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Pollen morphology of some species of Spermacoceae s.s. (Rubiaceae) of the Atlantic Forest, Rio de Janeiro, Brazil

ABSTRACT

This work aimed to morphologically characterize 22 species of Spermacoceae s.s found in the Atlantic Forest in the state of Rio de Janeiro with the purpose of improving palynological knowledge of the studied species. The studied species included six species of Borreria and Denscantia cymosa, Emmeorhiza umbellata, Hexasepalum apiculatum, H. radula, H. teres, Manettia fimbriata, Mitracarpus eichleri, M. frigidus, M. hirtus, M. lhotzkyanus, M. megapotamicus, Oldenlandia salzmannii, Pentodon pentandrus, Richardia brasiliensis, Richardia scabra and Spermacoce rubescens. Acetolysed pollen grains were analyzed using light (LM) and scanning electron miscroscopy (SEM). The pollen grains varied in size (polar diameter) from medium to large, with the exception of Hexasepalum teres which was very large. Pollen grains were oblate spheroidal in most species, suboblate in six species, prolate spheroidal in Spermacoce rubescens and subprolate in Manettia fimbriata. All pollen grains were colporate, while number of apertures varied (three-19). The endoaperture was lolongate in most species and lalongate in four species. Sexine ornamentation varied among reticulate, reticulate-granulate, microreticulate, granulate with perforation, echinate-perforate and vermiculate. Pollen attributes were found to distinguish the studied species, but do not separate genera, confirming that Spermacoceae is euripolynic.

Keywords:
Gentianales; multivariate analysis; palynology; Rubioideae; systematics

Introduction

According to Angiosperm Phylogeny Group (APG IV 2016APG - Angiosperm Phylogeny Group IV. 2016. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants. Botanical Journal of the Linnean Society 181: 1-20. ), Rubiaceae is positioned in the order Gentianales and recent phylogenetic studies have suggested support for only three subfamilies: Cinchonoideae Raf., Ixoroideae Raf. and Rubioideae Verdc. It is a cosmopolitan, predominantly pantropical family (Taylor et al. 2007Taylor CM, Campos MTVA, Zappi D. 2007. Flora da Reserva do Ducke, Amazonas, Brasil: Rubiaceae. Rodriguésia 58: 549-616.). In Brazil, this family is represented by approximately 126 genera and 1415 species, of which 733 are endemic (Flora do Brasil 2020 2019Flora do Brasil 2020. 2019. Rubiaceae. Rio de Janeiro. Jardim Botânico do Rio de Janeiro. http://reflora.jbrj.gov.br/reflora/floradobrasil/FB210
http://reflora.jbrj.gov.br/reflora/flora...
), with significant representation in the Atlantic Forest and the Amazon Basin (Delprete & Cortes 2006Delprete PG, Cortés BR. 2006. A synopsis of the Rubiaceae of the states of Mato Grosso and Mato Grosso do Sul, Brazil, with a key to genera, and a preliminar species list. Review Biological Neotropical 3: 13-96.; Delprete & Jardim 2012Delprete PG, Jardim JG. 2012. Sistemática, taxonomia e florística das Rubiaceae brasileiras: uma visão geral sobre o status e desafios futuros. Rodriguésia 63: 101-128. ).

The Spermacoceae s.s. is monophyletic tribe belongs the subfamily Rubioideae is one of the most representative tribes in Brazil, about 11 genera and 30 species are registered in the state of Rio de Janeiro (Flora do Brasil 2020 2019Flora do Brasil 2020. 2019. Rubiaceae. Rio de Janeiro. Jardim Botânico do Rio de Janeiro. http://reflora.jbrj.gov.br/reflora/floradobrasil/FB210
http://reflora.jbrj.gov.br/reflora/flora...
).

As already emphasized by Bacigalupo & Cabral (1996Bacigalupo NM, Cabral EL. 1996. Infrageneric classification of Borreria (Rubiaceae-Spermacoceae) on the basis of American species. Opera Botanica Belgica 7: 207-308.); Pire (1996Pire SM. 1996. Palynologycal study of American species of Borreria (Rubiaceae-Spermacoceae). Opera Botanica Belgica 7: 413-423.); Dessein et al. (2002Dessein S, Huysmans S, Robbrecht E, Smets E. 2002. Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspect. Grana 41: 69-89., 2005Dessein S, Ochoterena H, Block PD, et al. 2005. Palynological characters and their phylogenetic signal in Rubiaceae. The New York Botanical Garden 71: 354-414.); Harwood & Dessein (2005Harwood R, Dessein S. 2005. Spermacoce australiano (Rubiaceae: Spermacoceae). I. Território do Norte. Botânica Sistemática Australiana 18: 297-365.); Cabral & Pire (2006)Cabral EL, Mari E, Pire SM. 2006. Borreria sect. Pseudodiodia (Rubiaceae), aportes taxonómicos y palinológicos. Bonplandia 15: 79-80. ; Souza & Lorenzi (2008Souza VC, Lorenzi H. 2008. Botânica sistemática. Guia ilustrado para identificação das famílias de angiospermas da flora brasileira baseado em APG II. Ed Plantarum, Nova Odessa.), the palynology in Spermacoceae has proven valuable whereas the diversity in the pollinic attributes provides important data for the systematics of the tribe. Variations in the pollen grains of Spermacoceae s.s. related, mainly, in number, type of aperture and ornamentation of the sexine were registered by these authors.

The importance of pollen characters in Rubiaceae taxonomy has been highlighted by several authors, such as Campo (1976Campo M. 1976. Patterns of pollen morphological variation within taxa. In The evolutionary significance of the exine. Linn. Soc. Symp. Ser. 1Ferguson, I. K. and Muller J . Eds. 125-137. Acad. Press, London.), Jansen et al. (1996 a Jansen S, Robbrecht E, Smets E. 1996a. The systematic value of endexine ornamentation in some Psychotrieae pollen (Rubiaceae-Rubioideae). Grana 35: 129-137.; bJansen S, Robbrecht E, Beeckman H, Smets E. 1996b. Gaertnera and Pagamea: Genera within the Psychotrieae or constituting the tribe Gaertnereae. A wood anatomical and palynological approach. Acta Botanica Brasilica 109: 466-476.) and Dessein et al. (2005Dessein S, Ochoterena H, Block PD, et al. 2005. Palynological characters and their phylogenetic signal in Rubiaceae. The New York Botanical Garden 71: 354-414.), among others that prove their use in the systematic of certain groups.

The following work intends the morphological pollinic characterization of 22 species of the Spermacoceae tribe found in Atlantic Forest in the state of Rio de Janeiro, with the purpose of offering a better palynological knowledge of the studied species, as well as provide subsidies for future molecular studies in Rubiaceae.

Materials and methods

Pollen grains of 22 species of the Spermacoceae (Rubiaceae) were examined. The samples were obtained from anthers of flower buds of specimens held in the Brazilian herbaria (GUA, R and RB). Acronyms are according to Thiers (2017Thiers B. 2017 continuously updated. Index herbariorim: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium. http://sweetgum.nybg.org/ih/.
http://sweetgum.nybg.org/ih/...
continuously updated).

Whenever possible, four specimens of each species were analyzed and compared in order to obtain accurate results (List S1. in supplementary material). The permanent slides of this study are deposited in Laboratório de Palinologia Álvaro Xavier Moreira, Departmento de Botânica da Universidade Federal do Rio de Janeiro / Museu Nacional.

Pollen grains were prepared by acetolysis (Erdtman 1952Erdtman G. 1952. Pollen morphology and plant taxonomy - Angiosperms. Stockholm, Almqvisit and Wiksell.), as modified by Melhem et al. (2003Melhem TS, Cruz-Barros MAV, Corrêa AMS, Makino-Watanabe H, Silvestre-Capelato MSF, Gonçalves-Esteves V. 2003. Variabilidade polínica em plantas de Campos de Jordão (São Paulo, Brasil). Boletim do Instituto de Botânica (São Paulo) 16: 9-104.) for light microscopy (LM). The scanning electron photomicrographs (SEM) were taken by using either a JSM-5310 microscope at the Hertha Meyer Laboratório de Ultraestrutura, Instituto de Biofísica, Universidade Federal do Rio de Janeiro or Jeol 6390 LV at the Centro de Microscopia Eletrônica, Departmento de Invertebrados do Museu Nacional, Universidade Federal do Rio de Janeiro.

One specimen of each species was chosen for statistical treatment and illustrations and is indicated by an asterisk (*) after the collector’s name (List S1. in supplementary material). Measurements in equatorial view (PD - polar diameter, ED - equatorial diameter) were taken on 25 pollen grains per sample. Means (¯x), and 95 % confidence intervals (CI) were calculated. For measurements of equatorial diameter from polar view (EDPV), apocolpium side (AS), apertures and exine thickness, the arithmetic means of 10 measurements were used. Ten similar measurements of pollen grains were made on additional material, from another collection, to check the stability of the data (treated further as comparison material). Whenever possible, comparison specimens were analyzed for confirmation of the obtained results (Souza et al. 2016Souza MA, Mendonça CBF, Esteves RL, Gonçalves-Esteves V. 2016. Pollen morphology of species of Graphistylis B. Nord. (Asteraceae) of Brazil Acta Botanica Brasilica 30: 138-146.; Marinho et al. 2018Marinho EB, Bove CP, Mendonça CBF, Esteves-Gonçalves V. 2018. Pollen morphology of Mourera (Podostemaceae). Palynology 42: 553-559.; Moreira et al. 2018Moreira GL, Cavalcanti TB, Mendonça CBF, Gonçalves-Esteves V. 2018. Pollen morphology of Brazilian species of Verbesina L. (Heliantheae - Asteraceae). Acta Botanica Brasilica 32: 128-134.; Sousa et al. 2018Sousa HCF, Santos JC, Gonçalves-Esteves V, Mendonça CBF. 2018. Taxonomic significance of pollen morphology in the tribe Helieae (Gentianaceae) from the Atlantic Forest, Brazil. Palynology 43: 373-382.).

The terminology used for pollen descriptions follows Punt et al. (2007Punt W, Blackmore S, Nilsson S, Thomas A. 2007. Glossary of pollen and spore terminology. Review of Paleobotany and Palynology 143: 1-81. ), which takes into consideration elements of size, shape, number of apertures, and sexine ornamentation. The descriptions of the polar area and aperture size were made according to the classification system established by Faegri & Iversen (1966Faegri G, Iversen J. 1966. Textbook of modern pollen analysis. Copenhagen, Scandinavian University Books.) for the polar area index.

Principal component analysis (PCA) and a cluster analysis were performed using PC-ORD 5.0 (McCune & Mefford 2006 McCune B, Mefford MJ. 2006. Pc-Ord, version 5.0, Multivariate analysis of ecological data. Glaneden Beach, MjM Solfware Desing.) software. The metrics for pollen grains were transformed by the power of 0.5 (square root) to standardize analyses.

Principal component analysis (PCA) was conducted to determine whether the pollen features could cluster species. The variance and covariance matrix (var-cov) was obtained from the means of the morphometric data of the palynological analysis, and the coordinates in the biplot graph are based on Euclidian distances and show the first and second principal components. The matrix of characters, the values of the vectors in each axis and the total of accumulative variance are presented in tables. The cluster analysis (AHC Clustering) was conducted with the aim of classifying the analyzed species into groups based on shared pollen variables (similarity). Two factors were considered in forming the groups from the set of analyzed variables: the percentage of information (variables) necessary to arrive at the groups related to the final number of groups formed. A dendrogram was made using Euclidian distances (Caccavari et al. 2008Caccavari MA, Naab OA, Tamame MA. 2008. Palynological and physicochemical characteristics of three unifloral honey types from central Argentina. Spanish Journal of Agricultural Research 6: 566-576.) and using Ward’s linkage method.

Results

The palynological descriptions are arranged according to the following pollen features: one. Dispersal unit, polarity and size, two. shape and polar area, three. number and type of apertures and four. Stratification of exine and ornamentation.

Pollinic descriptions of the species of Borreria brachystemonoides (Fig. 1A-C); B. capitata (Fig. 1D-F); B. latifolia (Fig. 1G-H); B. palustris (Fig. 1I), B. scabioisoides (Fig. 1J-L); B. verticillata (Fig. 2A-C); Denscantia cymosa (Fig. 2D-F); Emmeorhiza umbellata (Fig. 2G-I); Hexasepalum apiculatum (Fig. 2J-L); H. radula (Fig. 3A-C); H. teres (Fig. 3D-F); Manettia fimbriata (Fig. 3G-I); Mitracarpus eichleri (Fig. 3J-L); M. frigidus (Fig. 4A-C); M. hirtus (Fig. 4D-F); M. lhotzkianus (Fig. 4G-I); M. megapotamicus (Fig. 4J-L); Oldenlandia salzmannii (Fig. 5A-C), Pentodon pentandrus (Fig. 5D-F); Richardia brasiliensis (Fig. 5G-I); R. scabra (Fig. 5J-L); Spermacoce rubescens (Fig. 5M-O).

Figure 1
Photomicrographs and eletronphotomicrographs of pollen grains of species Spermacoceae (Rubiaceae) of the Atlantic Forest. Borreria brachystemonoides - A. polar view: optical section (LM); B. equatorial view: aperture (LM); C. surface detail (SEM). B. capitata - D. polar view: optical section (LM); E. polar view: general aspect (SEM); F. equatorial view: aperture (SEM). B. latifolia - G. polar view: general aspect (SEM); H. equatorial view: aperture detail (SEM). B. palustris - I. equatorial view: general aspect and aperture. B. scabioisoides - J. polar view: optical section (LM); K. equatorial view: general aspect and aperture (SEM); L. surface detail (SEM). Bars: A, B, E, G, K (= 5 µm); D, H, J (= 10 µm); F (=2 µm); C, I, L (= 1 µm).

Figure 2
Photomicrographs and eletronphotomicrographs of pollen grains of species Spermacoceae (Rubiaceae) of the Atlantic Forest. Borreria verticillata - A. polar view: optical section (LM); B. equatorial view: optical section and aperture (LM); C. equatorial view: aperture detail (SEM). Denscantia cymosa - D. polar view: optical section (LM); E. equatorial view: aperture (LM); F. equatorial view: general surface and aperture (SEM). Emmeorhiza umbellata - G. polar view: optical section (LM); H. polar view: general aspect (SEM); I. equatorial view: aperture (LM). Hexasepalum apiculatum - J. polar view: optical section (LM); K. polar view: general aspect (SEM); L. equatorial view: general aspect and aperture (SEM). Bars: C, F, H, K, L (= 5 µm); A, B, D, E, G, I, J (= 10 µm).

Figure 3
Photomicrographs and eletronphotomicrographs of pollen grains of species Spermacoceae (Rubiaceae) of the Atlantic Forest. Hexasepalum radula - A. polar view: optical section (LM); B. equatorial view: aperture (LM); C. equatorial view: general aspect and aperture (SEM). H. teres - D. polar view: optical section (LM); E. equatorial view: aperture (LM); F. surface detail (SEM). Manettia fimbriata - G. polar view: optical section (LM); H. equatorial view: general aspect and aperture (LM); I. surface detail (SEM). Mitracarpus eichleri - J. polar view: optical section (LM); K. polar view: general aspect (SEM): L. equatorial view: general aspect and aperture (SEM). Bars: C, K, L (= 5 µm); A, B, D, E, G, H, J (= 10 µm); F, I (= 1 µm).

Figure 4
Photomicrographs and eletronphotomicrographs of pollen grains of species Spermacoceae (Rubiaceae) of the Atlantic Forest. Mitracarpus frigidus - A. polar view: general aspect (SEM); B. equatorial view: aperture (LM); C. surface detail (SEM). M. hirtus - D. polar view: optical section (LM); E. equatorial view: aperture (LM); F. surface detail (SEM). M. lhotzkianus - G. polar view: general aspect (SEM); H. equatorial view: general view and aperture (SEM); I. surface detail (SEM). M. megapotamicus - J. polar view: optical section (LM); K. equatorial view: aperture (LM); L. surface detail (SEM). Bars: A, G, H (= 5 µm); B, D, E, J, K (= 10 µm); C, F, I, L (= 1 µm).

Figure 5
Photomicrographs and eletronphotomicrographs of pollen grains of species Spermacoceae (Rubiaceae) of the Atlantic Forest. Oldenlandia salzmannii. A. polar view: optical section (LM); B. equatorial view: aperture (LM); C. surface detail (SEM). Pentodon pentandrus - D. polar view: optical section (LM); E. equatorial view: aperture (LM); F. surface detail (SEM). Richardia brasiliensis - G. polar view: general aspect (SEM); H. equatorial view: general aspect and aperture (SEM); I. surface detail (SEM). R. scabra - J. polar view: optical section (LM); K. polar view: general aspect and aperture (SEM); L. surface detail (SEM). Spermacoce rubescens - M. polar view: optical section (LM); N. polar view: surface detail (SEM); O. equatorial view: aperture (LM). Bars: G, H, N (= 5 µm); A, B, D, E, J, K, M, O (= 10 µm); C, F, I, L (= 1 µm).

Dispersal unit, polarity and size

All species examined had isopolar and radially symmetrical pollen grains on monads. are generally medium-sized (ranging from 25.5 to 47.9 µm in polar diameter and 25.9-48.7 µm in equatorial diameter, in equatorial view), large in Hexasepalum apiculatum, H. radula, Manettia fimbriata, Richardia brasiliensis and R. scabra (51.6-91.0 µm in polar diameter and 59.5-82.8 µm in equatorial diameter, in equatorial view) and very large (103.2 µm in polar diameter and 114.9 µm in equatorial diameter) only in Hexasepalum teres (Tabs. 1 - 3).

Table 1
Morphological characters of the species of Spermacoceae tribe (Rubiaceae) of the Atlantic Forest.

Table 2
Measurements (in μm) pollen grains in equatorial view. of species of Spermacoceae tribe (Rubiaceae) of the Atlantic Forest. (n=25; *n=10) arithmetic mean (x̄); standard deviation (s); confidence interval (CI 95 %); PD/ED = relationship between the polar and equatorial diameters.

Table 3
Measurements (in μm) pollen grains in polar view: equatorial diameter (EDPV); apocolpus side (AS); polar area index (PAI) and the exine layers of species of Spermacoceae tribe (Rubiaceae) of the Atlantic Forest (n=10). Arithmetic mean (x̄).

Shape and polar area

Most species have oblate spheroidal shape (PD/ED 0.90-0.97), suboblate in Borreria palustris, Hexasepalum apiculatum, H. radula, Pentodon pentandrus, Richardia brasiliensis, R. scabra (PD/ED 0.77-0.87), prolate spheroidal in Spermacoce rubescens (PD/ED 1.02) or subprolate in Manettia fimbriata (PD/ED 1.32). Polar area was large in most species, very small in Borreria latifolia, B. palustris, Emmeorhiza umbellata, Hexasepalum radula, H. teres, small in Borreria brachystemonoides, Manettia fimbriata and Mitracarpus hirtus or very large in Borreria capitata, Denscantia cymosa, Mitracarpus megapotamicus and Richardia brasiliensis (Tabs. 1 - 3).

Apertures

Pollen grains are colporate with the variable number of aperture (three-19) (Tab. 1). Long ectoaperture in most species, very long in Denscantia cymosa and Richardia brasiliensis, short in most species, very long in n Borreria latifolia, B. palustris, Emmeorhiza umbellate, Hexasepalum radula, H. teres, long in Borreria brachystemonoides, Manettia fimbriata and Mitracarpus hirtus and very small in Borreria capitata, Denscantia cymosa, Mitracarpus megapotamicus and Richardia brasiliensis (Tab. 4). The ectocolpus membrane is psilated. The ectocolpus is wide (4.0-4.6 µm) in Borreria latifolia, B. palustris, Emmeorhiza umbellata, narrow (1.0-3.0 µm) in most species and narrowest (0.9 µm) only in H. radula (Tab. 4). The endoaperture is lolongate in most species and lalongate in Borreria brachystemonoides (Fig. 1B), Denscantia cymosa, Manettia fimbriata (Fig. 3H) and Pentodon pentandrus. Denscantia cymosa was the only species to present three endoapertures in each ectocolpus (Fig. 2E).

Table 4
Measurements mean (μm) of the aperture and exine layers of species of Spermacoceae (Rubiaceae) of the Atlantic Forest. (n=10).

Stratification of exine and ornamentation

Sexine reticulate was found in Denscantia cymosa (Fig. 2F), Manettia fimbriata (Fig. 3I), reticulate-granulate with gemmae in Borreria brachystemonoides (Fig. 1C), reticulate-granulate in Borreria latifolia (Fig. 1H), B. palustris (Fig. 1I), in the species of the genus Mitracarpus (Fig. 4C, F, I, L), in Oldenlandia salzmannii (Fig. 5C) and Pentodon pentandrus (Fig. 5F). Sexine microreticulate was found only in Emmeorhiza umbellata. Sexine granulate with perforation was recorded in B. capitata (Fig. 1E, F), B. scabiosoides (Fig. 1L), B. verticillata (Fig. 2C), Spermacoce rubescens (Fig. 5O). In E. umbellata (Fig. 3H) and M. lhotzkianus (Fig. 4G, I), the lumens of the reticulum diminish towards the poles and are considered perforations. Only in Manettia fimbriata are observed granules inside the lumens (Fig. 3I). In the genera Hexasepalum the sexine is echinate, being in H. apiculatum, H. radula, H. teres and Richardia scabra. Only in Richardia brasiliensis was observed vermiculate sexine (Fig. 5I).

Hierarchical cluster analysis (HCA)

The cluster analysis of species of Spermacoceae produced a dendrogram with a linkage value of 31.03 for explaining the data. Taking into account the percentage of information (variable) and the final number of groups, three groups were recognized when 75 % of the information was analyzed.

The cluster one grouped six genera and 13 species: Borreria brachystemonoides, B. capitata, B. scabiosoides, B. verticillata, Emmeorhiza umbellata, Mitracarpus eichleri, M. frigidus, M. hirtus, M. lhotzkianus, M. megapotamicus, Oldenlandia salzmannii, Pentodon pentandrus and Spermacoce rubescens, but did not group all species of Borreria. These species present sexine reticulate-granulate, granulate-perforate in S. rubescens, granulate-perforate in B. capitaita, reticulate-granulate with gemmae in B. brachystemonoides, granulate-perforate in B. scabiosoides and B. verticillata. The shape is oblate spheroidal with exception of P. pentandrus (suboblate) and S. rubescens (prolate spheroidal). All species have medium pollen size except for B. verticillata which has small pollen grains. Oldenlandia sazlmannii, M. eichleri and M. frigidus were not separated, as were B. scabiosoides (=Spermacoce scabiosoides) and S. rubescens. The cluster two grouped two species of Borreria (B. latifolia and B. palustres), D. cymosa, H. radula, M. fimbriata, R. brasiliensis and R. scabra. The two species of Borreria and Manettia fimbriata present reticulate-granulate sexine or with granules in the lumen. The genus Richardia and the species H. radula and Manettia fimbriata present large-sized pollen. We can conclude that the qualitative data do not corroborate with the quantitative data in the two cluster above. In cluster three only two species of the genus Hexasepalum (H. apiculatum and H. teres) were grouped by the quantitative data and were confirmed by the qualitative data such as number of apertures (13-19), large-size pollen grain and echine-perforated sexine (Fig. 6).

Figure 6
Cluster analysis performed with the measured variables of pollen from Spermacoceae. Abbreviations: Bbrac = Borreria brachystemonoides, Bcap = Borreria capitata, Blat = Borreria latifolia, Bpal = Borreria palustres, Bsca = Borreria scabioisoides, Bver =Borreria verticillata, Dcym = Denscantia cymosa, Eumb = Emmeorhiza umbellata, Hapi = Hexasepalum apiculatum, Hrad = Hexasepalum radula, Hter = Hexasepalum teres, Mfim = Manettia fimbriata, Meic = Mitracarpus eichleri, Mfri = Mitracarpus frigidus, Mhir = Mitracarpus hirtus, Mlho = Mitracarpus lhotzkyanus, Mmeg = Mitracarpus megapotamicus, Osal = Oldenlandia salzmannii, Ppen = Pentodon pentandrus, Rbra = Richardia brasiliensis, Rsca = Richardia scabra, Srub= Spermacoce rubescens.

Principal component analysis (PCA)

The first two axes of the PCA explain 94.78 % of the total variance, with first and second axes explaining 85.92 % and 8.89 % of the variance, respectively. The most significant variables of the first principal component are: polar diameter/PD, equatorial diameter/ED, EDPV. The most significant variables of the second axis is index of polar area/AS.

The negative side of axis one concentrated most species of different genera: Borreria (five species), Emmeorhyza, Mitracarpus (five species), Oldenlandia, Pentodon e Spermacoce. The positive side of axis one and two concentrated four species: genus Borreria latifolia, Hexasepalum (two species), Manettia and the negative side of axis one and two concentrated species of the genera Borreria (three species), and Mitracarpus (three species), Oldenlandia and Spermacoce. Hexasepalum teres and H. apiculatum were noted to be vertically opposite at the ends of axis one and two to the right, completely distant from all other species, H. teres on the positive side and H. apiculatum on the negative side. Borreria scabiosoides, Mitracarpus megapotamicus and Spermacoce rubescens were very similar. Borreria brachystemonoides was completely separated from the other species of the genus on the positive side of axis one and negative axis two, Borreria latifolia and B. palustris, although they were together, also distanced themselves from the other species of the genus being on the positive side of axis one and two, Descanttia and Richardia are close from eachother at the positive side of axis one and negative axis two (Fig. 7).

Figure 7
Principal component analysis performed with the mesured variables of pollen from species of Spermacoceae. Abbreviations: PD- polar diameter, ED- equatorial diameter, PD/PE - shape, LEC- ectoaperture length, WEC - ectoaperture largure, LEN - endoaperture length, WEN - endoaperture largure, SEX- sexine, NEX -nexine, EDPV- equatorial diameter from polar view, AS- apocolpium side.

Discussion and conclusion

The species Manettia fimbriata and Pentodon pentandrus, presented three-colporate pollen grains while the most species, had many apertures ranging from five to 19 colpore. With regard to endoaperture, the majority of the species presented lolongated endoaperture. As for the shape, the pollen grains ranged from suboblate, oblate spheroidal, subprolate to prolate spheroidal. Regarding the sexine ornamentation, the following types were recorded: a) reticulate-granulate with few gemmae (B. brachysatemonoides), b) reticulate with granules in lumen (Manettia fimbriata), reticulate-granulate (all species of Mitracarpus, Oldenlandia salzmannii and Pentodon pentandrus), c) microreticulate (Emmeorhyza umbellata), d) vermiculate Richardia brasiliensis), e) perforate with granules (Borreria scabiosoides), f) echinate-perforate (all species of Hexasepalum, Richardia scabra), g) granulate-perforate (Borreria capitata, B. latifolia, B. verticillata, Spermacoce rubescens), h) reticulate (Descantia cymosa).

The genus Borreria was palynologically described in several studies, among those is the study by Salgado-Labouriau (1973Salgado-Labouriau ML. 1973. Contribuição à Palinologia dos Cerrados. Academia Brasileira de Ciências, Rio de Janeiro.) that, through the species B. capitata, established the Borreria type as zonoaperturate, with six-eight pores and sexine retipilate; Carreira (1976Carreira L, Medeiros M. 1976. Pollen morphpology of woody plants of the camping. Acta Amazonica 6: 247-269.) described the pollen grains as small, six-seven-pantoporate, retipilate sexine. Cabral (1985Cabral EL. 1985. Valor taxonômico del pólen em lás espécies Argentinas del gênero Borreria, seccion Borreria (Rubiaceae). Sociedad Argentina de Botánica 24: 169-178.) considered pollen grains of B. capitata medium, with five-six pores and granulate ornamentation.

Authors such as Chávez et al. (1991Chávez RP, Ludlow-Wiechers B, Villanueva R. 1991. Flora palinológica de la reserva de la biosfera de Sian Ka´na, Quintana Roo, México. Centro de Investigaciones de Quintana Roo. México, Cioro.), Arreguín-Sánchez et al. (1995Arreguín-Sánchez ML, Palacios-Chávez R, Queiroz-Garcia DL. 1995. Morfologia de los granos de polen de la familia Rubiaceae del Valle de Mexico. Phytologia 78: 357-360.) and Dessein et al. (2002Dessein S, Huysmans S, Robbrecht E, Smets E. 2002. Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspect. Grana 41: 69-89.) analyzed the pollen grains of B. verticillata and, in general, the results found here are similar to the last author. The results differ from Chávez et al. (1991Chávez RP, Ludlow-Wiechers B, Villanueva R. 1991. Flora palinológica de la reserva de la biosfera de Sian Ka´na, Quintana Roo, México. Centro de Investigaciones de Quintana Roo. México, Cioro.) and Arreguín-Sánchez et al. (1995Arreguín-Sánchez ML, Palacios-Chávez R, Queiroz-Garcia DL. 1995. Morfologia de los granos de polen de la familia Rubiaceae del Valle de Mexico. Phytologia 78: 357-360.) regarding type of aperture and ornamentation of the sexine that Chávez et al. (1991Chávez RP, Ludlow-Wiechers B, Villanueva R. 1991. Flora palinológica de la reserva de la biosfera de Sian Ka´na, Quintana Roo, México. Centro de Investigaciones de Quintana Roo. México, Cioro.) described as six-colpate and sexine microreticulate. Arreguín-Sánchez et al. (1995Arreguín-Sánchez ML, Palacios-Chávez R, Queiroz-Garcia DL. 1995. Morfologia de los granos de polen de la familia Rubiaceae del Valle de Mexico. Phytologia 78: 357-360.) characterized as seven or more colpi and per-reticulate surface, whereas, in the present work, we found grains of pollen (six)-seven-(eight) colporate and perforate-granulate sexine.

Melhem et al. (2003Melhem TS, Cruz-Barros MAV, Corrêa AMS, Makino-Watanabe H, Silvestre-Capelato MSF, Gonçalves-Esteves V. 2003. Variabilidade polínica em plantas de Campos de Jordão (São Paulo, Brasil). Boletim do Instituto de Botânica (São Paulo) 16: 9-104.) analyzed 20 species of Rubiaceae for Campos do Jordão, São Paulo, Brazil. In this study the authors analyzed three species of Borreria of which B. capitata and B. verticillata. The results are different from those obtained when the type of aperture (pores in B. capitata) and the ornamentation of B. verticillata (perforated). These species were here considered to have colpori and sexine granulated-perforated.

Denscantia cymosa was the only species that presented three endoapertures by ectoaperture. This species was analyzed by Cabral (1985Cabral EL. 1985. Valor taxonômico del pólen em lás espécies Argentinas del gênero Borreria, seccion Borreria (Rubiaceae). Sociedad Argentina de Botánica 24: 169-178.), Pire & Cabral (1992Pire SM, Cabral EL. 1992. El valor del polen em la revalidacion de Galianthe (Spermacoceae-Rubiaceae). Darwiniana 31: 1-10.) and Silveira-Junior et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.). The results here obtained (sexine reticulate) differed from the previous analyzes by regarding the surface da sexine (granulate-perforate).

The pollen grains of Hexasepalum were characterized by Silveira-Junior. et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.) who also analyzed the species and the results found here were similar regarding ornamentation of sexine and aperture number. Hexasepalum radula and H. teres were analyzed by Dessein et al. (2005Dessein S, Ochoterena H, Block PD, et al. 2005. Palynological characters and their phylogenetic signal in Rubiaceae. The New York Botanical Garden 71: 354-414.) who considered the pollen grains of H. teres to be very large. Our results agree with those of the author and differ from the descriptions made by Delprete & Cortés (2006Delprete PG, Cortés BR. 2006. A synopsis of the Rubiaceae of the states of Mato Grosso and Mato Grosso do Sul, Brazil, with a key to genera, and a preliminar species list. Review Biological Neotropical 3: 13-96.) that considered pollen grains H. radula with ornamentation foveolate-perforate sexine. The pollen data of H. teres presented in this study also differ from that described by Silveira-Junior. et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.) regarding the number of zonocolpate apertures (11) (-12)-four-(-15) (-16) and the echinate-granulate-perforate surface. It is believed that the differences in the ornamentation of the sexine between the present study and the previous ones are justified by the type of microscopy used.

The species Emmeorhiza umbellata has been described herein as having medium size, oblate spheroidal, eight-(nine) colporate pollen grains and the microreticulate surface. Pire & Cabral (1992Pire SM, Cabral EL. 1992. El valor del polen em la revalidacion de Galianthe (Spermacoceae-Rubiaceae). Darwiniana 31: 1-10.), Melhem et al. (2003Melhem TS, Cruz-Barros MAV, Corrêa AMS, Makino-Watanabe H, Silvestre-Capelato MSF, Gonçalves-Esteves V. 2003. Variabilidade polínica em plantas de Campos de Jordão (São Paulo, Brasil). Boletim do Instituto de Botânica (São Paulo) 16: 9-104.), Delprete & Cortes (2006Delprete PG, Cortés BR. 2006. A synopsis of the Rubiaceae of the states of Mato Grosso and Mato Grosso do Sul, Brazil, with a key to genera, and a preliminar species list. Review Biological Neotropical 3: 13-96.) and Silveira-Junior et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.) studied the E. umbellata pollen grains and the results found here differ from most of the authors cited above for surface (equinate-perforate). The results are similar only with those of Silveira-Junior. et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.) regarding surface (microreticulate).

Melhem et al. (2003Melhem TS, Cruz-Barros MAV, Corrêa AMS, Makino-Watanabe H, Silvestre-Capelato MSF, Gonçalves-Esteves V. 2003. Variabilidade polínica em plantas de Campos de Jordão (São Paulo, Brasil). Boletim do Instituto de Botânica (São Paulo) 16: 9-104.) described the pollen grains of the Mitracarpus species as medium, oblate spheroidal three-four-six-seven-nine-colpate, granulate exine. The results found here were similar differing in the type of aperture (colporate) and in ornamentation of sexine, described here as reticulate-granulate. Melhem et al. (2003Melhem TS, Cruz-Barros MAV, Corrêa AMS, Makino-Watanabe H, Silvestre-Capelato MSF, Gonçalves-Esteves V. 2003. Variabilidade polínica em plantas de Campos de Jordão (São Paulo, Brasil). Boletim do Instituto de Botânica (São Paulo) 16: 9-104.) considered it as granulate and Souza and Lorenzi (2008Souza VC, Lorenzi H. 2008. Botânica sistemática. Guia ilustrado para identificação das famílias de angiospermas da flora brasileira baseado em APG II. Ed Plantarum, Nova Odessa.) reported the presence of five-10-colporate pollen grains. The data of Delprete & Cortés (2006Delprete PG, Cortés BR. 2006. A synopsis of the Rubiaceae of the states of Mato Grosso and Mato Grosso do Sul, Brazil, with a key to genera, and a preliminar species list. Review Biological Neotropical 3: 13-96.) are different regarding type of aperture (colporate) and ornamentation of the exine.

Pentodon pentandrus was described in the present work as medium, suboblate, three-colporate pollen grains and reticulate-granulate surface, by the bibliographic survey, shows that the species was analyzed for the first time in the present study.

Authors such as Erdtman (1952Erdtman G. 1952. Pollen morphology and plant taxonomy - Angiosperms. Stockholm, Almqvisit and Wiksell.), Salgado-Labouriau (1973Salgado-Labouriau ML. 1973. Contribuição à Palinologia dos Cerrados. Academia Brasileira de Ciências, Rio de Janeiro.), Delprete & Cortés (2006Delprete PG, Cortés BR. 2006. A synopsis of the Rubiaceae of the states of Mato Grosso and Mato Grosso do Sul, Brazil, with a key to genera, and a preliminar species list. Review Biological Neotropical 3: 13-96.) and Silveira-Junior et al. (2012Silveira-Junior CEA, Saba MD, Jardim JG. 2012. Pollen morphology of Rubiaceae Juss. Species occurring in área of caatinga (dryland) vegetation in Bahia State, Brazil. Acta Botanica Brasilica 26: 444-445.) studied species of the genera Richardia, and the results found were different when compared to the size, shape, type and number of aperture and ornamentation of sexine.

Based on the results obtained here, it can be concluded that the pollen attributes such as size, shape, number of apertures and ornamentation of the sexine were important to define species but did not characterize the genera. However, in relation to ornamentation, some species of a genus presented the same type, for example: Hexasepalum species, echinate-perforate; Mitracarpus species reticulate-granulate confirming that the group is euripolynic.

Borreria, Mitracarpus, Spermacoce and Oldenlandia were grouped togheter in the PCA and HCA analysis, showing that the pollinic characteristics didn’t resolve those genera. Borreria palustre and B. latifolia are exceptions, forming a cluster different from the other species of the genera.

Hexasepalum are separeted in the PCA, due to apocolpium side and lenght ectolpus measures, but the species H. apiculatum and H. teres are agrouped in a cluster, from the HCA perspective.

From the multivariate analysis used here it can be concluded that the quantitative traits are not enough to completely separate the species of Spermacoceae.

It can be affirmed that the pollinic characteristics can be useful in taxonomic studies of the Spermococeae. The application of pollen knowledge is an additional tool for describing and identifying taxa to increase understanding of tribe systematics, presently established infrageneral categories arrangements for reassessment of circumscriptions, and a better understanding of the phylogenetic lineages of Spermacoceae.

Acknowledgements

The first and the last authors thanks CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for a research grant. This study received financial support from the CNPq, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and from the Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ).

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Publication Dates

  • Publication in this collection
    05 June 202030 July 2020
  • Date of issue
    Apr-Jun 2020

History

  • Received
    21 Aug 2019
  • Accepted
    18 Nov 2019
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