RESEARCH ARTICLE

AMERICAN JOURNAL OF BOTANY

Ornithophily for the nonspecialist: Diff erential pollination effi ciency of the Macaronesian island paleoendemic Navaea phoenicea (Malvaceae) by generalist passerines1

Alejandro G. Fernández de Castro 2,4, Juan Carlos Moreno-Saiz 3 , and Javier Fuertes-Aguilar2

PREMISE OF THE STUDY: A bird pollination syndrome exists in the archipelago across independent lineages despite the absence of specialist birds. The pollination effi ciency of current fl oral visitors remains unknown for many plant despite this being a fundamental factor in test- ing hypotheses about the origin of the syndrome. Here, we studied the components of pollination effi ciency in the paleoendemic Navaea phoenicea , a species exhibiting conspicuous anatomical modifi cations associated with bird pollination.

METHODS: We measured the components of the pollination effi ciency (PE) of species foraging on fl owers. The measured quantitative components were visitation frequency patterns to and individual fl owers. The qualitative components were the contributions to the fi tness of male and female func- tions (pollen removal and deposition and fruit set).

KEY RESULTS: Pollination by warbler species was highly effi cient, but visit frequency was low; conversely, Canarian chiff chaff s had high visit frequency and low effi ciency. Overall PE was almost 0 for blue tits due to disruptive behavior. We also found insects acting as nectar robbers.

CONCLUSIONS: Pollination effi ciency of three of the four bird species visiting fl owers of Navaea phoenicea may be high enough to maintain selective pres- sure on fl oral traits of a relict pollination syndrome. The behavior of these birds plays a crucial role in their pollination effi ciency. Perching, by generalist when visiting N. phoenicea fl owers, is the most effi cient habit. The frequency and PE of insect visits calls into question their role as legitimate visitors.

KEY WORDS Canary Islands; foraging behavior; Macaronesian bird–fl ower element; Navaea phoenicea ; Lavatera ; Malvaceae; opportunistic nectar feeding; ornithophily; pollination effi ciency; pollen limitation

Th e role played by fl oral visitors in the evolution of angiosperms, 2017 ). In plant–pollinator mutualisms, eff ectiveness accounts for where reproductive fi tness is pollen-limited, is governed by their the individual eff ects of the interactions of visitors with male and pollination eff ectiveness ( Herrera, 1987 , 1989 ; Gómez, 2002 ). Pol- female functions of the fl ower(s) occurring at a given frequency; lination eff ectiveness refers to the total contribution to plant fi tness these have been measured in a variety of ways ( Stebbins, 1970 ; by each species that visits and may pollinate fl owers (Herrera, 1987, Spears, 1983; Schemske and Horvitz, 1984; Herrera, 1987, 1989 ; 1989 ). Th e total eff ect of this contribution is normally estimated as Inouye et al., 1994; Stone, 1996; Mayfi eld, 2001), incorporating pre- a function of the product of (1) the frequency of the interaction and postzygotic eff ectiveness as well ( Alonso et al., 2012 ). Delayed (quantitative component, QNC) and (2) the eff ect of that interac- postinteraction measures, i.e., the number of viable seeds that are tion when it occurs (qualitative component QLC) ( Schupp et al., produced, are also considered as part of pollination eff ectiveness ( Schupp et al., 2017 ). 1 Manuscript received 29 May 2017; revision accepted 31 August 2017. Th e isolated condition of oceanic islands hinders colonization 2 Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2. 28014 Madrid, Spain; and and dispersal processes, leading in many instances to a disharmonic 3 Departamento de Biología (Botánica), Calle Darwin 2, Campus de Cantoblanco, Universidad Autónoma de Madrid 28049 Madrid, Spain and impoverished assemblage of the biota. As a consequence, 4 Author for correspondence (e-mail: [email protected]) plant–pollinator networks are frequently more depauperate in oce- https://doi.org/10.3732/ajb.1700204 anic islands than in the continental biota (Trøjelsgaard and Olesen, 1556 • AMERICAN JOURNAL OF BOTANY 104 (10): 1556 – 1568 , 2017; http://www.amjbot.org/ © 2017 Fernández de Castro et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons Attribution License (CC-BY-NC). OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.— POLLINATION OF NAVAEA PHOENICEA • 1557

2013 ; Castro-Urgal and Traveset, 2014). In addition, mutualistic Rodríguez-Rodríguez et al., 2013, 2015 ). Likewise, a molecular interactions on oceanic islands oft en present supergeneralist spe- phylogeny for Lotus species revealed a recent origin of the four cies, i.e., species that interact with multiple groups of species ( Olesen Macaronesian species visited by birds ( Ojeda et al., 2013 ). In Ana- et al., 2002 ) favoring new plant–pollinator interactions (Traveset gyris L. (Fabaceae), evidence of de novo mutualism with Phyllosco- et al., 2015 ). pus has been presented both for Macaronesia and for the European Bird–fl ower interactions are particularly prevalent in the tropics, continent ( Ortega-Olivencia et al., 2005 ; Valtueña et al., 2008 ; where specifi c mutualistic pollination interactions occur between Ortega-Olivencia and Catalán, 2009), the fi rst evidence of recent fl owers and nectar-feeding specialist birds, such as hummingbirds, ornithophilous adaptations in Europe. Such de novo mutualism sunbirds, and honeycreepers (Anderson, 2003). However, in tem- has also been demonstrated for three Mediterranean Scrophu- perate areas, generalist passerines have been reported occasionally laria species (Ortega-Olivencia et al., 2012; Valtueña et al., 2012; visiting fl owers in search of nectar ( da Silva et al., 2014 , 2017 ). Th e Navarro-Pérez et al., 2013). Th us, the occurrence of interactions incorporation of nectar into the diet of generalist passerines may with generalist passerines also in the European continent is a phe- serve as a food complement when resources are scarce. Th is phe- nomenon that occurs with food scarcity, although the ecological nomenon has been reported to be more frequent on islands than in implications of these interactions are not well understood ( da Silva mainland areas ( Valido et al., 2004 ) and has been interpreted as the et al., 2014 ). response of birds to trophic niche expansion and density com- In this paper, we study the quantitative and qualitative compo- pensation ( Olesen and Valido, 2003 ). nents of pollination effi ciency of generalist passerines on Navaea Among the examples of islands where visits to fl owers by gener- phoenicea (Malvaceae) to provide further insights on the evolution alist passerines have been observed, the Macaronesian archipelagos of the bird pollination syndrome in Macaronesia. Ornithophily is have been the subject of particular scientifi c interest in the last not infrequent within the Eumalvoideae (Malvaceae s.s.), although decade (Ojeda, 2013). Visits to fl owers by generalist passerines this type of mutualistic interaction by specialist bird lineages (hum- have been reported to be frequent for at least 17 endemic plant mingbirds and sunbirds) is concentrated in the tribes Hibisceae taxa belonging to the genera L. and Musschia Dumort. and Malvavisceae (Hibiscus , Pavonia , and Malvaviscus) and, within (), Isoplexis Lindley and Scrophularia L. (Scrophu- these, among neotropical and East African species. In the tribe lariaceae), Echium L. (Boraginaceae), Lotus L. and Anagyris L. Malveae, the vast majority of ornithophilous species are concen- (Leguminosae), and Navaea Webb & Berthel. (Malvaceae). Th e as- trated in the neotropical members of the subtribe Abutilinae, e.g., semblage of these plant species is known as the Macaronesian bird Abutilon Mill. (Bayer and Kubitzki, 2003) with a few cases in the fl ower element (MBE). Th ey share common fl oral traits that appear subtribe Malvinae, e.g., Anisodontea C.Presl from the Cape region to be adapted to bird pollination, such as copious, dilute nectar; red, ( Bates, 1969 ). Th e monotypic genus Navaea ( Fuertes-Aguilar et al., conspicuous corollas; open corollas; a lack of scent; and specifi c ori- 2003 ), included in the subtribe Malvinae, is endemic to entations depending on the pollinator ( Faegri and Van der Pijl, Island in the Canarian archipelago and encompasses two impor- 1979 ; Dupont et al, 2004 ; Cronk and Ojeda, 2008 ). Hence, they are tant features relevant to untangling the establishment of ornithoph- considered an example of a bird pollination syndrome that has ily in the Canaries. First, it is the only lineage among those included evolved independently in separate plant lineages ( Olesen, 1985 ). in the MBE that has developed highly specialized fl oral structures Early analyses of the MBE led to the proposal of two possible adapted to bird pollination, i.e., nectar storage and high production explanations for the evolution of this insular syndrome in the of dilute nectar, two features found only in Malvaceae among bird- absence of nectarivorous specialized lineages ( Vogel et al., 1984 ; pollinated taxa. Second, its phylogenetic position suggests a paleo- Olesen, 1985; Valido et al., 2004). Depending on the age of each endemic placement as a sister taxon to the Malva generic alliance, ornithophilous lineage, authors have discriminated between lin- an essentially entomophilous lineage (Escobar García et al., 2009). eages with a recent origin (de novo hypothesis), in which the lin- Unlike for other taxa, for Navaea phoenicea , the role of fl oral visi- eage is the result of a recent radiation, and older lineages with an tors in the evolution of fl oral traits has not been intensely studied. ancestral African origin (e.g., Navaea phoenicea and Canarina ca- Indeed, this species has been included only in general studies of the nariensis ). For instance, the genus Canarina has two additional MBE Valido et al., 2004; Ojeda, 2013). Previous observations of the older species (C. eminii , C. abyssinica ) in east continental Africa mating system of Navaea phoenicea show that the species is self- that have been dated to 7–8 Ma ( Olesen et al., 2012 ; Mairal et al., compatible, but reproductive success relies mainly on outcrossing 2015 ). For Canarina taxa, which are then considered relict lineages (Fernández de Castro et al., unpublished manuscript). Th ese fi eld associated with African Rand fl ora, ornithophily evolved in conti- observations revealed not only bird visits but also a high frequency nental ancestors in response to specialized nectarivorous sunbirds of visits by several insect species (E. Portellano and J. Fuertes- ( Olesen et al., 2012 ). Although fossil hummingbirds are known Aguilar, personal observations) for a northern hemisphere winter- from the Eocene in Europe ( Mayr, 2004 ) with evidence of bird– fl owering species. fl ower interactions in the same period (Mayr and Wilde, 2014), a Th e paleoendemic condition of Navaea phoenicea places the major drawback for this hypothesis is the lack of specialized sun- evolution of the bird pollination syndrome under the relict hypoth- bird fossils in the fossil record of Macaronesia (Valido et al., 2004). esis. Th is biogeographic characteristic suggests a hypothetical sce- Alternatively, the de novo hypothesis posits a local development nario in which the Navaea phoenicea continental ancestor may of ornithophily in response to nonspecialist bird fauna (Valido have developed an ornithophilous syndrome adapted to specialized et al., 2004 ). Several studies exploring the evolution of ornithophilous sunbirds. Th is syndrome then remained as an exaptation aft er the traits or the pollination effi ciency (PE) of generalist passerines sup- arrival to the Canaries and was maintained by generalist birds, port this hypothesis in several species: for Isoplexis canariensis , the which would provide suffi cient pollination effi ciency to ensure the effi ciency of the bird species Phylloscopus canariensis as a pollinator demographic viability of the species. However, the phylogenetic has been confi rmed (Rodríguez-Rodríguez and Valido, 2008, 2011 ; isolation of the species prevents the evolutionary interpretation of 1558 • AMERICAN JOURNAL OF BOTANY

the development of adaptations to bird pollination. Within the Mature individuals can produce up to 300 fl owers during the fl ow- closest lineage, the Malva alliance, Lavatera acerifolia, which is also ering season. Flowers are large and reddish-orange (petal length = endemic to the Canarian archipelago along with its sister taxon, the 29–32 mm, staminal column length = 36–39 mm) and are displayed Mediterranean L. maritima , presents an entomophilous pollination singly or in terminal racemes that last on average 6.5 d. Th e number system. To date, the only other single species in the Malva generic of ovules is 21. Th e staminal column produces on average 110 an- alliance, Lavatera assurgentifl ora, is visited by hummingbirds in the thers containing on average 100 pollen grains that are viable for Channel Islands (California, A. G. Fernández de Castro, personal 10 d. Pollen to ovule ratio is therefore 520, which places the species observation). Visits to Lavatera arborea , Abutilon sp., and Malva within the group of facultative autogamous species according sylvestris by Sylvia spp. ( da Silva et al., 2014 ) and to L. maritima by to Cruden’s classifi cation (Cruden, 1977). Flowers produce high sparrows (Passer domesticus L., A. G. Fernández de Castro et al., amounts of dilute nectar, which is off ered in the receptacles at the personal observation) have also been reported. basis of the petals. Sugar composition is invariably glucose and In addition to its evolutionary distinctiveness, Navaea phoenicea fructose (A. G. Fernández de Castro et al., unpublished manu- is a threatened taxon ( Rodríguez Núñez et al., 2004 ) with low popu- script), a typical trait of passerine-pollinated fl owers. Nectar pro- lation numbers. It is therefore important to ascertain the extent of duction is independent of time of day. Fruits, a dry schizocarp with reproductive success mediated by current fl ower visitors. In this no adaptations to zoochory, ripen within 26 d ( Fig. 1C ). work, our goals are dual: (1) From a macroevolutionary perspec- Manipulation experiments and qualitative components of polli- tive, we aim to provide an understanding of the ecology of bird nator effi ciency were conducted during three consecutive fl owering pollination contributing to the maintenance of the selection of orni- periods between 2006 and 2009. Th e quantitative components of thophilous floral traits. (2) From a microevolutionary and con- pollinator effi ciency were measured in 4 years between 2005 and servation perspective, our goal is to measure the components 2009. of pollination efficiency and to determine whether current visi- tors provide sufficient reproductive fitness to ensure population Pollen limitation experiments— We tested for pollen limitation by survival. applied the following treatments to randomly selected buds before For this purpose, we conducted a set of experiments and obser- opening: (1) control (C), nonmanipulated fl owers; (2) autogamy vations to analyze the components of pollination effi ciency. Our (A), bagged fl owers and no anther manipulation; (3) nonsupple- results allowed a direct comparison with other species of the MBE mented xenogamy (X), emasculated flowers; (4) supplemented and to distinguish individually the pollination effi ciency of each xenogamy I (RXI), emasculated fl owers before anthesis and subse- visitor at every stage of the pollination process: pollen removal, pol- quently pollinated with stored pollen from another plant from the len deposition, and the delayed outcomes of fruit set and seed fi t- same mountain massif (Teno or Anaga) upon stylar branch exser- ness ( Schupp et al., 2017 ). We tested the following hypotheses: (1) tion and bagged to control pollen source; and (5) supplemented Because Navaea phoenicea has ornithophilous traits, birds are sig- xenogamy II (RXII) was manipulated as in RXI, but pollen was nifi cantly more effi cient pollinators than insects. (2) An exaptation added from the opposite mountain massif (Anaga vs Teno) to test might be currently occurring and fl owers are thus actually adapted for diff erences in reproductive success due to outbreeding. Pollen to African sunbirds; pollen limitation is therefore expected due used in cross-pollination treatments was conserved in Eppendorf to lower pollination effi ciency by the current visitors. (3) Present tubes at 10° C until used for fertilization and always used within 18 h fl ower visitors are responsible for suffi cient reproductive fi tness in of the following day in RXII and within a day in RX-I. Treatments male and female functions to maintain the selection of ornithophi- 2 and 5 included bagging fl owers with tulle boxes. Following the lous characters in an insular environment. restrictions imposed by the fi eldwork permit given by the Canarian Government, each treatment was applied every year to two fl owers per individual in fi ve individuals in eight populations ( Fig. 1B ). In a MATERIALS AND METHODS preliminary treatment with chicken wire to exclude birds but allow the entry of insects, any wire diameter small enough to exclude Study site and species— Navaea phoenicea (Fig. 1A) has a restricted birds also seemed to deter insect visits; therefore, we did not do this distribution on the northern cliff s of the oldest volcanic mountain experiment. Fruits were collected if produced. Fruit set was consid- massifs in the East (Anaga, 11–5.5 million years ago [Ma]) and ered as the proportion of fl owers setting fruits. West (Teno, 8–3 Ma) of Tenerife Island, accounting for 15 popula- To evaluate diff erences in fruit set between treatments, fruit set, tions ( Fig. 1B ). Th e steepness of the areas where the populations number of viable seeds per fruit and seed mass were measured as an live made access extremely diffi cult. Th us, several experiments were estimate of fi tness. Seed viability was assessed directly through ger- limited to the eight populations with easier conditions for fi eld- mination aft er manual scarifi cation by carefully slicing the coat of work. Population size ranged from 15 individuals to 48. Th e pri- the seed with a scalpel. Failure or success in germination was con- mary habitat is thermophilous shrubland dominated by perennial sidered as the response variable. Generalized linear mixed models sclerophyllous species, mainly Pistacia lentiscus , Jasminum odorat- (GLMM) using the lme4 package (Bates et al., 2015) implemented issimum, and Rhamnus crenulata. Some populations are found in in R soft ware (R Core Team, 2013) were applied to test for signifi - the transition zones to the more humid laurel forest dominated by cant diff erences between treatments. Models included treatments, Myrica faya and Erica arborea . Th e species is a large shrub or small population and year as fi xed factors and individual plants as a ran- tree with its fl owering period spanning from the end of the aestival dom factor to control for potential individual variability in the re- dry period in September until January when fl owering overlaps in sponse variables. We used a binomial distribution with a logit link time and space with early spring species such as Canarina canarien- function for germinating seeds and an exponential error distribu- sis . Young plants start to produce fl owers when they reach a height tion for seed masses. Diff erences between treatment means were of at least 1.2 m and a diameter at the base of the stem of 2.2 cm. tested pairwise through multiple comparisons using the multcomp OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.—PASSERINE POLLINATION OF NAVAEA PHOENICEA • 1559

FIGURE 1 (A) Flowers of Navaea phoenicea. (B) Distribution of Navaea phoenicea populations in Tenerife island. Populations where fi eld study was car- ried out are marked in bold blue. Asterisks denote those populations where beehives were found less than 1 km nearby. Longitude and latitude UTM coordinates are below the population name. (C) Ripening schizocarp of N. phoenicea .

package ( Hothorn et al., 2008 ). Degrees of freedom were obtained visits of birds and insects to estimate these variables. Visitors were with the Satterthwaite approximation for degrees of freedom. censused over 4 years (2005–2009) across the eight populations on diff erent days spanning the whole fl owering period to account for Quantitative component of PE: Frequency of visitors— Quantita- variation between populations or caused by weather conditions. tive components of PE considered were the number of fl owers vis- Daily censuses encompassed the whole sunlight period. Th e number ited per plant and the number of visits/30 min. We monitored the of observation hours each day ranged from 13 h at the beginning of 1560 • AMERICAN JOURNAL OF BOTANY

the fl owering season in September to 10 at the end in January as the sources of diff erences in reproductive success. To check for pollen daylight period became shorter. Eleven night censuses were per- deposition on the stylar branches, we emasculated and bagged formed in 2005 to rule out nocturnal visits. Before the systematic fl owers before style exsertion and left them open when styles were censuses of visitors, we closely observed fl owers ad hoc for 5 whole exserted. Pollen grains on the stigmas were counted with a fi eld days in 5 diff erent populations, plus odd hours during fi eldwork magnifi er and gently removed with a soft brush to allow further tasks to become familiar with the insect visitors. Individual plants observations on the same style. Style length was measured to ac- were observed with binoculars from a discreet point distant enough count for variation in deposition effi ciency. Signifi cant diff erences to avoid interference with visitors but close enough to allow us to in pollen grains between visitors were tested with generalized linear unequivocally identify insect species. Five plants could be easily ob- models (GLM) fi tted with a Poisson distribution. Th e length of the served simultaneously. For every visit, we registered the time of the monitored style was incorporated in the model as a predictor vari- day, the species, the length of the visit, weather conditions, and able to account for possible effects of length on the number of the type of visit, which included hovering, hanging, or perching in grains deposited. In total, we spent 389 h monitoring pollen depo- the case of birds and hovering or landing in the case of insects. Th e sition, accounting for at least 20 measures per year for each bird eff ect of weather conditions on the frequency of visits of each insect species. species was tested with GLMM with monitored individuals treated as random factors. Additionally, the daily pattern of visits was as- Qualitative components of PE: Seed viability and fruit set— To as- sessed with the overlap R package (Meredith and Ridout, 2013). sess the pollination eff ectiveness of each visiting species, fl owers Th e total observation time per group of 5 plants spanned at least were emasculated, bagged, and then the bags were removed when 124 h in each of the eight populations per year for 4 years for at least stylar branches were exserted. Aft er a single visit of an animal, fl ow- 12 observation days in each population. In total, we spent 3988 h ers were bagged again, and any fruits were collected. For other ob- observing fl oral visitors. servations, the procedure was carried out every year until 40 fl owers Because the honeybee Apis mellifera is a frequent introduced fl o- per visitor species were monitored. Th e experiment was carried out ral visitor, fi eldwork also included a survey of existing beehives in in the eight selected populations in 10 diff erent individuals per the area to test the relationship between nearby hives (less than population and at least four random fl owers per individual. Fruit 1 km) and the frequency of visits by A. mellifera . sets were recorded, and seeds were weighed and subsequently sowed aft er manual scarifi cation to determine germination success. Qualitative components of PE: Pollen removal, transportation, GLMs were used to test the eff ect of the visitor species on the num- and deposition— Pollen removal was measured as the percentage ber of viable seeds per fruit with a Poisson distribution and seed of anthers from which pollen was removed during a single visit of mass of viable seeds with a normal distribution. Since insect visits an animal. We selected and tagged buds before opening. Before an- did not produce any fruits, they were excluded from the analyses thesis, we counted the number of anthers of each selected fl ower (see Results). and started monitoring immediately aft er the opening of the an- thers. We monitored fi ve fl owers simultaneously. Aft er every visit Pollination effi ciency and pollinator landscape— Portions of the of a bird or insect to a fl ower, we counted with a fi eld magnifi er the experiments and observations of quantitative and qualitative com- number of empty anthers (less than 10 pollen grains) and the num- ponents allowed us to use the same approach for Isoplexis canarien- ber of anthers containing pollen. We made at least 40 observations sis in Tenerife ( Rodríguez-Rodríguez et al., 2013 ). Th e analysis was every year for each fl oral visitor distributed in fi ve visits per popula- based on the methodology developed by Reynolds and Fenster tion. Diff erences between visitors in the percentage of pollen re- (2008) . Th e quantitative component (QNC) is considered as the moved were tested with GLMs fi tted with gamma distributions. product of the number of visits/plant × 30 min and the number of Th e pollen loads carried by fl oral visitors were determined in fl owers/plant × visit. Th e qualitative component (QLC) is the sum two ways. Insects were captured aft er the visit to the fl ower, stored of the proportion of grains removed/anther (male function) plus in a glass tube, and then immobilized with forceps during a manual the product of fruit set/visit and number of viable seeds/fruit × visit count of pollen grains with a fi eld magnifi er. Insects were then re- (female function). Pollination effi ciency is considered as the prod- leased. Th ese captures were made simultaneously with bird cap- uct of QLC and QNC. Th e pollination landscape therefore has two tures. Birds were captured with mist nets placed between Navaea coordinates corresponding to QNC and QLC, so that the pollina- individuals. Th e diffi culty in placing the nets due to steep habitats tion landscape can be shown in a two-dimensional plot. Every data severely limited capture sessions and the number of nets that could set for each QLC and QNC was resampled 5000 times. Pollination be set. Captured birds were tagged with individually numbered effi ciency was then calculated as the product of the mean values per rings plus color rings to create unique combinations, allowing sub- sample. Th is procedure was repeated 5000 times to complete a data sequent visual diff erentiation of individuals. Pollen loads were ob- set of mean PE values to account for mean and 95% confi dence in- tained from birds’ heads with adhesive tape and eventually from terval (CI) limits. Th e procedure has been completely described by throats if grains were detected, which was infrequent. Pollen grains Rodríguez-Rodríguez et al. (2013) . were subsequently counted in the laboratory with a magnifi er di- rectly from the tapes. Diff erences in the number of grains were tested by means of GLMs fi tted with gamma distributions. RESULTS Finally, we measured the number of grains deposited in the sty- lar branches by each species. Although this measure is not part of Pollen limitation experiments— Th ere were signifi cant diff erences the calculation of the pollination effi ciency value (see next section), for both seed mass and number of viable seeds per fruit between × −16 it is a measure taken before the postpollination measure included treatments (F 3, 5485 = 51.30, P < 2 10 , n = 5766; F 3, 375 = 5.482, P < 0.001, in the pollination effi ciency equation and can serve to assess the n = 389, respectively) but not between populations (F 7, 65.5 = 1.150, OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.—PASSERINE POLLINATION OF NAVAEA PHOENICEA • 1561

× −6 P = 0.343; F 7, 75.81 = 1.14, P = 0.344) or years (F 1, 4819.6 = 1.193, P = and TEN populations (z = 4.89, P = 8.39 10 ). No bats or lizards 0.274; F 1, 372 = 0.196, P = 0.719). Both supplemented cross pollina- were detected. tion treatments yielded signifi cantly higher values for seed mass Behavior among diff erent visitor classes diff ered signifi cantly. than for the nonsupplemented outcrossing and controls (z = 9.801, Birds had two clear activity peaks, starting at dawn in the morning P = 1e−04 , Fig. 2A). Mean values of seed mass were 0.8 mg for treat- until 11:00 AM and a secondary short peak before dusk ( Fig. 3 ). ment A, 1.24 mg for C, 2.51 mg for X, 3.6 mg for RXI and 3.62 for Modes of visit diff ered signifi cantly (Χ 2 = 47,006, df = 21, P < 2 × RXII. Likewise, there were signifi cant diff erences in the number of 10−16 ) between P. canariensis and C. teneriff ae . Phylloscopus canar- viable seeds per fruit (z = 3.612, P = 0.00032, means: treatment A, iensis either hovered (65% of visits) or perched (35%). Cyanistes 1.75, C, 2.94, X, 4.75, RXI, 6.51, RXII, 6.6, Fig. 2B ). Both results in- teneriff ae hung from either the fl ower pedicel or the staminal col- dicated pollen limitation. Th e similar values obtained for nonsup- umn in most of the visits (78.74%) rather than perching (21.26%). plemented outcrossing and controls along with lower seed yields of Both Sylvia species perched in all observations. Insects always used autogamy indicated that fruit set occurred mostly by outcrossing. lower petals as a landing platform and moved directly toward nec- tar receptacles. Insects tended to concentrate most visits at noon, Quantitative components of PE: Frequency of visitors— Visit fre- possibly because temperature conditions (on average, 8.2° C at ° ° quency varied widely among species (F 7, 280 = 8.78, P < 0.001, Table dawn, 13.4 C at noon, and 10.1 C at dusk) allowed better insect 1A, B). Among birds, Phylloscopus canariensis and Cyanistes tener- movement. iff ae made more frequent visits than did Sylvia spp. and insect spe- cies. Th e number of fl owers visited on one plant (geitonogamous Qualitative components: Pollen removal, transportation, and de- visits) was signifi cantly higher for C. teneriff ae than for other bird position— Th e mean proportion of anthers from which pollen was species (Table 1A). Regarding insects, bumblebees and honeybees removed in a single visit ranged from more than 50% for bird spe- foraged repeatedly on the same plant before leaving ( Table 1A, B ). cies to zero in P . xiphioides ( Table 1C , Fig. 4A ). Bird species re-

One visit of Serinus canarius was recorded during the observation moved greater proportions of pollen than insects (F 7, 1022 = 226.61, period. Cyanistes teneriff ae had more geitonogamous visits. Insects P < 2 × 10 −16 ). Post hoc tests revealed no diff erences in pollen re- accounted for 35% of the visits. Th e frequency of visits varied be- moval between species within either of the two functional groups, × −16 tween species (F 3, 1156 = 97.87, P = 2 10 ) and varied with tempera- birds and insects. × −16 ture (F 1, 1156 = 14191.10, P = 2 10 ) and moisture (F 1, 1156 = 97.87, Bird ringing during the fl owering season yielded low sample P = 2 × 10 −16). Additionally, species × temperature and species × mois- sizes for the analysis of pollen transportation ( Fig. 4B , Table 1D ). In ture interactions incorporated in the linear model were signifi cant total, we captured in mist nets 17 C. teneriff ae, 11 P. canariensis , × −16 × −16 (F 3, 1156 = 56.98, P = 2 10 , F 3, 1156 = 13.69, P = 2 10 ), indicating one S . melanocephala , and no specimens of S. atricapilla . Th e num- that species visitation rate diff ered depending on the environmen- ber of pollen grains recovered from bird heads varied widely. For C. tal conditions. No insect activity below 11° C or above 85% humidity teneriff ae , pollen loads ranged from 0 to 1476 grains and for P. ca- was detected, which indicated that weather was not limiting for nariensis from 0 to 106 grains. Fift y-eight percent of C. teneriff ae birds. Th e activity of A . mellifera was positively correlated with the specimens captured had pollen, while 100% of P. canariensis had distance to neighboring domestic beehives in the TOP, REP, TAB, pollen ( Fig. 4B ). Conversely, most insects captured had very low

FIGURE 2 Contribution of each treatment to (A) seed mass of fruit sets yielded by fl owers and (B) number of viable fruits set by each fl ower. The com- plete fl ower set manipulated in each pollination treatment is accounted for, independent of whether the fl ower set fruit. A = autogamy; C = control; X = xenogamy; RXI = reinforced xenogamy I; RXII = reinforced xenogamy II. 1562 • AMERICAN JOURNAL OF BOTANY

TABLE 1. Summary of the pollinator landscape components by species. Observation C) Anthers with D) Number of E) Pollen grains F) Fruit set achieved time= 3988 h pollen removed (%) grains on the body deposited in one visit A) Visits/30 B) Flowers/ Fruit Seed Viable Visitor min plant n Mean ± SD n Mean ± SD Max n Mean ± SD Max n set (%) mass (mg) seeds/fruit Birds C. teneriff ae 0.69 ± 0.71 5.13 ± 1.45 150 53.62 ± 22.92 17 171.39 ± 454.6 1476 596 13.90 ± 10.57 45 188 3.87 13 ± 36 1.55+4.36 P. canariensis 0.75 ± 0.84 1.27 ± 0.12 150 51.12 ± 21.56 10 21.6 ± 35.95 106 619 13.76 ± 10.49 49 43 15.17 28.2 ± 51 3.62+6.33 S. atricapilla 0.16 ± 0.07 2.1 ± 1.01 121 47.48 ± 30.72 0 NA NA 73 15.21 ± 17.52 67 57 18.42 33.6 ± 58 4.59+7.17 S. melanocephala 0.25+0.17 2.0 ± 1.01 121 55.07 ± 28.16 1 15 15 63 10.69 ± 12.21 40 41 18.75 30.5 ± 54.7 4.07+6.51 Insects A. haematodes 0.19 ± 0.15 1.1 ± 0.0 120 6.45 ± 6.11 23 3.22 ± 3.1 6 22 1.27 ± 1.60 5 NA NA NA NA A. mellifera 0.32 ± 0.43 8.3 ± 1.7 128 3.76 ± 3.75 34 2.26 ± 2.87 8 35 0.62 ± 0.91 3 NA NA NA NA B. canariensis 0.30 ± 0.18 9.1 ± 0.6 120 2.06 ± 3.31 19 2.37 ± 3.02 8 33 2.40 ± 2.61 8 NA NA NA NA Pa. xiphioides 1.1 ± 0.1 0.11 ± 0.04 121 0 11 2.7 ± 3.16 0 16 1.5 ± 1.75 5 NA NA NA NA

Notes: A. = Apis ; B. = Bombus ; C. = Cyanistes ; P . = Phylloscopus ; Pa. = Pararge ; S. = Sylvia

pollen loads or no pollen. Pollen loads registered for C. teneriff ae of viable seeds per fruit (F 3, 60 = 4.562, P = 0.006) indicated signifi - and P. canariensis were signifi cantly higher than in insects, but cant diff erences between bird species. Tukey’s honestly signifi cant sample numbers were lower ( Table 1D ). diff erence (HSD) post hoc tests indicated that visits of C. teneriff ae Birds deposited signifi cantly more pollen grains than did insects yielded lower fruit set rates than the other species (P adjusted = 0). × −16 ( F 7, 1446 = 19.61, P < 2 10 ¸ n = 1457, Fig. 4C ). Style length did not infl uence the number of pollen grains deposited (F 7, 1446 = 19.61, P = Qualitative components: Pollinator effi ciency — Th e effi ciency of 0.584). However, the number of pollen grains varied greatly in birds diff ered widely between species and effi ciency components every species, and numerous contact events with the stigma left no and was low in all cases ( Fig. 5 ). Th e bootstrap estimation for aver- pollen ( Table 1E ). Again, post hoc pairwise tests did not reveal dif- aging PE showed a coeffi cient of variation of 6.45% across itera- ferences among bird species or among insect species. tions. Quantity components were high in general; conversely, quality components were low. Phylloscopus canariensis had the Qualitative components: Fruit set— No fl owers visited by insects highest PE score (0.028) with intermediate values for qualitative yielded fruits and were thus excluded from subsequent analyses. In and quantitative components and higher variation in the computed birds, single bird visits yielded fruit: 3.87% for C. teneriff ae , 15.17% values. Sylvia melanocephala (PE = 0.014) and S. atricapilla (PE = for P. canariensis , 18.42% for S. atricapilla , and 18.75% for S. mela- 0.014) had higher qualitative scores and lower quantitative scores. nocephala ( Fig. 4D , Table 1F ). Th e GLMs for fruit set (F 3, 334 = 5154, Cyanistes teneriff ae had the highest quantitative value and the low- × −16 × −6 P < 2 10 ), seed mass (F 3, 700 = 53.22, P = 2.27 10 ) and number est qualitative score (PE = 0.012) and was the least effi cient pollinator, due to the high rate of geitonogamous visits and to the low fruit set rate. Th e insect PE score was in all cases zero due to the null qualitative contribution to reproductive fi tness.

DISCUSSION

Our results demonstrate that species of non- specialized passerines act as legitimate polli- nators of Navaea phoenicea, a species highly adapted to ornithophily. We assessed the en- tire spectrum of the components involved in the pollination process and the implications of each element for reproductive fi tness. Th e strong diff erences in both quantitative and qualitative aspects of the pollination landscape can be interpreted on the basis of pollinator behavior. Our results provide compelling evi- dence for excluding insects as legitimate pol- linators but allow the detection of asymmetric values of the role of birds in ecosystem polli- nation services. Furthermore, these results demonstrate that a visitor census is insuffi - FIGURE 3 Daily visitation patterns of (A) bird species and (B) insect species in 24 h. Visits are ex- cient to ascertain the pollination ecology of pressed as the density of visits in a given time. the species; more research is needed because OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.—PASSERINE POLLINATION OF NAVAEA PHOENICEA • 1563

Insects did not contribute to the reproductive success of Navaea. All insect species were found to remove, transport, and deposit very low amounts of pollen, which resulted in repro- ductive failure. Navaea phoeni- cea fl owers are large and open, so in most visits the insects were able to land directly on the lower petals and move toward the nectar receptacles without contacting the staminal column or stylar branches. Hence, pollination ac- curacy ( Armbruster et al., 2009 ) was null for insect species. Be- cause pollen grains are shed rap- idly aft er anthesis, some of them fall onto the nectar receptacles. Th erefore, the small amount of pollen grains carried by insects are attached aft er contact at the basal surface of the petal and not to the staminal column.

Contributions to pollination ef- fi ciency — Th e results of fl ower manipulation experiments and the quantifi cation of the six vari- ables of pollination effi ciency diff ered greatly across the ele- ments analyzed. At one extreme, FIGURE 4 Contribution of each fl ower visitor to (A) male fi tness, expressed as the percentage of pollen removed both Sylvia species had the high- from anthers per visit; (B) number of pollen grains on the bodies of captured animals; (C) number of grains de- est QLC score, while C. teneriff ae posited on stigmas per visit; (D) percentage of fl owers setting fruits after one single visit of a pollinator. Letters made the most visits. Manipula- above boxplots denote signifi cantly diff erent groups. Insects: A.h. = Ancistrocerus haematodes; A.m. = Apis mel- tion experiments yielded higher lifera; B.c. = Bombus canariensis; P.x. = Pararge xiphioides . Birds: C.t. = Cyanistes teneriff ae; P.c. = Phylloscopus ca- fruit sets in pollen-supplemented nariensis ; S.a. = Sylvia atricapilla ; S.m. = Sylvia melanocephala . treatments than with outcross- ing alone, indicating pollen lim- the global pollination effi ciency varies depending on the results itation. When visitation patterns and frequency were considered, obtained for visit frequency ( Rodríguez-Rodríguez et al., 2013 ). the four observed species generally had two clear peaks of activity at Assessments of pollinator shifts across plant lineages, pollina- dawn and dusk. Th is pattern is at odds with the pattern found for tion syndromes and networks based only on visit observations Isoplexis canariensis, with P. canariensis visiting more frequently in or incomplete knowledge of plant–pollinator interactions may the evening (Rodríguez-Rodríguez et al., 2013). Sylvia atricapilla be biased. and S. melanocephala visited much less frequently than P . canariensis and C . teneriff ae did, likely because they are estimated to be less Pollination effi ciency of insects — Th e analysis of pollination eff ec- abundant in Tenerife ( Martín and Lorenzo, 2001 ; Carrascal and tiveness components showed very diff erent results for birds and Palomino, 2005 ). Compared to other ornithophilous species, insects. Insect species were revealed as mere nectar robbers with a P. canariensis and C. teneriff ae had higher visitation frequencies null contribution to reproductive fi tness. Th e visit frequency was, than when foraging on Canarina canariensis (Rodríguez-Rodríguez however, higher for native Bombus and introduced Apis , which and Valido, 2011 ), Isoplexis canariensis ( Rodríguez-Rodríguez and occurred only in populations close to hives. Th ese results are relevant, Valido, 2008), Lotus (Ollerton et al., 2009), or Scrophularia spp. as reddish fl owers may be cryptic for insects. While a consistent (Ortega-Olivencia et al., 2012). Outside of the Macaronesian region, daily pattern of visits was found with the activity limited to the visit rates in the continental Anagyris latifolia by Sylvia species were warmest hours in the middle of the day, activity was dependent on higher ( Valtueña et al., 2008 ). weather conditions. However, the days were humid and cool dur- Beyond the abundance of visitors, several factors may explain ing the autumn–winter blooming of Navaea phoenicea . Th ese con- diff erences between species belonging to the MBE, including food ditions do not favor insect visits, even though the frequency of availability for generalist passerines. Insects and fruits are scarcer visits was the highest recorded for an MBE species ( Rodríguez- for passerines during the fl owering period of Navaea phoenicea Rodríguez and Valido, 2008 , 2011 ). (northern hemisphere winter), such that species rely more on the 1564 • AMERICAN JOURNAL OF BOTANY

small sample of captured birds. We did not fi nd those high amounts for P. canariensis , prob- ably due to sampling limitations; during visit censuses, large pollen loads on the head of P. canariensis could be observed. Th is phenom- enon was observed for Sylvia spp. but with a lower frequency. The four species had similar values of pollen removal and deposition, indicating that their effi ciency for both variables was similar. All species were able to completely remove all pollen on the staminal column in one visit, although the amount of pollen removed varied widely. A similar fi nding was obtained for the number of grains deposited on stylar branches, which was also not signifi cantly diff erent among bird species. However, the mean number of grains deposited was more frequently below the mean number of ovules in the fl owers (21), indicating that most of the visits likely did not assure fruit set. Th is result is consistent with the reproductive success results. FIGURE 5 Mean (± SD) pollination effi ciency (PE) values for the eight species for the quantitative component (x For the deposited grains, S. atri- axis) and qualitative component (y axis). Gray lines denote isolines for PE values. Dates indicate the earliest capilla yielded the highest fruit ages for colonization of the Canarian archipelago for the three main visitors as estimated by molecular set rate; C. teneriff ae yielded very dating. low fruit set. With similar quality components to other species and nectar supply from fl owers. Indeed, during our 4 years of fi eldwork, very high pollen loads, there was no a priori expectation for signifi - we observed little coincidence with other plant food sources such as cantly lower rates of fruit set by C. teneriff ae . Th is failure can be fruits or other fl owers potentially available to birds. Th erefore, interpreted on the basis of its foraging behavior. On most of the those fl oral traits may be better perceived in the isolation of the winter visits, the birds hung by their legs from the pedicel to manipulate plant landscape. Unexpectedly, the fl owers of Navaea phoenicea, un- the petals and the staminal column to reach all the nectar recep- like other MBE species ( Valido et al., 2004 ), did not attract species of tacles ( Fig. 6A ), which is an extended foraging behavior of Cya- Gallotia lizards living in the same habitats. nistes species ( Carrascal et al., 1994 ). Unlike the pedicels described Cyanistes teneriff ae and Phylloscopus canariensis showed a simi- for ornithophilous fl owers ( Cronk and Ojeda, 2008 ), the pedicels of lar frequency of visits, with the calculated quantitative component these fl owers appeared to be weak and unlikely to withstand heavy of the PE much higher in the former due to geitonogamous visits. bird weights or manipulations. Consequently, the visits of C . tener- Although these visits are considered in the fi nal product of the PE, iff ae damaged fl owers, precluding the completion of the pollination geitonogamy is not necessarily qualitatively adaptive for plant fi t- and fruit set process given the evidence of the success of a single ness, as it does not promote outcrossing. Indeed, previous results visit. While visits of other species would cumulatively increase the show that outcrossing facilitated a higher genetic diversity despite probability of fruit set, visits of C. teneriff ae would damage a polli- low eff ective numbers and geographical disjunction limiting gene nated fl ower. Interestingly, the position of fl owers on branches ap- fl ow. pears to favor perching rather than hovering, as fl owers are most commonly orientated upward in racemes. Further, they have a Qualitative contribution to pollination efficiency— The quality slight but noticeable zygomorphy, as the staminal column is components of the pollination landscape diff ered strongly between curved slightly upward ( Cronk and Ojeda, 2008 ), along with a fl o- bird species. Th e diffi culties in capturing birds in the habitat yielded ral orientation that can aff ect visitor behavior ( Fenster et al., 2009 ). poor sample sizes, which limited the information about pollen While hovering has been traditionally attributed to nectarivorous loads, although this measure was not used to calculate the pollina- specialist birds, mostly exclusive to hummingbirds, it is not an in- tion effi ciency values. Th e majority of birds carried pollen on the frequent behavior among passerines in the Old World (Wester, forehead, but the quantities varied greatly. Pollen load was greater 2014 ) as we observed across Iberian and Macaronesian ornithophi- in C. teneriff ae, with pollen grain counts in the thousands twice in a lous species in both P. canariensis and Sylvia spp. Even sunbirds OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.—PASSERINE POLLINATION OF NAVAEA PHOENICEA • 1565

show eff ective hovering behavior ( Janeček et al., 2011 ). Here, hov- to only one functional group, birds, while Isoplexis included both ering is the most frequent pattern for P . canariensis but not for Syl- lizard and bird groups. Among birds, three of four species were via spp. ( Fig. 6B ), which are unable to sustain hovering for the time legitimate pollinators, although using diff erent strategies across the needed to drink nectar. On captured birds, pollen loads on P. ca- pollinator landscape. An immediate outcome of this redundancy in nariensis were notably limited to the forehead, while pollen was species is the mitigation of a possible breakdown of plant–pollina- also found on the beak and throat of C. teneriffae, probably as a tor networks. result of this behavior pattern. Foraging behavior differences A set of issues concerning some of the variables estimated should may then drive the different fruit set rates found between Sylvia be considered: (1) Th e visitation frequency is infl uenced by several species and P. canariensis . Th e latter had a higher PE score, but it ecological constraints that are not related to attraction cues and the remains unknown whether ecological conditions allowing a higher adaptation of fl oral traits, such as local variation in pollinator den- density of Sylvia spp. would increase the reproductive fi tness in N. sity (e.g., Gómez and Zamora, 1999 ; Herrera, 2005 ). (2) As argued phoenicea . above, geitonogamous visits do not necessarily yield higher fi tness In sum, the analyses of qualitative and quantitative components for the population, although they certainly function as a way of allowed for a direct comparison between fl oral visitors, but inter- ensuring pollination. (3) Th e proportions of viable seeds in plants pretation of diff erences between plant species should be made with are dependent on other factors not related to fl ower visitors; self- caution. Pollination effi ciency values for all fl oral visitors to Navaea compatibility can indeed infl uence seed viability, and geitonog- phoenicea were much lower than for those visiting Isoplexis fl owers amy may thus again not be adaptive in self-incompatible species. ( Rodríguez-Rodríguez et al., 2013 ), and the distribution of the two (4) Even if the proportion of pollen removed is accounted for as an components differed substantially. Whereas values for Isoplexis accurate measure of the fi tness of the male function, there is no cor- were moderate for both components, Navaea had much lower qual- responding measure to estimate the female function, because the itative scores and much higher quantitative scores. Th e most impor- numbers of pollen grains deposited on the stigmas by the same visi- tant diff erence was the restriction of pollinators to N . phoenicea tors are not comparable among diff erent taxa.

FIGURE 6 Photographs of bird visitors (A) Blue tit Cyanistes teneriff ae hanging on the fl ower in the search for nectar; (B) captured blue tit carrying pollen grains on the head ( n = 1476); (C) Sardinian warbler Sylvia melanocephala foraging in Navaea phoenicea in a perching position; (D) Canarian chiff chaff Phylloscopus canariensis perching to drink nectar. Image credits: A, B: A. G. Fernández de Castro; C, D: José Juan Hernández. 1566 • AMERICAN JOURNAL OF BOTANY

Insights for the origin of the Macaronesian bird–fl ower element— provide additional evidence for the paleoendemic Navaea . Th e Not all visitors exhibited a mutualistic behavior with Navaea stronger performance as pollinators of Sylvia is associated with a phoenicea. Our results present the fi rst evidence of the decisive foraging pattern, which indeed is equal to the behavior of African contribution of generalist passerines to the reproductive success of Nectariniidae. Th us, the syndrome also likely originated in the Af- the study species, the most threatened fl agship species of the MBE. rican continent. Th is trophic niche and bird pollination syndrome One relevant fi nding is the antagonistic role of C . teneriff ae , which were then translated into the Macaronesian archipelagos through can be discarded as playing a role in the evolution of the bird pol- island colonization, which would have facilitated selective pressure lination syndrome. Foraging episodes by C . teneriff ae can be con- on other plant lineages and plant visitors, extending ornithophily. sidered as an opportunistic behavior, such as the ones detected for Th e current outcome is an assembly of species where mainly orni- other Cyanistes species across islands and continents (Peters et al., thophilous species coexist with those that have mixed pollination 1995 ; Kvist et al., 2005 ). Nectarivory in this genus should be inter- syndromes ( Ojeda, 2013 ). preted as part of the generalist trophic condition, rather than a con- Th e in-depth study of ecological interactions can off er very use- sequence of trophic niche expansion due to density compensation ful information to interpret evolutionary patterns addressed by on islands. other disciplines such as biogeography. For achieving a more com- Conversely, the three mutualistic species, P . canariensis and Syl- prehensive vision of the evolution of the MBE, the evolution of via spp., showed very diff erent qualitative and quantitative contri- fl oral traits in each of the plant lineages should be assessed. In par- butions to the reproductive success of the plant. First, P . canariensis ticular, the assessment of characters such as color or nectar produc- was shown to be a legitimate and predictable visitor and, to a cer- tion can provide additional accurate information on how and why tain extent, effi cient pollinator. As a general rule, previous works generalist passerines currently visit such a diverse ensemble of en- centered on the Mediterranean and Macaronesian ornithophi- demic plants. lous taxa consistently identifi ed P . canariensis as a true pollinator ( Rodríguez-Rodríguez and Valido, 2008 , 2011 ; Ortega-Olivencia et al., ACKNOWLEDGEMENTS 2012 ). Th e high redundancy provided by the three passerine spe- cies, together with their diff ering roles across the pollinator landscape Th is article is dedicated to the memory of the late Efraín Hernández for each of the plant species, suggests that this level of pollinator and Elena Portellano, who established the basis for this study. We generalization can be considered as the most adaptive (Gómez thank Juan Carlos Illera, Beatriz Rumeu, M. Candelaria Rodríguez, et al., 2007 ). If this is the case, the selective pressure on fl oral traits Lola Guil, Laura García, Juan Carlos Atienza, Marcus Ljunqvist, and of Navaea may be the result of the joint action executed by the as- David P. Padilla for invaluable help in bird ringing. We appreciate semblage of the three true pollinators rather than by the individual Manuel Nogales (IPNA-CSIC) for his help and hospitality during reproductive contribution of each bird species alone. Th e extent to fi eldwork. Gobierno de Canarias provided us with permits for which the pollination eff ectiveness of true pollinators is suffi cient to sampling. We also thank Juan Antonio Calleja and Luis Suárez for maintain viable populations in this endangered plant species in the additional help during fi eldwork and Gemma Andreu for assistance future remains an open question. Although in some species of in laboratory work. Rubén Milla and Isabel Marques revised earlier the MBE the persistence of the population is ensured, the case of versions of this manuscript. We also thank two anonymous reviewers the endangered N. phoenicea highlights the need for a demographic for their exhaustive and constructive revisions. Th is research was assessment to determine the contribution of pollinators to the sur- funded by CSIC Intramural project 2006-3-OI-028, Spanish Ministry vival of natural populations. of Science and Innovation research project CGL2007-66516 to J.F.A. From a macroevolutionary perspective, this study provides evi- and a personal grant FPI 0266/2005 from Madrid Regional Govern- dence to address some of the questions regarding the origin of the ment (European Social Fund) to A.G.F.d.C. MBE. Th e phylogeny for the tribe Malveae dates Navaea phoenicea as an ancient insular species (6.7 Ma, Fuertes-Aguilar et al., 2003; LITERATURE CITED Escobar García et al., 2009; I. Villa et al., unpublished manuscript), which accounts for the relict origin of its ornithophilous condition Alonso , C. , C. M. Herrera , and T. L. Ashman . 2012 . A piece of the puzzle: A as the most plausible. Our fi ndings support the idea that generalist method for comparing pollination quality and quantity across multiple spe- passerines are currently able to maintain the selective pressure on cies and reproductive events. Th e New Phytologist 193 : 532 – 542 . Anderson , S. H. 2003 . The relative importance of birds and insects as pol- traits related to bird pollination. However, a paradox arises, as the linators of the New Zealand flora. New Zealand Journal of Ecology 2 7 : origin of the Macaronesian endemic avifauna is generally recent 83 – 94 . ( Illera et al., 2012 ). For example, phylogeographic evidence reveals Armbruster , W. S. , T. F. Hansen , C. Pélabon , R. Pérez-Barrales , and J. Maad . that the Canarian Sylvia species colonized the archipelago only 2009 . Th e adaptive accuracy of fl owers: Measurement and microevolution- 3.0–0.3 Ma (Dietzen et al., 2008). Th e colonization of Cyanistes oc- ary patterns. Annals of Botany 103 : 1529 – 1545 . curred 1.5 to 0.3 Ma (Kvist et al., 2005) and that of Canarian Phyl- Bates , D. M. 1969 . Systematics of the South African genus Anisodontea Presl loscopus canariensis occurred as early as 2.5 Ma (Helbig et al., 1996). (Malvaceae) . Gentes Herbarum 10 : 215 – 383 . Th ere is therefore a chronological breach between the stem ages of Bates , D. M. , B. Maechler, S. Bolker, and , and Walker. 2015 . Fitting linear paleoendemics such as Navaea and Canarina and the arrival of pol- mixed-eff ects models using lme4 . Journal of Statistical Soft ware 67 : 1 . linators to the archipelago. Even if the gap remains unexplained, Bayer , C. , and K. Kubitzki . 2003 . Malvaceae . In K. Kubitzki [ed.], Th e families and genera of vascular plants, vol. 5, 225–311. Springer, Berlin, Germany. this new evidence has contributed to the comprehension of the https://doi.org/10.1007/978-3-662-07255-4_28. evolution of the MBE. Carrascal , L. M. , E. Moreno , and A. Valido . 1994 . Morphological evolution and Canarina provides an indication of the roots of ornithophily in changes in foraging behaviour of island and mainland populations of Blue the Macaronesian islands, as the genus originated in Africa with Tit (Parus caeruleus )—A test of convergence and ecomorphological hypoth- ornithophily associated with nectarinids. Th e fi ndings of this study eses. Evolutionary Ecology 8 : 25 – 35 . OCTOBER 2017 , VOLUME 104 • FERNÁNDEZ DE CASTRO ET AL.—PASSERINE POLLINATION OF NAVAEA PHOENICEA • 1567

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