Preliminary insight of pollen morphology of the Amazonian members of subtribe Columneineae (Gesneriaceae): details of morphological variation and pollen types

Abstract

Columneinae, a morphologically diverse Neotropical group of Gesneriaceae, is strongly supported as a monophyletic, but many of the genera are weekly supported and lack diagnostic characters at the generic level. Predominantly distributed in the Andes and Amazon biomes, this group has not been evaluated from a palynological context. To address this gap and initiate comprehensive pollen studies within Columneinae, we conducted analyses on 28 species belonging to the genera Centrosolenia Benth., Chrysothemis Decne., Columnea L., Drymonia Mart., Glossoloma Hanst., Lesia J.L.Clark & J.F.Sm., Nautilocalyx Linden., and Trichodrymonia Oerst. Pollen grains were subjected to acetolysis, measurement, and light microscopy, with select species also examined under scanning electron microscopy. Quantitative data underwent statistical analysis tailored to the sample size and multivariate analysis. Variability was observed in size, shape, aperture type, aperture number, ectoaperture length, endoaperture shape, and exine ornamentation. The diverse morphological features led to the identification of distinct pollen types within Columneinae clades. Our findings suggest that eurypalynous pollen grains define most Columneinae and that stenopalynous pollen grains define Chrysothemis ; however, comprehensive palynological studies involving a broader array of species within the subtribe are imperative to enhance our understanding of its palynology.

Full text

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Preliminary insight of pollen morphology of the
Amazonian members of subtribe Columneineae
(Gesneriaceae): details of morphological variation
and pollen types
Ana Carolina Venancio Lopes
USP FFCLRP: Universidade de Sao Paulo Faculdade de Filosoa Ciencias e Letras de Ribeirao Preto
https://orcid.org/0000-0002-6381-1877
Cintia Neves Souza
UNESP FCAV: Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e
Veterinarias
John L. Clark
Marie Selby Botanical Gardens
Lorrayne Albernaz D. C. Landi
UNESP FCAV: Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e
Veterinarias
Eduardo Lopes Soares
USP FFCLRP: Universidade de Sao Paulo Faculdade de Filosoa Ciencias e Letras de Ribeirao Preto
Isaura de Paula Cerdan
USP FFCLRP: Universidade de Sao Paulo Faculdade de Filosoa Ciencias e Letras de Ribeirao Preto
Eduardo Custódio Gasparino
UNESP FCAV: Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e
Veterinarias
Research Article
Keywords: Gesnerioideae, multivariate analysis, palynology, pollen types
Posted Date: September 3rd, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4824106/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Columneinae, a morphologically diverse Neotropical group of Gesneriaceae, is strongly supported as a
monophyletic, but many of the genera are weekly supported and lack diagnostic characters at the
generic level. Predominantly distributed in the Andes and Amazon biomes, this group has not been
evaluated from a palynological context. To address this gap and initiate comprehensive pollen studies
within Columneinae, we conducted analyses on 28 species belonging to the genera
Centrosolenia
Benth.,
Chrysothemis
Decne.,
Columnea
L.,
Drymonia
Mart.,
Glossoloma
Hanst.,
Lesia
J.L.Clark & J.F.Sm.,
Nautilocalyx
Linden., and
Trichodrymonia
Oerst. Pollen grains were subjected to acetolysis,
measurement, and light microscopy, with select species also examined under scanning electron
microscopy. Quantitative data underwent statistical analysis tailored to the sample size and multivariate
analysis. Variability was observed in size, shape, aperture type, aperture number, ectoaperture length,
endoaperture shape, and exine ornamentation. The diverse morphological features led to the
identication of distinct pollen types within Columneinae clades. Our ndings suggest that eurypalynous
pollen grains dene most Columneinae and that stenopalynous pollen grains dene
Chrysothemis
;
however, comprehensive palynological studies involving a broader array of species within the subtribe
are imperative to enhance our understanding of its palynology.
1 Introduction
The Amazon rainforest is one of the planet's most diverse, dense, and complex biomes. Its dynamic
interaction with the environment and inherent ability to control climate elements render it a vital
contributor to the Earth's ecological equilibrium (Capobianao et al. 2001). The South American continent
boasts mega biodiverse forests within its borders, with the Amazon rainforest representing the last
continuous stretch of humid tropical forests. Remarkably, it harbors 70% of the global genetic stock,
encompassing an estimated 60 thousand species, including over 30 thousand higher plant species, of
which 2500 are trees (Abigail 2001; Nobre 2014). Regrettably, over 15% of the Amazon region's forest
cover has been degraded due to res, agricultural expansion, and unsustainable logging practices
(Albagli 2001).
The Gesneriaceae Rich & Juss. ex DC., a family comprising approximately 150 genera and 3,700+ (Weber
et al. 2020), has garnered signicant attention. Classied within the Euasterid I in the order Lamiales
(APG IV 2016), this family's center of diversity spans from Colombia to Ecuador, encompassing the
Central American region and southeastern Brazil (Perret et al. 2003). In Brazil alone, the Gesneriaceae
family hosts 32 genera, and more than 232 species, of which 155 are endemic, all belonging to the
subfamily Gesnerioideae (Flora e Funga do Brasil 2023).
Columneinae, formerly treated as the tribe Episcieae, comprises morphologically diverse species with
distribution in Central and South America, covering the Amazon basin, Guiana Shield, Choco, and the
Andes, especially in northern South America (Weber et al. 2013; Mora and Clark 2016). However, its
trilacunar nodal anatomy, axillary owers, and chromosome count have established it as a monophyletic

Page 4/27
group, embracing 26 genera and over 700 species (Wiehler 1983; Weber 2004b; Clark et al
.
2006; Clark
2009).
While
Paradrymonia
,
Nautilocalyx
Linden ex Hanst., and
Chrysothemis
Decne. form a clade within
Columneinae; their relationships remain a subject of ongoing research. Robust phylogenetic analyses
incorporating extensive morphological characteristics are necessary for accurate circumscription (Clark
et al. 2006; 2012; Mora and Clark 2016). Despite synapomorphies dening other genera within the
subtribe,
Paradrymonia
,
Nautilocalyx
, and
Chrysothemis
rely on symplesiomorphic characters. Clark et
al. (2012) conrmed the non-monophyly of traditionally dened
Paradrymonia
and the recent updated
circumscripton of
Trichodrymonia
, supporting previous ndings and advocating for taxonomic
adjustments (Clark et al. 2006; 2012).
A phylogenetic study by Mora and Clark (2016) identied a clade within Columneinae, they referred to as
the "Paradrymonia alliance," encompassing
Paradrymonia
,
Chrysothemis
,
Nautilocalyx
,
Trichodrymonia
Oerst., and
Centrosolenia
Benth. The updated classication from Mora and Clark (2016) recognized
Nautilocalyx
as a paraphyletic genus, highlighting the non-monophyly of a major clade of the subtribe
Columneinae.
While the pollen morphology of Gesneriaceae has been extensively studied (Erdtman 1952; Campos
1962; Melhem and Mauro 1973; Nowicke 1974; Howard 1975; Skog 1976; Williams 1978; Felice et al.
1981; Fritze and Williams 1988; Luegmayr 1993a; 1993b; Zhi-Jian et al. 1995; 1997; Weigend and
Edwards 1996; Xifreda 1996; Melhem et al. 2003; Weber 2004b; Fourny et al
.
2010; Gasparino et al. 2011;
2013; 2014; 2021; Landi 2017; Souza et al. 2018; Dutra 2018; Belonsi 2018; Bellonzi and Gasparino 2022;
Souza 2022), records on Columneinae species remain scarce. Despite their morphological diversity,
more information is needed on the palynology of this subtribe. This study seeks to contribute a
preliminary investigation into the pollen characterization of Columneinae species, focusing on those
found in the Amazonian region with an emphasis on the
Paradrymonia
alliance.
2 Material and methods
Plant material -Pollen grains from 28 Amazonian species belonging to the genera
Centrosolenia,
Chrysothemis
,
Columnea
L.,
Drymonia
Mart.,
Glossoloma
Hans.,
Lesia
J.L.Clark & J.F.Sm.,
Nautilocalyx
,
and
Trichodrymonia
were examined. Floral buds were sourced from exsiccates available at the SP and
INPA herbaria (Table 1). Whenever possible, more than one specimen per species was analyzed.
Pollen analysis -For light microscopy analysis (LM), anthers extracted from oral buds were hydrated
with glacial acetic acid, macerated, and subjected to the classic acetolysis technique (Erdtman 1960)
with modications (Melhem et al. 2003). Post-processing, slides were mounted with glycerinated gelatin
for subsequent measurements and photomicrographs. These slides were integrated into the Plant
Morphology and Palynology Laboratory Pollen collection at the Department of Biology, Unesp-FCAV,
Jaboticabal, SP, Brazil.

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Quantitative data were derived from measurements of pollen grains. Pollen diameters (
n
=25) were
measured under LM within seven days of sample preparation (Salgado-Labouriau et al
.
1965). Additional
measurements (polar area, apertures, and exine thickness) were taken from 10 pollen grains per
specimen using a microscope with a camera attached to the microcomputer.
A descriptive statistical analysis was performed to obtain the arithmetic mean (x), standard deviation of
the mean (sx), sample standard deviation (s), coecient of variability (CV), and the 95% condence
interval (Vieira 2011; Zar 1999). Measurements were conducted using an OLYMPUS CX22LED and BEL
Photonics light microscopy.
Microsoft ExcelÒgraphs were employed to compare pollen grain diameters and represent the average
P/E ratio indicating pollen shape. Principal Component Analysis (PCA) was conducted using 11 metric
variables: polar diameter in equatorial view (PDEV), equatorial diameter in equatorial view (EDEV),
equatorial diameter in polar view (EDPV), colpus length (CLEN), colpus width (CWID), tectum (TECT),
sexine (SEXI), nexine (NEXI), exine (EXIN), shape (P/E), and width colpus index (WCI).
For scanning electron microscopy analysis (SEM), representative species from the genera were chosen
to observe pollen grain ornamentation details. SEM analysis followed the protocol proposed by Melhem
et al
.
(2003) for non-acetolyzed pollen grains.
Pollen terminology adheres to Punt et al. (2007), and Halbritter et al. (2018), and pollen descriptions
follow Bellonzi et al. (2020). Faegri and Iversen (1966) were consulted for the polar area index, Erdtman
(1952) for pollen shape, Soares et al. (2021) for endoaperture denitions, and Gasparino et al. (2013)
and Dutra et al. (2023) for colpus width index and colpus length index, respectively. Photomicrographs of
pollen in LM were obtained using a Leica IM50 light microscope, while SEM images were captured using
a scanning electron microscope JEOL JSM5410 from the Electronic Microscopy Laboratory, Unesp,
Jaboticabal, Brazil.
3 Results
General Description -The pollen grains of Columneinae were characterized as either monads, isopolar,
small to medium, medium, or medium to large size, hexagonal, subcircular, circular, subtriangular or
triangular amb; very small, small, large or very large polar area; suboblate, oblate spheroidal, prolate
spheroidal, subprolate or prolate; 6-heteroaperturate (3-colporate and 3-colpate), 3-colpate or 3-
colporate; short (brevicolpate), planaperturate, circulaperturate or angulaperturate; long or very long
colpi, narrow, wide or very wide, in some species the ectoaperture have bridges or margines; circular or
lolongate endoapertures. Exine character were dened as tectate perforate or fossulate; or semitectate
microreticulate or reticulate; thin, very thin, or thick exine.
The species analyzed here were grouped into three pollen types based on pollen grain aperture
variations. In some cases, within the pollen types, subtypes were dened by observing the
ornamentation of the pollen grains.

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Pollen type I – 6-heteroaperturate and perforate pollen grains (Fig. 1; Tables 2-3)
Pollen grains in monads, isopolar, medium size (Table 2), hexagonal amb (Fig. 1a, c), small polar area
(0.34 µm), prolate spheroidal (1.04 µm – Table 3); 6-heteroaperturate (3-colpate and 3-colporate, Fig. 1b,
d-e), planaperturate, long and very wide colpus (4.57 µm - Fig. 1b, e), colpus membrane with reticulum
(Fig. 1d) or colpus membrane with granulations (Fig. 1e), lolongate endoaperture (Fig. 1b); exine tectate,
perforate (Fig. 1f), sexine thicker than nexine (Table 3), thin exine.
Studied species:
Columnea strigosa
Benth.
Pollen type II, subtipo A – 3-colpate and microreticulate pollen grains (Fig. 2a-j; Tables 2-3)
Pollen grains in monads, isopolar, small to medium (
Chrysothemis melittifolia
) or medium size (Table 2),
subcircular (Fig. 2), circular (Fig. 2) or subtriangular amb (
C. dichroa
- Fig. 2a), very small, small or large
polar area (Table 3), oblate spheroidal (0.90-0.95 µm), or prolate (
C. melittifolia
- 1.62 µm – Table 2); 3-
colpate, planaperturate (
C. dichroa
) or circulaperturate, very long, long (
Centrosolenia porphyrotricha
) or
short (brevicolpate -
C. dichroa
), very wide or narrow colpi (Table 3), with ornamented colpi membrane
(Fig. 2b,); exine semitectate, microreticulate (Fig. 2), sexine thicker than nexine (Table 3), very thin exine
(
C. melittifolia
) or thin exine.
Studied species:
Chrysothemis dichroa
Leeuwenb.,
Chrysothemis melittifolia
(L.) M.M. Mora and J.L.
Clark,
Drymonia serrulata
(Jacq.) Mart., and
Centrosolenia porphyrotricha
(Leeuwenb.) M.M.Mora &
J.L.Clark.
Pollen type II, subtype B – 3-colpate and reticulate pollen grains (Fig. 2k-p; Tables 2-3)
Pollen grains in monads, isopolar, medium size (Table 2), circular (Fig. 2k) or triangular amb (Fig. 2), very
large polar area (Table 3), oblate spheroidal (
Centrosolenia hirsuta
) or subprolate (
Glossoloma
tetragonoides
); 3-colpate, planaperturate (Fig. 2) or circulaperturate, short (brevicolpate), wide or narrow
colpi (Table 3), with ornamented colpi membrane (Fig. 2); exine semitectate, reticulate (g. 2), sexine
thicker than nexine or nexine thicker than sexine (Table 3), thin exine.
Studied species:
Centrosolenia hirsuta
Benth. and
Glossoloma tetragonoides
(Mansf.) J.L.Clark
Pollen type II, subtype C – 3-colpate and fossulate pollen grains (Fig. 3; Tables 2-3)
Pollen grains in monads, isopolar, medium size (Table 2), circular amb (Fig. 3a, c, g, j, l, n), small or large
polar area (Table 3), oblate spheroidal; 3-colpate, circulaperturate, very long (
Drymonia conchocalyx
),
long or short (brevicolpate –
D. doratostyla
), very wide, wide, or narrow colpi (Table 3), with margo (Fig.
3b, d, i, k, n, p); exine tectate, fossulate (Fig. 3b-f, i, m, p), sexine thicker than nexine or nexine thicker than
sexine (
D. coccinea
– Table 3), thin or very thin exine (
D. semicordata
– Table 3).
Studided species:
Drymonia coccinea
(Aubl.) Wiehler,
Drymonia conchocalyx
Hanst.,
Drymonia
doratostyla
(Leeuwenb.) Wiehler,
Drymonia pendula
(Poepp.) Wiehler,
Drymonia semicordata
(Poepp.)

Page 7/27
Wiehler.
Pollen type III, subtype A – 3-colporate and microreticulate pollen grains (Fig. 4; Tables 2-3)
Pollen grains in monads, isopolar, medium size (Table 2), subcircular (Fig. 4, u, x), circular (
Columnea
rubra
– Fig. 4a), subtriangular (Fig. 4i, l, o, r), or triangular amb (
Nautilocalyx fasciculatus
– Fig. 4h), very
small or small polar area (Table 3), subprolate (
Nautilocalyx pictus
), prolate spheroidal, oblate
spheroidal, or suboblate (Table 3); 3-colporate, planaperturate (Fig. 4h, l, r), angulaperturate (Fig. 4o) or
circulaperturate (Fig. 4a, u, x), very long and very wide, wide or narrow colpi (
Trichodrymonia hypocyrta
–
Table 3), with brigde (constriction – Fig. 4m, s,), circular or lolongate endoaperture (Table 3); exine
semitectate, microreticulate (Fig. 4, m, n, o, p, q, s, t, u, v, w, x, y), sexine thicker than nexine or nexine
thicker than sexine (
C. ulei
,
Nautilocalyx
sp1 – Table 3), thin exine.
Studied species:
Columnea rubra
C.V.Morton,
Columnea ulei
Mansf.,
Lesia savannarum
C.V.Morton)
J.L.Clark & J.F.Sm.,
Nautilocalyx fasciculatus
L.E. Skog & Steyerm.,
Nautilocalyx pallidus
(Sprague)
Sprague,
Nautilocalyx pictus
(Hook.) Sprague,
Nautilocalyx
sp1,
Nautilocalyx
sp3, and
Trichodrymonia
hypocyrta
Wiehler.
Pollen type III, subtype B – 3-colporate and reticulate pollen grains (Fig. 5; Tables 2-3)
Pollen grains in monads, isopolar, small to medium (
Glossoloma herthae
), medium, or medium to large
size (
Paradrymonia densa
– Table 2), subcircular (Fig. 5d, o), circular (Fig. 5a), or subtriangular amb (Fig.
5f, i, l), small to large polar area (Table 3), oblate spheroidal (
Columnea sanguinea
and
Nautilocalyx
forgetti
) or subprolate (Table 3); 3-colporate, planaperturate (Fig. 5f, i, l) or circulaperturate (Fig. 5a, d, o),
very long, long (Table 3) or short (brevicolporate –
G. herthae
), very wide, wide and narrow colpi (Table
3), with margo (in
P. ciliosa
and
P. densa
– Fig. 5i, o-p) or brigde (in
N. forgetti
– Fig. 5h), lolongate
endoaperture (Table 3); exine semitectate, reticulate (Fig. 5a-d, f-g,), sexine thicker than nexine (Table 3),
very thin (
N. forgetti
) or thin exine.
Studied species:
Columnea sanguinea
(Pers.) Hanst.,
Glossoloma herthae
(Mansf.) J.L.Clar
k
,
Nautilocalyx forgetti
(Sprague) Sprague,
Paradrymonia ciliosa
(Mart.) Wiehler and
Paradrymonia densa
(C.H.Wright) Wiehler.
Pollen type III, subtype C – 3-colporate and fossulate pollen grains (Fig. 6; Table 2-3)
Pollen grains in monads, isopolar, medium size (Table 2), subcircular (Fig. 6a) or subtriangular amb (Fig.
6d), small polar area (Table 3), suboblate (
Drymonia strigosa
) or prolate spheroidal (
Nautilocalyx
sp2); 3-
colporate, planaperturate (Fig. 6d) or circulaperturate (Fig. 6a), very long or long, very wide, or wide colpi
(Table 2 – Fig. 6b-c, e), with ornamented colpi membrane (Fig. 6b-c), lolongate endoaperture (Table 3);
exine tectate, fossulate (Fig. 6a, f), sexine thicker than nexine (Table 3), very thin (
D. strigosa
) or thin
exine.
Studied species:
Drymonia strigosa
(Oerst.) Wiehler and
Nautilocalyx
sp2.

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Articial pollen key to Amazonian species of Columneinae
1. Heteroaperturate pollen grains …..………..……. Pollen type I –
Columnea strigosa
1’. Homoaperturate pollen grains ……………….…….…………………………...… 2
2. Tricolpate pollen grains …………………….………….………….. 3 (Pollen type II)
2’. Tricolporate pollen grains ………………….………..…………. 13 (Pollen type III)
3. Exine semitectate ………………………….………………….. 4 (Subtypes A and B)
3’. Exine tectate …………………..…………..…..…….. 9 (Pollen type II, Subtype C)
4. Microreticulate pollen grains …………………….…… 5 (Pollen type II, subtype A)
4’. Reticulate pollen grains ……………………………… 8 (Pollen type II, subtype B)
5. Planaperturate pollen grains ………………………....……….
Chrysothemis dichroa
5’. Circulaperturate pollen grains ………………….……...…………………………..6
6. Thin exine ……………………..…….…..………………..
Chrysothemis
melittifolia
6’. Very thin exine …………………...………….………………………………….….7
7. Small polar area ……………………..…………..…….
Centrosolenia
porphyrotricha
7’. Large polar area …………………………………...…………..
Drymonia serrulata
8. Circular amb and narrow colpi ……………………………….
Centrosolenia hirsuta
8’. Triangular am and wide colpi ………………….….…….
Glossoloma tetragonoides
9. Nexine thicker than sexine ………..………………….…………
Drymonia coccinea
9’. Sexine thicker than nexine ……………..………...….…………………………...10
10. Small polar area …………………...……………….…………………………….11
10’. Large polar area ………………………………….………………………...……12
11. Very long and wide colpi ……………...…………….………
Drymonia conchocalyx
11’. Short and narrow colpi …………………………..…….…….
Drymonia doratostyla
12. Subcircular amb and very wide colpi …………………………..
Drymonia pendula

Page 9/27
12’. Circular amb and wide colpi ……………….………..…….
Drymonia semicordata
13. Exine semitectate ..................................................................... 14 (Subtypes A and B)
13’. Exine tectate …………………………..………….. 27 (Pollen type III, subtype C)
14. Microreticulate pollen grains ……………..……...… 15 (Pollen type III, subtype A)
14’. Reticulate pollen grains …………………...……… 23 (Pollen type III, subtype B)
15. Nexine thicker than sexine ………………………….……………..………………16
15’. Sexine thicker than nexine ……………………………………………….……..17
16. Subcircular amb and circulaperturate pollen grains ……….……..…
Columnea ulei
16’. Subtriangular amb and planaperturate pollen grains …………….
Nautilocalyx
sp1
17. Very small polar area …………………………………….……………….………..18
17’. Small polar area ………………………………………….…………..………….19
18. Circular endoaperture, colpi with bridge ……………………
Nautilocalyx pallidus
18’. Lolongate endoaperture, colpi without bridge …………...……..
Columnea rubra
19. Subtriangular amb ……………………………………….........................…....……20
19’. Subcircular amb …………………………………...……………………..………..21
20. Lolongate endoaperture e suboblate pollen grains .........................
Lesia savannarum
20’. Circular endoaperture e subprolate pollen grains ........................
Nautilocalyx pictus
21. Suboblate pollen grains with circular endoaperture …………..….
Nautilocalyx
sp3
21’. Oblate spheroidal pollen grains with lolongate endoaperture ……...…………... 22
22. Wide colpi …………...………………………………….
Nautilocalyx fasciculatus
22’. Narrow colpi .....................................................................
Trichodrymonia hypocyrt
23. Oblate spheroidal pollen grains ................................................................................24
23’. Subprolate pollen grains ………………………………………………..…………25
24. Circular amb, wide colpi and thin exine …………………..…
Columnea sanguinea

Page 10/27
24’. Subtriangular amb, very wide colpi and very thin exine …….
Nautilocalyx forgetti
25. Planaperturate with subtriangular amb .....................................
Paradrymonia ciliosa
25’. Circulaperturate with subcircular amb ……………...………………..………… 26
26. Large polar area, short and wide colpi ……………..…………..
Glossoloma herthae
26’. Small polar area, very long and narrow colpi …………..…..
Paradrymonia densa
27. Circulaperturate, subcircular amb, very long and wide colpi ……
Drymonia strigosa
27’. Planaperturate, subtriangular amb, long and very wide colpi …..
Nautilocalyx
sp2
Quantitative pollen data -The diameters of pollen grains from the Amazonian species of Columneinae
exhibited a broad range, spanning from 17.50 µm to 62.50 µm (Table 2). Consequently, the observed
pollen sizes varied from small to medium, medium, or medium to large (Fig. 7; Table 3). For
Drymonia
doratostyla
, complete diameter measurements (n < 25) were not feasible, and only the arithmetic mean
(Table 2) without standard deviations (Fig. 7) was presented.
Most analyzed species demonstrated polar diameter in equatorial view (PDEV) values falling within the
range of 25.00–50.00 µm (Fig. 7A – medium size).
Paradrymonia densa
was the exception, presenting
PDEV values exceeding 50.00 µm (Fig. 7A; Table 2). In contrast, the equatorial diameter in equatorial
view (EDEV) values of the analyzed species were generally smaller than the PDEV (Fig. 7). Notably,
Chrysothemis melittifolia
presented EDEV values below 25.00 µm (small size, CI = 19.58-21.62 – Fig. 7B;
Table 2), while other species exhibited mean and standard deviation within the medium size range (Fig.
7B).
The diameter values of pollen grains played a pivotal role in dening their shape, as indicated by the P/E
ratio (Fig. 8; Table 3). Spheroidal pollen shapes (oblate spheroidal or prolate spheroidal – Fig. 8) were
predominant among the analyzed species. For species such as
Chrysothemis melittifolia
,
Glossoloma
herthae
,
Paradrymonia densa
,
G. tetragonoides
,
Paradrymonia ciliosa
, and
Nautilocalyx pictus
, where
PDEV values were higher than EDEV, prolate pollen grains were dened (Fig. 8; Table 3). Conversely,
Columnea ulei
,
Drymonia strigosa
,
Nautilocalyx
sp3, and
Lesia savanarum
exhibited EDEV values higher
than PDEV, dening an oblate shape (Fig. 8; Table 3).
Based on the analysis of pollen grain shape, species with subprolate or prolate pollen grains were
classied as Pollen type II, subtype A (
Chrysothemis melittifolia
), Pollen type II, subtype B (
Glossoloma
tetragonoides
), Pollen type III, subtype A (
Nautilocalyx pictus
), and Pollen type II, subtype B (
G. herthae
,
Paradrymonia ciliosa
and
P. densa
). Suboblate pollen grains were categorized as Pollen type III, subtype
A (
Columnea ulei
,
Lesia savannarum
, and
Nautilocalyx
sp3) and Pollen type III, subtype C (
Drymonia
strigosa
).

Page 11/27
Principal Component Analysis (PCA) was conducted to identify species groupings based on 11 metric
variables of their pollen grains (Table 4): polar diameter in equatorial view (PDEV), equatorial diameter in
equatorial view (EDEV), equatorial diameter in polar view (EDPV), colpus length (CLEN), colpus width
(CWID), tectum (TECT), sexine (SEXI), nexine (NEXI), exine (EXIN), shape (P/E), and width colpus index
(WCI).
The PCA provides an exploratory analysis summarizing 80.66% of the total data variability across its two
main axes. Axis 1, expressing 52.41% of the data, shows that species with the highest width colpus
index (WCI) and colpus width (CWID) values are concentrated on the positive side (Figure 9). Notably,
Paradrymonia densa
appears isolated on the positive side of the axis due to its high value for the most
signicant variable on the axis (Figure 9).
Axis 2, representing 28.25% of the total variation, identies colpus length (CLEN) as the most signicant
variable (Table 4). On the positive side of the axis, species with the lowest CLEN values are concentrated,
with
Columnea rubra
and
Centrosolenia hirsuta
respectively exhibiting the highest and lowest colpus
length values among the studied species.
Regarding pollen types, the PCA primarily contributes to the separation of pollen type II, subtype B,
formed by
Centrosolenia hirsuta
and
Glossoloma tetragonoides
. This separation is attributed to these
species having the lowest colpus length values among the studied species (Figure 9). Other pollen types
were not distinctly separated using the analyzed pollen grain metric variables.
The analysis also facilitates the separation of species within their respective genera, as observed with
Chrysothemis
,
Columnea
,
Drymonia
,
Nautilocalyx
, and
Paradrymonia
(Figure 9). Therefore, in addition to
qualitative variables, quantitative variables prove valuable for effectively distinguishing between the
studied species.
4 Discussion
Only some studies on the pollen morphology of Columneinae species (Gesneriaceae) are available in the
literature, and this number is even smaller when it comes to Amazonian species of the subtribe. Thus,
this study presents new pollen morphological data for these species. Williams (1978) analyzed pollen
grains of
Drymonia serrulata
and
Centrosolenia porphyrotricha
, and Fritze and Williams (1988) analyzed
pollen grains of
Columnea rubra
.
According to Williams (1978), the pollen grains of species in the
Columnea
alliance clade vary in shape
and pollen ornamentation, and pollen data are useful in dening pollen types and delimiting genera.
Williams (1978) describes reticulate pollen grains with thin exine for
Drymonia serrulata
and
Centrosolenia porphyrotricha
; our study identied pollen grains microreticulate with a very thin exine for
these species, following the index of Faegri and Iversen (1966).

Page 12/27
Fritze and Williams (1988) describe a diverse morphological pollen variation for the 67 Columneinae
species. The authors describe variations in the shape, ornamentation, and size of colpi, and they use
these data to characterize genera. For
Columnea rubra
, Fritze and Williams (1988) observed pollen
grains with perforate ornamentation long and narrow colpi with rounded ends. We observed similar data
for the species regarding the length and the ends of colpi; however, the pollen ornamentation is
microreticulate, and the colpi are narrow.
The separation by pollen types adopted in this study conrms the morphological data already reported
for the subtribe; that is, Columneinae is made up of morphologically diverse species (Wiehler 1983;
Weber 2004a; 2004b; Clark et al. 2006; Clark 2009). It was possible to verify that even species with
similar external morphology (positioned in the same genus) differ in pollen grain morphology. In this way,
the pollen types dened here can corroborate with the taxonomy of genera in dening groups and
subgenera, as has already been veried for other genera in Gesneriaceae (Gasparino et al. 2013).
The morphological variation of pollen in
Drymonia
species is quite pronounced. Heteroaperturate pollen
grains were observed for
D. teuscheri
(Pollen type I), colpate pollen grains in
D. serrulata
(Pollen type II,
subtype A – microreticulate),
D. coccinea
,
D. conchocalyx
,
D. doratostyla
,
D. pendula
,
D. semicordata
(Pollen type II, subtype C – fossulated), and colporate pollen grains in
D. strigosa
(Pollen type III, subtype
C – fossulated). The same occurs for the pollen grains of
Nautilocalyx
species, with variations in the
type of apertures (colpus or colporus – Pollen type II, subtype A – Pollen type III, subtypes A, B, and C)
and in the ornamentation of the exine (microreticulate, reticulate, or fossulate).
Pollen grains with reticulate ornamentation were observed for
Glossoloma
species; however,
G.
tetragonoides
has colpate pollen grains (Pollen type II, subtype B), and
G. herthae
has colporate pollen
grains (Pollen type III, B). The opposite is seen for the pollen grains of
Columnea
and
Paradrymonia
, as
despite being colporate (Pollen type III),
C. rubra
,
C. ulei
, have microreticulate pollen grains (subtype A),
C. sanguinea
,
P. ciliosa
, and
P. densa
reticulate pollen grains (subtype B).
The species of
Chrysothemis
analyzed here have morphologically similar pollen grains in terms of
apertures and ornamentation, colpate and microreticulate pollen grains (Pollen type II, subtype A), and
the species present variations in terms of shape, amb, size of the polar area, length, and width of the
colpi, and thickness of the exine. For Gesneriaceae, similar pollen grains in species of the same genus
have already been observed for Gloxiniinae (Souza et al. 2018) and
Sinningia
(Dutra 2018; Gasparino et
al. 2021); other studies conrm the morphological diversity of the family's genera such as
Nematanthus
(Gasparino et al. 2013; Landi 2017), and in Beslerieae (Belonsi 2018).
The analyses by Clark et al. (2006), Clark et al. (2012), and Mora and Clark (2016) pointed out the need
for taxonomic reorganization for the clade composed of
Paradrymonia
,
Nautilocalyx
, and
Chrysothemis
since these are probably non-monophyletic genera, and the morphology of the pollen grains of their
species can be used, together with other data, to assist the taxonomy of the group. Thus, other studies
involving the palynology of Columneinae may clarify the taxonomic relationships of the subtribe.

Page 13/27
Based on the palynology of the Columneinae species analyzed here, we can suggest that only
Chrysothemis
is a stenopalynous genus, while the others have pollen diversity that can contribute to the
delimitation of the species. Although the study involves a small number of Columneinae species, the
results obtained here suggest the eurypalynous character of the group.
Declarations
The authors declare that have no conict of interest.
Acknowledgments This study is funded by FAPESP (The São Paulo Research Foundation – Grant
2021/10717-6), A.C.V. Lopes is funded by CNPq (#130147/2018-4), and E.C. Gasparino is funded by
CNPq (#309555/2021-3). The authors thank the curators of Herbaria INPA and SP for kindly allowing
pollen sampling for the present study.
Author contributions ACVL, CNS, LADCL, and ECG contributed to the study conception and design.
Material preparation, data collection, and analysis were performed by ACVL, CNS, and ECG. Statistical
analyses, graphs, and tables were carried out by ACVL, ELS, and IPC. JLC and ECG contributed to the
supervision of the study and review of the manuscript. All authors commented on previous versions and
read and approved the nal manuscript.
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Tables
Tables 1 to 4 are available in the Supplementary Files section.
Figures

Page 17/27
Figure 1
Photomicrographs and scanning electron micrographs (SEM) of the pollen grains of Amazonian species
of Columneinae – Pollen type I. a – f.
Columnea strigosa.
a. Optical section in polar view. b. Detail of the
apertures in equatorial view. c. Detail of apocolpium in polar view, in SEM. d.Equatorial view, detail of
aperture membrane, in SEM. e. Equatorial view, detail of endoaperture, in SEM. f.Ornamentation detail, in
SEM. (Scale bars: f = 3 µm; a – e = 10 µm).

Page 18/27
Figure 2
Photomicrographs of the pollen grains of Amazonian species of Columneinae – Pollen type II, subtype A
(a – k) and B (l – p). a, b.
Chrysothemis dichroa
. a. General aspect, polar view. b. Equatorial view,
aperture detail. c, d, e.
Chrysothemis melittifolia
. c. General aspect, polar view. d. Equatorial view,
aperture detail. e. Ornamentation in high (e’) and low (e”) focus. f, g.
Drymonia serrulata
. f.General
aspect, polar view. g. Equatorial view, aperture detail. h, i, j.
Centrosolenia porphyrotrichus
. h. General
aspect, polar view. i. Equatorial view, aperture detail. j. Ornamentation in high (j’) and low (j”) focus. k, l,

Page 19/27
m.
Centrosolenia hirsuta
. k. Optical section in polar view. l. Equatorial view, aperture detail. m.
Ornamentation in high (m’) and low (m”) focus. n, o, p.
Glossoloma tetragonoides
. n. General aspect,
polar view. o. Equatorial view, aperture detail. p. Ornamentation in high (p’) and low (p”) focus. (Scales
bars: e, j, m, p = 3 µm; a – d, f – i, k, l, n, o = 10 µm).
Figure 3
Photomicrographs and scanning electron micrographs (SEM) of the pollen grains of Amazonian species
of Columneinae – Pollen type II, subtype C. a, b.
Drymonia coccinea
. a. General aspect, polar view. b.

Page 20/27
Equatorial view, aperture detail. c – f.
Drymonia coccinea
. c.Polar view in SEM. d. Equatorial view,
aperture membrane, in SEM. e.Equatorial view, detail of aperture, in SEM. f. Ornamentation detail, in SEM.
g – i.
Drymonia conchocalyx
. g. General aspect, polar view. h. Equatorial view. i. Equatorial view, aperture
detail and membrane. j, k.
Drymonia doratostyla
. j.General aspect, polar view. k.Equatorial view, aperture
detail. l, m.
Drymonia pendula
. l. Optical section in polar view. m. Equatorial view, aperture detail. n – p.
Drymonia semicordata
. n. General aspect, polar view. o. Equatorial view. p. Equatorial view, aperture
detail. (Scales bars: c – f = 3 µm; a, b, g – p = 10 µm).
Figure 4

Page 21/27
Photomicrographs and scanning electron micrographs (SEM) of the pollen grains of Amazonian species
of Columneinae – Pollen type III, subtype A. a, b.
Columnea rubra
. a. General aspect, polar view. b.
Equatorial view, aperture detail. c – e.
Columnea ulei
. c. Optical section in polar view. d.Equatorial view,
aperture detail. e. Ornamentation detail, in SEM. f, g.
Lesia savannarum
. f. Optical section in polar view.
g.Equatorial view, aperture detail. h - k.
Nautilocalyx fasciculatus
. h, i. Optical section in polar view. j, k.
Equatorial view, aperture detail. l – n.
Nautilocalyx pallidus
. l.Optical section in polar view. m.Equatorial
view, aperture detail. n. Ornamentation detail, in SEM. o – q.
Nautilocalyx pictus
. o.General aspect, polar
view. p. Equatorial view, aperture detail. q. Ornamentation detail, in SEM. r – t.
Nautilocalyx
sp1. r.
General aspect, polar view. s. Equatorial view, aperture detail. t. Ornamentation detail, in SEM. u – w.
Nautilocalyx
sp3. u. General aspect, polar view. v.Equatorial view, aperture detail. w. Ornamentation
detail, in SEM. x, y.
Trichodrymonia hypocyrta.
x. General aspect, polar view. y.Equatorial view, aperture
detail. (Scales bars: e, n, q, t, w = 3 µm; a – d, f – m, o, p, r, s, u, v, x, y = 10 µm).

Page 22/27
Figure 5
Photomicrographs and scanning electron micrographs (SEM) of the pollen grains of Amazonian species
of Columneinae – Pollen type III, subtype B. a – c.
Columnea sanguinea
. a. General aspect, polar view. b.
Equatorial view, aperture detail. c. Ornamentation detail, in SEM. d, e.
Glossoloma herthae
. d. General
aspect, polar view. e. Equatorial view, aperture detail. f – h.
Nautilocalyx forgetti
. f.General aspect, polar
view. g.Equatorial view, ornamentation detail. h.Equatorial view, aperture detail. i – n.
Paradrymonia
ciliosa.
i, j. General aspect, polar view. k, l. Equatorial view, aperture detail. m. Ornamentation in high (m’)

Page 23/27
and low (m”) focus. n. Ornamentation detail, in SEM. o, p.
Paradrymonia densa
. o. General aspect, polar
view. p. Equatorial view, aperture detail. (Scales bars: c = 3 µm; m’ – m” = 5 µm; a, b, d – l, n – p = 10 µm).
Figure 6
Photomicrographs and scanning electron micrographs (SEM) of the pollen grains of Amazonian species
of Columneinae – Pollen type III, subtype C. a – c.
Drymonia strigosa
. a. General aspect, polar view. b.
Equatorial view, aperture detail. c. Equatorial view, endoaperture detail. d – f.
Nautilocalyx
sp2. d.General
aspect, polar view. e.Equatorial view, aperture detail. f. Ornamentation detail, in SEM. (Scales bars: f = 3
µm; a – e = 10 µm).

Page 24/27
Figure 7
Representation of condence interval of mean in 95 % of the pollen grains of Amazonian species of
Columneinae. A. Polar diameter in equatorial view. B. Equatorial diameter in equatorial view. The higher
and lower boundaries showing the condence interval; the average circle showing the arithmetic mean.
The values are in µm. (Cen hir =
Centrosolenia hirsuta
Benth.; Cen por =
Centrosolenia porphyrotricha
(Leeuwenb.) M.M.Mora & J.L.Clark.; Chr dic =
Chrysothemis dichroa
Leeuwenb.; Chr mel =
Chrysothemis

Page 25/27
melittifolia
(L.) M.M. Mora and J.L. Clark; Col rub =
Columnea rubra
C.V. Morton.; Col san =
Columnea
sanguinea
(Pers.) Hanst.; Col ule =
Columnea ulei
Mansf.; Dry coc =
Drymonia coccinea
(Aubl.) Wiehler.;
Dry com =
Drymonia conchocalyx
Hanst.; Dry dor =
Drymonia doratostyla
(Leeuwenberg) Wiehler; Dry
pen =
Drymonia pendula
(Poepp.) Wiehler.; Dry sem =
Drymonia semicordata
(Poepp.) Wiehler.; Dry ser =
Drymonia serrulata
(Jacq.) Mart.; Dry str =
Drymonia strigosa
(Oerst) Wiehler.; Col str =
Columnea
strigosa
Benth.; Glo her =
Glossoloma herthae
(Mansf.) J.L.Clark,; Glo tet =
Glossoloma tetragonoides
(Mansf.) J.L.Clark; Les sav =
Lesia savannarum
(C.V.Morton) J.L.Clark & J.F.Sm.; Nau faz =
Nautilocalyx
fasciculatus
L.E.Skong & Steyerm.; Nau for =
Nautilocalyx forgetti
(Sprague) Sprague; Nau pal =
Nautilocalyx pallidus
(Sprague) Sprague; Nau pic =
Nautilocalyx pictus
(Hook.) Sprague; Nau sp1 =
Nautilocalyx
sp1; Nau sp2 =
Nautilocalyx
sp2; Nau sp3 =
Nautilocalyx
sp3; Par cil =
Paradrymonia ciliosa
(Mart.) Wiehler; Par den =
Paradrymonia densa
(CH Wright) Wiehler; Tri hyp =
Trichodrymonia hypocryta
Wiehler.
Figure 8
Increasing distribution of shape values (Polar diameter/Equatorial diameter) of the pollen grains of
Amazonian species of Columneinae. (Cen hir =
Centrosolenia hirsuta
Benth.; Cen por =
Centrosolenia
porphyrotricha
(Leeuwenb.) M.M.Mora & J.L.Clark.; Chr dic =
Chrysothemis dichroa
Leeuwenb.; Chr mel
=
Chrysothemis melittifolia
(L.) M.M. Mora and J.L. Clark; Col rub =
Columnea rubra
C.V. Morton.; Col san
=
Columnea sanguinea
(Pers.) Hanst.; Col ule =
Columnea ulei
Mansf.; Dry coc =
Drymonia coccinea
(Aubl.) Wiehler.; Dry com =
Drymonia conchocalyx
Hanst.; Dry dor =
Drymonia doratostyla
(Leeuwenberg)
Wiehler; Dry pen =
Drymonia pendula
(Poepp.) Wiehler.; Dry sem =
Drymonia semicordata
(Poepp.)
Wiehler.; Dry ser =
Drymonia serrulata
(Jacq.) Mart.; Dry str =
Drymonia strigosa
(Oerst) Wiehler.; Col str =

Page 26/27
Columnea strigosa
Benth.; Glo her =
Glossoloma herthae
(Mansf.) J.L.Clark,; Glo tet =
Glossoloma
tetragonoides
(Mansf.) J.L.Clark; Les sav =
Lesia savannarum
(C.V.Morton) J.L.Clark & J.F.Sm.; Nau faz
=
Nautilocalyx fasciculatus
L.E.Skong & Steyerm.; Nau for =
Nautilocalyx forgetti
(Sprague) Sprague; Nau
pal =
Nautilocalyx pallidus
(Sprague) Sprague; Nau pic =
Nautilocalyx pictus
(Hook.) Sprague; Nau sp1 =
Nautilocalyx
sp1; Nau sp2 =
Nautilocalyx
sp2; Nau sp3 =
Nautilocalyx
sp3; Par cil =
Paradrymonia ciliosa
(Mart.) Wiehler; Par den =
Paradrymonia densa
(CH Wright) Wiehler; Tri hyp =
Trichodrymonia hypocryta
Wiehler.
Figure 9
Principal component analysis performed with the pollen metric variables of Amazonian species
Columneinae (Gesneriaceae). (Cen hir =
Centrosolenia hirsuta
Benth.; Cen por =
Centrosolenia
porphyrotricha
(Leeuwenb.) M.M.Mora & J.L.Clark.; Chr dic =
Chrysothemis dichroa
Leeuwenb.; Chr mel
=
Chrysothemis melittifolia
(L.) M.M. Mora and J.L. Clark; Col rub =
Columnea rubra
C.V. Morton.; Col san
=
Columnea sanguinea
(Pers.) Hanst.; Col ule =
Columnea ulei
Mansf.; Dry coc =
Drymonia coccinea
(Aubl.) Wiehler.; Dry com =
Drymonia conchocalyx
Hanst.; Dry dor =
Drymonia doratostyla
(Leeuwenberg)
Wiehler; Dry pen =
Drymonia pendula
(Poepp.) Wiehler.; Dry sem =
Drymonia semicordata
(Poepp.)
Wiehler.; Dry ser =
Drymonia serrulata
(Jacq.) Mart.; Dry str =
Drymonia strigosa
(Oerst) Wiehler.; Col str =
Columnea strigosa
Benth.; Glo her =
Glossoloma herthae
(Mansf.) J.L.Clark,; Glo tet =
Glossoloma
tetragonoides
(Mansf.) J.L.Clark; Les sav =
Lesia savannarum
(C.V.Morton) J.L.Clark & J.F.Sm.; Nau faz
=
Nautilocalyx fasciculatus
L.E.Skong & Steyerm.; Nau for =
Nautilocalyx forgetti
(Sprague) Sprague; Nau
pal =
Nautilocalyx pallidus
(Sprague) Sprague; Nau pic =
Nautilocalyx pictus
(Hook.) Sprague; Nau sp1 =
Nautilocalyx
sp1; Nau sp2 =
Nautilocalyx
sp2; Nau sp3 =
Nautilocalyx
sp3; Par cil =
Paradrymonia ciliosa

Page 27/27
(Mart.) Wiehler; Par den =
Paradrymonia densa
(CH Wright) Wiehler; Tri hyp =
Trichodrymonia hypocryta
Wiehler.
Supplementary Files
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Table1.docx
Table2.docx
Table3.doc
Table4.docx