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Research Article
Pollen grain evolution in Zornia evidences the homoplastic nature of a stenopalynous genus of Leguminosae
expand article infoHigor Antonio-Domingues‡§|, Cynthia Fernandes Pinto da Luz§, Mônica Lanzoni Rossi#, Rafael Felipe de Almeida|, Adriana Pinheiro Martinelli#, Gwilym Peter Lewis|, Ana Paula Fortuna-Perez¤|
‡ Programa de Pós-graduação em Biodiversidade Vegetal e Meio Ambiente, Instituto de Pesquisas Ambientais, São Paulo, Brazil
§ Laboratório de Palinologia PALINO_IPA, Instituto de Pesquisas Ambientais, São Paulo, Brazil
| Royal Botanic Gardens, Kew, Richmond, United Kingdom
¶ University of the Witwatersrand, Johannesburg, South Africa
# Universidade de São Paulo, Piracicaba, Brazil
¤ Universidade Estadual Paulista, Botucatu, Brazil

Corresponding author: Higor Antonio-Domingues ( higor.domingues@hotmail.com )

Academic editor: Huasheng Huang
© 2026 Higor Antonio-Domingues, Cynthia Fernandes Pinto da Luz, Mônica Lanzoni Rossi, Rafael Felipe de Almeida, Adriana Pinheiro Martinelli, Gwilym Peter Lewis, Ana Paula Fortuna-Perez.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Antonio-Domingues H, Pinto da Luz CF, Rossi ML, de Almeida RF, Martinelli AP, Lewis GP, Fortuna-Perez AP (2026) Pollen grain evolution in Zornia evidences the homoplastic nature of a stenopalynous genus of Leguminosae. Plant Ecology and Evolution 159(1): 79-94. https://doi.org/10.5091/plecevo.160614
Open Access

Abstract

Background and aims – Zornia is the only pantropical genus in the Adesmia clade (Dalbergieae), subdivided into two clades by previous phylogenetic studies. Zornia remains the only genus of the Adesmia clade to be palynologically understudied. We present a comprehensive palynological study for this genus, testing the systematic relevance of pollen morphology in a phylogenetic context.

Materials and Methods – Standard acetolysis was performed on all pollen grains of the Zornia species, alongside advanced microscopic techniques (LM, SEM, and TEM). Additionally, a principal components analysis was performed to elucidate patterns of variation in quantitative data among species. Using the most recent phylogenetic framework existing for the taxa, we scored and coded 13 micromorphological characters to test for secondary homologies.

Key results – A comprehensive pollen characterisation enabled a complete description of Zornia as stenopalynous, with some differences in pollen grain size and ultrastructure of the operculum, margo, and sexine. The thickness of the pollen nexine (> 0.5 μm) is considered a synapomorphy for the genus Zornia, as shown by our character reconstruction analysis.

Conclusions – The presence of colpate apertures is a unifying pollen character of the genus Zornia. In addition, the thickness of the pollen nexine (> 0.5 μm) was recovered as a synapomorphy for the genus, while Zornia Clade A was supported by two homoplasies correlated with ultrasculpture of the apocolpium (psilate-perforate) and sexine thickness (< 0.5 μm) and Zornia Clade B by the exoaperture width (5–10 μm). The stenopalynous nature of Zornia pollen grains is corroborated here by the large number of homoplasies recovered.

Keywords

Dalbergia clade, Fabaceae, palynology, pantropical, pollen morphology

Introduction

Zornia J.F.Gmel. is a genus within the Adesmia clade (tribe Dalbergieae) of the legume family (Leguminosae; Lavin et al. 2001). It currently comprises approximately 90 species, primarily distributed across pantropical regions (POWO 2025). The genus is characterised by its herbaceous or shrubby habit, flowers arranged in spiciform inflorescences, paired peltate bracteoles protecting each flower, and stipules resembling bracteoles (Fortuna-Perez et al. 2013; Zeferino et al. 2025). Zornia was validly published by Gmelin in 1791, based on Zornia bracteata J.F.Gmel., and has traditionally been divided into two subgenera based on inflorescence type and three sections according to leaflet morphology (Mohlenbrock 1961).

The genus was recently resolved as monophyletic and closely related to Poiretia Vent. and Amicia Kunth, forming the Zornia-Amicia-Poiretia clade (ZAP clade). This clade is characterised by secretory cavities in leaflets, with anatomical and morphological functional traits adapted to colonising dry environments (Fortuna-Perez et al. 2021). However, molecular studies do not support the monophyly of the subgenera and sections of Zornia (Lavin et al. 2001; Fortuna-Perez et al. 2013, 2021). Fortuna-Perez et al. (2013) instead recognised two major clades and one subclade within Zornia. Clade A includes most species with 4-foliolate leaves, 1–4 articles per loment, and non-bristled articles, predominantly occurring in north-eastern Brazil in dry forests and savannas. Clade B comprises species with bifoliolate leaves, up to 15 articles per loment, and bristled articles, distributed across both the New and Old Worlds. Within Clade B, Subclade B1 is restricted to Africa, Asia, and Australia, although it lacks support from any morphological synapomorphy.

Previous studies describe Zornia pollen grains as having a distinctive morphology within the Adesmia clade, characterised by a markedly prolate shape and 3-colpate (rarely 3-colporate) apertures (Melhem 1966; Salgado-Labouriau 1973; Pire 1974; Carreira et al. 1996; Silvestre-Capelato and Melhem 1997; Moreti et al. 2007; Buril et al. 2011; Luz et al. 2013; Silva et al. 2016). However, there are inconsistencies among these studies regarding aperture type, specifically, colpate versus colporate apertures in some species. Over the last three years, the palynology of the Adesmia clade has been extensively studied, with only Zornia remaining to be described in a comprehensive and standardised way to support a pollen evolutionary study (Antonio-Domingues et al. 2022a, 2022b, 2023, 2024). We analysed the pollen morphology, ultrasculpture, and ultrastructure of Zornia species to (1) complete the palynotaxonomic study on the Adesmia clade, (2) confirm the aperture morphology, and (3) test if pollen morphology recovers any apomorphy for Clades A and B.

Material and methods

Unopened mature floral buds were obtained from 18 herbarium specimens belonging to 15 species of Zornia (Z. adenophora (Domin) Mohlenbr., Z. areolata Mohlenbr., Z. bracteata J.F.Gmel., Z. brasiliensis Vogel, Z. capensis Pers., Z. contorta Mohlenbr., Z. crinita (Mohlenbr.) Vanni, Z. gardneriana Moric., Z. glochidiata Rchb. ex DC., Z. harmsiana Standl., Z. myriadena Benth., Z. pardina Mohlenbr., Z. setosa Baker f., Z. thymifolia Kunth, and Z. virgata Moric.). Specimens were sampled from the K, MBM, SP, and UEC herbaria (acronyms follow Thiers 2025). We follow the guidelines for herbarium sampling recently proposed by Almeida et al. (2023), with all specimens examined being listed in the Suppl. material 7. Samples were labelled with a three-letter abbreviation of the species epithet plus the last two digits of the herbarium barcode or of the collection number (see Suppl. materials 1, 7). The selected Zornia species represent the full extent of the genus’s morphological and geographical diversity, including taxa from both the New and Old Worlds, as well as representatives from each of the clades sampled in the phylogeny of the genus proposed by Fortuna-Perez et al. (2013).

Pollen grains were processed for acetolysis analysis (Erdtman 1960), following the modifications proposed by Melhem et al. (2003). Measurements were performed under light microscopy (LM) using an Olympus OSM-4 (10×) micrometre drum coupled to the eyepiece of an Olympus BX50 binocular microscope. The polar and equatorial axes in equatorial views were measured on 25 randomly selected pollen grains from each taxon. These measurements were statistically analysed for the arithmetic mean (x), average standard deviation (Sx), sample standard deviation (s), coefficient of variability V%, and 95% confidence interval (CI) (Zar 2010; Vieira 2016). Ten measurements of the prominent pollen morphometric parameters were also made: length and width of the colpus and the thickness of the nexine and sexine layers. Exine measurements were performed in the mesocolpium region. The arithmetic means and the range were calculated for each pollen grain size class and exine (sexine + nexine). Photomicrographs were obtained using an Olympus BX-50 light microscope equipped with an Olympus U-CMAD-2 video camera and Olympus CellSens 1.5 software. Permanent LM slides were deposited in the pollen reference collection of Laboratório de Palinologia PALINO-IPA, Instituto de Pesquisas Ambientais, São Paulo State, Brazil.

For scanning electron micrographs (SEM), non-acetolysed pollen grains were rinsed in an ethanol series (50–100%), placed on a metal stub, and sputter-coated (Leica EM ACE 600, Vienna, Austria) with gold (80 nm). Samples were observed under a JEOL JSMIT300LV microscope (Tokyo, Japan) operating at a 20 kV electron beam, and the images were digitised.

For transmission electron microscopy (TEM), mature anthers were removed from flower buds of five species (Zornia brasiliensis, Z. bracteata, Z. contorta, Z. crinita, Z. myriadena) and fixed in a modified Karnovsky solution (2% glutaraldehyde, 2% paraformaldehyde, 5 mM calcium chloride in 0.05 M sodium cacodylate buffer, pH 7.2, Karnovsky 1965) for at least 24 h, under refrigeration. They were rinsed with sodium cacodylate buffer (0.1 M) and immediately post-fixed in osmium tetroxide (1% in 0.1 M sodium cacodylate) for 1 h at room temperature. Samples were then rinsed in a saline solution (0.9% sodium chloride) and pre-stained with uranyl acetate (2.5%), followed by dehydration in an acetone series (35–100%) for 20 min each. Samples were then gradually infiltrated with Spurr resin (Spurr 1969) and embedded in pure resin for 48 h at 70°C. Ultrathin sections (60 nm) were obtained with an ultramicrotome (Sorvallter Blum MT2, Norwalk, CT, USA), deposited on copper grids and poststained in aqueous uranyl acetate (2.5%), followed by lead citrate (0.1%) (Reynolds 1963). Images were obtained using a transmission electron microscope (JEOL JEM-1400, Tokyo, Japan) at an acceleration voltage of 80 kV.

Pollen terminology was based on Punt et al. (2007) and Halbritter et al. (2018). Pollen shape classes and amb types follow Erdtman (1952), and colpus length categories follow Mark et al. (2012). The endoaperture classes and index of sexine and nexine thickness were proposed by Antonio-Domingues et al. (2022a). TEM descriptions of the exine infratectum followed the terminology proposed by Halbritter et al. (2018) and Ferguson and Skvarla (1983).

Principal component analysis (PCA) was performed to investigate whether pollen grain characteristics enabled the grouping of genera and species. Seven metric variables (polar axis [PA], equatorial axis [EA], colpus length [CL], colpus width [CW], nexine thickness [N], sexine thickness [S = infratectum + tectum], exine thickness [Ex = N + S]) and two classes/indices (shape class [P/E = PA/EA], index of sexine and nexine thickness [S/N]) were analysed using FITOPAC (Shepherd 1996) and PC-ORD v.7 (McCune and Mefford 2016).

Sequences of the Internal Transcribed Spacer (ITS, including ITS1 and ITS2 spacer regions and the 5.8S ribosomal subunit) were downloaded from GenBank for all ingroup and outgroup species sampled by Fortuna-Perez et al. (2013). A maximum likelihood analysis was run using Raxml-GUI (Edler et al. 2021). Character coding followed the recommendations of Sereno (2007) for morphological analyses. Primary homology hypotheses (De Pinna 1991) were proposed for pollen shape, size, ornamentation, exine structure, and apertures. A total of 13 pollen characters were scored for Zornia and outgroups (Suppl. material 5). All characters were optimised on the concatenated tree using the Maximum Likelihood function (mk1 model) using Mesquite v.2.73 (Maddison and Maddison 2006) and visualised with Winclada (Nixon 1999).

Results

General description

Zornia J.F.Gmel.

Pollen grains in monads, small to medium-sized (Suppl. material 1), radially symmetrical, isopolar, oblate-spheroidal to prolate, circular to ellipsoidal in equatorial view, amb circular to subtriangular, angulaperturate (Figs 1B, 1E, 1H, 1K, 1N, 3B, 3E, 3H, 3K, 3N, 5B, 5E, 5H, 5K; Suppl. material 1); polar area small to very small, 3-zonocolpate, with long to very long, narrow, non-constricted colpus (Figs 1A, 1D, 1G, 1J, 1M, 3A, 3D, 3G, 3J, 3M, 5A, 5D, 5G, 5J) with pointed apices (Figs 2C, 2I, 4F, 4I, 4L, 6C, 6I, 6O); colpi margins are broad, psilate-perforate or rugulate-perforate (Figs 2B, 4E, 6B, 6H, 6N), fastigium not observed. Operculum areolate-granulate, rugulate-perforate, perforate, psilate-perforate or psilate.

Figure 1. 

Light microscopy of Zornia species. A–C. Zornia adenophora. A. General view of the colpus, operculum, and surface, equatorial view. B. Apertures 3-colpate, pointed apices colpi and apocolpium surface. C. Optical section, equatorial view. D–F. Zornia areolata. D. General view of the colpus, surface, and operculum, equatorial view. E. Apertures 3-colpate, pointed apices colpi and apocolpium surface. F. Optical section, equatorial view. G–I. Zornia bracteata. G. General view of the colpus, surface, and operculum, equatorial view. H. Apertures 3-colpate, pointed apices colpi and apocolpium surface. I. Optical section, equatorial view. J–L. Zornia brasiliensis. J. General view of the colpus, surface, and operculum, equatorial view. K. Apertures 3-colpate, pointed apices colpi and apocolpium surface. L. Optical section, equatorial view. M–O. Zornia capensis. M. General view of the colpus, surface, and operculum, equatorial view. N. Apertures 3-colpate, pointed apices colpi and apocolpium surface. O. Optical section, equatorial view. Scale bars: A–G, I, J, L, M, O = 5 μm; H, K, N = 2 μm.

Figure 2. 

Scanning electron microscope images of Zornia species. A–C. Zornia adenophora. A. General view of colpus, operculum, margo, and mesocolpium, equatorial view. B. Detail of mesocolpium, margo, colpus membrane, and operculum, equatorial view. C. Detail of apocolpium, polar view. D–F. Zornia areolata. D. General view of colpus, operculum, margo, and mesocolpium, equatorial view. E. Detail of mesocolpium, equatorial view. F. Detail of apocolpium, oblique polar view. G–I. Zornia bracteata. G. General view of colpus, operculum, margo, and mesocolpium, equatorial view. H. Detail of mesocolpium, equatorial view. I. Detail of apocolpium, polar view. J–L. Zornia brasiliensis. J. General view of colpus, operculum, margo, and mesocolpium, equatorial view. K. Detail of mesocolpium, equatorial view. L. Detail of apocolpium, polar view. M–O. Zornia capensis. M. General view of mesocolpium, equatorial view. N. Detail of apertural area, equatorial view. O. Detail of apocolpium, oblique polar view. Scale bars: A, D, G, J, O = 5 μm; B, C, E, F, H, I, K, M, N = 2 μm; L = 1 μm.

Figure 3. 

Light microscopy of Zornia species. A–C. Zornia contorta. A. General view of the colpus, operculum, and surface, equatorial view. B. Apertures 3-colpate, pointed apices colpi and apocolpium surface. C. Optical section, equatorial view. D–F. Zornia crinita. D. Detail of the colpi, endoaperture, surface, and operculum, equatorial view. E. Apertures 3-colpate, pointed apices colpi and apocolpium surface. F. Optical section, equatorial view. G–I. Zornia thymifolia. G. Detail of the colpi, surface, and operculum, equatorial view. H. Apertures 3-colpate, pointed apices colpi and apocolpium surface. I. Optical section, equatorial view. J–L. Zornia gardneriana. J. Detail of the colpi, surface, and operculum, equatorial view. K. Apertures 3-colpate, pointed apices colpi and apocolpium surface. L. Optical section, equatorial view. M–O. Zornia glochidiata. M. Detail of the colpi, surface, and operculum, equatorial view. N. Apertures 3-colpate, pointed apices colpi and apocolpium surface. O. Optical section, equatorial view. Scale bars: A, C–O = 5 μm; B = 2 μm.

Figure 4. 

Scanning electron microscopy of Zornia species. A–C. Zornia contorta. A. General view of colpus, operculum, margo, and mesocolpium, equatorial view. B. Detail of mesocolpium, equatorial view. C. Detail of apocolpium, oblique polar view. D–F. Zornia crinita. D. General view of mesocolpium, colpus, operculum, and margo, equatorial view. E. Detail of mesocolpium, equatorial view. F. Detail of apocolpium, polar view. G–I. Zornia thymifolia. G. General view of colpus, operculum, margo, and mesocolpium, equatorial view. H. Detail of apertural area and mesocolpium, equatorial view. I. Detail of apocolpium. J–L. Zornia gardneriana. J. General view of colpus, operculum, margo, and mesocolpium, equatorial view. K. Detail of mesocolpium and apertural area, equatorial view. L. Detail of apocolpium. M–O. Zornia glochidiata. M. General view of mesocolpium and apertural areas, equatorial view. N. Detail of mesocolpium. O. Detail of apocolpium, polar view. Scale bars: A, D, G, J, M = 5 μm; B, I, K, O = 1 μm; C, E, F, H, L, N = 2 μm.

Figure 5. 

Light microscopy of Zornia species. A–C. Zornia harmsiana. A. General view of the colpus, operculum, and surface, equatorial view. B. Apertures 3-colpate, pointed apices colpi and apocolpium surface. C. Optical section, equatorial view. D–F. Zornia pardina. D. General view of the colpus, operculum, and surface, equatorial view. E Apertures 3-colpate, pointed apices colpi and apocolpium surface. F. Optical section, equatorial view. G–I. Zornia setosa. G. General view of the colpus, operculum, and surface, equatorial view. H. Apertures 3-colpate, pointed apices colpi and apocolpium surface. I. Optical section, equatorial view. J–L. Zornia virgata. J. General view of the colpus, operculum, and surface, equatorial view. K. Apertures 3-colpate, pointed apices colpi and apocolpium surface. L. Optical section, equatorial view. Scale bars: A–C, H = 2 μm; D–G, I–L = 5 μm.

Figure 6. 

Scanning electron microscopy of Zornia species. A–C. Zornia harmsiana. A. General view of colpus, operculum, margo, and mesocolpium, equatorial view. B. Detail of mesocolpium and apertural area, equatorial view. C. Detail of apocolpium, polar view. D–F. Zornia myriadena. D. General view of colpus, operculum, margo, and mesocolpium, equatorial view. E. Detail of mesocolpium and apertural area, equatorial view. F. Detail of apocolpium, oblique polar view. G–I. Zornia pardina. G. General view of colpus, operculum, margo, and mesocolpium, equatorial view. H. Detail of mesocolpium and apertural area, equatorial view. I. Detail of apocolpium, polar view. J–L. Zornia setosa. J. General view of colpus, operculum, margo, and mesocolpium, equatorial view. K. Detail of mesocolpium and apertural area, equatorial view. L. Detail close to the apocolpium, oblique equatorial view. M–O. Zornia virgata. M. General view of colpus, operculum, margo, and mesocolpium, equatorial view. N. Detail of mesocolpium and apertural area, equatorial view. O. Detail of apocolpium, polar view. Scale bars: A–F, J–L = 1 μm; G–I, M–O = 2 μm.

Sexine with nanoreticulate to reticulate-perforate, perforate, rugulate-perforate or psilate ornamentations, network-like pattern with predominantly rounded lumina, rarely polygonal, straight to curved muri, simple columellate, continuous or interrupted, with wall connections at one level. Sexine is 1.2 to 2.7 times thicker than the nexine and sometimes thickened in the apocolpium or mesocolpium areas; exine 1.3 to 2.6 μm. A summary of the measurements is shown in Suppl. material 1, and additional observations and descriptions are provided in Suppl. material 2.

Exine ultrastructure

The exine has an electron-translucent (e-translucent) compact tectum that varies from semitectate to tectate-perforate. The tectum is smooth or undulated with an electron-dense (e-dense) or e-translucent pollenkitt (Fig. 7). The infratectal structures are e-translucent, and we observed two infratectal structures: 1) granules somewhat organised in a narrow, irregular row; and 2) simple, complete, and/or incomplete columellar. Combinations of the two types occurred in all analysed species; however, the infratectal type is more evident in the apertural area. We also observed an e-translucent and e-dense infratectal pollenkitt (Fig. 7). The thin foot layer was compact, with channels, and irregular, from continuous to discontinuous and absent in the apertural area. The e-dense endexine is compact, continuous, thin, and costate. Near the aperture, it is spongy, thick, endosculptured, and 2-layered. Bi-layered e-dense ektintine and an e-translucent endointine (Fig. 7).

Figure 7. 

Transmission electron microscopy of Zornia species. A–B. Zornia bracteata. A. Section near to the aperture. B. Section in a non-apertural area. C–D. Zornia brasiliensis. C. Section near to the aperture. D. Section in a non-apertural area. E–F. Zornia contorta. E. Section in the aperture. F. Section in a non-apertural area. G–I. Zornia crinita. G. General view. H. Section near to the aperture. I. Section in a non-apertural area. J–L. Zornia myriadena. J. General view. K. Section in the aperture. L. Section in a non-apertural area. Pollenkitt (*). Scale bars: A–F, H, I, K–L= 1 μm; G = 2 μm, J = 5 μm.

1. Zornia adenophora (Domin) Mohlenbr.

Pollen grains are small to medium-sized, oblate spheroidal to subprolate. Colpus with areolate-granulate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.4 to 2 times thicker than nexine; exine 1.6–1.9 μm. Mesocolpium nano- to microreticulate-perforate and nanoreticulate-perforate apocolpium (Figs 1A–C, 2A–C).

2. Zornia areolata Mohlenbr.

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with areolate-granulate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.3 to 1.8 times thicker than nexine; exine 1.3–1.5 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate (Figs 1D–F, 2D–F).

3. Zornia bracteata J.F.Gmel.

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with areolate-granulate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.5 to 2.3 times thicker than nexine; exine 1.5–1.9 μm. Mesocolpium micro- to reticulate and apocolpium nano- to microreticulate (Figs 1G–I, 2G–I, 7A–B).

4. Zornia brasiliensis Vogel

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with psilate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.3 to 2 times thicker than nexine; exine 1.4–1.8 μm. Mesocolpium nano- to microreticulate-perforate and apocolpium perforate (Figs 1J–L, 2J–L, 7C–D).

5. Zornia capensis Pers.

Pollen grains are small to medium-sized, prolate. Colpus with rugulate-perforate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.3 to 2 times thicker than nexine; exine 1.5–1.7 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate. (Figs 1M–O, 2M–O).

6. Zornia contorta Mohlenbr.

Pollen grains are small to medium-sized, prolate. Colpus with areolate-granulate operculum, granulate membrane, psilate-perforate margo. Sexine 1.5 to 2.3 times thicker than nexine; exine 1.4–1.8 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate (Figs 3A–C, 4A–C, 7E–F).

7. Zornia crinita (Mohlenbr.) Vanni

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with perforate operculum, granulate membrane, rugulate-perforate margo. Sexine 1.4 to 2 times thicker than nexine; exine 1.6–2.1 μm. Mesocolpium nano- to microreticulate-perforate and apocolpium nanoreticulate-perforate (Figs 3D–F, 4D–F, 7G–I).

8. Zornia gardneriana Moric.

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with psilate-perforate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.6 to 2.6 times thicker than nexine; exine 1.7–2.3 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate (Figs 3J–L, 4J–L).

9. Zornia glochidiata Rchb. ex DC.

Pollen grains are small to medium-sized, prolate spheroidal to prolate. Colpus with areolate-granulate, granulate membrane, psilate-perforate membrane. Sexine from 1.5 to 2.3 times thicker than nexine; exine 1.6–2.1 μm. Mesocolpium rugulate-perforate to rugulate and apocolpium rugulate-perforate (Figs 3M–O, 4M–O).

10. Zornia harmsiana Standl.

Pollen grains are small-sized, prolate spheroidal to subprolate. Colpus with psilate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.3 to 2.1 times thicker than nexine; exine 1.2–1.7 μm. Mesocolpium nano- to reticulate and apocolpium psilate (Figs 5A–C, 6A–C).

11. Zornia myriadena Benth.

Colpus with areolate-perforate operculum, granulate membrane, psilate-perforate margo. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate. Note: this species was analysed only in SEM and TEM. (Figs 6D–F, 7J–L).

12. Zornia pardina Mohlenbr.

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with psilate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.3 to 1.7 times thicker than nexine; exine 1.5–2 μm. Mesocolpium nano- to microreticulate-perforate and apocolpium nanoreticulate-perforate (Figs 5D–F, 6G–I).

13. Zornia setosa Baker f.

Pollen grains are small to medium-sized, prolate spheroidal to subprolate. Colpus with psilate-perforate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.2 to 2.7 times thicker than nexine; exine 1.7–1.9 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate (Figs 5G–I, 6J–L).

14. Zornia thymifolia Kunth

Pollen grains are small to medium-sized, subprolate to prolate. Colpus with psilate operculum, granulate membrane, psilate perforate margo. Sexine 1.5 to 2 times thicker than nexine; exine 1.3–1.5 μm. Mesocolpium and apocolpium nanoreticulate-perforate (Figs 3G–I, 4G–I).

15. Zornia virgata Moric.

Pollen grains are medium-sized, subprolate to prolate. Colpus with psilate-perforate operculum, granulate membrane, psilate-perforate margo. Sexine from 1.7 to 2.6 times thicker than nexine; exine 2.1–2.6 μm. Mesocolpium nano- to microreticulate and apocolpium nanoreticulate-perforate (Figs 5J–L, 6M–O).

Principal component analysis (PCA)

The PCA revealed the relationship between species based on seven metric variables and two classes/indices (Fig. 8). The correlation coefficients are presented in Suppl. material 3. The first two axes accounted for 88.14% of the total variability of the quantitative data analysed. The first axis explained 74.31% of the variance and was mainly associated with EA, CW, and N. The second axis summarised 13.83% of the total variability, with PA, P/E, CL, S, E, and S/N being the variables that contributed most to this axis.

Figure 8. 

Principal component analysis biplot of the pollen grain metric variables and classes/indices of Zornia specimens.

Zornia pardina (par65), Z. crinita (cri96), Z. areolata (are58), Z. bracteata (brac14), and Z. thymifolia (thy79) were located on the upper left side of the PCA due to their highest CL, PA, and EA values. Morphologically, Z. pardina and Z. crinita are very similar; however, Z. crinita can be distinguished by its densely villous stems and branches, whereas Z. pardina has glabrous branches. In addition, the loment of Z. crinita bears 1 mm long bristle-like trichomes, which are absent in Z. pardina (Zeferino et al. 2025). In contrast, Z. areolata, Z. bracteata, and Z. thymifolia are not morphologically similar. Zornia virgata (vir46, vir61) and Z. adenophora (ade52) showed high and low P/E and were located on the bottom left side of the PCA. The species Z. adenophora and Z. virgata are not morphologically similar.

Conversely, due to their lower CW, N, S, E, S/N, and high P/E values, Zornia capensis (cap39), Z. contorta (con26), and Z. brasiliensis (bras24, bras72) were placed on the upper right side of the PCA. Specimens with the smallest pollen grains were located mainly on the bottom right side of the graph. These specimens also had the lowest CL, PA, EA, and P/E values: Z. gardneriana (gar35), Z. glochidiata (glo67), Z. setosa (set22), Z. brasiliensis (bras73), and Z. harmsiana (har25). These species are not morphologically similar, but they share leaves with four leaflets.

Cluster analysis (UPGMA and Euclidean distance)

We identified two groups of specimens with 0% similarity (Fig. 9). One group had a similarity greater than 75% and comprised two specimens of Zornia virgata (vir46, vir61), which exhibited the most significant values for pollen grains, colpus dimensions, and exine thickness. Interestingly, Z. virgata also presents the biggest fruit in the genus (Fortuna-Perez pers. comm.). Inversely, the remainder of the species with smaller pollen grains, colpus dimensions and exine thickness had a similarity greater than 55%: Z. adenophora (ade52), Z. areolata (are58), Z. thymifolia (thy79), Z. crinita (cri96), Z. pardina (par65), Z. bracteata (brac14), Z. capensis (cap39), Z. contorta (con26), Z. gardneriana (gar35), Z. glochidiata (glo67), Z. setosa (set22), and Z. brasiliensis (bras24, bras72, bras73) (except for Zornia harmsiana (har25) with about 35%).

Figure 9. 

Dendrogram built from the cluster analysis (Euclidean distance) of Zornia specimens, similarity information remaining (%).

Character mapping

All lineages from the molecular phylogeny were recovered with at least one or more homoplasies/apomorphies, except for Zornia brasiliensis, Z. contorta, and Z. areolata (Fig. 10, Suppl. material 6). The genus Poiretia was recovered supported by two synapomorphies regarding the endoaperture size (width > 10 μm, length < 10 μm) and form (lalongate), and one homoplasy regarding the operculum ornamentation (areolate-granulate). Zornia was palynologically supported by one synapomorphy related to the thickness of the nexine (> 0.5 μm) and two other homoplasies regarding the thickness of the sexine (> 0.5 μm) and exine (> 1.5 μm). Zornia myriadena was recovered, supported by one homoplasy associated with the exoaperture length (< 20 μm) and one synapomorphy linked to operculum ornamentation (areolate-perforate). Zornia Clade A was supported by two homoplasies correlated with ultrasculpture of the apocolpium (psilate-perforate) and sexine thickness (< 0.5 μm); Zornia Clade B by the exoaperture width (5–10 μm) and Zornia Subclade B1 by operculum ornamentation type (areolate-granulate).

Figure 10. 

Molecular phylogeny and pollen character mapping of Zornia and the outgroup Poiretia. Character mapping tree: red circles represent apomorphies (synapomorphies and autapomorphies); white circles represent homoplasies. The numbers above the circles represent the number of the pollen character; the numbers below the circles represent the number of pollen character states that have been reconstructed.

Our results indicate that the presence of colpate apertures is a consistent feature found in all Zornia species analysed, thus representing a unifying character for the genus. This trait may be related to the occurrence and adaptation of Zornia species in dry environments. In addition to this feature, the thickness of the pollen nexine (> 0.5 μm) also emerges as a unifying morphological trait across the genus.

Discussion

Pollen morphology, ultrasculpture, and ultrastructure of Zornia

Of the species analysed here, previous palynological analyses had been performed on Zornia bracteata, Z. brasiliensis, Z. myriadena, and Z. virgata by Ohashi (1971), Pire (1974), Carreira et al. (1996), Moreti et al. (2007), Buril et al. (2011), and Silva et al. (2016). Most of the results of these studies are supported by our analyses. However, there are some differences in the classification of pollen size, aperture type (colpus/colporus), operculum presence, and size of reticulum. Supplementary material 4 compares our results and previous pollen descriptions available in the literature.

One significant divergence is the detail of the pollen aperture. In the present study, all Zornia representatives are 3-colpate (absent endoaperture); this is confirmed by previous studies of pollen grains of the genus (Ohashi 1971; Pire 1974; Buril et al. 2011; Silvestre-Capelato and Melhem 1997; Luz et al. 2013). The presence of endoaperture (colporate) previously has been reported both for species analysed by us and for species not analysed (Carreira et al. 1996; Moreti et al. 2007; Silva et al. 2016; RCPol 2025) and is probably a morphological artefact caused by acetolysis, resulting in misinterpretation of the fractured operculum/membrane (Hesse and Waha 1989).

The absence of an operculum is another divergence between present and earlier studies (Carreira et al. 1996; Moreti et al. 2007; Buril et al. 2011; Silva et al. 2016). This discrepancy has already been reported and discussed for other representatives of the Adesmia clade. It is a morphological artefact caused either by the removal of this structure during acetolysis or by the misinterpretation of the operculum and membrane. The operculum is a structure covering the aperture and has ecological importance for minimising water loss. It has also been mentioned in the context of the systematic relationship between representatives of the Dalbergia and Adesmia clades (Antonio-Domingues et al. 2022a, 2022b, 2022c, 2023, 2024; Hesse and Waha 1989).

The systematic relevance of pollen morphology to Zornia clades and genus relatives

Based on the previous palynological studies of other Adesmia taxa, Zornia is shown to be less diverse in pollen morphology and ultrasculpture (Antonio-Domingues et al. 2022a, 2022b, 2023, 2024), and the quantitative (morphometrical) and qualitative (morphology and ultrasculpture) characters do not support the delimitation of pollen types within the genus. Thus, future studies that include more species of Zornia should aim to confirm if these invariant trends are maintained in Zornia.

The palynological traits of Zornia (nexine < 0.5 μm, sexine < 0.5 μm, and exine < 1.5 μm) diagnosed here, in addition to the other vegetative and reproductive characters (digitate leaves and spiciform inflorescences, except for Z. echinocarpa (Moric. ex Meisn.) Benth.and Z. myriadena), morphologically support the genus (Fortuna-Perez et al. 2013). The infrageneric relationships in Zornia, as defined by Fortuna-Perez et al. (2013), are reinforced in the present study. Clade A includes species from the Zornia sect. Zornia (sensu Mohlenbrock 1961) is supported by psilate-perforate ultrasculpture in the apocolpium region and sexine thicker than 0.5 μm, leaves 4-foliolate, flowers arranged in spiciform racemes; each lomentum with 1–4 articles and geographically distributed in caatinga, campo rupestre, and cerrado vegetation of Brazil. Clade B includes species from different traditional sections, with pollen colpi width between 5–10 μm, most of the taxa with leaves 2-foliolate, fruits with many articles per lomentum (up to 15), each with many bristles, and a pantropical distribution. Subclade B1 comprises species occurring in Africa, Asia, and Australia, which have an areolate-granulate operculum. Previously, Subclade B1 had no morphological trait described to support its delimitation.

In the present study, Zornia myriadena is recovered in a separate clade, supported by colpi length greater than 20 μm and the operculum ultrastructure being areolate-perforate. However, previous studies reported this species within Clade A, although with a different morphology (e.g. flowers solitary vs flowers grouped in inflorescences, presence of elaborate stellate or echinate trichomes on the fruit articles vs. absence of bristles on the articles, Fortuna-Perez et al. (2013, 2015, 2021).

Conclusion

According to palynological standards of pollen morphology variation, Zornia can be categorised as stenopalynous, as all species exhibit the same pollen type, with some subtle differences between pollen grains, such as ornamentation, shape, size, and thickness of the exine. However, the presence of colpate apertures is a unifying pollen character of the genus Zornia. In addition, the thickness of the pollen nexine (> 0.5 μm) is a synapomorphy for Zornia, while Zornia Clade A was supported by two homoplasies correlated with ultrasculpture of the apocolpium (psilate-perforate) and sexine thickness (< 0.5 μm) and Zornia Clade B by the exoaperture width (5–10 μm). Finally, the stenopalynous nature of Zornia pollen grains is corroborated here by the large number of homoplasies recovered.

Acknowledgements

The authors are grateful to the curators of the BOTU, ICN, K, MBM, SP, and US herbaria for providing samples from their herbarium specimens. We also thank “Laboratório de Microscopia Eletrônica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo”, coordinated by Prof. Dr Elliot Watanabe Kitajima, who made available their scanning electron microscope, and the “Centro de Microscopia e Imagem, Faculdade de Odontologia de Piracicaba, Universidade de Campinas”, coordinated by Prof. Dr Pedro Duarte Novaes, for the use of their transmission electron microscope. We thank Dr Carin Stanski, who provided flower bud samples from the MBM herbarium. This study was financed in part by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” – Brazil (CAPES) – Finance Code 001 (MSc Grant), awarded to HAD (process no 88882.444252/2019-01); and by the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq), for a fellow researcher awarded to CFPL under Grant no 307607/2022-4 and APM under Grant no 312602/2019-7. APFP thanks FAPESP (process no 2022/10636-9), CNPq (process no. 457911/2013-1; 400567/2016-4, PQ 313945/2021-7 and PROTAX 445824/2024-7), and CAPES Print (process no 8887.373155/2019-00, code 001) for funding a collecting expedition in Brazil and visits to analyse samples in national and international herbaria.

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Supplementary materials

Supplementary material 1 

Zornia pollen grain dimensions (μm) in equatorial view using light microscopy. Specimens were identified by a voucher ID (see Suppl. material 7). Confidence Interval at 95% probability of the lowest sample values (IC-) and highest sample values (IC+), arithmetic mean (x), average standard deviation (sx), sample standard deviation (s), coefficient of variability (V%).

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Supplementary material 2 

Morphology and ultrasculpture of Zornia pollen grains using light and scanning electron microscopy. PT, pollen type; S, small, M, medium; --, not conclusive.

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Supplementary material 3 

Pearson and Kendall coefficients of pollen grain metric variables and classes/indices from the first two ordination axes of the principal component analysis (PCA) of Zornia species. PA, polar axis; EA, equatorial axis; P/E = PA/EA; CL, colpus length; CW, colpus width; N, nexine thickness; S, sexine thickness (S = infratectum + tectum); E, exine thickness (Ex = N + S); s/n, index of sexine and nexine thickness.

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Supplementary material 4 

Pollen morphology data from previously published studies are compared with the present study. M, medium; S, small; PS, present study; O 1971, Ohashi (1971); P 1974, Pire (1974); M 1966, Melhem (1966); C 1996, Carreira et al. (1996); M 2007, Moreti et al. (2007); B 2011, Buril et al. (2011); S 2016, Silva et al. (2016).

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Supplementary material 5 

List of morphological characters of pollen grains and their character states for the species of Zornia and outgroups sampled.

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Supplementary material 6 

List of homoplasies and apomorphies of pollen grains and their character states for the species of Zornia and outgroups sampled.

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Supplementary material 7 

Specimens investigated, with their collection data, herbarium code, and voucher ID used in the principal component analysis. The specimen of Zornia myriadena (Fortuna-Perez et al. 190, UEC) was only analysed in SEM and TEM.

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