﻿Mazusmotuoensis (Mazaceae), a new species from Xizang, China

﻿Abstract Mazusmotuoensis W.B.Ju, Bo Xu bis & X.F.Gao is a newly described species found in Xizang Autonomous Region, China. Morphologically, this species differs from all the other known Mazus species by having erect perennial herb form with a rhizome, presence of multicellular hairs, without basal leaves, opposite arrangement of stem leaves, and corolla lobes with erose-toothed margins. Molecular phylogenetic analysis using nuclear and cpDNA genes suggests that this new species occupies a basal position within Mazus. In conclusion, both morphological evidence and molecular phylogenetic analyses support that this species belongs to Mazus and represents an as-yet-unreported new species with distinct differences from other species within the genus.

In 2022, a field survey was conducted in Motuo County, located within the Xizang Autonomous Region of southwest China, the authors discovered an unknown species of Mazaceae in an evergreen broad-leaved forest.Through careful comparison with specimens, related literature, and phylogenetic analysis of Mazaceae, it was concluded that this species represents a new addition to the Mazus.

Morphological analysis
One population of this new species was rediscovered in Mar 2022 in Xizang Autonomous Region, China.Morphological observations of the new species were conducted using living plants collected from the type locality, as well as type specimens deposited at CDBI.Detailed photographs of morphological features, such as rhizomes, multicellular hairs, stems, leaves, inflorescences, and flowers, were taken using a digital camera and stereoscope.Measurements were carried out on both wild plants and pressed specimens using a ruler and a metric vernier caliper.Digital herbarium images of Mazus specimens were sourced from diverse outlets, including JSTOR Global Plants (http://plants.jstor.org/), the Global Biodiversity Information Facility (https://www.gbif.org/zh/), the Chinese Virtual Herbarium (https://www.cvh.ac.cn/), and Europeana (https://www.europeana.eu/en/search).A thorough examination and comparison of these images with the new species ensued.Subsequently, the morphological attributes of the species were meticulously described in accordance with the guidelines provided by the Flora of China (Hong et al. 1998).

Assessment of conservation status
In the field, we conducted an estimation of the population size of the new species and evaluated the factors posing threats to its existence.In order to determine the conservation status of the new species, we applied the established criteria as outlined by the International Union for Conservation of Nature (IUCN 2019) Red List.

DNA sequencing and outgroup selection
We extracted total DNA from silica gel-dried leaves of the new species using a modified CTAB protocol (Doyle and Doyle 1987).To determine the phylogenetic position of the new species within the Mazus genus, we employed two datasets for our analysis.The first dataset consisted of a combined matrix of two cpDNA regions (rbcL, trnL-trnF), while the second dataset was nrITS.The DNA sequences were amplified and sequenced following the methods described by Deng et al. (2019), using the primers specified in their study.Based on previous phylogenies (Deng et al. 2019;Xiang et al. 2021) 28 accession of the relatives of M. motuoensis were selected as ingroups.Additionally, we chose five species from three different genera as outgroups.The related sequences were obtained from NCBI (https://www.ncbi.nlm.nih.gov/).The GenBank accession numbers for the new species are OQ383430 (trnL-F), OQ383431 (rbcL) and OP720888 (ITS).A comprehensive list of all species included in the phylogenetic analysis, along with their respective accession numbers, can be found in Table 1.

Phylogenetic analysis
The sequence chromatograms were visually inspected on Sequencher 5.2.4 (Gene Codes Corporation) and integrated into a single sequence.All sequences were then aligned with MUSCLE in MEGA 7.0.14(Kumar et al. 2016) and manually adjusted.Phylogenetic analyses were performed based on the combined cpDNA dataset (rbcL and trnL-trnF) and the nrITS dataset using both the maximum likelihood (ML) and Bayesian inference (BI), respectively.We did not combine the cpDNA and nrITS datasets for analysis because of the different sampling of taxa in the datasets.Settings of parameters during analysis follow those presented in Deng et al. (2019) and Xiang et al. (2021).

Morphological analysis
Morphologically, the new species has intermediate characteristics of Mazus and Puchiumazus.The new species has characteristics such as rhizomes, erect stems, and stem leaves opposite similar to Puchiumazus.However, the plant of this new species is covered with multicellular hair and has stems that are not quadrangular and leaf blade elliptic-ovate, which distinguishes it from the only known species, Puchumazus lancefolius (Hemsley) Bo Li, D.G.Zhang, and C.L.Xiang (Xiang et al. 2021).There are also perennial herb species with erect stems and opposite cauline leaves in the genus of Mazus, such as M. caducifer Hance (1882).But the new species has a series of ray characteristics not commonly seen in Mazus, including single erect unbranched stems without basal leaves, stem leaves many and opposite, petioles nearly absent, lobes margin erose-toothed.In conclusion, based on the morphological key provided by Hong et al. (1998), the new species is classified morphologically within the Mazus and represents an anomalous existence.

Phylogenetic analysis
The phylogenetic tree was generated using a combined cpDNA matrix, consisting of 34 aligned sequences and comprising 2197 characters (rbcL: 1318 bp; trnL-trnF: 879 bp).Additionally, the nrITS matrix included 28 aligned sequences and comprised 703 characters.Due to differences in taxon sampling between the cpDNA and nrITS datasets, they were not combined for analysis.Both maximum likelihood (ML) and Bayesian inference (BI) methods yielded congruent topologies.Therefore, only the results of the ML trees are presented (Figs 1, 2: MLBS: 100%, BIPP: 1.00; all support values follow this order hereafter).

Taxonomic treatment
Distribution and habitat.Mazus motuoensis is currently known from Nibi Valley, Motuo County, Xizang, China.It can be found under evergreen broadleaved forest at altitudes of 2253 m.
Phenology.Flowering was observed from May to June.

Figure 1 .
Figure 1.Phylogenetic relationships of M. motuoensis and related species inferred from ML and BI analyses based on the nrITS dataset.Numbers on the branches indicate the bootstrap support of the ML and the posterior probability of BI analyses.

Figure 2 .
Figure 2. Phylogenetic relationships of M. motuoensis and related species inferred from ML and BI analyses based on the combined dataset of rbcL and trnL-trnF.Numbers on the branches indicate the bootstrap support of the ML and the posterior probability of BI analyses.

Figure 3 .
Figure 3. Living images of M. motuoensis A habit B inflorescence in frontal view C inflorescence in rear view D rhizome.

Figure 4 .
Figure 4. Morphology of M. motuoensis A plant B leaves C old stems D multicellular hairs attached to stem E flower F unfolded corolla, showing limb upper lip and lower lip G bract H, I calyx J anthers K ovary and style.

Figure 5 .
Figure 5. Line drawings of M. motuoensis A whole plant B multicellular hairs attached to stem C leaves D inflorescence E flower F unfolded corolla G calyx H ovary and style.Drawn by Mr. Zhen-long Liang.