Most pollen grains of Clerodendrum species investigated here are spheroidal or subprolate in equatorial view. Subprolate or prolate pollen grains observed in some species (C. bungei, C. intermedium Chamisso, C. phlomidis) and reported in previous studies (Raj 1983; Perveen and Qaiser 2007) were probably affected by the acetolysis treatment, as the pollen shape of Lamiaceae is easily affected during hydration and/or fixation (Sebsebe and Harley 1992). On account of the broken colpus membranes, oblate or suboblate pollen grains are easily incorrectly considered as subprolate or prolate (Harley 1992). Raj (1983) described pollen of C. speciosissimum C. Morren as distinct with three to four to six apertures, which was the first report of non-triaperturate pollen in Clerodendrum (without illustration). In our study, in contrast, only triaperturate pollens were observed from Clerodendrum (Figs 1–5). Based on the numbers of apertures and the characters of exine sculpture, Liu (1985) considered that pollen grains of most species of Clerodendrum (excepting C. fortunatum L., C. paniculatum L., C. trichotomum Thunb., and C. yunnanense Hu ex Hand.-Mazz.) are tricolpate-spiny, characterized by large or very large size, tricolpate, dense or sparse spine over the microreticulate exine ornamentation. Overall, our results are mostly consistent with the finding of Liu (1985). However, C. fortunatum, C. paniculatum, C. trichotomum and C. yunnanense pantocolpate pollen grains are as described by Liu (1985). This type is characterized by spheroidal grains of large size, with pantocolpate (6 to 8) and spiny exine. In contrast, all pollens grains of Clerodendrum (including C. fortunatum, C. paniculatum and C. trichotomum) observed in this study are tricolpate with dense or sparse spine in the exine (Figs 1–5).

The infrageneric classification system applied in Flora of China (Chen and Gilbert 1994) is not fully supported by our results. In comparison to the pollen shape and size, the exine sculpture appears to be a more taxonomically valuable and steady character. There is a clear distinction in pollen exine sculpture between sect. Siphonanthus and sect. Clerodendrum. The obtuser spine exine sculpture is only found in C. indicum (Fig. 5M–O), the sole species of sect. Siphonanthus. However, consistency between morphology and exine sculpture is hardly found in taxa belonging to sect. Clerodendrum. For example, species in Ser. Axilliflorae Schauer have different exine sculpture types: C. griffithianum C. B. Clarke is spine-tectum perforatum (Fig. 2P–R); C. fortunatum is microspine-tectum perforatum (Fig. 4A–C) and Volkameria inermis (=C. inerme (L.) Gaertn.) belongs to the spine-tectum imperforatum type (Fig. 2A–C). A similar phenomenon is observed in Ser. Densiflora Schauer: C. bracteatum Wall. ex Walp. and C. bungei belong to spine-tectum perforatum type, while C. lindleyi Decne. ex Planch. and C. chinense var. simplex (Moldenke) S. L. Chen. belong to spine-tectum imperforatum type (Table 2); In Ser. Penduliflorae Schauer: C. cyrtophyllum, C. garrettianum, C. wallichii Merr. and C. longilimbum Pei belong to the microspine-tectum perforatum type, C. hainanense Hand.-Mazz. and C. kwangtungense Hand.-Mazz. belong to the spine-tectum perforatum type and C. henryi Pei belongs to microspine-tectum imperforatum type (Table 2); In Ser. Paniculata Schauer: C. colebrookianum Walp., C. mandarinorum Diels, C. trichotomum and C. villosum Blume belong to the spine-tectum imperforatum type (Table 2), C. brachystemon C. Y. Wu et R. C. Fang belongs to the spine-tectum perforatum type and C. kaichianum Hsu belongs to the microspine-tectum perforatum type (Table 2); In Ser. Squamata Schauer: C. japonicum (Thunb.) Sweet belongs to the microspine-tectum perforatum type (Table 2), whereas C. paniculatum belongs to the microspine-tectum imperforatum type (Table 2). In conclusion, the pollen morphology of Chinese Clerodendrum species investigated in this study does not provide obvious evidence for infra-sectional classification.

Pollen characteristics have been proven to be useful in species delimitation in some genera of Lamiaceae (Erdtman 1952; Abu-Asab and Cantino 1989, 1994; Abu-Asab 1991; Trudel and Morton 1992; Large and Mabberley 1995; Moon et al. 2008a, 2008b; Özler et al. 2011; Badamtsetseg et al. 2012; Ma et al. 2016). According to our results, the varieties could be easily distinguished from the original variety. For instance, the pollen tectum of Clerodendrum trichotomum is imperforated, whereas that of C. trichotomum var. fargesii (Dode) Rehder is perforated. In addition, species that are difficult to distinguish from one another based on external morphology can be discerned at the pollen level. For example, C. wallichii and C. henryi share many similarities such as 4-angled branchlets, white corolla, ovate lobes, exserted stamens, and style (Chen and Gilbert 1994). The two species are challenging to differentiate at first glance due to their overlapping habitats, similar plant height, and leaf shape. The primary identification characteristic relies on the length of the petiole: which in the case of C. wallichii is typically about 1 cm, while C. henryi generally exceeds 1 cm in length. Additionally, young branches of C. wallichii may exhibit winged branchlets, whereas this characteristic is absent in C. henryi (Chen and Gilbert 1994). Our study suggests that morphology of pollen grains can help to distinguish C. wallichii (with perforatum tectum; Fig. 5J–L), from C. henryi (imperforated tectum; Fig. 3J–L). Morphologically, Clerodendrum lindleyi is very similar to C. bungei and shares some characteristics: leaf blade broadly ovate to cordate; terminal inflorescences, dense, capitate, corymbose cymes; corolla pinkish to purple, lobes obovate; drupes blue-black and subglobose (Chen and Gilbert 1994). However, our study indicates that they are easily distinguished from each other because the exine sculpture type pollen of C. bungei has a perforated tectum (Fig. 2G–I), whereas that of C. lindleyi is imperforated (Fig. 1M–O).

Pollen grains of Lamiaceae are commonly monad, isopolar, and there is a significant relationship between the number of pollen colpi in the subfamilies of Lamiaceae (Abu-Asab and Cantino 1989; Large and Mabberley 1995; Ma et al. 2016). The tectum of the pollen grains in Ajugoideae usually exhibits projections and spinules. For example, supratectal projections are conical in Cardioteucris C.Y. Wu and Amethystea L. (Abu-Asab and Cantino 1989); spinose in Tripora P.D. Cantino et al. (1998) and Trichostema Gronov. (Harley et al. 2004); spinulose in Caryopteris Bunge (Abu-Asab et al. 1993) and Teucrium L. (Harley et al. 2004). Perveen and Qaiser (2007) described the ornamentation of Clerodendrum phlomidis (C. phlomidis) as Clerodendrum phlomidis-type (Tectum reticulate with spinules or reticulate-rugulate). Our results complement these previous findings.

Clerodendrum is currently placed within Ajugoideae of Lamiaceae based on molecular phylogenetic evidence (Wagstaff and Olmstead 1997; Wagstaff et al. 1998). Recently, Zhao et al. (2021) further divided Ajugoideae into four tribes (Ajugeae, Clerodendreae, Teucrieae and Rotheceae) and assigned Clerodendrum and Volkameria in Clerodendreae. In our palynological study, the pollen grains of V. inermis (Fig. 2) are spine-tectum imperforatum, which is the same as found in several species of Clerodendrum (C. villosum, C. trichotomum, C. mandarinorum, C. colebrookianum and C. lindleyi; Fig. 1; Table 2). Therefore, our palynological data support the close relationship between Clerodendrum and Volkameria (Zhao et al. 2021). Barrabe et al. (2015) suggested that Clerodendrum has a close relationship with Amasonia, Kalaharia, Tetraclea, and Volkameria, and that they are sister taxa to the alliance encompassing Hosea, Aegiphila, Oxera, and Faradaya. Zhao et al. (2021) found that Clerodendrum was grouped together with Oxera and Volkameria. Here, pollen morphology supports Barrabe et al.’s (2015) and Zhao et al.’s (2021) findings. Palynological evidence has revealed that pollen characteristics of Clerodendrum (Figs 1–5; Table 2) and Volkameria (Fig. 2; Table 2), are similar to those of Aegiphila, Amasonia, Faradaya, Hosea, Kalaharia, Tetraclea and Oxera (Raj 1983). Most species exhibit radiosymmetric, tricolpate, monads, spiny ornamentation and spheroidal or subprolate shapes. Although palynological evidence supports that Clerodendrum is closely related to other eight genera, accurate relationships among those genera require a more comprehensive study.

The authors have declared that no competing interests exist.

No ethical statement was reported.

This work is supported by the National Natural Science Foundation of China (Grant No. 31970220 and 32260047), the Natural Science Foundation of Guangxi Province (Grant No. 2018GXNSFAA281132 and 2023GXNSFAA026346), and the Foundation of Guangxi Key Laboratory of Sugarcane Biology (GXKLSCB-202004).

Xiakai Huang: Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing - Original Draft;Rui Wu: Data Curation, Writing - Original Draft;Zheng Xiong: Software;Zhonghui Ma: Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review & Editing.

Xiakai Huang https://orcid.org/0000-0003-4515-5199

Rui Wu https://orcid.org/0000-0003-0484-3761

Zheng Xiong https://orcid.org/0000-0003-4295-2432

Zhonghui Ma https://orcid.org/0000-0002-3898-3079

All of the data that support the findings of this study are available in the main text.