Pollinator and Floral Odor Specificity Among Four Synchronopatric Species of Ceropegia (Apocynaceae) Suggests Ethological Isolation That Prevents Reproductive Interference

doi:10.21203/rs.3.rs-1424836/v1

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Pollinator and Floral Odor Specificity Among Four Synchronopatric Species of Ceropegia (Apocynaceae) Suggests Ethological Isolation That Prevents Reproductive Interference

https://doi.org/10.21203/rs.3.rs-1424836/v1

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Possession of flowers that trap dipteran pollinators is a phylogenetically conserved trait within the genus Ceropegia, in which pollination systems can be generalized or highly specialized. However, little is known about the role of plant–pollinator interactions in maintaining reproductive isolation between plant species. This study examined the degree of specificity in plant-pollinator interactions and identified the mechanisms responsible for specificity among four synchronopatric species of Ceropegia. These species showed significant differences in floral scents, floral morphology, colour patterns, and presence of other functional structures, e.g., vibratile trichomes, whose essential role in fly attraction was experimentally demonstrated here. Similarity in chemical compositions of the floral scents to that of crushed Cletus trigonus bugs living in the same habitat suggests kleptomyiophily in at least some of the species studied. This is the first study to empirically demonstrate that mechanical isolation plays no discernable part in floral isolation in Ceropegia, but instead that a combination of olfactory and visual cues differentially influenced pollinator preferences and hindered heterospecific visitation, ensuring specificity through pollinator constancy, thereby driving ethological isolation among these congeneric sympatric species. We also showed experimentally that specificity was not maintained outside these plants’ native range, where other fly species occur.

Closely related plant species that co-occur in the same community and flower in synchrony, i.e. synchronopatric species, share similar habitat and may compete for both biotic and abiotic resources¹. Synchronopatric congeners may exhibit either positive or negative interactions mediated by pollen vectors. Co-flowering species may enjoy greater reproductive success by attracting more pollinators through larger floral displays^2,3. Alternatively, co-flowering may lead to reproductive interference, i.e. interspecific sexual interaction that negatively affects fitness⁴ of one or more of the co-flowering species due to the increasing competitive interactions among plants for pollinators and the costs of interspecific pollen transfer and gametic wastage⁵. Nevertheless, these costs may be negligible when reproductive isolation in plants occurs through multiple barriers that restrict reproductive interference.

Maintenance of species boundaries typically results from the combination of pre- and postzygotic mechanisms. However, their relative contributions are highly variable among plant groups and depend on specific characteristics of their reproductive biology^6,7. Asclepiadoideae (Apocynaceae) and Orchidaceae are characterized by pollen grains packaged into pollinia, often associated with a variety of accessory structures, forming a pollinarium—the functional unit (two to eight in orchids, five in asclepiads) removed by the pollinators⁸. Consequently, only a few visits may result in the removal of all pollen en masse from a flower. The cost of reproductive interference through pollen loss is thus particularly high for these plants. Asclepiads and orchids are expected to be under strong selective pressure to impede inaccurate pollinia transfer to sympatric heterospecific plants through prezygotic pre-pollination isolation mechanisms. Indeed, these barriers have been reported in many orchids^9–11, but have been little investigated in asclepiads^12,13. Pollinator specificity is of primary importance in maintenance or reinforcement of reproductive barriers. Pollinator-mediated reproductive isolation may be effected by floral isolation, either mechanical (morphological fits between pollinators and flowers) or ethological (pollinator attraction), resulting from differential visitation or constancy among pollinators driven by variable floral traits^7,12,14. Divergent floral traits between coexisting populations of congeneric species may occur as a by-product of allopatric speciation or may result from selection for reproductive isolation per se (especially in cases of reproductive interference)^15,16.

The genus Ceropegia L. (Apocynaceae, Asclepiadoideae) is specialized for pollination by small Diptera^17–19 despite large variation in floral traits across species. Most species are considered to rely on deceptive pollination, trapping flies for one or two days before releasing them with a pollen load. The level of specialization in pollinator attraction is highly variable among species, some taxa being specialists attracting flies from a single genus or family, such as C. sandersonii and C. pachystelma that use respectively Desmometopa spp. (Milichiidae) and Forcipomyia sp. (Ceratopogonidae) as main pollinators^20,21, and others being generalists attracting flies of various genera or families, such as C. ampliata, which uses flies in the families Anthomyiidae, Lauxaniidae, Muscidae, Sarcophagidae and Tachinidae^21,22. Moreover, these associations may be conservative, i.e., constant over most or all of the plant’s native range¹⁸ and even when plants are transported out of their native ranges and grown in greenhouses²³, or less conservative, wherein different pollinator assemblages are used by individual plants of the same species within different parts of their native range or outside their range^18,22,24. To date, it is still unknown whether this variability is due to spatial variation in local pollinator communities or whether there also exist other factors or strategies influencing the degree of specificity of interactions. Likewise, the extent to which plant species affect one another’s pollination through reproductive interference is unknown. Where habitats of co-flowering Ceropegia species overlap, as do those of different pollinating fly species, can interspecific visitation be avoided by differential attraction to pollinators through specific floral traits? In addition to a low number of pollen transfer opportunities (only five pairs of pollinia) per flower, individual Ceropegia plants display very few flowers, increasing the cost of pollen loss to heterospecific flowers.

The role of volatile organic compounds (VOCs) of Ceropegia flowers in pollinator attraction has been extensively investigated, mostly in South African species, as olfactory signals are considered to be the most important ones for attracting flies from a distance^,20,21. Based on these olfactory cues, it has been suggested that plants exploit pollinator feeding preferences. The involvement of VOCs in prey mimicry strategies attracting kleptoparasitic flies—flies feeding on the food or prey of predatory arthropods—as pollinators has been evidenced in a small number of Ceropegia species^20,21. This pollination strategy, the attraction of kleptoparasitic flies for deceptive pollination, is called kleptomyiophily²⁵. Chemical composition of floral scents was found to be highly variable among species and not constrained by phylogeny^20,21. Therefore, we can expect that floral scents play a crucial role in the attraction of a limited set of pollinator species. In addition, physical matching, involving relative sizes of fly and flower, has also been hypothesized to drive pollinator specificity (i.e., mechanical isolation) ^18,19,26. Moreover, the attractiveness of various visual cues remains undeciphered in Ceropegia. The most prominent of the putative visual cues are scintillant and motile structures termed flickering bodies (“Flimmerkoerper” sensu Vogel, 1954, 2001) ^27,28.

The region comprised by the Indian subcontinent and South-East Asia is one of the most species-rich areas for Ceropegia, but information on pollination systems in this region so far remains sparse^18,29,30 relative to other centers of diversity^{18, 19}. In Pha Taem National Park, located in eastern Thailand, four rare and narrowly endemic Ceropegia species—C. acicularis, C. boonjarasii, C. citrina and C. tenuicaulis—coexist in close sympatry. Although their rarity and their occurrence in small populations necessarily limit sample sizes, their sympatry offers a unique opportunity for studying the mechanisms of reproductive isolation among Ceropegia spp. The first aim of our study was to evaluate the risk of reproductive interference among these species. For this, we verified that the four species are synchronopatric and share the same pollination strategy. The second aim was to identify putative pre-pollination barriers that could have evolved in response to reproductive interference. As the four species studied are endemic to this area, we consider it highly unlikely that pre-pollination barriers are a mere by-product of allopatric speciation. Instead, we consider that pre-pollination barriers must have been driven by selection for (at least reinforcing) reproductive isolation per se. To identify pre-pollination barriers, we measured pollinator specificity among the four Ceropegia species and investigated mechanical (size matching) and ethological (VOCs from flowers and floral morphology) isolation mechanisms.

Pollinator assemblages are distinct among Ceropegia species. Floral visitors found within the corolla tubes of the four Ceropegia species were exclusively small Diptera (n = 168). The morphomolecular approach allowed delineating 16 species, belonging to the families Chloropidae (n = 97, 11 species) and Milichiidae (n = 71, five species) (Table 1, Fig. S2). COI sequences for the 41 individuals were deposited on Genbank with accession numbers OK138486-OK138526 (Fig. S2). All of these flies were female, except for two males of Chloropidae sp. 2 found in C. tenuicaulis.

Table 1

Flies trapped in the flowers of the four *Ceropegia* species sampled in Pha Taem National Park between 2016 and 2019. All flies were female, except otherwise indicated. n = number of flowers sampled for each species, M = male, ^(#poll) = number of flies carrying pollinaria.
	C. acicularis	C. boonjarasii	C. citrina	C. tenuicaulis	Indicator species
	(n = 59)	(n = 36)	(n = 3)	(n = 20)	IV	P
Milichiidae
Milichiella sp.1	11^(4poll)			2
Milichiella sp.2				34^(10poll)	0.93	<0.001
Neophyllomyza sp.1			11^(1poll)		1	<0.001
Neophyllomyza sp.2			12^(2poll)		1	<0.001
Neophyllomyza sp.3				1
Chloropidae
Chloropidae sp. 1		1
Chloropidae sp. 2		6^(5poll)		2M^(1poll),1
Chloropidae sp. 3		2^(1poll)
Chloropidae sp. 4	1
Chloropidae sp. 5		8^(1poll)
Chloropidae sp. 6		5^(5poll)
Chloropidae sp. 7	34 ^(12poll)				0.82	0.03
Chloropidae sp. 8		33^(13poll)		1	0.83	<0.001
Chloropidae sp. 9		1
Chloropidae sp. 10		1^(1poll)
Chloropidae sp. 11	1
Total flies	47	57	23	41

The frequencies of flies of the two families varied significantly among the four Ceropegia species (test of homogeneity, χ² = 118.678, df = 3, P < 0.001), with C. citrina and C. tenuicaulis attracting mostly Milichiidae, and C. acicularis and C. boonjarasii mostly Chloropidae. Of all flies trapped within the flowers, 56 (33.3%) carried pollinaria (Table 1). All flies that were found with pollinaria attached carried them on the mouthparts, the corpuscular groove of the pollinarium being caught on the membranous posterior part of the rostrum (Fig. S3). Out of 16 fly species, 13 were found in only one Ceropegia species. The three others were found in two Ceropegia species, but with a highly unequal distribution between the two species (Table 1).

The four Ceropegia species appeared significantly distinct by NMDS based on pollinator assemblages in individual flowers (Fig. 2; PERMANOVA F_3,43 = 15.192, P < 0.001). Pollinator assemblage of each of the four Ceropegia species was significantly different from those of the other three (PERMANOVA, P < 0.05). However, C. boonjarasii and C. tenuicaulis slightly overlapped. The result of the indicator species analysis identified the insect species that were significantly representative of the pollinators of each species (Table 1).

Flowers are synchronopatric and activity patterns of pollinators overlap. During continued observations from 2014 to 2019, no plant individuals with floral morphology intermediate between the congeneric species were found in the area. All species flowered once a year with overlapping blooming seasons extending from May to August, or even into October in some years. Flowering was maximum in July. The blooming period of C. acicularis preceded that of the others by about one month, but there was still some overlap. Each plant of C. citrina and C. tenuicaulis bore only a single flower at a time at anthesis on their unbranched stems, while C. acicularis and C. boonjarasii, which produced branched stems, could have respectively up to six and three open flowers simultaneously.

Video recordings showed flies of the families Chloropidae and Milichiidae landing on the flowers and either walking readily (for C. acicularis) or falling (for C. boonjarasii, C. citrina and C. tenuicaulis) into the corolla tube (e.g., Supplementary Information Video 1). Video recordings inside flowers showed flies running toward `light windows` at the bottom of the corolla produced by the light contrast between the gynostegium and the surrounding internal structure (e.g., Supplementary Information Videos 2 and 3). Flies then probed nectar (around the gynostegium) and eventually removed or deposited pollinaria. For a full description of floral anthesis and pollinator behavior based on video recordings, see Supplementary Information S1 and Fig. S4.

Flies visited flowers between 08:00 and 19:00. Among Ceropegia species, temporal range of floral visits over the day overlapped largely, but peak activity differed (Fig. 3). Two distinct patterns could be recognized. Visits of C. boonjarasii and C. acicularis by Chloropidae and of C. citrina by Neophyllomyza peaked in late afternoon (Fig. 3A, C, D), when temperature and sunlight dropped and relative humidity increased (Fig. 3E, F). On the contrary, visits of C. tenuicaulis and C. acicularis by Milichiella peaked with highest temperature and light intensity, and lowest relative humidity (Fig. 3A, B, E, F).

Non-natural pollinators can provide pollination services. We tested the efficiency of five fly species as non-natural pollinators of three Ceropegia species (Table 2) by introducing the flies into the flowers manually and checking for removal of pollinia. To work as an effective pollinator, a fly should be capable of removing a pollinarium and keeping it attached to the mouthparts long enough for deposition in another flower of the same species. As a whole, in our experiment, 28 pollinaria were removed by 41 flies that had been introduced. However, only 15 flies had pollinaria attached to the mouthparts at the end of the experiment, most of the other pollinaria being found free within the flowers (they probably fell off the flies). Nevertheless, all fly species tested, including Drosophila melanogaster laboratory strains, were capable of providing pollination service (Table 2).

Table 2

Scheme and results of the crossed-pollinators experiment. For flowers with a number of pollinaria removed higher than the number of flies carrying pollinaria, the difference corresponds to pollinaria found free inside the flower, if not otherwise stated by a footnote.
Recipient flower	Fly source	Fly species	No. of flies introduced	No. of flies carrying pollinaria^a	No. of polliniaria removed^a
C. acicularis #1	Lab strain	Drosophila melanogaster	2	1	2
C. acicularis #2	Lab strain	D. melanogaster	4	1	4
C. acicularis #3	C. tenuicaulis	Unidentified^b	1	0	2
C. acicularis #3	Lab strain	D. melanogaster	3	1	2
C. acicularis #4	Lab strain	D. melanogaster	2	2	3
C. acicularis #5	Lab strain	D. melanogaster	4	0	4
C. acicularis #6	C. tenuicaulis	Milichiella sp. 2	1	0	0
C. acicularis #7	C. tenuicaulis	Milichiella sp. 2	2	2	2
C. citrina #1	C. boonjarasii	Chloropidae sp. 5	1	1	3^c
C. citrina #1	C. boonjarasii	Chloropidae sp. 6	2	1	3^c
C. citrina #2	C. boonjarasii	Chloropidae sp. 4	1	1	4
C. citrina #2	C. boonjarasii	Chloropidae sp. 6	3	2	4
C. tenuicaulis #1	Lab strain	D. melanogaster	8	0	0
C. tenuicaulis #2	Lab strain	D. melanogaster	3	0	0
C. tenuicaulis #3	C. boonjarasii	Chloropidae sp. 6	4	3	4
^a At the end of the experiment.
^b The fly was too damaged to be identified at the end of the experiment.
^c One of the flies had two pollinaria attached to its mouthparts.

Although pollinaria had been removed from the gynostegium and flies within flowers had pollinaria attached to the mouthparts, no pollinaria were found inserted between the guide rails, indicating that recently removed pollinaria are unlikely to effect self-pollination.

Floral morphology differs among species but is not correlated with pollinator size. Overall, floral morphological characters differed significantly between species (Table 3, MANOVA, F_3,58 = 124.56, P < 0.001). The first two axes of the PCA, explaining a total of 90.7% of variance, clearly discriminated the species, except C. tenuicaulis and C. boonjarasii, which showed substantial overlap (Fig. 1E). The size of flies ranged from 1.4 to 2.4 mm in length and 0.5 to 0.9 mm in width (Fig. S2, Table S1A). Overall, there were no significant relationships between fly size, either in length or in width, and the size of different parts of flowers (Table S1B).

Table 3

Measurement of floral characters (mean ± SE; mm) compared between the four *Ceropegia* species
Flower traits	C. acicularis (n = 10)	C. boonjarasii (n = 10)	C. citrina (n = 9)	C. tenuicaulis (n = 10)
total flower length	12.3 ± 0.6	37.5 ± 0.9	41.9 ± 2.0	54.0 ± 2.2
corolla tube length	7.5 ± 0.4	24.8 ± 0.7	29.6 ± 1.2	17.1 ± 0.7
smallest diameter of corolla tube	2.6 ± 0.1	2.5 ± 0.1	4.1 ± 0.2	2.7 ± 0.1
basal inflation length	5.7 ± 0.3	13.0 ± 0.5	9.1 ± 0.2	9.7 ± 0.5
widest diameter of basal inflation	4.5 ± 0.62	6.1 ± 0.2	7.6 ± 0.3	5.9 ± 0.2

Floral scents are distinct among Ceropegia species. A total of 39 VOCs, mostly aliphatic compounds, were detected from the four Ceropegia species and the co-occurring Cletus trigonus bug (Hemiptera: Coreidae), among which 23 occurred in relative amounts above 0.5% in at least one species (Table 4). The number of major compounds (abundance ≥ 5%) was only three to five in each species (Cletus trigonus and the four Ceropegia). None of these major compounds were species-specific and each was shared by two to five species. The prominent compounds were hexyl butyrate for C. acicularis, isoamyl acetate for C. citrina, and (E)-2-octenyl acetate for C. boonjarasii and C. tenuicaulis. These three compounds were present in the chemical extract of Cletus trigonus bugs. However, the chemical odour of this species was dominated by hexanal.

Table 4

Average relative amounts of volatile organic compounds (VOCs) (mean ± SE) detected in floral scents collected from the flowers at first day of anthesis of the four *Ceropegia* species and from crushed *Cletus trigonus* bugs. The unknown VOCs are listed with important ions at various mass-to-charge ratios (m/z). n = number of samples; KRI = Kovats Retention index; SE = standard error. Indicator compounds are VOCs with a significant observed indicator value (IV). Bold-faced compounds are characteristic for the respective species. ^a : compound identity verified through authentic standard.
		C. acicularis			C. boonjarasii			C. citrinaa			C. tenuicaulis			Cletus trigonus			Indicator
VOCS	KRI	(n = 7)			(n = 7)			(n = 4)			(n = 6)			(n = 5)			compounds
Major compound (relative amount > 5%)		4			5			4			5			3			IV	P
Aliphatic compounds
Ethyl acetate	614				0.14	±	0.17				0.19	±	0.22
3-methylbutanal	654				0.32	±	0.31	0.11	±	0.07				0.26	±	0.29
1-butanol	676	0.16	±	0.16							0.16	±	0.17	4.74	±	1.77	0.86	0.002
4-methylheptane	769	0.3	±	0.3	1.47	±	0.98				1.06	±	1.3
Isobutyric acid	791										2.85	±	3.27				0.82	0.003
Hexanal	799	0.15	±	0.07	0.37	±	0.18				0.5	±	0.61	61.01	±	9.15	0.99	<0.001
2.4-dimethyl-1-heptene	841	1.52	±	1.49	18.3	±	8.19	0.18	±	0.12	4.34	±	4.66				0.87	0.002
Isovaleric acid	863										3.01	±	3.54	0.37	±	0.41
1-hexanol	871	9.79	±	6.41							2.77	±	1.89	2.45	±	1.31
Isoamyl acetate ^a	878				1.04	±	0.53	43.93	±	15.5	10.01	±	11.61	4.61	±	5.15
Unknown m/z: 57,43,85,71,41,99	978										0.3	±	0.32
Butyl butyrate ^a	996	24.64	±	5.37	1.55	±	1.84	1.39	±	0.75	0.23	±	0.28	0.67	±	0.44	0.93	<0.001
Hexyl acetate ^a	1012	1.7	±	1.00	3.31	±	1.98	0.27	±	0.21	7.24	±	4.26	13.91	±	6.48
(E)-2-hexenyl acetate	1017	0.25	±	0.25	4.42	±	2.27				4.37	±	3.23
2-ethyl-1-hexanol	1031	0.13	±	0.13	8.43	±	3.31	1.84	±	0.67	8.63	±	9.64
2-nonanone ^a	1091							0.13	±	0.05							1.00	<0.001
Nonanal ^a	1103				1.85	±	1.29	0.91	±	0.49	0.41	±	0.51
Hexyl butyrate ^a	1191	54.04	±	7.61				29.54	±	19.17				4.04	±	1.23
(Z)-3-octen-1-yl-acetate	1196	5.39	±	4.16	0.13	±	0.1				1.83	±	0.92
(Z, E)-5-octen-1-ol.	1207													0.31	±	0.32	0.77	0.005
(E)-2-octen-1-yl acetate ^a	1217	0.21	±	0.16	36.03	±	11.51	5.45	±	2.27	34.7	±	13.7	0.69	±	0.23
Tridecane ^a	1300				0.12	±	0.14	0.39	±	0.23							0.62	0.05
Hexyl hexanoate	1384													6.95	±	3.33	1.00	<0.001
Octyl butyrate	1388	1.71	±	0.44													0.93	<0.001
(E)-5-decen-1-yl acetate	1395							11.08	±	5.32	0.29	±	0.23				0.85	0.002
(E)-2-decen-1-yl acetate	1407				5.68	±	3.62	0.8	±	0.38	15.08	±	10.3
Hexadecanoic acid	1969				4.63	±	5.48	1.3	±	0.12
Terpenoids
Monoterpenes
6-methyl-5-heptene-2-one	987				0.58	±	0.26	0.53	±	0.3
Linalool ^a	1101				2.71	±	3.2	0.62	±	0.62	0.3	±	0.34
(E)-linalool oxide (pyranoid) ^a	1177				2.43	±	1.4	0.2	±	0.06
Sesquiterpenes
(E)-geranyl acetone	1453				0.27	±	0.23	0.37	±	0.2
Others unknown compounds
m/z: 59, 123	1073				0.51	±	0.37	0.18	±	0.11	0.15	±	0.06
m/z: 58,73	1171				5.06	±	1.93				0.39	±	0.47				0.96	<0.001
m/z: 43,99,117,114	1471										0.46	±	0.25				0.82	0.002
m/z: 99,155,191	1517							0.16	±	0.08	0.23	±	0.25
m/z: 44, 67, 91, 134, 202, 161	1556							0.15	±	0.11							0.71	0.017
m/z: 43, 57, 73, 91, 105, 218, 189	1771							0.17	±	0.08							1.00	<0.001
m/z: 55, 43, 69, 83, 97, 218, 189	1882				0.4		0.38	0.15	±	0.07	0.5	±	0.44
m/z: 69, 43, 138, 109, 218	1899				0.23		0.1	0.16	±	0.14

The VOC profiles of the five species were clearly separated in the NMDS (Fig. 4), as confirmed by the PERMANOVA (F_4,28 = 7.10, P < 0.001). VOCs of each of the five species were significantly different from those of the other four (PERMANOVA, pairwise comparisons, P < 0.05). According to the results of the indicator species analysis, some compounds could be identified as significantly representative of the odor of each species (Table 4). Among these, only two were designated as dominant compounds for some species: 2,4-dimethyl-1-heptene for C. boonjarasii, 9-decenyl acetate and (E)-5-decen-1-yl acetate for C. citrina, and Hexyl hexanoate and hexanal for Cletus trigonus.

Vibratile trichomes of C. boonjarasii play a role in pollinator attraction. The number of flies in flowers of C. boonjarasii with intact vibratile trichomes [5.0 ± 1.8 (mean ± SE), n = 7] was significantly higher than that in the flowers from which trichomes were removed [1.9 ± 0.9 (mean ± SE.), n = 11] (GLM, F_1,17 = 5.910, P = 0.04). A significant effect of individual plants was also detected (F_6,17 = 3.802, P = 0.03).

As for many Ceropegia species^{18,21,23,24,29,30}, pollinators of the four species we investigated are Diptera of two families, Chloropidae and Milichiidae, the latter represented by two genera, Milichiella and Neophyllomyza. As many species of Choropidae and Milichiidae are kleptoparasites, attracted by various types of prey, frequently true bugs, killed by other arthropods, kleptomyiophily has repeatedly been proposed as the pollination strategy of plants pollinated by these families^{19–21, 25}. Most often, only females are kleptoparasitic^23,31, perhaps because they need specific compounds from particular insects for egg maturation. Interestingly, we found almost exclusively female flies in the investigated Ceropegia flowers.

Several of the major VOCs identified in the four Ceropegia species have been detected in the secretions of various bug families: 1-hexanol and hexyl acetate in Coreidae^32,33 and Miridae^25,34−36, (E)-2-decen-1-yl acetate in Pentatomidae³⁷, and many other compounds, including (Z)-3-octen-1-yl acetate, (E)-2-octen-1-yl acetate, octyl butyrate and butyl butyrate, in Miridae^25,34,35. In addition, a species of Milichiella was reported to be attracted to various crushed Coreidae and Pentatomidae³⁶. Certain VOCs have also been reported in other Ceropegia species, e.g., (E)-2-hexenyl acetate in C. sandersonii²⁰, and in other fly-pollinated families, e.g., hexyl acetate, hexyl butyrate, (E)-2-octen-1-yl acetate, octyl butyrate and butyl butyrate in Aristolochia rotunda (Aristolochiaceae)²⁵. Notably, A. rotunda was shown to be kleptomyiophilous, attracting Chloropidae by imitating the scent of a Miridae bug. In our study, we found, for each Ceropegia species, at least one major compound (relative abundance > 5%) similar to that found in the scent of the bug Cletus trigonus (Coreidae), which occurred in the same habitat as the plants. It is likely that Cletus trigonus is a mimicry model of one or several Ceropegia species in Pha Taem. Further investigation should determine whether each Ceropegia species mimics a particular insect species or whether each species mimics a specific part of the bouquet of VOCs from a single bug species.

All these clues argue in favor of kleptomyiophily as the pollination strategy of the four Ceropegia species studied. However, alternative strategies should not be overlooked. Many flowers described as deceptive have been found to provide some rewards, e.g., nectar and nutritive pseudopollen in Orchidaceae^38,39 or shelter in Araceae⁴⁰. Flowers provide flies with physical protection from predators or unfavorable weather conditions^41,42. In the case of C. acicularis, the facts that its very small flowers with a short corolla tube were less efficient in trapping insects than those of the three other species and that its main pollinator visited flowers at the end of the day, suggest that flies might seek the flowers of this species as overnight shelter.

Overall, the new data we produced on the pollination biology of four species of Ceropegia endemic to Pha Taem National Park in Thailand allow us to propose a scenario for guidance—and the associated cues—of pollinating flies to pollinia. Flies are most likely attracted at long distance by floral scent. They were observed to walk or slip into the corolla tube through a window-like aperture, probably guided by the floral scents originating from the epithelial osmophores on the corolla lobes, as shown for various species in the genus^26,29, and/or attracted by vibratile trichomes, as we showed for C. boonjarasii. Similar floral motile appendages are known in various other Asclepiadoideae^18,26,28 and in unrelated angiosperm families^25,28,43 pollinated by Diptera. The function of these structures has long been questioned^17,18. Chemical and visual cues of Ceropegia flowers are likely to act in concert in attracting pollinators.

While detained inside the corolla tube, flies are attracted toward the bottom of the corolla by light windows around the gynostegium, as described for many Ceropegia species^17,19,26,44. When doing so, they detect the nectar cups located on the five sides of the gynostegium below the guide rails, and actively probe them for nectar while moving around the gynostegium. While the fly is feeding on nectar, the soft membrane at the base of the proboscis gets stuck between the guide rails, leading to pollinarium removal. This uniform placement of pollinaria on fly mouthparts has been shown to be a phylogenetically conserved trait in the entire tribe Ceropegieae^{17–19, 26,45}. In addition, nectar maintains detainees alive^39,44. Finally, the flower exploits the positive phototaxis of flies for pollination.

The biology of the four Ceropegia species in Pha Taem shows a high potential for sharing pollinators among species. First, they have largely overlapping flowering phenologies. Second, the four species are found in the same habitat, and individuals are close to each other (see Fig. S1). Third, they seem to share the same pollination strategy and attract flies of only two families (Chloropidae and Milichiidae). Fourth, flowers are receptive all day long, pollinators are all diurnal and their activity patterns, although varying according to species, largely overlap (see Supplementary Information S1). In addition, Meve and Liede-Schumann (2007)⁴⁶ documented that hybridization mediated by small flies was responsible for morphological transitions between the taxa of Ceropegia and those of the closely related Brachystelma (recently merged into the former genus), and one of the authors (M. Kidyoo) found putative hybrids in various Ceropegia populations across Thailand. Thus, natural hybridization in the genus is not rare. Nevertheless, during several consecutive years of field observations, no putative hybrid plants have ever been found in Pha Taem National Park, indicating efficient reproductive isolation among the four species. Whether or not post-zygotic reproductive isolation occurs, we expect pre-zygotic isolation mechanisms to have evolved because of the relatively high value of each pollinarium (only five per flower and few flowers per plant) and the high potential for pollinators to switch from one Ceropegia species to another. Although Ceropegia species in Pha Taem National Park were not all highly specialized (each used two to eight species of pollinators, sometimes of two families), their pollinators were highly specific: most fly species were found in flowers of only one plant species, and those found in flowers of two species had a highly unequal distribution between the plant species.

Pre-zygotic isolation can be achieved through ‘mechanical isolation’, requiring morphological fit between flower and pollinator. It has been suggested that the association between Ceropegia and their pollinators is regulated by a match in fly and flower size¹⁹, as both were correlated in a study based on 60 Ceropegia taxa¹⁸. We did not find such a correlation in Pha Taem National Park, although the four Ceropegia species could be distinguished based on floral morphometrics. In addition, all flies identified as pollinators were small enough to enter any of the four Ceropegia flowers. Furthermore, our enforced crossed-pollinators experiment showed that non-natural pollinators were capable of providing pollination services by efficiently removing pollinaria from the gynostegium. Interestingly, even Drosophila melanogaster which have been reared for several decades for hundreds of generations under laboratory conditions, implying no acquired preferences from previous foraging or oviposition experience, was able to remove pollinaria. In all cases, placement of pollinaria was restricted to the flies’ mouthparts, similar to the placement observed on the specific natural pollinators. Therefore, pollinator specificity is not explained by a morphological fit between flowers and flies.

Pre-zygotic isolation can also be achieved through ‘ethological isolation’, implying manipulation of pollinators via specific sensory cues. Olfactory cues have been suggested to play a key role in attracting pollinators and to underlie the specialized pollination system of Ceropegia^18,19,21. Although most compounds in the floral bouquet of the four Ceropegia species in Pha Taem are of the same chemical class, i.e., aliphatic compounds, mainly esters, mixtures of VOCs significantly differed among species. Distinct floral scents are thus likely to contribute to pollinator specificity, in combination with other cues such as visual cues. Although flowers of the four Ceropegia species are rather similar in basic color appearance and Bauplan¹⁸, there are sharp differences in shape, color pattern and ornaments such as vibratile trichomes, whose role in pollinator attraction was proven here in one species.

We showed that the four synchronopatric species of Ceropegia endemic to Pha Taem National Park have distinct communities of pollinators, validating our initial hypothesis that limited opportunities of pollen transfer combined with a high potential for sharing pollinators, have driven the evolution of pre-zygotic isolation. Selective pressure drove divergent floral traits, resulting in fine-tuned attraction of specific pollinators. Floral traits resulted from local adaptation of the plant community to the fly community. Local pollinator guilds are known to constrain floral adaptation^18,47. What would happen to plant populations in the presence of a distinct fly community? Unpublished field data from the authors indicate that flowers of the four sympatric species of Pha Taem National Park and of several other Ceropegia species each endemic to different National Parks in Thailand (e.g., C. foetidiflora Kidyoo, C. graminea K. Suwann. & Kidyoo, C. suddeei Kidyoo and C. thailandica Meve) grown in the greenhouse in Bangkok, 600 km away from Pha Taem National Park and 600 to 700 km from the native habitats of the other endemic Ceropegia species mentioned above, often shared pollinators. Although some of these plants could set fruits, pollinator specificity was lost. If these plants had been growing in an actual disjunct population, separated from the fly communities to which they are adapted, the cost of pollen loss would have been potentially very high. Concomitantly, several other Ceropegia species have been found to be strict specialists in their native range but are more generalist when grown elsewhere^17,18. Ceropegia species that have natural populations distant from each other are either specialized on a few specific pollinators conserved across geographical locations²³, or are more generalist and less conservative^18,19,22. It would be worth characterizing pollination strategy and Ceropegia and fly communities in various geographical locations to test for the effects of these factors on the evolution of pollinator specificity.

Study site and species. Research was conducted in Pha Taem National Park, Ubon Rachathani Province, eastern Thailand (15°23'56" N, 105°30'27" E, 220 m a.s.l.), where four Ceropegia species, C. acicularis, C. boonjarasii, C. citrina and C. tenuicaulis (Fig. 1A-D), all endemic to the park, occur in sympatry in sandy soil in open areas of dry deciduous dipterocarp forest (see Fig. S1 for more details on study site and species)^48–50. The permissions to collect flowers of Ceropegia species studied were obtained from Department of National Park Wildlife and Plant Conservation. (DNP), Thailand. All the plant experiments were in compliance with relevant institutional, national, and international guidelines and legislation. Voucher specimens of the four Ceropegia species were deposited in BCU (Thailand): C. acicularis Kidyoo: THAILAND. Ubon Ratchathani: Pha Morn, Pha Taem National Park, 240 m, 13 July 2016, M. Kidyoo 1645 (BCU); C. boonjarasii Kidyoo: THAILAND. Ubon Ratchathani: Pha Morn, Pha Team National Park, 240 m, 5 July 2017, M. Kidyoo 1648 (BCU); C. citrina Kidyoo & A. Kidyoo: THAILAND. Ubon Ratchathani: Pha Morn, Pha Taem National Park, 240 m, 2 August 2017, M. Kidyoo 1651 (BCU); C. tenuicaulis Kidyoo: THAILAND. Ubon Ratchathani: Pha Morn, Pha Taem National Park, 240 m, 5 July 2017, M. Kidyoo 1647 (BCU).

Census of floral visitors. A total of 118 samples of Ceropegia flowers were collected in 70% ethanol in the daytime during 2016‒2019. The insects found inside (which were exclusively Diptera) were identified to family or genus based on morphology using the identification keys in Marshall (2012)⁵¹ and the website http://milichiidae.info curated by I. Brake, the world specialist of the Milichiidae.

Specimens were classified into morpho-molecular taxonomic units most likely to correspond to species. Sequences of the cytochrome c oxidase subunit I (COI, 658 bp) were obtained for 41 individual flies (out of 168 collected in total). Specimens were selected to include several individuals of each morphospecies whenever possible. However, amplification failure resulted in four morphospecies not being sequenced (see Fig. S2 for details). DNA was amplified using the primer pairs LCO-1490: 5’-ggtcaacaaatcataaagatattgg-3’ and HCO-2198: 5’-taaacttcagggtgaccaaaaaatca-3’ ⁵². Maximum likelihood phylogeny constructed with PhyML 3.0 online (http://www.atgc-montpellier.fr/phyml/)⁵³ was used as a guide to refine morphospecies delineation. Differential morphological diagnosis was defined for each final morpho-molecular taxonomic unit and used to classify the specimens that were not sequenced.

Each fly was checked for attached pollinaria. Body length and width of the major pollinators were measured. Specimens are kept in the collections of A. Kidyoo at Chulalongkorn University, Bangkok, Thailand, and Rumsaïs Blatrix at CEFE, Montpellier, France. Non-Metric Multidimensional Scaling (NMDS) based on presence-absence of fly species was applied on flower samples, using the Sørensen distance. Differences among plant species were tested using permutational multivariate analysis of variance (PERMANOVA). Pairwise PERMANOVAs were run and p-values were adjusted for multiple comparisons using the FDR method⁵⁴. Indicator Species Analysis⁵⁵ was applied to the fly species (10,000 permutations) in order to identify those specific to each Ceropegia species.

Behavior of floral visitors. Temperature, relative humidity and light intensity were recorded hourly at the study site during 29–31 July 2017 and 14–19 July 2019, using data loggers (iButton DS1923, Maxim Integrated, San Jose, California, USA; HOBO Pendant Temp/Relative Light Two Channel and HOBO U23-001A Temperature/Relative Humidity Data Logger, Onset Computer Corporation, Bourne, Massachusetts, USA). Temporal patterns of flower visitation and behavior of pollinators were recorded with NV-GS75 digital cameras (Panasonic, Matsushita Electric Industrial Co., Ltd., Japan) on the flowers on the first day of anthesis of C. acicularis (n = 10), C. boonjarasii (n = 9) C. citrina (n = 3) and C. tenuicaulis (n = 10) from 06:00 to 18:00 continuously. As visitation of C. boonjarasii flowers did not cease after sunset, additional recording from 18:00 to 06:00 was also made on them. Visitation rate was computed from videos made during the same time period as the recordings of atmospheric data. Only flies that entered the flowers were counted as visits. As morphospecies assignment was not possible from video recordings, fly identity was noted at the family level or occasionally at the genus level when possible.

In addition, behavior of the insects confined inside the basal inflated portion of the flowers of C. acicularis (n = 3, with 3 to 5 hours of observation per flower) and C. tenuicaulis (n = 6, with 1 to 2 hours of observation per flower) was recorded by inserting the tip of a commercial mini camera into the corolla tubes cut transversely just above the basal inflation.

Crossed pollinators experiment. To test whether morphological fit between flowers and flies (mechanical isolation) is involved in pollinator specificity, flies visiting one Ceropegia species were transferred to another Ceropegia species with which they are not naturally associated. Flies were collected from C. boonjarasii and C. tenuicaulis flowers. Those with pollinaria attached to the mouthparts were screened out. In addition to natural pollinators, we used Drosophila melanogaster laboratory strains, which are similar in size to natural pollinators.

Pre-anthesis flowers (recipient flowers) of C. acicularis (n = 7), C. citrina (n = 2) and C. tenuicaulis (n = 3) were bagged (mesh size 0.5 x 0.65 mm) to preclude uncontrolled visitation. At anthesis, the target flies were gently dropped into each flower using a moistened paintbrush, and the neck of the corolla tube was plugged with cotton to prevent insects from escaping and additional pollinators from entering. After the manipulated flowers wilted, they were collected in 70% ethanol. The number of pollinaria removed from the gynostegium and the number of insects carrying pollinaria were counted in the laboratory.

Morphometric analysis of interspecific variation in floral morphology. To evaluate the differences between plant species in quantitative floral morphological traits that may function to restrict access by insects, we measured the following characters on 10 flowers from different plant individuals per species (nine for C. citrina): total flower length, corolla tube length, smallest diameter of corolla tube, basal inflation length and widest diameter of basal inflation. Interspecific variation in floral morphology was tested with multivariate analysis of variance (MANOVA), prior to which the data were standardized, with the five floral characters as dependent variables and plant species as the main factor. The degree of morphological overlap between the plant species was assessed by a principal components analysis (PCA). Moreover, the relationship between the body length and width of insects and the size of different floral characters measured was investigated by a regression analysis with Holm adjusted p-values.

Floral and bug scents. The volatile organic compounds (VOCs) were collected from the flowers on the first day of anthesis (n = 4 to 7 individuals per species) during the time when insect visitation was peak for each Ceropegia species by the dynamic headspace technique^30,56,57. Details about the method of collection and analyses of plant VOCs are presented in Kidyoo et al. (2021)³⁰. The cycle of 30 min.-accumulation plus 5 min.-air pulling was repeated two times (thus three cycles in total) for C. boonjarasii and C. citrina, the floral odours of which were hardly detectable by the human nose. In parallel, control extractions for ambient contaminant compounds were carried out using empty bags. The volatile extracts were subsequently analyzed by GC–MS at the ‘Platform for Chemical Analyses in Ecology’ (PACE, Montpellier) technical facilities of the LabEx CeMEB (‘Centre Méditerranéen pour l’Environnement et la Biodiversité,’ Montpellier, France) following the method of Kidyoo et al. (2021)³⁰ and Souto-Vilarós et al. (2018)⁵⁷.

During field study, we regularly observed Cletus trigonus⁵⁸ bugs (Coreidae) living in the same habitat as Ceropegia plants. This bug species has a very large distribution. When crushed, this bug smells like the flower of C. acicularis to the human nose. As bugs have been reported as possible models of mimetic odors used by flowers to attract kleptoparasitic fly pollinators, individuals of this species were collected for VOCs analysis and comparison with flowers. Each bug individual (n = 5) was enclosed in a PET (polyethylene terephthalate) bag and crushed from the outside to simulate attack by a predator. The odor was left to accumulate for 15 min. Otherwise, samples were processed as for flowers.

Data were processed using MZmine™ version 2.18⁵⁹ adapted to GC data processing (customized software available on demand), using the same automated protocol ensuring the consistency of peak integration. By comparing samples to the controls collected on the same day, potential contaminant compounds were subtracted from the samples. We discarded all the VOCs that had a relative amount < 0.1%. The retention times of a series of n-alkanes (standard solution of alkanes, 04070, Sigma Aldrich®, Munich, Germany) were used for converting retention times to Kovats retention indices. Identity of VOCs was determined by comparing the mass spectra with those in the NIST database (NIST 2007 library; Wiley, 9th edition), and by comparing the calculated retention indices with those reported in the literature⁶⁰. Moreover, when possible, the identity of VOCs was confirmed by comparison with mass spectra and retention times of authentic standards, using the same equipment and method. Non-Metric Multidimensional Scaling (NMDS) based on standardized relative proportions of VOCs was applied on flower samples, using the Bray-Curtis distance. Differences among plant species on their volatile profiles were tested using PERMANOVA (10000 permutations). Pairwise PERMANOVAs were run and p-values were adjusted for multiple comparisons using the FDR method⁵⁴. Indicator Species Analysis⁵⁵ was applied to the VOCs (10,000 permutations) in order to identify those specific to each species.

Role of vibratile trichomes of C. boonjarasii flowers in pollinator attraction. We tested whether the vibratile trichomes that remarkably ornament the distal end of the flowers of C. boonjarasii (Fig. 1B) are involved in pollinator attraction by comparing visitation rates between shaved and intact flowers. From 18 pre-anthesis flowers, 11 had the vibratile trichomes completely pulled out by hand and seven were kept intact for use as controls. Flowers were covered with nylon bags (mesh size 0.5 x 0.65 mm) to prevent uncontrolled visitation. Bags were removed during the next day to allow normal visitation of the flowers. At the end of the day, before the flowers bend down and release the insects trapped inside^17,29 flowers were collected in 70% ethanol for census of flies inside. The effect of experimental shaving was tested using a Generalized Linear Model (GLM) with number of flies trapped in the flowers as the dependent variable and presence of vibratile trichomes and the individual plants as fixed factors.

Statistical analyses were performed with R 4.0.4⁶¹.

Data availability

All DNA sequences obtained during this study are publicly available in NCBI GenBank at http://www.ncbi.nlm.nih.gov/genbank/, under the accession number OK138486-OK138526. All data generated or analysed during this study are included in this published article and its Supplementary Information files.

Acknowledgements

We thank Dr. Obchant Thaithong for her continued support and Mr. Phataravee Prommanut for valuable help during our fieldwork. We also thank Nicolas Barthes and Bruno Buatois for help during the chemical analysis conducted at the ‘Platform for Chemical Analyses in Ecology’ (PACE), platform of the LabEx CeMEB (Centre Méditerranéen Environnement et Biodiversité, Montpellier), an ANR ‘Investissements d’avenir’ program (ANR-10-LABX-04-01). The authors declare that there is no conflict of interest. This work was supported by ‘Franco-Thai Mobility Programme (PHC SIAM, France) 2018–2019’ [(Project No. 40533RA], by the programme ‘International Emerging Actions’ of the French National Centre for Scientific Research, France [Project FOOLFLY 2020-2021] and by ‘Grant for Research, Rachadaphiseksomphot Endowment Fund 2020 [CU-GR_63_62_23_06] of Chulalongkorn University.’

Author contributions

A.K., D.M., M.K and R.B. conceived the ideas and designed methodology; A.K., D.M., M.K, N.U., P.W. and R.B. conducted fieldwork; M.K. identified plant species; A.K., M.K, M.P., N.U., P.W. and R.B. collected the data; A.K., G.D., M.P., P.E. and R.B. analyzed the data; A.K., D.M., M.P., M.K. and R.B. wrote the manuscript. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Pollinator and Floral Odor Specificity Among Four Synchronopatric Species of Ceropegia (Apocynaceae) Suggests Ethological Isolation That Prevents Reproductive Interference

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