Plant species
Lamium album L. var. barbatum (Lamiaceae) is a perennial herb that grows along forest edges throughout East Asia [47]. It produces creamy white, two-lipped, entomophilous, and self-incompatible flowers [40, 41]. The flowers are frequently visited by various bumblebee species, and in Japan, the corolla tube length of the flowers may correlate with the size (proboscis length and head length) of their bumblebee pollinators. Flower–pollinator morphological matching has been reported to improve seed set in a population of L. album var. barbatum located near the populations of this study [42]. A bumblebee visiting a flower of L. album var. barbatum inserts its tongue into the inner part of the corolla tube to forage for nectar and in the process rubs its head and thorax against the anthers and the stigma. In addition to bumblebees, honeybees and wild bees have been observed to visit L. album var. barbatum flowers, but in Japan bumblebees are their main pollinators [40].
Study site
Populations of L. album var. barbatum were surveyed at 12 sites in two mountain areas in Matusmoto, Nagano Prefecture, the central Japan Alps. All surveys were conducted between April and July, during the flowering season of each population, in 2018 or 2019. The two mountain areas were around Mt. Norikura, west of the Matsumoto basin (the "west area"), and around the Utsukushigahara highland, which is east of the basin (the "east area") (Fig. 3). Each population of L. album var. barbatum was a geographically cohesive group of densely distributed plants located along a forest road in deciduous broad‐leaved forest. The distance between the populations ranged from 0.4 to 52.4 km.
Corolla tube length measurement
First, 18–170 individuals were haphazardly selected from each population. Then, following the method of Hattori et al. (2015) [40], we measured the corolla tube length of 1–6 flowers per individual plant with a digital caliper (precision, 0.01 mm). The corolla tube length was defined as the distance from the flower's base at the stem to its tip (Fig. 4). Preliminary measurements showed that the variation of corolla tube length among flowers on an individual plant was less than the variation among plants. Therefore, we used the average value of the measured corolla tube lengths of 1–6 flowers on an individual plant as the corolla tube length of that plant. We also measured plant height, as a proxy for plant size, of 20 haphazardly selected individuals in each population. Average corolla tube lengths were compared between populations by using Tukey's honestly significant difference (HSD) test. In addition, we used the Moran's I test for spatial autocorrelation to determine to what degree correlations could be explained by the sampling of populations in close proximity to one another. For this test, we used the moran.test function in the "spdep" package in the R software environment ver. 4.0.2 [48].
Pollinator assemblages and size variation
To observe the pollinator assemblages of L. album var. barbatum, we selected the largest patch of plants (ranging in area from about 10 to 200 m2) in each of the 12 populations and haphazardly established a 1 m × 1 m quadrat (about 100 individuals) within the patch on each census day (Table 1). We then recorded the insects that visited the flowers in this quadrat. Observations were made on several days between 8:00–14:00 local time, when flower visitors were active in each population. At each location, we observed all flower visitors for a total of 90–660 minutes spread over 1–4 days during the peak flowering period. Since bumblebee species (Bombus spp.) can be easily distinguished while they are visiting a flower, the species of each bumblebee was recorded as they visited a flower, and the observed species were regarded as the bumblebee pollinator assemblage. In contrast, it is difficult to distinguish among Eucera spp. and species of small bees during their flower visits, so we estimated the species-level pollinator assemblage of these taxa from capture survey results (see below).
To define the size of each pollinator species, we measured morphological traits of each species relevant to the pollinating behavior of that species. For this survey, flower-visiting insects were haphazardly captured following their flower visitation, and the size of each of the selected traits was measured with a digital caliper (precision, 0.01 mm). We collected visiting insects following their flower visitation. Bombus spp., Eucera spp., and Apis cerana japonica (hereafter, "large bees") are "thrust pollinators"; they forage for nectar by thrusting their heads into flowers and extending their tongues. Thus, we defined the pollinator size of large bees as the sum of the tongue length and the head length. (Fig. 4). In contrast, Ceratia spp., Lasioglossum spp., and Andrena spp. (hereafter, "small bees") are "whole-body pollinators"; they forage for nectar by crawling into the corolla tube. The small bees first land at the entrance to the flowers (upper or lower lip), and then crawl into the flowers to forage, moving through the anthers and stigma to the nectary. As a result, pollen grains become attached to both the head and the ventral side of the abdomen of small bees; thus, we defined the pollinator size of small bees as the body length from the tip of its tongue to the caudal end of the abdomen (Fig. 4). Nectar robbers (Apis mellifera, Bombus hypocrita, Xylocopa appendiculata circumvolans) and small bees on which we did not observed attached pollen grains (Euodynerus nipanicus, Lasioglossum nipponense, L. occidens, Nomada comparata at Onosawa, Nomada spp. at Onosawa) were excluded from this calculation of average pollinator size. We checked for attached pollen grains soon after a bee's visit to a flower and identified L. album var. barbatum pollen grains under a microscope (× 2–10). Pollinator size was measured separately for each plant population, even for insects of the same species. Although B. diversus workers were observed in the quadrat surveys at Onosawa and Norikura, and B. honshuensis workers at Ohmizusawa, they were not captured and their sizes in those populations were not measured. Therefore, the mean size of all B. diversus (B. honshuensis) individuals captured from the other populations was used as the size of B. diversus at Onosawa and Norikura (B. honshuensis at Ohmizusawa).
As the average pollinator size for each plant population, the weighted arithmetic mean was calculated from the relative abundance of each pollinator species in the pollinator assemblage and the size of that species:
where n = the total number of insect species visiting a L. album var. barbatum population (patch), Pi = mean size of the ith insect species, Ni = the number of flowers in the patch that the ith insect species visited, and Nt = the number of flowers in the patch that any of the insect species visited. Thus, Ni/Nt is the relative abundance of the ith insect species visiting the population. For each population, average pollinator size was calculated for three groups of flower visitors: all flower visitors, only large bees, and only small bees.
Factors influencing local corolla tube length
To examine factors influencing corolla tube length, we used a generalized linear model (GLM) with a Gaussian error distribution and identity as the link function. In this analysis, average corolla tube length of each population was the objective variable, and the average pollinator size (all pollinators), average pollinator size (only large bees), plant height, and the altitude of each population were predictive variables. The calculated average pollinator size of only small bees differed little among populations, and we judged their size variation to be not meaningful because of their crawling style of flower visitation. Therefore, average pollinator size (only small bees) was excluded from the GLM analysis. Plant height (mean of 20 individuals in each population) was included as an indicator of plant size, and altitude was included as a proxy for clinal abiotic environmental changes (e.g., meteorological changes). The Shimashima I population was excluded from the GLM for the average pollinator size of only large bees, because only small bees visited flowers of that population. The GLM analysis was performed with the glm function in the R software environment ver. 4.0.2 [48]. First, we performed model selection on the entire dataset, starting from a global model including corolla tube length, small bee size, large bee size, and plant height. We then compared the global model with all simpler models using the dredge function in the "MuMIn" package in the R software environment ver. 4.0.2 [48]. This function returned the model with the lowest Akaike information criterion (AIC), and we adopted this model (Additional file 2: Table S2). The results of this model selection procedure informed which average pollinator size variable (all pollinators or only large bees) was used in a least-squares regression analysis. Using these results, therefore, we explored covariation between corolla tube length and the average pollinator size of only large bees across populations by a least-squares regression analysis.
Genetic similarities of Lamium album var. barbatum populations
To examine the genetic structure of L. album var. barbatum, we used 10 polymorphic microsatellite primers originally developed for L. album [49] (Additional file 3: Table S3). For this analysis, fresh leaf material was collected randomly from 8–16 individual plants in each of the 12 L. album var. barbatum populations during 2018–2019. DNA was extracted by the CTAB method [50], and the extracted DNA was diluted or concentrated to a final concentration of 10 μg/ml.
Each of the forward microsatellite primers was synthesized after adding one of four different universal fluorescent sequences: 5’-GCCTCCCTCGCGCCA-3’, 5’-GCCTTGCCAGCCCGC-3’, 5’-CAGGACCAGGCTACCGTG-3’, or 5’-CGGAGAGCCGAGAGGTG-3’ [51]. Polymerase chain reaction (PCR) analyses were performed in a thermal cycler using a reaction mixture consisting of 1 μl template DNA, 3 μl of 2 × Type-it Microsatellite PCR Kit (QIAGEN, Valencia, California, USA), 0.7 μl of 0.1 μM forward primer, 0.7 μl of 0.2 μM reverse primer, and 0.7 μl of 0.1 μM fluorescent-labeled universal primer. The DNA amplification program consisted of an initial denaturation step of 5 min at 95 °C, followed by 35 cycles at 95 °C for 30 s, 60 °C for 90 s, and 72 °C for 30 s, and final elongation at 60 °C for 30 min. The PCR products were detected by using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA) and GeneScan™ 500 LIZ™ dye Size Standard (Applied Biosystems). Fragment lengths were calculated with GeneMapper version 4.0 software (Applied Biosystems).
A cluster analysis of the fragment length datasets was performed with STRUCTURE software version 2.3.4 [52, 53]. Simulations were conducted with 100 k burn-in iterations and 100 k Markov chain Monte Carlo repetitions. The number of genetic clusters (K) was calculated 10 times for each of 1–12, and the ΔK value [54] was used as the criterion for selecting the appropriate number of clusters, that is, the number of genetic clusters from which the 12 populations of L. album var. barbatum were derived.
In addition, we tested analysis of molecular variance (AMOVA) models estimating the percentage of molecular variance accounted for by each level of the nested sampling hierarchy, in which the 12 populations were grouped according to the mountain area (east or west areas). AMOVA was run using Arlequin ver 3.5.2.2 [55]. The significance of variance components in the AMOVA models was tested by 1000 random permutations.