Beilschmiedia turbinata: A Newly Recognized but Dying Species of Lauraceae from Tropical Asia Based on Morphological and Molecular Data

China took great efforts to reforestation, even turned the long-term forest loss into a net gain, but this cannot hide the loss of species diversity due to destruction of primary forests, habitat loss, invasion of alien species, and over exploitation. Here we provide such a case by recording a dying tree species of Lauraceae from the evergreen forests of SE Yunnan of China and adjoining Vietnam. We made field collections and observations for four consecutive years from 2009 to 2012. Phylogenetic analyses were conducted based on a combined dataset from nrITS and plastid trnL-trnF region, rpl16 intron, and psbA-trnH spacer. The results indicate that the Asiatic Beilschmiedia and Syndiclis are reciprocally monophyletic with Endiandra as a sister group, and both morphology and molecular phylogeny clearly suggest that the new species belongs to Beilschmiedia. Thus Beilschmiedia turbinata Bing Liu et Y. Yang is illustrated and described as new to science, color plates, line drawings, distribution map and comparison with related species are provided. This new species is similar to B. yunnanensis in the small and ferruginous-brown tomentose terminal buds, elliptic to oblong-lanceolate and alternate or subopposite leaves bearing the fine veinlet reticulation, but differs from the latter by the smaller flowers, the eglandular stamens of the third whorl, and the large turbinate furfuraceous fruits.


Introduction
Global biodiversity loss is a serious problem due to habitat loss, overexploitation, biological invasion, pollution, and global climate change according to the Millennium Ecosystem Assessment [1], and it is the case in China as well. Though China took great efforts in reforestation, and even turned the long-term forest loss into a net gain, the increased forest cover mainly consists of non-native tree crops but not regenerating natural forest [2]. The primary forest was continuously cut down and transformed into monoculture plantations of non-native economic trees [2][3][4]. After privatization of former collective forests since 2008, smallholders often cut natural forests for immediate income, then plant monoculture tree crops for long-term investment [2]. Because loss of native species diversity is masked under the increasing reforestation, it is more serious than what people thought [2,5], especially in areas with high species richness, e.g. Yunnan of China.
Southeast Yunnan of China, adjoining to Vietnam and Laos, preserves the northernmost tropical rainforests in Asia, and harbors extremely high diversity of plant species [6], with more than 7000 native species of flowering plants [7], and falls within one of the biodiversity hotspots in the world [8], but experienced a fast loss of species due to farming, logging, habitat loss, and vegetation destruction. Many species were thus eliminated from the earth before they were recognized and described. Here we describe a critically threatened tree species of Lauraceae from the evergreen forest of SE Yunnan of China based on field investigations in SE Yunnan and N Vietnam in the last four years and a detailed comparative study including morphology, anatomy, and molecular systematics analyses.
The Lauraceae are typical elements of SE Asia, and are also well represented in SE Yunnan of China [9][10]. The pantropical genus Beilschmiedia Nees is one of the largest genera within the family, containing ca. 250 species worldwide [11][12][13], and includes common trees in tropical Asia [9]. In addition to old publications of Liou [14] and Kostermans [15], a few regional revisions on this genus have recently been completed, e.g. America [16], Borneo [17], Madagascar [13], and China [18].
It is difficult to distinguish Beilschmiedia from its close relatives due to overlapping characters [19][20], e.g. Potameia (Madagascar), Endiandra (mostly in Australia, three species in China), Syndiclis (mostly in China), Sinopora (Hong Kong), and Yasunia (S America), which together belong to the Beilschmiedia group [20]. The genus Beilschmiedia has been recognized based on a combination of characters: cymose-paniculate inflorescences with the lateral flowers of the cymes not strictly opposite but somewhat alternate [21], trimerous flowers bearing usually nine but sometimes six fertile stamens, anthers 2-celled, and small tepals rarely persistent at the base of fruits. None of these characters, however, can distinguish this genus from its close relatives [13,20]. The minor venation pattern is a useful character to delimit Beilschmiedia species, and could be divided into two groups: fine type and coarse type [16]. Cuticular characters were sometimes used to determine generic ascription [19][20]22], and indeed, leaf cuticular morphology is a good marker to define Syndiclis [22]. We tried several times to extract cuticles of our new species, but failed, because the epidermis of this new species is strongly thickened, in which it differs from all other known Asiatic species of the Beilschmiedia group [22].
To determine the generic ascription of our new species, we sampled twelve species of Asiatic Beilschmiedia, two Endiandra, and four Syndiclis, which is the most extensive sampling of the Beilschmiedia group thus far, no DNA analysis has been applied in Lauraceae to determine the taxonomic ascription of newly described species thus far. Both nuclear ribosomal ITS and chloroplast DNA fragments (trnL-trnF, rpl16 and psbA-trnH) were sequenced. This phylogenetic study is helpful to determine the taxonomic position of the newly recorded species in addition to morphological evidence.

Ethics Statement
The field investigations were carried out in collective forests in Sumawan of China and Thanh Thuy of Vietnam, which are owned by the local village, but not protected area of state-owned land. The village head of Sumawan, Mr. Maorong Tian, gave us permissions to conduct the investigations and recommended two villagers as guides for us. Dào Thiêu assisted us collecting in Thanh Thuy of Vietnam.

Morphology and Anatomy
We surveyed three sites which cover the whole known distribution area of the new species. The voucher specimens, FAA-pickled materials, measurements, and photographs of vegetative and reproductive characters were obtained in the field. Anatomical observations of leaflet venation pattern and floral structure were taken under light microscope (Zeiss Axio Imager A1) in LSEB (State Key Laboratory of Systematic and Evolutionary Botany), IBCAS (Institute of Botany, the Chinese Academy of Sciences). Beilschmiedia yunnanensis Hu is close to the new species in morphology, and the two species were compared in detail.

Assessment of Conservation Status
We estimated the population size in the field, investigated the threat factors, and assessed the endangered category according to IUCN red list criterion [34]. The distribution map and calculation of EOO (extent of occurrence) were made by usingArgGIS ver. 9.3 [35].

Molecular Systematics
To determine the systematic position of our new species, thirtytwo samples belonging to nineteen species of four genera (Beilschmiedia, Endiandra, Syndiclis, and Cryptocarya) were included in this study. All but the new one were recorded in Flora of China [18]. Leaf materials for DNA extraction were dried with silica gel. Vouchers of samples (Tab. 1) were deposited in the National Herbarium (PE), IBCAS. Cryptocarya calcicola H. W. Li is selected as the outgroup of Beilschmiedia, Endiandra, and Syndiclis. Due to lacking DNA samples and sequences in the Gene Bank, Beilschmiedia species outside China were not integrated into our analyses.
Total DNA was extracted from silica-gel-dried leaves using TiangenH extraction kits. Polymerase chain reaction amplification was accomplished using Prime Taq DNA Polymerase with annealing temperatures of 50uC. The ITS region was amplified with primer pair ITS4, ITS5 [36] and C26A, 18S [37]. The chloroplast trnL-trnF region was amplified using primers designed by Taberlet et al. [38]. For some special species, the sequences of Beilschmiedia and related genera were obtained by our own designed primers, and the suitable annealing temperature is 50uC. The rpl16 intron and psbA-trnH spacer were amplified using primers of Sang et al. [39] and Kelchner et al. [40], respectively. All the primers used in this study are listed in Tab. 2.
Sequences were manually aligned using the Sequence Alignment Editor (Se-Al Carbon) v2.0 [41]. Bayesian inference (BI) and Maximum parsimony (MP) analyses based on the combined datasets of nuclear ribosomal ITS and chloroplast trnL-trnF, rpl16, psbA-trnH were carried out in MrBayes 3.1.2 [42] and PAUP 4.0 [43]. The poly-T and poly-A regions were removed from the sequences during analyses.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to IPNI, from where they will 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: PubMed Central, LOCKSS. 1.5 mm in length, 3 mm in diam.; pedicels 2-2.5 mm, sparsely tomentose; perianth lobes 6, ovate or broadly ovate, 1.261 mm, pubescent on both surfaces. Fertile stamens nine in three whorls, eglandular, subequal, ca. 1 mm long, filaments short but present and densely pubescent; anthers 2-celled, cells of the first and second whorls of stamens introrse, and those of the third whorl extrorse, the tip of anthers glabrous; staminodes of fourth series long-triangular and pubescent, ca. 0.6 mm long; pistils pyriform, pubescent, style short, ca. 0.3 mm. Each infrutescence usually bearing one single mature fruit; fruits maturing in two years. Young fruits of the first year ovoid, 2.5-361. 5 (Fig. 3).

Description of the New Species
Habit Conservation. There is only one population with ca. 30 adult trees across the boundary of China and Vietnam. These mature individual trees are dispersed in the evergreen broad-leaved forest of ca. 20 km 2 (EOO). The trees were cut down for construction leading to the population decline. Moreover, increasing human economic activities, e.g. the surrounding reclamation of farmlands, construction of border roads, and rubber plantations, are resulting in the fragmentation of the population, and threatening the existence of the new species and its living habitat. Our field observation found dozens of seedlings nearby the parent trees, but no young sapling exists. Alien species (e.g. Chromolaena odorata (L.) R. M. King et H. Rob. and Crassocephalum rubens (Jacq.) S. Moore) are also found nearby. Consequently, we here tentatively consider the new species to be ''Critically Endangered'' (CR Blab (v)+D), according to IUCN red list category and criterion [34].
Etymology. The specific epithet ''turbinate'' of this new species is from its turbinate shape of fruits.
Additional specimens examined.

Systematics
Partition homogeneity test between nrITS and chloroplast sequences was carried out using PAUP and the P value is 0.07. Therefore, we combined them into one dataset, representing 2618 characters, including 163 characters that were parsimony-uninformative and 135 characters that were parsimony-informative. Analyses of nrITS, trnL-trnF, rpl16, psbA-trnH and combined regions were carried out using MP (heuristic search) and the results are shown in Tab. 3, and the Bayesian consensus tree based on combined sequence data is shown in Fig. 4.
The result indicates that Syndiclis and Beilschmiedia are reciprocally monophyletic with the monophyletic Endiandra as outgroup, and Beilschmiedia turbinata clearly belongs to Beilschmiedia, though the position of the species is not well resolved within the genus (Fig. 4). In addition, this new species has typical Beilschmiedia morphology such as the trimerous flowers, nine fertile stamens in three whorls, fruits lacking persistent perianth lobes. This suggests that the new species is a Beilschmiedia but markedly differs from Syndiclis and Endiandra. Beilschmiedia turbinata shows great similarities to B. yunnanensis in morphology, but it is not conclusive whether B. turbinata and B. yunnanensis are basal to other Beilschmiedia species or not due to the low bootstrap value (,50%).

Morphological Comparison
Beilschmiedia turbinata possesses a unique set of morphological characters, e.g. the small type of terminal buds densely pubescent, elliptic to lanceolate elliptic leaves having fine veinlet reticulations, the trimerous flowers bearing nine eglandular fertile stamens in three whorls, and the large ferruginous-furfuraceous turbinate fruits. This new species is similar to B. yunnanensis in the small type and ferruginous-brown tomentose terminal buds, alternate or subopposite elliptic to oblong-lanceolate leaves, the elevated midrib, and the fine veinlet reticulation (Fig. 5), but differs from the latter by the smaller flowers (3 mm in diam. in the new species vs. up to 8 mm in B. yunnanensis), the eglandular stamens of the third whorl (vs. each of the three fertile stamens of the third whorl bearing two sagittate glands in B. yunnanensis), and the ovoid to turbinate, large, 5.4-7.263.8-4.6 cm, and densely ferruginousfurfuraceous (vs. ellipsoid to globose or subglobose, smaller, 2-461.5-2.7 cm in B. yunnanensis) (Fig. 5).
A few species from Southeast Asia also bear large fruits, e.g. Beilschmiedia macrocarpa A. Chev. ex H. Liou from S Vietnam, with the ovoid fruits up to 6.2 cm [14]; it differs from B. turbinata by its large terminal buds (vs. small terminal buds) and coarse veinlet reticulation (vs. fine veinlet reticulation). Beilschmiedia gigantocarpa Kosterm. from Sulawesi, B. glauciphylla Kosterm., B. gynotrochioides Kosterm. and B. kinabaluensis Kosterm. from Borneo, all bear fruits larger than 5 cm in diam. [17,44]. Beilschmiedia turbinata can be easily distinguished from them by the turbinate shape of fruits vs. spindle-shaped shape of fruits in B. glauciphylla and B. gynotrochioides, globose to subglobose shape of fruits in B. gigantocarpa and B. kinabaluensis.

Phylogenetic and Morphological Implications
Beilschmiedia is mixed with allied genera (e.g. Potameia and Endiandra) according to previous studies [24,29]. Sampling in these studies is poor, there are only six species of Beilschmiedia, two of Potameia, and one of Endiandra in Chanderbali et al. [24], seven species of Beilschmiedia, one of Potameia, and three of Endiandra in Rohwer & Rudolph [29], and these samplings are common in lacking Asiatic species. We sampled nineteen Asiatic species of the group (mostly from Yunnan) with the highest sampling density of the Beilschmiedia group so far. Our new phylogenetic analysis suggested that the new species belongs to the genus Beilschmiedia.
Morphology of the terminal buds is useful in classification of the genus Beilschmiedia [16][17][18][45][46]. Hooker [44] even divided Beilschmiedia into two sections mainly by the type of terminal buds: Sect. I, leaves opposite or alternate, terminal buds very small, pubesent or tomentose, not enclosed in coriaceous scales; Sect. II, leaves usually opposite, terminal buds enclosed in large glabrous coriaceous scales. According to the phylogenetic tree of this study, species bearing large type of terminal buds do constitute a wellsupported clade (Fig. 4), and are considered to be derived from the species of small type. Beilschmiedia yunnanensis and B. turbinata, occupying the basal position of this genus, both have the small type terminal bud. Worldwide sampling of the Beilschmiedia group is necessary to test and verify whether this character is useful for subgeneric classification or not.

Conservation Significance
Conservation status of B. turbinata warrants our attention. The new species is an endangered species and listed as CR, which is attributed to population decline, habitat loss, and alien species invasions because of increasing human activities in the area. Large trees of the species were cut down for construction. Expansion of farmlands and rubber plantations results in deforestation, and of course, habitat loss and fragmentation of population of the species. Road building causes fragmentation of population of the new species. Alien species invasions had potential impacts on both seed germination and growth of seedlings, and young seedlings might be difficult to grow up because of the lean soils and pressure of competition from both native and alien species. These factors are also major threats of regional biodiversity in Tropical Asia [5,9].
Yunnan Province is located in the SW China, harbors more than half the total species in China, and is the province with the highest floristic richness [47]. Within Yunnan Province, the  Table 3. Summary of parsimony analyses and data properties for nuclear ribosomal internal transcribed spacer (nrITS), chloroplast trnL-trnF, rpl16, psbA-trnH, and combined regions.  The local governments have made great efforts to develop cash crops in the tropical areas, especially in southern Yunnan and lowlands of southeast Yunnan. Plantations occupy a large area previously occupied by old-growth primary forests, leading to deforestation and environmental destruction much more serious than several decades ago. Wang [49] took part in the China-Russia joint expedition to southern Yunnan in 1950's, and revisited the same area in 1990's. According to his observation in the air and on-the-spot investigation, both appearance and composition of the forests in southern Yunnan had greatly changed within the 40 years. The canopy color of the forest was dark green with very thick forests in 1950's, but was changed into light green with many open places in 1990's. Many of the primary forests with abundant species were cut down and transformed into economic monoculture forests (e.g. rubber), and the local biodiversity in south Yunnan was precipitously reduced. Transformation of forest structure has negative effects not only on plant species, but also on insects [3]. The rubber plantations are less than 5 kilometers away from the forest where our new species was collected. Local villagers are unaware of protection, but still utilize the timbers by logging the large trees and substituting the forests with economic trees. The rapid economic development mode at the price of destruction of native vegetation was called into questions and even caused a lot of criticism [2,[49][50]. Vegetation shifts caused by human disturbance have led to changes of floristic components and severe species loss [4]. Beilschmiedia turbinata, probably once a more widespread species, but became a dying species in SE Yunnan, sounds the alarm again that conservation of the primary forests is urgent. Nyssa yunnanensis W.Q. Yin ex H.N. Qin & Phengklai of Nyssaceae also experienced a similar process [51]. As a result, conservation of primary vegetation and endangered species of this biodiversity hot spot is quite worrying, conservation action is necessary. This finding furthers that botanical investigations are urgently needed before the primary forests are cut down and replaced by economic crops, e.g. rubber, eucalyptus, and banana.