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.

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.

References in Appendix

^a Zuniga JD, Mulcahy DG, Coddington JA (2017) DNA barcodes from Tibetan plant genera. [Unpublished.]

Albach DC, Li HQ, Zhao N, Jensen SR (2006) Molecular systematics and phytochemistry of Rehmannia (Scrophulariaceae). Biochemical Systematics and Ecology 35(5): 293–300. https://doi.org/10.1016/j.bse.2006.11.003

Arroyo MTK, Pérez F, Jara-Arancio P, Pacheco D, Vidal P, Flores MF (2018) Ovule bet-hedging at high elevation in the South American Andes: Evidence from a phylogenetically controlled multispecies study. Journal of Ecology 107(2): 668–683. https://doi.org/10.1111/1365-2745.13069

^b Jiang CC, Zhang CC, Wei SS, Huang HH, Huang ZZ (2018) Identification of Weeds Based on DNA Barcoding. [Unpublished.]

Beardsley PM, Olmstead RG (2002) Redefining Phrymaceae: The placement of Mimulus, tribe Mimuleae, and Phryma. American Journal of Botany 89(7): 1093–1102. https://doi.org/10.3732/ajb.89.7.1093

Bremer B, Bremer K, Heidari N, Erixon P, Olmstead RG, Anderberg AA, Källersjö M, Barkhordarian E (2002) Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels. Molecular Phylogenetics and Evolution 24(2): 274–301. https://doi.org/10.1016/S1055-7903(02)00240-3

^c Xu Y, Zeng CX, Zhang YQ, Ren Y, Ge XJ (2018) DNA barcoding the Flora of Qinling Mt. in China. [Unpublished.]

Chi X, Wang J, Gao Q, Zhang F, Chen SL (2018) The complete chloroplast genomes of two Lancea species with comparative analysis. Molecules (Basel, Switzerland) 23(3): e602. https://doi.org/10.3390/molecules23030602

^d Smissen RD, Millar RT, Heenan P (2015) Phylogenetic Tree of New Zealand vascular genera. [Unpublished.]

Deng T, Zhang XS, Kim C, Zhang JW, Zhang DG, Volis S (2016) Mazus sunhangii (Mazaceae), a new species discovered in Central China appears to be highly endangered. PLoS ONE 11(10): e0163581. https://doi.org/10.1371/journal.pone.0163581

Deng T, Lin N, Huang X, Wang H, Kim C, Zhang D, Zhu W, Yusupov Z, Tojibaev ShK, Sun H (2019) Phylogenetics of Mazaceae (Lamiales), with special reference to intrageneric relationships within Mazus. Taxon 68(5): 1037–1047. https://doi.org/10.1002/tax.12150

^e Zhou QM, Jensen SR, Liu GL, Wang S, Li HQ (2014) Familial placement of Wightia (Lamiales). [Unpublished.]

Kuzmina ML, Braukmann TWA, Fazekas AJ, Graham SW, Dewaard SL, Rodrigues A, Bennett BA, Dickinson TA, Saarela JM, Catling PM, Newmaster SG, Percy DM, Fenneman E, Lauron-Moreau A, Ford B, Gillespie L, Subramanyam R, Whitton J, Jennings L, Metsger D, Warne CP, Brown A, Sears E, Dewaard JR, Zakharov EV, Hebert PDN (2017) Using herbarium-derived DNAs to assemble a large-Scale DNA barcode library for the vascular plants of Canada. Applications in Plant Sciences 5(12): e1700079. https://doi.org/10.3732/apps.1700079

Oxelman B, Kornhall P, Olmstead RG, Bremer B (2005) Further disintegration of Scrophulariaceae. Taxon 54(2): 411–425. https://doi.org/10.2307/25065369

Refulio-Rodriguez NF, Olmstead RG (2014) Phylogeny of Lamiidae. American Journal of Botany 101(2): 287–299. https://doi.org/10.3732/ajb.1300394

Schaefer H, Hardy OJ, Silva L, Barraclough TG, Savolainen V (2011) Testing Darwin’s naturalization hypothesis in the Azores. Ecology Letters 14(4): 389–396. https://doi.org/10.1111/j.1461-0248.2011.01600.x

Schäferhoff B, Fleischmann A, Fischer E, Albach DC, Borsch T, Heubl G, Müller KF (2010) Towards resolving Lamiales relationships: Insights from rapidly evolving chloroplast sequences. BMC Evolutionary Biology 10(1): e352. https://doi.org/10.1186/1471-2148-10-352

Tkach N, Ree RH, Kuss P, Röser M, Hoffmann MH (2014) High mountain origin, phylogenetics, evolution, and niche conservatism of arctic lineages in the hemiparasitic genus Pedicularis (Orobanchaceae). Molecular Phylogenetics and Evolution 76: 75–92. https://doi.org/10.1016/j.ympev.2014.03.004

Tsukaya H, Eukuda T (2003) Hybridization and introgression between Callicarpa japonica and C. mollis (Verbenaceae) in central Japan, as inferred from nuclear and chloroplast DNA sequences. Molecular Ecology 12(11): 3003–3011. https://doi.org/10.1046/j.1365-294X.2003.01961.x

Umemoto H, Yokota M, Kokubugata G (2015) Reconsideration for Occurrence of Mazus goodenifolius (Phrymaceae) in Miyazaki Prefecture, Japan using Molecular and Morphological Data. Bulletin of the National Museum of Nature and Science, Series B 41: 61–67.

Vallejo-Marín M, Cooley AM, Lee MY, Folmer M, McKain MR, Puzey JR (2016) Strongly asymmetric hybridization barriers shape the origin of a new polyploid species and its hybrid ancestor. American Journal of Botany 103(7): 1272–1288. https://doi.org/10.3732/ajb.1500471

Wagstaff SJ, Olmstead RG (1997) Phylogeny of Labiatae and Verbenaceae inferred from rbcL sequences. Systematic Botany 22(1): 165–179. https://doi.org/10.2307/2419684

Wicke S, Müller KF, DePamphilis CW, Quandt D, Bellot S, Schneeweiss GM (2016) Mechanistic model of evolutionary rate variation en route to a nonphotosynthetic lifestyle in plants. Proceedings of the National Academy of Sciences of the United States of America 113(32): 9045–9050. https://doi.org/10.1073/pnas.1607576113

Wink M, Kaufmann M (1996) Phylogenetic relationships between some members of the subfamily Lamioideae (Family Labiatae) inferred from nucleotide sequences of the rbcL gene. Botanica Acta 109(2): 139–148. https://doi.org/10.1111/j.1438-8677.1996.tb00554.x

Xia Z, Wang YZ, Smith JF (2009) Familial placement and relations of Rehmannia and Triaenophora (Scrophulariaceae s.l.) inferred from five gene regions. American Journal of Botany 96(2): 519–530. https://doi.org/10.3732/ajb.0800195

Xia Z, Wen J, Gao Z (2019) Does the enigmatic Wightia belong to Paulowniaceae (Lamiales)? Frontiers of Plant Science 10: e528. https://doi.org/10.3389/fpls.2019.00528

Yi DK, Kim KJ (2016) Two complete chloroplast genome sequences of genus Paulownia (Paulowniaceae): Paulownia coreana and P. tomentosa. Mitochondrial DNA. Part B, Resources 1(1): 627–629. https://doi.org/10.1080/23802359.2016.1214546

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. https://doi.org/10.1186/s12915-020-00931-z