Botany School, Downing Street, Cambridge CB2 3EA, U.K. {Accepted 2 November 1981) SUMMARY

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1 New Phytol. (), - COMPETITION FOR POLLINATORS IN THE ECOLOGY OF CENTAUREA SCABIOSA L. AND CENTAUREA NIGRA L. IIL INSECT VISITS AND THE NUMBER OF SUCCESSFUL POLLINATIONS BY ANDREW J. LACK* Botany School, Downing Street, Cambridge CB EA, U.K. {Accepted November ) SUMMARY Observations are presented on: (a) the numbers of insects foraging on Centaurea scabiosa and Centaurea nigra per available capitulum of each species; (b) the numbers of insect visits to each capitulum of C. scabiosa and C. nigra in min intervals; (c) the numbers of insect visits to rayed capitula compared with rayless capitula; (d) the number of successful pollinations. The study was carried out on the Devil's Ditch, Cambridgeshire where C. nigra is rayless, and on Hod Hill, Dorset where C. nigra is rayed, with a few results from a garden trial set out in the Cambridge Botanic Garden. In the counts of insects in each area of the Devil's Ditch the numbers recorded feeding on each Centaurea species per capitulum were approximately proportional to the nectar reward of each species. On Hod Hill the number of insect visits to C. nigra plants from which rays had been removed was half to three-quarters of the number of visits to rayed capitula. This is very similar to the difference on the Devil's Ditch between the number of visits to C. scabiosa (rayed) and C. nigra (rayless), and this difference may therefore be a result simply of the possession of ray florets in C. scabiosa but not in C. nigra. The difference between the two species on Hod Hill was greater (C. scabiosa receiving two to three times as many visits as C. nigra) although C. nigra possesses ray florets and therefore resembles C. scabiosa. Some bees were observed visiting C. scabiosa selectively on Hod Hill but none did so on the Devil's Ditch. On the Devil's Ditch both species had similar numbers of styles successfully fertilized on nearly all dates. Hod Hill on most dates a similar number of styles was fertilized in each species, but on a few dates C. scabiosa had more successful fertilizations than C. nigra. It is concluded that competition for pollinators is not a strong selective factor in Centaurea on the Devil's Ditch but may be a factor promoting the later flowering and the possession of ray florets in C. nigra on Hod Hill. Competition for pollinators may have been more important as a selection pressure in the past when less suitable habitat was available. INTRODUCTION The results presented in papers I and II (Lack, a, b) suggest that Centaurea scabiosa and Centaurea nigra may be competing for the visits of pollinating insects in some parts of their range where they grow in mixed populations. This paper reports investigations on the relative number of visits of pollinating insects to the two species, and the results of the visits in the numbers of styles successfully pollinated. If competition for pollinators between C. scabiosa and C. nigra is important as a selection pressure influencing C. nigra then those capitula of C. nigra * Present address: Department of Botany and Microbiology, University College, Swansea SA PP, U.K. -X//OO + S./ ^ The New Phytologist

2 A. J. LACK produced at the time of maximum compeition with C. scabiosa (likely to be around its peak flowering) must set less seed than those produced at other times in the flowering season. Those plants which flower at other times will then be favoured. There are clearly many factors which will affect seed set other than the number of successful pollinations, but comparative observations on the numbers of styles pollinated in both species at different stages in the flowering season will give an indication whether pollination is one of the limiting factors. The numbers of insects which were seen feeding on the two species through the flowering seasons of, and are presented, with the number of visits which each capitulum received. This was carried out in the two main study areas, the Devil's Ditch, Cambridgeshire and Hod Hill, Dorset (for details of sites see paper I, Lack, a). Assessment ofthe number of successful pollinations in the two species was made at both sites at several different times through the flowering season. Except for occasional individuals both species are said to be self-incompatible (Marsden-Jones and Turrill, ; Lack, ) and in Compositae incompatibility is sporophytic. In this system a plant's own pollen will normally not germinate on its own stigma (or one of similar genotype) or, if it does germinate, the pollen tube is blocked by a callose plug as it enters the stigma (Heslop-Harrison, ; Nettancourt, ). If a pollen grain can be seen to have germinated and entered the stigma without a callose plug to block it we can assume that the floret has been successfully pollinated by pollen from a different genotype. The two study sites have different forms of C. nigra. On Hod Hill and throughout much of the calcareous grassland in central and western southern England, it occurs as ssp. nemoralis and possesses sterile ray florets, and on the Devil's Ditch, and through most of eastern England, the plants are intermediate in morphology between ssp. nigra and ssp. nemoralis and do not possess ray florets (see Marsden-Jones and Turrill, ; Lack, ). The possession of ray florets makes the capitula larger (about cm across compared with - to cm for rayless capitula), and perhaps more attractive to insects. It also causes the capitula of C. nigra to resemble those of C scabiosa and I have suggested earlier (Lack, ) that rayed C. nigra may be 'mimicking' C. scabiosa. To investigate the importance of the ray florets for the insects, on Hod Hill the number of insect visits to untouched rayed capitula was compared with the number of capitula from which the rays had been removed. For the purposes of this paper the records of visits for all bumblebee species {Bombus spp. and Psithyrus spp.) have been combined (referred to hereafter as Bombinae) since all the common species except Bombus hortorum (L.) and B. ruderatus (Fabr,) were feeding mainly on Centaurea species from mid-july onwards (Lack,, and in preparation). B. hortorum and B. ruderatus were rare on the Devil's Ditch by late July and not recorded after this. They were not recorded in the observations on Hod Hill. Other insects recorded visiting the two Centaurea species were less numerous than the Bombinae, and probably much less important as pollinators of these species since, in general, they flew less from plant to plant and carried much less pollen. Results for all insects except Bombinae from the Devil's Ditch have been combined, including Apis mellifera L. which occurred in very small numbers after mid-july. On Hod Hill A. mellifera was common, potentially important as a pollinator, and is treated separately.

3 Pollination in Centaurea. ///. Insects METHODS Counts of insects on the Devil's Ditch Counts of insects were made from July to September between. and. h (B.S.T.). All Hymenoptera, Lepidoptera and Syrphidae (Diptera) which were feeding on the fiowers of Centaurea and other major bee-pollinated species (such as Onobrychis viciifolia, Scabiosa columbaria, Chamaenerion angustifolium and Rubus fruticosus agg.) were recorded, with the plant species on which each was feeding. This study was carried out on all three areas of the Devil's Ditch. Before August, when there were rather few insects on the Devil's Ditch, records were made over larger areas on the exposed southwest aspect and northeast aspect (including the main areas). The counts after August took between and min (depending on the number of insects), and before August a few counts on the larger areas took up to min. The number of available capitula of C. scabiosa and C. nigra (all open capitula not yet discoloured) in each study area was counted each week (see paper I, Lack, a). Counts of insects were made in the same way from July to September in the sheltered soutwest aspect area but no records were kept of the number of available capitula. In insects were counted in the same way on four dates in August and the capitula were counted each week, as in. Smaller numbers of insects (particularly of Lepidoptera and Syrphidae) were recorded in strong wind or rain but nearly all counts were made under good weather conditions. Numbers of insect visits to each individual capitulum The number of insect visits which each capitulum received was counted by observing individual capitula of both Centaurea species over min periods. Usually capitula of each species were observed simultaneously. Observations were made in each study area of the Devil's Ditch and on Hod Hill on several dates through and. Experimental comparison of rayed and rayless capitula The significance of the ray florets, which occur in C nigra on Hod Hill, was investigated by removing the ray florets from some C. nigra capitula and counting insect visits to these and, at the same time, to an adjacent group of untouched C. nigra capitula. Insect visits were counted over min on two groups of capitula. As a control measure for most comparisons, observations were made first at the two groups before any rays had been removed. Only if there was no significant difference between the two groups of capitula in this first observation was the second observation made. The tables present only the second observation for each group. A few observations were made in a similar way on C scabiosa (normally rayed throughout its range). Number of successful pollinations In plants of both species, grown in pots, were placed in an insect-proof greenhouse and artificially selfed and crossed. The styles were collected h later ( August) and h later ( August) for examination. Styles were collected in the field from the Devil's Ditch and Hod Hill on several dates in and. On each date each style was collected from a different plant and these included some from small and somewhat hidden capitula. On the Devil's

4 A. J. LACK Ditch the three study areas are treated separately. On Hod Hill a fairly exposed area near the top of the hill ('exposed') is separated from a more sheltered area on the southern side ('sheltered'). All styles were fixed initially in formalin-acetic acidalcohol (::) for storage. They were then softened in saturated sodium hydroxide for approximately h and stained with aniline blue for at least min. They were then squashed on a microscope slide under a coverslip and observed, normally at times magnification, using incident ultraviolet light (mercury vapour lamp with filters) which makes the aniline blue fluoresce (technique after Martin, ; Dr D. J. Ockendon pers. comm.). Aniline blue stains callose clearly and it is possible to count the number of pollen tubes which have grown down the styles. RESULTS Counts of insects on the DeviVs Ditch The numbers of capitula of C. scabiosa and C. nigra available through in the three study areas are presented in Figure and a summary of the insect visits in Figure. Figures and give this information for with insect visits on the sheltered southwest aspect in. Table gives the total numbers of insects observed. In Figures and all results are given as percentages of the total number of visits recorded on each aspect on each date. When the total number of visits was less than four the percentages are not shown (for a few dates results from adjacent or near-adjacent dates with less than four visits of a particular insect group could be combined; results from and September have been combined with those from September throughout).. At nearly all times there were many more bumblebees visiting Centaurea species than all other insects combined. Of the bumblebees, B. lapidarius (L.) was the most abundant visitor and smaller numbers of B. terrestris (L.), B. lucorum (L.) and B. pascuorum (Scopoli) and Psithyrus spp. were recorded. Several species of Syrphidae (particularly Eristalis species) and some Lepidoptera (particularly Pieris spp., Pyronia tithonus L.) were the commonest other insects recorded (see Lack, ). The three areas showed a similar pattern, the two Centaurea species receiving the majority of visits of all insects after the beginning of August. More insects were recorded visiting C. scabiosa than C nigra at all times when there were more C scabiosa capitula available than C. nigra, and on the exposed southwest aspect and the northeast aspect more were visiting C. scabiosa even at the flowering peak of C. nigra. On the sheltered southwest aspect more insects visited C. nigra by the end of August but there were many more capitula of C. nigra available than C. scabiosa (Fig. ). Compared with the Bombinae other insects visited more flowers of species other than Centaurea (e.g. Scabiosa columbaria), particularly on the sheltered southwest aspect. The number of insects per capitulum of each Centaurea species was calculated for each count, and in each area these varied as much between counts within day (mainly smaller numbers in the morning and evening) as between dates. A mean with % confidence limits was calculated for each area of all counts between August and September (Table ), the narrow confidence limits indicating the narrow range of variation through the season. The proportion of bumblebees visiting the two species differed on the different aspects (C. scabiosa had times as many bees as C. nigra on the sheltered southwest aspect, only - times as many on the exposed southwest aspect and * times as many on the northeast aspect).

5 Pollination in Centaurea. ///. Insects (a) r \ \ \ \ \ \ - ^ N IOO I Lr Q. o (b) - / \ \ \ \ \ IOO / /'' L-^ - - II July August Sept. Fig.. Nunaber of available capitula of Centaurea scabiosa ( ) and C. nigra { ); Devil's Ditch,, (a) sheltered southwest aspect, (b) exposed southwest aspect, (c) northeast aspect. Other insects also visited C. scabiosa more than C. nigra but the difference was less marked than for the bumblebees. and. In C. nigra received a greater proportion of visits in all three areas than in. The number of bees visiting both species was very high earlier in the season than in, and the mean number of bees per capitula is given in Table. There were approximately twice as many bees per capitulum of Centaurea spp. in as in, even though there were slightly more Centaurea capitula. The difference between C. scabiosa and C. nigra in numbers of bees per capitulum was less on all aspects in {e.g. - times as many on C. scabiosa as C. nigra on the sheltered southwest aspect). There were similar numbers of insects other than Bombinae per capitulum in and but, as with Bombinae, C. nigra received a greater share ofthe visits in, and on the exposed southwest aspect there were more visiting C. nigra per capitulum than visiting C. scabiosa. In the pattern was similar to that shown in the other years, though the

6 A. J. LACK Table. Total numbers of insects recorded visiting fiowers in the three study areas Devil's Ditch Bombinae Other insects Sheltered SW aspect Exposed SW aspect Sheltered SE aspect Exposed SW aspect July July July July Aug. Aug. Aug. Aug. Aug. Aug. Sept. Sept. Sept. Sept. Sept. Sept. Sept. Aug. Aug. Aug. Aug. Sheltered SW aspect only Bombinae Other insects Bombinae Other insects July July July July Aug. Aug. Sept. Sept. Sept. Sept. numbers of Bombinae on C. scabiosa increased earlier than in and C. nigra received a greater proportion of the visits during September than in. Number of insect visits to each capitulum Tables and show the numbers of insect visits to each capitulum in each -min count, and the mean for each aspect of the Devil's Ditch and for Hod Hill for each year. In nearly all counts from the Devil's Ditch C. scabiosa received more visits by Bombinae than C nigra, and in, on average, received just under twice as many visits on the two southwest aspects and - times as many on the northeast aspect where more visits overall were recorded. The numbers did not vary very much through the season. The other insects visited in much smaller numbers than the Bombinae and visited the two species more or less equally. In there were overall many more visits by bees than in (confirming the results from the

7 Pollination in Centaurea. ///. Insects Bombinae Other insects I i July August Sept. July August Sept. Fig.. Percentage of the total number of visits by Bombinae and other insects to Centaurea scabiosa (El), C. nigra (U) and other species (Q : particularly Onobrychis viciifolia and Rubus fruticosus agg. in July; Scabiosa columbaria, Knautia arvensis and Chamaenerion angustifolium in August and September). Devil's Ditch,, (a) sheltered southwest aspect, (b) exposed southwest aspect, (c) northeast aspect. insect counts) and the difference between the two Centaurea species was less than in, with - times as many visits to C. scabiosa as to C. nigra on the two southwest aspects and - times as many on the northeast aspect (- times as many discounting the anomalous result on August; this 'corrected' result shown in brackets). Other insects showed similar patterns in the years.

8 A. J. LACK Table. Number of insects per capitula of Centaurea scabiosa and C. nigra: means and % confidence limits of all results from between August and September, and all results from, DeviVs Ditch Species Bombinae Others Sheltered SW aspect Exposed SW aspect Centaurea scabiosa C. nigra C. scabiosa C. nigra C. scabiosa C. nigra - ±- l-±o ± ±- - ± ±- - ±- -±- Sheltered SW aspect Exposed SW aspect C. scabiosa C. nigra C. scabiosa C. nigra C. scabiosa C. nigra - ±- - ± ± l-±- - ±- - ±- - ±- - ±- On Hod Hill the Bombinae were much less numerous than on the Devil's Ditch, though the numbers increased in the second half of the season. B. terrestris, B. lucorum, B. lapidarius and B. pascuorum were the only species recorded, with B. pascuorum the most abundant species during September (fairly even numbers before this). They visited C. scabiosa more often than C. nigra on most dates. On average C. scabiosa received - times as many visits by Bombinae in. Apis mellifera was common on Hod Hill and varied considerably in numbers on the different days, probably mainly as a result of the weather. They visited C. scabiosa more often than C. nigra at all times when many were visiting the fiowers, and on average in C. scabiosa received * times as many visits as C. nigra. Other insects were not common visitors except during September when Eristalis species were numerous and visited mainly C. scabiosa. The results from were very similar to though there were slightly fewer Apis mellifera and more Bombinae. Comparison of rayed and rayless capitula Table shows the comparisons of rayed and rayless capitula from Hod Hill. On to August, when honeybees were numerous, they visited rayless capitula about half as much as rayed, and the same behaviour was shown by bumblebees when they were numerous on September. At lower densities (on the other dates) there was little difference between rayed and rayless C. nigra for either insect group. Overall the difference was highly significant statistically for both insect groups when analysed by two-way analysis of variance (Bombinae F = -, d.f. =,, P < -; Apis F = -, d.f. =,, P <^ -). The honeybees visited rayless C. scabiosa less than rayed on August but bumblebees showed no difference on either date on which comparison was studied. All other insects on these plants were not sufficiently numerous for definite conclusions to be made (two-way analysis of variance for C. nigra visits, F =, d.f. =, ).

9 Pollination in Centaurea. ///. Insects - (c) / \ _ / / \^ / / \^ / / V / V / ' Ni ^ r j.-f ^ July August Sept. Fig.. Number of available capitula of Centaurea scabiosa ( ) and C. nigra { ); Devil's Ditch,, (a) sheltered southwest aspect, (b) exposed southwest aspect, (c) northeast aspect. Number of successful pollinations Results from the artificial pollinations are shown in Table. This shows that on both dates pollen tubes had grown into the cross-pollinated styles by the time of collection (about h after pollination on August). No pollen tubes had grown m self-pollinated styles of either species except two in C. nigra plant A. Many of these self-pollinated styles had ungerminated pollen grains nearby, and some had a few germinated grains blocked with a callose plug as they entered the stigma. The styles collected in the field (Table ) have been divided into those with no pollen tubes, those with one to three tubes, four to nine and or more. In on each date from the Devil's Ditch there were similar numbers of styles in each category for both species, though there were quite large differences between dates. ANP

10 O (a) Bombinae A. J. LACK Other insects iiiiiiii i:ii:ii:iii!:iiiii iii!^^^^^^^^^^p Hiiiiiiiiiiiiiiiiiiii Hiiliiiiiliiiiliiilllllliliiiilsliiiiiiiiiiiiiiiiyiyiiii-n July August Sept. - o "o - ««:::::::::::::: ill iii :;;-:-:;:::;;::; August August Fig.. Percentage of the total number of visits by Bombinae and other insects to Centaurea scabiosa (), C. nigra (M) and other species (D); Devil's Ditch, (a) sheltered southwest aspect ; (b) sheltered southwest aspect, (c) exposed southwest aspect, (d) northeast aspect.

11 Pollination in Centaurea. ///. Insects Table. Numbers of insects visiting each capitulum /Centaurea scabiosa (C.s.) and C. nigra (C.n.) in min mean and % confidence limits of capitula except where stated. Devil's Ditch Bombinae Other insects C.s. C.n. C.s. C.n. Sheltered SW aspect Aug. Aug. Sept. Sept. Mean ±- - ±- - ± ±- -±l- - ±- - ±- l-±l ±- -±l- -±O- - ±- l-±l- - ±- - ±- -±l-l - ±- - ±- - ±- - ±- - ± ±- ( C. s.) ( C. s.) ( C. s., min) Exposed SW aspect Aug. Aug. Sept. Sept. Mean ± ± ±l- - ±- -±l- - ±- - ±- - ±- - ±- l-±o- - ±- - ±- - l-±l- - ±- - ±- - ±- -±M - ± ±- - ±- - ±- - ±- -±- ( C. n.) ( C. s.) Aug. Aug. Sept. Sept. Mean ±- - ±- - ±- -± ±- - ±- -±l-l - ± ±l-l - ±- - ±- - ±- - ±- - ± ±- l-±t) ±- - ± ±- - ±- ( min) Sheltered SW aspect Aug. Aug. Aug. Mean..... ll-± ±- -±-l ll-±- -±- - ±- -± ±- - ±- -±l- - ± l-±l- - ±- - ±- - ±- - ±- - ±- - ±- - ±- - ±- Exposed SW aspect H Aug. Aug. Aug. Mean ± ±-O - ±- - ± ll-o±- - ±- - ± ±- - ±- - ±- - ±- - ±- l-±l ±- n Aug. Aug. Aug. Mean ± ll-±- - ±- (-±-) - ± ±- - ±- -±l- - ±- - - ±- - ± ±- - - ±- (C.s.) -

12 A. J. LACK Table. Numbers of insects visiting each capitulum of Centaurea scabiosa (C.s.) and C. nigra. (Cn.) in min-mean and % confidence limits of capitula Hod Hill Bombinae Apis mellifera Other insects C.s. C.n. C.s. C.n. C.s. C.n. Aug. Aug. Aug. Sept. Sept. Mean ±- ±M ± - ±- - ±- - ± ±- - ±- - ± ± - ±- - ±- - ±- - ±- - ±- - ±- - ±- - ±- - ± ±- - ± ±- - ± ±- ll-± ± ±- - ±- - ±- - ± ±l ±- - ±- - ±- - ±- - ± ±- -O±l- -±l-l - ±- - ±- - ±- - ±- - ±- - ±- - ±- l-±l- - ±- Aug. Aug. Sept. Mean ±- - ±- - ± ±- - ±- - ± ± ± ± ±l- - ±- ±l ±- ±l ±l- - ± ±- - ±- - ± ±- -± l-±- - ±- - ± ±- - ±- - ±- - ±- - ± ±- - ±- l-±l- - ± ±- l-±l- - ±- - ± - - ±- - ±- - ±- - ±- -±- - ±- l-±l- - ±- l-±l- - ±- - ±- - ±- - ±- - ±- l-±l- - ±- l-±- Table. Numbers of insects visiting rayed capitula and capitula with rays removed, in min - capitula used for each mean. Hod Hill, Bombinae Apis mellifera Other insects Rayed Rayless Rayed Rayless Rayed Rayless Aug. Aug. Aug. Sept l-±l- Centaurea ±- nigra ± ± ±- - ± l-±' - ±- - ±- Aug. Sept ±- C. scabiosa -+- -±l-l ±- - -

13 Pollination in Centaurea. ///. Insects Table. Numbers of styles penetrated by given numbers of pollen tubes from artificial pollinations of Centaurea scabiosa and C. nigra; Botanic Garden, Crossed Selfed Plant Date > tubes - tubes tubes > tubes - tubes tubes A A B Aug. Aug. Aug. Centaurea scabiosa A B C D Aug. Aug. Aug. Aug. Aug. Aug. Aug. Aug. C. nigra August and September were fine days and very nearly all the styles of both species had several penetrating pollen tubes (collected in the afternoon). The results from August and September show a greater proportion of styles of both species with very few or no penetrating pollen tubes, particularly on the more exposed sites. The weather was poor on both dates with light rain through the night of to August (styles collected between. and. h) and on September a heavy overcast day followed a wet night (styles collected between. and. h). On Hod Hill most of the styles of both species had several penetrating pollen tubes on all dates in. All were fine days and the styles were collected in the afternoon. On August there was a higher proportion of C nigra styles with no pollen tubes than C scabiosa {x^ =, - > P > -), the only date in with a significant difference between the species. In, on the Devil's Ditch on August and September, both fine days, nearly all the styles of both species (collected in the afternoon) had several pollen tubes. On the northeast aspect of the Devil's Ditch on September, and on Hod Hill on August, also fine days (styles collected in the afternoon), C. scabiosa styles had several penetrating tubes but there was a considerable number of C. nigra styles with none or only one to three pollen tubes. DISCUSSION Insect visits Bees are clearly the most abundant and important insect pollinators of C. scabiosa and C. nigra. Bees use visual stimuli as the main attractant to flowers from a distance (Proctor and Yeo, ). The importance of visual attraction in Centaurea was demonstrated in the comparisons of rayed and rayless capitula of C. nigra on Hod Hill, since in all respects except for the possession of ray florets the rayless plants were identical to the rayed. The difference between the number of visits to rayed and rayless C. nigra on Hod Hill was very similar to the difference m number of visits on the Devil's Ditch to C. scabiosa (rayed) and to C. nigra

14 A. J. LACK Table. Numbers of styles penetrated by given numbers of pollen tubes of Centaurea scabiosa and C. nigra collected in the field C. scabiosa (no. of tubes) C (no.. nigra of tubes) Area > - - > - - Aug. Sept. Aug. Aug. Sept. Sept. Sheltered SW aspect Exposed SW aspect SW aspect (combined) Sheltered SW aspect Exposed SW aspect Sheltered SW aspect Exposed SW aspect Sheltered SW aspect Exposed SW aspect Sheltered SW aspect Exposed SW aspect Devil's Ditch Aug. Aug. Aug. Sept. Combined Exposed Sheltered Exposed Sheltered Exposed Sheltered Hod Hill (rayless). Perhaps the difference in number of visits to the two species on the Devil's Ditch is a consequence solely of difference in visual attraction. All bees which I followed individually on the Devil's Ditch fed at both species indiscriminately. On Hod Hill the capitula of both Centaurea species have ray florets and are of similar size and shape, but bumblebees and honeybees both visited C. scabiosa more often than C. nigra. As on the Devil's Ditch many individual bumblebees fed on each capitulum encountered regardless of species, but several bumblebees and many honeybees fed exclusively on C. scabiosa. When they approached capitula of C. nigra they circled over the capitula and flew on without settling to feed, only settling when they encountered a capitulum of C. scabiosa. It is evident that they can discriminate between the two species on Hod Hill, perhaps on scent, which is usually the stimulus to alight and feed (Free, ; Proctor and Yeo, ). Since they can discriminate on Hod Hill it is likely that they are able to discriminate on the Devil's Ditch too, where C. nigra is rayless and looks different from C. scabiosa- It suggests that it may be advantageous for them not to discriminate on the Devil's Ditch.

15 Pollination in Centaurea. ///. Insects One possible reason for the lack of discrimination by visiting insects on the Devil's Ditch may be that they occur in large numbers relative to the number of available capitula. Heinrich () found, in an area in Maine, that the plant species which produced most nectar were depleted fastest and, with a large bee population, may provide less food reward per visit than a plant which overall produces less nectar. In uncovered flowers of both Centaurea species on the Devil's Ditch it was never possible to extract nectar (see paper II, Lack, b), i.e. the bees are depleting the resource almost totally. On Hod Hill there were fewer insects per capitulum and a greater difference between the species in quantity of nectar (paper II, Lack, b) and therefore discrimination between the Centaurea species may be of advantage. In on Hod Hill no measurements were made on nectar, but bumblebees, particularly B. lapidarius, were abundant (Lack, ). In the only bumblebees I saw in days on Hod Hill in mid-august were three B. terrestris (or B. lucorum). The severe drought of summer followed by the cold wet spring of probably contributed to this decline and it seems that the numbers were only beginning to build up again in and. In the bumblebees were discriminating in favour of C. scabiosa but the honeybees in favour of C. nigra (Lack, ), a different picture from and and perhaps this varies with the numbers of insects present. The weather has an effect on the numbers of bees visiting flowers (particularly honeybees) and all my counts were done on fine days (though often quite cool and cloudy, particularly in ). On wet days the numbers would undoubtedly be lower and perhaps patterns of discrimination different, though most days in the two summers had conditions similar to those on the counting days. There are rather different results from the counts of insects on the Devil's Ditch than from the numbers of insect visits to each capitulum. The much greater difference in numbers of insects feeding on the two species indicates that each visit to C nigra must, on average, be shorter than the visits to C. scabiosa. During C. scabiosa produced about three to four times as much nectar as C nigra on average (paper II, Lack, b). The number of bees per C. scabiosa capitulum was about five times that per C. nigra capitulum on the northeast aspect where hoth species occur as large plants intermixed. It seems that the total time that the hees spend on each species is roughly proportional to the nectar reward. On the exposed southwest aspect most bees were foraging in the most sheltered part near the base where large C. nigra plants grow, most C. scabiosa plants occurring on the exposed slope. This may explain the relatively smaller number of bees per capitulum of C. scabiosa (- times as many as C. nigra). On the sheltered southwest aspect there is a large group of very large C. nigra plants producing most of the recorded capitula. Many bees visited C. nigra on this aaspect (Fig. ) but per capitulum the number of bees on C. scabiosa was - times the number per C. nigra capitulum, probably because of the particularly large number and high density of capitula of C. nigra. In the bees started visiting C scabiosa in large numbers earlier in the season than in, perhaps because many more capitula of C. scabiosa were produced in ( was the peak number in, in ; paper I, Lack, a). The subsequent lower number of bees on C. scabiosa in favour of C. nigra during late August and September is surprising (there were similar numbers of C. nigra capitula in the years), but results collected on nectar production in (paper JI, Lack b) indicated that C. scabiosa plants were producing very much less nectar at the end of the season than they were earlier in or all through.

16 A. J. LACK This may be the reason for the reduced number of bee visits. In the higher proportion of visitors on C. nigra may be a reflection of the higher total number of bees since, with an increased number of bees, they will be able to extract very little nectar from either species (perhaps none from some capitula) and either species is equally likely to contain some. It will then be advantageous to visit all capitula encounted, and the proportion of the total number of insects recorded on C. nigra is likely to be greater. In general it appears that the bees, much the most important pollinators of Centaurea spp., can discriminate between the species, but will only do so where it is advantageous for them, and this will depend on the relative numbers of capitula and insects, which vary in each place from year to year. The bees are very sensitive to precise differences or changes in the nectar reward available, and will visit capitula in a way which maximizes their own gain. Success of pollination Only one ovule is produced per floret and so theoretically only one pollen tube is necessary for fertilization. It has been shown, however, that more than one pollen grain per ovule may be necessary for full seed set in some species (Jennings and Topham, ; Cruden, ) perhaps owing to a growth factor produced by germinating grains (Brewbaker and Majumder, ). Ter-Avanesjan () suggested that more uniform offspring are produced with a greater number of pollen grains. In the insect-proof greenhouse the unfertilized styles remained receptive for at least a week before withering, whereas fertilized styles withered within days of pollination. Under field conditions unfertilized styles would probably last less than a week, but this suggests that the styles collected for this study, which were fresh and fully expanded, were nearly all newly mature and the resultant figures for number of pollen grains penetrating them are biased towards the smaller numbers. In addition, in the developing head of fruits it is likely that some fruits will abort, since C scabiosa produced a mean of - fertile florets (mean of collected on August ; range to ) and Dickie () found a mean of - seeds in uninfected ripe capitula of C scabiosa in on the Devil's Ditch (range to ) and a mean of - (range to ) in. C. nigra produced a mean of " fertile florets (range to ) on the same date but I have no data on seed set. Taking the factors mentioned in the last two paragraphs into account, it is apparent that both species of Centaurea had very similar numbers of styles fertilized on each data, and on most dates this was near %. There were quite large differences between some dates in both species, which suggests that factors such as the weather are the most important factors limiting pollination in both species, not interaction between the two Centaurea species. On most fine days nearly all the styles had been successfully fertilized in both species, although the insects appeared to discriminate to some extent. The rain which immediately preceded the collection on August and September would have severely depressed insect activity and possibly affected the styles adversely. On these dates both species were affected to the same extent. On September, in addition to the overcast humid conditions, a cold north wind was blowing, perhaps the reason for the particularly large number of styles with no penetrating tubes from the northeast aspect. On August, on Hod Hill there were very few plants of C. nigra flowering (markedly fewer than on the Devil's Ditch on August) whereas

17 Pollination in Centaurea. ///. Insects C. scabiosa was flowering abundantly, and this probably explains the difference between the species on this date (more C nigra were flowering in the sheltered area on Hod Hill than elsewhere and this is reflected by the higher proportion of pollinations there). In the difference between the two species on the northeast aspect of the Devil's Ditch on September is not easy to explain. It was a fine sunny day similar to September when on the southwest aspect there was almost % pollination of both species. On Hod Hill in no observations were made on the numbers of visiting insects but it was evident from casual observation that honeybees were the most important visitors and were visiting mainly C. scabiosa, and bumblebee numbers were very low indeed. This probably explains the very great difference between the Centaurea species on August on Hod Hill (C. nigra pollinated much less than C scabiosa). A discussion of all three papers follows this paper. ACKNOWLEDGEMENTS This study was financed by a research studentship from the Natural Environment Research Council. Drs P. F. Yeo and D. Briggs helped greatly with all aspects of the study. I am greateful to Mr J. Symonds and the staff of the University Botanic Garden, Cambridge, for their help, and Professor R. G. West for providing facilities at the Botany School, Cambridge. C. O'Toole and O. E. Prys-Jones helped with identification of the insects. I am most grateful to Dick and Margaret Harthan for their hospitality in Dorset. Drs P. F. Yeo and D. Briggs critically read drafts of this paper. REFERENCES BREWBAKER, J. L. & MAJUMDER, S. K. (). Cultural studies of the pollen population effect and the self-incompatibility inhibition. American Journal of Botany,, -. CRUDEN, R. W. (). Pollen-ovule ratios: a conservative indicator of breeding systems in flowering plants. Evolution,, -. DICKIE, J. B. (). The reproduction and regeneration of some common chalk grassland perennials. Ph.D. thesis. University of Cambridge. FAGERSTROM, T. & AGHEN, G. I. (). Theory for coexistence of species differing in regeneration properties. Oikos,, -. FREE, J. B. (). Insect Pollination of Crops. Academic Press, London, New York. GENTRY, A. H. (). Bignoniaceae of southern Central America: distribution and ecological specificity. Biotropica,, -. HARPER, J. L. (). Population Biology of Plants. Academic Press, London, New York. HEINRICH, B. (). The foraging speciahzations of individual bumblebees. Ecological Monographs., -. HESLOP-HARRISON, J. (). Incompatibility and the pollen-stigma interaction. Annual Review of Plant Physiology,, -. JENNINGS, D. L. & TOPHAM, P. B. (). Some consequences of raspberry pollen dilution for its germination and for fruit development. New Phytologist,, -. LACK, A. (). Competition for pollinators and evolution in Centaurea. New Phytologist,, -. LACK, A. J. (). Pollination ecology of Centaurea scabiosa and C. nigra. Ph.D. thesis. University of Cambridge. LAC K, A. J. (a). Competition for pollinators in the ecology of Centaurea scabiosa L. and Centaurea nigra L.. Variation in flowering time. New Phytologist,, -. LACK, A. J. (b). Competition for pollinators in the ecology of Centaurea scabiosa L. and Centaurea nigra. L.. Observations on nectar production. New Phytologist,, -. MARSDEN-JONES, E. M. & TURRILL, W. B. (). British Knapweeds. Ray Society, London. MARTIN, F. M. (). Staining and observing pollen tubes in the style by means of fluorescence. Stain Technology,,. NETTANCOURT, D. DE (). Incompatibility in Angiosperms. Springer-Verlag, Berlin.

18 ^ A. J. LACK OSTLER, W. K. & HARPER, K. T. (). Floral ecology in relation to plant species diversity in the Wasatch Mountains of Utah and Idaho. Ecology,, -. PROCTOR, M. & YEO, P. (). The Pollination of Flowers. Collins, London. READER, R. J. (). Competitive relationships of some bog ericads for major insect pollinators. Canadian Journal of Botany,, -. TER-AVANESJAN, D. V. (). The role of the number of pollen grains per flower in fertilization in plants. Bulletin of Applied Botany, Genetics and Plant Breeding, Leningrad,, - (in Russian). WASER, N. M. (a). Competition for hummingbird pollination and sequential flowering in two Colorado wildflowers. Ecology,, -^. WASER, N. M. (b). Interspecific pollen transfer and competition between co-occurring plant species. Oecologia {Berlin),, -. GENERAL DISCUSSION AND CONCLUSIONS FROM PAPERS I TO HI Centaurea nigra ssp. nigra appears to flower earlier than ssp. nemoralis in the parts of the country where the subspecies are distinct (paper I). Where they are not distinct, in eastern England, the populations are similar morphologically but may differ considerably in flowering time, and local ecological factors appear to exert a strong influence on the flowering time of particular populations. Some populations of C nigra in Cambridgeshire growing in isolation from C. scabiosa flower much earlier than C. scabiosa, but the populations of C. nigra growing sympatrically with C. scabiosa are among the latest C. nigra populations to flower, and all flower after C. scabiosa. Heinrich () suggested that the poorer competitor of two species competing for pollinators is likely to be at greatest advantage if it flowers slightly after its more attractive rival, and perhaps this may be the situation with C. nigra in sympatry with C. scabiosa. In addition, although C. scabiosa and C. nigra attract the great majority of visiting insects in late summer, plants such as Onobrychis viciifolia and Hippocrepis comosa which attract many insects (Lack, ) flower earlier than C. scabiosa. If C. nigra were to flower earlier than C scabiosa in these populations it may compete for pollinators with other plants. At present on the Devil's Ditch there is a large number of insects visiting the two Centaurea species in their flowering seasons, and this leads to nearly % pollination of both species under most environmental conditions, although the species differ in the quantity of food reward (nectar) which they produce. Since pollination appears not to be a limiting factor in eastern England there is no selection for a change in flowering time in C. nigra or for the incorporation of sterile ray florets into the C. nigra populations, and they are rayless. In southwest England there appeared to be a greater difference in nectar production between the species and there were fewer visiting insects per capitulum, with some insects visiting C. scabiosa only. Ray florets are present in these populations of C. nigra, increasing the size of the capitula (making them more attractive to visiting insects) and making the capitula resemble those of C scabiosa. It is likely that there would be greater discrimination by visiting insects if C. nigra did not possess ray florets, and competition for pollinators may be the selection pressure maintaining them in these populations. There was a difference between the species in the numbers of styles successfully fertilized on some dates particularly in, but on most dates in, when there were more insects than in, there were similar numbers of styles fertilized in the two species. In there were fewer insects than in or so this competition is perhaps of minor importance. Competition for pollinators, and consequently specialization in mode of pollination or time of flowering, is likely to be most obvious in floristically rich plant

19 Pollination in Centaurea. ///. Insects communities (Gentry, ; Ostler and Harper, ) and chalk grassland is, floristically, one of the richest of Britain's plant communities. If competition for pollinators is important as a selection pressure seed production will be limited, but there are many other factors which may affect seed production. In Centaurea in particular, the parasite Euribia solstitialis (L.) (= Urophora jaceana auctt.), the knapweed gall-fiy, is very common and may infect most capitula in some years often destroying all the fruits in the capitulum (Dickie, ). Selection must be acting on many other parts of the regeneration cycle (seed dispersal, seed longevity in the soil, germination, establishment) and any of these stages may be critical (Harper, ). Fagerstrom and Agren () have emphasized that a flowering time difference between two species may be maintained by factors involved in other parts of the regeneration cycle (e.g. seed germination). It is clear that there are many factors influencing successful regeneration and in Britain, since insect numbers fluctuate very markedly between years (C. O'Toole pers. comm., and my own observations on Hod Hill) it is perhaps unlikely that competition for pollinators would be a uniform selection pressure influencing these plant populations. Other environmental features such as climate, disturbance, predation and parasitism are likely to be the overriding influences in British vegetation (Harper, ). Frequent interspecific pollen transfer may be an important factor limiting fruit set in Centaurea. Reader () and Waser (a, b) suggested that this was more important than competition for pollinators in their studies in temperate North America. Centaurea scabiosa pollen will germinate on and penetrate a C. nigra stigma, and vice versa. The effect of this on seed set is not known, but there were frequent interspecific flights by the visiting insects which must lead to frequent pollen transfer between the species. Competition for pollinators may have been a more important selective influence in primeval times since there would have been less suitable habitat for either Centaurea species, and less suitable habitat for their bee pollinators. Centaurea scabiosa and C. nigra are pioneer species of open habitats and thus, particularly when open habitats did not remain as static as they do today, successful seed set would have been very important. Today, in southwest England, competition for pollinators may be a minor selective factor maintaining not only a flowering time difference between the species, but also the presence of ray florets in C nigra, characteristics which were established in much earlier times.

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