Gerbera hieracioides (Kunth) Zardini of the Gerbera-complex (Mutisieae, Asteraceae/Compositae) is distributed in Ecuador and Peru. This perennial herb was first named as Onoseris hieracioides Kunth and was later recognised as Trichocline hieracioides (Kunth) Ferreyra. Now it is generally treated as Gerbera hieracioides (Kunth) Zardini but it has never been included in any section of Gerbera. In this study, the position of Gerbera hieracioides is assessed based on morphology and a molecular phylogeny that includes G. hieracioides and 28 other species from the Gerbera-complex. Morphologically, G. hieracioides bears leaves with the adaxial epidermal surface without stomates but with soft thin trichomes, bracteate scapes, trimorphic capitula and inner ray florets with the corolla shorter than the style. These characters suggest that the species is most closely related to Chaptalia rather than to Gerbera or Trichocline. Furthermore, the phylogenetic results based on two nuclear (ITS and ETS) and two chloroplast (trnL–trnF and trnL–rpl32) sequences strongly support the placement of G. hieracioides nested within Chaptalia. As both morphological characters and the molecular phylogenetic results support the transfer of G. hieracioides to Chaptalia, this enigmatic taxon is recognised as Chaptalia hieracioides (Kunth) X.-D. Xu & W. Zheng.

Compositae , Gerbera hieracioides , Trichocline hieracioides , Chaptalia hieracioides , Gerbera-complex, SEM, stomata, South America, Africa, Asia

Gerbera hieracioides (Kunth) Zardini (Mutisieae, Asteraceae) is a species belonging to the Gerbera-complex (Gerbera L., Leibnitzia Cass., Uechtritzia Freyn, Amblysperma Benth., Chaptalia Vent., Trichocline Cass., Perdicium L. and Lulia Zardini). The species is distributed in Ecuador and Peru. This perennial herb was first named as Onoseris hieracioides Kunth in 1818. It was transferred to Trichocline hieracioides (Kunth) Ferreyra in 1944. In 1974, Zardini moved this species out of Trichocline because it did not have the characters which were used to define that genus. The apex of achenes is truncate in Trichocline but tapering or beaked in G. hieracioides (Zardini 1974, Hansen 1990). Zardini (1974, 1975) moved it into Gerbera because it had bracteate scapes, uniseriate ray florets, achenes rostrate at the apex and slender achene hairs. However, Gerbera and Chaptalia were found to share the same traits such as achenes rostrate at the apex (Katinas 2004) and the transfer of Trichocline hieracioides to Gerbera remained controversial (Hansen 2006).

Gerbera currently contains about 32 species, which belong to six sections: the three African sections: Gerbera (8 species), Parva H.V.Hansen (1 species) and Lasiopus (Cass.) Sch.Bip. (6 species), the Aisan section Isanthus (Less.) C. Jeffrey (7 species), the Madagascar section Pseudoseris (Baill.) C. Jeffry (8 species) and section Piloselloides Less. (2 species, one of which is widespread from Asia, Africa and Australia: Hansen 1985a, 1985b, 1988, Johnson et al. 2014, Funk et al. 2016). However, Zardini (1974) did not include G. hieracioides in any section of Gerbera, she only compared it with two species in sect. Lasiopus (Hansen 1988): G. jamesonii Bolus ex Adlam and G. ambigua Sch. Bip. Although G. hieracioides has the trimorphic capitula similar to those of G. sect. Lasiopus (Hansen 1985a) from Africa, it has bracteate scapes, suggesting that it is perhaps related to Gerbera sect. Isanthus (Hansen 1988) from Asia. Furthermore, the SEM studies showed that the achene hairs of G. hieracioides possess a significantly lower L/W ratio than that in either sect. Isanthus or sect. Lasiopus of Gerbera (Hansen 1990). Therefore, it was still difficult to place G. hieracioides into an existing section (or a new section) of the genus Gerbera.

Gerbera is an Old World genus, whereas Chaptalia, Trichocline and the enigmatic G. hieracioides are New World groups (Nesom 2004b, 1995). Recently, phylogenetic analyses of the Gerbera-complex based on molecular data showed that Chaptalia was placed between Trichocline and Gerbera (Baird et al. 2010, Funk et al. 2014, Pasini et al. 2016). This suggested to the authors that the New World G. hieracioides may be a species of Chaptalia.

In this study, the authors seek to determine the correct generic placement of G. hieracioides by sampling 28 congeneric species using both molecular (two nuclear and two chloroplast markers) and morphological data (leaf adaxial surface, scape and floral morphology).

A total of 29 species from four genera (Gerbera, Amblysperma, Chaptalia and Trichocline) of the Gerbera complex and Adenocaulon chilense (outgroup) were sampled for this study. Most of the specimens were sampled from the United States National Herbarium (US) of the Smithsonian Institution (Tables 1, 2).

Adaxial leaf epidermal morphology. Lamina (0.5–1.0 cm²) were placed with the adaxial side exposed on carbon tape over stubs for the scanning electron microscopy (SEM), without soaking the material in different solutions prior to SEM. The stubs bearing leaves 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). The 22 samples were subsequently observed and photographed under SEM. Images of the leaves were captured using the proprietary software associated with the Philips SEM. Images of at least 15 different areas of the adaxial leaf surface were captured.

Floret morphology. The florets and scapes of 20 herbarium specimens were examined in the United States National Herbarium, Smithsonian Institution, using an optical microscope.

DNA extraction, amplification and sequencing. The molecular work was performed in the Laboratory of Analytical Biology (LAB) of NMNH (Smithsonian Institution). DNAs of 16 samples (15 species, including two samples of Gerbera hieracioides) were extracted using the AutoGen. Herbarium leaf samples, along with 1.0 and 2.3 mm diameter beads, were dipped in liquid nitrogen then immediately shaken for 30 seconds at 18000 rpm. About 500 ml of CTAB was added to the tubes, vortexed and incubated overnight (500 rpm at 45 °C). Then 300 µl of the supernatant was transferred to an AutoGen plate. AutoGen was run according to the manufacturer’s default settings (AutoGen, Inc., Holliston, MA, USA).

Four markers including two nuclear ribosomal (ITS and ETS) and two chloroplast intergenic spacers (trnL–trnF and trnL–rpl32) 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) and trnL–rpl32 spacer primers by Timme et al. (2007) (Table 3). The PCR reaction mixture had a total volume of 25 µl, comprising 14.05 µl nuclease free water, 2.5 µl 10× buffer, 2 µl dNTPs, 1.25 µl MgCl₂, 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 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, USA) and 2.0 µl 5× Big Dye buffer and 4.2 µl water.

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). The PCR reactions were performed in a Veriti PCR Thermal Cycler. The amplification protocols for all markers are summarised in Table 3. Sequences were aligned by using MAFFT (Katoh and Standley 2013) using Geneious 10.0.9. (Biomatters Ltd., Auckland, New Zealand) and checked manually. A total of 57 newly generated sequences from the 16 samples were deposited in GenBank (Table 2).

A total of 37 sequences of 16 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 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 using 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.

Adaxial leaf epidermal morphology. The results of the SEM work (Table 1) showed that the two tested samples of Gerbera hieracioides have no stomates but have soft, thin and appressed trichomes on the adaxial leaf surface (Figure 1G). These adaxial leaf morphological traits differ from the Gerbera species: (1) they are different from Gerbera sections sampled [sect. Lasiopus (4 species), sect. Piloselloides (2 species) and sect. Pseudoseris (1 species)] which have stomates and stiff, straight, upright trichomes. Figure 1 has images of one sample for each section: G. ambigua (Fig. 1A), G. piloselloides (Fig. 1B) and G. perrieri (Fig. 1D), respectively. (2) they are different from the members of Gerbera sect. Gerbera which have stomates and soft, thin and appressed trichomes. Three species from South Africa were examined and represented by G. crocea (Fig. 1C). (3) they are different from the Asian Gerbera sect. Isanthus which have no stomates and no trichomes based on this study of five species of sect. Isanthus that were examined in the study and are represented by G. maxima (Fig. 1E): the authors’ observations agree with Lin et al. (2008) for the Asian species G. delavayi. Additionally, the morphological traits of G. hieracioides differ significantly from those of the Trichocline species, which have many stomates with guard cells sunken on the leaf surface, illustrated by T. catharinensis (Fig. 1H). However, the two tested G. hieracioides samples share the same adaxial leaf epidermal characters such as soft, thin and appressed trichomes, epidermal cell shape and striations and absence of stomates, with the three examined Chaptalia species, as represented by C. pringlei (Fig. 1F). Therefore, based on the adaxial leaf epidermal morphology, G. hieracioides is most closely related to Chaptalia rather than to Gerbera or Trichocline.

Figure 1. 

Adaxial leaf epidermal surface morphology of Gerbera hieracioides and the related species. A G. ambigua (sect. Lasiopus) B G. piloselloides (sect. Piloselloides) C G. crocea (sect. Gerbera) D G. perrieri (sect. Pseudoseris) E G. maxima (sect. Isanthus) F Chaptalia pringlei G G. hieracioides H Trichocline catharinensis. Arrows point to the soft thin trichomes. Scale bar=50 μm.

Scape and floret morphology. The results (Table 1) showed that the two examined samples of Gerbera hieracioides have bracteate scapes and trimorphic capitula which have the inner rays with corollae shorter than the styles (Fig. 2G, H). The above morphological traits also differ from those of the Gerbera species: (1) Gerbera sect. Lasiopus, sect. Piloselloides and sect. Pseudoseris have ebracteate scapes and trimorphic capitula and the inner rays have corollae as long as the styles or longer. Gerbera jamesonii (Fig. 2A) and G. ambigua (Fig. 2B) belong to sect. Lasiopus and G. cordata (Fig. 2C) for sect. Piloselloides. (2) they are different from Gerbera sect. Gerbera and sect. Isanthus, which have bracteate scapes but dimorphic capitula without inner rays of florets. Three South African species and five Asian species were examined and are illustrated by G. crocea (Fig. 2D), G. nivea (Fig. 2E) and G. gossypina (Fig. 2F). The two tested G. hieracioides samples share the traits of bracteate scapes and trimorphic capitula which have inner rays with corollae shorter than the styles with the three tested Chaptalia species, represented by C. meridensis (Fig. 2I) and C. mandonii (Fig. 2J). Therefore, based on the scape and floret morphology, G. hieracioides should be best considered as a species of Chaptalia rather than Gerbera.

Figure 2. 

Scape and floret morphology of Gerbera hieracioides and the related species. A G. jamesonii (sect. Lasiopus) B G. ambigua (sect. Lasiopus) C G. cordata (sect. Piloselloides) D G. crocea (sect. Gerbera) E G. nivea (sect. Isanthus) F G. gossypina (sect. Isanthus) G G. hieracioides (Ecuador) H G. hieracioides (Peru) I Chaptalia meridensis J C. mandonii. The arrows mark the styles of inner ray florets.

Phylogenetic analysis. The MrBayes analysis of the combined nuclear markers and two plastid genes showed four clades of the sampled species of the Gerbera-complex, all with a strong biogeographic signal (Fig. 3): (1) the African and Australian species of the Gerbera complex (African Gerbera species are sister to the Australian Amblysperma), (2) the American genus Chaptalia and the South American Gerbera hieracioides, (3) the Asian Gerbera species and (4) the South American genus Trichocline. However, there is no well-supported resolution amongst the first three clades mentioned above, so no conclusions can be made about the monophyly of Gerbera at this time.

Figure 3. 

Phylogeny of Gerbera hieracioides and the related species. The phylogeny is based on the MrBayes analysis of the combined ITS and ETS, trnL–trnF and trnL–rpl32 markers. The posterior probabilities support values are shown next to branches.

Both samples of Gerbera hieracioides were nested within the Chaptalia clade. Gerbera hieracioides is sister to Chaptalia pringlei; then the G. hieracioides-C. pringlei clade is sister to the other Chaptalia species with strong support (posterior probability of 1.00). Therefore, the molecular data also support the placement of G. hieracioides in Chaptalia.

The placement of Gerbera hieracioides within Chaptalia is strongly supported by both the molecular sequence data (two nuclear markers ITS and ETS and two chloroplast markers trnL–trnF and trnL–rpl32) and the morphology of the scape, capitula and the leaf adaxial epidermal surface. Therefore, Gerbera hieracioides has been transferred to Chaptalia and it is recognised as Chaptalia hieracioides (Kunth) X.-D. Xu et W. Zheng.

This work was supported by the National Natural Science Foundation of China (no. 31560086), the China Scholarship Council, the Social Science Foundation of Kunming University of Science and Technology (no. kkz3201655009) and the Laboratory of Analytical Biology of the Smithsonian Institution. We are grateful to Marinda Koekemoer (South African National Biodiversity Institute) and Yousheng Chen (Institute of Botany, the Chinese Academy of Sciences, China) for providing samples and AJ Harris, Stanley Yankowski, Scott Whittaker, Carol Kelloff, Harold Robinson and Gabriel Johnson (all of the Smithsonian Institution) and Yuan Xu (South China Botanical Garden, China), Sayed Afzal Shah (Quaid-i-Azam University, Pakistan) and Zhumei Ren (Shanxi University, China) for their assistance with experiments, data analyses and helpful discussions. We also appreciate the suggestions and advice from two reviewers and the subject editor Alexander Sukhorukov.