Home at last III: Transferring Uechtritzia and Asian Gerbera species into Oreoseris (Compositae, Mutisieae)

Abstract Recently the Asian Gerbera species were shown to form a clade that was not the sister group of the African Gerbera. In this study, the position of the Asian Gerbera species was further assessed based on morphology and molecular phylogenetic analyses that included six Asian Gerbera and 26 other species from the Gerbera-complex. Morphological results showed that the six Asian Gerbera species, which were sampled, bear leaves with the adaxial epidermal surface lacking stomates, possess bracteate scapes and lack inner ray florets. These characters suggest that the Asian Gerbera species are most closely related to the species of Uechtritzia, which also share similar pollen grain size and shape with the Asian Gerbera, rather than to the African Gerbera. Furthermore, the phylogenetic results based on two nuclear (ITS and ETS) and three chloroplast (trnL–trnF, trnL–rpl32 and trnC–petN) sequences strongly support the Asian Gerbera and Uechtritzia forming a clade, with the latter nested within the Asian Gerbera species. Both morphological and molecular phylogenetic data thus confirmed the taxonomic identity of the Asian Gerbera and Uechtritzia. The authors herein formally treat the nine species of the Asian Gerbera and the three species of Uechtritzia as members of the genus Oreoseris, which is the earliest generic name of this lineage and has the nomenclatural priority.

(2 species, one of which is widespread in Asia and Africa) and the Asian sect. Isanthus (Less.) Jeffrey (6 species;Hansen 1985a, 1985b, 1988, Johnson et al. 2014. One South American species G. hieracioides (Kunth) Zardini was not included in any of the above-mentioned sections of Gerbera (Zardini 1974) and the authors have recently transferred it to Chaptalia based on both morphological and molecular data (Xu et al. 2018).
The Asian Gerbera section Isanthus is characterised mainly by campanulate involucres, naked receptacles and rostrate achenes (Hansen 1988). A recent molecular phylogenetic analysis showed that the Asian Gerbera species did not form a clade with the African species : the Asian Gerbera + Uechtritzia formed a clade and the African Gerbera and Amblysperma constituted another clade. Some earlier workers also suggested treating the Asian section as an entity separate from the African Gerbera (Candolle 1838, Jeffrey 1967. Hansen (1990), however, argued that, while the Asian Gerbera sect. Isanthus differed somewhat from the African Gerbera, it shared four apomorphies as well as 11 plesiomorphies with Uechtritzia and the three entities could not be discerned from one another.
Species of Uechtritzia have hemispherical involucres, fimbriate receptacles and slightly rostrate achenes (Hansen 1988). This genus contains three species, namely U. armena Freyn endemic to Turkey (Doganet al. 2016) and Armenia, U. kokanica (Regel et Schmalh.) Pobed. from Central Asia (Kazakhstan, Uzbekistan, Tajikistan, Kyrgyzstan, Turkmenistan to Afghanistan) and U. lacei (G.Watt) C.Jeffrey of the Himalayan region (Hansen 1988). Pasini et al. (2016) included one Uechtritzia species, U. kokanica and showed that the species was nested within the two sampled Asian Gerbera species based on nuclear (ITS) and chloroplast (trnL-trnF) sequence data. This result indicated the possibility that the Asian Gerbera may belong to the genus Uechtritzia. However, the phylogenetic position and the taxonomic identity of the Asian Gerbera need to be tested with an expanded taxon sampling by adding more Asian and African species of Gerbera and Uechtritzia before any taxonomic decisions can be made.
In this study, the phylogenetic position of the Asian Gerbera was tested by expanding the taxon sampling of the Asian and African Gerbera and the Uechtritzia species and using both molecular (two nuclear markers: ITS and ETS and three chloroplast markers: trnL-trnF, trnL-rpl32 and trnC-petN) and morphological data (leaf adaxial surface, pollen, scape and floral morphology).

Materials and methods
A total of 32 species from eight genera of the Gerbera-complex and Adenocaulon chilense (outgroup) were sampled for this study (Tables 1, 2). The morphological data were taken from specimens at the United States National Herbarium (US) and included characters of the leaf epidermis, pollen, flowers and scapes.
Adaxial leaf epidermal and pollen morphology. A small area of the leaf lamina (about 0.5-1.0 cm 2 ) was placed with the adaxial side exposed, on carbon tape over stubs for the scanning electron microscopy (SEM). For the pollen analysis, samples were dehydrated and were then placed on aluminium stubs using double-sided adhesive tape following Wen and Nowicke (1999). The stubs bearing the leaf sample and pollen were treated with gold-palladium to 16.6 μm thickness and were examined under a Philips XL-30 scanning electron microscope at the SEM Lab of the National Museum of Natural History (NMNH), Smithsonian Institution. The 22 samples were subsequently observed and photographed under the SEM using the proprietary software associated with the Philips SEM. Images of at least 15 different areas of the adaxial leaf surface were captured for each sample, as well as 20 pollen grains. The polar and equatorial axes of pollens were measured by ImageJ 1.8.0.
DNA extraction, amplification and sequencing. The DNA molecular work was undertaken in the Laboratory of Analytical Biology (LAB) of NMNH. DNA from 16 samples (15 species) was extracted through AutoGen (AutoGen Inc., Holliston, Massachusetts, USA) or the DNeasy Plant Mini Kit (Qiagen, Valencia, California, USA). Leaf tissue samples, along with 1.0 and 2.3 mm diameter beads, were dipped in liquid nitrogen then immediately shaken for 60 seconds at 1800 rpm by Tissuelyser. About 500 μl of the CTAB extraction buffer was added to the tubes, vortexed and incubated overnight (500 rpm at 55 °C). Then 300 μl of the supernatant was transferred to an AutoGen plate. The AutoGen was run according to the manufacturer's default settings.
Five markers including two nuclear ribosomal ITS and ETS and three chloroplast trnL-trnF, trnL-rpl32 and trnC-petN intergenic spacers were amplified. The ITS primers were designed by Downie and Katz-Downie (1996) and White et al. (1990), ETS primers by Baldwin and Markos (1998); trnL-trnF primers by Taberlet et al. (1991), trnL-rpl32 spacer primers by Timme et al. (2007) and trnC-petN spacer primers by Lee and Wen (2004) (Table 3). The PCR reaction mixture had a total of 25 μl volume, comprising 14.05 μl nuclease free water, 2.5 μl 10x buffer, 2 μl dNTPs, 1.25 μl MgCl 2 , 1 μl of both forward and reverse primers, 0.5 μl BSA, 0.2 μl Taq DNA polymerase and 2.5 μl of template DNA. The PCR reactions were performed in a Veriti PCR Thermal Cycler. The amplification protocols for all markers are summarised in Table 3. The amplified products were purified with ExoSapIT enzyme with activation at 37 °C and deactivation at 95 °C. 4 μl of the purified product and same primers (1 μl, 1 μM) were cycle-sequenced in a mixture containing 0.8 μl Big Dye (Applied Biosystems, Foster City, California, USA) and 2.0 μl 5x Big Dye buffer and 4.2 μl water. Notes: + designates those mentioned present; − designates those mentioned absent; N represents data not available.  The cycle sequencing programme was 30 cycles of 95 °C for 30 s, 50 °C for 30 s and 60 °C for 4 min. The resultant product was sephadex-filtered and sequenced through an ABI 3730 automated sequencer (Applied Biosystems, Foster City, USA). Sequences were aligned by using MAFFT (Katoh and Standley 2013) in Geneious 10.0.9. (Biomatters Ltd., Auckland, New Zealand) and checked manually. A total of 90 newly generated sequences from the 23 samples were deposited in GenBank (Table 2).
A total of 16 sequences of eight species were retrieved from NCBI for the related taxa within the tribe Mutisieae (Table 2). Phylogenetic relationships were inferred based on the concatenated ITS+ETS+trnL-rpl32+trnL-trnF+trnC-petN data with MrBayes v. 3.2.2 (Ronquist et al. 2012) by using the substitution model of GTR based on the best-fitting model determined by jModelTest 2.1.6 (Posada 2008), the chain length of 10,000,000, rate variation of gamma, gamma categories of 4, heated chains of 4, heated chain temp of 0.2, subsampling freq. of 200 and burn-in length of 100,000. Tracer v. 1.5 (Rambaut and Drummond 2009) was used to confirm that the effective sample size (ESS) for all relevant parameters was > 200. After discarding the trees as burn-in, a 50 % majority-rule consensus tree and posterior probabilities (PP) for node support were calculated using the remaining trees.

Results
Adaxial leaf epidermal morphology. The results of the SEM work (Table 1) showed that the six tested Asian Gerbera species have no stomates on the adaxial leaf surface (Figure 2A, B, C, D). This adaxial leaf morphological trait differs from that of the African Gerbera species: (1) Three East African Gerbera sections sampled [sect. Lasiopus (4 species), sect. Piloselloides (2 species) and sect. Pseudoseris (2 species)] have stomates and stiff, straight, upright trichomes on the adaxial surface. Figure 1 has representative images for each of the above sections: G. ambigua (Fig. 1A), G. piloselloides (Fig. 1B) and G. perrieri (Fig. 1C), respectively. (2) Members of the South African sect. Gerbera have stomates.  Five species were examined and the epidermal characters are represented by G. serrata (Fig. 1D) and G. crocea (Fig. 1E). Furthermore, the adaxial leaf morphological traits of the Asian Gerbera species also deviate from two Asian-American disjunct Leibnitzia species, which have stomates on the adaxial leaf epidermal, as represented by L. nepalensis (Fig. 1F). Nevertheless, the Asian Gerbera samples share similar adaxial leaf epidermal characters of lacking stomates with the two examined Uechtritzia species, U. kokanica (Fig. 2E) and U. lacei (Fig. 2F). Based on the adaxial leaf epidermal morphology, the Asian Gerbera is most closely related to Uechtritzia rather than to the African Gerbera. Pollen morphology. The pollen grains of the examined species of the Gerberacomplex are very similar to one another, differing only in the size of the grains as well as the granules on the surfaces (Figs 3 and 4). They are tricolporate, have a granule exine and are prolate and subprolate in shape. The ratios of the polar axis and equatorial axis (P/E) are given in Table 1. For Gerbera and Uechtritzia, the P/E ratios are between 1.2-1.4. The average polar axis of the Asian Gerbera and Uechtritzia pollen grains is 50.05-55.80 μm. For the African Gerbera, however, the average polar axis of pollen grains is 42.09-45.77 μm in sects. Lasiopus, Piloselloides and Pseudoseris and 47.01-53.83 μm in sect. Gerbera. The P/E ratio of the pollen grains of the Asian Gerbera and Uechtritzia ( Table 1) differs from that of the East Asian-North American Leibnitzia and the Australian Amblysperma, which fall between 1.10-1.20 (Fig. 5). Furthermore, the average polar axis of the Asian Gerbera and Uechtritzia pollen grains is higher than that of Leibnitzia species, which has the range of 32.16-37.33 μm.    (6) the South American genus Trichocline. The three samples of Uechtritzia (two species of U. kokanica and U. lacei) were clearly nested within the Asian Gerbera clade (Fig. 6).

Discussion
Based on this study, the Asian Gerbera and the Uechtritzia species share several morphological characters, including bracteate scapes, absence of inner ray florets, no stomates on the adaxial leaf surface and similar pollen size and shape (Table 1). Hansen (1990) also commented that the Asian Gerbera (i.e. sect. Isanthus) is morphologically similar to Uechtritzia and presented a key to distinguish Gerbera sect. Isanthus, Uechtritzia and Leibnitzia. The differences between the Asian Gerbera sect. Isanthus and Uechtritzia were minor. Uechtritzia species generally have hemispherical heads, alveo- Figure 6. Phylogeny of the Gerbera-complex. The phylogeny is based on the Bayesian inference of the combined ITS and ETS, trnL-trnF, trnL-rpl32 and trnC-petN markers. The posterior probabilities are shown next to branches. lar receptacles that are fimbriate-ciliate; margins of involucral bracts (at least the upper part) often with reddish hairs; achenes that are slightly or indistinctly tapering with hairs that are long-villose, ca. 1 mm long (Katinas 2004) and sericeous (Hansen 1988). Gerbera sect. Isanthus has heads campanulate; a receptacle that is alveolate and naked; the margins of involucral bracts are without reddish hairs; the achenes are tapering and pilose glabrous, with hairs that are shorter, tapered and not sericeous (Hansen 1988). The heads of Uechtritzia were reported as hemispherical, in contrast to the heads of the Asian Gerbera sect. Isanthus which are campanulate. However, the species of U. armena (the type species of the genus) from Turkey showed the heads as campanulate in the fresh plants (Dogan et al. 2016), which is the same as the Asian Gerbera sect. Isanthus (e.g. G. delavayi; Zheng et al. 2017).
Some previous workers argued that the species of Asian Gerbera (sect. Isanthus) should be treated as an entity, separate from African Gerbera (Candolle 1838, Jeffrey 1967. The results presented here show that the Asian Gerbera sect. Isanthus differs from the African Gerbera sect. Lasiopus, sect. Piloselloides and sect. Pseudoseris in the ebracteate scapes, presence of inner ray florets, stomates on the adaxial leaf surface and smaller pollen size of the African Gerbera compared with the Asian Gerbera. Although the Asian Gerbera sect. Isanthus shares the traits of bracteates scapes, absence of inner ray florets and similar pollen size with Gerbera sect. Gerbera, the Asian species have no stomates on the adaxial leaf surface. Hansen (1990) stated that the Asian Gerbera sect. Isanthus shows style-arms laterally dilated and truncate achenes; in contrast, the African Gerbera sect. Gerbera has the style-arm slender and achenes tapering or beaked. Additionally, most species of the African Gerbera sect. Gerbera grow in open areas, have leathery leaves and flower only in the spring and summer (Manning et al. 2016), whereas the Asian species of Gerbera sect. Isanthus often grow in forest habitats, have herbaceous leaves and flower in the winter (Gao et al. 2011).
The two Uechtritzia species sampled in the molecular phylogeny ( Fig. 6) were nested within the Asian Gerbera species based on two nuclear markers (ITS and ETS) and three chloroplast markers (trnL-trnF, trnL-rpl32 and trnC-petN). This result, based on the authors' expanded taxon and character sampling, is consistent with the findings of Pasini et al. (2016). This study included two of the three species of Uechtritzia (Hansen, 1988) and six of the nine Asian Gerbera sect. Isanthus taxa (Gao et al. 2011). The phylogenetic analysis clearly supports the species of Uechtritzia as nested within the Asian Gerbera and this clade is the sister group of Leibnitzia with strong support (PP=0.99) (Fig. 6).
Leibnitzia is a genus containing about six species with a disjunct distribution: four species in Asia (Gao et al. 2011) and two species in Mexico (Baird et al. 2010). It shows the same characters of bracteate scapes and no inner ray florets as the Asian Gerbera + Uechtritzia. It differs from the latter by the presence of stomates on the adaxial leaf surface and smaller pollen size (polar axis of 32.16-37.33 μm) compared with Asian Gerbera + Uechtritzia (polar axis of 50.05-55.80 μm). Furthermore, Leibnitzia has two generations of heads (a vernal generation with chasmogamous capitula and an aestival generation with cleistogamous capitula), subseriate involucral bracts, slender style-arm, anthers of the ray flowers reduced to threads or wanting and achenes that are tapering or beaked. The Asian Gerbera + Uechtritzia, on the other hand, have one generation of heads, imbricate involucral bracts, laterally dilated style-arms, a fully developed apex (and base) on the anthers in the ray flowers and truncate achenes (Hansen, 1990).
Based on the molecular phylogenetic results, the Asian Gerbera species are closest to Uechtritzia, with the latter nested within the Asian Gerbera species. Leibnitzia shows significant morphological differences to the Asian Gerbera + Uechtritzia. The taxonomic identity of Uechtritzia and the Asian Gerbera is strongly supported by the morphology of inflorescences, scapes, capitula, pollen and the lack of stomates on the adaxial leaf surface. Therefore, the authors herein include the nine Asian Gerbera species and the three Uechtritzia species in Oreoseris DC. which is the earliest available name for the expanded Eurasian genus.

Taxonomic synopsis with nomenclatural changes
In trying to determine the correct genus name for the Eurasian clade, it is necessary to investigate three relevant generic names. Gerbera L. was described in 1758; Arnica gerbera L. is the basionym of the African species G. linnaei Cass., the conserved type of Gerbera L. (lectotype designated by Hansen 1985a). Gerbera was named after Traugott Gerber, a German naturalist who died in 1743. Oreoseris DC. was described in 1838 and its type species is O. nivea DC. which was designated by Hansen in 1988. While de Candolle (1838) did not say why he named the genus, Oreo is from the Greek oreos for mountain and, in his description, de Candolle says that the genus is a "… perennial herb from the mountains of eastern India (translated)." Uechtritzia Freyn was described in 1892; the type species is U. armena Freyn (lectotype designated by Pobedimova, 1963). The genus was named in honour of Rudolf Karl Friedrich von Uechtritz (1838-1886), a botanist from Wroclaw, Poland (ex-Breslau) (Freyn 1892).
When Oreoseris nivea DC. was absorbed into Gerbera, the priority was given to Gerbera because the latter was the older generic name and, as long as this species stayed in Gerbera, the name Oreoseris was not available. Uechtritzia was described later in 1892; and, as long as O. nivea remained in Gerbera, then Oreoseris continued to be unavailable.
However, as soon as Gerbera nivea from Asia was removed from Gerbera and a separate genus was formed from the Asian species of Gerbera + Uechtritzia, then the name Oreoseris became available and it is the oldest available name. Hence, these species have been transferred into Oreoseris.