of the pollinating cockroach B. bisignata (the proportions approximated from the figures shown in Xiong et al. 2020). In contrast, the flowers of V. sangyojarniae measure only 6-6.5 mm in diameter, which is about one (nymphs) to two (adult) time(s) smaller than the length of the visiting cockroach Margattea sp. (Fig. 3E-F ,Video 3 & 4). For an insect to be able to dislodge a pollinarium from the anthers and carry it, a body part of the insect must engage the groove in the corpusculum, and the insect must be strong enough to free the body part and dislodge the pollinarium before leaving the flower (Wyatt and Broyles, 1994). Therefore, this preliminary comparison between the overall sizes of cockroaches and flowers suggests that Margattea sp. is likely too large to effect pollination in V. sangyojarniae. These observations strongly suggest that size of floral parts constitutes a mechanical filter in V. sangyojarniae.

The cecidomyiid flies that pollinated V. sangyojarniae have a short, stout labium (Fig. 4A,D), and no structures or appendages around the proboscis, other than the two maxillary palpi, can engage the corpuscular groove. This is due to the suitable reciprocal relation of the sizes, forming a lock-and-key relationship [START_REF] Barad | Pollination of stapeliads[END_REF][START_REF] Kunze | Structure and function in asclepiad pollination[END_REF], between these insect and flower structures. The fact that the corpuscular groove is usually attached to a maxillary palpus emphasizes the essential role of this organ of the insect in the detection of nectar sources, as found in other Diptera (e.g., Syed & Leal 2007 and references therein). Also, it seems that whilst searching for nectar, the insect bends its maxillary palpi so that the dorsal side of the second segment slips into the corpuscular groove. We thus suggest that the flower of V. sangyojarniae manipulates its cecidomyiid pollinators by exploiting this nectar-foraging behavior.

These findings suggest that floral morphological traits mechanically prevent the transport of pollinaria by many visitors to V. sangyojarniae flowers. In this manner, traits of the flower are specialized for pollination by cecidomyiid flies. Interestingly, while the pollination system of the plant is thus narrowly specialized, its floral traits allow visits by a wide range of insects. The flower is disk-shaped, and its open corona offers open access to the centre (see [START_REF] Ollerton | Corona structure in Cynanchum: linking morphology to function[END_REF]Yamashiro et al. 2008 for different categories of corona). The five staminal lobes are completely separate, and conspicuous interstaminal parts are lacking. There is thus no connate basal portion to store nectar. Rather, nectar accumulates in a shallow cup-shaped corolla tube, where it can be freely exploited not only by pollinating cecidomyiid flies but also by numerous other visitors that do not pollinate. Although some nectar is thus "wasted", the morphological traits of the reproductive organs avoid wastage of pollinaria [START_REF] Wiemer | Functional morphology and wasp pollination of two South American asclepiads (Asclepiadoideae -Apocynaceae)[END_REF], a loss that would probably have a much higher fitness cost. Therefore, in species like V. sangyojarniae that offer open access to floral parts and rewards, the functional traits associated with mechanical fits between flower and pollinator are probably key in shaping pollinator specialization. Such mechanical fits have been revealed in many asclepiads [START_REF] Kunze | Structure and function in asclepiad pollination[END_REF][START_REF] Ollerton | Corona structure in Cynanchum: linking morphology to function[END_REF][START_REF] Wiemer | Functional morphology and wasp pollination of two South American asclepiads (Asclepiadoideae -Apocynaceae)[END_REF][START_REF] Shuttleworth | Entering through the narrow gate: A morphological filter explains specialized pollination of a carrion-scented stapeliad[END_REF].

Although corona morphology and arrangement of V. sangyojarniae do not restrict access to rewards, they may contribute to guiding the maxillary palpi of pollinators towards the guide rails and corpuscula. When the insects approach the flower and attempt to reach the nectar, their maxillary palpi are driven to the guide rails by the positioning of the five staminal corona lobes -each of the five sinuses directs the maxillary palpus to the guide rail and the corpusculum above (Figs. 2A). In addition, although nectar robbing by non-pollinating visitors may incur a cost for the plant [START_REF] Endress | Diversity and Evolutionary Biology of Tropical Flowers[END_REF], such fully exposed nectar presentation may enhance visual signals that attract the plant's nocturnally foraging nectar-feeding pollinators. Visually, flowers can be detected by insects against the background (soil, leaves, sky) due to chromatic contrastcolour (spectral reflectance pattern)-or achromatic contrast-luminance (intensity or brightness) [START_REF] Olsson | Chromatic and achromatic vision: parameter choice and limitations for reliable model predictions[END_REF]. The dark red coloration, as found in our night blooming V. sangyojarniae, has been suggested to reduce visits by unwelcome visitors [START_REF] Mochizuki | Pollination by fungus gnats and associated floral characteristics in five families of the Japanese flora[END_REF]), e.g., moths that have a preference for white or pale flowers [START_REF] Goyret | Why do Manduca sexta feed from white flowers? Innate and learnt colour preferences in a hawkmoth[END_REF], rather than to increase attractiveness to the fly pollinators. It has been proposed that achromatic visual cues, e.g., the amount of reflected light, may be crucial for flowers pollinated at night (van der Kooi & Kelber 2022). In flowers of V. sangyojarniae, a reflective ring of nectar present on the corolla surface around the gynostegium might contribute to such visual attraction, and deserves further exploration by experiments.

The present results support the findings of Ollerton et al. (2019) that almost three-quarters of Apocynaceae species are functionally specialized on pollination by a single major group of insects, and also support the general trend in pollination systems relative to corona structure in the genus Vincetoxicum s.l. suggested by Yamashiro et al. (2008). Flowers that lack the interstaminal parts (making the entrances to the guide rails fully exposed to floral visitors), traits found in V. sangyojarniae, are frequently pollinated by small Diptera with a short proboscis, for example V. aristolochioides and V. japonica, pollinated by small flies of the families Cecidomyiidae and Sciaridae, respectively (Yamashiro et al. 2008). By contrast, flowers with interstaminal parts forming a cup-shaped structure concealing the entrances to the guide rails (increasing the difficulty of access for small insects) tend to be pollinated by insects with a long proboscis, for example V. inamoenum, pollinated by flies of the family Empididae, and V. ambiguum, pollinated by moths (Yamashiro et al. 2008).

Nectar sugar composition in relation to pollinator types / phylogenetic constraints

Nectar is an energy-rich resource for insect pollinators. The nutritional value of nectar owes mostly to the presence of three sugars, i.e. sucrose and its component hexoses, glucose and fructose, although both interspecific and intraspecific variation in proportions of these sugars often occur [START_REF] Baker | A brief historical review of the chemistry of floral nectar[END_REF][START_REF] Nicolson | Sweet solutions: nectar chemistry and quality[END_REF]. Nectar sugar composition may greatly influence the preference of insect foragers [START_REF] Roy | Review: Nectar biology: From molecules to ecosystems[END_REF][START_REF] Balduino | To each their own! Nectar plasticity within a flower mediates distinct ecological interactions[END_REF]. The flowers of Vincetoxicum sangyojarniae secrete a small volume of highly viscous nectar, in which sucrose is the only sugar contained in the samples from all study sites. Considering the sucrose/hexose ratiothe ratio by weight of sucrose to the combined hexose sugars, glucose and fructose [START_REF] Baker | The predictive value of nectar chemistry to the recognition of pollinator types[END_REF])-the nectar of V. sangyojarniae can be categorized as sucrose-dominated. It has been suggested that the nectar sugar profile might primarily be shaped by phylogenetic constraints. The sucrose/hexose ratio has been found to be associated with plant family affinity [START_REF] Percival | Types of nectar in angiosperms[END_REF][START_REF] Stiles | Patterns in floral nectar characteristics of some bird-visited plant species from Costa Rica[END_REF][START_REF] Petanidou | Sugars in Mediterranean floral nectars: an ecological and evolutionary approach[END_REF]. The sucrose-dominated nectar of V. sangyojarniae is in accord with records from other species of Asclepiadoideae whose nectar is dominated by sucrose, e.g., tribe Asclepiadeae: Asclepias spp. [START_REF] Percival | Types of nectar in angiosperms[END_REF][START_REF] Southwick | Nectar biology and nectar feeders of common milkweed, Asclepias syriaca L[END_REF], Cynanchum harlingii, Ditassa spp., Scyphostelma spp. (Wolff 2005); tribe Marsdenieae: Hoya verticillata (A. Kidyoo, unpubl. data); and more than ten additional Asclepiadoideae species in Wolff (2005). In contrast, records from previous studies of two Ceropegia species (tribe Ceropegieae) in eastern Thailand do not follow this tendency: their nectars contain no sucrose, but fructose (60%) and glucose (40%) (A. Kidyoo, unpubl. data). However, despite an exception observed in certain Ceropegia species, the data suggest that sucrose-dominated nectar is predominant in Asclepiadoideae and hence, that there is probably a strong phylogenetic signal of nectar sugar composition traits.

Sucrose/hexose ratios have also been linked to different pollinator types. High-sucrose nectar has been found to be mostly associated with hummingbirds, butterflies (but see [START_REF] Petanidou | Sugars in Mediterranean floral nectars: an ecological and evolutionary approach[END_REF], and long-tongued bees. By contrast, flies have been suggested to prefer high-hexose nectar [START_REF] Percival | Types of nectar in angiosperms[END_REF][START_REF] Pyke | The production of dilute nectars by hummingbird and honeyeater flowers[END_REF][START_REF] Baker | Chemical constituents in nectar in relation to pollination mechanisms and phylogeny[END_REF]. However, each of these pollinator classes may be a heterogeneous assemblage, including smaller taxonomic groups with nectar preferences varying from low-to high-sucrose, e.g., within the long-tongued bee class, Apidae prefer low-, Anthophoridae prefer medium-, whilst Megachilidae prefer high-sucrose nectar [START_REF] Petanidou | Sugars in Mediterranean floral nectars: an ecological and evolutionary approach[END_REF]. In light of this fact, it is not surprising that our result on V. sangyojarniae with its sucrose-dominated nectar does not conform to the general trend for fly-pollinated flowers.

Analyses of nectar in the present study also showed that the sugar mass per flower was significantly higher in nectar samples collected during night than those collected early morning of the next daywhen the light intensity increases, and the air humidity decreases at dawn (Fig. 5). This fact suggests diel patterns of nectar production within flowers. Nectar secretion dynamics appear to be synchronized with flower displaythe corolla lobes opening during night and closing during the whole day, which may be an advantageous strategy in excluding certain illegitimate visitors from the dish-shaped flowers (hence with open access to floral resource) of Vincetoxicum sangyojarniae. Given that insect visits were excluded in the present study, the reduction in nectar sugar masses from night to early morning might be due to nectar reabsorption for resource recovery [START_REF] Pacini | Nectar production and presentation[END_REF]Nepi & Stpiczynska 2008a;[START_REF] Nicolson | Sweet solutions: nectar chemistry and quality[END_REF]. The reabsorption of nectar might also have an ecological function, allowing modulation of nectar concentration that facilitates flowers' control of resource use in response to their pollinator visitations (Nepi & Stpiczynska 2008b;[START_REF] Prieto-Benítez | Circadian rhythm of a Silene species favours nocturnal pollination and constrains diurnal visitation[END_REF]) Such temporal variation of nectar concentration within a flower has also been found in plants of other families, e.g., Cucurbita pepo (Cucurbitaceae) [START_REF] Edge | Diel nectar secretion rhythm in squash (Cucurbita pepo) and its relation with pollinator activity[END_REF], Silene spp. (Caryophyllaceae) [START_REF] Prieto-Benítez | Circadian rhythm of a Silene species favours nocturnal pollination and constrains diurnal visitation[END_REF], as well as in other asclepiads, which are much less studied compared to other plant families, e.g. Hoya carnosa (Marsdenieae) [START_REF] Matile | Circadian rhythmicity of nectar secretion in Hoya carnosa[END_REF].

Floral volatiles: signalling of rewards

In many plant-pollinator interactions, pollinator attraction over long distances is mediated by floral scents (Raguso 2002;[START_REF] Hossaert-Mckey | Floral scents: their roles in nursery pollination mutualisms[END_REF]. The flowers of Vincetoxicum sangyojarniae released VOCs that are very different from those of V. hirundinaria, the only species of the genus whose floral volatiles have been previously investigated [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]. Vincetoxicum hirundinaria has flowers with cream-coloured corolla and corona. It is a generalist, mostly pollinated by large flies, e.g., Tachinidae (Diptera) (Timonin & Savitsky 1997), and by some Hymenoptera and Lepidoptera (Nymphalidae and Noctuidae) [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]. Mosquitoes (which do not pollinate it) were observed to get trapped and die in the guide rails [START_REF] Leimu | Variation in the mating system of Vincetoxicum hirundinaria (Asclepiadaceae) in peripheral island populations[END_REF][START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]. The floral scents of V. hirundinaria as perceived by the human nose are sweetish and sourish, and their chemical composition is heavily dominated by benzenoid compounds (> 97 %), particularly phenylacetaldehyde, methyl benzoate, acetophenone, benzaldehyde and methyl salicylate [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]). Among these compounds, only methyl salicylate was detected in V. sangyojarniae. In contrast, the floral scents of V. sangyojarniae contained mostly aliphatic and terpenoid compounds, with (E)--ocimene (about 44 %) as the most abundant compound. (E)--ocimene is widespread in various flowering plant families [START_REF] Knudsen | Diversity and distribution of floral scent[END_REF], and frequently present in large proportions in the floral scents of Asclepiadoideae and Secamonoideae (Apocynaceae) [START_REF] Jürgens | Floral scent composition in early diverging taxa of Asclepiadoideae, and Secamonoideae (Apocynaceae)[END_REF]).

However, it was absent from the floral volatiles of V. hirundinaria [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]. This compound is known to attract various insects seeking nectar rewards, and is frequently present in butterfly-and moth-pollinated plants, as documented in many species of Asclepiadoideae and Secamonoideae [START_REF] Jürgens | Chemical diversity of floral volatiles in Asclepiadoideae-Asclepiadeae (Apocynaceae)[END_REF]2010) and in some species of Rauvolfioideae (Apocynaceae) [START_REF] Knudsen | Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa[END_REF], as well as in other plant families, e.g. Caryophyllaceae, Rubiaceae, Solanaceae and Zingiberaceae [START_REF] Knudsen | Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa[END_REF]. It may also be involved in food-deceptive strategies, attracting moth pollinators to the nectar-less flowers of Plumeria alba (Apocynaceae, Apocynoideae) [START_REF] Knudsen | Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa[END_REF], or Nematocera flies (mainly Sciaridae) to the nectar-less flowers of Riocreuxia torulosa (Asclepiadoideae) [START_REF] Heiduk | Fly pollination of kettle trap flowers of Riocreuxia torulosa (Ceropegieae-Anisotominae): A generalized system of floral deception[END_REF]). The homoterpene (E)-DMNT is a widespread herbivoreinduced VOC released from damaged leaves (Tholl et al. 2011) and commonly known as a floral scent component (Kaiser 1911;[START_REF] Knudsen | Diversity and distribution of floral scent[END_REF], It was also reported in flowers of Asclepiadoideae, e.g. in moth-pollinated Hoya carnosa (Kaiser 1911) and fly-pollinated Riocreuxia torulosa and Ceropegia spp. [START_REF] Heiduk | Fly pollination of kettle trap flowers of Riocreuxia torulosa (Ceropegieae-Anisotominae): A generalized system of floral deception[END_REF].

In contrast to (E)--ocimene, known to be attractive to various nectar-feeding pollinators of rewarding flowers, the other main VOCs emitted by the flowers of V. sangyojarniae, i.e., N-(3methylbutyl)acetamide and 1-octen-3-ol, are often related to false signals, mimicking respectively food sources and mating or brood sites, used in deceptive pollination strategies. N-(3-methylbutyl)acetamide is unusual among floral scents, but common in insect glandular secretions, e.g., in secretions of the prothoracic glands of lacewings (Chrysopidae, Neuroptera) [START_REF] Aldrich | Prothoracic gland semiochemicals of green lacewings[END_REF] or in the pheromones of many species, e.g., of tephritid fruit flies [START_REF] Scolari | Tephritid fruit fly semiochemicals: current knowledge and future perspectives[END_REF]. It is also known to be released from the venom gland of social vespid wasps, and used by kleptoparasitic flies (i.e., flies that exploit food items killed by predatory arthropods) of the genus Desmometopa (Milichiidae) as kairomones to locate their protein-rich food meal (by following odours of vespids and/or their freshly killed prey) required for egg production and development (Zhang et al. 2022). Similar compounds are also emitted by so-called kleptomyiophilous flowers of Ceropegia dolichophylla (Apocynaceae, Asclepiadoideae) that mimic VOCs of insects to lure their kleptoparasitic milichiid fly pollinators [START_REF] Heiduk | Deceptive Ceropegia dolichophylla fools its kleptoparasitic fly pollinators with exceptional floral scent[END_REF].

There is growing evidence of kleptomyiophilous pollination strategies both in Apocynaceae [START_REF] Heiduk | Floral scent and pollinators of Ceropegia trap flowers[END_REF]) and other plant families, e.g., Aristolochiaceae [START_REF] Oelschlägel | The betrayed thief -the extraordinary strategy of Aristolochia rotunda to deceive its pollinators[END_REF], wherein other compounds are also involved in attraction.

However, so far almost all of the previously described kleptoparasitic flies belong to the Brachycera and Cyclorrhapha. This strategy is very rare in Nematocera, where it has only been documented in some Cecidomyiidae [START_REF] Sivinski | A kleptoparasitic Cecidomyiid and other flies associated with spiders[END_REF]. The presence of N-(3methylbutyl)acetamide in floral volatiles of V. sangyojarniae is thus curious. Whether it is related to kleptomyiophily, and if so how, is unclear. Little is known about biology and ecology of tropical fly pollinators [START_REF] Larson | Flies and flowers: taxonomic diversity of anthophiles and pollinators[END_REF][START_REF] Raguso | Don't forget the flies: Dipteran diversity and its consequences for floral ecology and evolution[END_REF]) and even less is known about Nematocera than about other pollinating flies. In addition, information about floral scents in plants from tropical Asia, outside Asclepiadoideae, pollinated by cecidomyiid or ceratopogonid flies has been provided in only a few studies (see e.g., [START_REF] Gardner | A flower in fruit's clothing: Pollination of jackfruit (Artocarpus heterophyllus, Moraceae) by a new species of gall midge, Clinodiplosis ultracrepidata sp. nov. (Diptera: Cecidomyiidae)[END_REF]. Further advances in our understanding of their biology will improve our ability to understand the pollination strategies of V. sangyojarniae and other Vincetoxicum species. Interestingly, N-(3methylbutyl)acetamide and 1-octen-3-ol also occur in combination in the aroma profile of a particular red wine [START_REF] Fedrizzi | Changes in wine aroma composition according to botrytized berry percentage: A preliminary study on amarone wine[END_REF]. These compounds contribute to vinegar and mushroom notes, respectively [START_REF] Fedrizzi | Changes in wine aroma composition according to botrytized berry percentage: A preliminary study on amarone wine[END_REF]. The production of both compounds has been found to be associated with fungal infection and occurs during the fermentation of infected grapes [START_REF] Fedrizzi | Changes in wine aroma composition according to botrytized berry percentage: A preliminary study on amarone wine[END_REF][START_REF] Azzolini | Selection of Botrytis cinerea and Saccharomyces cerevisiae strains for the improvement and valorization of Italian passito style wines[END_REF]. These findings may be linked to another deceptive aspect, more plausible than kleptomyiophily, which will be discussed in the following.

The compound 1-octen-3-ol, also known as mushroom alcohol, is characteristic of fungal volatiles and associated with the earthy, yeasty odours of mushrooms [START_REF] Johnson | Convergent evolution and faecal scent mimicry in fly pollinated angiosperm flowers and a stinkhorn fungus[END_REF][START_REF] Kecskeméti | Fungal volatiles as olfactory cues for female fungus gnat, Lycoriella ingenua in the avoidance of mycelia colonized compost[END_REF]. This compound has also been found in earthy (often foul) floral scents in plants of various families, most often in species of Orchidaceae, Araceae and Aristolochiaceae [START_REF] Knudsen | Diversity and distribution of floral scent[END_REF] whose main pollinators are mycophagous flies (i.e., Diptera that feed on fungi), e.g., the orchid Dracula lafleurii [START_REF] Policha | Disentangling visual and olfactory signals in mushroom-mimicking Dracula orchids using realistic three-dimensional printed flowers[END_REF] and Anthurium salvadorense (Araceae) [START_REF] Schwerdtfeger | Anthecology in the neotropical genus Anthurium (Araceae): A preliminary report[END_REF], both pollinated by flies of the family Drosophilidae, and the orchid Malaxis monophyllos, visited by drosophilids, mosquitoes, and fungus gnats of the families Mycetophyilidae and Sciaroidae [START_REF] Jermakowicz | The floral signals of the inconspicuous orchid Malaxis monophyllos: How to lure small pollinators in an abundant environment[END_REF]. Flies use fungi for various resources, including food, mating sites or brood sites. They may feed on fungal hyphae (Kirk-Spriggs & Sinclair 2017) or obtain nectar-like sugary fluid produced by fungi [START_REF] Policha | Fungi, plants, and pollinators: Sex, disease, and deception[END_REF]. This association is exploited by many fly-pollinated flowers, which have been described as deceptive, mimicking olfactory cues (often combined with visual cues) of fungi to attract their pollinators [START_REF] Johnson | Convergent evolution and faecal scent mimicry in fly pollinated angiosperm flowers and a stinkhorn fungus[END_REF][START_REF] Kaiser | Flowers and fungi use scents to mimic each other[END_REF][START_REF] Policha | Fungi, plants, and pollinators: Sex, disease, and deception[END_REF][START_REF] Raguso | Don't forget the flies: Dipteran diversity and its consequences for floral ecology and evolution[END_REF]).

In the present study, we did not observe any oviposition behaviour of cecidomyiid or ceratopogonid flies upon their arrival at the flowers. Therefore, we suggest that the nightblooming flowers of V. sangyojarniae attracts its fly pollinators by emitting floral volatiles bearing olfactory notes associated with the presence of fungi or, less likely, of prey captured by predatory arthropods (the food sources of its pollinators), but offers a nectar reward upon insect arrival. This mismatch between the advertisement and the actual reward is most similar to that found in the wasp-pollinated orchid Epipactis helleborine (protein as the expected reward in this case) [START_REF] Brodmann | Orchids mimic green-leaf volatiles to attract prey-hunting wasps for pollination[END_REF]. By the way, in Ceropegia, which is often described as a genus of deceptive flowers, nectar is also produced and seems to play a role in manipulating pollinator (including cecidomyiids) behaviour, such that they are positioned to pick up pollinaria on their mouthparts [START_REF] Ollerton | Diversity of Diptera families that pollinate Ceropegia (Apocynaceae) trap flowers: an update in light of new data and phylogenetic analyses[END_REF]. Nectar in flowers of V. sangyojarniae might function in a similar way. Our results also suggest that in addition to olfactory signals, floral traits such as flowering time act to exclude some insects from visiting the flowers. If illegitimate visitors do visit the flowers, other floral features prevent them from detaching pollinaria, e.g., the morphological fit between the body parts of insects and the shape and size of the guide rails and/ or corpuscular groove of V. sangyojarniae. Further phylogenetic and comparative studies on the chemical composition of floral scents, the micromorphology of the functional traits of the flowers in relation to their respective pollinators, and the structure of the plant-pollinator networks of the Vincetoxicum species in Thailand, will provide a full understanding of floral evolution and plant-pollinator interaction specialization in the genus and in related taxa. Photographs by Manit Kidyoo.

  flowers of Vincetoxicum sangyojarniae opened only at night and closed during the day, completely filtering out diurnal visitors. Although individual flowers are small, several stems and branches of different plant individuals often twisted around each other, so that multiple inflorescences were disposed near one another, enhancing the floral display. As shown in previous studies of other Vincetoxicum species (Yamashiro et al. 2008; Xiong et al. 2020), nectar

  Fig. S1. Boxplots comparing the number of visits per inflorescence per day observed in the two study sites (n = 43 and 26 inflorescences at the Surin and the Bangkok sites, respectively), for insect visitors whose relative abundance was > 5%.

Fig. S2 .

 S2 Fig. S2. Boxplots comparing the mean sugar (sucrose) mass (in mg) per flower between the nectar samples collected at night and those collected the next morning (n = 3 for each treatment).

  Vincetoxicum sangyojarniae. The mean values were obtained from three samples. Each sample was extracted from multiple inflorescences (each from a distinct plant individual): two samples collected at the Surin site (n = 30 and 20 flowers from two and three inflorescences, respectively) and one sample collected at the Bangkok site (n = 65 flowers from five inflorescences). For each unknown VOC, main mass fragments are listed with the base peak first, followed by other fragments in order of decreasing abundance. VOCs within each class are listed in order of their retention index on the HP-5MS column (HP-5MS obs ), values of which were compared with published data on a similar type of column (DB-5 lit / ZB-5 lit ) when available. n/a = compounds for which published data on retention index on the HP-5MS column or a similar type of column are not available; SE = standard error. Compounds in boldface type are main constituents of the floral bouquet (relative amount > 5%); Refs = references.

Fig. 1 .

 1 Fig. 1. Photographs of Vincetoxicum sangyojarniae. (A) An inflorescence. (B) A close-up of two flowers, one in top view showing nectar accumulating in a shallow corolla tube that encircles the central gynostegium (yellow arrow) and the other in lateral view showing nectar running down from the corolla tube, which can be seen as a glossy film on a corolla lobe (blue arrow).

Fig. 2 .

 2 Fig. 2. Scanning electron microscopy images showing floral microstructures of Vincetoxicum sangyojarniae. (A) Gynostegium with five staminal corona lobes and the surrounding shallow cup-shaped corolla tube where the nectar is present (black asterisks). Note that there is a broken part of maxillary palpus caught inside the front guide rail (green arrow) and a pollinium inserted left at the entrance of the guide rail at 4 o'clock position before the insect could introduce it to the top of the stigmatic chamber (yellow arrow). (B) Higher magnification of the entrance to the guide rail seen in photo A (bottom view of the area inside square indicated by a long white arrow). (C) Part of the pollinarium with one pollinium inserted into the guide rail. Also, a part of a maxillary palpus of the fly which had been caught by the corpuscular groove was left. (D) A sector of gynostegium from which the anther wings were removed (remnant can be seen), showing an inserted pollinium inside the stigmatic chamber. Part of translator arm that has been broken from the pollinarium can be seen. (E) Pollinarium. (F) Higher magnification of the pollinarium in photo E, showing the basal entrance of corpuscular groove (blue arrow) and the points where the translator arms usually break off (white asterisks). (G) Two inserted pollinia with pollen tubes (red arrows) entering the style-head. ap = anther appendage; aw = anther wing; c = corpusculum; cg = corpuscular groove; cl = corolla lobe; cs = staminal corona lobe; ct = corolla tube; gr = guide rail; ov = ovary; p = pollinium; ps = pollen sac; s = style; sc = stigmatic chamber; sh = style-head; tr = translator arm. Photographs by Manit Kidyoo.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Insect visitors of Vincetoxicum sangyojarniae. (A) Cecidomyiidae morphosp. 2, whose mouthparts are trapped in the corpuscular groove. (B) The same individual Cecidomyiidae morphosp. 2 with a pollinarium (which was dislodged when the insect freed itself subsequently to the photo in A) attached to its mouthparts. (C) Cecidomyiidae morphosp. 1. (D) Ceratopogonidae morphosp. 1 caught on a sticky trap. (E) Nymph of cockroach. (F) Adult of cockroach. Photographs by Manit Kidyoo.

Fig. 5 .

 5 Fig. 5. The visiting activity of cecidomyiid fly pollinators to inflorescences in relation to weather factors, comparing the two study sites. (A, B) Information on average hourly ambient temperature, solar radiation and relative humidity obtained from data loggers installed at the Surin and Bangkok sites, respectively. (C, D) Number of visits per inflorescence per hour observed respectively at Surin (n = 43) and Bangkok (n = 26); the error bars represent standard errors.

Table 1 .

 1 The identity and relative importance of floral visitors as pollinators of Vincetoxicum sangyojarniae. The pollination importance index (PII) is based on pollination importance value (PIV), which is determined by relative abundance (A), pollen-carrying capacity (PCC), host fidelity (F) and pollination efficiency (PE).

	1
	2
	3
	Visitors	Number of	A	PCC F PE PIV PII	Pollinarium
		individuals							placement site
		observed/
		collected
	SURIN SITE
	Diptera
	Nematocera
	Cecidomyiidae morphosp. 1	29(5 * 1 ** )	0.31 0.21 1	1 0.07 0.76 maxillary palpus
	Ceratopogonidae morphosp. 1	6(1 * )	0.06 0.17 1	1 0.01 0.12	proboscis
	Psychodidae morphosp. 1	1	0.01	0	.	1	0	0	.
	Culicidae morphosp. 1	1	0.01	0	. 0.5	0	0	.
	Culicidae morphosp. 2	9	0.10	0	.	1	0	0	.
	Other Nematocera	1	0.01	0	.	1	0	0	.
	Blattodea
	Ectobiidae
	Pseudophyllodromiinae
	Nymph of cockroach Margattea sp. 1	25(1 * )	0.27 0.04 1	1 0.01 0.12	legs
	Adult of cockroach Margattea sp. 1	1	0.01	0	.	1	0	0	.
	Hymenoptera
	Ant morphosp. 1	13	0.14	0	.	1	0	0	.
	Ant morphosp. 2	1	0.01	0	. 0.5	0	0	.
	Lepidoptera
	Moth morphosp. 1	1	0.01	0	.	0	0	0	.
	Moth morphosp. 2	2	0.02	0	.	0	0	0	.
	Orthoptera
	Grasshopper	4	0.04	0	. 0.5	0	0	.
	BANGKOK SITE
	Diptera
	Nematocera
	Cecidomyiidae morphosp. 2	105(8 * 18 ** 4 *** ) 0.97 0.29 1	1 0.28	1	mouthparts
	Culicidae morphosp. 1	1	0.01	0	. 0.5	0	0	.
	Culicidae morphosp. 2	2	0.02	0	.	1	0	0	.

Table 2 .

 2 Average relative amounts of volatile organic compounds (VOCs) (mean ± SE) detected in floral scents collected in situ from inflorescences containing flowers at anthesis stage of

ACKNOWLEDGEMENTS

We are thankful to the two reviewers for their insightful comments and suggestions that substantially improved the manuscript. We are grateful to Assoc. Prof. Dr. Obchant Thaithong for her continued support and to Asst. Prof. Pacharaporn Sangyojarn for help during fieldwork.

We are thankful to Rajamangala University of Technology Isan Surin Campus for granting us permission to collect samples and to conduct fieldwork in the area. We thank Wuttipon Pathomwattananurak for assistance with cockroach identification. This research was funded by 'Thailand Science Research and Innovation Fund Chulalongkorn University' and was partly conducted in the framework of the International Research Project (IRP) SPECIFLY, of the Institut Ecologie et Environnement (INEE), Centre National de la Recherche Scientifique (CNRS), France.

DECLARATION OF COMPETING INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS


Frédéric Pourraz

Alain Duclos

Gregory Coubat

CRISTAL: A FRAMEWORK AND TOOLS FOR RESPONSIBLE DECISION-MAKING

Keywords: Decision-making, criteria, vigilance mode, avoiding any surprise effect

As avalanche triggering is impossible to predict fairly and accurately, risk assessment is still too much influenced by so-called "human factors". Such cognitive biases often prevent us from seeing obvious dangers. The CRISTAL approach aims at helping us make a conscious decision, from conception at home to technical choices on the field. Based on six observable and tangible criteria leading to four distinct vigilance modes, this iterative approach has four key steps: (i) the evaluation of the six criteria is factual, to avoid integrating any unexplained "feeling", and must also be independent of the group size and its expertise level; (ii) it leads to the determination of a minimal vigilance mode to be adopted on the field, which evolves along the route; (iii) the analysis completes the identification of the vigilance mode by also considering some human factors (experience, objective, group size, ...), as well as the topographical environment (terrain traps, ...). Such an analysis is in itself quite subjective because the resulting decision is bounded for each vigilance mode; (iv) the decision phase then ends the process... before it is restarted for the rest of the project. Developed and practiced for about two decades, this approach is now subject to research studies. A fuzzy rule-based system is being validated so that this transition from the six criteria to the adapted vigilance mode is no longer totally delegated to the user end.

INTRODUCTION

Initiated in the 90s by Werner Munter, several methods have been developed to attempt to reduce risk while navigating avalanche terrain.

From the inception of the first Professional and Elementary Reduction Methods [Munter, 1997], the main focus was to establish a gradual scale of "frequentable" slope steepness based on the danger level from the snow bulletin. A whole range of methods were then constructed following this premise: Graphic Reduction Method [START_REF] Harvey | Practical Risk Assessment and Decision Making in Avalanche Terrain. An Overview of Concepts and Tools in Switzerland[END_REF], After Ski Method [Brattlien, 2008]; although sometimes supplemented by other criteria for certain methods: Stop or Go [Larcher, 1999], SnowCard [START_REF] Engler | Die weisse gefahr: schnee und lawinen[END_REF], Avaluator [START_REF] Haegeli | Avaluator avalanche accident prevention card[END_REF], Quantitative Reduction Method [START_REF] Schmudlach | Quantitative Risk Reduction Method (QRM), a datadriven avalanche risk estimator[END_REF].

*Corresponding author address:

Frédéric Pourraz LISTIC -University Savoie Mont Blanc 5 chemin de bellevue 74944 Annecy-le-Vieux FRANCE tel: +33 450 666 002, email: frederic.pourraz@univ-smb.fr However, as depicted in Figure 1, the distribution of avalanches based on slope steepness is very similar, whether by danger levels 2, 3, or 4. As stated in [Ortovox, 2023], for all three danger levels, the average slope steepness is 38°, "Therefore, it would be wrong to assume that a lower danger level means a greater slope steepness is required for an avalanche to be triggered.". The initial premise generally adopted is thus strongly challenged.

Another type of method is initiated by Ian Mc-Cammon in the early 2000s. The stated aim is to counter biases induced by human factors, based on the observation that our decisions can be heavily influenced by cognitive biases, "This results in ballistic behavior where people appear to ignore obvious clues that they are making a mistake." [McCammon, 2000].

The systematic evaluation of tangible and observable criteria then becomes necessary, initiated by the Obvious Clues Method [McCammon, 2006] and later taken up in the Avaluator [START_REF] Haegeli | Avaluator avalanche accident prevention card[END_REF]. This new paradigm is further refined later on with the Bounded Vigilance Model, which "assumes that engaged awareness of a relatively small number of carefully-chosen cues will guide users to higher levels of situational awareness and more consciously formulated decisions regarding avalanche risk." [McCammon, 2009].

This approach was expanded upon and complemented by [Duclos, 2012], whose primary objective remains to reduce risk-taking, but above all, to avoid the element of surprise by characterizing four distinct vigilance modes. After undergoing extensive refinement to mature the approach, it is now called CRISTAL and has multiple objectives, namely:

1. Prevent the element of surprise by limiting the influence of cognitive biases.

2.

Decrease risk based on clear foundations.

3.

Completely avoid danger if that is the desired outcome.

4.

Become aware of the unpredictable factor.

CRISTAL, the subject of this article, is based on six tangible and observable criteria, which will be presented in Section 2. We will then focus on explaining the four vigilance modes in Section 3. The limitations of the approach and ongoing efforts to develop it into a fully-fledged method will be discussed in Section 4, before concluding in Section 5.

THE SIX CRITERIA

As humans and therefore fallible, our judgment and decisions are daily influenced by our cognitive biases, which sometimes blind us and prevent us from seeing obvious signs of danger. One way to guard against this is to objectively and systematically measure and observe our surrounding environment. As this phase is iterative: at home or in the refuge, during the approach, and throughout the route, we can only consider a limited number of criteria. The complexity of the phenomenon requires simplification, as our brains have limited capabilities for multi-criteria analysis. As seen in the introduction, this simplification cannot be confined to the danger level and slope steepness alone. The CRISTAL approach is therefore based on six criteria that either play a key role in the physics of avalanche triggering or reflect ongoing instability. To make them easier to memorize and to systematize their evaluation, we have created a sticker representing these six criteria (see Figure 2) to be affixed to the tips of skis. Graphically represented in the form of a fresh snow crystal (hence the name of the approach), these six criteria can be assessed in a random order but must be evaluated in their entirety. In the field, it's all about awakening our senses (sight, hearing, and touch), as we will see in the presentation of these six criteria.

Slopes steeper than 30°A

slope steeper than 30°is a recognized axiom for avalanche triggering. It is now easy to consult online maps and digital terrain models (albeit fraught with uncertainties) to assess the slope steepness right from the preliminary phase of the project. Also accessible in the field via a Smartphone, this information can be validated or corrected on-site using ski poles or an inclinometer. It is important not to focus solely on the slope directly underfoot but to have an overall view of the surrounding slopes. Indeed, in the case of a remote trigger, slopes above or just below the route can also reach or carry away the group. Spontaneous avalanches, likely to start more or less far above the route, must also be considered. Sometimes, a map showing the avalanche history of the area (such as the CLPA map in France) can provide valuable information about the potential extent of an avalanche. As we will see later (see Section 3), this notion of potential scale can influence our decision-making.

Recent avalanches observed

As Bruce Tremper stated in The Avalanche Review, "the best sign of avalanche danger are avalanches". Being aware of avalanches observed in the days preceding a field outing provides crucial information for decision-making. This is indeed the least equivocal criterion among the six.

It informs us about the type of avalanche we can expect, the presence of sensitive weak layers, the potential scale of upcoming avalanches, and the critical altitudes and aspects of the moment. This information is not always accessible and is often incomplete, but it would be a missed opportunity to ignore it given its richness. In France, this is the purpose of the data-avalanche association: www.dataavalanche.org/now, which compiles and disseminates all avalanche observations reported by practitioners. The limitation of this criterion primarily occurs during and just after disturbed episodes when few or no avalanches have yet been observed and recorded.

A possible buried weak layer

This is likely the most difficult criterion to grasp, but it is also one of the most important (there are no slab avalanches without weak layers, except in the case of gliding slab releases). Learning to perceive the presence of a weak layer within the snowpack is thus an essential skill for moving more confidently in snowy and potentially exposed terrain (see criterion 2.1). Regularly probing the snowpack with our ski pole helps detect potential heterogeneity (layers of different hardnesses), a sign of the probable presence of a weak layer. This should clearly alert us to the potential instability of the traversed slope. However, the mere presence of a weak layer is not enough: it also requires that the dynamic stress induced by one or more skiers can trigger its collapse and that this collapse propagates within it. We have seen that recent avalanche observations were an accessible way for everyone to be informed about the presence of this phenomenon. Another indication is the perception of a "whoomph" sound (a collapse has just occurred and propagated). For more experienced practitioners, snow tests are also a way to characterize this phenomenon.

Temperature increase, snow thawing

A rise in temperature within the snowpack always leads to a change in the mechanical properties of the snow, sometimes resulting in avalanche flooding. This can involve spontaneous releases due to wetting of the snowpack, as well as slab releases, with or without tangible wetting. While wetting is easily detectable in situ, the temperature rise within the snowpack, without melting, is more difficult to comprehend, as it is not always correlated with air temperature (as felt by the practitioner). For example, measurements from automatic stations at around 3000 meters altitude in France show differences exceeding 20°C between air temperature and snow surface temperature.

New overloading

Whether caused by wind-driven snow accumulation, by fresh snow associated with a weather disturbance, or even by rain, overloading the snowpack is almost always an aggravating factor. It is primarily the increase in the slab's mass, above a weak layer, that facilitates the propagation of its collapse. In addition to this overloading effect, rain changes the mechanical properties of the slab by disrupting its structure (see the previous criterion). Data from snow and meteorological stations are very useful for assessing this criterion. Depending on the type of station, two snow depth sensors are sometimes installed, one on the windward side and the other on the leeward side, to better assess the amount of transported snow [START_REF] Bourgeois | Double measurements of snow depth: Innovation for better management of avalanche risk in real time on the roads of Savoy (France)[END_REF]. Indeed, a simple wind speed sensor is not sufficient to determine whether or not snow has been transported, as it also strongly depends on the hardness and type of surface grains. Some stations, equipped with FlowCapt-type acoustic sensors, provide this indication directly.

Danger level

The danger level is considered as an input in most avalanche danger assessment methods, and practitioners are accustomed to referring to it. That's why it was essential to include it in the CRISTAL approach. However, it only illustrates the average situation of a mountain range and can conceal a highly heterogeneous situation (fatal accidents have occurred at both danger levels 1 and 2, sometimes due to large-scale avalanches). Although presented as one of the six criteria, it stands out due to its non-observable nature (it's already a synthesis of analysis by a third party). However, the text of the associated bulletin often contains information that can inform one or more of the other five criteria. The six criteria we have just described serve as the entry point for the CRISTAL approach. Their systematic evaluation, as objectively as possible, is essential for making informed and conscious decisions. This is the first phase of the approach, which consists of three phases (see Figure 3): Detect, Analyze, and finally Decide, as this is the ultimate goal.

The first detection phase aims to be factual and entirely independent of the group's level and size, terrain traps, objectives, or even weather conditions. It relies solely on the observation and evaluation of the six criteria. Although work is underway (see Section 4) to partially automate this process, the task is currently dedicated to humans. The aim is to determine the appropriate vigilance mode for the situation, which should be the same for a given situation, whether one is a professional or an amateur. It's important to note that this mode may fluctuate throughout the route, depending on the evolution of each of the six criteria. At this point, human factors are mentioned in the sense of [Mccammon, 2004], to always keep in mind that they can influence our evaluation of the criteria and our perception of the environment.

The second phase is analysis. Once the vigilance mode is determined, this phase allows us to consider elements other than avalanche danger. After all, we move in a complex environment, often with companions who are also complex. This analysis phase is therefore always indispensable. This is where subjectivity comes into play, as our desires and risk acceptance can vary greatly. Depending on the vigilance mode, this analysis focuses on the possibility or desire to completely avoid danger or to reduce risk. In all cases, the prior evaluation of the six criteria allows us to carry out this analysis with full knowledge of the facts.

The third, essential phase is the decision phase, which is not subjective but rather bounded for each vigilance mode. However, its implementation requires the skills of the group of practitioners (discussed during the analysis phase). Paradoxically, it often turns out, after an accident, that this phase was neglected.

Relaxed

Detect: The Relaxed mode means that no signs of danger have been detected, and therefore, no avalanche is possible in the frequented area. This is the case, for example, when no slope exceeds 30°. If this is not the case, it means that all other criteria have been assessed without consequences, implying that no dangerous snowpack instability is present.

Analyse: This is then very straightforward and only focuses on the group's desires. Decide: At this moment, all choices are still on the table until a potential mode change.

Alert

In Figure 3, the second mode is the Suspicious mode. We chose to present it last because this mode is specific, and gradually, the Alert mode follows the Relaxed mode.

Detect:

The Alert mode means that one or more signs of danger have been detected, and therefore, an avalanche is possible in the planned area. However, the potential scale of the avalanche remains moderate.

Analyse:

We are aware that the avalanche danger is real, and therefore, we are exposed to risk if we proceed on this section of the route. It's up to each individual to accept or decline this risk, depending on our objectives. If accepted, the challenge is to minimize this risk as much as possible so that, in the worst-case scenario, only one person would be affected by an avalanche. The analysis focuses on reduction possibilities: the presence of safe gathering zones (before and after the section), a safe acceptable distance, etc. As mentioned earlier, factors like level and group size, terrain traps, weather conditions, come into consideration. For instance, if the route overhangs a rocky ledge, regardless of the practitioners' skill levels, the outcome would be the same; however, if the passage is technically challenging, the analysis would consider the group members' abilities to navigate confidently. Visibility and the time required to implement this risk reduction would also be critical points to analyze. If this reduction is not possible or not desired, proceeding with the project would entail transitioning into the Gamble mode.

Decide: Having accepted the commitment, the decision is bounded to risk reduction. All actions discussed during the analysis phase need to be implemented, and the group should be prepared to rescue a potential victim.

Gamble

Detect: Similar to the Alert mode, the Gamble mode indicates that one or more signs of danger have been detected, and therefore, an avalanche is possible in the frequented area. The distinctiveness here is that in case of an avalanche, it would be of significant scale, and a severe accident would be unavoidable.

Analyse:

No more analysis is possible. If we proceed, we can only rely on luck.

Decide: Let's make the wise decision to change the plan while there's still time. This decision could also have been prompted by the analysis phase of the Alert mode, for instance, in the case of a potential avalanche of moderate or even small scale, but in an extremely unfavorable environment (e.g., a rocky ledge or a stream immediately downstream).

Suspicious

Detect: In this mode, the focus shifts from examining the six parameters to assess danger where I am currently, to where I could be in the short term, assuming any possibility of danger in the area is not ruled out (unlike the Relaxed mode). The concept of spatio-temporal "buffer" that we introduce extends the examination of conditions both in time and space: where I might be later. We've arbitrarily depicted it as a disk (illustrated by the orange circle in Figure 4), centered on my current location. Its diameter varies (from a few meters to several hundred) depending on the direction of movement (ascent/descent), group size, or visibility. In the example provided (see Figure 4), we base this on the GPS track of an ascent progression (backcountry skiing) in potentially dangerous snow conditions (new overloading and possible buried weak layers). The Suspicious mode was necessary for the highlighted track section in red (from the point marked at 2335 meters) to (i) avoid danger and transition into Alert mode while progressing between short inclined slopes exceeding 30°( which was done correctly); (ii) consider whether or not to continue the project at the end of the section, necessitating a transition into Alert or Gamble mode (the choice, highly debatable, was to continue the project). Even if I am relaxed when stationary and no immediate danger threatens me, the buffer intersects objective dangers that require a mode change. As soon as I start moving, I must constantly pay attention to my path to either avoid these dangers or transition into a higher vigilance mode (Alert or Gamble). Analyse: We are aware that objective dangers are nearby and that we must remain attentive to anticipate and avoid them if we wish to avoid risk. In this case, the analysis focuses on the possibilities of avoiding the danger. If avoidance is not possible or not desired, we will need to change modes and transition into the Alert or Gamble mode. Avoidance of danger is typically the best option during ascent (reducing risk can be challenging). Transitioning into the Alert mode is often acceptable during descent (risk reduction is often feasible due to greater agility, and significant gains can be made if the chosen objective knowingly includes steep slope sections).

Decide: Having chosen not to expose ourselves to danger, the decision is therefore limited to avoiding all critical zones.

DISCUSSION

As we've just seen, the CRISTAL approach provides a framework for making decisions consciously rather than being driven by emotions or feelings. Applicable throughout the itinerary, the approach is also suitable for all stages of the 3x3 [Munter, 1997]. Ultimately, our goal is to transform this approach into a fully-fledged decision-support method. To achieve this, we need to reduce the subjectivity induced by human evaluation of the six criteria and the successive vigilance modes adopted throughout the journey. Aware of the current limitations of the approach, we have already begun the process of transformation.

The first step was to formalize a graduated scale of values for each criterion, except the danger level. Therefore, we focused solely on five criteria. With each criterion having 3 to 4 gradual values, the number of possible combinations reaches 432 [START_REF] Pagnier | A Global System for Avalanche Risk Assessment[END_REF]. In order to represent the uncertain and imprecise nature of evaluating these criteria, we formalized them using fuzzy set theory [Zadeh, 1965]. Consequently, we also expressed the rules for combining these five criteria in order to automatically deduce the appropriate vigilance mode for the situation. These fuzzy rules have been developed through expert analysis of concrete situations (like the example in Figure 4) which, among other things, led to the emergence of the spatio-temporal "buffer" (as discussed in Section 3.4).

Although each criterion is tangible and observable, none are trivial. This was evident with the criterion of slope steepness exceeding 30°, which encompasses the slope directly underfoot, slopes above the route and if an avalanche triggered there can reach us, slopes just below us and if an avalanche triggered there can sweep us away, the historical behavior of that slope (has it previously avalanched?), and finally, the potential extent of an avalanche if triggered (as discussed in Section 2.1). These components are what we refer to as sub-criteria. As each of the five criteria can be decomposed into sub-criteria, we also formalized these using fuzzy subsets and then, once again, defined fuzzy rules to combine them and obtain a fuzzy value for each criterion. Thus, there are different levels of combination, one for transitioning from sub-criteria to associated criteria, and another for transitioning from the five criteria to the four vigilance modes [START_REF] Pagnier | A Global System for Avalanche Risk Assessment[END_REF].

This entire process is now largely automated but requires a crucial and significant validation step before it can be more reliably used by practitioners. All these expert rules need to be tested on a wide range of known historical situations to be validated or challenged. The ultimate goal of all these efforts is, for a given itinerary, to achieve the most automated evaluation possible of all subcriteria using all the information available through our online tool: SYNTHESIS (which is the specific subject of another article in this ISSW issue). After applying the various levels of combination, a semi-automated evaluation of the appropriate vigilance mode to adopt will be provided to the user. Unlike current deep learning approaches, the advantage of using fuzzy logic is that, at the end of this step, the result (vigilance mode) is explainable. This means that the most influential sub-criteria in achieving this mode can be explicitly described to the end user. If desired, the user can then re-evaluate some or all of these sub-criteria to refine the result and incorporate their own expertise into evaluating the appropriate vigilance mode. This semi-automation will only affect CRISTAL's detection phase. The analysis and decision-making phases will remain human-driven, as CRISTAL is only intended to be a decision-support method.

CONCLUSION & PERSPECTIVES

The CRISTAL approach, now formalized and supported by the graphical representation provided earlier (see Figure 3), is based on three distinct phases: Detect, Analyze, and Decide.

The first detection phase aims to systematize the objective assessment of six tangible and observable criteria to avoid surprise and limit the influence of cognitive biases in decisionmaking. This assessment results in the detection of an appropriate vigilance mode to adopt in the field, which can evolve throughout the journey.

The Relaxed and Gamble modes bracket the Suspicious and Alert modes. These latter modes respectively allow for complete avoidance of danger (if desired) and reduction of risk on clear bases. The Suspicious mode also introduces the innovative concept of spatio-temporal "buffer". This "buffer" embodies evaluating danger where I could be in the short term, rather than only where I am, ensuring continuous anticipation and, again, avoiding surprises. The analysis phase then takes into account all other elements complementary to avalanche danger to make the most suitable decision while remaining consistent with our objectives.

This CRISTAL approach should ultimately become a comprehensive decision-support method.

Through the work already done, the formalization of criteria and rules for detecting the appropriate vigilance mode have been established using fuzzy logic (a mathematical tool for considering notions of gradualness, imprecision, and uncertainty). One of our main goals is to maintain a "white-box" approach, ensuring complete explainability of the result. Already largely tooled, these efforts are at the validation stage, on numerous concrete cases, before being offered to the mountaineering community.

Figure 1 :

 1 Figure 1: Distribution of avalanches, regarding slope steepness, for each danger level ORTOVOX -Safety Academy -Lab Snow) (Source: S. Harvey)

Figure 2 :

 2 Figure 2: CRISTAL's sticker of the six criteria (printable version can be found on cristal-avalanche.org)

Figure 3 :

 3 Figure 3: CRISTAL's tool supporting the framework

Figure 4 :

 4 Figure 4: Spatial and temporal buffer of the suspicious mode


Thi Hong

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Silicon slotted photonic crystal cavities fabricated by deep-ultraviolet lithography