Meiogyne oligocarpa (Annonaceae), a new species from Yunnan, China

Meiogyne oligocarpa sp. nov. (Annonaceae) is described from Yunnan Province in Southwest China. It is easily distinguished from all previously described Meiogyne species by the possession of up to four carpels per flower, its bilobed, sparsely hairy stigma, biseriate ovules and cylindrical monocarps with a beaked apex. A phylogenetic analysis was conducted to confirm the placement of this new species within Meiogyne. Meiogyne oligocarpa represents the second species of Meiogyne in China: a key to the species of Meiogyne in China is provided to distinguish it from Meiogyne hainanensis. Paraffin sectioning was undertaken to study the anatomy of the corrugations on the inner petals of Meiogyne oligocarpa to verify whether they are glandular.

Yunnan Province is located in southwestern China and harbors more than 19,000 plant taxa, accounting for over 50% of China's overall floristic diversity (Qian et al., 2020). Plant diversity in Yunnan faces continuous threats with the deterioration of ecology and environment, however. In order to constrain the rapid loss of biodiversity, the Chinese Academy of Science and Yunnan Provincial government jointly launched a project named Tropical Plant Resource Conservation and Sustainable Use from 2000 to 2004. In this project, more than 6,000 plant species collected from tropical areas of China and nearby countries were preserved in 35 living collections in Xishuangbanna Tropical Botanical Garden. These include about 100 Annonaceae collections, among which twelve small trees have been continuously flowering and fruiting in recent years. These treelets were propagated from 20 seeds collected from one unidentified Annonaceae tree in He-kou County, Yunnan Province in 2001 by a field collection team leading by Professor Guo-Da Tao. The flower morphology indicates that it belongs to Meiogyne, but it differs from all previously described species. It is readily distinguished from other Meiogyne species by a combination of the following characters: flowers with bilobed stigmas, up to four carpels per flower, ovaries with ovules attached in two rows and cylindrical monocarps with a beaked apex. Morphological comparisons and phylogenetic analyses based on seven chloroplast regions indicate that the treelets represent a hitherto undescribed species, which we describe and name here as Meigoyne oligocarpa.
Meiogyne is characterized by inner petals with a longitudinally grooved or verrucose base to the adaxial surface and innermost stamens with tongue-shaped apical prolongations (van Heusden, 1994;Thomas et al., 2012;Xue et al., 2014;Johnson et al., 2019). The elaborate inner petal corrugations are synapomorphic for Meiogyne, and have often been referred to as ''glands'' (van Heusden, 1992;Saunders, 2010), although glandular function has never been confirmed as no liquid secretions have been observed (Xue et al., 2017). Shao & Xu (2015) similarly failed to observe apertures for secretion on the surface of the corrugations (''strumae'') on inner petals of Meiogyne hainanensis (as ''Oncodostigma''). They found polysaccharides on the strumae, suggesting that the structure may provide nutrition to floral visitors as well as a protected site for mating, oviposition, brooding and larval feeding (Shao & Xu, 2015). This is consistent with the ''food body'' hypothesis in Sapranthus (Schatz, 1987) or ''nutritious tissues'' hypothesis (Gottsberger & Webber, 2018). Moreover, Shao & Xu (2015) also introduced a third hypothesis, the ''brood-site'' hypothesis. The study failed to establish that the observed floral visitors (thrips) are effective pollinators, however.
In this study, paraffin sectioning was undertaken to investigate the anatomy of the corrugations at the base of the adaxial surface of the inner petals, with the aim to testing the alternative hypotheses regarding the function of the corrugations.

Ethics statements
The new species reported in this study was collected from Xishuangbanna Tropical Botanical Garden, Yunnan Province, China, which permitted our field work in the Garden. Since this species is currently undescribed, it is not currently included in the China Species Red List (Wang & Xie, 2004).

Nomenclature
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix ''http://ipni.org/''. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Material collection
The new species has been monitored in Xishuangbanna Tropical Botanical Garden by the authors continuously since 2014. Flowering and fruiting specimens were collected for morphological study. Mature flowers for anatomical study were fixed in FAA (70% alcohol, formaldehyde and glacial acetic acid in a ratio of 90: 5: 5) for 24 h and then transferred to store in 70% alcohol. Leaf materials for DNA extraction were collected and dried using silica gel in the field.

Morphological observations
Morphological description of the new species was based on careful examination of materials collected. Comparisons with other similar Meiogyne species were based on the existing literature (van Heusden, 1994;Li & Gilbert, 2011;Thomas et al., 2012;Johnson et al., 2019) as well as the study of herbarium specimens and digitized images (mainly from HITBC, IBSC, KEP, KUN, PE and SING herbaria).
Flower samples in 70% alcohol were prepared for scanning electron microscopy by dehydration and critical-point drying. Carpels, stamens and pollens were then mounted on metal stubs, sputter-coated with gold, and examined using scanning electron microscope (SEM) as in Xue et al., 2017. Flowers fixed in 70% alcohol were also dissected for anatomical observations using paraffin sectioning following Xue et al., 2017.  (matK, ndhF, ndhF-rpl32, rbcL, rpl32-trnL, trnL-F and ycf1) were sequenced for the three species.

Molecular phylogenetic analyses
These newly generated sequences were added to the seven-region dataset compiled by Xue et al. (2014). The final dataset therefore included 73 Annonaceae accessions, with the ingroup consisting of 30 accessions (representing 26 species) of Meiogyne. For detailed information regarding DNA extraction, PCR amplification, primer sequences and sequence alignment, refer to Thomas et al. (2012) and Xue et al. (2014). The sampled species, voucher information, and GenBank accession numbers are listed in Appendix S1 .
The phylogenetic trees were reconstructed using maximum parsimony (MP) (Swofford, 2003) and Bayesian inference (BI) (Ronquist & Huelsenbeck, 2003) based on the seven combined regions. MrModeltest ver. 2.3 (Nylander, 2004) was used for best-fit likelihood model selection for each region under Akaike Information Criterion: the general timereversible model with a gamma distribution of substitution rates (GTR+G) was chosen for the matK, trnL-F and ndhF-rpl32 regions; and the GTR+I+G model with a proportion of invariant sites was selected for the ndhF, rbcL, rpl32-trnL and ycf1 regions. For detailed methods in tree reconstruction, refer to Thomas et al. (2012) and Xue et al. (2014).

Phylogenetic analysis
The seven-region concatenated alignment of the 73-accesion dataset consisted of 8,923 characters. The resultant MP and BI topologies based on the concatenated alignment are similar. The BI tree with both posterior probabilities and MP bootstrap values for each clade is shown as Fig. 1. The new species, Meiogyne oligocarpa, is deeply nested within the Meiogyne clade (PP = 1, MPBS = 92%). Although these results confirm that the new species unequivocally belongs to the genus Meiogyne, limitations in internal resolution and support preclude any definitive conclusion regarding which species is phylogenetically closest to M. oligocarpa.
To conclude, both the molecular and morphological data support the placement of the new species in Meiogyne. It differs from all previously described species, and therefore unequivocally represents a new species.   to Meiogyne kanthanensis, but differs in having smaller leaves with more secondary veins, shorter pedicels, stigmas that are bilobed and distinctly less hairy, and cylindrical monocarps with a beaked apex. Description. Small trees to 5 m tall, ca. 4 cm dbh. Bark grayish. Young twigs green, yellowish puberulent, soon become grayish and glabrous. Petioles 3-5 mm long, 1-2 mm in diameter, pubescent; leaf laminas lanceolate, or narrowly elliptic or narrowly oblong, length:width ratio 3.5-4.5, 12-20 × 3-4.2 cm, base slightly asymmetrical, oblique to rounded, apex acute to acuminate, papery, slightly glossy above in vivo, drying dull greyish-green, concolorous beneath, glabrous above, beneath base of leaf margin and midrib pilose, secondary veins sparsely pilose; midrib impressed and glabrous above, raised and hairy below; secondary veins 8-13 on each side of the leaf, parallel, diverging at 45-60 • from midrib, upturned and gradually diminishing towards apex, connecting to subsequent secondary veins by series of cross veins, and lacking prominent marginal loops, distinctly raised below; tertiary veins scalariform, prominent abaxially (Fig. 4A). Inflorescences axillary; one flower per inflorescence (Figs. 2B, 4A), greenish to yellowish     In our extensive study of Annonaceae herbarium collections in China, we find only one specimen from Man-hao, Ge-jiu city (Ge-jiu Forestry Bureau exped. 94111, KUN) representing this species collected from wild populations. One of the authors, Yun-Hong Tan, has undertaken extensive field survey in He-kou County and adjacent regions, but has failed to locate any wild populations. Primary forests in He-kou have been under severe pressure from agricultural expansion over recent decades, and most unprotected forests at low elevation have been replaced by banana plantations. Additional field surveys are required to locate wild populations of this species in Yunnan. This study highlights the essential role of botanical gardens on biodiversity conservation. Ecology and phenology. In evergreen forests. Flowering and fruiting from March to September.

Anatomy of inner petal corrugations
The elaborate inner petal corrugations are synapomorphic for Meiogyne. Although these structures have previously often been referred to as ''glands' ' (van Heusden, 1992;Saunders, 2010), glandular function has never been confirmed (Xue et al., 2017;Saunders, 2020). Shao & Xu (2015) examined the inner petal of Meiogyne hainanensis (as ''Oncodostigma'') but found no evidence of secretion. It is therefore doubtful whether the corrugations represent true glands, although additional species should be studied. During our continuous field observations on M. oligocarpa, no liquid secretions were observed to form on the inner petals, although strong fruity scents were emitted during anthesis. The anatomical results also indicate that the corrugations are not glandular. The anatomical organization of nectar glands often consist of four distinct tissues (Nepi, 2007;Xue et al., 2017): epidermis; sub-epidermal secretory parenchyma, comprising several layers of small cells with densely staining cytoplasm; ground parenchyma, comprising several layers of larger cells, more loosely packed than those of the secretory parenchyma; and vascular bundles. The anatomy of the inner petal corrugations of M. oligocarpa (Figs. 5C, 5D), however, consist of an epidermis, several layers of homogeneous parenchyma and a few vascular bundles, and do not differ from adjacent non-corrugated parts of the inner (Fig. 5E) and outer petals (Fig. 5F). The inner petal corrugations are therefore not glandular.
Shao & Xu (2015) studied the corrugations of Meiogyne hainanensis. Based on their observations, they raised two alternative hypotheses regarding the function of the inner petal corrugations, noting that polysaccharides may provide nutrition for floral visitors, and hence the corrugations may function as a ''food body '' as in Sapranthus (Schatz, 1987), aligned with the ''nutritious tissues'' hypothesis (Gottsberger & Webber, 2018).
In our study, we observed numerous curculionid beetles within the flowers of M. oligocarpa, with evidence of gnawing of the petals, although this was present on both the corrugated and non-corrugated parts of the inner petals. We also observed starch and tannin in both whorls of petals (Figs. 5C-5F), which may provide food for beetles (Gottsberger & Webber, 2018). A more detailed histochemical study is nevertheless required, as well as additional field observations on the distribution of gnawing marks to verify the ''nutritious tissues'' hypothesis.
The second hypothesis raised by Shao & Xu (2015) is that strumae may provide floral visitors with a protected site for mating, oviposition, brooding and larval feeding, thus functioning as a ''brood-site''. Although this might represent an example of pollinator brood-site adaptations of petals in Annonaceae, Shao & Xu (2015) failed to establish that thrips are the effective pollinator (Saunders, 2020). This also requires further study. It would also be interesting to check whether beetle larvae are present on the petals of M. oligocarpa.

Meiogyne species in China
According to the Flora of China (Li & Gilbert, 2011), only one Meiogyne species is recorded from China: M. kwangtungensis Li, known from only two fruiting collections from Hainan Province. Rainer & Chatrou (2006) suggest that M. kwangtungensis might be better accommodated within Pseuduvaria or Mitrephora, although its placement in Meiogyne is debatable due to the lack of flowers. Fortunately, with new flowering and fruiting collections recently collected from Hainan Province, we have been able to confirm that M. kwangtungensis should be placed in Pseuduvaria (Wang et al., 2021)  The only currently accepted Meiogyne species in China is M. hainanensis (Merr.) Bân from Hainan Province (Tsiang & Li, 1979;van Heusden, 1994;Rainer & Chatrou, 2006;Xue et al., 2014), although Li & Gilbert (2011) treated this species as Chieniodendron hainanense (Merr.) Tsiang & P.T. Li . The transfer of Chieniodendron to Meiogyne has been widely accepted ( Bân 1973;van Heusden, 1994) and supported in recent molecular phylogenetic studies (Thomas et al., 2012;Xue et al., 2014).