Michaelmoelleria (Gesneriaceae), a new lithophilous dwelling genus and species with zigzag corolla tube from southern Vietnam

Michaelmoelleria (Gesneriaceae), a new lithophilous dwelling genus and species with zigzag corolla tube from southern Vietnam
expand article infoFang WenZi-Bing XinLong-Fei FuShu LiLan-Ying SuStephen MaciejewskiZhang-Jie HuangTruong Van DoYi-Gang Wei

Citation: Wen F, Xin Z-B, Fu L-F, Li S, Su L-Y, Maciejewski S, Huang Z-J, Do TV, Wei Y-G (2020) Michaelmoelleria (Gesneriaceae), a new lithophilous dwelling genus and species with zigzag corolla tube from southern Vietnam. PhytoKeys 146: 89-107.

Michaelmoelleria, a new genus from southern Vietnam is described with a single species, M. vietnamensis. The new genus is morphologically most similar to Deinostigma and Tribounia but it differs from the latter two by having four fertile stamens. Nuclear ribosomal internal transcribed spacer (ITS) region and plastid trnL-F intron spacer (trnL-F) DNA sequence data from the new genus and eighty-seven species representing 42 genera within tribe Didymocarpeae are used to resolve its generic placement. The molecular evidence reveals that it is most closely related to Cathayanthe rather than Deinostigma and Tribounia. The chromosome number is counted as 2n = 36 that further clarified its distinction comparing to the related genera within tribe Didymocarpeae. A global conservation assessment is also performed and classifies Michaelmoelleria vietnamensis as Critically Endangered (CR).
Cathayanthe, cliff-dwelling, DeinostigmaDidymocarpoideae, flora of Vietnam, IUCN, phylogeny, Tribounia
Morphological observations and specimens examined
A study of the new genus Michaelmoelleria and the only currently known species, M. vietnamensis, from southern Vietnam, was undertaken. All available specimens of Michaelmoelleria are stored in the following herbaria in China and Vietnam: IBK and VNMN (herbarium acronyms according to Index Herbariorum; Thiers 2016, 2019). All morphological characters were studied using a dissecting microscope (SZX16, Olympus, Tokyo, Japan). Characteristics were described using the applicable terminology presented by Wang et al. (1998). The morphological comparison with other species was based on the study of living plants in the field, in cultivation in the VNMN and the GCCC, and herbarium specimens.
Genomic DNA extraction, PCR amplification, and Sequencing
To confirm the placement of this new plant, we performed phylogenetic inference of DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) region and the plastid trnL-F intron spacer (trnL-F). Eighty-seven species representing 42 genera as in-group and two species representing one genus as an out-group, including nearly all genera within tribe Didymocarpeae, were sampled. DNA extraction, PCR amplification, and sequencing were performed, following Wei et al. (2013). Sequences obtained from this study and GenBank are listed in the Appendix I.
Phylogenetic analysis
Sequence data were edited and assembled using Lasergene Navigator 7.1 (DNAstar, Madison, Wisconsin, USA). Cleaned sequences were aligned with Geneious R11 (Kearse et al. 2012). Regions of ambiguous alignment and sites with more than 80% missing data were excluded during analyses (Sun et al. 2018). Phylogenetic analyses were conducted using Bayesian inference (BI) and maximum likelihood (ML) methods. ITS and trnL-F datasets were used to construct the ML tree independently to evaluate the congruence between two makers. As there were no hard incongruences (Nishii et al. 2015), we performed the following analysis using a combined dataset. Best-fit DNA substitution models were selected using the Akaike Information Criterion (AIC) in Modeltest v 2.7 (Posada and Crandall 1998). Modeltest determined the best models GTR + G + 1 for the combined dataset. BI analyses were based on a Markov chain algorithm implemented in MRBAYES 3.2.6 (Huelsenbeck and Ronquist 2001). Four chains of the Markov chain Monte Carlo (MCMC) simulation were performed for 4,000,000 generations, each with trees sampled every 100 generations. After discarding the first 25% of the trees, the retained ones were used to calculate the node probability (posterior probability). ML analyses with 1000 bootstrap resampling were conducted using an online version of RAxML-HPC2 v8.2.10 (Stamatakis et al. 2008), available at (http://www.phylo.org/index.php/portal/) (Miller et al. 2010) with the gamma model of rate heterogeneity.
Chromosome preparations
Leaf cuttings yielded new root tips when grown hydroponically for 2–3 weeks. The new root tips were then pretreated with a solution of 0.002 mol·L-1 8-hydroxyquinoline at 13 °C for 4–5 h. After fixation for 24 h by Carnoy solution (3:1 ethanol: acetic acid) at 4 °C, dissociate, stain, and squash methods followed (Jong and Möller 2000, Christie et al. 2012). The chromosome numbers were determined in at least 20 cells from 10 different root tips with well-spread chromosomes in metaphase and captured using a light microscope (Leica DM 2500, camera Leica DFC420).
Michaelmoelleria vietnamensis
 Figure 5. Photos of Cathayanthe biflora Chun, the related genus/species of Michaelmoelleria vietnamensis F. Wen, Z.B. Xin & T.V. Do A habitat B cyme and frontal view of flower C cyme and lateral view of flower D fruit. Photos by Fang Wen, arranged by Wen-Hua Xu.
Figure 6. Photos of Deinostigma W.T.Wang & Z.Y.Li (A–E) and Tribounia D.J.Middleton (F–I), the morphologically similar genera and species of Michaelmoelleria F.Wen, Y.G.Wei & T.V.Do (M. vietnamensis F.Wen, Z.B.Xin & T.V.Do) Deinostigma eberhardtii (Pellegr.) D.J.Middleton & H.J.Atkins B D. tamiana (B.L.Burtt) D.J.Middleton & H.J.Atkins C D. cycnostyla (B.L.Burtt) D.J.Middleton & H.J.Atkins D D. cicatricosa (W.T.Wang) D.J.Middleton & Mich.Möller ED. cyrtocarpa (D.Fang & L.Zeng) Mich.Möller & H.J.Atkins F–G Tribounia venosa (Barnett) D.J.Middleton H–I T.grandiflora D.J.Middleton. A–E photos by Fang Wen F–I photos by David Middleton, arranged by Wen-Hua Xu.
In March 2018, a plant having bluish-purple flowers of Gesneriaceae was collected in southern Vietnam. Neither the collectors nor the researchers on the family were able to allocate it to any known genus at that time. Collectors once thought that it might be a member of Deinostigma because this genus is distributed from South China to Central Vietnam (Möller et al. 2016), and shows superficial similarities to Deinostigma in general appearance. For example, the caulescent habit (D. cicatricosaD. cyrtocarpaD. minutihamata(D.Wood) D.J.Middleton & H.J.Atkins), usually more and less fleshy leaves (in most Deinostigma species except three species as mentioned above) and fleshy stems, but differs in long and zigzag narrowly infundibuliform corolla tube, four fertile stamens and two lingulate stigmas. The other morphological similar genus is Tribounia, a genus endemic to Thailand (Middleton and Möller 2012). The two genera share the characters of zigzag corolla tube and rounded corolla lobes but can be distinguished by the number of fertile stamens. Based on those morphological characters above, we confirmed that it does not belong to Deinostigma or Tribounia. Furthermore, after consulting the related literature (Burtt 1954, 1963; Wang et al. 1990, 1998; Li and Wang 2005; Weber et al. 2011c, 2013), we also could not find any genus in which to place this unknown species.
The phylogenetic relationship was largely congruent with previous studies (Möller et al. 2009, 2011a, 2016; Middleton et al. 2015, 2018). Michaelmoelleria vietnamensis and its morphologically similar genus Deinostigma is recovered within a polytomy but the phylogenetic relationship of them is distant. It also shows a more distant relationship between Michaelmoelleria vietnamensis and the other similar genus Tribounia. However, our analyses of DNA sequence data suggest that Michaelmoelleria vietnamensis is closely related to the genus Cathayanthe with strong support (PP = 1, BS = 100) and both of them sister to the clade comprised of the genera of AllocheilosGyrocheilosLiebigia, and Didymocarpus with strong support of BI analysis (PP = 0.99) but weak support of ML analysis (BS = 69). Morphologically, Michaelmoelleria vietnamensis, which represents this new genus endemic to Vietnam, can be easily distinguished from the genera of CathayantheAllocheilosGyrocheilosLiebigia, and Didymocarpus by these distinct characters: fleshly stem and leaves alternate on elongated aerial stem. In addition, only two genera’s chromosome numbers among above-mentioned genera were reported (Liebigia speciosa (Blume) DC. in Liebigia2n = 28 or 32Didymocarpus ssp.: 2n = 2022242832364454) (Möller and Pullan 2015onwards; Yang et al. 2019). Furthermore, the chromosome numbers of Allocheilos W.T. Wang (two species, both endemic to China) and Gyrocheilos W.T. Wang (six species and two varieties, distributed from South China to North Vietnam), which are the relative genera of Michaelmoelleria, had never been reported before (Li and Wang 2005; Middleton 2015). The cytological evidence showed 2n = 36, which is the difference from above genera, except Didymocarpus pedicellatus R.Br. (Mehra and Vasudevan 1972; Vasudevan 1976). Building on these, we treat Michaelmoelleria vietnamensis as a distinct genus of tribe Didymocarpeae. All distinguishing characters for identification of MichaelmoelleriaCathayanthe (Fig. 5), Deinostigma (Fig. 6A–E) and Tribounia (Fig. 6F–I) are listed in Table 1.

Thanks to: Mr. Wen-Hua Xu for his attractive arrangement of photos; Mr. Wuf and Ms. Banbino and VietNam’s Garden (the owner of the Facebook account) for their helpful information about this species and Dr. David Middleton from Singapore Botanic Gardens for his constructive suggestions and photos. This study was financially supported by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.03-2019.308, the Fund of Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain (19-050-6), the National Natural Science Foundation of China (31860047), the Natural Science Foundation of Guangxi (2017GXNSFAA198006), Science Research Foundation of Guangxi Academy of Sciences (2017YJJ23022), Guilin Science and Technology Foundation (20180107-6), the Key Sci. & Tech. Research and Development Project of Guangxi (Guike AB16380053). We also wish to thank Michael LoFurno (Adjunct Professor, Temple University) from Philadelphia, USA for his editorial assistance.
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