Big fruits with tiny tepals: An unusual new species of Lauraceae from southwestern China

Abstract We collected an unusual new plant of Phoebe (Lauraceae) from southeastern Yunnan, China, which possesses more or less oblong leaves, paniculate inflorescences with strictly opposite lateral cymes, trimerous flowers with 4-locular stamens, and large fruits with tiny, equal, persistent tepals. Our molecular phylogenetic study based on nrITS, LEAFY and plastid matK sequences suggests that this species belongs to a clade of Phoebe including P. puwenensis, P. megacalyx, and P. macrocarpa. However, this species differs from the latter three species by subglabrous twigs, leaves and inflorescences (vs. pubescent twigs, leaves and inflorescences in the latter three species), larger fruits (5–8 cm long vs. 1–4 cm long in the latter three species), and smaller tepals (1–2.5 mm long vs. 5–15 mm long in the latter two species). As a result, Phoebe jinpingensis sp. nov. is described and illustrated here as new to science.


Introduction
The Lauraceae are woody plants, except for the hemiparasitic climber genus Cassytha L. (Linnaeus 1753), and have more than 3,000 species mainly distributed in tropical regions (Rohwer 1993). This family is notorious for its complicated taxonomy. Many Lauraceae are large trees, which makes it difficult to collect specimens in the field. Good quality specimens are rarely represented in herbaria, many species being known from only one or a few imperfect specimens lacking floral or fruiting characters. The integration of morphological and molecular evidence represents a promising way to better understand the diversity of the tropical family Lauraceae.
Recently, we collected both flower and fruit materials of one notable tree species while conducting field investigations in southeastern Yunnan, China. Further morphological and molecular phylogenetic studies indicate that this species is a new species of Phoebe. We thus describe it here as new to science.

Morphology and Anatomy
We conducted field collections and morphological observations, obtained voucher specimens, preserved flowers in FAA, and dried leaf materials with silica gel. Photographs of vegetative and reproductive characters were taken with a Nikon camera (D700). The preserved flowers were dissected and observed, and photographs were taken under a stereo microscope (Leica S8APO).

Phylogeny
To determine the systematic position of our new species, we conducted a phylogenetic study using nrITS, LEAFY and plastid matK sequences. This phylogeny included 40 species from five genera of the Persea group (Alseodaphne, Alseodaphnopsis, Dehaasia, Machilus and Phoebe). Litsea acuminata (Blume) Sa. Kurata (Kurata 1968) and Litsea akoensis Hayata (Hayata 1911) were selected as the outgroup. Markers used in this phylogenetic study were either sequenced in this study or obtained from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Vouchers and accession numbers of sequences are listed in Table 1.
Total DNA was extracted from silica-gel-dried leaves using Tiangen Plant Genomic DNA kits. To amplify the nrITS, LEAFY and plastid matK fragments, we followed Li et al. ( , 2011b and Sang et al. (1997) in the primers and the PCR amplification.

Figure treatments
The line drawing was done manually with a black ink pen. Illustrations and photos showing morphological characters were edited and merged in Adobe Photoshop CS2 ver. 9.0. Phylogenetic trees were browsed and adjusted in FigTree ver. 1.4.0 (Rambaut 2012) and then improved with Adobe Illustrator CS ver. 11.0.0. A distribution map was generated with ArcGis ver. 10.0 (ESRI, Redlands, CA, USA; http://www.esri.com).

Red list assessment
Extinction risk was assessed using IUCN categories and criteria (IUCN 2012). Population data and area of occupancy were obtained/estimated according to our field investigations.  (Lee et al. 1979) in the large fruits over 3 cm in diam., but differs from the latter two species by the subglabrous leaves being more or less oblongelliptic and the larger fruits having smaller tepals.
Distribution. So far, this species has only been found in southeastern Yunnan, China (Fig. 4).
Habitat. This species occurs in montane evergreen forests at an altitudinal range of 900-980 m. It blooms in April, and the fruits mature from September to December.

Conservation.
There is only one population with 10 mature individuals occupying ca. 400 m 2 . Fewer than 10 juvenile individuals were found. All the individuals have not been protected in any nature reserve, and a rubber plantation exists nearby the population. Based on IUCN Red List Categories and Criteria (IUCN 2012), the new species is categorized as "Critically Endangered" (CR Blab (v); D).

Discussion
Modern taxonomy is based on phylogeny. There is no phylogeny with full sampling of Phoebe, only a few molecular phylogenetic studies including partial sampling of the genus (Rohwer et al. 2009;Li et al. 2011a, b;Song et al. 2017). Frey (2015) stated that the genus Phoebe is polyphyletic but provided no evidence. Li et al. (2011a, b) reconstructed a phylogeny based on nrITS and nuclear LEAFY indicating that Phoebe as a monophyletic group receives low bootstrap value (<50%) but high posterior probability (Li et al. 2011a, b). Our new phylogenetic study using three markers reaches a similar conclusion. However, Song et al. (2017) reconstructed a phylogeny of Phoebe based on 15 highly variable regions of the chloroplast genome and found that the genus as a clade receives high support (BS: 100%; PP: 1.00). These phylogenetic studies consistently suggested that the traditional subdivision of the genus into two sections, i.e. sect. Phoebe and Caniflorae Meisn. (Meissner 1864), is unreasonable because some species    Li et al. 2008;Mo et al. 2017). A few species formerly assigned to Phoebe with less indurate tepals slightly recurving at the apex and globose fruits were transferred to Machilus based on molecular phylogenetic studies (Li et al. 2011a). Traditional taxonomic studies have suggested that the genus Phoebe is characterized by fruits with persistent appressed tepals (van der Werff 2001; Li et al. 2008). Our new species, P. jinpingensis, clearly belongs to the genus Phoebe, because of its fruits having persistent and appressed tepals in fruits (Fig. 1E). Our phylogenetic study also confirmed that the new species belongs to Phoebe.
Phoebe jinpingensis is unusual in the genus Phoebe. First, the fruit is very large and avocado-shaped. Second, the tepals of P. jinpingensis are quite tiny (1-2.5 mm long) and concealed and not obvious in well-developed swollen fruits (Fig. 1E). Third, the leaf shape of Phoebe is usually oblanceolate, while our new species possesses oblong to oblanceolate leaves which is unusual in the genus and more similar to Alseodaphne/Alseodaphnopsis.
The genus Phoebe usually possesses small fruits ca. 1 cm, the largest fruits being seen in P. megacalyx (ca. 3.2 cm long) and P. macrocarpa (3.5-4.2 cm long) (Wei and van der Werff 2008). Our new species is most similar to P. megacalyx and P. macrocarpa in its large fruits, but differs from the latter two species by the subglabrous twigs, leaves and inflorescences (vs. pubescent or tomentose twigs, leaves, and inflorescences), the bigger fruits (5-8 cm long vs. 3-4 cm long in the latter two species), and the smaller tepals (1-2.5 mm long vs. >5 mm long in the latter two species). Phoebe puwenensis also belongs to the same clade as P. jinpingensis in our phylogenetic trees. Our new species differs from P. puwenensis in the subglabrous twigs and leaves (vs. tomentose twigs and leaves in the latter species) and the bigger fruits (5-8 cm long vs. 1-2 cm long in the latter species). For taxonomic purposes, we provide a new key to these closely related species of clade I of the genus Phoebe.
Key to species of clade I of the genus Phoebe