{"paper_id":"76837cc0-6787-4b9c-8e38-36b4365263d3","body_text":"Mixed-vertebrate pollination traits and pollinators of Haageocereus acranthus 1 \n(Cactaceae) in a Lomas desert ecosystem of coastal Peru  2 \n 3 \nBernardo Garcia-Simpson1* 4 \nBraulio A. Prado1,2 5 \nJuan J. Pellón3 6 \nAlfonso Valiente-Banuet4 7 \n 8 \n1Círculo de Investigación en Taxonomía, Florística y Ecología Vegetal, Departamento de Biología, 9 \nFacultad de Ciencias, Universidad Nacional Agraria La Molina, Lima, Perú 10 \n2Círculo de Investigación en Mamíferos del Perú, Departamento de Biología, Facultad de Ciencias, 11 \nUniversidad Nacional Agraria La Molina, Lima, Perú 12 \n3Laboratorio de Ecología y Conservación de Vertebrados Terrestres, Instituto de Ecología, Universidad 13 \nNacional Autónoma de México, Ciudad de México, México 14 \n4Laboratorio de Ecología de Comunidades, Instituto de Ecología, Universidad Nacional Autónoma de 15 \nMéxico, Ciudad de México, México 16 \n 17 \nRunning title: Mixed-vertebrate pollination in Haageocereus acranthus 18 \n*For correspondence. E-mail: bernardogarcia2303@gmail.com 19 \nKeywords: Bat pollination, floral traits, hummingbird pollination, intraspecific variation, morphology, 20 \nphenology. 21 \n 22 \nKey message: In a Lomas desert ecosystem of coastal Peru, the columnar cactus Haageocereus acranthus 23 \nshows traits and interactions consistent with a mixed -vertebrate pollination system (hummingbirds and 24 \nbats), with no detectable differences between flower color morphotypes in either traits or interactions. 25 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nAbstract 26 \n(1) Cacti are key components of arid ecosystems and, being mostly self -incompatible, rely on animal 27 \npollination. Although bee pollination is ancestral, systems supported by birds, moths, bats, and mixed 28 \nstrategies have evolved. Despite the ecological uniquenes s and extreme seasonality of the fog -dependent 29 \nLomas of coastal Peru, cacti pollination remains unstudied. This work examines Haageocereus acranthus, 30 \na characteristic columnar cactus of this ecosystem, in a population where individuals produce either white 31 \nor pink-red flowers. It was hypothesized that white flowers would be associated with bat pollination and 32 \npink-red flowers with hummingbird pollination, reflected in differences in floral morphology, phenology 33 \nand pollinator visitation. 34 \n(2) Year-round monitoring of the population was conducted to characterize flowering phenology. Floral 35 \nmorphology, daily anthesis patterns, and nectar production were quantified and compared between color 36 \nmorphs. Floral visitor frequency and behavior were recor ded using camera traps to test for pollinator 37 \npreference. 38 \n(3) Floral phenology, morphology, and nectar characteristics were broadly consistent with vertebrate 39 \npollination, showing traits associated primarily with bat pollination but also compatible with hummingbird 40 \npollination. These floral traits did not differ between color morphs. Hummingbirds were the most frequent 41 \nvisitors, followed by bats; yet, neither group showed preference for a specific flower color. 42 \n(4) Findings support a mixed pollination system  involving both hummingbirds and bats  in an ecosystem 43 \nwhere the availability of pollinators can shift over geographic or temporal scales . This pollinator 44 \nunpredictability may ensure consistent reproductive success and reduce vulnerability to the absence or 45 \ndecline of specific pollinator groups. 46 \n 47 \n 48 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nIntroduction  49 \nPollination is one of the most important mutualistic plant-animal interactions and has played a central role 50 \nin angiosperm diversification (Vamosi & Vamosi 2010; Ballesteros-Mejia et al. 2016). These interactions 51 \nreflect the evolutionary innovations of flowers in response to different ecological conditions (Armbruster 52 \n2017; Opedal 2019). In this context, species sharing pollinators have been under similar selective pressures, 53 \nresulting in a convergence of floral traits known as pollination syndromes (Faegri & van der Pijl 1979; 54 \nFenster et al. 2004; Dellinger 2020). 55 \nPollination syndromes have frequently been considered a principle governing plant -pollinator 56 \ninteractions and used to infer functional pollinator groups based on flower traits (Proctor et al. 1996; Fenster 57 \net al. 2004). However, as the understanding of pollination has expanded, increasing evidence supports that 58 \nthese patterns are more complex than the traditional syndromes hypothesis suggests (Ollerton  et al. 2009; 59 \nRosas-Guerrero et al.  2014; Dellinger 2020). Pollinator assemblages can be  highly variable across the 60 \ndistribution of a particular plant species (Thompson 2002), and interactions often show geographical or 61 \ntemporal changes (Olesen & Jordano 2002; Burkle & Alarcon 2011). Also, intraspecific variation in floral 62 \ntraits may contribute to deviations from consis tent classic syndromes, as different morphotypes within a 63 \nspecies distribution range can attract distinct pollinators groups (e.g., Schlumpberger et al. 2009; Cardona 64 \net al. 2020; Wenzell et al. 2025). These geographic patterns in interactions, along with intraspecific floral 65 \nvariation, can drive local adaptation, divergence in pollination strategies, and potentially lead to speciation 66 \n(Kay & Sargent 2009). 67 \nCacti are distributed across the Americas and constitute crucial components of arid and semi -arid 68 \necosystems (Fleming et al. 2001; Fleming & Valiente-Banuet 2002). In addition, these are obligate out -69 \ncrossers that depend on animals for pollination  (Mandujano et al.  2009) and show adaptations mostly 70 \nconsistent with classic pollination syndromes (Grant & Grant 1979; Rowley 1980; Schlumpberger 2011). 71 \nWhile bee pollination is ancestral in cacti, specialized systems for bird, moth, and bat pollination have 72 \nevolved; including mixed pollination strategies (Schlumpberger 2011; Lendel 2013 ) (e.g., Sahley 1996; 73 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nBustamante et al. 2010; Walter 2010). This diversity of strategies makes cacti an excellent group to explore 74 \nand test hypotheses on pollination syndromes. 75 \nPeru is a cacti species -rich country with ~250 spp.  distributed mainly in coastal and Andean 76 \necosystems (Ulloa et al.  2004; Arakaki  et al.  2006). However, although Peruvian cacti have received 77 \nconsiderable attention at taxonomic, distributional and genetic studies (e.g., Ostolaza 1996, 2014; Arakaki 78 \net al. 2006, 2007, 2021; Calderón et al. 2007), scarce effort has been made to understand the ir ecological 79 \ninteractions (e.g., Sahley 1996; Novoa et al. 2005, 2022; Ceroni et al., 2007).  80 \nHaageocereus acranthus (Tribe Trichocereeae) is a representative and abundant member of the 81 \ngenus, occurring predominantly in arid river valleys of Peru (Calderón et al. 2007). Flowers of H. acranthus 82 \nare relatively large and robust, funnel - to tubular- shaped, single-opening, nocturnal, white colored and 83 \nnectar-abundant. These traits have been putatively associated with bat and sphingid moth pollination by 84 \nCalderón et al. (2007), based on classic syndromes proposal by Faegri & van der Pijl (1979). However, this 85 \nspecies exhibits high variability in floral traits, including flower size, shape, and pigmentation ranging from 86 \nwhite to pink-red. This variation, along with extended anthesis times (crepuscular and matutinal), suggests 87 \npotential pollination by other groups, such as hummingbirds. While Rodophis vesper (Oasis hummingbird) 88 \nand Platalina genovensium (Peruvian long-tongued bat) have been reported visiting Haageocereus flowers 89 \n(Grillo & Arana 2016; Maguiña & Amanzo 2016), no further studies have an alyzed these interactions in 90 \ndetail. Also, c omparable traits occur in Weberbauerocereus weberbaueri , a closely related species 91 \npollinated by both bats and hummingbirds on the southern Peruvian coast (Sahley 1996). 92 \nThis study examined the floral morphology,  phenology, and pollinator assemblage  of 93 \nHaageocereus acranthus in a Lomas ecosystem of the Peruvian central desert coast, where the species 94 \nexhibits two distinct flower colors: white and pink-red, each expressed by different individuals. We 95 \nhypothesized that H. acranthus  flowers would generally display traits associated with ve rtebrate 96 \npollination, particularly by bats and hummingbirds, and that these visitors would act as effective pollinators. 97 \nBased on classic pollination syndromes, we further predicted intraspecific differences linked to flower 98 \ncolor: white flowers would be more closely associated with bat pollination, whereas pink-red flowers would 99 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nshow a stronger tendency toward hummingbird pollination. We anticipated that these flower types would 100 \noccupy distinct regions of floral morphological space and exhibit different phenological patterns, with pink-101 \nred flowers showing a broader anthesis and nectar secretion period that extend s into diurnal hours. 102 \nConsequently, we also expected differences in visitation frequency between bats and hummingbirds across 103 \nflower types. 104 \nMaterials and Methods  105 \nStudy area and species 106 \nThis study was conducted in Cardal (Pachacamac, Lima, Peru) (12°11'S, 76°50'W; 215 m a.s.l.), a location 107 \nwith a previously documented H. acranthus population (Ostolaza 1996; Calderón et al. 2007) (Fig. 1A). 108 \nThis area is part of the Lomas, a unique ecosystem dependent on winter fogs, which is mainly restricted to 109 \ncoastal Peru and Chile, and characterized by two contrasting seasons. The wet season (May –Oct), with 110 \nlower temperatures (13℃–16℃) and high relative humidity (95%–100%), promotes the growth of seasonal 111 \nherbs. In contrast, during the dry season (Nov –Apr), with relative humidity ranging from 80% –90% and 112 \ntemperatures between 20℃ –25℃, most herbs die off, leaving only perennial xerophytes ( Dillon et al. 113 \n2011). Fieldwork was conducted from January 2022 to March 2023. 114 \nH. acranthus is a columnar cactus up to 1.5 m tall, with multiple vertical stems branching at the 115 \nbase and flowers often growing from areoles at the branch apex (Calderón et al. 2007) (Fig. 1B).  Aside 116 \nfrom observations of  flowering from November to  January, no detailed phenological information is 117 \navailable (Calderón et al. 2007; Maguiña & Amanzo 2016 ). Two flower types are present in this area: 118 \ntypical white flowers of H. acra nthus subsp. acranthus and pink-red flowers of H. acranthus  var. 119 \nolowinskianus f. rubriflorior (Ostolaza 1996), currently a synonym of H. acranthus subsp. acranthus 120 \n(Calderón et al. 2007; POWO 2024). Flower types are hereafter referred to as white and pink-red floral 121 \nmorphs.  122 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nAlthough H. acranthus dominates the site, other cacti co-occur at much lower densities, including 123 \nH. pseudomelanostele (Werderm. & Backeb.) Backeb. and Loxanthocereus acanthurus (Vaupel) Backeb. 124 \nThe former is morphologically and phenologically similar to H. acranthus (Ostolaza 1996; Calderón et al. 125 \n2007), whereas the latter exhibits a hummingbird-pollination syndrome, flowering at a different time of the 126 \nyear (B. Garcia-Simpson, pers. obs.). 127 \nAnnual phenology 128 \nWe monthly monitored 30 tagged individuals throughout one year (Jan –Dec 2022). For every individual, 129 \nwe recorded the number of floral buds, open flowers and fruits (unripe and ripe). Unripe and ripe fruits 130 \nwere differentiated by coloration and firmness (firm and green for unripe, red and soft for ripe). Due to field 131 \nlimitations, monitoring was conducted exclusively on individuals with white flowers. However, qualitative 132 \nobservations from recurrent prior and subsequent visits to the study area (years 2021  to 2025; B. Garcia-133 \nSimpson, pers. obs.) suggest that the phenological pattern of the white morphotype is representative of the 134 \noverall population.  135 \nFloral traits – morphology 136 \nWe randomly collected 15 fully open flowers of each floral morph from different individuals. Longitudinal 137 \ncut sections were photographed in the field with an iPhone XR camera (12MP, f/1.8 aperture, 26mm focal 138 \nlength) (Apple, California, USA) against a black background with a reference scale.  Total length (TL), 139 \nperianth width (PW), tube length (TuL), tube width (TuW), stigma exsertion (StiE), stamen exsertion 140 \n(StaE), ovary length (OL), ovary width (OW), nectar chamber length (NL), and nectar chamber width (NW) 141 \n(Fig. 2A–D; terminology modified from Nassar et al. 1997) were measured from photos using ImageJ 142 \nv.1.54f (Schneider et al. 2012). 143 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFloral traits – daily phenology 144 \nTo characterize flower aperture and nectar production, we measured the perianth width of 23 flowers ( 11 145 \nwhite, 12 pink-red) from different individuals at eight stages of anthesis (15:00, 17:00, 19:00, 23:00, 03:00, 146 \n05:00, 07:00, 09:00) using a 0.05 mm resolution mechanical caliper (Uyustools, Hangzhou, China). Nectar 147 \nvolume, sugar concentration, and energy content were assessed at five anthesis stages (15:00, 19:00, 23:00, 148 \n03:00, 06:00) using bagged flowers from different individuals. Nectar volume was measured in 34 flowers 149 \n(18 white, 16 pink-red), while sugar content and energy supply were evaluated from a subset of 17 flowers 150 \n(10 white, 7 pink-red). Nectar volume was quantified, following Ibarra-Cerdeña et al. (2005), by extracting 151 \nall possibl e nectar using a capillary tube inserted into the nectar chamber. Total nectar volume was 152 \ncalculated by multiplying the column length by the specified cross-sectional area of the capillary tube. A 153 \nfraction of the nectar was used to determine sugar concentr ation with a handheld refractometer (BRIX30) 154 \nwith automatic temperature compensation; readings were expressed as sucrose percentage following Dafni 155 \n(1992) and energy supply was calculated using the following formula: 156 \n𝐽 = 16.8[( 𝑆\n100) ∗ 𝑉𝐷] 157 \nwhere J is energy in joules, S is the sugar percentage, V is nectar volume in microliters, and D is the density 158 \nof sucrose at the observed concentration (Dafni 1992; Ibarra-Cerdeña et al. 2005). 159 \nFrequency and behavior of floral visitors 160 \nWe monitored floral visitors using two Bushnell 24MP Core Low Glow Trail Cameras (Bushnell 161 \nCorporation, Kansas, USA) placed 1 –2.5 meters from flowers to capture interactions with vertebrate and 162 \nlarge invertebrate visitors. A total of 22 ( 11 white, 11 pink-red) flowers from different individuals were 163 \nobserved over 11 non -consecutive days during the flowering season (Sep 2022 –Mar 2023), totaling 378 164 \nobservations hours (~17 hours per flower). Monitoring started in the early afternoon and continued until 165 \nmid-morning the following day. Based on Ibarra -Cerdeña et al. (2005), we recorded visitor species and 166 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nnumber of legitimate visits (i.e., involving contact with reproductive structures) for each flower. Additional 167 \nvisitors were documented through direct observation throughout anthesis. 168 \nPreliminary pollinator exclusion experiments 169 \nTo evaluate how the absence of different pollinator groups influenced fruit initiation, we implemented five 170 \ntreatments: (i) control, with flowers left fully exposed; (ii) bat exclusion, covering flowers at night with a 171 \ncylindrical metallic mesh; (iii) hummingbird exclusion, applying the same mesh only during the daytime; 172 \n(iv) nocturnal exclusion, fully bagging flowers at night to prevent all nocturnal visitors; and (v) diurnal 173 \nexclusion, fully bagging flowers during the daytime to block diurnal visitors. We monitored each flower 174 \nover the following days to record whether it abscised or initiated ovary expansion. Due to fieldwork 175 \nconstraints, sample sizes were limited to 12 flowers per morph in the control treatment and six flowers per 176 \nmorph in each exclusion treatment. Therefore, results are presented descriptively as preliminary evidence, 177 \nwithout formal statistical analyses. 178 \nData analyses 179 \nAll statistical analyses were conducted in R v.4.4.1 (R Core Team 2024) using RStudio (Posit Team 180 \n2024). 181 \nIntraspecific morphological variation. We evaluated the effect of flower morph (white vs pink-red) on 182 \nall morphometric variables using a MANOVA (Pérez -Barrales et al. 2007). Variables were standardized 183 \n(mean = 0, standard deviation = 1) for comparability. To avoid multicollinearity, we examined correlations 184 \namong morphometric variables: total length, tube length, and nectar chamber length were highly correlated 185 \n(Pearson’s r > 0.8), therefore, we excluded tube length and nectar chamber length from the test. The analysis 186 \nwas performed with the manova function. 187 \nModelling flower anthesis and nectar production. We fitted Generalized Linear Mixed-Effects 188 \nModels (GLMMs) to examine the effects of time after midday (hours) and flower morphotype (white vs 189 \npink-red) on four response variables: (i) flower aperture, (ii) nectar volume, (iii) nectar sugar concentration, 190 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nand (iv) nectar energetic content. In the absence of specific hypotheses regarding differences among 191 \nmorphotypes (e.g., response magnitude or peak times), we opted for an exploratory model selection 192 \napproach by generating a set of biologically plausible candidate models and selecting the best fit. 193 \n Model selection was conducted separately for each response variable. Candidate models were built 194 \nusing the glmmTMB package (Brooks et al. 2017), with Flower ID included as a random effect to account 195 \nfor individual variability. Time after midday (T) and flower morphotype (M) were included as fixed effects, 196 \ntesting first-, second-, and third-order orthogonal polynomial terms for time to capture potential curvilinear 197 \nrelationships. Fixed effects combination in candidate models included: T, T + M, T * M, T 2, T2 + M, T2 * 198 \nM, T 3, T 3 + M, T 3 * M. When a model includes a polynomial of degree n, it inherently incorporates all 199 \nlower-order terms (e.g., a T³ model also includes T and T²). Here, ‘+’ indicates additive effects, while ‘*’ 200 \ndenotes additive and interaction effects. Flower aperture and sugar content were modeled using a Gaussian 201 \ndistribution, while nectar volume and energetic content were modeled with a Tweedie distribution, chosen 202 \nfor its suitability in handling continuous data with a high proportion of zeros. Model diagnostics; presence 203 \nof over/under-dispersion, outliers, and zero inflation, were assessed using the DHARMa package (Hartig 204 \n2022). Model selection indices were computed using compare_performance from the performance package 205 \n(Lüdecke et al. 2021) and model.sel from the MuMIn package (Barton 2024). Final selection was based on 206 \nthe Akaike Information Criterion cor rected for small sample sizes (AICc), with models differing by ≥  2 207 \nAICc units considered significantly better fits (Burnham & Anderson 2002).  208 \nFrequency and behaviour of floral visitors.  To visualize the temporal activity patterns of main 209 \nfloral visitors, we used a kernel density estimation (KDE) with the density function (bandwidth = 1). 210 \nDensities were scaled by the total number of visits to make activity patterns visually comparable across 211 \nvisitor groups with differing number of visits.  212 \nTo dete rmine if the number of visits varied among floral visitors and flower types, we built 213 \nGeneralized Linear Mixed-effects Models (GLMMs) with a Poisson distribution (log-link). Flower ID was 214 \nconsidered as a random effect, while visitor type (bat, hummingbird or sphingid) and flower morphotype 215 \nwere considered as fixed effects. We evaluated three models including effects of (i) visitor type (V), (ii) 216 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nvisitor type and flower morphotype (V + M), and (iii) visitor type, flower morphotype, and their interaction 217 \n(V * M). To account for differences in evaluation time among individual flowers, all models included the 218 \nlog-scaled evaluation time of each flower as an offset variable. Model fitting, diagnostics, and selection 219 \nwere performed as previously described. Based on  the best -supported model, post-hoc pairwise 220 \ncomparisons of visitation frequencies among floral visitors were conducted using the emmeans package 221 \n(Lenth & Lenth 2018). Pairwise differences were evaluated with the pairs function, applying a Tukey 222 \nadjustment to account for multiple comparisons. 223 \nResults 224 \nAnnual phenology 225 \nH. acranthus from Cardal displayed a single flowering peak during the year. Bud and flower production 226 \noccurred primarily from January to April, followed by a sharp decline. From May to September, plants 227 \nremained mostly vegetative with minimal reproductive activity. Bud production resumed in October, 228 \npeaking in November, resulting in a high number of open flowers. Fruit production followed this pattern 229 \nbut with a slight delay due to the s equential nature of these reproductive stages.  Overall, reproduction 230 \noccurred during the dry season (Nov–Apr), while the vegetative phase in the humid season (May–Oct) (Fig. 231 \n3A–B; Table S1).  232 \nFloral traits – morphology 233 \nH. acranthus  flowers were funnelform to tubular in shape and radially symmetric , but occasionally 234 \nexhibited a certain degree of zygomorphy (Fig. 2B). The flower tube exhibited a constriction above the 235 \nnectar chamber, which limits access to nectar. Perianth color varied from white and white/green to pink-236 \nred, but remained consistent within individuals (Fig. 2C–D). Overall morphometry of the two floral morphs 237 \ndid not differ (Table S2), as supported by the MANOVA results (Pillai’s Trace = 0.221, F (8, 23) = 0.813, 238 \nP = 0.598). 239 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFloral traits – daily phenology 240 \nUnless otherwise indicated, model estimates are reported with 95% confidence intervals (mean ± 95% CI). 241 \nFlower aperture. The top-ranked model included time up to the cubic term (T, T², T³) and its 242 \nrespective interactions with morphotype (AICc = 467.29, weight = 0.560). The second -best model 243 \nadditionally included the main effect of morphotype (ΔAICc = 2.31, weight = 0.176) (Table S3). The strong 244 \nsecond-order polynomial effect confirmed the parabolic pattern in flower aperture over time, showing the 245 \nexpected non-linear pattern of anthesis. The interaction terms, present in the final model but with a weak 246 \neffect, revealed that this parabolic relationship varied between morphs, highlighting slight differences in 247 \nhow aperture responds to time. Both floral morphs reached maximum aperture around 23:00 (white: 6.51 248 \n± 0.3 cm, pink-red: 5.9 ± 0.3 cm) and differences between morphs were  observed in the early stages of 249 \nanthesis, where the pink-red morph showed higher estimates than the white morph, indicating a slightly 250 \nearlier aperture initiation (white: 14:34, pink-red: 12:81) (Fig. 4A, Table 1A). Visually, styles and stigmas 251 \nremained turgid throughout anthesis. 252 \nNectar volume. The best model for nectar volume included time up to the third-order polynomial 253 \nterm but excluded interactions with morphotype and its main effect (AICc = 1025.65, weight = 0.920). The 254 \nsecond-best model included interactions with morphotype (ΔAICc = 5.46, weight = 0.060). The main effect 255 \nof morphotype was present in models of lower rank (Table S 4). The second- and third-order polynomial 256 \nterms together indicated a parabolic pattern in nectar volume over time, with an initial rise in late afternoon 257 \nto a peak up to 93.5 ± 13.5 µL at 21:00 followed by a less steep decline, where lower quantities of nectar 258 \nwere available until the following morning (Fig. 4B, Table 1B).  259 \nSugar content. The three best -ranked models showed ΔAICc < 2 among them ; we selected the 260 \nsimpler model, which included the linear effect of time without the interaction with morphotype nor its 261 \nmain effect (AICc = 164.00, weight = 0.265). This model showed equivalent support compared to more 262 \ncomplex models that included the second and third-order effect of time (AICc = 163.28, weight = 0.379), 263 \nor the main effect of morphotype and its interactions (AICc = 165.08, weight = 0.154) (Table S 5). The 264 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nmodel estimates an initial content of sugars of 23.5 ± 0.5  % at 18:30, showing a linear decline over time, 265 \nreaching lower values of 15.4 ± 0.8 % at 07:40 (Fig. 4C, Table 1C). 266 \nEnergy content. The best model for nectar energ y content included time up to the third -order 267 \npolynomial term but excluded morphotype effects (AICc = 540.06, weight = 0.738). The second -best 268 \nmodel, which added interactions with morphotype, had less support (ΔAICc = 3.05, weight = 0.160). The 269 \nmain effect of morphotype was included in models of lower rank (Table S6). Energetic supply followed a 270 \nsimilar pattern to nectar secretion, with an initial rise in late afternoon to a peak up to 529.8 ± 77.8 J at 271 \n20:30 followed by a less steep decline lasting until the following morning (Fig. 4D, Table 1D).  272 \nFrequency and behavior of floral visitors 273 \nWe recorded 11 invertebrate and two vertebrate species of floral visitors; two nocturnal, five diurnal, and 274 \nsix active during both periods (Table 2). Nine did not contact the reproductive structures, making them 275 \nunlikely pollinators due to their incompatibility with floral morphology. Two species occasionally made 276 \ncontact with the reproductive structures, while the remaining two consistently made eff ective pollinator 277 \nvisits, regularly contacting both reproductive structures while accessing floral resources (Table 2). 278 \nAmong the small flower visitors, two ant species (Formicidae) were observed visiting flowers at 279 \nnight, primarily for nectar and often in large numbers (> 20 individuals). These ants continued harvesting 280 \nnectar until the flowers senesced (Fig. S1A–C). One sap beetle species (Carpophilus sp., Nitidulidae) was 281 \nfound inside flowers, likely feeding on pollen or plant tissue, and was more abundant in older flowers (Fig. 282 \nS1D). Three spider species (Anyphaenidae, Thomisidae , and Salticidae) were recorded on the flowers, 283 \noccupying the external zones of the floral tubes and perianth, acting as predators (Fig. S1E–F; Fig. S2A–284 \nB). Two Diptera species were documented; one was abundant and found inside the floral tubes (Phoridae) 285 \n(Fig. S2C), and a syrphid was observed once feeding on pollen. The common bee ( Apis mellifera) was 286 \nfrequently observed collecting pollen from anthers but rarely contacting the stigma, except when gathering 287 \nin larger groups (> 5 individuals) (Fig. S2D), while the metallic-green bee Caenohalictus sp. (Halictidae) 288 \nwas also recorded (Fig. S1E–F). 289 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nRegarding large flower visitors, the hummingbird Rhodopis vesper (Trochilidae) was the most 290 \nfrequent (Fig. 5 A–B). Their activity started in the late afternoon (17:00) until sunset (18:30 –19:00) and 291 \nresumed in the early morning (05:00) until the end of anthesis  (9:00–11:00) (percentage of total visits: 292 \nwhite: 71.4%, pink-red: 81.4%). They hovered or, in occasions landed on flowers to access nectar, always 293 \ncontacting stamens and stigma. The Peruvian Long-tongued Bat, Platalina genovensium (Phyllostomidae), 294 \nwas the second most frequent visitor (Fig. 5C–D), active at night (20:00–03:00), peaking at 22:00 (white: 295 \n20.6%, pink-red: 16.3%). Unlike R. vesper, bats made single contacts to flowers while hovering but never 296 \nlanded on them. Visits by h awkmoths (Manduca sp., Sphingidae, Lepidoptera) were rare and recorded at 297 \ndusk or at night (white : 7.9%, pink-red: 2.3%). They either hovered or landed on flowers, but their body 298 \nnot always contacted reproductive structures (illegitimate visits = 2, legitimate visits = 4) (Fig. 5E–F).  299 \nThe model including only the effect of flower visitor was selected as the best-fitting model (AICc 300 \n= 209.6, weight = 0.717). The additive (ΔAICc = 2.32, weight = 0.225) and multiplicative models received 301 \nsignificantly less support ( ΔAICc = 5.05, weight = 0.057) (Table S 7). In consequence, we compared 302 \nvisitation frequencies among visitors based on the first model (Table 1E); which showed sphingids had 303 \nsignificantly lower visitation rates compared to both P. genovensium (estimate = -1.20, SE = 0.465, P = 304 \n0.0262) and R. vesper (estimate = -2.59, SE = 0.423, P < 0.001). Additionally, P. genovensium exhibited 305 \nlower visitation rates than Rhodopis vesper (estimate = -1.39, SE = 0.250, P < 0.001). 306 \nPreliminary pollinator exclusion experiments 307 \nControl flowers showed the highest fruit  initiation rates (white: 41.7%; pink -red: 33.3%). Bat exclusion 308 \nreduced fruit initiation in the white morph but not in the pink-red morph (white: 16.7%; pink-red: 33.3%). 309 \nHummingbird exclusion lowered fruit initiation in both morphs (white: 16.7%; pink-red: 16.7%). Nocturnal 310 \nexclusion produced moderate initiation in the white morph but low initiation in the pink-red morph (white: 311 \n33.3%; pink -red: 16.7%), and diurnal exclusion resulted in the same pattern (white: 33.3%; pink -red: 312 \n16.7%). In general, fruit initiation was low across all treatments. When morphs were pooled, most 313 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\ntreatments showed a fruit initiation rate of 25%, except for the hummingbird -exclusion treatment, which 314 \nshowed 17%. The control flowers exhibited the highest fruit initiation rate at 37.5%. 315 \nDiscussion 316 \nAnnual phenology 317 \nThe Lomas formations are marked by strong seasonality in floral resources availability, with most plant 318 \nspecies reproducing during the wet season (Tovar et al. , 2018). In contrast, our data show s that 319 \nHaageocereus acranthus flowers in the dry season, with a single reproductive peak. Similar dry -season 320 \nflowering has been reported at Lachay National Reserve  by Maguiña & Amanzo (2016), and Calderón et 321 \nal. (2007) describe flowering in January and fruiting in February. This recurring pattern across different 322 \nLomas populations of H. acranthus suggests that rising temperatures (Servicio Nacional de Meteorología 323 \ne Hidrología del Perú - SENAMHI; Figure S1), and reduced humidity may act as cues for bud development, 324 \nalthough this requires further testing. Flowering during resource-limited periods positions H. acranthus as 325 \na potential key species in Lomas ecosystems, providing an important resource for associated fauna. 326 \nFloral morphology, phenology, and expected pollination syndrome 327 \nThe floral dimensions of H. acranthus are consistent with those of other vertebrate -pollinated cacti. Its 328 \nlarge, sturdy flowers can withstand landings and manipulations by relatively large visitors, while the broad 329 \nfrontal area (~6.3 cm diameter) likely enhances detection by bats through echo location, as shown for 330 \nPachycereus (González-Terrazas et al., 2016). This surface also provides ample contact for pollen transfer. 331 \nEach flower bears 200 –400 stamens (Calderón et al., 2007), indicating high pollen output and potential 332 \nseed set. Comparable b at-pollinated species produce 10⁵ –2×10⁵ pollen grains per flower (Nassar et al., 333 \n1997), with Pilosocereus reaching ~500 stamens, ~2000 ovules, and ~1100 seeds per fruit (Martins et al., 334 \n2020). Together, these traits suggest that H. acranthus depends on pol linators with high pollen -carrying 335 \ncapacities to ensure reproductive success within its short floral lifespan (< 24 h). 336 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nThe floral tube is determinant in cacti plant-pollinator interactions (Schlumpberger 2011). In H. 337 \nacranthus, an average tube length of 4.8 cm restricts nectar access to visitors with sufficiently long feeding 338 \nstructures, establishing a threshold for effective foraging. Some species with shorter feeding structures can 339 \nstill exploit the resource by inserting their head or rostrum into the funnel-shaped tube, which allows deeper 340 \naccess than in narrower flowers (e.g., R. vesper; Fig. 5B). Experimental studies further show that tube width 341 \ninfluences foraging behavior: wider tubes extend the feeding reach of nectar -feeding bats (Winter & von 342 \nHelversen 2003; Nicolay & Winter 2006), whereas narrower tubes increase hummingbird handling times 343 \n(Smith et al. 1996). Finally, visitors with feeding structures exceeding ~6.8 cm (tube length plus stamen 344 \nexsertion) may obtain nectar without contacting the rep roductive organs, thereby acting as nectar robbers 345 \n(e.g., sphingid moths; Fig. 5F). 346 \nMonitoring of flower phenology confirmed that H. acranthus flowers open only once, with peaks 347 \nin aperture, nectar secretion, and energy availability occurring at night (Fig. 4A –D). Total nectar 348 \nproduction, sugar content, and energetic supply were high, comparable to values reported for cacti 349 \npollinated by hummingbirds and bats (e.g., Rowley 1980; Grant & Grant 1980; Schlumpberger et al. 2009, 350 \nSchlumpberger 2011; Albuquerque-Lima et al. 2023). Although flower opening varied among morphotypes 351 \n(pink-red flowers opened slightly earlier than white flowers), nectar characteristics did not differ, providing 352 \ninsufficient evidence for phenological divergence among morphs. These ch aracteristics suggest an 353 \nassociation with pollination by nocturnal vertebrates or large invertebrates (Baker 1975; Medel et al. 2022). 354 \nHowever, anthesis extended into the late  afternoon and morning, whe n small amounts of nectar were 355 \navailable (Fig. 4B), allowing diurnal vertebrates to forage flowers and potentially contribute to pollination 356 \n(Baker 1961; Miyake & Yahara 1999). 357 \nAlthough pink-red and white pigmentation are traditionally linked to diurnal hummingbird and 358 \nnocturnal bat/sphingid pollination, respectively, our analyses did not reveal differences among white and 359 \npink-red floral morphs. Besides slight differences between flower aperture patterns, floral morphology and 360 \nphenology were consistent across morphs. Overall, the observed floral traits strongly support the hypothesis 361 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nof primary adaptation to bat pollination, with characteristics also associated to hummingbird pollination. In 362 \ncontrast, the relatively short floral tube indicates only limited adaptation to sphingid pollination. 363 \nEmpirical evidence of flower visitors and pollinators 364 \nVisitors are here  grouped into three categories based on morphology, behavior, and contact with 365 \nreproductive organs: (1) ants, sap beetles, flies, and arachnids, whose foraging rarely involves stigma or 366 \nstamen contact and thus contribute little to no pollination; (2) bees and sphingid moths, which may 367 \noccasionally transfer pollen but more often act as nectar or pollen thieves; and (3) vertebrates, whose traits 368 \nalign more closely with floral morphology and are therefore expected to be the most effective pollinators. 369 \nAnts, sap beetles, flies and arachnids. Ants were frequently observed in large groups on flowers, 370 \nbut their small size, unlikely stigma contact, and foraging behavior suggests negligible contributions to  371 \npollen transfer (Rico -Gray 1989; Fagua & Ackerman 2011; LeVan et al. 2014). Similarly, Carpophilus 372 \nbeetles, common in both wild and cultivated cacti, ac t as pollen thieves  they feed on nectar, lay eggs in 373 \nbuds, and their larvae consume decaying flowers  (Grant & Connell 1979; Miranda -Jácome et al. 2021). 374 \nDipterans frequently visited H. acranthus flowers but rarely contacted r eproductive organs due to their 375 \nsmall size, making them ineffective pollinators (Rowley 1980; McIntosh 2005; Schlumpberger et al., 2009). 376 \nFinally, spiders were exclusively predatory visitors, typically using flowers as hunting grounds  (Su et al. 377 \n2020), though occasional resource use has been reported (e.g., Nelson 2023). 378 \nBees and sphingid moths.  Bees are frequent visitors due to their pollen -collecting behavior 379 \n(Westerkamp 1996; Thorp 2000) and, when morphologically compatible, can be effective cross-pollinators 380 \n(Armbruster et al. 1989; Solís‐Montero & Vallejo‐Marín 2017). In H. acranthus, however, the large flowers 381 \nand exserted stigmas limit bee -stigma contact,  which could reduce their effectiveness as pollinators. 382 \nAlthough not quantified in our study, studies on tropical cacti with similar floral traits and growth habit 383 \nsuggest that the contribution of bees and other small insects to pollination is low or null (e.g., bees, flies, 384 \nand butterflies on Weberbauerocereus in Sahley 1996; A. mellifera on Pilosocereus in Rivera-Marchand & 385 \nAckerman 2006; Xylocopa grisescens on Pilosocereus in Rocha et al. 2019; A. mellifera on Cipocereus in 386 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nMartins et al. 2020). In contrast, in temperate zones bees often act as effective pollinators, even in species 387 \nwith floral traits associated with bats or moths, reflecting more generalized pollination strategies (Valiente-388 \nBanuet et al. 1996; Fleming et al. 2001). 389 \nThe traits of H. acranthus may also suggest sphingid moth pollination (e.g., white pigmentation, 390 \nnocturnal anthesis, nectar rewards). However, sphingids accounted for less than 10% of visits and were 391 \nobserved both landing on flowers or accessing nectar without contacting the stamens or stigma. This 392 \nbehavior has been previously documented in moths, as it allows t hem to feed from the relatively short -393 \ntubed flowers of while avoiding close approaches that increase predation risk (Wasserthal 2001). By 394 \ncontrast, species adapted to sphingid pollination usually possess much longer floral tubes (e.g., > 10 cm in 395 \nGrant & Grant 1979; > 15 cm in Silva & Sazima 1995; > 15 cm in Schlumpberger et al. 2009; ~24 cm in 396 \nAlbuquerque-Lima et al. 2023). Thus, the role of hawkmoths as pollinators of H. acranthus results unlikely. 397 \nBats and hummingbirds.  Hummingbird foraging overlapped with anthesis only during 398 \ncrepuscular and early morning hours, yet their visits accounted for a major proportion of total observations. 399 \nThe high frequency of R. vesper  visits highlights its potential role as a pollinator, ev en though nectar 400 \navailability at these times was often low or undetectable . This can be explained by the fact that nectar 401 \nmeasurements involved repeated extraction throughout anthesis; therefore, morning measurements 402 \nreflected nectar levels under continuous extraction rather than natural overnight accumulation and cannot 403 \nrule out nectar persistence in flowers unvisited during the night.  404 \nIn Cactaceae, hummingbird -pollinated flowers are typically red to orange, less robust, 405 \nzygomorphic, and tubular (e.g., Loxanthocereus, Borzicactus, Matucana spp.) ( Grant & Grant 1979; 406 \nRowley 1980; Schlumpberger 2011). Although the morphology of H. acranthus does not fully match this 407 \nbauplan, it exhibits secondary compatibility with R. vesper to access nectar while ensuring contact with 408 \nreproductive structures. Visitation rates did not differ between morphs, indicating that both floral color 409 \nvariants are equally attractive to hummingbirds. Overall, the frequent and effective visits of R. vespe r 410 \nsupport its role as a compatible pollinator, consistent with reports of this species visiting Peruvian cacti 411 \nlacking typical ornithophilous traits (Sahley 1996; Novoa et al. 2022). 412 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nThe synchronization of H. acranthus phenology with the nocturnal activity  of P. genovensium, 413 \ncoinciding with peak flower aperture and nectar availability is consistent with bat pollination (e.g., Valiente-414 \nBanuet et al. 1996; Nassar et al. 1997; Ibarra-Cerdeña et al. 2005; Rocha et al. 2019; Martins et al. 2020; 415 \nAlbuquerque-Lima et al. 2023). Morphologically, P. genovensium is well-suited to exploit these flowers, 416 \nas its long feeding structures match the floral tube. Observations of individuals carrying Haageocereus 417 \npollen (Maguiña & Amanzo 2016), along wit h its status as a cactus specialist (Sahley & Baraybar 1996), 418 \nfurther support its role as a pollinator. Although bats were not the most frequent visitors, they exhibited the 419 \ngreatest morphological fit to the flowers, suggesting higher pollen transfer efficiency per visit compared to 420 \nhummingbirds (Muchhala & Thomson 2010) . Thus, P. genovensium  may represent the most effective 421 \npollinator of H. acranthus, as Leptonycteris yerbabuenae does for several columnar cacti in North America 422 \nbelow 22º latitude (e.g., Tremlett et al. 2020).  423 \nPreliminary pollination exclusion experiments in other Lomas (Lachay, northern Lima) suggest 424 \nthat H. acranthus is primarily pollinated at night (Grillo & Arana 2016). In Lachay, hummingbirds visited 425 \nflowers frequently, but bat activity was slightly higher. Combined with our observations, this evidence 426 \nindicates that H. acranthus is mainly adapted to bat pollination, though the predominance of chiropterophily 427 \nmay depend on the presence of P. genovensium. This bat species is highly dependent on cactus flowers and 428 \nintact arid habitats, and its distribution is restricted to well -preserved areas that are rapidly declini ng in 429 \nLima (Ossa et al. 2020; Lambert 2021). Accordingly, P. genovensium has not been reported from urban 430 \nenvironments, where only generalist bats persist by feeding on exotic plants (Pellón et al. 2021). The peri-431 \nurban setting of our study site, surrounde d by towns and agriculture, contrasts with Lachay, a conserved 432 \nNational Protected Area (Dourojeanni 2018), likely explaining the greater prevalence of hummingbird 433 \nvisitation at Cardal. In addition, our preliminary exclusion experiments showed that , although fruit 434 \nproduction been generally low, all exclusion treatments tended to produce equal or lower fruit initiation  435 \nthan open flowers, suggesting that both diurnal and nocturnal pollinators contribute to fruit set. Together, 436 \nthese patterns support that the m ixed-vertebrate pollination system of H. acranthus  may facilitate its 437 \npersistence even where its ideal bat pollinators decline or are absent. 438 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nOur findings partially align with those of Sahley (1996) for Weberbauerocereus weberbaueri in 439 \nArequipa (southern Peru). This closely related species (Tribe Trichocereeae) shares similar morphological 440 \nand phenological floral traits and is also primarily visited by P. genovensium and R. vesper (although in W. 441 \nweberbaueri the wine-colored flowers are strongly z ygomorphic, unlike in H. acranthus). Sahley (1996) 442 \ndocumented interannual shifts in pollinator dominance, with bats prevailing in one year and hummingbirds 443 \nin other, a pattern linked to El Niño events that alter rainfall, vegetation, and bat abundance. We propose 444 \nthat H. acranthus may follow a similar strategy, with mixed floral traits enabling dual pollination. Such 445 \nflexibility is likely advantageous in the seasonal Lomas ecosystem, where extreme climatic events like El 446 \nNiño strongly influence resource availability (Ferreyra 1993; Cano et al. 1999). The hypothesized migratory 447 \nbehavior of P. genovensium (Sahley 1996; Sahley & Baraybar 1996; Ossa et al. 2020) further underscores 448 \nthe value of maintaining alternative pollinators: hummingbirds may provide reliable service during periods 449 \nof reduced bat activity, thereby supporting reproductive success under fluctuating conditions (Muchhala & 450 \nThomson 2010). Finally, the disjunct distribution of H. acranthus  along the Peruvian coast, with 451 \npopulations separated by large distances, aligns with bat-mediated pollination, as bats are highly effective 452 \nlong-distance pollen vectors, promoting genetic connectivity despite habitat fragmentation (Fleming et al. 453 \n2009).  454 \nMixed-pollination strategies in a broader context 455 \nThe most effective pollinator principle states that floral traits should evolve toward the selective optimum 456 \nimposed by the pollinator contributing the most to reproductive success ( Stebbins 1970), but this is not 457 \nalways observed. When no single guild imposes strong or consistent selection, floral phenotypes matching 458 \nmultiple guilds can be favored because their combined contributions exceed those provided by 459 \nspecialization. Such mixed strategies often emerge when main pollinator abundances, and thus their relative 460 \ncontributions to plant fitness, shift across space or time (Kay & Anderson, 2025).  461 \nIn some cases, mixed pollination occurs even in species with clear floral syndromes. For example, 462 \nMarginatocereus marginatus  in Mexico , although exhibiting traits associated with hummingbirds, 463 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\ncompetition with co-flowering columnar cacti reduced the reliability of individual pollinator guilds, and its 464 \ndual nocturnal -diurnal anthesis allowed reliance on both bats and hummingbirds (Dar et al. 2006) . In 465 \ncontrast, we consider it unlikely that H. pseudomelanostele  or L. acanthurus  generate meaningful 466 \npollination interference for H. acranthus at our site, as both species occur at much lower densities, and L. 467 \nacanthurus shows little to no flowering overlap with H. acranthus. However, this is a possibility in several 468 \nother Lomas populations of coastal Peru, where this cactus coexists with multiple hummingbird- and bat-469 \npollinated species, often showing reproductive compatibility even across genus (Arakaki et al. 2020). 470 \nGeographic variation in pollination systems has been documented in cacti. Schlumpberger et al. 471 \n(2009) found shifts from bee pollination (short, morning-opening, low-nectar flowers) sphingid pollination 472 \n(long-tubed, dusk-opening, nectar-rich flowers) across populations of Echinopsis ancistrophora along an 473 \naltitudinal gradient in Argentina. In Mexico, Stenocereus thurberi also showed spatial differences: northern 474 \nand central populations relied mainly on bats with no pollen limitation, whereas southern populations on 475 \nmixed pollinators and showed pollen limitation, likely due to temporal variation in pollinator availability 476 \n(Bustamante et al. 2010). Pachycereus pecten -aboriginum also displayed geographic differentiation in 477 \nanthesis and nectar secretion that aligns with nocturnal versus mixed nocturnal -diurnal pollination in 478 \nValiente-Banuet et al. (2004). This tendency to more generalized pollination systems has also been 479 \ndocumented in other Pachycereeae  such as Carnegia gigantea (Fleming et al., 1996 ); supporting that 480 \ngeneralized strategies appear to be more reliable in zones where bats predictability is lower (e.g., northern 481 \nMexico versus central and Southern regions, Rojas-Martínez et al. 1999). These dynamics remain unstudied 482 \nin Peruvian cacti. The mixed pollination strategy we propose for H. acranthus may be shaped by local 483 \nfactors such as habitat condition, pollinator abundance, and the presence or absence of co-flowering cacti. 484 \nHowever, since our data come s from a single site and one sampling year, it cannot reveal if pollination 485 \nmechanisms vary across space or time, or whether such variation explain s possible differences among 486 \npopulations. Wider geographic coverage and long-term studies will be essential to address these questions. 487 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nConclusions 488 \nOur study shows that H. acranthus  exhibits floral traits aligned with nocturnal bat pollination while 489 \nremaining compatible with diurnal hummingbird pollination, with both likely serving as the main effective 490 \npollinators. In contrast, bees and sphingids mismatched key floral traits and likely acted primarily as nectar 491 \nor pollen thieves. We found no differences in floral traits or visitation frequencies between color morphs, 492 \nproviding no support for our initial hypothesis. Overall, our findings indicate a mixed vertebrate pollination 493 \nsystem involving both bats and hummingbirds. In the strongly seasonal Lomas ecosystem, where pollinator 494 \navailability can shift across space and time, maintaining interactions with multiple pollinator guilds may 495 \nenhance reproductive stability. Our results highlight the need for broader spatial and temporal studies of H. 496 \nacranthus reproduction. 497 \nAcknowledgements 498 \nWe thank A. Ceroni and M. Flores (Weberbauer Herbarium -MOL) for their support in obtaining 499 \nfunding and for their contributions during the design of the study ; C. Reynel and S. Terreros (Forestry 500 \nHerbarium-MOLF) for receiving the botanical samples; M. Alvarado and E. Medina (Department of 501 \nEntomology-UNMSM) for receiving the arthropod samples and assisting with their taxonomic 502 \nidentification. This research was funded by the ‘XI Concurso de fondos de investigación para círculos de 503 \ninvestigación 2021’ from La Molina National Agrarian University (Lima, Peru). 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PLoS One , 13, e0190572.  812 \nhttps://doi.org/10.1371/journal.pone.0190572 813 \n  814 \nTremlett C.J., Moore M., Chapman M.A., Zamora‐Gutierrez V., Peh K.S.H. (2020) Pollination by bats 815 \nenhances both quality and yield of a major cash crop in Mexico. Journal of Applied Ecology, 57, 450 –459. 816 \nhttps://doi.org/10.1111/1365-2664.13545 817 \n 818 \nUlloa C., Zarucchi J.L.,  León B. (2004) Diez años de adiciones a la flora del Perú: 1993 -2003. Arnaldoa, 819 \nTrujillo, Peru: 242 pp. 820 \n 821 \nValiente‐Banuet A., Molina‐Freaner  F., Torres A., Arizmendi M.D.C., Casas A. (2004) Geographic 822 \ndifferentiation in the pollination system of the columnar cactus Pachycereus pecten‐aboriginum . American Journal 823 \nof Botany, 91(6), 850-855. https://doi.org/10.3732/ajb.91.6.850 824 \n 825 \nValiente-Banuet A., Arizmendi M.D.C., Rojas -Martínez A., Domínguez -Canseco L. (1996) Ecological 826 \nrelationships between columnar cacti and nectar -feeding bats in Mexico.  Journal of Tropical Ecology , 12, 103–119. 827 \nhttps://doi.org/10.1017/S0266467400009330 828 \n 829 \nVamosi J.C., Vamosi S.M. (2010) Key innovations within a geographical context in flowering plants: 830 \ntowards resolving Darwin’s abominable mystery.  Ecology Letters , 13, 1270 –1279. https://doi.org/10.1111/j.1461-831 \n0248.2010.01521.x 832 \n 833 \nWalter H.E. (2010) Floral biology of Echinopsis chiloensis ssp. chiloensis (Cactaceae): evidence for a mixed 834 \npollination syndrome.  Flora - Morphology, Distribution, Functional Ecology of Plants , 205, 757 –763. 835 \nhttps://doi.org/10.1016/j.flora.2009.12.038 836 \n 837 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nWesterkamp C.H. (1996) Pollen in bee‐flower relations some considerations on melittophily.  Botanica 838 \nActa, 109, 325–332. https://doi.org/10.1111/j.1438-8677.1996.tb00580.x 839 \n 840 \nWasserthal L.T. (2001) Anpassungen bei Sphingiden zur Vermeidung von Spinnen-und Fledermausattacken. 841 \nVerhandlungen Westdeustchen Entomologentag Dusseldorf, 2000, 13–30. 842 \n 843 \nWenzell K.E., Neequaye M., Paajanen P., Hill L., Brett P., Byers K. J. (2025) Within-species floral evolution 844 \nreveals convergence in adaptive walks during incipient pollinator shift.  Nature Communications , 16, 2721. 845 \nhttps://doi.org/10.1038/s41467-025-57639-3 846 \n 847 \nWinter Y., von Helversen O. (2003) Operational tongue length in phyllostomid nectar-feeding bats. Journal 848 \nof mammalogy, 84, 886–896. https://doi.org/10.1644/BWG-032 849 \n 850 \n 851 \n 852 \n 853 \n 854 \n 855 \n 856 \n 857 \n 858 \n 859 \n 860 \n 861 \n 862 \n 863 \n 864 \n 865 \n 866 \n 867 \n 868 \n 869 \n 870 \n 871 \n 872 \n 873 \n 874 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigures 875 \nFigure 1. (A) Geographic location and composition of the study area in Peru, showing the study area (SA) 876 \nwithin the Lomas ecosystem (LE), near the Lurin  River (LR), and surrounded by agricultural and semi -877 \nurban land. (B) Individual of H. acranthus in the study area. 878 \n 879 \n 880 \n 881 \n 882 \n 883 \n 884 \n 885 \n 886 \n 887 \n 888 \n 889 \n 890 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigure 2. (A) Longitudinal cut of H. acranthus flowers indicating measured morphometric variables. (B) 891 \nSize and shape variation in flowers of  both morphotypes. Frontal view showing perianth color and 892 \nreproductive structures of (C) white and (D) pink-red flower morphotypes. 893 \n 894 \n 895 \n 896 \n 897 \n 898 \n 899 \n 900 \n 901 \n 902 \n 903 \n 904 \n 905 \n 906 \n 907 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigure 3. Monthly mean counts of (A) floral buds and flowers, and (B) unripe and ripe fruits observed 908 \nthroughout 2022. Vertical bars indicate the standard error of the mean. Shaded gray areas correspond to the 909 \nhumid Lomas season. 910 \n 911 \n 912 \n 913 \n 914 \n 915 \n 916 \n 917 \n 918 \n 919 \n 920 \n 921 \n 922 \n 923 \n 924 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigure 4.  Temporal variation in (A) flower aperture, (B) nectar volume, (C) nectar sucrose content, and 925 \n(D) nectar energy production as a function of time after midday.  926 \nSolid lines represent model predictions, and dotted and dashed lines indicate 95% confidence intervals. In 927 \npanel (A), the model includes the effect of morphotype, providing separate estimates and confidence 928 \nintervals for each form. Shaded gray areas correspond to the nocturnal period. 929 \n 930 \n 931 \n 932 \n 933 \n 934 \n 935 \n 936 \n 937 \n 938 \n 939 \n 940 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigure 5.  Floral visitors of H. acranthus: (A–B) Rhodopis vesper (Trochilidae); ( C–D) Platalina 941 \ngenovenisum (Phyllostomidae); (E–F) Manduca sp. (Sphingidae). 942 \n 943 \n 944 \n 945 \n 946 \n 947 \n 948 \n 949 \n 950 \n 951 \n 952 \n 953 \n 954 \n 955 \n 956 \n 957 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nFigure 6. Scaled density of floral visits of Rhodopis vesper, Platalina genovensium, and sphingid moths 958 \nover time for (A) white and (B) pink-red flower morphotypes. Ring plots indicate the percentage of total 959 \nvisits contributed by each visitor group for each morphotype. Shaded gray regions indicate the nocturnal 960 \nperiod. Shaded gray areas correspond to the nocturnal period. 961 \n 962 \n 963 \n 964 \n 965 \n 966 \n 967 \n 968 \n 969 \n 970 \n 971 \n 972 \n 973 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nTables 974 \nTable 1. Summary of the best -fitting models for each response variable: (A) flower aperture, (B) nectar 975 \nvolume, (C) nectar sugar concentration, (D) nectar energy content, and (E) visitation frequency.  Fixed 976 \neffects are expressed as estimates with their standard errors (SE), Z-values (Z), and p-values (P). Estimates 977 \nare relative to white flowers for morphotype and to sphingid moths (Manduca sp.) for floral visitors. 978 \n           \nResponse variable Fixed effect Estimate SE Z P \n(A) Flower aperture Intercept 4.11 0.17 24.88 <0.001 \n  T -0.97 1.59 -0.61 0.540 \n  T2 -18.83 1.58 -11.92 <0.001 \n  T3 4.34 1.54 2.82 0.005 \n  T : Morph red-pink -4.32 2.26 -1.91 0.056 \n  T2 : Morph red-pink 4.89 2.24 2.18 0.029 \n  T3 : Morph red-pink -2.61 2.25 -1.16 0.247 \n(B) Nectar volume Intercept 3.33 0.15 22.45 <0.001 \n  T -0.11 2.05 -0.05 0.957 \n  T2 -9.28 1.76 -5.29 <0.001 \n  T3 8.14 1.81 4.51 <0.001 \n(C) Nectar sugar content Intercept 20.39 0.43 47.27 <0.001 \n  T -13.77 1.60 -8.58 <0.001 \n(D) Nectar energy Intercept 4.33 0.22 19.69 <0.001 \n  T -1.19 2.55 -0.47 0.639 \n  T2 -7.71 2.06 -3.74 <0.001 \n  T3 8.54 1.79 4.78 <0.001 \n(E) Visitation frequency Intercept -5.63 0.72 -7.83 <0.001 \n  Visitor P. genovenisum 1.20 0.47 2.59 0.01 \n  Visitor R. vesper 2.59 0.42 6.12 <0.001 \n´:´ indicates an interaction term. ´T´ represents time after midday (in hours). \n 979 \n 980 \n 981 \n 982 \n 983 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint \n\nTable 2. Summary of registered floral visitors, including visitor group, taxonomic classification, visit type, 984 \nfloral resource utilized, and activity period. 985 \n              \nVisitor group Order Family Species Visit \ntype \nFloral \nresource Activity period \nBees Hymenoptera           \n    Apidae Apis mellifera I/L Pollen Diurnal \n    Halictidae - I Pollen Diurnal \n Ants Hymenoptera           \n    Formicidae Solenopsis sp. I Nectar Diurnal/Nocturnal \n    Formicidae Linepithema sp. I Nectar Diurnal/Nocturnal \n Flies Diptera           \n    Phoridae - I Pollen Diurnal \n    Syrphidae - I Pollen Diurnal \nBeetles Coleoptera           \n    Nitidulidae Carpophilus sp. I Pollen, plant \ntissue Diurnal/Nocturnal \nSpiders Araneae           \n    Salticidae - I Hunting \nground Diurnal/Nocturnal \n    Anyphaenidae - I Hunting \nground Diurnal/Nocturnal \n    Thomisidae - I Hunting \nground Diurnal/Nocturnal \nSphingids Lepidoptera           \n    Sphingidae Manduca sp. I/L Nectar Nocturnal \nHummingbirds Apodiformes           \n    Trochillidae Rhodopis vesper L Nectar Diurnal \nBats Chiroptera           \n    Phyllostomidae Platalina \ngenovensium L Nectar Nocturnal \n´I´ and ´L´ represent illegitimate and legitimate visits to flowers, respectively. \n 986 \n 987 \n 988 \n 989 \n 990 \n 991 \n 992 \n 993 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 9, 2026. ; https://doi.org/10.64898/2026.01.08.698271doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}