Morphological analysis of pollen grains in Machaerium species from São Paulo, BrazilAbstract
Pollen morphology serves as a valuable tool for taxonomic characterization and evolutionary studies. In this study, we conducted a detailed morphological analysis of pollen grains from 16 species of Machaerium Pers. (Fabaceae) occurring in São Paulo, Brazil. Pollen samples were obtained from herbarium specimens and subjected to both qualitative and quantitative analyses using light microscopy and scanning electron microscopy. Our results confirm the stenopalynous character of Machaerium pollen, characterized by monads, isopolar pollen grains, 3-colporate with narrow colpi, and microreticulate exine. Quantitative measurements of colpus length and width, and endoaperture width, proved to be relevant for species differentiation. Additionally, qualitative characteristics such as pollen size, amb, shape, aperture position, presence of fastigium, and the thickness of the exine layer were identified as distinguishing factors among species. Despite variations observed, our analysis did not reveal distinct pollen features for taxonomic sections within the genus, indicating a high degree of pollen similarity among species. These findings contribute to our understanding of pollen morphology in Machaerium species and highlight the importance of pollen characteristics in taxonomic studies and evolutionary research.
Keywords: Cerrado; Fabaceae; Multivariate analysis; Palynology; Papilionoideae
Introduction
Machaerium Pers. (Papilionoideae DC., Fabaceae Lindl.) is one of the largest tropical arboreal genera in Papilionoideae, comprising approximately 130 species (Klitgaard & Lavin, 2005). Among these, 74 are found in Brazil, with 44 being endemic and 18 present in the state of São Paulo (Filard et al., 2024). The genus belongs to the tribe Dalbergieae De Candolle and includes shrubs, trees, or lianas commonly found in forest formations. The arboreal species of the genus are popularly known as “jacarandas” due to the quality of the wood they provide and are predominantly found in the Atlantic Forest (41 species), Amazon (33 species), and Cerrado (22 species) regions (Filard et al., 2024).
The monophyly of Machaerium has been supported by phylogenetic analyses related to chromosomal DNA and ITS regions (Ribeiro et al., 2007). This monophyly is further confirmed by the morphology of its samara fruits, characterized by a basally positioned indehiscent seed chamber with a wing (Lima, 1990). The genus is divided into five sections: Lineata Benth., Oblonga (Benth.) Taub., Acutifolia Taub., Reticulata (Benth.) Taub., and Penninervia (Benth.) Taub (Taubert, 1891). Recent analyses of the ITS region and morphological data concluded that Machaerium is paraphyletic concerning the Aeschynomene L. sect. Ochopodium Vogel clade (Lewis et al., 2012). Lewis et al. (2012) proposed a clade comprising Aeschynomene sect. Ochopodium and Machaerium species with cladodromous venation, and another clade of Machaerium species with brochidodromous venation. Therefore, the pattern of secondary venation in leaflets is a crucial character for Machaerium's infrageneric classification (Bentham, 1860; Rudd, 1987).
Pollen grains of Machaerium have been described in a limited number of studies, including Barth (1964, 1989), Freitas & Carvalho (2012), Barreto et al. (2013), Silva (2014), Ybert et al. (2017), Fernandez et al. (2021), Escobar-Torrez et al. (2024) and RCPol (2024). Barth (1964) described pollen grains of 12 species, such as Machaerium aculeatum Raddi, M. paraguariense Hassl., and M. stipitatum Vogel. The pollen grains differed in shape, while their small size, three operculate colpi, and tectate and reticulate sexine ornamentation were common features among the studied species.
Freitas & Carvalho (2012) classified Machaerium pollen as monads, isopolar, radiasymmetric, subprolate, 3-colporate, with long colpi and circular endoapertures, and scabrate to microreticulate exines. Barreto et al. (2013) described Machaerium pollen as monads, isopolar, small, radially symmetrical, subtriangular in amb., 3-colporate, with narrow colpi and reticulate ornamentation.
The pollen grains of Machaerium hirtum (Vell.) Stellfeld were characterized as monads, isopolar, small to medium, radially symmetrical, oblate-spheroidal to prolate-spheroidal, subtriangular in amb, 3-colporate, with long colpi, lalongate endoaperture, exine perforate. Machaerium acutifolium pollen was characterized by as monads, isopolar, small, radially symmetrical, spheroidal to subprolate, subtriangular in amb, 3-colporate, with long colpi, lalongate endoaperture, and exine microreticulate, according to the Online Pollen Catalog Network (RCPol 2024).
Divergences in shape, amb, ornamentation, and, occasionally, the type of apertures of Machaerium pollen grains can be observed (Barth, 1964; Silva, 2014; Ybert et al., 2017). Given the genus's significancein all Brazilian phytogeographic domains (Filard et al., 2024), the limited number of pollen studies for this genus, and the lack of knowledge regarding pollen morphology variation and its relation to group taxonomy, this study aimed to characterize the pollen of Machaerium species occurring in the State of São Paulo, Brazil. This includes native species found in forest remnants northwest of São Paulo, identifying diagnostic morphological characters for species or species groups within the genus. This study complements pollen analyses of Fabaceae (Soares et al., 2021, 2022, 2024) and other families in the area (Souza & Gasparino, 2014; Belonsi & Gasparino, 2015; Dutra & Gasparino, 2018; Landi & Gasparino, 2018; Souza et al., 2019; Bellonzi et al., 2020; Dutra et al., 2020; Landi et al., 2021; Lopes et al., 2022; Landi et al., 2022; Torrati-Guioti et al., 2023; Cerdan et al., 2024).
Material and Methods
The present study investigated pollen grains of 16 species (Table 1) of Machaerium Pers. (Fabaceae) occurring in the State of São Paulo, according to Filardi et al. (2024). Species such as M. incorruptibile (Vell.) Benth. and M. reticulatum (Poir.) Pers., also listed for the State of São Paulo, were not analyzed due to a lack of available pollen material.
Voucher specimens of Machaerium (Papilionoideae, Fabaceae) from Brazilian forest patches (In parentheses is the Herbarium Code and the registration numbers in the collection). * Standard material.
Pollen materials were obtained from SJRP and SP herbaria (abbreviations according to Thiers, 2024). For pollen morphology analysis, floral buds close to anthesis were used. For each species, pollen grains from more than one specimen were analyzed.
For light microscopy analysis (LM), pollen grains were acetolyzed using the classical acetolysis technique (Erdtman, 1960), with modifications proposed by Melhem et al. (2003). Diameter measurements were taken randomly (n = 25) over one week (Melhem & Matos, 1972; Salgado-Labouriau 1973). For other characters, such as apertures and exine layers, 10 measurements were taken. The obtained slides were deposited in the pollen collection of the Department of Biology, Faculty of Agricultural and Veterinary Sciences of Jaboticabal (FCAV), UNESP - Campus of Jaboticabal - SP, as voucher material.
Scanning Electron Microscopy (SEM) analysis was conducted to elucidate pollen grain ornamentation, following the methodology described in Melhem et al. (2003) for non-acetolyzed pollen grains.
Photomicrographs of pollen grains were taken using an optical microscope (Leica IM50 optical microscope) with a video camera attached to a computer, and electron micrographs were obtained using the JEOL JSM5410 scanning electron microscope. Plates illustrating the analyzed pollen grains were prepared from these images.
Quantitative data obtained were used to calculate the arithmetic mean (x̅), standard deviation of the mean (Sx), sample standard deviation (s), coefficient of variability (CV), and 95% confidence interval (CI) (Zar, 2010; Vieira, 2011). Comparisons of means and confidence intervals of diameters, shape means, and standard deviations among the studied species were performed using graphs created in Excel. With the help of the Fitopac program (Shepherd, 2010) for transforming pollen grain metric measurements by natural logarithm [log(x+1)], and subsequently, the PC-ORD version 7 program (McCune & Mefford, 2011), a Principal Component Analysis (PCA) was conducted using a covariance matrix. This aimed to evaluate the influence of quantitative pollen grain data on species ordination. For PCA, 14 metric variables common to Machaerium species pollen grains were used: equatorial diameter in polar view (EDPV), polar diameter in equatorial view (PDEV), equatorial diameter in equatorial view (EDEV), colpus length (CLEN), colpus width (CWID), endoaperture length (ELEN), endoaperture width (EWID), colpus width index (WCI), polar area index (PAI), exine (EXIN), sexine (SEXI), nexine (NEXI), tectum (TECT), and margin (MARG).
A Hierarchical Cluster Analysis (HCA) was generated in the PC-ORD program, and a dendrogram was obtained from the Jaccard similarity coefficient using the Group Average Linkage Method (Chung et al., 2019; Cerdan et al., 2024). Qualitative variables indicated as absent (0) or present (1) for Machaerium pollen grains were used in the analysis. The qualitative characteristics included size (small, medium, small to medium), amb (hexagonal, subtriangular, subcircular), polar area (small, large), ectoaperture location (angulaperturate, circulaperturate, planaperturate), ectoaperture length (short, long), fastigium, ectoaperture constriction, bridge, ectoaperture membrane ornamentations, endoaperture (elongated, circular), exine thickness (thin, thick), and exine layers thickness (sexine thinner or equal to nexine).
Pollen descriptions followed Bellonzi et al. (2020), and terminology was based on the glossaries of Punt et al. (2007) and Halbritter et al. (2018). The classification of endoapertures followed Punt et al. (2007), based on Erdtman (1952), using the calculation proposed by Soares et al. (2021). Size and shape classes of pollen grains followed Erdtman (1952), while polar area index (PAI) classes followed Faegri & Iversen (1966), and colpus width index followed Gasparino et al. (2013). The relationship of apertures to the amb followed Walker & Doyle (1975). We define it as a constriction in the colpus (ectoaperture) when there is an approximation of the margins of the colpus in the medial region, whereas the term bridge was defined according to Punt et al. (2007) and Halbritter et al. (2018).
Results
General description
Machaerium pollen grains are monads, isopolar, small to medium size (Tables 2, 3), hexagonal, subcircular and subtriangular amb, small to large polar area, oblate spheroidal to subprolate (Table 3); 3-colporate, angulaperturate, circulaperturate or planaperturate, short to long and narrow colpi, tapered in the ends, with margo, fastigium, constriction, and bridge in some species, and ornamented membrane; circulate and lalongate endoaperture (Table 3). Semitectate exine, microreticulate, thin to thick, and sexine thinner than nexine or the same thickness (Table 3).
Quantitative data from pollen grains of Machaerium (Papilionoideae, Fabaceae) species in equatorial view, n = 25. (x̅ = mean (µm); Sx = standard deviation (µm); S = standard error (µm); CI = confidence interval in 95% (µm); V = coefficient of variability (%)).
Quantative and qualitative data of Machaerium (Papilionoideae, Fabaceae) species. (S = Small; M = Medium; S/M = small to medium; P/E = Ratio between polar and equatorial diameter; OS = Oblate spheroidal; PS = Prolate spheroidal; SP = Subprolate; ENDO = endoaperture; CIR = Circular; LA = Lalongate; ECLE = length of ectoaperture; ECWI = width of ectoaperture; MAR = margo; ENLE = length of endoaperture; ENWI = width of endoaperture; WCI = Width colpus index; PAI = Polar area index;; EX = Exine thickness; TE = Tectum thickness; SE = Sexine thickness; NE = Nexine thickness; ETI = exine thickness index). Measurements in µm; Values are the averages of n=10; x̅= mean (µm); Sx = standard deviation (µm)
Species description of Macherium
Machaerium acutifolium Vogel (Fig. 1A-C’, Tables 2-3)
Pollen grains of small size (21.25 x 21.10 µm), subtriangular amb (Fig. 1 A ), small polar area, prolate spheroidal (P/E=1.01); 3-colporate (15.63 x 1.69 µm), angulaperturate, long and narrow colpi, with fastigium (Fig. 1 A -B); lalongate endoaperture (Fig. 1 B ’). Semitectate exine, microreticulate (Fig. 1 C-C’ ), thick (2.32 µm), and sexine thinner than nexine.
Photomicrographs of the pollen grains of Machaerium (Papilionoideae, Fabaceae) species. A-C’. M. acutifolium Vogel.; A. Optical section in polar view; B. Optical section in equatorial view; B’. Detail of aperture; C-C’. Ornamentation in high (C) and low (C’) focus. D-F’. M. amplum Benth.; D. Optical section in polar view; E. Optical section in equatorial view; E’. Detail of aperture; F-F’. Ornamentation in high (F) and low (F’) focus. G-I’. M. brasiliense Vogel.; G. Optical section in polar view.; H. Optical section in equatorial view; H’. Detail of aperture; I-I’. Ornamentation in high (I) and low (I’) focus. J-L’. M. cantarellianum Hoehne.; J. Optical section in polar view.; K. Optical section in equatorial view; K’. Detail of aperture; L-L’. Ornamentation in high (L) and low (L’) focus. Scale bars: 5 µm.
Machaerium amplum Benth. (Fig. 1D-F’, Tables 2-3)
Pollen grains of small size (19.85 x 18.60 µm), subcircular amb (Fig. 1 D ), small polar area, prolate spheroidal (P/E=1.07); 3-colporate (18.93 x 2.39 µm), circulaperturate, long and narrow colpi, with fastigium (Fig. 1 D ); lalongate endoaperture (Fig. 1 E’ ). Semitectate exine, microreticulate (Fig. 1 F-F’ ), thin (1.81 µm), and sexine thinner than nexine.
Machaerium brasiliense Vogel (Fig. 1G-I’, Tables 2-3)
Pollen grains of small size (22.65 x 20.30 µm), subcircular amb (Fig. 1 G ), small polar area, prolate spheroidal (P/E=1.12); 3-colporate (19.22 x 1.41 µm), circulaperturate, long and narrow colpi; circular endoaperture (Fig. 1 H’ ). Semitectate exine, microreticulate (Fig. 1 I-I’ ), thick (2.14 µm), and sexine thinner than nexine.
Machaerium cantarellianum Hoehne (Fig. 1J-L’, Tables 2-3)
Pollen grains of small size (23.65 x 22.00 µm), subcircular amb (Fig. 1 J ), small polar area, prolate spheroidal (P/E=1.08); 3-colporate (17.60 x 2.56 µm), circulaperturate, long and narrow colpi; lalongate endoaperture (Fig. 1 K’ ). Semitectate exine, microreticulate (Fig. 1 L-L’ ), thin (2.10 µm), and sexine thinner than nexine.
Machaerium debile (Vell.) Stellfeld (Fig. 2A-C’, Tables 2-3)
Pollen grains of medium size (34.65 x 31.90 µm), subcircular amb (Fig. 2 A ), small polar area, prolate spheroidal (P/E=1.09); 3-colporate (22.05 x 4.13 µm), circulaperturate, long and narrow colpi, with fastigium; lalongate endoaperture (Fig. 2 B’ ). Semitectate exine, microreticulate (Fig. 2 C-C’ ), thin (3.28 µm), and sexine thinner than nexine. Under SEM, it is possible to observe an ornamented membrane under the ectoaperture and endoaperture (Fig. 2 B ).
Photomicrographs and electron micrographs of the pollen grains of Machaerium (Papilionoideae, Fabaceae) species. A-C’. M. debile (Vell.) Stellfeld.; A. Polar view in SEM; B. Equatorial view and detail of aperture in SEM; B’. Detail of aperture in LM; C-C’. Ornamentation in high (C) and low (C’) focus. D-F’. M. declinatum (Vell.) Stellfeld.; D. Polar view in SEM; E. Optical section in equatorial view; E’. Detail of aperture; F-F’. Ornamentation in high (F) and low (F’) focus. G-I’. M. hatschbachii Rudd.; G. Optical section in polar view; H. Equatorial view and detail of aperture in SEM; H’. Detail of aperture in LM; I-I’. Ornamentation in high (I) and low (I’) focus. J-L’. M. hirtum (Vell.) Stellfeld.; J. Polar view in SEM; K. Equatorial view and detail of aperture in SEM; K’. Detail of aperture in LM; L-L’. Ornamentation in high (L) and low (L’) focus. Scale bars: 2 µm = A, B, D, H, J and K; 5 µm = other images.
Machaerium declinatum (Vell.) Stellfeld (Fig. 2D-F’, Tables 2-3)
Pollen grains of small size (23.45 x 21.05 µm), subtriangular amb (Fig. 2 D ), small polar area, prolate spheroidal (P/E=1.11); 3-colporate (15.10 x 1.61 µm), angulaperturate, long and narrow colpi, constricted in the medial region (Fig. 2 E’ ), membrane of colpi ornamented (Fig. 2 D ); lalongate endoaperture (Fig. 2 E’ ). Semitectate exine, microreticulate (Fig. 2 D, F-F’ ), thin (2.07 µm), and sexine thinner than nexine.
Machaerium hatschbachii Rudd (Fig. 2G-I’, Tables 2-3)
Pollen grains of small to medium size (30.70 x 24.75 µm), hexagonal amb (Fig. 2 G ), small polar area, subprolate (P/E=1.24); 3-colporate (19.47 x 1.21 µm), planaperturate, long and narrow colpi, sometimes with a bridge, difficult to visualize; circular endoaperture (Fig. 2 H’ ). Semitectate exine, microreticulate (Fig. 2 I-I’ ), thin (2.53 µm), and sexine thinner than nexine.
Machaerium hirtum (Vell.) Stellfeld (Fig. 2J-L’, Tables 2-3)
Pollen grains of small size (19.80 x 15.80 µm), subcircular amb (Fig. 2 J ), small polar area, subprolate (P/E=1.25); 3-colporate (17.62 x 2.72 µm), circulaperturate, long and wide colpi, with ornamented membrane; lalongate endoaperture (Fig. 2 K’ ). Semitectate exine, microreticulate (Fig. 2 L-L’ ), thick (1.67 µm), and sexine of the same thickness as nexine.
Machaerium lanceolatum (Vell.) J.F.Macbr. (Fig. 3A-C’, Tables 2-3)
Pollen grains of small to medium size (30.00 x 25.55 µm), subcircular amb (Fig. 3 A ), small polar area, subprolate (P/E=1.17); 3-colporate (21.54 x 2.18 µm), circulaperturate, long and narrow colpi, with fastigium; lalongate endoaperture (Fig. 3 B’ ). Semitectate exine, microreticulate (Fig. 3 C-C’ ), thin (2.20 µm), and sexine thinner than nexine.
Photomicrographs and electron micrographs of the pollen grains of Machaerium (Papilionoideae, Fabaceae) species. A-C’. M. lanceolatum (Vell.) J-F.Macbr.; A. Optical section in polar view; B. Optical section in equatorial view; B’. Detail of aperture; C-C’. Ornamentation in high (C) and low (C’) focus. D-F’. M. nyctitans (Vell.) Benth.; D. Optical section in polar view; E. Optical section in equatorial view; E’. Detail of aperture; F-F’. Ornamentation in high (F) and low (F’) focus. G-I’. M. oblongifolium Vogel.; G. Optical section in polar view; H. Equatorial view and detail of aperture in SEM; H’. Detail of aperture in LM; I-I’. Ornamentation in high (I) and low (I’) focus. J-L’. M. paraguariense Hassl.; J. Optical section in polar view; K. Optical section in equatorial view; K’. Detail of aperture; L-L’. Ornamentation in high (L) and low (L’) focus. Scale bars: 2 µm = H (SEM); 5 µm = other imagens.
Machaerium nyctitans (Vell.) Benth. (Fig.3 D-F’, Tables 2-3)
Pollen grains of small to medium size (29.50 x 23.15 µm), subcircular amb (Fig. 3 D ), small polar area, subprolate (P/E=1.27); 3-colporate (19.75 x 2.09 µm), circulaperturate, long and narrow colpi, sometimes constricted, with fastigium (Fig. 3 D ); lalongate endoaperture (Fig. 3 E’ ). Semitectate exine, microreticulate (Fig. 3 F-F’ ), thin (2.40 µm), and sexine thinner than nexine.
Machaerium oblongifolium Vogel. (Fig. 3G-I’, Tables 2-3)
Pollen grains of small to medium size (24.60 x 23.65 µm), subcircular amb (Fig. 3 G ), large polar area, prolate spheroidal (P/E=1.04); 3-colporate (20.67 x 1.65 µm), circulaperturate, short and narrow colpi, sometimes with a bridge, difficult to visualize; lalongate endoaperture (Fig. 3 H’ ). Semitectate exine, microreticulate (Fig. 3 I-I’ ), thick (3.07 µm), and sexine of the same thickness as nexine.
Machaerium paraguariense Hassl. (Fig. 3J-L’, Tables 2-3)
Pollen grains of small size (19.40 x 17.75 µm), subcircular amb (Fig. 3 J ), small polar area, prolate spheroidal (P/E=1.09); 3-colporate (15.83 x 1.84 µm), circulaperturate, long and narrow colpi; lalongate endoaperture (Fig. 3 K’ ). Semitectate exine, microreticulate (Fig. 3 L-L’ ), thin (1.99 µm), and sexine of the same thickness as nexine.
Machaerium scleroxylon Tul. (Fig. 4A-C’, Tables 2-3)
Pollen grains of small size (20.50 x 17.30 µm), subcircular amb (Fig. 4 A ), small polar area, subprolate (P/E=1.18); 3-colporate (13.53 x 1.04 µm), circulaperturate, long and narrow colpi, with fastigium; lalongate endoaperture (Fig. 4 B’ ). Semitectate exine, microreticulate (Fig. 4 C-C’ ), thick (1.95 µm), and sexine thinner than nexine.
Photomicrographs of the pollen grains of Machaerium (Papilionoideae, Fabaceae) species. A-C’. M. scleroxylon Tul.; A. Optical section in polar view; B. Optical section in equatorial view; B’. and detail of aperture; C-C’. Ornamentation in high (C) and low (C’) focus. D-E. M. stipitatum Vogel.; D. Optical section in polar view; E. Optical section in equatorial view; E’. Detail of aperture. F-H’. M. uncinatum (Vell.) Benth.; F. Optical section in polar view; G. Optical section in equatorial view; G’. Detail of aperture; H-H’. Ornamentation in high (H) and low (H’) focus. I-K’. M. villosum Vogel.; I. Optical section in polar view; J. Optical section in equatorial view; J’. Detail of aperture; K-K’. Ornamentation in high (K) and low (K’) focus. Scale bars: 5 µm.
Machaerium stipitatum (DC.) Vogel (Fig. 4D-E, Tables 2-3)
Pollen grains of small size (18.05 x 18.15 µm), subcircular amb (Fig. 4 D ), small polar area, oblate spheroidal (P/E=0.99); 3-colporate (16.06 x 1.07 µm), circulaperturate, long and narrow colpi, sometimes constricted, with fastigium; lalongate endoaperture (Fig. 4 E’ ). Semitectate exine, microreticulate, thick (1.96 µm), and sexine thinner than nexine.
Machaerium uncinatum (Vell.) Benth. (Fig. 4F-H’, Tables 2-3)
Pollen grains of small to medium size (26.15 x 20.70 µm), subcircular amb (Fig. 4 F ), small polar area, subprolate (P/E=1.26); 3-colporate (19.98 x 1.45 µm), circulaperturate, long and narrow colpi, sometimes constricted, with fastigium; lalongate endoaperture (Fig. 4 G’ ). Semitectate exine, microreticulate (Fig. 4 H-H’ ), thin (2.08 µm), and sexine thinner than nexine.
Machaerium villosum Vogel. (Fig. 4I-K’, Tables 2-3)
Pollen grains of small size (24.40 x 23.75 µm), subcircular amb (Fig. 4 I ), small polar area, prolate spheroidal (P/E=1.03); 3-colporate (21.35 x 1.69 µm), circulaperturate, long and narrow colpi, with fastigium and sometimes with a bridge, difficult to visualize; circular endoaperture (Fig. 4 J’ ). Semitectate exine, microreticulate (Fig. 4 K-K’ ), thin (2.41 µm), and sexine thinner than nexine.
Statistical analysis
Quantitative data
Most studied Machaerium species exhibit pollen grains with small diameters (Fig. 5 A-B ), but they can also present medium diameters, as observed in M. debile (Fig. 5 A-B ), or vary between small and medium sizes (M. hatschbachii, M. lanceolatum, M. nyctitans, M. oblongifolium, and M. uncinatum; Fig. 5 A-B ). The shape varies from oblate spheroidal (M. stipitatum - Fig. 6), prolate spheroidal (most species), to subprolate (M. lanceolatum, M. scleroxylon, M. hatschbachii, M. hirtum, M. uncinatum, M. nyctitans - Fig. 6), considering the mean shape of pollen grains. However, within the sample range for each species (Fig. 6 - confidence interval bars), a variation in shape is observed within the species, with some having up to three types of pollen grain shapes, such as M. hirtum (Fig. 6); most other species predominantly exhibit two types of shapes.
Representation of confidence interval of mean in 95 % of the pollen grains of Machaerium (Papilionoideae, Fabaceae). A. Polar diameter in equatorial view. B. Equatorial diameter in equatorial view. The higher and lower boundaries showing the confidence interval; the average circle showing the arithmetic mean. The values are in µm. (M. acut = Machaerium acutifolium; M. ampl = M. amplum; M. bras = M. brasiliense; M. cant = M. cantarellianum; M. debi = M. debile; M. decl = M. declinatum; M. hats = M. hatschbachii; M. hirt = M. hirtum; M. lan = M. lanceolatum; M. nyct = M. nyctitans; M. oblo = M. oblongifolium; M. para = M. paraguariense; M. scle = M. scleroxylon; M. stip = M. stipitatum; M. unci = M. uncinatum; M. vill = M. villosum).
Graphical representation of the arithmetic means and sample standard deviation of the P/E ratio (shape) of pollen grains of Machaerium (Papilionoideae, Fabaceae). The bars represent the limits of the sample standard deviation, and the circles represent the arithmetic mean. (M. acut = Machaerium acutifolium; M. ampl = M. amplum; M. bras = M. brasiliense; M. cant = M. cantarellianum; M. debi = M. debile; M. decl = M. declinatum; M. hats = M. hatschbachii; M. hirt = M. hirtum; M. lan = M. lanceolatum; M. nyct = M. nyctitans; M. oblo = M. oblongifolium; M. para = M. paraguariense; M. scle = M. scleroxylon; M. stip = M. stipitatum; M. unci = M. uncinatum; M. vill = M. villosum).
For the definition of endoaperture types, the comparison of the limits of the sample standard deviation of endoaperture length and width was employed (Soares et al., 2021). Most species displayed lalongate endoapertures, except for M. brasiliense, M. hatschbachii, and M. villosum (Fig. 7 - indicated with *), which exhibited overlapping sample standard deviation ranges and were thus classified as circular endoapertures (Fig. 7 - indicated with bars).
Graphical representation of the arithmetic means and sample standard deviation of the length and width of the endoapertures of pollen grains of Machaerium (Papilionoideae, Fabaceae), in µm. The bars represent the limits of the sample standard deviation and the circles and squares represent the arithmetic mean. (M. acut = Machaerium acutifolium; M. ampl = M. amplum; M. bras = M. brasiliense; M. cant = M. cantarellianum; M. debi = M. debile; M. decl = M. declinatum; M. hats = M. hatschbachii; M. hirt = M. hirtum; M. lan = M. lanceolatum; M. nyct = M. nyctitans; M. oblo = M. oblongifolium; M. para = M. paraguariense; M. scle = M. scleroxylon; M. stip = M. stipitatum; M. unci = M. uncinatum; M. vill = M. villosum; * = circular endoaperture).
To compare all metric variables obtained from Machaerium pollen grains, a multivariate analysis of principal components (PCA - Fig. 8, Table 4) was performed to define which variables are most significant in distinguishing groups of studied species. In the analysis, the first two axes accounted for 77.27% of data variability, with Axis 1 showing the highest metric data variability at 44.46% (Fig. 8). The main variables distinguishing species were colpus length and width (CLEN and CWID) and endoaperture width (EWID) (Table 4). On the positive side of Axis 1, species with the lowest values for these three variables were located, with M. stipitatum and M. scleroxylon separated from the others by having the lowest values (Fig. 8). On the negative side of Axis 1, species with the highest values were placed, with M. debile having the highest values for colpus and endoaperture width (CWID and EWID), and M. oblongifolium having the highest colpus length value (CLEN). Although less significant, Axis 2 represented 32.81% of the variability, with colpus width index (WCI) being the most significant variable. M. hatschbachii and M. hirtum pollen grains showed the highest and lowest values, respectively, for this variable. In the PCA, the most significant variables for the studied species were related to ectoaperture and endoaperture measurements, such as colpus length, width, and colpus width index.
Pearson and Kendall correlation coefficients for pollen grain metric variables of the first and the second axis of principal component analysis ordination (PCA) of 16 species of Machaerium (Papilionoideae, Fabaceae).
Principal component analysis performed with the pollen metric variables of Machaerium (Papilionoideae, Fabaceae). (M. acut = Machaerium acutifolium; M. ampl = M. amplum; M. bras = M. brasiliense; M. cant = M. cantarellianum; M. debi = M. debile; M. decl = M. declinatum; M. hats = M. hatschbachii; M. hirt = M. hirtum; M. lan = M. lanceolatum; M. nyct = M. nyctitans; M. oblo = M. oblongifolium; M. para = M. paraguariense; M. scle = M. scleroxylon; M. stip = M. stipitatum; M. unci = M. uncinatum; M. vill = M. villosum).
Qualitative data
The analysis carried out with qualitative data (Table 5) shows that the largest groups formed, which showed less than 50% similarity, do not present qualitative characteristics that could differentiate them. The absence of qualitative characteristics that define the groups indicates the similarity of pollen grains among Machaerium species.
Absence (0) and presence (1) of qualitative descriptive characteristics of pollen grains of Machaerium species (Papilionoideae, Fabaceae), used in the Hierarchical Cluster Analysis (HCA). A - small size; B - medium size; C - small to medium size; D - subtriangular amb; E - subcircular amb; F - hexagonal amb; G - small polar area; H - large polar area; I - angulaperturate; J - circulaperturate; K - planaperturate; L - short ectoaperture; M - long ectoaperture; N - fastigium; O - constriction of ectoapertures; P - bridge; Q - membrane of the ectoaperture with ornamentations; R - circular endoaperture; S - lalongate endoaperture; T - thick exine; U - thin exine; V - sexine is thinner than nexine; W - sexine of the same thickness as nexine.
Machaerium debile distinguishes itself from the group consisting of M. hatschbachii, M. lanceolatum, M. nyctitans, M. oblongifolium, and M. villosum due to its medium-sized pollen grains and ornamented colpus membrane (Fig. 9 B , Q). Within this group, M. oblongifolium stands apart from the others by its short ectoaperture, thick exine, and sexine of equal thickness to the nexine (Fig. 9 L , T, W), whereas the other species exhibit a small polar area, thin exine, and sexine thinner than the nexine (Fig. 9 G , U, V). Machaerium lanceolatum, M. nyctitans, and M. villosum are grouped because of their subcircular amb, circulaperturate apertures, and the presence of fastigium (Fig. 9 E , J, N), while M. hatschbachii differs from this group due to its hexagonal amb and planaperturate pollen grains (Fig. 9 F , K).
Dendrogram produced from Cluster analysis (Jaccard’s Coefficient of Similarity) performed with the morphological characters of Machaerium (Papilionoideae, Fabaceae). (M. acut = Machaerium acutifolium; M. ampl = M. amplum; M. bras = M. brasiliense; M. cant = M. cantarellianum; M. debi = M. debile; M. decl = M. declinatum; M. hats = M. hatschbachii; M. hirt = M. hirtum; M. lan = M. lanceolatum; M. nyct = M. nyctitans; M. oblo = M. oblongifolium; M. para = M. paraguariense; M. scle = M. scleroxylon; M. stip = M. stipitatum; M. unci = M. uncinatum; M. vill = M. villosum. A - small size; B - medium size; C - small to medium size; D - subtriangular amb; E - subcircular amb; F - hexagonal amb; G - small polar area; I - angulaperturate; J - circulaperturate; K - planaperturate; L - short ectoaperture; N - fastigium; O - constriction of ectoapertures; P - bridge; Q - membrane of the ectoaperture with ornamentations; R - circular endoaperture; S - lalongate endoaperture; T - thick exine; U - thin exine; V - sexine is thinner than nexine; W - sexine of the same thickness as nexine.
Machaerium amplum, M. cantarellianum, and M. hirtum are grouped with a similarity greater than 87.5%. Machaerium hirtum stands out from the others due to the presence of an ornamented colpus membrane, thick exine, and sexine of equal thickness to the nexine (Fig. 9 Q , T, W), while the other species exhibit thin exine and sexine thinner than the nexine (Fig. 9 U , V). Machaerium amplum, characterized by its fastigium (Fig. 9 N ), separates from M. cantarellianum.
Machaerium paraguariense, with sexine of equal thickness to the nexine (Fig. 9 W ), differs from M. acutifolium, M. brasiliense, M. declinatum, and M. uncinatum, which have sexine thinner than the nexine (Fig. 9 V ). Within this group, M. acutifolium and M. declinatum are grouped due to their subtriangular amb and angulaperturate apertures (Fig. 9 D , I), while M. brasiliense and M. uncinatum exhibit a subcircular amb and circulaperturate apertures (Fig. 9 E , J).
The analysis subtly demonstrates the differentiation of species and groups through details such as pollen grain size, aperture characteristics, and exine thickness. Despite exhibiting features allowing for species separation, the analysis confirms that Machaerium pollen grains are stenopalynous.
Discussion
The Machaerium species analyzed share common characteristics in their pollen grains, including being monads, isopolar, 3-colporate, with narrow colpi, and microreticulate exine. These characteristics define a distinct pollen type for the genus, which has been utilized in applied palynology studies (Barth, 1989; Freitas & Carvalho, 2012; Barreto et al., 2013; Fernandez et al., 2021; Escobar-Torrez et al., 2024). The morphological similarities of pollen grains, such as pollen unit, number and type of apertures, and exine ornamentation, contribute to defining stenopalynous taxa, as these are significant morphological traits for taxonomy (Melhem et al., 2003).
The pollen description presented in this study identified discrete variations that aid in distinguishing species or groups of species. These variations include differences in size, outline in polar view, position of the apertures, presence of fastigium or ornamented membrane of the colpus, and thickness of the layers of the exine. Regarding the quantitative data measured in Machaerium pollen grains, we observed that especially the length and width of the colpus, and the width of endoaperture helped in grouping species.
Studies describing the pollen grains of Machaerium species are scarce in the literature, our study confirms the data observed in the pollen grains of the genus regarding the pollen unit, the type and number of apertures, and, in most cases, the exine ornamentation.
Only Ybert et al. (2017) reported small to medium and medium pollen grains for the genus. Escobar-Torrez et al. (2024) indicated Machaerium with medium pollen grains, citing values of approximately 16 µm for the genus, which defines small pollen grains according to Erdtman (1952).
The amb of pollen grains, defined by the outline in the polar view, is the most subjective qualitative characteristic in pollen grains (Gasparino, 2022). The pollen grains amb analyzed in this study were defined as hexagonal, subcircular, or subtriangular, confirming literature data, except for the study by Ybert et al. (2017), which defined as circular (M. fulvovenosum), and triangular (M. hirtum and M. lanceolatum).
One variable morphological characteristic in the Machaerium species analyzed here is the shape of the pollen. Our study verified variations from oblate spheroidal to subprolate pollen grains, as described in previous studies. However, for the species M. hirtum, M. uncinatum, and M. nyctitans, we observed prolate pollen grains when considering the confidence intervals of the P/E measurements.
In general, ectoapertures in Machaerium pollen grains are defined as long and narrow, and pollen grains with very long or syncolporate ectoapertures were sometimes observed (Barth 1989). In our study, only M. oblongifolium presents pollen grains with short ectoapertures. Regarding the endoapertures, Machaerium presents pollen with circular endoapertures (corroborating Barth, 1989; Freitas & Carvalho, 2012; Escobar-Torrez et al., 2024) or lalongate (Barth, 1964; Silva, 2014; Ybert et al., 2017). However, Ybert et al. (2017) also mention lolongate endoapertures for M. hirtum pollen grains, which differs from the data observed here. It is worth mentioning that some studies, especially in applied palynology (as in Freitas & Carvalho, 2012; and Escobar-Torrez et al., 2024), mistakenly cite endoapertures as pores, and, according to Punt et al. (2007) and Halbritter et al. (2018), pori and endoapertures differ due to the absence of different exine layers, and cannot be considered similar structures.
The exine ornamentation of the Machaerium pollen grains analyzed here was defined as microreticulate, a common characteristic for the species which helps to characterize Machaerium as stenopalynous. However, we observed divergence in the literature regarding this character. Barth (1964) cites reticulate pollen grains for M. aculeatum, M. paraguayensis, and M. stipitatum. Freitas & Carvalho (2012) and Fernandez et al. (2021) indicate ornamentation varying from scabrate to microreticulate for the Dalbergia-Machaerium type and Machaerium sp., respectively. Barreto et al. (2013) describe the Machaerium pollen type as reticulate. Escobar-Torrez et al. (2024) describe psilate pollen grains for the genus. This variation in ornamentation in Machaerium pollen may be associated with different definitions, especially in the distinction between reticulate and microreticulate pollen grains (Punt et al., 2007), or regarding the difficulty of visualization, since applied palynology studies normally do not perform electron microscopy analyses and/or chemical analysis of acetolysis.
According to Bentham (1860), Taubert (1891), and Rudd (1987), the Machaerium species analyzed here are distributed in four of the five sections described for the genus. In general, pollen data did not allow the distinction of these sections since the genus has high pollen similarity, and the discrete variations are not associated with taxonomic categorization. As for quantitative data, PCA analysis revealed the similarity of M. debile and M. oblongifolium, both from the Reticulata section; however, the species M. brasiliense and M. lanceolatum, also from the same section, were not grouped with the previous ones in this analysis. The PCA also revealed the similarity of M. scleroxylon and M. stipitatum; however, these species are in the Oblonga and Acutifolia sections. In the analysis of qualitative pollen data (cluster analysis), we did not observe distinctions and groupings of species positioned in the same taxonomic sections. This homogeneity of species from different sections in terms of pollen data confirms the stenopalynous character of the genus, and that the discrete variations observed are not significant for the definition of more than one pollen type for Machaerium.
The results obtained in this study confirm the stenopalynous character of Machaerium. Data such as measurements of the length and width of the colpus and the width of the endoaperture were relevant for the separation of species, reinforcing the importance of quantitative analysis of pollen data. Our analysis revealed that some pollen characteristics, such as size, amb, aperture details, type of endoaperture, and exine thickness, can be used to distinguish species or groups of species. In general, the observed data contribute to the definition of a pollen type for Machaerium.
Notably, our analysis did not reveal distinct pollen features for taxonomic sections within the genus, indicating a high degree of pollen similarity among species. This homogeneity across taxonomic sections supports the stenopalynous nature of Machaerium and suggests that the observed variations may not be taxonomically significant enough to define multiple pollen types within the genus.
Acknowledgments
The authors would like to thank the curators of the SJRP and SP herbarium for enabling the obtaining of pollen material and the Electron Microscopy Laboratory at FCAV, Unesp, Jaboticabal. V.H.B. Gusman is funded by FAPESP (The São Paulo Research Foundation - Grant 2016/25414-0) and E.C. Gasparino is funded by CNPq (#309555/2021-3).
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Publication Dates
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Publication in this collection
02 Dec 2024 -
Date of issue
2024
History
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Received
09 Feb 2024 -
Accepted
08 Sept 2024
