A second species of Pseuduvaria in China: the identity of the enigmatic species Meiogyne kwangtungensis

Abstract Meiogyne kwangtungensis is a rare species endemic to Hainan, China, known just from two fruiting collections made in the 1930s. Although it was published under the name Meiogyne in 1976, it was suggested that it might be better placed within Pseuduvaria or Mitrephora. For decades, this species was never collected again, thus its true generic affinity remained unresolved due to the lack of flowers. During a field exploration in Hainan, we re-discovered this species and collected a flowering specimen for the first time. The flower immediately confirmed its affinity with Pseuduvaria. Phylogenetic analyses of five chloroplast regions (psbA-trnH, trnL-F, matK, rbcL, and atpB-rbcL; ca. 4.2 kb, 70 accessions) also unambiguously placed Meiogyne kwangtungensis in the Pseuduvaria clade (PP = 1.00, ML BS = 99%). Morphologically, it is most similar to P. multiovulata which is endemic to Myanmar and Thailand, both with often-paired flowers, long pedicels and short peduncles, and often 1–2 monocarps. However, it differs in having smaller flowers with kidney-shaped glands on the inner petals, fewer stamens and carpels, smaller ovoid monocarps with an apicule and fewer seeds. On the basis of the combined molecular phylogenetic and morphological data, we propose a new combination, Pseuduvaria kwangtungensis (P.T.Li) Qing L.Wang & B.Xue. A full description including floral characters and a color plate are provided here for this species. A key to species in the genus Pseuduvaria in China is also provided.


Introduction
The genus Meiogyne Miq. is a medium-sized genus in tribe Miliuseae Hook.f. & Thomson of Annonaceae (Chatrou et al. 2012; Thomas et al. 2012;Xue et al. 2014;Guo et al. 2017). It consists of ca. 30 species of trees or shrubs, distributed in wet tropical lowland and lower montane rainforests across South-east Asia and the western Pacific (Thomas et al. 2012;Tan et al. 2014;Xue et al. 2014Xue et al. , 2017Turner and Utteridge 2015;Johnson et al. 2019). Species in Meiogyne are characterized by sub-equal petals, inner petals with corrugated or verrucose base of the adaxial surface and innermost stamens with tongue-shaped apical prolongations (van Heusden 1994; Thomas et al. 2012;Xue et al. 2014).
Meiogyne kwangtungensis Li was published in 1976, based on two fruiting collections (F. C. How 73305, IBSC, A, IBK and Z. Huang 33693, IBSC) from Hainan, China, in 1935and 1933respectively (Li 1976Tsiang and Li 1979). After that, it was not collected again. The long fruiting pedicle of this species (up to 50 mm in length) is unusual in Meiogyne, as most Meiogyne species have short flowering and fruiting pedicels (usually less than 30 mm in length, except Meiogyne chiangraiensis Chalermglin & M.F.Liu; Johnson et al. 2019). Rainer and Chatrou (2006) indicate that this species can also belong to Pseuduvaria or Mitrephora. Flower characters are essential for generic delimitation in Annonaceae, and the three genera, Meiogyne, Pseuduvaria and Mitrephora can be easily distinguished on that basis (van Hesuden 1992; Su and Saunders 2006). Therefore, flowers are required to confirm the correct generic placement of this species (Li and Gilbert 2011).
For the past few years, we have carried out several field explorations in Hainan to search for this species. The explorations finally resulted in new collections of Meiogyne kwangtungensis, including flowers and fruits. Based on the mature flowers, we are able to confirm that Meiogyne kwangtungensis should be placed in Pseuduvaria.
Pseuduvaria is a genus widely distributed in continental SE Asia and Malesia, extending from Indochina and the Philippines to New Guinea and NE Australia (Su and Saunders 2006). The only comprehensive taxonomic monograph recognizes 52 species in the genus (Su and Saunders 2006). Three new species and one new combination in Su et al. (2010) and one new species in Turner (2010) bring the total species recognized in Pseuduvaria to 57. The flowers of Pseuduvaria are often unisexual and it's unique in having inner petals apically connivent over the reproductive organs, forming a mitriform dome (Su and Saunders 2006;Su et al. 2008). Each inner petal is differentiated into a distinct blade and basal claw, which results in three lateral apertures between the petal claws, enabling access by floral visitors (Su and Saunders 2006). Moreover, the adaxial surface of the inner petals often bears one or two protruding glands (Su and Saunders 2006). In contrast, the flowers of Meiogyne are bisexual and the inner petals are spreading and corrugated at the base of the adaxial surface.
With the available flowering materials and silica-gel samples for DNA extraction, we clarify the generic placement of Meiogyne kwangtungensis based on morphological data and phylogenetic analysis in this study.

Morphological studies
The morphological characters were examined based on the living plants and specimens kept in the HITBC, IBSC, IBK, and KUN herbaria. Comparisons were also made against published Pseuduvaria species in the monograph and recent papers (Su and Saunders 2006;Su et al. 2010;Turner 2010;Li and Gilbert 2011).

Phylogenetic studies
Total DNA of the silica-gel dried material of Meiogyne kwangtungensis (Q. L. Wang 20200528002, IBSC) was extracted using a modified CTAB method (Doyle and Doyle 1987). Five chloroplast regions (psbA-trnH, trnL-F, matK, rbcL, and atpB-rbcL) were newly generated. For detailed information on PCR amplification and primer sequences we refer to Su et al. (2008). 54 Pseuduvaria species from Su et al. (2010) were included in this study. Monocarpia euneura Miq. and 14 species in the tribe Miliuseae were selected as outgroups based on the phylogenetic framework reported in previous studies (Chatrou et al. 2012;Chaowasku et al. 2014;Guo et al. 2017;Xue et al. 2018Xue et al. , 2020a. Sequences were downloaded from the nucleotide database of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The final data matrix comprised a total of 70 species of Annonaceae. The information on sequence alignment can be found in Xue et al. (2018).
Detailed information about the samples, localities and GenBank accession numbers are all listed in the Appendix 1.
Phylogenetic analyses were done using Bayesian Inference (BI) and maximum likelihood (ML) methods. The information on model selection of the sequence matrix constructed could refer to Xue et al. (2018). The best partition scheme suggested five partitions based on DNA region identity with GTR + I + Γ chosen for matK and rbcL; and GTR + Γ selected for atpB-rbcL, psbA-trnH and trnL-F regions. Detailed methods for tree reconstruction could refer to Xue et al. (2018) and Xue et al. (2020b).

Results
The morphological observation is illustrated in Figs 1, 2, and discussed in detail below.
The backbone of the tribe Miliuseae is not well resolved as in previous studies.

Discussion
With the new collections of the flowering specimens of Meiogyne kwangtungensis, the enigmatic identity of this species is resolved. The flowers of Meiogyne kwangtungensis are unisexual ( Fig. 1D-G). Both female and male flowers are characterized by having longer inner petals that are apically connivent over the reproductive parts to form a mitriform dome (Fig. 1B, D, F). The inner petals are differentiated into distinct blades and claws, with the adaxial surface of the claw of the inner petal bearing two protruding glands (Fig. 1E). The stamen has a flat-topped connective extending over the thecae ('uvarioid' sesu Prantl 1891; Mols et al. 2004) (Fig. 1E, 2D). These characters are completely different from that of the Meiogyne species. In contrast, flowers of Meiogyne are bisexual; both whorls of petals are sub-equal and similar in shape; the inner petals are not connivent either. Therefore, the flower morphology of Meiogyne kwangtungensis is consistent with that of Pseuduvaria, which immediately confirmed its affinity with Pseuduvaria.
The molecular phylogeny further supported the placement of Meiogyne kwangtungensis in the genus Pseuduvaria. It falls into the same clade with Pseuduvaria gardneri, P. fragrans and P. multiovulata (PP =1.00; ML BS = 91%) (Fig. 3). Morphologically, Meiogyne kwangtungensis is most similar to Pseuduvaria multiovulata (C.E.C.Fischer) J.Sinclair in Thailand, both with 1-2 flowers per inflorescence, with long pedicels and short peduncles, and often with 1-2 monocarps (Su and Saunders 2006;Gardner et al. 2015). However, the two species differ in the size of the flowers, the shape of the inner petal glands, the number of stamens and carpels, the shape of the apex of the monocarps and the number of seeds per monocarp (Table 1). Meiogyne kwangtungensis has small flowers (outer petal ca. 7 mm long, inner petal ca. 8mm long) whereas Pseuduvaria multiovulata has larger flowers (outer petal 7.5-11 mm long, inner petal 9-18.5 mm long; Su and Saunders 2006). Meiogyne kwangtungensis has two kidney-shaped to ellipsoid glands on adaxial surface of the inner petals (Fig. 1E), whereas the inner petals glands of Pseuduvaria multiovulata are square (Su and Saunders 2006). Meiogyne kwangtungensis has 20-30 stamens in male flower and 3 carpels in female flower (Fig. 1E, G), whereas Pseuduvaria multiovulata has 110-115 stamens in male flower and ca. 11 carpels in female flower (Su and Saunders 2006). The monocarps of Meiogyne kwangtungensis have an apiculate apex, with 5-10 seeds per monocarp, whereas the monocarps of Pseuduvaria multiovulata do not have apicule, with ca. 17 seeds per monocarp (Su and Saunders 2006). In China, only one Pseuduvaria species is recorded in Yunnan Province, i.e. Pseuduvaria trimera (Craib) Y.C.F.Su & R.M.K.Saunders (Li and Gilbert 2011) (Fig. 4). This species is relatively widely distributed, also occurring in Myanmar, Thailand and Vietnam. Meiogyne kwangtungensis and Pseuduvaria trimera could be easily differentiated from each other by the growth habit, the morphology of leaf, inflorescence, flower and fruit. Pseuduvaria trimera is a tree up to 20 m tall (Su and Saunders 2006;Li and Gilbert 2011), whereas Meiogyne kwangtungensis is a shrub to 4 m tall. The leaf laminas of Pseuduvaria trimera are subcoiaceous with 14-18 pairs of secondary veins (Fig. 4A), whereas leaf laminas of Meiogyne kwangtungensis are papery with ca. 10 pairs of secondary veins (Fig. 1A). The inflorescences of Pseuduvaria trimera are clustered on young branches with yellow flowers (Fig. 4B-G), whereas those of Meiogyne kwangtungensis are axillary with cream-colored or purple flowers (Fig. 1B-F). Pseuduvaria trimera is distinct in lacking any glands on the clawed inner petals (Fig. 4F), whereas Meiogyne kwangtungensis has a pair of glands on the adaxial surface of the inner petals (Fig. 1E). Pseuduvaria trimera has globose, stipitate monocarps with rugulose pericarps (Fig. 4H, I; Su and Saunders 2006; Li and Gilbert 2011), whereas Meiogyne kwangtungensis has ovoid, sessile monocarps with smooth pericarp (Fig. 1H).