Study materials were obtained from herbarium sheets of the HAST, TAI and TAIF herbaria and from living plants (herbarium acronyms follow Index Herbariorum (Thiers 2019, continuously updated). Type specimens of S. taipeiensis, deposited in the herbarium of the National Taiwan University (TAI), were also examined. Voucher specimens were deposited in the herbarium of the Taiwan Forestry Research Institute (TAIF). We examined leaf, floral and fruit morphology from dried and living materials. For living materials, we observed four populations of S. barbata and two of S. taipeiensis, including the type locality. Scutellaria barbata is widespread in Taiwan. Hence, herbarium sheets complement the fresh material gathered so that the variation, present in Taiwan, was represented in the study. For the population of S. taipeiensis, only few populations, including the type, have been recorded. All of these populations were located in Taipei City. Thus, our observation covered all populations in Taiwan. Observation of the nutlet sculpture of Hsiung et al. (2017) was applied here as a reference. The identification of S. barbata, S. taipeiensis and other Taiwanese species was according to the protologues of Huang et al. (2003) and other related literature (Hsieh and Huang 1995; Huang et al. 1998).

In order to revise the taxonomic state of S. taipeiensis, phylogenetic trees were reconstructed. The species, selected for analysis, were from Chiang et al. (2012) and Holmskioldia sanguinea was applied as outgroup, since it was closely related to Scutellaria (Bendiksby et al. 2011). Two nuclear (CAD, CHS) and three chloroplast DNA fragments (matK, ndhF-rpl32 and rpl32-trnL) were used by Chiang et al. (2012), amongst them, ndhF-rpl32 and rpl32-trnL being also applied in the study of Hsiung et al. (2017). Two chloroplast regions (ndhF-rpl32 and rpl32-trnL) were applied in the phylogenetic analysis of this study. In addition to the sequences from Chiang et al. (2012) and Hsiung et al. (2017), we sequenced the chloroplast DNA fragments of ndhF-rpl32 spacer from S. galericulata, S. incana and H. sanguinea and rpl32-trnL spacer from S. galericulata, S. incana, S. taiwanensis and H. sanguinea. These newly generated sequences were amplified following the procedure of Hsiung et al. (2017). All sequences, applied in this study, are listed in Table 1. These sequences were used for phylogeny reconstruction by Bayesian Inference (BI), Maximum Likelihood (ML) and Neighbour-Joining (NJ) approaches. The variable sites, parsimony-informative sites and substitution model were checked and selected by MEGA 7 (Kumar et al. 2016). The optimal model with the highest BIC and AIC values was selected for BI and ML analyses (Kumar et al. 2016) (Table 2). The BI reconstruction was conducted using Mr. Bayes 3.2.6 (Ronquist et al. 2012). Two independent runs were conducted with 10,000,000 generations, sampled every 1000 generations and a 10% dataset was discarded as burn-in. ML analysis was performed by PhyML 3.1 (Guindon et al. 2010). The substitution model of the two loci was the same as for BI analysis and the gamma distribution parameter was fixed at 1.52 and 0.35, respectively, according to the results of model selection. The tree topology search operation was set as the best of NNI and SPR (Guindon et al. 2010). The approximate likelihood ratio test non-parametric branch support was based on a Shimodaira-Hasegawa-like procedure (Guindon et al. 2010). NJ analysis was conducted using MEGA 7, with 1000 bootstrap resamplings. All of the phylogenetic trees were summarised and output by FigTree 1.4.4 (Rambaut 2012).

Leaves

Leaf morphology had been regarded as a diagnostic character for distinguishing S. barbata from S. taipeiensis (Huang et al. 2003). Leaf shape of S. barbata varies from suborbicular to narrowly lanceolate (Fig. 1A–D); in contrast, the leaf shape of S. taipeiensis varies from ovate to broadly ovate (Fig. 1E, F). The leaves of both species had sparse pubescence on the abaxial surface. The leaves of S. barbata are 1.1−2.8 cm long and 0.9−1.4 cm wide, the length-width-ratio from 1.1 to 2.0, while S. taipeiensis leaves are 1.0−1.7 cm long and 0.5−1.1 cm wide, the length-width-ratio from 1.5 to 2.0. The shapes and sizes of the leaves overlapped between the two species and thus were difficult for use as a diagnostic character to distinguish species.

Figure 1. 

Leaf variation of S. barbata (A–D) and S. taipeiensis (E, F). A New Taipei City, Gueishan rd. (Chao 4768) B Taipei City, Hsichou street (Chao 4762) C Ilan County, Ilan City (Chao 4787) D Ilan County, Sanhsing Township (Chao 4789) E Taipei City, campus of NCCU (Chao 4837) F Taipei City, Maukong (type locality, Chao 4838).

Inflorescence and flowers

The floral morphology of S. barbata (Fig. 2A, C) was very similar to that of S. taipeiensis (Fig. 2B, D). They both had terminal inflorescences and bilabiate flowers that were only slightly curved near base (Fig. 3), while other species of Taiwan have geniculate (e.g. S. austrotaiwanensis, S. indica etc.) or a strongly curved corolla (e.g. S. tashiroi). The corolla was bluish-purple, 0.8−1.3 cm long and pubescent on the outer surface.

Figure 2. 

Flower morphology of S. barbata (A, C)(Chao 4762) and S. taipeiensis (B, D)(Chao 4838). A, B lateral view C, D front view.

Figure 3. 

Inflorescence morphology of S. barbata (A) and S. taipeiensis (B).

Nutlets

According to the observations of Hsiung et al. (2017), the sculpture of S. barbata and S. taipeiensis are rounded concentric type and no other difference is found between them.

Molecular phylogeny

The best substitution model for both fragments, ndhF-rpl32 and rpl32-trnL, was HKY+G. The phylogenetic trees, reconstructed by ML, NJ and BI, revealed similar topologies with slight differences in the arrangement of non Taiwanese species (Figs 4, 5; Suppl. materials 1–4). In all analyses, all Taiwanese species formed a clade with moderate to high support (PP = 0.99, bootstrap = 0.65–0.91). Amongst them, S. barbata and S. taipeiensis formed a highly supported monophyletic group (PP = 1.0, bootstrap = 0.87–0.95), but neither S. barbata nor S. taipeiensis formed a single clade. Instead, S. taipeiensis was nested with S. barbata in the phylogenetic tree, i.e. neither species being monophyletic as currently delimited.

Figure 4. 

Phylogenetic tree reconstructed by Bayesian inference from chloroplast DNA sequence ndhF-rpl32. Only posterior probability > 0.85 was labelled on the branch. Scale bar represent substitutions.

Figure 5. 

Phylogenetic tree reconstructed by Bayesian inference from chloroplast DNA sequence rpl32-trnL. Only posterior probability > 0.85 was labelled on the branch. Scale bar represent substitutions.

Distribution and habitat

Scutellaria barbata grows throughout low altitudes, ca. 50–500 m, in Taiwan, but lower in southern parts. Scutellaria taipeiensis is found in Taipei City and New Taipei City. The two species were growing in similar habitats, such as grassland, roadside, riverbank or forest margin and often with high humidity. Some references regarded S. barbata as an aquatic plant in a broad sense (Chen 1990) due to its wetland habitat. This could also be true for S. taipeiensis, according to the field investigation. No apparent differentiation was observed in the distribution and habitat types between these two species.

Scutellaria taipeiensis was first described by Huang et al. (2003), based on the morphology of the leaves and the nutlets. According to the original description, the leaves of S. taipeiensis were triangular-ovate to broadly ovate and less than twice as long as the width. To confirm the similarity of the leaves, we collected and compared leaf morphology of the two species amongst several populations. The results showed that the variation within S. barbata was larger than the difference between S. barbata and S. taipeiensis. Additionally, the length-width-ratio of leaves is the same in both species. Therefore, leaf morphology could not be regarded as a diagnostic character for these two species.

With regard to the nutlets, the coat had also been used to distinguish S. barbata from S. taipeiensis. The former had a radiating, umbrella-like shape, while the latter was a rounded concentric type (Huang et al. 2003). However, Hsiung et al. (2017) reviewed this character on a population level and found no remarkable difference between the two species. The mature nutlets appear to the rounded concentric type in both species, such state was stable amongst populations (Hsiung et al. 2017). The umbrella-like appendage was found in immature nutlets only, which meant that it was a transitional state during nutlet development and could not provide a valid taxonomic value.

We further looked at other characters to separate them, such as floral morphology and DNA sequence data. Different sequence data revealed some phylogenetic incongruence amongst lineages S. alpina, S. altissima, S. bicalensis, S. diffusa, S. galericulata, S. salviifolia and S. zhongdianensis. Such incongruence may be due to uneven sampling, but the relationship of these species was not a concern in this study. Therefore, we will not discuss the evolutionary relationship between this group of species here. All Taiwanese species formed a highly supported clade in all trees. Scutellaria barbata and S. taipeiensis, which had very similar inflorescences and floral morphology, are phylogenetically nested within a monophyletic clade. Based on this evidence, S. taipeiensis was treated as a synonym of S. barbata, rather than a distinct species or on an intraspecific level.

According to the results and discussions, we established the following taxonomy: