Two new species of Alseodaphnopsis (Lauraceae) from southwestern China and northern Myanmar: evidence from morphological and molecular analyses

Abstract Alseodaphnopsis maguanensis and A. putaoensis, two new species of Alseodaphnopsis (Lauraceae) from southwestern China (Yunnan Province) and northern Myanmar (Kachin State), are here described and illustrated based on both morphological and molecular evidence. They are morphologically similar to Alseodaphnopsis rugosa and phylogenetically closely related to A. rugosa and A. hainanensis respectively. Their preliminary conservation status is also estimated according to the IUCN Red List Categories and Criteria.


Introduction
Alseodaphnopsis H. W. Li & J. Li, including nine species at present, is a recently described new genus of the Lauraceae (Mo et al. 2017a). Mo et al. (2017a) separated Alseodaphnopsis from the traditionally recognized tropical Asian genus Alseodaphne Nees based on both morphological and molecular evidence. The combination of principal morphologi-cal characters to distinguish the two genera (Alseodaphnopsis vs. Alseodaphne) includes: 1) twigs thick, 4-11 mm in diameter, not obviously whitish in color vs. thin, 2.5-4.5 mm in diameter, obviously whitish in color; 2) terminal buds perulate vs. not perulate; 3) perianth lobes persistent at least in young fruit vs. early deciduous; 4) inflorescences relatively large, 8.5-35 cm long, generally many-flowered, with 3-4 order of branching vs. 3-20 cm long, few-flowered, with 1-2 orders of branching; and 5) mature fruit relatively large, 3-5 cm vs. < 2.5cm in diameter (Mo et al. 2017a). In addition, Alseodaphnopsis species are distributed in the northern marginal zone of Asian tropics in southwestern China (also in Hainan island) and northern Vietnam while Alseodaphne species are mostly found in the tropics of south and southeast Asia (Kostermans 1973;Li et al. 2008;Mo et al. 2017a).
During recent field surveys in southwestern China (Maguan, Yunnan Province) and northern Myanmar (Putao, Kachin State), two unknown Lauraceae species were collected. Based on both morphological and molecular evidence, they were confirmed as new species of Alseodaphnopsis and closely related to Alseodaphnopsis rugosa (Merr. & Chun) H. W. Li & J. Li and A. hainanensis (Merr.) H. W. Li & J. Li respectively. In the work of Mo et al. (2017b), the specimens collected in Maguan (Yunnan Province, China) were treated as synonyms of Alseodaphnopsis rugosa. In this paper, these new Alseodaphnopsis species are described and illustrated.

Morphological studies
Morphological characters of the two new Alseodaphnopsis species were examined in detail based on dried specimens and fresh materials in field observations and compared with possible relatives based on the specimens from the HITBC, IBK, IBSC and KUN herbaria as well as images of specimens available on JSTOR Global Plants (http://plants.jstor.org/).

Molecular studies and phylogenetic analyses
Total genomic DNA was extracted from silica-gel dried leaf material using the Plant Genomic DNA Kit (Tiangen, Beijing, China). Two nuclear DNA fragments, internal transcribed spacer (ITS) and the second intron of LEAFY gene (LEAFY intron II), were amplified and sequenced following the work of Li et al. (2011). ITS and LEAFY intron II sequences of other possibly related species involved here were obtained from GenBank according to the works of Li et al. (2011) and Mo et al. (2017a). Species examined in this study, voucher information, collection locality and GenBank accession numbers for ITS and LEAFY sequences are given in Table 1. DNA sequences were aligned using Clustal X 2.1 (Larkin et al. 2007) and adjusted manually. The combined dataset including ITS and LEAFY intron II sequences for phylogenetic analysis was built according to the works of Li et al. (2011) and Mo et al. (2017a). Phylogenetic analyses were performed using the maximum parsimony (MP) and Bayesian inference (BI) methods. The MP analysis was performed using PAUP* 4.0b10 (Swofford 2003). The heuristic search was performed with 1000 random sequence addition replicates, tree-bisection-reconnection (TBR) swapping, MulTrees on, and all characters equally weighted. Bootstrap values of the internal nodes were obtained with 1000 bootstrap replicates. The BI analysis was performed using MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2003). Different DNA sequences were defined as separate data partitions. The evolutionary model for each partition (ITS: TVM+I+G; LEAFY intron II: HKY+G) was estimated using jModelTest v.2.1.10 (Darriba et al., 2012) with the Akaike information criterion (AIC) (Akaike 1974;Posada and Buckley 2004). The Markov chain Monte Carlo (MCMC) algorithm was run for 10 million generations with a sampling frequency of one every 1000 generations, and the first 25% trees were discarded as burn-in.

Results
The MP and BI analyses of the ITS + LEAFY intron II combined dataset generated congruent topologies. The Bayesian consensus tree with MP bootstrap (BS) and Bayesian posterior probability (PP) values is shown in Fig. 1. All Alseodaphnopsis individuals investigated in the present study formed a monophyletic clade that receives a BS of 86% and a PP of 1.00. Within the Alseodaphnopsis clade, two well-supported subclades are found, each consisting of four species. The new species Alseodaphnopsis maguanensis is sister to A. rugosa (BS 89%, PP 1.00) and new species A. putaoensis is sister to A. hainanensis (BS 88%, PP 1.00). Diagnosis. Alseodaphnopsis maguanensis is morphologically similar and phylogenetically closely related to A. rugosa, but can be distinguished by its much larger fruit (4-5 × 5-6 cm vs. ca. 2.5 × 3 cm), mature fruit color (brown vs. deep purple or black) and different fruiting phenology.
Phenology. Flowering from May to June and fruiting from July to September.
Etymology. The species is named after the type locality, Maguan County, in Yunnan Province, China.
Distribution and habitat. Currently known only from the type locality in Maguan, Yunnan Province, southwestern China. Tropical montane forests in valleys; ca. 800m.
Preliminary conservation status. Currently, A. maguanensis is only known from Maguan (Yunnan Province, China) with two populations, which are all located in a small natural reserve (ca. 71 km 2 ), each population with less than 50 mature individuals (seedlings can be found near the mature individuals), and no other occurrence in adjacent regions of SE Yunnan and N Vietnam. Thus, the preliminary conservation status for A. maguanensis is suggested as critically endangered (CR C2a (i) Diagnosis. Alseodaphnopsis putaoensis is morphologically similar to A. rugosa, but phylogenetically closely related to A. hainanensis. It can be distinguished from them by its fruit stalk characters (apex slightly dilated, not fleshy, red and warty when fresh vs. apex dilated, nearly cylindric, fleshy, red and warty when fresh), much larger fruit (6-6.5 × 7-10 cm vs. ca. 2.5 × 3 cm and 1.2-2 cm), mature fruit color (brown vs. deep purple or black) and different fruiting phenology.
Phenology. Individuals with immature or mature fruits have been collected in May, fruiting may be from April to June.
Etymology. The species is named after the type locality, Putao County, in Kachin State, Myanmar.

Distribution and habitat.
Currently known only from the type locality in Putao, Kachin State, northern Myanmar. Tropical montane forests on mountain slopes or in valleys; 600-1400m.
Preliminary conservation status. During the field survey in Putao (Kachin State, Myanmar), several populations of A. putaoensis were found and at least two of them with more than 50 mature individuals (seedlings could be found near the mature individuals) each. In future field surveys, potential populations and more individuals are expected to be found in Putao and adjacent regions. Currently, some localities of A. putaoensis have not been legally protected. The habitat fragmentation, as well as ongoing road construction and continuous logging, are threatening its survival. Thus, the preliminary conservation status for A. putaoensis is suggested as vulnerable (VU C12a(i)) according to the IUCN Red List Categories and Criteria (IUCN 2012).

Discussion
The close relationships of Alseodaphnopsis maguanensis, A. rugosa, A. putaoensis and A. hainanensis were indicated by the phylogenetic analyses. They formed a well-supported subclade within the Alseodaphnopsis clade, and A. maguanensis is sister to A. rugosa while A. putaoensis is sister to A. hainanensis (Fig. 1). Alseodaphnopsis maguanensis, A. putaoensis and A. rugosa are very similar in their vegetative characters (e.g. leaf and branchlet characters), but A. hainanensis can be easily distinguished from them by its narrowly elliptic and smaller leaves (vs. oblong-obovate or oblong-oblanceolate and larger leaves of A. maguanensis, A. putaoensis and A. rugosa) (Li et al. 2008). The fruit stalk and fruit characters are very important to distinguish Alseodaphnopsis maguanensis, A. putaoensis and A. rugosa. Alseodaphnopsis putaoensis has the largest fruit (6-6.5 × 7-10 cm, brown when mature) with the fruit stalk slightly dilated at the top (not fleshy and warty when fresh), while A. maguanensis and A. rugosa have relative smaller fruits (4-5 × 5-6 cm, brown when mature and ca. 2.5 × 3 cm, deep purple or black when mature) with the fruit stalks distinctly dilated at the top (nearly cylindric, fleshy and warty when fresh).
Alseodaphnopsis maguanensis, A. rugosa, A. putaoensis and A. hainanensis also have different fruiting phenologies. Alseodaphnopsis maguanensis is fruiting from July to September, both immature and mature fruits can be found in August. According to the work of Li et al. (2008), Alseodaphnopsis rugosa is fruiting from July to December. However, based on specimen and field observations, very few individuals with young fruits were collected in August, most fruiting individuals were collected from October to December. For Alseodaphnopsis putaoensis, individuals with immature or mature fruits have been collected in May, fruiting may be from April to June. Alseodaphnopsis hainanensis is fruiting from October to February (Li et al. 2008). A detailed comparison of the morphological differences among these four taxa, as well as their phenologies and distributions, is given in Table 2. Infructescence with only one well-developed fruit with one or several well-developed fruits with only one well-developed fruit with one or several well-developed fruits Fruit stalk apex dilated, 5-10 mm in diam., sometimes nearly cylindric, fleshy and warty when fresh apex dilated, nearly cylindric, 5-8 mm in diam., fleshy, red and warty when fresh apex slightly dilated, 5-6 mm in diam. apex dilated, nearly cylindric, 5-8 mm in diam., fleshy, red and warty when fresh Fruit oblate, 4-5 × 5-6 cm, brown when mature oblate, ca. 2.5 × 3 cm, deep purple or black when mature oblate, 6-6.5 × 7-10 cm, brown when mature globose or ovoid, 1.2-2 cm, deep purple or black when mature

Flowering Phenology
May-Jun Unfortunately, the flowers of Alseodaphnopsis putaoensis and A. rugosa remain unknown. The evidence from both phylogenetic and morphological analyses support the recognition of Alseodaphnopsis maguanensis and A. putaoensis as distinct species in the genus. With Alseodaphnopsis maguanensis, A. putaoensis and the recently described A. ximengensis H. W. Li & J. Li from Ximeng, Yunnan Province, China (Mo et al. 2017a) included, Alseodaphnopsis currently has only 11 recorded species, indicating that the species diversity of the genus is still in need of investigation and open to discovery. More new species of Alseodaphnopsis are expected to be discovered in the northern marginal zone of the Asian tropics when more field investigations are conducted in this region.