﻿ Mazusjiangshiense (Mazaceae), a new species from China: evidence from morphological and molecular analyses

﻿Abstract Utilising both morphological and molecular analyses, this study unveils Mazusjiangshiensesp. nov., a novel addition to the Mazaceae family, discovered in Shaowu County, Fujian Province, China. The comprehensive description and illustrations provided here are a result of a meticulous exploration of its morphological features. While bearing a resemblance to M.gracilis, this new-found species is distinguished by three distinct characteristics: its stems are delicately soft, its leaves possess a membranous quality and the ovary is notably villous at the apex. Integration of molecular evidence, derived from the nuclear ribosomal DNA (nrITS) and three plastid DNA sequences (rps16, rbcL and trnL-trnF), unequivocally supports the classification of M.jiangshiense as a distinct species. Notably, the molecular analysis positions it as a sister species to M.spicatus, underscoring the phylogenetic relationships within the genus Mazus. Our research not only introduces M.jiangshiense as a novel taxonomic entity, but also provides a nuanced understanding of its morphological differences and molecular affinities, enriching our comprehension of the diversity and evolutionary relationships of Mazaceae.


Introduction
Mazaceae Reveal (2011) stands as a recently established family within Lamiales, distinctively separated from Phrymaceae according to several phylogenetic studies (Albach et al. 2005;Xia et al. 2009;Schäferhoff et al. 2010).Comprising four genera and approximately 43 species (Xiang et al. 2021), Mazus Loureiro (1790) takes precedence as the largest genus, encompassing 39 accepted species primarily distributed in Asia and Australasia (Hong et al. 1998;Fischer 2004;Deng et al. 2016).In China, the Flora of China (Hong et al. 1998) originally recorded 25 species and three varieties, but subsequent reports introduced new species, such as M. tainanensis T.H.Hsieh (Hsieh 2000), M. sunhangii D.G.Zhang & T.Deng (Deng et al. 2016), M. somggangensis S.S. Ying (Ying 2019) (Xiang et al. 2021) and M. danxiacola Bo Li & B. Chen (Li et al. 2022).These additions suggest that China likely serves as the epicentre of distribution and diversity within Mazus (Yang 1979;Hsieh 2000).While investigating the background resources of protected areas in Fujian Province, south-eastern China, a distinctive Mazus species with a creeping stem and villous ovary was discovered.Through a meticulous analysis of flowering specimens and molecular data, it was unequivocally identified as a new species.Consequently, we hereby introduce and describe this novel entity as Mazus jiangshiense sp.nov.

Materials and methods
In June and August of 2022, a comprehensive exploration of the putative new species was conducted within Jiangshi Provincial Nature Reserve, Shaowu City.Digital photographs of fresh plants in the field were taken by a Canon digital camera (6D) and flowers and fruits were collected and deposited in the Herbarium of the College of Forestry, Fujian Agriculture and Forestry University (FJFC).A Canon digital camera (6D) was used to document the plants in the laboratory, while micro-morphological features were examined under a stereomicroscope (SZM-41, LAOSVI).
To ascertain the phylogenetic position of this newly-discovered species, a curated set of 21 taxa within the Mazus, as per Li et al. ( 2022), was employed.Additionally, three species-Dodartia orientalis Linnaeus (Linnaeus 1753), Lancea tibetica Hook.f.& Thomson (Hooker and Thomson 1857) and Puchiumazus lanceifolius (Hemsl.)Bo Li, D.G.Zhang & C.L.Xiang (Xiang et al 2021)-were chosen as outgroups.Detailed voucher information and GenBank accession numbers for all specimens are outlined in Table 1.The phylogenetic analyses were conducted employing three plastid DNA sequences (rbcL, rps16 and trnL-trnF) and nuclear ribosomal ITS (nrITS).A total of 24 DNA sequences of rps16, 23 of rbcL, 23 of trnL-trnF and 20 of ITS were successfully obtained.By using the CTAB procedure of Doyle (1987), total genomic DNA was obtained from silica-dried leaves and the DNA was re-suspended in double-distilled water and kept at -40 °C.In a thermocycler (Eppendorf Scientific, Inc., Westbury, NY, USA), DNA amplifications were performed.For three plastid DNA sequences (rbcL, rps16 and trnL-trnF) and nuclear ribosomal ITS (nrITS), the fragments were respectively amplified with primers RH1/Z1352RC (Olmstead et al. 2001), rps16-2F/rps16-R3 (Bremer et al. 2002), c/f of Taberlet et al. (1991) and ITS1 and ITS4 (Wendel et al. 1995).There were 50 μl of final volumes used for PCR reactions, along with 1 μl of each specific primer (10 μM each), 5 μl 10× PCR buffer, 35-50 ng template, 1 μl 10 mM dNTP (2.5 mM each) and 0.2 μl MDBio Taq DNA Polymerase (MDBio, Taipei, Taiwan).After purifying PCR products with the Tian quick Midi Purification Kit according to the manufacturer's instructions, they were sequenced directly.Sequences newly obtained have been deposited in GenBank.The evaluation of phylogenetic relationships utilised Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) techniques, executed on the CIPRES Science Gateway web server PAUP on XSEDE v.4.a165 and MrBayes on XSEDE v.3.2.7a) as described by Miller et al. (2010), with parameters aligned as outlined by Chen et al. (2019).(Forbes and Hemsley 1890), but it differs in the stem texture and whether the ovary is villous or not.It is also close to M. spicatus Vaniot (Vaniot 1905), but it can be easily distinguished by the growth habit of the stem and texture.Description.Perennials, densely villous.Stems creeping to 50 cm, soft, slender, branched, base woody, inter-node nearly 5 cm, often longer than or equal to leaves, nodal rooting.Basal leaves are several to numerous, often deciduous.Cauline leaves opposite or alternate, leaf blade subrounded or oblong, membranous, 2.5-5.5 cm long including petiole, 2.1-4.1 cm width, larger at base of the stem, adaxially green, abaxially greyish-green to silver grey, apex acute to obtuse, base truncate and tapering, margin coarsely crenate, both sides villous; lateral veins 3-4 pairs, fluted adaxially, elevated abaxially and conspicuous on both surfaces; petiole 0.5-2.5 cm, slender, villous.Racemes axillary or terminal, obliquely ascending, to 17.0 cm or more, villous, sparse, multiflowered to 20; pedicels slender, 0.6 cm in fruit, densely villous; Calyx funnelform, 3.0-4.0mm, slightly enlarged in fruit, villous on both surfaces, lobes 5, lanceolate, acute and longer than the tube.Corolla light purple or white, 0.9-1.1 cm long, dotted yellow on the palate and with sparse glandular-hair, tube cylindrical with glandular-hair, 0.4-0.5 cm long; limb 2-lipped, upper lip bilobed, lobes lanceolate, apex acute, slightly upturned; lower lip trilobed, middle lobe oblong, smaller and narrower than lateral lobes, lateral lobes broadly ovate, spreading away from middle lobes; plaits with sparse glandular hairs; palate comprising 2 longitudinal elevations, mostly hidden in the corolla tube, covered by sparse glandular hairs, clavate hairs, white to transparent, 0.6 mm long.Stamens 4, filaments protruding from the tube, appressed to the corolla tube, glabrous, included, anterior pair longer, curved, anthers positioned adjacent to the corolla tube on upper lip; Ovary villous, styles ca.0.5 cm long, stigma lamellate, included.Capsule oblong, ca.2.5 mm long, ca.1.5 mm diam., apex rounded and villous.Seeds brown-yellow, numerous.
Phenology.The flowering period is from June to July and the fruiting period is from August to September.
Etymology.The new species was named after the locality of Jiangshi Provincial Nature Reserve, where it was discovered.
Distribution and habitat.The species is distributed in Xiao Jiafang Town of Shaowu County, northwest Fujian Province, China and grows under a stone cave in a cliff at an elevation of approximately 395 m (Fig. 2A).
Phylogenetic analysis.The nucleotide sequence lengths for the new species nrITS, plastid rps16, rbcL and trnL-trnF are 550 bp, 806 bp, 1261 bp and 930 bp, respectively.Table 3 provides a summary of the characteristics of each dataset utilised in this study.The phylogenetic analyses are presented in the form of Maximum Likelihood (ML) trees and the support values, including bootstrap percentages (BS ML /BS MP ) and posterior probabilities (PP), are indicated near the respective nodes.Upon analysis of the combined dataset, the phylogenetic trees consistently demonstrated that the new species is closely related to M. spicatus, garnering robust support (BP ML = 78, PP = 0.99, BP MP = 68).The tree derived from the nrITS dataset (Fig. 1A) corroborates this relationship, with strong support for the new species being the sister to M. spicatus (BP ML = 82, PP = 0.99, BP MP = 75).However, the plastid-based phylogenetic tree introduces an intriguing twist, indicating that the new Mazus species is proximate to M. caducifer Hance (Hance 1882), M. spicatus and M. humilis Hand-Mazz (Hong et al. 1998).This forms a collapsed topology (Fig. 1B), suggesting a potentially intricate evolutionary relationship amongst these species.

Figure 1 .
Figure 1.The phylogenetic tree was constructed by the plastid, ITS and combined matrix using Maximum Likelihood, Maximum Parsimony and Bayesian Inference methods.The numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BP ML , PP, BP MP ).Separate combined results of nrITS (A) and plastid (B) are shown in the top left corner.A dash (-) indicates that the node is inconsistent between the Bayesian tree and the topology of the MP/ ML trees.The asterisk (*) node is 1.00 posterior probability or 100 bootstrap percentage.

Figure 2 .
Figure 2. Morphology of Mazus jiangshiense A, B habitat C flowering plant D roots and stolons E leaves F inflorescence G a mature inflorescence with flowers and fruits H corolla I pedicel, calyx, stamens and pistils J fruit K seed.

Figure 3 .
Figure 3. Mazus jiangshiense Y.B.Chen, Xin Y. Chen & Liang Ma A plant B leaves C-E corolla F stamens G pistils H calyx I seed.

Table 1 .
Taxon, vouchers and GenBank accession numbers of the specimens.The asterisk (*) indicates the sequences obtained in this study; the others are from GenBank.A dash (-) indicates missing data.

Table 2 .
Comparisons amongst M. jiangshiense and morphologically similar species.A dash (-) indicates missing data.

Table 3 .
Statistics from the three DNA datasets analysed.