Alphonsea glandulosa (Annonaceae), a New Species from Yunnan, China

Alphonsea glandulosa sp. nov. is described from Yunnan Province in south-west China. It is easily distinguished from all previously described Alphonsea species by the possession of glandular tissue at the base of the adaxial surface of the inner petals. Nectar was observed throughout the flowering period, including the pistillate phase and subsequent staminate phase. Small curculionid beetles were observed as floral visitors and are inferred to be effective pollinators since they carry pollen grains. A phylogenetic analysis was conducted to confirm the placement of this new species within Alphonsea and the evolution of the inner petal glands and specialized pollinator reward tissues throughout the family.

Two indigenous trees growing in Xishuangbanna Tropical Botanical Garden and in a small forest patch near Man-zhang Reservoir in Meng-la County, Yunnan Province, were easily identifiable as conspecific and belonging to Alphonsea, but could not be assigned to any previously described species. The two trees are readily distinguished from all other Alphonsea species as they have multiple flowers (often 5 to 9) in each inflorescence and have glandular tissue at the base of the inner petals. We propose that these two trees represent a hitherto a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 unlinked setting. MrModeltest ver. 2.3 [22] was used to determine the best-fit likelihood model for each locus and the concatenated matrix using the Akaike Information Criterion: the general time-reversible model with a gamma distribution of substitution rates (GTR+G) was chosen for the matK, psbA-trnH and trnL-F regions; and the GTR+I+G model with a proportion of invariant sites was selected for the ndhF, rbcL and ycf1 regions. Two independent Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run. Each search used three incrementally heated and one cold Markov chain, and was run for 10 million generations and sampled every 1,000th generation. The mean branch length prior was set from the default mean (0.1) to 0.01 (brlenspr = unconstrained: exponential (100.0)) to reduce the likelihood of stochastic entrapment in local tree length optima [23,24]. Convergence was assessed using the standard deviation of split frequencies, with values < 0.01 interpreted as indicating good convergence. The first 25% of samples (2,500 trees) were discarded as burn-in, and the post-burn-in samples summarized as a 50% majority-rule consensus tree.

Morphological observations
The morphological description of the new species was based on the examination of fresh materials and dried herbarium specimens. Morphological comparisons with other species in Alphonsea were based on studies of herbarium specimens (from herbaria HITBC, IBSC, K, KUN, PE and SING; institutional acronyms follow the Index Herbariorum [25]), specimen photographs and a literature survey.
Flowers were fixed in FAA (70% alcohol, formaldehyde and glacial acetic acid in a ratio of 90:5:5) for 24 hrs and subsequently stored in 70% alcohol. Samples examined using scanning electron microscopy were dehydrated using an ethanol series, and critical-point dried with a Leica EM CPD300 Automated Critical Point Dryer (Leica, Wetzlar, Germany). The dried materials were later attached to metal stubs using adhesive carbon tabs, sputter-coated with gold/palladium, and viewed using a JSM-6360LV scanning electron microscope (JEOL, Tokyo, Japan) at 5 kV.
For anatomical observations, the fixed inner petals were dissected and transferred into 70% ethanol, stained with Ehrlich's hematoxylin, dehydrated in ethanol series, infiltrated with xylene, embedded in paraffin wax and serially sectioned at thickness of 10 μm, using a rotary microtome. Subsequently, mounted slides were examined and photographed under a LEICA DM5500 B microscope equipped with a LEICA DFC550 digital camera.

Nomenclature
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In addition, the new name contained in this work has been submitted to IPNI, from where it will be made available to the Global Names Index. The IPNI LSID 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: PubMed Central and LOCKSS.

Phylogenetic analysis
The concatenated alignment of the 63-terminal dataset consisted of 7,399 characters. The MP heuristic search retrieved 24 most parsimonious trees of 3,968 steps (consistency index, CI = 0.66; retention index, RI = 0.70).
The MP and Bayesian analyses are topologically similar, differing mainly in the relative MP bootstrap (BS) and posterior probability (PP) values for particular groups (Fig 1). The new species, Alphonsea glandulosa, is deeply nested within the Alphonsea clade and retrieved as sister to A. elliptica. Although these results confirm that the new species unequivocally belongs to the genus Alphonsea, limitations in the extent of taxon sampling within the genus (nine out of 28 species; 32%) preclude any definitive conclusion regarding which species is phylogenetically closest to A. glandulosa.

Morphological comparisons
Alphonsea glandulosa flowers have saccate petals (Fig 2A-2C), 'miliusoid' stamens in which the connective does not extend over the thecae (Fig 3C), and hairy ovaries with up to 13 biseriate ovules ( Fig 3D). These characters corroborate its placement in the genus Alphonsea.
Alphonsea glandulosa can easily be distinguished from A. ventricosa, even from vegetative characters: A. ventricosa has large leaves (12-27 cm by 4-8 cm) that are distinctly thick and coriaceous, whereas the leaves of the new species are smaller (6-19 cm by 3-5.5 cm) and slightly coriaceous. A. ventricosa furthermore differs in having flowers with more stamens (40-50, in four whorls) and more carpels (10-12) per flower, and in having fruits with larger monocarps (up to 6 cm long, 4 cm in diameter) and longer stipes (ca. 3 cm). Alphonsea glandulosa has flowers with 26-35 stamens in three whorls and 4-7 carpels per flower, fruits that are 2-4 cm long and 1.5-3 cm in diameter, and stipes up to 10 mm long.
In contrast, Alphonsea glandulosa is vegetatively more similar to A. philastreana. The two species differ, however, in the number of secondary veins on each side of leaf, the indumentum of the abaxial surface of the flower buds, pedicel length, the number of carpels per flower, and the shape of the stigma. Alphonsea glandulosa has 8-18 nerves on each side of the leaf, whereas A. philastreana has 8-9 [2,27]. The abaxial surface of flower buds of A. glandulosa is greyish to yellowish pubescent, whereas that of A. philastreana is densely rusty tomentose [27,28]. Pedicel length and the number of carpels per flower are useful characters for species delimitation: Keβler [2], for example, frequently used these characters in his key to species of Alphonsea. Although the name A. philastreana was validated using a protologue description based on flower buds and hence difficulties were encountered when drawing comparisons, the difference in the length of the pedicel in the two species is nevertheless very significant when buds of a similar size are compared: the pedicel of flower buds that are ca. 3-4 mm in diameter, for example, is 10-20 mm long in A. glandulosa (Fig 2B and 2C) but only ca. 3 mm long in A. philastreana [2,28]. Although previous descriptions of A. philastreana state that it has (4-)6 carpels per flower and the stigma is globose [27][28][29], Keβler [2] counted only three carpels in all studied buds and found that the stigma is actually two-headed (deeply bilobed). The twoheaded stigma may have misled previous authors into counting six carpels from above. In contrast, A. glandulosa has between four and seven carpels (Figs 2B and 4B), and the stigma is globose to shallowly bilobed. Ovule numbers of the two species are also different: A. glandulosa has 10-13 ovules per carpel, whereas A. philastreana has 14-18(-20) [27][28][29].
Amongst the Alphonsea species sampled in our phylogenetic study, A. elliptica is retrieved as sister to A. glandulosa (Fig 1). The differences between these two species are nevertheless clear: the inflorescences of A. glandulosa are composed of (3-)5-9(-13) flowers, whereas those   [2,30]. Alphonsea glandulosa furthermore has stamens in three whorls, whereas A. elliptica has stamens in four whorls [2].
The most distinctive diagnostic character of Alphonsea glandulosa, however, is the possession of nectar glands at the base of the inner petals (Fig 4A-4D). The gland is clearly visible irrespective of preservation technique: in fresh (Fig 4A and 4B) and FAA-fixed (Fig 4C) material the gland is clearly ridge-shaped, and in dried specimens it is apparent as a distinct groove ( Fig 4D); it is even obvious in small flower buds.
Anatomically, the nectar glands consist of four distinct tissues (Fig 5A), similar to those described for other species [31][32][33]: (i) epidermis; (ii) subepidermal secretory parenchyma: several layers of small cells with densely staining cytoplasm; (iii) ground parenchyma: several layers of larger cells, more loosely packed than those of the secretory parenchyma; and (iv) vascular bundles. The anatomical structure is distinct from the non-glandular part of the inner petals (Fig 5B), which only consists of epidermis, several layers of homogeneous parenchyma and a few vascular bundles. The surface of the nectar glands also differs from the surrounding epidermis: nectar stomata, which are raised slightly above the epidermis with an aperture for nectar secretion [32], are found across the surface of the glandular tissue (Fig 3A and 3B), but are otherwise absent from the non-glandular part of the inner petals. The ultrastructure of the glands is similar to those of Pseuduvaria froggattii (F.Muell.) Jessup [34,35].
To conclude, both the molecular and morphological data support the placement of the new species in Alphonsea. It differs from all previously described species, and therefore represents a new species.

Evolution and function of nectar glands
Nectar glands have never previously been recorded in Alphonsea [2,36], and it therefore appears that within the genus nectar glands are autapomorphic for A. glandulosa.
Chatrou et al. [54] recognized four subfamilies in their recent subfamilial and tribal classification of the Annonaceae, viz. subfam. Anaxagoreoideae, Ambavioideae, Annonoideae and Malmeoideae (Fig 1). The genera mentioned above with glands or specialized tissues on the inner petals are distributed in all the subfamilies except subfam. Anaxagoreoideae. These genera are classified in up to seven different tribes, indicating that this character is likely to have evolved independently on multiple occasions (Fig 1). A summary of the occurrences of the glands and specialized tissues across the family is provided in  Fig 1).
Pollination ecology studies have been undertaken for several species that possess glandular or specialized inner petal tissues, viz.: Asimina obovata (Willd.) Nash and A. pygmaea (W. Bartram) Dunal, which are pollinated by large scarabaeid beetles [37]; Pseuduvaria froggattii, pollinated by Drosophilidae and other flies [34]; Pseuduvaria mulgraveana Jessup, pollinated by small diurnal nitidulid beetles [53]; and Sapranthus palanga R.E.Fr., pollinated by tenebrionid, nitidulid, and scarabaeid beetles and apid bees [45]. In Asimina obovata and A. pygmaea, beetles were observed to consume the corrugated inner petal tissues that were sometimes also observed to secrete a small volume of exudate [36]. In Pseuduvaria froggattii and P. mulgraveana, the secretion of nectar from the inner petal glands was recorded in Table 1. The occurrence of inner petal glands and specialized pollinator reward tissues in genera, tribes and subfamilies across the family Annonaceae.

Subfamilies
Tribes Genera detail and shown to be concomitant with the anthetic changes: nectar was present throughout the pistillate and staminate phases in hermaphroditic flowers of both species, and in staminate flowers of P. mulgraveana [34,53]. Pollinators were observed consuming the nectar [34,53], indicating that the nectar glands function as a food reward for pollinators throughout anthesis.

Ambavioideae -Tetrameranthus
As with all hermaphroditic-flowered Annonaceae species [55], Alphonsea glandulosa is protogynous. In A. glandulosa, abundant sweet nectar was observed to be secreted from the beginning of the pistillate phase until the end of the staminate phase (Fig 4A and 4B). Around three to eight small curculionid beetles were found inside the flowers (Fig 4E), and pollen was observed on their bodies, indicating that they are likely to be effective pollinators. The nectar is likely to provide a food reward to those beetles, as the beetles were observed to consume it. One opportunist bee was also observed visiting the flower (Fig 4F), although it is unlikely that the bee could effectively pollinate the flower because of the presence of the floral chamber formed by the inner petals, which would restrict access by the bees [56]. Diagnosis. Alphonsea glandulosa is unique amongst Alphonsea species in having a nectar gland at the base of the adaxial surface of each inner petal. It is most similar to A. philastreana (Pierre) Finet & Gagnep., but differs in having a greater number of secondary veins on each side of the leaf, greyish to yellowish pubescent flower buds, longer pedicels, a greater number of carpels per flower, a smaller number of ovules per carpel, and globose to shallowly bilobed stigmas.
Etymology. The specific epithet reflects the presence of nectar glands at the base of the adaxial surface of each inner petal. Distribution. Only known from two localities in Yunnan, China (Fig 7). Ecology and phenology. In evergreen forests. Flowering specimens collected in March to May, and fruiting specimens in June to July. IUCN Conservation Status. Only two individuals were found in Meng-la County, Yunnan Province. The primary forests in Xishuangbanna have been under severe pressure from agricultural expansion over the last 30 years, and below 900 m elevation most unprotected forest has been replaced by rubber plantations [57]. The tree growing in the forests close to Manzhang Reservoir in Meng-la County is located at the edge of a rubber plantation. One of the authors, Yun-Hong Tan, has undertaken an extensive field survey in Xishuangbanna, but was unable to locate other individuals. Perhaps because of the dearth of individuals, the level of fruitset in the two trees is poor. On the basis of current IUCN red list categories and criteria [58], we therefore recommend critically endangered status, CR D. Supporting Information S1 Table. GenBank accession numbers and voucher information for the materials used in this study. (XLSX)