Utilising both morphological and molecular analyses, this study unveils Mazus jiangshiense sp. 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.

Lamiales, Mazus, molecular markers, morphology, nrITS, phylogenetics, rbcL, rps16, trnL-trnF

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), M. fruticosus Bo Li, D.G.Zhang & C.L.Xiang (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.

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 (RAxML-HPC2 on XSEDE v.8.2.12, 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).

We extend our sincere appreciation to Jun-Ping Zhang and De-Wang Nie, the esteemed managers of Shaowu Jiangshi Provincial Nature Reserve, for their invaluable assistance during the fieldwork. Special gratitude is extended to Ming-Zhu Liu for his meticulous preparation of images and to Cheng-Yuan Zhou for his expertise and contributions to the molecular analyses. Their dedicated support significantly enriched the quality and depth of this research endeavour.