﻿Alseodaphnopsismaguanensis is conspecific with A.hokouensis (Lauraceae) based on morphological and molecular evidence

﻿Abstract Based on both morphological and molecular evidence, it is confirmed that Alseodaphnopsismaguanensis is conspecific with A.hokouensis. Hence, Alseodaphnopsismaguanensis is treated as a synonym of A.hokouensis here. The conservation status of Alseodaphnopsishokouensis is also re-evaluated according to the IUCN Red List Categories and Criteria in this study.


Introduction
As one of the largest basal angiosperm families, Lauraceae includes more than 50 genera and 2500 ~ 3000 species distributed in tropical and subtropical regions worldwide (Chanderbali et al. 2001).The delimitation, delineation and identification of Lauraceae species, especially for tropical species, are always complicated due to limited variation in floral and other morphological characters and insufficient specimen collection (van der Werff and Richter 1996;Rohwer 2000;Li and Li 2004;Yang and Liu 2015).
Alseodaphnopsis hokouensis (H.W. Li) H. W. Li & J. Li was first published as an Alseodaphne Nees species (Lee et al. 1979) and then it was transferred to Alseodaphnopsis H. W. Li & J. Li, a recently described genus of the Lauraceae (Mo et al. 2017).Since it was first collected in 1953, only three herbarium records (including two type specimens) of Alseodaphnopsis hokouensis can be found and no fruiting specimen has been collected.
During recent field surveys in SE Yunnan (SW China), some flowering and fruiting individuals of Alseodaphnopsis hokouensis were found and they resembled a recently-published Alseodaphnopsis species, A. maguanensis L. Li & J. Li (Li et al. 2020).Based on both morphological and molecular evidence, we confirm that Alseodaphnopsis maguanensis is conspecific with A. hokouensis.Therefore, we treat Alseodaphnopsis maguanensis as a synonym of A. hokouensis here.Based on the field survey data, we also re-evaluate the conservation status of Alseodaphnopsis hokouensis in this study.

Field surveys
We conducted field surveys in both Hekou (Hokou) County and Maguan County (Yunnan Province, China) from 2020 to 2022.Flowering specimens of Alseodaphnopsis hokouensis and A. maguanensis were collected from May to June and fruiting specimens were collected from July to September.

Morphological studies
Morphological characters of Alseodaphnopsis hokouensis and A. maguanensis were examined and compared in detail, based on fresh and preserved materials as well as dried specimens collected in the field.Herbarium specimens of these two species from the Herbaria HITBC, KUN and PE were also examined.

Molecular studies and phylogenetic analyses
Based on the work of Li et al. (2020), three individuals of Alseodaphnopsis hokouensis and two additional individuals of A. maguanensis were sampled in the present study.DNA extraction, fragment amplification and sequencing, as well as DNA sequence alignment, followed the work of Li et al. (2020).The newly-obtained DNA sequences were integrated into the ITS + LEAFY intron II combined dataset of the work by Li et al. (2020).Species examined in this study, voucher information, collection locality and GenBank accessions for ITS and LEAFY intron II sequences are given in Table 1.
The combined dataset including ITS and LEAFY intron II sequences was used for phylogenetic analysis according to the works of Li et al. (2011), Mo et al. (2017) and Li et al. (2020).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 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: GTR+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 parameters used in both MP and BI analysis followed the work of Li et al. (2020).

Results and discussion
During our field surveys in SE Yunnan, two populations of Alseodaphnopsis hokouensis were found in Huayudong and Qincaitang, Nanxi Town, Hekou County (Fig. 1), each with about 10 mature individuals.No individual was found at the type locality of Alseodaphnopsis hokouensis, Masike, Nanxi Town, Hekou County.Two populations of Alseodaphnopsis maguanensis, each with about 20 mature individuals, were also investigated in Gulinqing Provincial Natural Reserve (Maguan County) according to the work of Li et al. (2020).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. 2. As in the work of Li et al. (2020), all Alseodaphnopsis specimens sampled in the present study formed a well-defined clade (BS 88%, PP 1.00).Within the Alseodaphnopsis clade, there are two well-supported subclades which consist of four and five species, respectively.All individuals of Alseodaphnopsis hokouensis and A. maguanensis formed a strongly-supported clade (BS 100%, PP 1.00), closely related to A. rugosa.Within this clade, the individuals of these two species are mixed with each other and their relationships are poorly resolved.
The morphological characters of Alseodaphnopsis hokouensis, A. maguanensis and closely related A. rugosa were compared in detail, based on the data from type specimens and protologues (Li et al. 2008;Li et al. 2020).Morphological comparisons showed that Alseodaphnopsis maguanensis is almost the same as A. hokouensis, except that the leaves of the former are oblong-obovate or oblong-oblanceolate and those of the latter are elliptic to oblong (Table 2).After carefully checking the specimens collected from field surveys, we found that the leaves of both species can vary from elliptic, oblong to oblong-obovate or oblong-oblanceolate (Fig. 3).They are usually oblong-obovate or oblong-oblanceolate and occasionally elliptic to oblong.According to the newly-obtained fruiting specimens of Alseodaphnopsis hokouensis, the fruit characters of Alseodaphnopsis hokouensis are also consistent with those of A. maguanensis (Fig. 3).Additionally, the phenologies and habitats of these two species are quite similar and their distribution areas are adjacent as well (Fig. 1).To sum up, the results of both morphological comparison and phylogenetic analysis showed that Alseodaphnopsis maguanensis is conspecific with A. hokouensis.Therefore, we propose that Alseodaphnopsis maguanensis should be treated as a synonym of A. hokouensis.The insufficient specimen collection of Alseodaphnopsis hokouensis, especially the lack of fruiting specimens, hinders a comprehensive understanding of the species.The same situation is found in many other rare and endemic Lauraceae species in SE Yunnan, such as species of Alseodaphne, Beilschmiedia Nees, Caryodaphnopsis Airy Shaw, Cryptocarya R. Br., Endiandra R. Br., Machilus Rumph.ex Nees, Phoebe Nees and Syndiclis Hook.f., etc. (Li et al. 2008).Thus, more field surveys and specimen collections are suggested in SE Yunnan in order to improve the understanding of these rare and endemic Lauraceae species.Conservation status.Currently, Alseodaphnopsis hokouensis is known from Hekou Country and Maguan Country (Yunnan Province, China) with four populations (Fig. 1).Two populations found in Maguan Country are all located in Gulinqing Provincial Nature Reserve, each with about 20 mature individuals

Figure 3 .
Figure 3. Alseodaphnopsis hokouensis A flowering branchlet B fruiting branchlet C different shapes of leaf blades.Photographed by Lang Li.

Figure 4 .
Figure 4. Different habitats of Alseodaphnopsis hokouensis A in the forest of the Nature Reserve B on the roadside C in the strongly disturbed forest near the village.

Table 1 .
Species examined in this study, voucher information, collection localities and GenBank accession numbers for ITS and LEAFY sequences.

Alseodaphnopsis hokouensis (H. W. Li) H. W. Li & J. Li
Flowering from May to June and fruiting from July to September.Distribution and habitat.Hekou County and Maguan County, Yunnan Province, China.Tropical limestone forests in valleys, usually near streams, at an elevation of 150-850 m.