Research Article
Research Article
Rediscovery of Mazus lanceifolius reveals a new genus and a new species in Mazaceae
expand article infoChun-Lei Xiang, Hong-Li Pan§, Dao-Zhang Min|, Dai-Gui Zhang, Fei Zhao, Bing Liu#¤, Bo Li|
‡ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
§ Sichuan Academy of Forestry, Chengdu, China
| Jiangxi Agricultural University, Nanchang, China
¶ Jishou University, Jishou, China
# Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan, China
¤ Institute of Botany, Chinese Academy of Sciences, Beijing, China
Open Access


Mazus lanceifolius (Mazaceae) is a perennial herb with opposite leaves and endemic to central China that has not been collected for 130 years. Rediscovery of this enigmatic species in the wild allows for determination of its phylogenetic position within Mazaceae. Phylogenetic reconstruction of Mazaceae based on DNA sequences from four plastid markers (matK, rbcL, rps16 and trnL-trnF) and nuclear ribosome ITS consistently showed that Mazus was not monophyletic. Mazus lanceifolius is in the most basal clade within Mazaceae, as sister to the remaining species of three recognized genera Dodartia, Lancea and Mazus. These results support the separation of M. lanceifolius from Mazus as a new genus, which was established here as Puchiumazus Bo Li, D.G. Zhang & C.L. Xiang. Meanwhile, a collection from Shennongjia Forestry District of Hubei Province, China, misidentified as “M. lanceifolius” in previous molecular study, is here revealed to represent an undescribed species of Mazus, i.e., M. fruticosus Bo Li, D.G. Zhang & C.L. Xiang, sp. nov. Morphologically, Puchiumazus is clearly distinct from the other three genera by having quadrangular to somewhat ribbed stems, and obviously opposite leaves. In addition, we provide a taxonomic key to the four genera of Mazaceae.


Dodartia, Lamiales, Lancea, new genus, Puchiumazus


Mazaceae (Reveal 2011) is a small herbaceous family in Lamiales currently containing three genera: Dodartia L., Lancea Hook.f. & Thomson and Mazus Lour. (APG IV 2016; Olmstead 2016; Christenhusz et al. 2017). The monotypic genus Dodartia based on D. orientalis L., occurs mainly in southern Russia and western to central Asia (Fischer 2004) and is characterized by having scale-like leaves and much-branched stems. The genus Lancea is found only in the Qinghai-Tibetan Plateau (QTP) where it includes two species, L. tibetica Hook.f. & Thomson and L. hirsuta Bonati (Chi et al. 2018, 2019), of which the former species is widely used in traditional Tibetan medicine. Morphologically, Lancea is characterized by leaves in a rosette and a lower corolla lip with a distinct palate. Mazus is the largest genus in Mazaceae, including approximately 30 species of annual or perennial herbs (Hong et al. 1998; Deng et al. 2016) distributed in Asia, Australia and New Zealand (Li 1954; Barker 1991; Fischer 2004). China is considered to be the center of distribution and differentiation of the genus (Yang 1979; Hsieh 2000), with ca. 26 species and three varieties currently recorded (Hong et al. 1998; Deng et al. 2016). Species delimitation in Mazus has been problematic because of relatively high levels of morphological variation (Li 1954; Hong et al. 1998). In general, Mazus can be distinguished from the other two genera by a combination of morphological characters: a strongly two-lipped corolla (3/2-bilabiatae), a palate with two longitudinal plaits and a capsule enveloped in a persistent calyx (Fischer 2004; Deng et al. 2019).

Dodartia, Lancea and Mazus were once placed in the traditionally circumscribed Scrophulariaceae (e.g. Von Wettstein 1891) but variably affiliated with tribe Gratioleae (Von Wettstein 1891; Thieret 1954, 1967) or Mimuleae (Dumortier 1829; Burtt 1965; Argue 1984; Fischer 2004). However, Scrophulariaceae were found to be polyphyletic and some genera were subsequently transferred to existing families like Orobanchaceae, Plantaginaceae, Phrymaceae and Stilbaceae, and some genera were separated as small monophyletic families, including Calceolariaceae, Linderniaceae, Mazaceae, Paulowniaceae, Schlegeliaceae, and Wightiaceae (Olmstead and Reeves 1995; Oxelman et al. 1999, 2005; Olmstead et al. 2001; Beardsley and Olmstead 2002; Albach et al. 2005; Rahmanzadeh et al. 2005; Tank et al. 2006; Schäferhoff et al. 2010; Liu et al. 2020), then leaving a much reduced Scrophulariaceae s.s. To date, a number of genera have not yet been sequenced and are still unplaced.

When redefining Phrymaceae based on molecular phylogenetics, Beardsley and Olmstead (2002) had first shown that Mazus and Lancea formed a well-supported group that was weakly supported as sister to the rest of Phrymaceae. Consequently, they tentatively included the two genera in the redefined Phrymaceae and assigned them to a provisional subfamily “Mazoideae” (Beardsley and Olmstead 2002). However, subsequent studies did not recover the sister relationship between “Mazoideae” and the rest of Phrymaceae, and Mazus and Lancea were found to be sister to the Orobanchaceae+Paulowniaceae+Phrymaceae clade (Oxelman et al. 2005; Albach et al. 2009; Schäferhoff et al. 2010). Thus, a new family Mazaceae Reveal (2011) was established to accommodate this. When Dodartia was first included in a molecular analysis, Xia et al. (2012) found that this genus was closely related to Lancea and they together formed the sister clade of Mazus. Currently, Mazaceae Reveal (2011) with the inclusion of all these three genera has been widely accepted (Refulio-Rodriguez and Olmstead 2014; APG IV 2016; Olmstead 2016; Christenhusz et al. 2017). It was found to be a member of the clade comprising Lamiaceae, Mazaceae, Wightiaceae, Phrymaceae, Paulowniaceae and Orobanchaceae (Liu et al. 2020).

Within the genus Mazus, M. lanceifolius Hemsl. is a distinctive species through its quadrangular stems and narrowly lanceolate, mostly cauline, opposite leaves (Fig. 1). By contrast, the other species of Mazus have terete stems and leaves often in basal rosettes (Yang 1979; Hong et al. 1998). Therefore, M. lanceifolius was assigned to a monotypic section: sect. Lanceifoliae Bonati (1908), which was followed by Yang (1979). Since its description by Forbes and Hemsley (1890), M. lanceifolius has never been recorded by any specimens until two populations of the rare species were rediscovered in Sichuan Province of China in 2020. The rediscovery of M. lanceifolius after more than one century offers us a precious opportunity to test its phylogenetic position based on morphological and molecular data.

Figure 1. 

Puchiumazus lanceifolius (≡ Mazus lanceifolius) A lectotype deposited at K (A. Henry 5837, barcode K001079356) B habit C stem, showing the obtuse ribs D leaves E inflorescence F flower in lateral review G young fruits. Scale bars: 5 cm (B); 0.5 cm (C, F, G); 2 cm (D); 1 cm (E).

Since the establishment of the family Mazaceae (Reveal 2011), only one molecular phylogenetic study exclusively focused on its phylogeny (Deng et al. 2019), including one species from each Lancea and Dodartia, and 23 out of 30 species of Mazus. In that study, Deng et al. (2019) notably included two samples named as “Mazus lanceifolius”, and stated that “M. lanceifolius” can be easily distinguished from other Mazus species by having lanceolate leaves and a robust stem. After consulting the vouchers of “Mazus lanceifolius” (D.G. Zhang zdg6673, Fig. 2) sampled by Deng et al. (2019) as well as the type specimens (Henry 7250, K001079356!; Henry 5837, K001079356!) and the original description of M. lanceifolius, we found that the plants of “Mazus lanceifolius” used by Deng et al. (2019) have opposite to subopposite leaves, which may have led the authors to identify the plant as M. lanceifolius because this species is the only known Mazus species with opposite leaves. However, except for these opposite leaves, their “Mazus lanceifolius” is remarkably different from the type specimen of M. lanceifolius in many aspects. For example, the plants sampled by Deng et al. (2019) are robust shrubs having numerous and much branched stems, leathery leaves that are acutely serrate on the apical half and multiflowered inflorescences (Fig. 2; see also fig. 2C in Deng et al. 2019), while the type material of M. lanceifolius is a slender herb having several unbranched stems, submembranaceous and almost entire leaves and remarkably sparse inflorescences with no more than six flowers (Fig. 1). We therefore have to conclude that the specimen sampled as “M. lanceifolius” by Deng et al. (2019) was misidentified, with the identity of that sample needing to be confirmed.

Figure 2. 

Mazus fruticosus A voucher of “Mazus lanceifolius” sampled in Deng et al. (2019), deposited at JIU (the herbarium of Jishou University, Hu’nan, China) B habit and habitat C leaves D flower in frontal view, showing morphology of its lower lips E flower in frontal view, showing morphology of its upper lips F flowers in lateral view. Scale bars: 2 cm (C); 0.5 cm (D, E, F).

In the present study, we carried out an updated phylogeny of Mazaceae, in order to (1) investigate the phylogenetic placement of the distinct and enigmatic species M. lanceifolius based on its rediscovered populations; (2) confirm the identity of the misidentified M. lanceifolius by Deng et al. (2019); and (3) further contribute to a comprehensive phylogenetic framework for Mazaceae.

Material and methods

Field work, taxon sampling and data collection

Two populations of Mazus lanceifolius were rediscovered in June 2020 in Sichuan Province, China. One is located in the Qingchengshan Mountain near Dujiangyan City, and another was found in Qianfoshan Mountain near Mianyang City. Morphological observations were conducted based on wild individuals as well as the type specimens. Fresh leaves were collected in the field and dried with silica-gel for DNA extraction (Chase and Hills 1991). Voucher specimens are deposited in the herbarium of Shanghai Chenshan Botanical Garden (CSH).

In the present study, most DNA sequences are based on previous phylogenetic analyses (Deng et al. 2019), but some problematic sequences were excluded for analyses. For example, the trnL-trnF sequences of Mazus japonicus (Thunb.) Kuntze 3 (KX807207) in the study of (Deng et al. 2019) were actually under the name of M. pumilus (Burm. f.) Steenis in GenBank. Similarly, trnL-trnF sequences of two different species (i.e. Mazus sp., MK266435 and Mazus japonicus var. delavayi (Bonati) P.C. Tsoong, KX783521) are completely identical. Such kinds of sequences were excluded for analyses. In addition, two individuals of Dodartia orientalis and three individuals of Lancea tibetica were included for analyses. Thus, all genera (Mazus, Lancea and Dodartia) of the newly established family Mazaceae (Reveal 2011) were represented. Voucher information and GenBank accession numbers for taxa used in this study are provided in Appendix 1.

Based on previous studies (Schäferhoff et al. 2010; Refulio-Rodriguez and Olmstead 2014; Luna et al. 2019; Xia et al. 2019; Liu et al. 2020), 14 taxa representing 12 genera in five families (Pedicularis L., Rehmannia Libosch. ex Fisch. & C.A. Mey. and Striga Lour. [Orobanchaceae], Paulownia Siebold & Zucc. [Paulowniaceae], Erythranthe Spach, Mimulus L. and Phryma L. [Phrymaceae], Wightia Wall. [Wightiaceae], Callicarpa L., Lamium L., Premna L. and Vitex L. [Lamiaceae]) were selected as outgroups for the cpDNA dataset. While, because of the high divergence of nrITS sequences, only eight species from the above-mentioned families were selected as outgroups.

DNA extraction, amplification and sequencing

Total genomic DNA was obtained from silica-dried leaves using the CTAB procedure of Doyle and Doyle (1987). After extraction, the DNA was re-suspended in double-distilled water and kept at -40 °C for polymerase chain reaction (PCR) amplifications.

The DNA amplifications were performed in a thermocycler (Eppendorf Scientific, Inc., Westbury, NY, USA). Based on Deng et al. (2019), four cpDNA regions (matK, rbcL, rps16 and trnL-trnF) and nrITS were selected for phylogenetic reconstruction. Primers, protocols for PCR, sequencing followed those in Deng et al. (2019) and references therein.

Phylogenetic analysis

Sequences were initially assembled and edited with Geneious v.7.1.7 (Kearse et al. 2012) and aligned using MUSCLE (Edgar 2004) as implemented in Geneious v.7.1.7 (Kearse et al. 2012). The final alignments were manually adjusted in PhyDe v.0.9971 (Müller et al. 2010). The four chloroplast DNA regions were combined directly because the plastid genome is mostly uniparentally inherited (Soltis and Soltis 1998) and supposedly safe to be combined in phylogenetic analyses (Olmstead and Sweere 1994). Nuclear (ITS) and the combined plastid data set were analyzed separately using maximum likelihood (ML) and Bayesian inference (BI) methods.

ML analyses were performed using RAxML-HPC2 v.8.2.9 (Stamatakis 2014) as implemented on the CIPRES Science Gateway ( (Miller et al. 2010) under the GTRGAMMA model. The partitioned model (-q) was used for the concatenated plastid data, bootstrap iterations (-# | -N) set to 1000, and other parameters followed default settings.

BI analyses using Markov chain Monte Carlo (MCMC) methods (Yang and Rannala 1997) were performed with MrBayes v3.2.2 (Ronquist et al. 2012) and implemented on the CIPRES Science Gateway ( (Miller et al. 2010). The optimal substitution models were selected using Model Finder (Kalyaanamoorthy et al. 2017) plugin in PhyloSuite (Zhang et al. 2018). Model parameters were estimated directly during the runs. For each Bayesian analysis, four MCMC chains were run simultaneously for 20 million generations. Each run began with one random tree and sampled one tree every 1000 generations. At the end of the run, chain convergence and estimated sample size (ESS) parameters were assessed with Tracer v.1.6.0 (Rambaut et al. 2014). A 50% majority-rule consensus tree was calculated for each dataset after discarding the first 25% of the trees as burn-in. In the resulting summary tree, posterior probability values (PP) ≥ 0.95 were considered to be strongly supported (Suzuki et al. 2002).


Sequence and alignment characterization

Ten sequences were newly generated for this study (Appendix 1). The resulting combined and aligned cpDNA dataset contained 4514 positions (including gaps), of which 1287 positions belong to matK, 1266 to rbcL, 963 to the rps16 partition and 998 to the trnL-trnF spacer. Of these 1259 (27.89%) nucleotides were variable in the dataset (Table 1). The aligned nrITS dataset includes 641 nucleotides, of which 300 (46.80%) were variable (Table 1).

Table 1.

Properties and best-fitting models of data partitions used in this study.

Data matrix Aligned positions Variable characters GC content (%) AIC selected model
matK 1287 431 33.4% GTR+F+G4
rbcL 1266 172 43.8% GTR+F+I+G4
rps16 963 333 33.4% GTR+F+G4
trnL-trnF 998 323 35.4% GTR+F+G4
Combined cpDNA matrix 4514 1259 37.2% GTR+F+I+G4
nrITS 641 300 60.1% GTR+F+I+G4

Phylogenetic analysis of Mazaceae

In all analyses, the monophyly of Mazaceae was strongly supported (Figs 3, 4; ML BS: 100%, BI PP: 1.00; all values reported in this order below). Because the taxon sampling is different in the datasets of cpDNA and nrITS, we did not combine them for analyses.

Three subclades can be identified in the cpDNA (Fig. 3) as well as nrITS trees (Fig. 4). The two individuals of M. lanceifolius consistently form a clade sister to the rest of Mazaceae. Within the rest of the family, Dodartia-Lancea clade is sister to Mazus (Figs 3, 4). In both ML and BI analyses, a sister relationship between Lancea and Dodartia is well supported (87%, 1.00 in cpDNA tree; 92%, 1.00 in nrITS tree). Monophyly of Mazus is also strongly supported (97%, 1.00) based on cpDNA dataset while moderately supported in nrITS analyses (62%, 0.93). Relationships within the genus Mazus are not fully resolved (Figs 3, 4). The “M. lanceifolius” misidentified in Deng et al. (2019) was found to be grouped with M. sunhangii based on cpDNA analyses with low support values (Fig. 3), while emerging as an isolated lineage in nrITS analyses when ITS sequence of M. sunhangii was not available (Fig. 4).

Figure 3. 

Maximum Likelihood phylogram of Mazaceae as inferred from analysis of combined dataset of matK, rbcL, rps16 and trnL-trnF. Support values ≥ 50% BS or 0.90 PP are displayed near the branches following the order ML-BS/BI-PP.

Figure 4. 

Maximum Likelihood phylogram of Mazaceae as inferred from analysis of nrITS. Support values ≥ 50% BS or 0.90 PP are displayed near the branches following the order ML-BS/BI-PP.

Taxonomic treatment

Puchiumazus Bo Li, D.G. Zhang & C.L. Xiang, gen. nov.

Fig. 1


Puchiumazus lanceifolius (Hemsl.) Bo Li, D.G. Zhang & C.L. Xiang ≡ Mazus lanceifolius Hemsl., in: J. Linn. Soc., Bot. 26 (174): 181. 1890.


The new genus is characterized by having quadrangular to somewhat ribbed stems and opposite, narrowly lanceolate leaves (Figs 1, 5A1–A3). Puchiumazus is sister to a clade composed of Dodartia, Lancea and Mazus. Morphologically, it is most similar to Mazus, but it differs in having quadrangular stems, lanceolate leaves (vs. terete stems and usually obovate-oblong leaves).


Perennial herbs. Rhizomes fleshy, white, horizontal. Root thin, fibrous. Stems erect, unbranched, glabrous, up to 30 cm tall, old stems quadrangular, glabrous, young stems inconspicuously quadrangular to obtusely ribbed, minutely puberulent. Leaves opposite, petiole inconspicuous to nearly absent; leaf blade narrowly lanceolate, 5.5–8.5 × 0.8–1.1 cm, submembranaceous to papery, adaxially green, pubescent, abaxially pale green, (sub)glabrous, base cuneate, margin basally entire and apically sparsely serrate, apex acute to long acuminate; lateral veins 3–5 pairs, abaxially raised and adaxially slightly depressed. Racemes terminal, 3–6 cm, flowers remarkably sparse, less than 6; pedicels 4–7 mm, sparsely puberulent; bracts tiny, narrowly lanceolate to linear. Calyx funnelform, 4–6 mm, sparsely pubescent outside, subglabrous inside, 5-lobed; lobes narrowly triangular to lanceolate, as long as tube in length, midrib conspicuous, apex acute. Corolla creamy yellow, 1.8–2.2 cm long, densely puberulent outside; tube straight, cylindric, long exserted from calyx, gradually dilated; limb 2-lipped, reddish in throat, posterior lip bilobed, lobes orbicular, anterior lip trilobed, lobes subequal, rounded. Stamens 4, didynamous, inserted on corolla tube, included, anterior pair longer; anthers bithecal, locules divergent, apically connivent; filaments filiform, glabrous. Styles included, glabrous, persistent; stigma 2-lamellate. Capsule ovoid, ca. 2 × 3 mm, glabrous.


The generic name is derived from “Puchiu” (in honor of Prof. Pu Chiu Tsoong (1906–1981), who was a prominent Chinese taxonomist specializing in the taxonomy of Scrophulariaceae in the traditional sense) and “mazus”, indicating that the new genus was separated from Mazus and is morphologically similar to it.

Common name

(assigned here). Bu Qiu Cao Shu (补求草属; Chinese name).


According to our data, this genus is endemic to Central China. It is known only from Hubei (Jianshi), Sichuan (Dayi and Dujiangyan) and Chongqing (Wushan) and can be found under evergreen broad-leaf forest at elevations of 600–1250 m.

Puchiumazus lanceifolius (Hemsl.) Bo Li, D.G. Zhang & C.L. Xiang, comb. nov.

Fig. 1

Mazus lanceifolius Hemsl., in: J. Linn. Soc., Bot. 26(174): 181. 1890. Lectotype (designated here): China. Hubei province (Hupeh): Jianshi (Chienchih), March 1889, A. Henry 5837 (K barcode K001079356 [photo!]). Basionym.


Flowering and fruiting from March to July.

Common name

(assigned here). Bu Qiu Cao (补求草; Chinese name).

Additional specimens examined

China. Sichuan Province (Szechuen): South Wushan, March 1889, A. Henry 7250 (K barcode K001079357 [photo!]); Duajiangyan City, Qingchengshan Mountain, under evergreen broad-leaf forest, 1200 m elev., 3 June 2020, X.X. Zhou et al. LB1067; Mianyang City, Dayi County, Qianfoshan Mountain, 850 m elev., 8 June 2020, X.X. Zhou et al. LB1067-2.


In the protologue of Mazus lanceifolius, two collections from Sichuan (A. Henry 7250) and Hubei (A. Henry 5837), China, respectively, were simultaneously listed without exact type designation because that was not the practice in the 19th century. After checking all floras and literature dealing with Mazus in China, we are certain that M. lanceifolius has not been lectotypified before. Thus, we here propose the specimen A. Henry 5837 (Kew barcode: K001079356) as lectotype of M. lanceifolius (Fig. 1A) in accordance with article 9.3 of the International Code of Nomenclature for Algae, Fungi, and Plants (Shenzhen Code) (Turland et al. 2018).

Mazus fruticosus Bo Li, D.G. Zhang & C.L. Xiang, sp. nov.

Fig. 2


China. Hubei Province: Shennongjia Forestry District, Laoyaya to Luoboxi, on rocky cliffs, 110°29'07.98"N, 31°19'23.92"E, 1282 m elev., 6 June 2012, D.G. Zhang zdg6673 (Holotype: JIU!).


Mazus fruticosus differs from all other conspecific taxa by being a shrub with numerous and much branched stems and having opposite to subopposite leathery leaves that are acutely serrate on apical half.


Shrubs, 25–55 cm tall. Stems woody, numerous branched, old stems greyish brown, terete, leafless, glabrous, young stems and branchlets brown, densely puberulent. Leaves nearly fascicled on the top of branchlet, opposite to subopposite, subsessile; lamina lanceolate, leathery, 3.5–5.5 × 0.7–1.1 cm, adaxially green, subglabrous to sparsely puberulent, abaxially light green, subglabrous, puberulent on veins, apex acute to acuminate, base cuneate, margin acutely serrate on apical half; midrib conspicuous abaxially, lateral veins inconspicuous; petioles nearly absent, densely puberulent. Racemes terminal, ascending to 7.5 cm long, lax, multiflowered; pedicels slender, 1–1.5 cm long, puberulent; bracts narrowly lanceolate, 3–4 mm long, puberulent. Calyces broadly campanulate, ca. 5 mm long, slightly enlarged in fruit, 5-veined, pubescent outside, pubescent to subglabrous inside; lobes 5, broadly triangular, as long as tube, apex acute, midrib conspicuous, lateral veins inconspicuous. Corolla purple, dotted yellow on palate, 1.6–1.9 cm long, puberulent to subglabrous outside, tube cylindric, 1.1–1.3 cm long, exserted from calyx; limb 2-lipped, upper lip bilobed, erect, lobes triangular ovate; lower lip trilobed, middle lobe narrowly ovate, ca. 3 mm long, smaller than lateral lobes, lateral lobes spreading away from middle lobe, broadly ovate to rectangular; palate comprising 2 longitudinal elevations extending from point of filament fusion to base of lower lobes, with sparse erect hairs. Stamens 4, didynamous, glabrous, inserted at the same level in distal part of tube, included; anterior pair longer, curved, appressed to corolla tube, posterior pair spreading; anthers bithecal, positioned adjacent to corolla tube on upper lip; filaments filiform, glabrous. Styles 1.4–1.7 cm long, included, exserted beyond anthers, stigma 2-lamellate. Capsule globose, ca. 4 mm in diam, apex rounded, included by persistent calyx.


The epithet of the new species refers to its shrubby habit.

Common name

(assigned here). Guan Zhuang Tong Quan Cao (灌状通泉草; Chinese name).

Distribution and habitat

Mazus fruticosus is currently known only from Shenlongjia Forest District in Hubei Province, central China. It frequently occurs on rocky cliffs or near evergreen mixed forests at an elevation of 1100–1250 m.

Additional specimens examined

China. Hubei Province: Shennongjia Forestry District, 29 March 2012, D.G. Zhang y1071 (JIU!); 11 May 2012, D.G. Zhang zdg00023 (JIU!); 17 August 2012, D.G. Zhang 00006 (JIU!); 21 May 2013, D.G. Zhang 130521012 (JIU!); 23 April 2015, D.G. Zhang 0423007 (JIU!).

Key to the four genera of Mazaceae

1 Stems quadrangular or somewhat ribbed; leaves opposite Puchiumazus
Stems not quadrangular; leaves rosette, alternate or rarely opposite to subopposite 2
2 Stems much branched; leaves reduced, scale-like; lower corolla lip without palate Dodartia
Stems inconspicuous or unbranched, rarely much branched in Mazus; Leaves not reduced; lower lip with distinct palate 3
3 Fruit usually completely enclosed in calyx when mature Mazus
Fruit half enclosed by calyx when mature Lancea


We here reconstruct the phylogeny of Mazaceae based on a combined cpDNA dataset of four markers (matK, rbcL, rps16 and trnL-trnF), and nrDNA ITS dataset, which have been used previously to infer relationships within Mazaceae (Deng et al. 2019; Yamamoto 2020) and among Lamiales (Refulio-Rodriguez and Olmstead 2014; Liu et al. 2020). The monophyly of Mazaceae is recovered as reported in previous work (Deng et al. 2019) relying on the same molecular markers. The major difference is that the third clade identified in the present study was not sampled by Deng et al. (2019).

Based on our analyses (Figs 3, 4), Mazaceae is composed of four genera (Fig. 5), including the new genus Puchiumazus described here. Three major clades can be identified for a re-circumscribed Mazaceae, and the cladogram is accompanied by some general morphological characters and geographical distribution patterns. The first clade is composed of two individuals of the new monotypic genus Puchiumazus (Figs 1, 5A1–A3), which is currently only known from three provinces in central China. Morphologically, the new genus can be distinguished clearly from other genera by having quadrangular to somewhat ribbed stems and opposite, narrowly lanceolate leaves.

Figure 5. 

Morphological comparisons of the four genera of Mazaceae A Puchiumazus lanceifolius B Dodartia orientalis C Lancea tibetica D Mazus stachydifolius A1, B1, C1, D1 habits A2, B2, C2, D2 flowers A3, B3, C3, D3 fruits.

The second clade consists of Dodartia (Fig. 5B1–B3) and Lancea (Fig. 5C1–C3). Both genera have broader distribution area than Puchiumazus, with Lancea always found at high elevations in QTP and Dodartia distributed in southern Russia and western to central Asia; it is cultivated as medical herb which has increased its distribution. Morphologically, both genera have small scale-like leaves (with a basal rosette of larger leaves in Lancea). Another important character is that ca. half of the capsule is enclosed by fruiting calyx and that calyx-teeth are much shorter than the fruit (Fig. 5B3, C3). In Puchiumazus, the style is persistent and ca. 2/3 of the fruit is enclosed in the fruiting calyx with calyx-teeth being much longer than the fruit. Calyx of Mazus is usually at least 1–2 times longer than capsule (e.g., Fig. 5D3).

Species of Mazus comprise the third clade, which is well supported in the cpDNA tree (94%, 1.00; Fig. 3), but moderately supported in the nrITS phylogeny (62%, 0.93; Fig. 4). Mazus is the largest genus of Mazaceae and it is widely distributed in East Asia and Australia. It can be distinguished from the other three genera by the more or less secund inflorescences and a corolla with a palate on the lower lip. Using the same DNA markers, Deng et al. (2019) produced a fully resolved phylogeny of Mazus in which five clades of the genus were highly supported (see Fig. 4 of their study). The interesting finding is that we cannot recover a similar topology, although the data of most species come from their dataset. Part of the reason for this may be that some sequences generated for their study were wrongly submitted to GenBank (see samples in Material and methods). Another possible reason is that they did not consider the topology incongruence between cpDNA and nrITS sequences, but concatenated the data for their analyses.

Phylogenetic analyses in our study did not support the sectional classification (i.e. Lanceifoliae, Mazus and Trichogymus) of Mazus proposed by Hong et al. (1998). At that time, Mazus lanceifolius was placed within Mazus, which we here recognize as a new genus. In addition, monophyly of the remaining two sections was also not supported, which was also the case in the study of Deng et al. (2019). Accordingly, they proposed a new infrageneric classification of Mazus, with two subgenera, Mazus and Notomazus T. Deng, N. Lin & H. Sun. Subgenus Mazus comprises most of the species and is native to Asia, while subgenus Notomazus comprises all species native to Australia and New Zealand. However, the monophyly of the two subgenera were not supported in our study. In both cpDNA and nrITS trees, Mazus radicans (Hook.f.) Cheeseman from subgenus Notomazus is deeply nested in subgenus Mazus, indicating it is necessary to redefine subgenus Notomazus. Given the discordance between the trees presented here and the one presented in Deng et al. (2019), on the basis of the same sequence data, we think some additional checking of the data, perhaps even resampling of M. radicans, is needed before any revision is made to the subgeneric classification of Mazus. In addition, a future study including more individuals of each species and more DNA markers (especially single and/or low copy nuclear genes) is necessary to clarify internal relationships within Mazus.

Previously, all species of Mazus are described as herbs (Yang 1979; Hong et al. 1998; Fischer 2004), but five species (M. caducifer Hance, M. celsioides Hand.-Mazz., M. spicatus Vaniot, “M. lanceifolius” [described as M. fruticosus in the present study], and sp.) were recorded as having “no herbaceous stem” in Deng et al.’s (2019) study. Actually, M. caducifer, M. spicatus, M. celsioides have rigid stems that look woody, but are not actually forming wood, thus these should be recognized as having a herbaceous habit. The new species described in the present study is probably the only species with a shrubby habit in the genus Mazus. This interesting find will help us to better understand the character evolution of Mazus. If Mazus sp. in Deng et al.’s (2019) also has a shrubby habit, we can speculate this character originated independently at least twice within the genus.

The abovementioned findings mean that more intensive field collections are necessary even in the post-Flora time. Yang (1979) have noticed the morphological difference between Puchiumazus lanceifolius (≡ Mazus lanceifolius) and other Mazus species. He pointed out that the quadrangular stem is only found in this species, and the nearly entire lanceolate leaves are also rare in Mazus, thus he suggested that this species probably is generically distinct. At the same time, he also emphasized that, because no fully developed flowers could be investigated based on specimens, he placed this species within Mazus. In this study, the rediscovery of this species offers an opportunity to investigate morphological characters of P. lanceifolius and provide a chance to extract DNA for molecular phylogenetic analyses, which led to the establishment of the new genus in the present study.

In recent years, many plants of Lamiales were rediscovered from biodiversity hotspots of China, including Aeschynanthus monetaria Dunn (Gesneriaceae; Hu et al. 2020), Ombrocharis dulcis Hand.-Mazz. (Lamiaceae; Chen et al. 2016), Wenchengia alternifolia C.Y. Wu & S. Chow (Lamiaceae; Li et al. 2012) and Pedicularis humilis Bonati (Orobanchaceae; Li et al. 2016). Most of these species had only been collected once before. The new genus described in the present study was also only known from the type collections (A. Henry 5837, 7250) before it was rediscovered. The type specimens of this were, until recently, the only known collections, and as a result, studies on the species since the original 1890 publication have been wanting. The re-investigation of this species is not only providing a chance to amend its description, but also a chance for a recognition of a new genus and redefinition of the family. The study highlights the important roles of field collections for systematic and biodiversity studies, which are often neglected in this age of biodiversity informatics (Wen et al. 2015).


We are grateful to Dr. Hong-Bo Ding (Xishuangbanna Tropical Botanical Garden, CAS) for sharing photos of Mazus fruticosus, to Dr. Maarten J. M. Christenhusz (Curtin University) and Dr. Su Liu (Shanghai Chenshan Botanical Garden) for their comments on nomenclature and improving the manuscript, and to Dr. Alan Paton for his comments on lectotypification of Mazus lanceifolius. We thank three peer reviewers and academic editors for their input in helping to improve the manuscript. The research was supported by the National Natural Science Foundation of China (31900181) granted to Bo Li, the Sino-Africa Joint Research Center, Chinese Academy of Sciences, CAS International Research and Education Development Program (SAJC201613) granted to Bing Liu, the CAS “Light of West China” program, Yunnan Fundamental Research Projects (2019FI009) and the “Ten Thousand Talents Program of Yunnan” (YNWR-QNBJ-2018-279) granted to Chun-Lei Xiang, the Major Program on Technology Innovation of Hubei Province (2018ACA132) and the Hubei Key Laboratory of Shennongjia Snub-nosed Monkey Conservation Fund (2018SNJ0009) granted to Dai-Gui Zhang, and Postdoctoral Directional Training Foundation of Yunnan Province granted to Fei Zhao.


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Appendix 1

Table A1.

Source publications and GenBank accession numbers of DNA sequences used in this study. If papers were not published, then indicated using superscript, references were listed below the table. GenBank accession numbers of the newly sequenced are marked in bold face. An n-dash (–) refers to a missing sequence.

Taxon References GenBank No.
matK rbcL rps16 trnL–trnF ITS
Dodartiao rientalis 1 Schäferhoff et al. (2010) FN773539 FN794091 FN794057
Dodartiao rientalis 2 Deng et al. (2019) MK392230 JQ342984 JQ342982 JQ342981 JQ342980
Lancea tibetica 1 Deng et al. (2019) MK266276 KX783467 KX807200 KX807205 MK192678
Lancea tibetica 2 Xia et al. (2009); Zuniga et al. (2017)a MF786907 a MF786661 a FJ172699 FJ172685 FJ172736
Lancea tibetica 3 Chi et al. (2018) MF593117 MF593117 MF593117 MF593117
Mazus reptans Refulio-Rodriguez and Olmstead (2014); Beardsley and Olmstead (2002) HQ384502 HQ384872 HQ385147 AF479004 AF478940
Mazus alpinus 1 Deng et al. (2019) MK266256 KX783481 KX783501 KX783520 MK192641
Mazus alpinus 2 Deng et al. (2019) KX783480 KX783500 KX783519 MK192642
Mazus caducifer 1 Deng et al. (2019) MK266277 KX783477 KX783497 KX783516 MK192664
Mazus caducifer 2 Deng et al. (2019) KX783487 KX783506 KX783526 MK192659
Mazus celsioides Deng et al. (2019) KX783486 MK266366 KX783525
Mazus fauriei 1 Deng et al. (2019) MK266255 KX783499 MK266420 MK192640
Mazus fauriei 2 Deng et al. (2019) LC034207
Mazus gracilis Xia et al. (2009) FJ172729 FJ172701 FJ172687 FJ172738
Mazus humilis 1 Deng et al. (2019) MK266367 MK266421
Mazus humilis 2 Deng et al. (2019) MK192667
Mazus japonicus var. delavayi Deng et al. (2019) MK266257 KX783482 KX783502 KX783521
Mazus japonicas Xia et al. (2009); Deng et al. (2019) MK266259 FJ172728 FJ172700 FJ172686
Mazus fruticosus 1 Deng et al. (2019) MK266261 KX783470 KX783490 KX783509 MK192660
Appendix 1 Continued
Mazus fruticosus 2 Deng et al. (2019) MK266254 KX783471 KX783491 KX783510 MK192649
Mazus longipes 1 Deng et al. (2019) MK266267 KX783474 KX783494 KX783513 MK192652
Mazus longipes 2 Deng et al. (2019) MK192654
Mazus miquelii 1 Deng et al. (2019) KX783475 KX783495 KX783514 MK192637
Mazus miquelii 2 Deng et al. (2019) MK266271 KX783476 KX783496 KX783515 MK192655
Mazus miquelii 3 Deng et al. (2019) MK266272 KX783483 KX783503 KX783522 MK192656
Mazus miquelii 4 Umemoto et al. (2015) LC027734
Mazus novaezeelandiae Deng et al. (2019) MK266278 KX783469 KX783489 KX783508 MK192676
Mazus omeiensis 1 Deng et al. (2019) MK266252 KX807209 KX807203 KX807208 MK192636
Mazus omeiensis 2 Xia et al. (2009); Deng et al. (2019) FJ172731 FJ172702 FJ172688 MK192663
Mazus pulchellus Deng et al. (2019) KX783472 KX783492 KX783511 MK192638
Mazus pumilus 1 Deng et al. (2019); Jiang et al. (2018)b; Xu et al. (2018)c MH265198 b MK266346 KX807201 KX807206 MH711724 c
Mazus pumilus 2 Xia et al. (2009); Schaefer et al. (2011); Deng et al. (2016) HM850959 HM850162 KX807202 KX807207 FJ172737
Mazus pumilio Deng et al. (2019) MK266277 KX783468 KX783488 KX783507 MK192671
Mazus radicans Deng et al. (2019); Smissen et al. (2015)d KT626738 d MK266381 MK192635
Mazus spicatus 1 Xia et al. (2009) MK266251 FJ172730 FJ172703 FJ172689 FJ172740
Mazus spicatus 2 Deng et al. (2019) MK192681
Mazus surculosus Deng et al. (2019) KX783473 KX783493 KX783512
Mazus sunhangii 1 Deng et al. (2016) KX783485 KX783505 KX783524
Mazus sunhangii 2 Deng et al. (2016) KX783484 KX783504 KX783523
Mazus xiuningensis 1 Deng et al. (2019) MK266348 MK266383
Mazus xiuningensis 2 Deng et al. (2019) MK266349 MK266384 MK266430
Mazus procumbens Deng et al. (2019) MK266261 KX783478 KX783498 KX783517 MK192647
Puchiumazus lanceifolius 1 This study MW373735 MW373737 MW373739 MW373741 MW364623
Puchiumazus lanceifolius 2 This study MW373736 MW373738 MW373740 MW373742 MW364624
Paulownia tomentosa Xu et al. (2018)c; Deng et al. (2019) MK392226 KX783466 KX807199 KX807204 MH711291 c
Paulownia coreana Yi and Kim (2016) NC_031435 NC_031435 NC_031435 NC_031435
Lamium purpureum Wink and Kaufmann (1996); Oxelman et al. (2005); Refulio-Rodriguez and Olmstead (2014) HQ384493 Z37403 HQ385141 AJ608588
Callicarpa mollis Tsukaya et al. (2003); Refulio-Rodriguez and Olmstead (2014) HQ384498 HQ384868 HQ385145 HQ412928 AB099648
Vitex agnuscastus Refulio-Rodriguez and Olmstead (2014); Wagstaff and Olmstead (1997) HQ384496 U78716 HQ385143 HQ412926
Premna odorata Refulio-Rodriguez and Olmstead (2014) HQ384494 HQ384866 HQ385142 HQ412925
Wightia speciosissima Xia et al. (2019); Zhou et al. (2014)e MK381318 MK381318 MK381318 MK381318 KJ563189 e
Mimulus sp. Zhao et al. (2021) MT473772 MT473772 MT473772 MT473772
Phryma leptostachya Wagstaff and Olmstead (1997); Bremer et al. (2002); Xu et al. (2018)c AJ429341 U28881 AJ609150 AJ430928 MH711667 c
Erythranthe lutea Vallejo-Marín et al. (2016); Arroyo et al. (2019) NC_030212 NC_030212 NC_030212 NC_030212 MH781192
Erythranthe guttata Refulio-Rodriguez and Olmstead (2014); Kuzmina et al. (2017) KJ161979 KJ161981 KJ161978 KJ161975 MG219646
Striga hermonthica Wicke et al. (2016) KU212372 KU212372 KU212372 KU212372
Appendix 1 Continued
Rehmannia elata Oxelman et al. (2005); Albach et al. (2006); Refulio-Rodriguez and Olmstead (2014) HQ384505 HQ384874 DQ856490 AJ608572 DQ069315
Pedicularis groenlandica Refulio-Rodriguez and Olmstead (2014); Tkach et al. (2014) HQ384503 HQ384873 HQ385148 HQ412930 HG424130

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Zhao F, Chen YP, Salmaki Y, Drew BT, Wilson TC, Scheen AC, Celeop F (accepted for publication) Bräuchler c, Bendiksby M, Wang Q, Ming DZ, Peng H, Olmstead RG, Li B, Xiang CL (2021) An updated tribal classification of Lamiaceae based on plastome phylogenomics. BMC Biology.

Supplementary material

Supplementary material 1 

Figures S1, S2

Chun-Lei Xiang, Hong-Li Pan, Dao-Zhang Min, Dai-Gui Zhang, Fei Zhao, Bing Liu, Bo Li

Data type: Phylogenetic tree

Explanation note: Figure S1. Bayesian Inference (BI) phylogram of Mazaceae based on the combined cpDNA dataset (matK, rbcL, rps16, and trnL-F). Bayesian posterior probabilities are shown near the branches. Figure S2. Bayesian Inference (BI) phylogram of Mazaceae based on the nrITS dataset. Bayesian posterior probabilities are shown near the branches.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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