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Pollen morphology in Brazilian species of Codonanthe (Mart.) Hanst. and Nematanthus Schrader (Gesneriaceae)

Eduardo Custodio Gasparino, Maria Amelia Vitorino Da Cruz-Barros & Alain Chautems

To cite this article: Eduardo Custodio Gasparino, Maria Amelia Vitorino Da Cruz- Barros & Alain Chautems (2013) Pollen morphology in Brazilian species of Codonanthe (Mart.) Hanst. and Nematanthus Schrader (Gesneriaceae), Grana, 52:4, 258-274, DOI: 10.1080/00173134.2013.826274 To link to this article: https://doi.org/10.1080/00173134.2013.826274

Published online: 27 Sep 2013.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=sgra20 Grana, 2013 Vol. 52, No. 4, 258–274, http://dx.doi.org/10.1080/00173134.2013.826274

Pollen morphology in Brazilian species of Codonanthe (Mart.) Hanst. and Nematanthus Schrader (Gesneriaceae)

EDUARDO CUSTODIO GASPARINO1, MARIA AMELIA VITORINO DA CRUZ-BARROS2 &ALAINCHAUTEMS3

1Faculdade de Ciências Agrárias e Veterinárias, UNESP - Universidade Estadual Paulista, Jaboticabal, Brazil, 2Núcleo de Pesquisa em Palinologia, Instituto de Botânica, São Paulo, Brazil, 3Conservatoire et Jardin Botanique de La Ville de Genève, Geneva, Switzerland

Abstract Pollen morphology of 24 Brazilian species of Codonanthe and Nematanthus (Gesneriaceae) was investigated by means of light microscopy, scanning and transmission electron microscopy, in search of new characters that might contribute to taxonomic characterisation of the genera. The pollen grains are tricolpate or tricolporate, semitectate, reticulate, with simplicolumellate muri, sometimes with microreticulum and perforations, or duplicolumellate muri ring-shaped around lumina. The species studied were grouped into five pollen types on the basis of aperture characteristics and details of surface ornamentation. Sub-types were defined with reference to presence or absence of microreticulum and perforations around the lumina. Based on analysis of the morphology and measurements of pollen grains, we verified a close relationship among the genera, but can nevertheless discriminate their species.

Keywords: Brazil, Episcieae, palynology, pollen grains

The large and diverse tribe Episceae (Gesnerioideae, Codonanthe are not monophyletic as pointed out as Gesneriaceae), comprising about 22 genera and over early as Clark and Zimmer (2003). Nematanthus 700 species, is restricted to the Neotropics. The is distributed with 31 species in the Atlantic for- species are characterised by axillary flowers derived est along the Brazilian coast (Chautems, 1988; from a pair-flowered cyme inflorescence by reduc- Chautems et al., 2005). The genus consists of tion, a three-trace trilacunar node with split lateral sub-shrubs, epiphytes or lithophytes, with fleshy bundles, superior ovaries, mostly with a haploid fruits with loculicidally dehiscing capsules, and a chromosome number of n = 9[n = 8inCodonanthe haploid chromosome number of n = 8 (Chautems (Mart.) Hanst. and Nematanthus Schrader] and by & Matsuoka, 2003). The presence or absence of the presence of sepals connate at the base and an resupinate flowers was used by Moore (1973a) urceolate cup or funnel-shaped calyx (Wiehler, 1983; and Chautems (1988) to separate groups of Clark et al., 2006). Molecular data shows that the species within Nematanthus. Thus, floral morphol- tribe represents a monophyletic clade with a hetero- ogy has been used to assist in the classifica- geneous morphology within the Gesneriaceae (Smith tion of Nematanthus. Chautems (1988) recognised & Carroll, 1997; Smith, 2000a, 2000b; Zimmer 26 species and distinguished three groups: species et al., 2002; Clark et al., 2006, 2012). with non-resupinate flowers, with resupinate flowers, According to Clark et al. (2006), some generic lim- and with pendent resupinate flowers. Weber (2004) its of Episcieae are not well-defined, although the defined four types of flowers for the genus: hypocyr- Nematanthus, Codonanthe and Codonanthopsis Mansf. toid non-resupinate, hypocyrtoid resupinate, pen- clade is well-supported, the genera Nematanthus and dent resupinate and large campanulate white flowers.

Correspondence: Eduardo C. Gasparino, Departamento de Biologia aplicada à Agropecuária, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Universidade Estadual Paulista, UNESP, via de acesso Prof. Paulo Castellane, s/n, CEP 14884-900, Jaboticabal, SP, Brazil. E-mail: [email protected]

(Received 19 March 2013; accepted 1 July 2013) © 2013 Collegium Palynologicum Scandinavicum Codonanthe and Nematanthus pollen morphology 259

However, Clark et al. (2006) followed Chautems morphological features, pollen grains allow the dif- (1998) arrangement and treated the fourth group as ferentiation of genera and even species. white campanulate non-resupinate flowers. The aim of this study was to characterise the pollen Codonanthe has 18 species, growing epiphytically grains of native Brazilian species of Codonanthe and or lithophytically, with corollas tubular-campanulate Nematanthus (Gesneriaceae), in attempt to recognise to slightly ventricose, and as in Nematanthus,ahap- qualitative and quantitative characteristics that can loid chromosome number of n = 8 (Moore, 1973b; contribute to useful taxonomic characterisation for Wiehler & Chautems, 1990; Chautems & Matsuoka, these genera. 2003; Weber, 2004). The Amazonian and Central American species of the genus present extrafloral nectaries on the abaxial leaf side and, according to Material and methods Prance (1973), ants can assume an important role in seed dispersal. Fritsch (1894) used an anther feature, Pollen from dried herbarium specimens was supplied i.e. the broad connective causing a spatial separa- by the following herbaria: Universidade Estadual de tion of the pairs of thecae, to separate the species Campinas, Campinas, Brazil (UEC), Instituto de of Codonanthe from other genera of the subtribe Botânica, São Paulo, Brazil (SP) and Universidade Codonanthinae. de São Paulo, São Paulo, Brazil (SPF). Five out Currently, based on the fruit type, the genus is of 19 described species of Codonanthe,17out divided into two subgenera, Codonanthella (includ- of 31 species of Nematanthus and two hybrids ing species distributed in Central America and the from a total of 35 specimens were studied by Caribbean with fleshy capsules, as in Nematanthus) transmitted light microscopy (LM), scanning elec- and Codonanthe (with species that occur in Brazil tron microscopy (SEM) and transmission electron and the Amazon Basin). The species of the sub- microscopy (TEM). Pollen of 16 of these species genus Codonanthe are divided into two sections: had not been studied previously. The pollen sam- Spathuliformae occurring in eastern and north- ples were acetolysed according to the method of ern Brazil, Ecuador and Central America, and Erdtman (1960), with modifications by Melhem Codonanthe including the endemic species of the et al. (2003), and measured within seven days after Atlantic forest sympatric with species of Nematanthus their preparation (Salgado-Labouriau et al., 1965). (Chautems, 1988; Wiehler & Chautems, 1990). Permanent light microscopy slide preparations are There are only a few studies on pollen morphol- held in the pollen reference collection of the Instituto ogy of species of New World Gesneriaceae, and de Botânica, São Paulo, Brazil. papers dealing with Brazilian species are restricted to The polar axis (P) and equatorial diameter (E)of the studies of Campos (1962), Melhem and Mauro 25 pollen grains were measured per sample; other (1973), Melhem et al. (2003), Fourny et al. (2010) measurements (apocolpia, sexine, nexine thickness, and Gasparino et al. (2011). According to these lumina, muri, apertures and ornamentation) were taken from ten pollen grains per specimen. Statistical analyses were conducted to obtain the means, stan- Table I. Pollen grain metric variables used in PCA and cluster dard deviations, confidence intervals (95%), ampli- += = analysis ( present; – absent). tudes (minimum and maximum values) and variabil- Pollen grains ity coefficients (Vieira, 1981;Zar,1996). A principal Variables Tricolpate Tricolporate component analysis (PCA) was performed to ver- ify whether the pollen data permitted the grouping APOC (Apocolpium) ++of the species included here. This analysis was per- CLEN (Length of colpus) ++formed using the programs FITOPAC (Shepherd, CWID (Width of colpus) ++ EDEV (Equatorial diameter in ++1996) and PC-ORD (McCune & Mefford, 1999). equatorial view) To determine whether the pollen characteristics pro- EDPV (Equatorial diameter in ++vided additional discrimination among the analysed polar view) Codonanthe and Nemantanthus species, we compared PDEV (Polar diameter in ++the obtained pollen grains data by a cluster analy- equatorial view) ELEN (Length of endoaperture) −+sis (UPGMA and Euclidean distance) using version EWID (Width of endoaperture) −+7.0 of the STATISTICA software package (StatSoft. EXIN (Exine) ++Inc., Tulsa, OK, USA). For these analyses, we used SHAP (Shape) ++13 metric variables of the pollen grains (Table I). NEXI (Nexine) ++To calculate the width index of colpi, we divided the SEXI (Sexine) ++ TECT (Tectum) ++equatorial diameter by the width of the colpi. Indices below 5.0 were obtained for very large colpi, between 260 E. C. Gasparino et al.

Figure 1. Pollen grains of Codonanthe (Type I). A–F. Codonanthe carnosa: A. Polar view (LM); B. Equatorial view (LM); C. High focus in apocolpium (LM); D. Low focus in apocolpium (LM); E. Polar view, apocolpium (SEM); F. Equatorial view (SEM). G–I. Codonanthe gracilis: G. Polar view (LM); H. Equatorial view (LM); I. Section through pollen wall (TEM). J–M. Codonanthe venosa: J. Polar view (LM); K. Equatorial view (LM); L. High focus in apocolpium (LM); M. Low focus in apocolpium (LM). Scale bars – 10 µm (A, B, E, G, H, J, K); 5 µm (C, D, F, L, M); 500 nm (I).

5.0 and 7.0 for large colpi and above 7.0 for narrow and TEM, following Sabatini et al. (1963)and colpi. Haddad et al. (1998). For further details of ornamentation and exine The palynological terminology follows Barth and ultrastructure, non-acetolysed pollen grains were Melhem (1988) and Punt et al. (2007), and Fægri used under SEM, according to Melhem et al. (2003), and Iversen (1966) for the polar area index (PAI). Codonanthe and Nematanthus pollen morphology 261

Figure 2. Pollen grains of Codonanthe (Type II). A–D. Codonanthe cordifolia: A. Polar view (LM); B. Equatorial view (LM); C. High focus in mesocolpium (LM); D. Low focus in mesocolpium (LM). E–J. Codonanthe devosiana: E. Polar view (LM); F. Equatorial view (LM); G. High focus in mesocolpium (LM); H. Low focus in mesocolpium (LM); I. Apocolpium (SEM); J. Equatorial view, apertural membrane (SEM). Scale bars – 10 µm (A, B, E, F); 5 µm (C, D, G–J).

Results defined with reference to the presence or absence of a micro-reticulum and perforations around lumina. General description All species studied here have medium to large, isopo- Key to pollen types and taxa studied lar and symmetrical pollen grains. They are oblate spheroidal, prolate spheroidal, prolate or subprolate; 1. Pollen grains colporate circular to subtriangular in polar view, with small 2. Endoaperture lolongate, sexine reticulate, muri or very small polar areas; have long and narrow, simplicolumellate ...... TypeI large or very large colpi with a granular aperture (Codonanthe carnosa, C. gracilis, C. venosa) membrane; and are tricolpate or tricolporate. The 2’. Endoaperture lalongate sexine is reticulate, heterobrocate, lumina are slightly 3. Muri simplicolumellate, columellae irregularly smaller on apocolpium and aperture. The sexine is distributedaroundlumina ...... TypeII thicker or of the same thickness as the nexine. Five (Codonanthe cordifolia, C. devosiana) pollen types could be distinguished on the basis of 3’. Muri duplicolumellate, ring-shaped and col- aperture characteristics and details of the surface umellaeincirclesaroundlumina.....TypeIII ornamentation. Only in one case, sub-types could be (Nematanthus wettsteinii) 262 E. C. Gasparino et al.

Figure 3. Pollen grains of Nematanthus wettsteinii (Type III). A. Polar view (LM); B. Equatorial view, endoaperture (LM); C. Polar view, apocolpium (SEM); D. Equatorial view, aperture (SEM); E. Ornamentation in mesocolpium (SEM). Scale bars – 10 µm(A,B);5µm (C, D); 2 µm(E).

1’. Pollen grains colpate (5.2–6.6 × 3.7–5.5 µm); sexine reticulate, hetero- 4. Sexine reticulate, muri duplicolumellate, muri brocate, straight and simplicolumellate muri, ring-shaped and columellae in circles around lumina slightly smaller on apocolpium and aperture lumina ...... TypeIV (Figure 1C–F, L–M). (Nematanthus bradei, N. fornix, N. gregarius, Comments: The largest pollen grains were N. sericeus, N. strigillosus, N. villosus) observed in Codonanthe gracilis (TablesII, III). Among 4’. Sexine reticulate, muri simplicolumellate, col- the species studied, only C. carnosa presented a sub- umellae irregularly distributed around lumina triangular amb and large muri in the reticulum...... TypeV No supratectal elements had been observed (Figure 5. Microreticulum and perforations present 1E,F).InC. gracilis, it was observed under the aroundlumina ...... Sub-typea TEM as thick, continuous and imperforate tectum, (Nematanthus fritschii, N. maculatus, thick columellae with different diameter in the infra- N. × mattosianus, N. × kuhlmannii, N. striatus) tectal layer (Figure 1I). The nexine-1 was thin and 5’. Microreticulum and perforations absent discontinuos and nexine-2 thicker and continuous. aroundlumina ...... Sub-typeb Species included: Codonanthe carnosa, C. gracilis, (Nematanthus brasiliensis, N. crassifolius, C. venosa. N. fissus, N. fluminensis, N. jolyanus, N. monanthos, N. tessmannii) TypeII – Colporate-lalongate-simplicolumellate (Figure 2, Tables II, III). — Pollen grain size medium, tri- colporate, 25.3–31.0 × 25.8–31.3 µm; prolate sphe- Description of pollen types roidal to oblate spheroidal; amb circular (Figure Type I – Colporate-lolongate-simplicolumellate (Figure 1, 2A, E); long and narrow colpi (22.2–22.3 × Tables II, III). — Pollen grain size medium, tri- 2.9–3.1 µm), endoaperture lalongate (5.4–6.7 × colporate, 25.8–39.0 × 28.1–35.7 µm; prolate sphero- 7.7–10.4 µm); sexine reticulate, heterobrocate, idal to oblate spheroidal; amb circular or sub- straight to sinuous simplicolumellate muri, lumina triangular (Figure 1A, G, J); long and narrow colpi slightly smaller on apocolpium and aperture regions (23.1–29.9 × 3.2–4.1 µm), endoaperture lolongate (Figure 2C,D,G–J). Codonanthe and Nematanthus pollen morphology 263

Figure 4. Pollen grains of Nematanthus (Type IV). A. Nematanthus bradei, polar view (LM). B–D. Nematanthus fornix: B. Equatorial view (LM); C. High focus in mesocolpium (LM); D. Low focus in mesocolpium (LM). E–G. Nematanthus gregarious: E. Equatorial view (SEM); F. Ornamentation in mesocolpium (SEM); G. Section through pollen wall (TEM). H–K. Nematanthus sericeus: H. Polar view (LM); I. Equatorial view (LM); J. High focus in mesocolpium (LM); K. Low focus in mesocolpium (LM). L–N. Nematanthus strigillosus: L. Equatorial view (LM); M. High focus in mesocolpium (LM); N. Low focus in mesocolpium (LM). O. Nematanthus villosus, polar view (LM). Scale bars – 10 µm (A, B, E, H, I, L, O); 5 µm (C, D, J, K, M, N); 2 µm (F); 1 µm(G).

Comments: Codonanthe cordifolia presented the Species included: Codonanthe cordifolia, C. smallest pollen grains and ornamentation consist- devosiana. ing of smaller lumina (> 38/25 µm2; Table IV). The endoapertures were not constricted in C. devosiana. TypeIII – Colporate-lalongate-duplicolumellate (Figure 3, Under SEM, a granular apertural membrane was Tables II, III). — Pollen grain size medium, tri- observed, ornamented in the endoaperture, and colporate, 33.3–43.6 × 34.9–41.9 µm; prolate a reticulum with sinuous muri in C. devosiana spheroidal; amb subtriangular (Figure 3A); long and (Figure 2J). large colpi (29.3 × 5.8 µm), endoaperture lalongate 264 E. C. Gasparino et al.

Figure 5. Pollen grains of Nematanthus (Type V, sub-type a). A–D. Nematanthus fritschii: A. Polar view (LM); B. Optical section (LM); C. High focus in mesocolpium (LM); D. Low focus in mesocolpium (LM). E–G. Nematanthus × kuhlmannii: E. Equatorial view, mesocolpium (SEM); F. Ornamentation in mesocolpium (SEM); G. Ornamentation in apocolpium (SEM). H, I. Nematanthus maculatus: H. Polar view (LM); I. Equatorial view (LM). J–M. Nematanthus × mattosianus: J. Polar view (LM); K. Equatorial view (LM); L. High focus in mesocolpium (LM); M. Low focus in mesocolpium (LM). N. Nematanthus striatus, polar view (LM). Scale bars – 10 µm (A, E, H–K, N); 5 µm(B–D,L,M);2µm(F,G).

(6.8 × 7.0 µm); sexine reticulate, heterobrocate, that described for N. wettsteinii, but this species muri duplicolumellate, columellae distributed in was the only one among the taxa analysed for this circles around lumina, lumina slightly smaller on genus that had tricolporate pollen as in Codonanthe apocolpium and aperture regions (Figure 3C). (Figure 3B). The pollen characteristics described Sexine thicker than the nexine. for N. wettsteinii were similar in the analyses made Comments: Other species of Nematanthus also for other specimens (e.g. F. Barros et al. 1891, had pollen grains with similar ornamentation to M. Kirizawa et al. 2660 and R. Mello-Silva et al. Codonanthe and Nematanthus pollen morphology 265

Figure 6. Pollen grains of Nematanthus (Type V, sub-type b). A, B. Nematanthus crassifolius: A. Polar view (LM); B. Ornamentation in mesocolpium (SEM). C. Nematanthus fissus, equatorial view (LM). D. Nematanthus fluminensis, polar view (LM). E. Nematanthus jolyanus, equatorial view (LM). F, G. Nematanthus monanthos: F. High focus in apocolpium (LM); G. Low focus in apocolpium (LM). H–M. Nematanthus tessmannii: H. Polar view, optical section (LM); I. Equatorial view (LM); J. High focus in apocolpium (LM); K. Low focus in apocolpium (LM); L. Polar view, apocolpium (LM); M. Section through pollen wall (TEM). Scale bars – 10 µm (A, C–E, H, I, L); 5 µm (F,G,J,K);2µm(B);1.5µm(M).

890). Under SEM, apertures with densely granulated Type IV – Colpate-duplicolumellate (Figure 4, Tables membranes could be observed. The muri were large, II, III). — Pollen grain size medium to large, tri- sparsely perforated, with irregular and crenulated, colpate, 29.9–55.9 × 29.9–48.6 µm; prolate to sometimes sparse granulations within the lumina oblate spheroidal; amb circular or subtriangular (Figure 3C–E). (Figure 4A, H, O); long and large or narrow Species included: Nematanthus wettsteinii. colpi (29.0–39.6 × 3.2–6.6 µm); sexine reticulate, 266 E. C. Gasparino et al.

Figure 7. Principal component analysis performed with the pollen metrical variables from Codonanthe and Nematanthus. Ccarno = Codonanthe carnosa, Ccordi = C. cordifolia,Cdevos= C.devosiana,Cgraci= C.gracilis, Cvenos = C. venosa,Nbrade= Nematanthus bradei,Nbrasi= N. brasiliensis,Ncrass= N. cras- sifolius, Nfissu = N. fissus, Nforni = N. for nix, Nfrits = N. fritschii, Ngrega = N. gregarius, Njolya = N. jolyanus,Nxkuhl= N. × kuhlmannii, Nmacul = N. macu- latus,Nxmatt= N. × mattosia- nus, Nmonan = N. monanthos, Nseric = N. sericeus,Nstria= N. striatus,Nstrig= N. strigilo- sus, Ntessm = N. tessmannii, Nvillo = N. villosus, Nwetts = N. wettstennii; •=Codonanthe,  = Nematanthus.

heterobrocate, straight and duplicolumellate muri, (31.4–41.9 × 3.4–7.1 µm); sexine reticulate, hetero- columellae distributed in circles around lumina brocate, simplicolumellate muri, straight or slightly (Figure 4C, D, M, N), lumina usually slightly smaller sinuous, with microreticulum and perforations on apocolpium and aperture region. around lumina; lumina usually smaller on apoc- Comments: Nematanthus bradei had pollen grains olpium and aperture region (Figure 5C–G,L,M). with smaller diameter, thin exine and narrower colpi Comments: In some cases, the apertural mem- when compared with other species of the genus brane in the pollen grains of Nematanthus × kuhlman- (Tables II and III). The lumina of the reticulum nii broke and looked like an endoaperture. In this had similar shapes and sizes in both apocolpium and subtype, the species had pollen grains with sinu- mesocolpium in the pollen grains of N. fornix, N. gre- ous muri in the mesocolpium (Figure 5F,L,M). garius and N. sericeus. Under SEM, N. gregarius pre- In the pollen grains of Nematanthus × kuhlmannii sented a densely granulated apertural membrane and under SEM, mesocolpia with large lumina granu- some granulations within the lumina (Figure 4E). lated internally and colpi with granular membranes The pollen grains of this species, when analysed and psilate margos were observed (Figure 5E). under TEM, showed an exine composed of a con- Species included: Nematanthus fritschii, Nematan- tinuous tectum, undulating internally and externally, thus × kuhlmannii, N. maculatus, Nematanthus × mat- and incomplete columellae. The nexine consisted of tosianus, N. striatus. nexine-1 (thinner and darker) and nexine-2 (thicker and clear). Intine thick (Figure 4G). Type V – Colpate-simplicolumellate (Tables II, III). Sub- Species included: Nematanthus bradei, N. fornix, N. type b – Muri with microreticulum and perforations gregarius, N. sericeus, N. strigillosus, N. villosus. (Figure 6). — Pollen grain size medium to large, tri- colpate, 31.2–58.9 × 32.9–54.9 µm; oblate sphero- idal to subprolate; amb circular, subcircular or sub- Type V – Colpate-simplicolumellate (Tables II, III). Sub- triangular (Figure 6A,D,H,L);longandverylarge, type a – Muri with microreticulum and perforations large or narrow colpi (27.1–44.9 × 3.6–11.7 µm); (Figure 5). — Pollen grain size medium to large, sexine reticulate, heterobrocate, simplicolumellate tricolpate, 38.2–55.4 × 34.4–51.5 µm; subprolate to muri, straight or slightly sinuous, without microretic- oblate spheroidal; amb subcircular or subtriangular ulum and perforations (Figure 6B), lumina decreas- (Figure 5A, H, J, N); long and large or narrow colpi ing on apocolpium and aperture region (Figure 6L). Codonanthe and Nematanthus pollen morphology 267

Figure 8. Cluster analysis (UPGMA, Euclidean distance) performed with the pollen morphological characters of Codonanthe and Nematanthus species. Abbreviations: see caption to Figure 7.

Comments: Among all species of Nematanhus analysed (Figure 7). The first component of the anal- studied, this subtype had pollen grains with the ysis explained 56.5% of the variance and had a high longest (N. tessmannii) and shortest colpi (N. positive influence for most variables measured except jolyanus). Nematanthus fluminensis had the largest col- for the dimension measurements of the endoaper- pus width, being the only species of the genus classi- ture and the thickness of the tectum (negative side fied as with ‘very large’ colpi, as well as a thicker exine of the axis; Figure 7). On the left of the graph (Table III). As also reported for N. × kuhlmannii, are the ordinates for all species of Codonanthe and some species had the aperture membranes present also Nematanthus wettsteinii and N. bradei, possess- even after acetolysis, which break and appeared like ing higher values for the width of the endoaperture an endoaperture (e.g. N. crassifolius, N. fluminen- (EWID) and length of endoaperture (ELEN). All sis and N. monanthos). Under SEM and LM, a the other species of Nematanthus are grouped on the difference was observed between apocolpium and right, based on their higher values for almost all other mesocolpium ornamentation in pollen grains of N. variables (Table V). crassifolius (Figure 6B, L). The apocolpium pre- The second component explained 17.9% of the sented straight muri and smaller lumina, while in variability of the data, in which the width of colpi the mesocolpium the lumina were larger, heteroge- (CWID) and shape of the pollen grains (SHAP) were neous with sinuous muri. Under TEM, the pollen the most important variables for ordination of species grains of N. tessmannii had a slightly wavy exine, (Table V). Most species were placed on the posi- with discontinuous tectum as thick as the nexine, col- tive side of the second axis; however, it was observed umellae of different diameters with a thickened apex that a group of species, which had high P/E values, (Figure 6M). appeared positioned on the negative side of axis 2. Species included: Nematanthus brasiliensis, N. cras- The CA produced a dendrogram (Figure 8), sifolius, N. fissus, N. fluminensis, N. jolyanus, N. where two distinct clusters can be recognised: The monanthos, N. tessmannii. first group contained all species of Codonanthe and the second group only Nematanthus species. The PCA and the CA presented similar results when Principal component and cluster analysis we analysed the relationships between the measures Relationships among the genera included, based of pollen grains of the species studied. The rela- on pollen morphological data, were explored using tionship evidenced between the three Nematanthus PCA and cluster analysis (CA). The first two axes species (N. bradei, N. fluminensis and N. wettsteinii) contained 74.4% of the total variability of the data and the species of Codonanthe by the cluster grouping 268 E. C. Gasparino et al. VC (%) CI 25). s = n Equatorial diameter 0.46 2.28 5.05 44.21–46.11 0.47 2.33 5.50 41.37–43.31 0.50 2.50 6.39 38.11–40.17 0.29 1.45 3.37 42.40–43.60 0.41 2.05 4.27 47.28–48.86 0.43 2.13 5.08 40.97–42.75 0.44 2.18 4.34 49.39–51.21 0.46 2.28 5.89 37.68–39.58 0.38 1.91 4.14 45.38–46.94 0.36 1.81 4.83 36.82–38.30 0.37 1.85 4.45 40.77–42.29 0.42 2.11 5.18 39.94–41.68 0.69 3.46 9.69 34.26–37.10 0.45 2.26 5.27 41.91–43.77 x 0.22 1.11 3.50 31.27–32.17 0.39 1.97 5.17 37.31–38.91 0.38 1.89 5.99 30.80–32.26 0.47 2.37 6.05 38.11–40.05 0.20 1.02 3.53 28.63–29.45 0.41 2.04 4.61 43.52–45.20 0.22 1.12 4.02 27.48–28.38 0.33 1.63 3.76 42.76–44.12 0.20 1.02 3.10 32.43–33.25 0.37 1.85 4.29 42.32–43.84 s ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± x s ), variability coefficient (VC) and confidence interval (CI), ( VC (%) CI Variation s 1.69 4.09 40.74–42.14 40.50–46.50 43.08 pollen grains in equatorial view. Polar diameter ), sample standard deviation ( 0.34 0.37 1.85 4.16 43.62–45.14 40.63–50.00 45.16 0.48 2.41 5.79 40.63–42.61 39.38–47.63 42.34 0.42 2.12 5.90 35.07–36.81 32.88–43.50 39.14 0.27 1.36 3.40 39.53–40.65 39.38–46.75 43.00 0.43 2.16 4.46 47.67–49.45 43.00–51.50 48.12 0.33 1.65 4.17 38.85–40.21 38.13–47.88 41.86 0.34 1.72 3.76 45.20–46.60 46.13–54.88 50.30 0.48 2.42 5.72 41.27–43.25 34.25–43.50 38.63 0.41 2.06 4.47 45.26–46.94 42.88–49.50 46.16 0.47 2.33 5.96 38.08–40.02 34.88–41.88 37.56 0.33 1.63 3.66 43.69–45.05 38.75–44.75 41.53 0.41 2.03 4.09 48.81–50.49 37.50–44.88 40.81 0.50 2.52 7.21 33.97–36.03 29.88–41.50 35.68 0.54 2.72 5.05 52.72–54.94 39.25–47.38 42.84 0.24 1.18 3.93 29.40–30.38 29.75–34.50 31.72 0.45 2.25 4.34 50.79–52.65 34.25–42.13 38.11 x 0.31 1.56 4.35 35.24–36.52 28.13–35.75 31.58 0.42 2.08 4.07 50.23–51.97 34.38–43.13 39.08 0.26 1.29 4.70 26.84–27.92 27.24–31.32 29.04 0.27 1.35 3.35 39.74–40.86 40.25–48.63 44.36 0.22 1.12 3.85 28.59–29.49 25.82–29.49 27.93 0.30 1.49 3.36 43.79–45.03 38.63–46.00 43.44 0.29 1.46 4.93 28.92–30.12 30.57–34.65 32.84 x s s ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Nematanthus x and Codonanthe Variation 38.25–44.63 41.44 40.50–47.63 44.38 38.38–48.38 41.62 31.25–40.25 35.94 37.50–43.00 40.09 43.63–52.38 48.56 36.63–43.38 39.53 42.63–49.13 45.90 38.13–47.63 42.26 42.88–50.88 46.10 33.25–43.63 39.05 41.50–47.38 44.37 45.50–55.50 49.65 29.88–41.50 35.00 49.63–58.88 53.83 27.63–32.00 29.89 47.50–55.88 51.72 33.13–39.00 35.88 47.13–55.38 51.10 25.32–30.15 27.38 37.75–42.50 40.30 26.24–30.99 29.04 41.63–48.25 44.41 25.82–32.24 29.52 x ), medium standard deviation ( mattosianus kuhlmannii × × Note: Arithmetic average ( Nematanthus Nematanthus maculatus Nematanthus Nematanthus jolyanus Nematanthus gregarius Nematanthus fritschii Nematanthus fornix Nematanthus fluminensis Nematanthus fissus Nematanthus crassifolius Nematanthus wettsteinnii Nematanthus brasiliensis Nematanthus villosus Nematanthus bradei Nematanthus tessmannii Codonanthe venosa Nematanthus strigillosus Codonanthe gracilis Nematanthus striatus Codonanthe devosiana Nematanthus sericeus Codonanthe cordifolia Nematanthus monanthos Table II. Measures (in micrometres) of Species Codonanthe carnosa Codonanthe and Nematanthus pollen morphology 269 10.40 7.74 5.55 3.78 10.14 7.00 — — — — — — — — — — — — — — — — — × × × × × × Endoaberture Width culpus index legth x width 25). Polar area index (PAI), exine = n width 3.55 11.50 6.39 6.55 3.59 11.93 11.74 4.28 3.36 11.34 6.41 6.03 3.44 11.36 5.40 8.55 6.65 6.67 6.71 6.19 6.04 7.19 3.23 11.05 — 6.85 6.29 4.00 7.98 6.70 7.09 6.37 3.20 9.87 5.40 6.55 6.46 3.10 9.37 6.64 5.76 6.80 2.94 9.50 5.21 5.31 8.10 4.11 7.99 6.64 6.84 7.04 5.80 6.48 6.96 × × × × × × × × × × × × × × × × × × × × × × × × × Colpus 36.25 29.26 length / E P 1.04 1.22 39.60 0.941.01 28.98 1.26 44.93 0.91 37.26 1.35 38.45 1.09 32.85 1.31 41.93 1.00 34.84 0.91 31.73 1.07 37.13 1.02 36.99 0.98 29.25 0.96 33.25 0.94 23.96 0.98 34.11 1.13 29.93 0.98 31.43 0.94 22.27 0.92 27.11 1.04 22.37 0.93 30.29 0.90 23.12 > > > > > > > > > > = > > > > > > > > = > > > > 10). = ), ( n / E P s ), variability coefficient (VC) and confidence interval (CI), ( / equatorial diameter ratio ( VC (%) CI PAI Ex Sex:Nex s pollen grains in polar view, exine and apertures. 0.33 1.65 4.40 36.78–38.14 0.30 2.15 0.44 2.20 5.11 42.16–43.98 0.26 2.13 0.42 2.12 4.36 47.77–49.51 0.22 2.16 0.46 2.32 5.32 42.67–44.57 0.42 1.87 0.62 3.12 6.17 49.25–51.81 0.23 2.12 0.30 1.48 3.04 47.93–49.17 0.21 2.70 0.49 2.44 5.37 44.39–46.41 0.21 2.11 0.42 2.09 5.18 39.47–41.21 0.19 2.52 0.47 2.37 5.22 44.55–46.49 0.22 2.00 0.32 1.60 3.39 46.44–47.76 0.24 2.46 0.39 1.95 4.46 43.00–44.60 0.42 2.28 0.44 2.18 4.90 43.56–45.38 0.12 2.41 0.40 1.98 4.49 43.30–44.94 0.15 2.32 0.45 2.35 6.62 34.59–36.45 0.29 1.63 0.40 1.98 4.62 42.07–43.71 0.23 2.11 0.31 1.57 5.07 30.23–31.51 0.16 1.82 0.44 2.20 4.78 45.15–46.97 0.24 2.04 0.26 1.32 4.07 31.82–32.90 0.22 1.90 0.32 1.60 3.86 40.95–42.27 0.26 2.33 0.27 1.36 4.80 27.82–28.94 0.19 1.92 0.46 2.28 5.79 38.37–40.27 0.27 2.21 0.27 1.33 4.92 26.56–27.68 0.24 1.81 0.28 1.39 3.44 39.77–40.93 0.35 2.15 0.36 1.82 5.99 29.62–31.10 0.28 1.90 x ), sample standard deviation ( s x s ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Nematanthus x Equatorial diameter and Codonanthe 34.13–41.00 37.46 44.38–51.13 48.64 39.00–47.63 43.07 40.25–47.63 43.62 46.00–56.13 50.53 46.25–52.25 48.55 39.38–50.50 45.40 34.75–44.13 40.34 41.50–50.63 45.52 44.00–49.88 47.10 40.25–49.00 43.80 40.63–49.50 44.47 40.50–48.00 44.12 31.63–41.13 35.52 39.38–48.63 42.89 28.00–34.13 30.87 42.25–50.25 46.06 30.13–35.13 32.36 39.38–46.00 41.61 25.66–30.90 28.38 35.75–43.88 39.32 24.91–29.82 27.12 38.38–43.00 40.35 27.07–33.49 30.36 Range of variation x ), medium standard deviation ( mattosianus kuhlmannii × × Note: Arithmetic average ( Nematanthus wettsteinnii Nematanthus fritschii Nematanthus villosus Nematanthus fornix Nematanthus tessmannii Nematanthus fluminensis Nematanthus strigillosus Nematanthus fissus Nematanthus striatus Nematanthus crassifolius Nematanthus sericeus Nematanthus brasiliensis Nematanthus monanthos Nematanthus bradei Nematanthus Codonanthe venosa Nematanthus maculatus Codonanthe gracilis Nematanthus Codonanthe devosiana Nematanthus jolyanus Codonanthe cordifolia Nematanthus gregarius Table III. Measures (in micrometres) of Species Codonanthe carnosa thickness (Ex), ratio of thickness between sexine and nexine (Sex:Nex), polar axis 270 E. C. Gasparino et al.

Table IV. Measures (in micrometres) of lumina, muri and number of lumina (apocolpium and mesocolpium) of Codonanthe and Nematanthus pollen grains.

Apocolpium Mesocolpium Lumina Muri Lumina Muri Species no./25 µm2 Variation Variation no./25 µm2 Variation Variation

Codonanthe carnosa 28–30 0.59–1.42 0.54–0.87 25–30 0.70–1.57 0.57–1.06 Codonanthe cordifolia 39–44 0.48–1.39 0.32–0.58 38–44 0.58–1.47 0.36–0.67 Codonanthe devosiana 27–34 0.51–1.40 0.32–0.62 28–32 0.40–1.89 0.36–0.60 Codonanthe gracilis 28–33 0.42–1.32 0.30–0.55 26–32 0.48–1.58 0.36–0.54 Codonanthe venosa 22–23 0.55–1.39 0.34–0.57 25–28 0.46–1.24 0.30–0.68 Nematanthus bradei 38–44 0.57–1.05 0.40–0.82 11–13 0.95–2.65 0.67–1.60 Nematanthus brasiliensis 35–40 0.51–1.41 0.51–0.81 16–23 0.73–2.69 0.64–1.18 Nematanthus crassifolius 31–36 0.47–1.57 0.53–0.81 18–21 0.79–2.65 0.42–0.99 Nematanthus fissus 42–48 0.57–1.36 0.40–0.74 6–9 1.28–3.45 0.99–2.04 Nematanthus fluminensis 24–26 0.67–2.09 0.46–0.85 12–18 0.70–2.47 0.68–1.01 Nematanthus fornix 9–10 1.02–2.35 0.81–1.27 5–9 1.74–3.46 1.35–1.74 Nematanthus fritschii 39–43 0.58–1.79 0.40–0.80 13–19 0.77–2.76 0.85–1.20 Nematanthus gregarius 9–14 0.75–3.27 0.80–2.05 3–4 2.75–4.58 1.78–3.29 Nematanthus jolyanus 50–57 0.41–1.03 0.36–0.80 7–9 1.21–2.54 1.24–2.00 Nematanthus × kuhlmannii 30–36 0.54–1.96 0.33–1.09 9–13 0.69–4.40 0.54–1.14 Nematanthus maculatus 20–23 0.70–1.57 0.47–1.39 4–6 0.92–3.59 0.60–2.13 Nematanthus × mattosianus 31–37 0.52–1.82 0.41–0.95 9–13 0.72–5.82 0.52–1.42 Nematanthus monanthos 35–38 0.63–1.55 0.41–1.06 3–4 1.96–4.72 1.08–2.93 Nematanthus sericeus 4–5 1.44–3.68 0.89–1.98 3–4 1.71–3.97 1.15–2.55 Nematanthus striatus 26–32 0.69–1.62 0.52–1.15 3–5 2.15–4.08 1.63–3.00 Nematanthus strigillosus 24–29 0.92–1.71 0.57–1.38 4–5 2.36–5.33 1.49–3.09 Nematanthus tessmannii 28–30 0.63–1.63 0.41–1.26 3–6 1.63–3.48 1.05–2.30 Nematanthus villosus 33–37 0.54–1.71 0.46–1.06 4–6 1.80–3.30 0.81–2.69 Nematanthus wettsteinnii 27–31 0.80–1.80 0.43–1.38 3–4 2.49–3.58 1.72–2.95

confirmed the ordination data shown by the PCA. in Nematanthus. The colpus of colpate pollen grains Similarly, for the remaining species of Nemantanthus; is covered by a membrane, which persists even after a group with N. striatus, N. strigillosus, N. tessman- acetolysis. This membrane is granulated, especially nii and N. villosus was also retrieved in the PCA. in the centre of the colpus where the granulation For some species, the quantitative data used in the is denser, appearing like colporus (endoaperture). CA confirmed the pollen types based on qualitative According to Barth and Melhem (1988) and Punt data, for example: Codonanthe venosa and C. carnosa et al. (2007), the colpus is an elongate aperture (Type I), C. devosiana and C. cordifolia (Type II), formed by the absence of sexine, and the colporus N. maculatus and N. fritschii, N. × mattosianus and is an aperture composed of an ectoaperture and an N.× kuhlmannii (Type V, sub-type a), N. monanthos endoaperture characterised by the absence of a nex- and N. brasiliensis (Type V, sub-type b). The PCA ine. The pollen grains of the Nematanthus species showed that N. fluminensis, N. bradei and N. wettsteinii studied here were classified as colpate by visualisation do not group close to the other Nematanthus species. of the nexine in optical section. Thus, only the pollen grains of N. wettsteinii were considered tricolporate, as well as the pollen grains of all the Codonanthe Discussion species included here. Gesneriaceae is characterised as a europalynous fam- Several studies of palynological data for some ily, based mainly on the apertures and ornamentation Codonanthe and Nematanthus species have been pub- of the pollen grains of its members. However, in lished previously (e.g. Williams, 1978; Melhem & some cases, the pollen morphology tends to be more Mauro, 1973; Roubik & Moreno, 1991; Melhem consistent. In this study, we verified a close relation- et al., 2003; Fourny et al., 2010). Williams (1978) ship among the genera by analysis of the morphology studied the pollen grains of C. luteola Wiehler from and the dimensions of pollen grains, although it is Panama. According to that author, the species has possible to discriminate the species. oblate spheroidal to oblate pollen grains, reduced The apertures generally represent a good diag- mesocolpium and sexine finely reticulated with very nostic character in pollen grains. However, it was small lumina. The five species of Codonanthe studied difficult to distinguish between colpus of colporus here showed oblate spheroidal, prolate spheroidal Codonanthe and Nematanthus pollen morphology 271 or subprolata, reticulate and heterobrocate pollen species studied are easily recognised by external grains. However, comparing the illustrations in morphological characteristics such as leaf shape, Williams (1978) with this paper, it was found that the corolla shape and colour of filaments (Chautems & reticulum of C. luteola pollen grains was smaller there Matsuoka, 2003). The pollen types defined here may than that described here. According to Perret et al. help distinguish these taxa once species with similar (2013) despite a common origin of the Codonanthe morphological characters (such as C. carnosa and C. lineage in South American rainforests, there was a devosiana or C. cordifolia and C. gracilis) have distinct vicariance event between the Brazilian Atlantic for- pollen grains (with different types of endoaperture). est and the Amazonian taxa afforded by the recent The pollen grains of Nematanthus bradei, N. fis- formation of a dry corridor among these habitats. sus, N. fluminensis and N. fritschii were studied by Therefore, the small pollen morphology differences Melhem and Mauro (1973), who described them as can be related to the divergence time for this group. pilate in apocolpium and reticulate in mesocolpium. Roubik and Moreno (1991) studied ten species of Here, we observed reticulate ornamentation in both Gesneriaceae from Barro Colorado Island (Panama) apocolpium as in well as mesocolpium. In relation and observed that Codonanthe crassifolia (Focke) to the apertures, the pollen grains of N. bradei and Mart. and C. uleana Fritsch. have pollen grains N. fissus were described by those authors as tricolpo- with circular amb, suboblate, tricolpate, long colpi, rate, and N. fluminensis as tricolporoidate, diverging reticulate, homobrocate, simplicolumellate muri and from our description that was tricolpate. This fact lumina with granules inside. These two species were can be explained by the difficulty in distinguish- separated by the authors based exclusively on quan- ing the apertures as aforesaid. When the shape and titative data, i. e. larger pollen grains in C. uleana amb were analysed, there was also a small discor- (c. 50–70 mm) and smaller ones in C. crassifolia dance between the results reported by these authors (c. 35–40 mm). Our results for Codonanthe are very and this paper. According to Melhem and Mauro similar to data reported by previous studies. They (1973), N. bradei has a suboblate shape and subtri- only differ by the presence of a tricolporate aper- angular amb, N. fissus has a prolate spheroidal shape ture and the absence of granulations inside the and circular amb, N. fluminensis is spherical with lumina. triangular amb, whereas N. fritschii has an oblate Codonanthe species analysed here have been sepa- spheroidal shape and triangular amb. We found rated mainly by lalongate endoapertures (C. carnosa, that the amb and shape were circular and oblate C. gracilis and C. venosa – type I) and lolongate spheroidal in N. bradei, subtriangular and oblate endoapertures (C. cordifolia and C. devosiana –type spheroidal subprolate in N. fissus, and subcircular II). In the lalongate species, the exine thickness is and oblate spheroidal to prolate spheroidal in N. flu- another character, which contributed to the differ- minensis and N. fritschii. For these data, we found that entiation of types within Codonanthe. The type of they may have variations in the amb and shape of the aperture and the lowest values for most variables pollen grains for these species. measured contributed to the distinction between the Melhem and Mauro (1973) also studied Codonanthe and Nematanthus pollen grains (except Nematanthus gregarius under the name Hypocyrta for N. wettsteinii). Taxonomically, the Codonanthe radicans Kl. et Hanst. The data obtained here agreed with the general description of the authors regarding the shape, amb, apertures, ornamentation and Table V. Pearson and Kendall correlation coefficients for pollen grains metric variables of the first and the second axis of PCA diameter values of the pollen grains. ordination in Codonanthe and Nematanthus. The description of the pollen grains presented by Melhem et al. (2003)forNematanthus fornix is Variables Principal components similar to the results we found, particularly in rela- APOC 0.137 0.443 tion to shape, aperture and exine ornamentation. CLEN 0.106 −0.054 However the authors described the amb as subtrian- CWID 0.111 0.793 gular whereas here we observed circular pollen grains EDEV 0.110 0.003 for this species, confirming once again, the variation EDPV 0.115 0.127 observed in the amb among Nematanthus species. PDEV 0.110 0.003 ELEN −0.670 0.128 Fourny et al. (2010) also observed differences in EWID −0.680 0.164 the types of aperture and ornamentation in pollen EXIN 0.031 0.115 grains of Codonanthe and Nemanthus. Microreticulate SHAP 0.007 0.121 and colporate pollen grains were described for C. NEXI 0.077 0.122 carnosa, C. devosiana and C. gracilis. Our results for SEXI 0.041 0.122 TECT −0.038 −0.003 Codonanthe species were similar, differing only in relation to the ornamentation (reticulate). For the 272 E. C. Gasparino et al. four species of Nematanthus analysed by the authors N. sericeus, N. strigillosus and N. villosus). Based on (N. brasiliensis, N. crassifolius, N. fissus and N. flu- the results of Perret et al. (2013), these characteris- minensis) our results differ in the type of aperture tics may have evolved repeatedly in the Codonanthe- for N. brasiliensis, N. crassifolius and N. fissus (col- Nematanthus clade. pate). Variations in the shape of pollen grains can Reticulate pollen grains, tricolpate or tricolpo- be observed in the species studied by Fourny et al. rate, such as we observed here were also found in (2010) and in the present study. Brazilian neotropical species of other genera than Nematanthus × kuhlmannii, a natural and fertile studied here (Campos, 1962; Melhem & Mauro, hybrid between N. fritschii and N. villosus, has pollen 1973; Melhem et al., 2003; Fourny et al., 2010; grains significantly smaller than the parent species. Gasparino et al., 2011) and some genera of pale- This hybrid, however, had ornamentation like that otropical Gesneriaceae (Luegmayr 1993a, 1993b; of N. fritschii: reticulum with simplicolumellate muri Palee et al., 2003). However, the peculiar features in the mesocolpium and columellae irregularly dis- such as a reticulum with large lumina surrounded by tributed around the lumina (Type V, sub-type a). a microreticulum and perforations, and also dupli- The reticulum of N. villosus has ring-shaped dupli- columellate muri, had not been documented for the columellate muri and columellae in circles around pollen grains of species of Gesneriaceae analysed to the lumina (Type IV). Nematanthus × mattosianus,a date. Codonanthe and Nematanthus pollen features hybrid between N. fissus and N. fritschii, has pollen can thus be used in the separation of taxa of these two grains with simplicolumellate muri as observed in genera (with a few exceptions), and combined with the parent species (Type V), but the pollen grains of other morphological and molecular characters can N. × mattosianus and N. fritschii had large lumina, in contribute to a better understanding of these genera most cases, surrounded by a microreticulum and per- improving their taxonomical placement. forations (Type V, sub-type a), which did not occur in N. fissus (Type V, sub-type b). The tricolporate aperture of Nematanthus Acknowledgements wettsteinii is a distinctive pollen feature among Nematanthus species that could suggest a possi- The authors wish to thank the directors and cura- ble close relationship of this species with other tors of the herbaria UEC (Universidade Estadual Codonanthe species. This was shown in our PCA de Campinas, Campinas, Brazil), SP (Instituto de and CA results. Pollen ornamentation with sim- Botânica, São Paulo, Brazil) and SPF (Universidade plicolumellate or duplicolumellate reticulum, de São Paulo, São Paulo, Brazil). The original ring-shape muri around lumina, and the presence manuscript benefited from comments by Fábio de of a microreticulum and perforations in muri were Barros and Angela Maria da Silva Correa (Instituto helpful features in the delimitation of groups of de Botânica, São Paulo). Finally, the authors would species with similar pollen grains (see pollen types like to express their gratitude for the techni- defined in the results). It is worth mentioning that cal assistance of Priscila Rodrigues (Instituto de the quantitative data have also contributed to sepa- Botânica, São Paulo), Silvia Regina Galleti (Instituto rate pollen grains of Nematanthus species. The CA Biológico, São Paulo) and Mauro Peixoto. Financial and PCA show that some species of Nematanthus are support was received from the Coordenação de placed near Codonanthe species, such as N. wettsteinii Aperfeiçoamento de Pessoal do Ensino Superior and N. bradei. The high ratio of colpus width in N. (CAPES). fluminensis contributed to the distant placement of this species in relation to the other species in the analysis. Specimens investigated Based on molecular phylogenetic studies, Clark Codonanthe carnosa (Gardner) Hanst. Brazil, Rio de Janeiro, et al. (2006, 2012) and Perret et al. (2013) Guanabara. A.P. Duarte 5297 (UEC); Brazil, Rio de Janeiro, reported the close relationship of Nematanthus aus- Rio de Janeiro. F.C. Hoehne 184 (SP); Brazil, São Paulo, tralis Chautems and N. wettsteinii to the genus Salesópolis. P. Fiaschi et al. 737 (SP); Brazil, São Paulo, Codonanthe, with both species nesting within Ubatuba. M. Kuhlmann 3828 (SP). Codonanthe cordifolia Chautems. Brazil, São Paulo, Cunha. Codonanthe. Through pollen grains observations of A.Chautems & M. Peixoto 392 (SP). N. wettsteinii, it was found that the pollen mor- Codonanthe devosiana Lem. Brazil, Paraná, Paranaguá. R.M. phology supported such a placement. However, the Britez 24885 (UEC); Brazil, Rio de Janeiro, Guapimirim. pollen grains of N. wettsteinii had duplicolumellate C.B. Costa et al. 515 (SP); Brazil, São Paulo,Cananéia. ring-shaped muri with columellae in circles around A.Chautems & M.M.R.F. Melo 135 (SP); Brazil, São Paulo, Caraguatatuba. M. Kuhlmann 3837 (SP); Brazil, São Paulo, the lumina, sharing these features with other species Iporanga. M. Kirizawa et al. 2649 (SP); Brazil, São Paulo, São of Nematanthus (N. bradei, N. fornix, N. gregarius, Sebastião. O. Handro 1103 (SP); Brazil, São Paulo, Ubatuba. Codonanthe and Nematanthus pollen morphology 273

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