Palynomorphological peculiarities of representatives of tribes Lindenbergieae and Cymbarieae and pollen evolution in early-branching lineages of Orobanchaceae

Pollen morphology of four species belonging to four genera of the tribe Cymbarieae and two species of Lindenbergia of the tribe Lindenbergieae (Orobanchaceae) was studied using light and scanning electron microscopy. Pollen grains in Lindenbergieae are 3-colporate (rarely 2-colporate), prolate, spheroidal and oblate-spheroidal, small-sized, with reticulate exine sculpture. Pollen grains in Cymbarieae are 3-colpate, rarely 4-colpate, prolate, spheroidal and oblate-spheroidal, medium-sized. Exine sculpture in Cymbarieae is retipilate and rugulate-retipilate. Pollen characters of Cymbarieae and Lindenbergieae are compared with pollen patterns in Paulowniaceae and crown clades of Orobanchaceae. The earliest-branching clade of Orobanchaceae (Lindenbergieae) is palynomorphologically similar to Paulowniaceae (the clade sister to all Orobanchaceae) and to Wightia. These palynomorphological findings confirm the phylogenetic patterns recently revealed in basal Orobanchaceae and their closest relatives. It is concluded that the colporate (most probably 3-colporate) type of pollen grains could be ancestral in Orobanchaceae. Pollen diversity in crown clades of Orobanchaceae evolved on the base of a few pollen types and subtypes, which were peculiar to hypothetical ancestors of Orobanchaceae and are probably preserved in the extant taxa of Paulowniaceae and Lindenbergieae.

Our earlier studies and analysis of pollen morphology in basal clades of Scrophulariaceae sensu stricto allowed us to outline the main trends of morphological pollen evolution in the family in its new circumscription and to hypothesize on possible ancestral pollen types in the group (Mosyakin, Tsymbalyuk, 2015. Because of that, we may expect that a comprehensive analysis of pollen morphology in early-branching clades of Orobanchaceae could bring comparable results and shed light on main trends of pollen evolution in that group as well.
The purpose of the present research was to study and analyze the morphological features of pollen grains of representatives of Cymbarieae and Lindenbergieae in the updated taxonomic circumscriptions of these tribes, and to compare the pollen patterns with existing systems and molecular phylogenetic data.

Materials and methods
Pollen of four species belonging to four genera of Cymbarieae (Bungea, Cymbaria, Cymbochasma, and Siphonostegia) was sampled in the National Herbarium of Ukraine (KW -herbarium of the M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kyiv, Ukraine). Pollen grains of two species of Lindenbergia were sampled in the herbarium of the Missouri Botanical Garden (MO; St. Louis, Missouri, USA). Data of the studied specimens are cited exactly according to the label information, in English translation and in original languages.
In general, the methods used in the present study are essentially the same as those we used earlier (see Mosyakin, Tsymbalyuk, 2015a, b, 2017. Pollen morphology was studied using light microscopy and scanning electron microscopy. For light microscopy studies (LM, Biolar, × 700), the pollen was acetolyzed following Erdtman (1952). For size determinations, 20 measurements were taken along the polar (P) and equatorial (E) axes for each species. For scanning electron microscopy (SEM, JSM-6060LA), pollen grains were treated with 96%-ethanol, then these samples were sputter-coated with gold and investigated at the Center of Electron Microscopy of the tribe Stemodieae Reveal, which was positioned in his system close to Gratioleae, in the same subfamily. Fischer (2004) placed Lindenbergia in Scrophulariaceae trib. Stemodieae. Now the genus is placed in Orobanchaceae trib. Lindenbergieae T. Yamaz. (= Lindenbergiaceae Doweld, 2001).
Terminology used in descriptions of pollen grains mainly follows the glossaries by Tokarev (2002) and Punt et al. (2007) with some necessary minor adjustments.

Comparative pollen morphology of genera of Cymbarieae and Lindenbergieae
In general, our data are in good agreement with the results of previous studies (Inceoğlu, 1982;Minkin, Eshbaugh, 1989;Lu et al., 2007). Analysis of our original palynomorphological data and literature records demonstrated that pollen grains of representatives of all genera of Cymbarieae are characterized by the 3-colpate type of apertures, mainly with retipilate sculpture (Table 2) Minkin, Eshbaugh, 1989 Original data are those reported here and in Table 2; "-" means no data reported.  Minkin, Eshbaugh, 1989 the exine is thinner (0.7-1.1 µm thick), and exine layers are invisible (indistinct).

Comparison of palynomorphological and molecular phylogenetic evidence
According to molecular phylogenetic studies (Bennett, Mathews, 2006;McNeal et al., 2013), the clade of Cymbarieae is subdivided into two subclades. The subclade that includes Bungea, Cymbaria, Cymbochasma, and Monochasma is characterized by larger pollen grains and wider colpi, as compared to the subclade of Siphonostegia (including Lesquereuxia) and Schwalbea.
The earliest-branching clade of Lindenbergia is sister to the clade containing all other members of Orobanchaceae. Small-sized 3-colporate pollen grains with reticulate exine revealed in all studied taxa of Lindenbergia are similar to pollen of some representatives of Plantaginaceae, in particular, those of Gratioleae (Tsymbalyuk, Mosyakin, 2013aTsymbalyuk, 2016). In our opinion, that superficial similarity does not reflect direct phylogenetic relationships of these taxa, but rather some recurrent patterns (plesiomorphic characters) appearing is several clades of Lamiales.
The genus Paulownia Siebold & Zucc. is currently placed phylogenetically as a group sister to Orobanchaceae (Olmstead et al., 2001;Oxelman et al., 2005;Bennet, Mathews, 2006;Schäferhoff et al., 2010;McNeal et al., 2013). Pollen grains of Paulownia are 3-colporate, with reticulate exine (Erdtman, 1952;Chen, 1983;. In these characters, Paulownia is rather similar to Lindenbergia. However, Paulownia differ from Lindenbergia in having small-and medium-sized pollen grains (small in Lindenbergia), trilobate and sub-triangular in outline (only trilobate in Lindenbergia), with distinct orae (indistinct in Lindenbergia) and the colpus membrane smooth and granulate (only smooth in Lindenbergia). Some similarity with pollen of Paulownia and Lindenbergia is also observed in pollen grains of the phylogenetically still problematic genus Wightia Wall. (Zhou et al. 2014), which also has 3-colporate pollen with reticulate exine sculpture (Wei, 1989;Tsymbalyuk, , 2016. However, in Paulownia and Lindenbergia orae are circular and colpi are tapered to acute ends, while Wightia has eliptical orae and colpi expanded to rounded ends. Thus, the earliest-branching clade of Orobanchaceae (Lindenbergieae) is palynomorphologically similar to the clade sister to all Orobanchaceae (Paulowniaceae) Pollen grains in Cymbochasma borysthenica are 3-colpate and occasionally 4-colpate (Tsymbalyuk, 2011). They have the thinnest exine (0.7-1.3 μm) among pollen grains of the studied species of the tribe. Pollen grains of Cymbochasma borysthenica are smaller as compared to pollen of Cymbaria dahurica and C. mongolica, and also differ from species of Cymbaria sensu stricto in the pollen shape and outline (see Table 2). Thus, pollen characters may provide additional evidence in favor of recognition of Cymbochasma as a separate genus.
The two studied species of Cymbaria sensu stricto are similar in their pollen size, shape, and outline; however, they differ in their colpi structure and exine sculpture. In pollen grains of Cymbaria dahurica, colpi are medium-length, wider than in C. mongolica, and exine sculpture is retipilate (caput of pila 0.41-0.90 μm) or rugulate-retipilate, while C. mongolica has narrower and longer colpi (as compared to those in C. dahurica) and retipilate exine sculpture (caput of pila 0.16-0.40 μm) (Lu et al., 2007, and original data).
The smallest sizes are characteristic of pollen grains of Siphonostegia chinensis (Table 1). This species also has the narrowest colpi among all studied species. Columellae in all species studied here are mainly indistinct, while in Siphonostegia chinensis those are distinct, thin, and arranged more or less regularly.
Pollen grains of Siphonostegia syriaca (Lesquereuxia syriaca) (Inceoğlu, 1982) and S. chinensis are similar in their shape, outline, size, exine sculpture, and length of colpi; however, in S. syriaca the colpi ends are acute, while in S. chinensis they are obtuse (with blunt ends).
Pollen grains of Schwalbea (Minkin, Eshbaugh, 1989) are similar to those of Siphonostegia in their outline, size, and exine sculpture. Unfortunately, the characters reported by Minkin and Eshbaugh (1989) are insufficient for a more detailed comparative analysis.
The two studied species of Lindenbergia are similar to each other in having the 3-colporate aperture type, reticulate exine sculpture, and long and narrow colpi. They, however, differ by the exine thickness: in L. philippensis the exine is 1.1-1.6 µm thick, the tectum is nearly equal to the infratectum, and columellae are indistinct or distinct, while in L. sinaica and to the currently phylogenetically unplaced (?) genus Wightia. These palynomorphological findings confirm the phylogenetic patterns currently revealed in basal Orobanchaceae and their closest relatives.
Thus, available palynomorphological data are well consistent with the phylogenetic patterns in early-branching Orobanchaceae (Lindenbergieae and Cymbarieae) and their relatives, which are currently revealed by molecular phylogenetic evidence (Bennett, Mathews, 2006;McNeal et al., 2013). The considerable pollen diversity in crown clades and subclades of Orobanchaceae evolved on the base of a few pollen types and subtypes, which were peculiar to hypothetical ancestors of Orobanchaceae and are probably preserved until now in the extant taxa of Paulowniaceae and Orobanchaceae trib. Lindenbergieae.