DOI: 10.5061/dryad.0cfxpnwf5
title: Ecological niche differentiation mediates near-complete premating reproductive isolation within the <em>Gladiolus carneus</em> (Iridaceae) species complex

description: Ecological niche differentiation is well associated with intraspecific
 divergence of functional traits, which may lead to the evolution of
 premating reproductive isolation. However, the link between the ecological
 niches, trait divergence, and premating isolation remains poorly
 understood. This is particularly pertinent in hyper-diverse areas such as
 the Cape Floristic Region (CFR) of South Africa, where fine-scale
 ecological heterogeneity has been hypothesised as a major driver of
 speciation. Using the polymorphic geophyte Gladiolus carneus, endemic to
 the CFR, we test whether ecological niche differentiation mediates
 premating reproductive isolation. We first tested whether
 putative ecotypes of G. carneus were distinct based on their floral and
 vegetative morphology. Next, we documented each putative ecotype’s abiotic
 niche, flowering phenology, and pollination niche and further tested
 whether any resulting niche differentiation causes premating reproductive
 isolation. Seven distinct ecotypes were identified. Using niche modelling
 and multivariate analyses, we found that these ecotypes occupied distinct
 abiotic niches, resulting in strong ecogeographic isolation. They also had
 distinct flowering times, causing varying strengths of phenological
 isolation. For the pollinator niche, we found that all sampled populations
 were pollinated by one of three highly effective functional pollinators;
 however, at the ecotypic level, there were no consistent trends leading to
 varying strengths in pollinator-mediated isolation. Across all ecotypes,
 ecogeographic isolation was the strongest gene flow barrier, which,
 combined with phenological and pollinator-mediated isolation, caused
 near-complete premating reproductive isolation. These results suggest that
 ecological niche differentiation between G. carneus ecotypes may be
 contributing to incipient speciation within the species complex and
 further suggest that ecological niche differentiation may be a major
 driver of speciation in the hyper-diverse Cape Floristic Region.
description: # Ecological niche differentiation mediates near-complete premating
 reproductive isolation within the *Gladiolus carneus* (Iridaceae) species
 complex Dataset DOI: [10.5061/dryad.0cfxpnwf5](10.5061/dryad.0cfxpnwf5) ##
 Description of the data and file structure This file ‘README.txt’ was
 generated on 07/08/2025 ### GENERAL INFORMATION: 1. Author Information:
 Katharine L. Khoury, Shelley Edwards, and Ethan Newman 2. Date of data
 collection: 2020 to 2024 ### DATA AND FILES Our dataset has been collected
 to investigate whether ecological niche differentiation mediates premating
 reproductive isolation in the *Gladiolus carneus* species complex. We
 tested (1) whether putative ecotypes of *G. carneus* are morphologically
 distinct from one another; (2) whether they occupy distinct abiotic,
 phenological, and pollinator niches; and (3) whether differences in these
 ecological niches result in premating reproductive isolation. #### File 1:
 1._G._carneus_morphology_2020_-_2023.csv Description: All morphological
 measurements from all *G. carneus* ecotypes. (A) Number of variables: 18
 (B) Number of rows: 650 (C) Variable list: Date: Date of the measurement
 Variety: *G. carneus* ecotype Site: Site of the measurement tube_length:
 tube length (mm) flower_gape: flower gape (mm) petal_size: petal size (mm)
 flower_width: flower width (mm) style_length: style length (mm)
 flower_depth: flower depth (mm) inflourescence_height_cm: inflorescence
 height (cm) inflourescence_display_mm: inflorescence height (mm)
 total_flowers: total number of buds, open and senesced flowers no_leaves:
 total number of leaves length_longest_leaf_cm: leaf length (cm)
 width_longest_leaf: leaf width (mm) nectar_volume_ul: nectar volume (ul)
 nectar_conc: nectar concentration (%) nectar_welling: the height of the
 nectar welling in the corolla (mm) (D) Missing data codes: NA (E)
 Abbreviations used: None (F) Other relevant information: None #### File 2:
 2._G._carneus_spectra_new_spec.csv Description: Spectral measurements of
 *G. carneus* ecotypes taken from 2022 and 2023. Files 2 to 4 include all
 spectral data from the *G. carneus* ecotypes and the *Eristalis tenax*
 spectral sensitivities used in the fly colour vision models. (A) Number of
 columns: 710 (B) Number of rows: 3649 (C) Variable list: Wavelength:
 wavelength measurement Individual spectral measurements: the reflectance
 at a particular wavelength (D) Missing data codes: None (E) Abbreviations
 used: Naming system for spectral measurements Ecotype: The *G. carneus*
 ecotype from which the measurement was taken. \[number]: The number
 associated with each *G. carneus* individual morphology: The part of the
 flower from which the measurement was taken (tepal - tepal, Mcentre -
 centre of the median tepal, Mguide - guide of the median tepal, Lcentre -
 centre of the lateral tepal, Lguide - guide of the lateral tepal) (F)
 Other relevant information: NA #### File 3:
 3._G._carneus_spectra_old_spec.csv Description: Spectral measurements of
 *G. carneus* ecotypes taken from 2020. Files 2 to 4 include all spectral
 data from the *G. carneus* ecotypes and the *Eristalis tenax* spectral
 sensitivities used in the fly colour vision models. (A) Number of columns:
 552 (B) Number of rows: 2049 (C) Variable list: Wavelength: wavelength
 measurement Individual spectral measurements: the reflectance at a
 particular wavelength (D) Missing data codes: None (E) Abbreviations used:
 Naming system for spectral measurements Ecotype: The *G. carneus* ecotype
 from which the measurement was taken. \[number]: The number associated
 with each *G. carneus* individual morphology: The part of the flower from
 which the measurement was taken (tepal - tepal, Mcentre - centre of the
 median tepal, Mguide - guide of the median tepal, Lcentre - centre of the
 lateral tepal, Lguide - guide of the lateral tepal) (F) Other relevant
 information: NA #### File 4: 4._Eristalis_spectral_sensitivities.csv
 Description: *Eristalis tenax* spectral sensitivities. Files 2 to 4
 include all spectral data from the *G. carneus* ecotypes and the
 *Eristalis tenax* spectral sensitivities used in the fly colour vision
 models. (A) Number of columns: 5 (B) Number of rows: 402 (C) Variable
 list: Wavelength: wavelength Individual Receptor Sensitivities: The
 efficiency of detection for a particular wavelength of light. Four
 receptors are given - R7p, R7y, R8p, R8y (D) Missing data codes: None (E)
 Abbreviations used: None (F) Other relevant information: This data set was
 provided by Adrian Dyer. #### File 5: 5._G._carneus_inat_occurance.csv
 Description: File 5 includes all occurrence data for all *G. carneus*
 ecotypes mined from iNaturalist observations. These observations were used
 to quantify the fundamental and realised niches of the *G. carneus*
 ecotypes. (A) Number of variables: 5 (B) Number of rows: 1247 (C) Variable
 list: quality_grade: the quality of the observation, which was either
 research (iNat research grade) or own (provided by KLK) latitude:
 latitudinal coordinates longitude: longitudinal coordinates
 positional_accuracy: the accuracy (m) of the observation field:varieties:
 *G. carneus* ecotype (D) Missing data codes: None (E) Abbreviations used:
 *G. carneus* ecotypes AL - albidus BL - blandus CL - callistus HA -
 high-altitude LB - langeberg MC - macowanianus PS - prismatosiphon (F)
 Other relevant information: None #### File 6:
 6._G._carneus_flowering_times.csv Description: File 6 includes all
 phenological flowering data for all *G. carneus* ecotypes mined from
 iNaturalist observations. The data were used to quantify the phenological
 niche of the ecotypes. (A) Number of variables: 8 (B) Number of rows: 1231
 (C) Variable list: date_observed: the full observation date day: the day
 of the month of the observation month: the month of the observation (1 -
 12) year: the year of the observation day_num: the day of the year (1 -
 365) of the observation degrees: the day of the year converted to degrees
 radians: the day of the year converted to radians Varieties: the *G.
 carneus* ecotypes (D) Missing data codes: None (E) Abbreviations used:
 None (F) Other relevant information: None #### File 7:
 7._G._carneus_visitation_rates.csv Description: Visitation rates of
 functional pollinators at *G. carneus* populations. Files 7 and 8 contain
 visitation rates and pollen deposition of the functional pollinators at
 *G. carneus* populations. This data was used to quantify the pollinator
 niche for each population and ecotype. (A) Number of variables: 7 (B)
 Number of rows: 36 (C) Variable list: Date: the date of the observations
 Variety: *G. carneus* ecotype Site: The site of the observations. The
 first letter indicates the ecotype. Func_pollinator: the functional
 pollinators Visits.flower.hour: the number of visits/flower/hour
 No_flowers_obs: the total flowers observed at the site Decminal_hours:
 total hours observed at the site (D) Missing data codes: None (E)
 Abbreviations used: Functional pollinators MTFs - medium-tongued flies
 LTFs - long-tongued flies (F) Other relevant information: None #### File
 8: 8._G._carneus_pollen_deposition.csv Description: Pollen deposition on
 functional pollinators at *G. carneus* populations. Files 7 and 8 contain
 visitation rates and pollen deposition of the functional pollinators at
 *G. carneus* populations. This data was used to quantify the pollinator
 niche for each population and ecotype. (A) Number of variables: 18 (B)
 Number of rows: 51 (C) Variable list: Date_Collected: Date the pollinator
 was caught Variety: the *G. carneus* ecotype Site: The site of the
 observations. The first letter indicates the ecotype. Insect_Family:
 Family-level identification of the pollinator Func_pollinator: the
 functional pollinator designation Polliantor_species: Species-level
 identification of the pollinator Ext_proboscis_length: the extended
 proboscis length of the pollinator (mm) body_length: the body length of
 the pollinator (mm) thorax_width: the thorax width of the pollinator (mm)
 thorax_depth: the thorax depth of the pollinator (mm) Visitor - whether
 the pollinator was observed visiting flowering *G. carneus* individuals
 (Y) or whether it was caught in the surrounding environment (N)
 Glad_thorax_pollendep: number of *G. carneus* pollen grains on the thorax
 of the pollinator. Glad_head_pollendep: number of *G. carneus* pollen
 grains on the head of the pollinator. Glad_abdomin_pollendep: number of
 *G. carneus* pollen grains on the abdomen of the pollinator.
 Glad_total_pollen: total number of *G. carneus* pollen grains on the
 pollinator. foreign_thorax_pollendep: number of foreign pollen grains on
 the thorax of the pollinator. foreign_head_pollendep: number of foreign
 pollen grains on the head of the pollinator. foreign_abdomin_pollendep:
 number of foreign pollen grains on the abdomen of the pollinator. (D)
 Missing data codes: NA (E) Abbreviations used: Functional pollinators MTFs
 - medium-tongued flies LTFs - long-tongued flies (F) Other relevant
 information: Pollen loads on each part of the pollinator were only counted
 to 1000 grains. #### File 9:
 9._G._carneus_tube_length___flower_gape_morphology.csv Description:
 Measurements of tube length and flower gape for *G. carneus* populations,
 which are used to test for correlations between pollinator and floral
 morphology. (A) Number of variables: 5 (B) Number of rows: 650 (C)
 Variable list: Date: the date of the measurement Variety: the *G. carneus*
 ecotype Site: The site of the observations. The first letter indicates the
 ecotype. tube_length: tube length (mm) flower_gape: flower gape (mm) (D)
 Missing data codes: NA (E) Abbreviations used: None (F) Other relevant
 information: None #### File 10: 10._G._carneus_ecogeographic_binary.csv
 Description: Binary occurrence records for *G. carneus* ecotypes used to
 calculate ecogeographic isolation. Files 10 to 12 contain the data used to
 calculate ecogeographic, phenological, pollinator isolation, and total
 premating reproductive isolation. (A) Number of columns: 8 (B) Number of
 rows: 1 048 576 (C) Variable list: id: the ID of a specific place in the
 niche model predictions. Individual *G. carneus* ecotypes: binary
 occurrence predictions of the ecotypes, with 1 indicating the prediction
 was above the ETSS threshold, 0 indicating the prediction was below the
 ETSS threshold, and NA indicating there was no predicted niche. (D)
 Missing data codes: NA (E) Abbreviations used: None (F) Other relevant
 information: All NA values are converted to 0 in the R script to
 facilitate the calculation of ecogeographic isolation. #### File 11: Name:
 11._G._carneus_phenology_binary.csv Description: Binary phenological
 records for *G. carneus* ecotypes used to calculate phenological
 isolation. Files 10 to 12 contain the data used to calculate
 ecogeographic, phenological, pollinator isolation, and total premating
 reproductive isolation. (A) Number of columns: 8 (B) Number of rows: 366
 (C) Variable list: day_num: the day of the year Individual *G. carneus*
 ecotypes: binary occurrence predictions of the ecotypes, with 1 indicating
 the flowering individuals were documented and 0 indicating no flowering
 individuals were documented. (D) Missing data codes: None (E)
 Abbreviations used: None (F) Other relevant information: None #### File
 12: 12._G._carneus_pollinator_binary.csv Description: Binary pollinator
 records for *G. carneus* ecotypes used to calculate pollinator isolation.
 Files 10 to 12 contain the data used to calculate ecogeographic,
 phenological, pollinator isolation, and total premating reproductive
 isolation. (A) Number of variables: 7 (B) Number of rows: 7 (C) Variable
 list: FuncPollinator: the functional pollinator Individual *G. carneus*
 ecotypes: binary records showing whether a particular functional
 pollinator was documented for an ecotype, with 1 indicating that the
 functional pollinator was documented, and 0 indicating the functional
 pollinator was not documented (D) Missing data codes: None (E)
 Abbreviations used: None (F) Other relevant information: Blandus is
 missing from the data set due to a lack of pollinator data #### File 13:
 Gladiolus_carneus_Ecological_Niche_Differentiation.Rmd Description: The
 annotated R code used to analyse all of the data.  ## Code/software All R
 scripts used to analyse the data have been included in an R Markdown
 document. All data analysis was conducted in R v.4.1.2.  ## Access
 information Other publicly accessible locations of the data: * Worldclim
 ([https://www.worldclim.org/data/worldclim21.html](https://www.worldclim.org/data/worldclim21.html)) * Cramer MD, Wootton LM, van Mazijk R, Verboom GA. 2019. New regionally modelled soil layers improve prediction of vegetation type relative to that based on global soil models. *Diversity & Distributions* 25: 1736–1750. doi:10.1111/ddi.12973