Summary: Mutualism between long corolla tubed plants and their potential pollinators, long‐tongued flies, is a classic example of coevolution, but to date, has only been reported from the regions of southern Africa. Many plant species from theHimalayas also show botanical characteristics that could be consistent with pollination by long‐tongued flies. Here, we seek the evidence of the “long‐tongued‐long tubed fly/flower” mutualism out of Africa, in a different continent and climatic region, the Himalayas. Floral traits ofHimalayan region endemic alpine genus,Roscoea, indicate possible mutualism with long‐tongued flies for pollination success; however, effective pollinators of this genus are yet unknown. This study investigates whether long‐tongued flies andRoscoea purpureain NepalHimalayas show exclusive mutualism for their survival/reproduction. We made extensive observations of floral visitors ofR. purpureaand food source ofPhiloliche longirostrisacross their wide ranges of populations in Nepal Himalayas for three consecutive years (2012–2014). To confirm the obligate reliance ofR. purpureauponP. longirostrisfor pollination success, manipulated pollination experiments were conducted at two populations for 2 years. Similarly foraging behavior, visitation frequency, and pollination efficiency ofP. longirostriswere assessed at two populations for 2 years, and its contribution for the reproductive success ofR. purpureawas evaluated. Our results indicate thatR. purpureais self‐compatible but lacks autonomous selfing and obligatorily relies onP. longirostrisfor reproductive success. Across all populations,P. longirostriswas observed as an exclusive and highly efficient pollinator ofR. purpurea,whileP. longirostrisexclusively depends up onR. purpureafor food source. Out of Africa, this study provides the first evidence of long‐tongued fly pollination system and indicates the possibility of additional instances of such a rare phenomenon in theHimalayas. Finding of specialized pollinator ofRoscoeaonly at its evolutionary center indicates thatRoscoeaspecies are originally pollinated by long‐tongued flies. Spatial mismatch with specialized pollinators may have induced the evolution of autonomous selfing in North Indochinese clades ofRoscoea. This finding thus substantiates how geographic disjunction causes the shifting of pollination mechanism in closely related plant species.

Multiple barriers may contribute to reproductive isolation between closely related species. Understanding the relative strength of these barriers can illuminate the ecological factors that currently maintain species integrity and how these factors originally promoted speciation. Two Himalayan alpine gingers, Roscoea purpurea and R. tumjensis, occur sympatrically in central Nepal and have such similar morphology that it is not clear whether or how they maintain a distinct identity. Our quantitative measurements of the components of reproductive isolation show that they are, in fact, completely isolated by a combination of phenological displacement of flowering, earlier for R. tumjensis and later for R. purpurea, and complete fidelity of visitation by different pollinator species, bumblebees for R. tumjensis and a long‐tongued fly for R. purpurea. Furthermore, the nectar of R. tumjensis flowers is available to the shorter‐tongued bumblebees while R. purpurea nectar is less accessible, requiring deep probing from long‐tongued flies. Although flowering phenology is a strong current barrier that seemingly obviates any need for pollinator discrimination, this current pattern need not reflect selective forces occurring at the initial divergence of R. tumjensis. There has been considerable pollinator switching during the radiation of the Himalayan Roscoea, and the association of flowering time with type of pollinator in these sympatric species may have originated among the earliest or latest flowering individuals or populations of an ancestor in order to exploit either bumblebee activity early in the breeding season or long‐tongued fly abundance later in the season. These two sympatric Roscoea species add to accumulating evidence of the primacy of pre‐zygotic pollination traits in speciation among angiosperms even in the absence of post‐zygotic incompatibility. This article is protected by copyright. All rights reserved

The pollination syndromes have been widely used to predict effective pollinators of plant species and provide the mechanistic explanation of floral diversity. However, in recent years, the predictive applicability of pollination syndromes has been frequently questioned. The accuracy of the syndromes among the closely related plant species may vary temporally and spatially. This suggests the need for the standardized, comprehensive evaluation of factors influencing the matching of a pollination syndrome of a plant species to the predominant pollinator community. We studied the pollination biology of three geographically/phenologically isolated alpine gingers (Roscoea auriculata,Roscoea capitata, andRoscoea tumjensis) exhibiting the correlated suites of floral traits that suggest long‐tongued insects as major pollinators, to test for the predictive power of the respective pollination syndrome. We also tested if geographical and temporal isolation affects interspecies breeding system and extent of pollinator generalization. We demonstrate that the threeRoscoeaspecies are self‐compatible but lack autonomous selfing, and thus completely rely on pollinators for pollination success. Five years of observations demonstrate that only diurnal insect visitors foraged on the flowers of the threeRoscoeaspecies. Estimates of the pollinator importance (pollen deposition) of the observed floral visitors indicate that morphologically mismatched bumblebees contribute more than 90% of pollination service in all the threeRoscoeaspecies, while long‐tongued butterflies and moths function as nectar robbers. We found that geographical isolation and temporal variation in flowering period does not affect the breeding system and pollinator generalization in the threeRoscoeaspecies. The three geographically and phenologically isolated HimalayanRoscoeaspp., with long‐tongued insect pollination syndrome, exhibit generalized pollination system and primarily rely on the morphologically mismatchedBombusspecies for pollination success. While, for threeRoscoeaspp., our result suggests that pollination syndromes are a weak predictor of efficient contemporary pollinator community, it also substantiates the role of non‐pollinator agents for the evolution of floral traits. The evolution ofBombuspollination in these alpine gingers suggests how the mismatch between plant and pollinator phenology can lead to the emergence of novel reproductive strategies among the congeners, which ultimately seems to play a key role for the speciation and diversification of the genusRoscoeain the Himalayas.

Environmental stress at high altitudes drives the development of distinct adaptive mechanisms in plants. However, studies exploring the genetic adaptive mechanisms of high-altitude plant species are scarce. In the present study, we explored the high-altitude adaptive mechanisms of plants in the Himalayas through whole-genome resequencing. We studied two widespread members of the Himalayan endemic alpine genus Roscoea (Zingiberaceae): R. alpina (a selfing species) and R. purpurea (an outcrossing species). These species are distributed widely in the Himalayas with distinct non-overlapping altitude distributions; R. alpina is distributed at higher elevations, and R. purpurea occurs at lower elevations. Compared to R. purpurea, R. alpina exhibited higher levels of linkage disequilibrium, Tajima’s D, and inbreeding coefficient, as well as lower recombination rates and genetic diversity. Approximately 96.3% of the genes in the reference genome underwent significant genetic divergence (F_ST ≥ 0.25). We reported 58 completely divergent genes (F_ST = 1), of which only 17 genes were annotated with specific functions. The functions of these genes were primarily related to adapting to the specific characteristics of high-altitude environments. Our findings provide novel insights into how evolutionary innovations promote the adaptation of mountain alpine species to high altitudes and harsh habitats.

The Himalayan endemic alpine genus Roscoea, like other members of ginger family, exhibits the combination of floral traits that would fit pollination by long distant foragers such as bees, birds or flies. We studied the pollination biology of Roscoea alpina, observed potential floral visitors and determined their foraging behaviour, visitation frequency and pollination efficiency, to seek evidence in support of the pollination syndrome hypothesis. We also measured the floral spectra of R. alpina flowers to evaluate if signals fit with the currently known framework for observed floral visitors. We found that R. alpina have autonomous selfing and pollinator-mediated crossing, but lack apomixis. We observed that a beetle (Mylabris sp.), and a moth (Macroglossum nycteris) visit the flowers of R. alpina for pollen and nectar feeding respectively. Our field observations, the stigmatic pollen count and fruit set data indicated that the visit by the beetle was legitimate, while that of the moth was illegitimate. Emasculated flowers visited by beetles set as many fruits and seeds/fruit as auto-selfed and naturally pollinated flowers, while emasculated flowers excluded from beetle visits did not set fruit and seed; indicating that a single visit of a beetle to the flowers of R. alpina can facilitate pollination. We found that flower spectral signal of R. alpina does not fit typical spectra previously reported for beetle or bee-visited flowers. Our results suggest that, to ensure reproductive success in alpine habitat, R. alpina has evolved autonomous selfing as a predominant mode of reproduction, while beetle pollination would promote genetic diversity of this plant species. The visitation of beetles to the flowers of R. alpina, despite floral signal mismatch with the classically associated beetle vision, suggests that a different visual processing may operate in this plant-pollinator interaction at high altitudes.