General description of nutlet micromorphology
The nutlet's morphology and ultrastructure characteristics such as shape size, appendages, and surface sculpturing, varied among the studied taxa. The Rochelieae nutlets' shapes were different across samples; while some had ovoid shapes (ovoid, ovoid-triangular, and ovoid-rectangular), some other samples had pyramid shape (Fig. 1). In the out-groups, the shape of Asperugo procumbense was semicircular, and the Myosotis sylvatica was of ellipse shape. These two genera belong to Asperugeae and Myosotideae tribes, respectively.
Nine qualitative characters were selected for the morphological evaluation of nutlets. The results obtained from nutlet-ultrastructure investigations are described below and illustrated in Fig. 1. Generally, six different surface types were recognized among studied taxa based on nutlet ultrastructure characters:
Type I: Heterocaryum and Pseudolappula (Syn: L. siniaca)
There was no glochid or appendage on the nutlet disk, but a row of glochid (Heterocaryum) or glochid-like (Pseudolappula) on the nutlet edge. The glochids were distributed sparsely on the edges of the nutlets of Pseudolappula. The nutlet disk ornament of Heterocaryum was “papilla verrucose with verrucae minutely muricate” (called complex papilla), the nutlet disk ornament of Pseudolappula was “papilla with aggregate verrucose in the center”. It appeared that each of the microcapillaries found in Psudolappula were complex in the Heterocaryum, and each formed warts (verrucose), leading to a more complex and denser status.
Type II: Lappula (L. barbata, L. microcarpa and L. semiglabra).
Glochids in different sizes and rows can be seen at the nutlet edge and sometimes on the nutlet disk surface. Glochids had an anchor with 2–4 branches at the apex, and their surface was smooth. The ultrastructure of the nutlet emergencies was stellar-aculeate, and in some case the appendage, prickles, or tubercles could be seen (scattered or collected) on the surface of the nutlet disk and edge. The glochid stem was composed of fusiform cells, and there were tubercles with 2 to 5 mineralized spines on the stem. These tubercles were also present on the entire surface of the nutlet with a different distribution.
Type III: Rochelia (R. disperma, R. sessiflora = L. sessiflora).
The prickles were arranged in a stellate pattern and scattered throughout the surface of the nutlet. The surface of the prickles was not glossy and was of verrucose. The tubercles often had more than 2 spines, and the emergencies were stellar-aculeate (similar to type II). Nonetheless, the nutlet surface of R. sessiflora was similar to type II (presence of glochid on the nutlet edge). Moreover, the prickles and the accumulation of tubercles with more than 5 spines around each prickle shared more resemblance with type III.
Type IV: L. ceratophora and L. spinocarpus
There was not any glochid, tubercle, or prickles on the nutlet surface. The nutlet surface of L. ceratophora was not smooth, and the papilla appeared as verrucose-like. The ultrastructure of the nutlet in L. spinocarpus had a similar appearance to papilla with flowerlike verrucose. Also, the tubercles appeared as verrucose lacking any spines.
Type V: Asperugo
The nutlet surface lacked any glochid and prickles. Papilla appeared dome-shaped in different sizes, with verrucose at the base of it.
Type VI: Myosotis
The surface of the nutlet was smooth, and there was no ornamentation (nonexpressiate).
Evolution of nutlet’s microstructural characters
We did not observe topological contradiction for the Rochelieae tribe in the analysis that were performed by constructing gene trees from the nr-DNA ITS, concatenated trnL-F–rpl32-trnL(UAG), and concatenated nr-DNA ITS–trnL-F–rpl32-trnL(UAG) data sets. This was consistent with what was reported by Khoshsokhan-Mozaffar et al. (2018). Therefore, for preventing sloppiness, we selected nr-DNA ITS because of better resolution. Details of the ML analyses based on the nr-DNA ITS data set confirmed the phylogenetic relationships of some species of Rochelieae and then formed a well-supported lineage encompassing two clades. This is totally in agreement with the topology published by Khoshsokhan-Mozaffar et al. (2018). One clade was composed of sub-tribe Heterocaryinae (Heterocaryum-Suchtelenia), with high support (BS = 0.99). The next clade contained most subtribe Eritrichiinae (BS = 0.85) comprising Lappula, Eritrichium, Lepechiniella, Hackelia, and Rochelia. Within this clade, Lappula sinaica formed the distinct branch and nested far away from other Lappula species. Moreover, Lappula sessiliflora was nested with a subclade of Rochelia species (Fig. 2A).
The resulting ancestral state reconstruction and the proportional likelihoods for character states are shown in Fig. 2B. Two out-group species Asperugo and Myosotis were unique with the bilaterally flattened and ellipse with a smooth surface, respectively. Tracing the evolution of nutlet micromorphology indicated that the glochids are not an ancestral character.
Arrangement of glochid character
The status of ancestral taxa (with or without glochids) was unclear, and the proportional likelihoods of all three characters were almost equal (node A). Transition to the glochids character occurred in the genus Lappula (L. semiglabra, L. microcarpa, and L. barbata) (node H).
The appendage on nutlet disk character
The status “without appendage” and “dump-shape papilla” in Myosotis and Asperugo (the proportional likelihood 1) differentiated these two tribes from each other and Rocheliea tribe. The tubercle and prickles on the disk were ancestral characters (the proportional likelihood 0.43). While the ancestor of these characters was unclear in node A, the status “tubercle and prickles on disk” was of more proportional likelihood in A and then C, D, F group. Transition to the “lack of appendage” status occurred in the L. ceratophora, L. spinocarpus; node G).
Prickles surface character
Tracing of character “prickles surface” showed the ancestral status of “lack of prickles” in node A that to be followed with less proportionality in nodes B, C, and D. Transition to the “glossy prickles” status occurred in the genus Lappula in node H. Moreover, the transition to the simple and complex “verrucose prickles” status was observed in genus Rochelia in node F.
Surface emergence
Tracing of surface emergence character was unclear in node A. Though, the proportional likelihoods of “stellar-aculeate” status had the highest node C ratio (0.72).
In node G, the transition to “verrucose-subverrucose” status (the proportional likelihoods 0.99) was stabilized as a synapomorphy.
Other traits were studied regarding evolutionary tracing that did not show clear evolutionary signals in the nodes, such as the shape of the nutlet, the lamella type, the shape of the nutlet, the lamella type, and the centerline of the nutlet disk.
Close boundary of nutlet microstructures between L. barbata and L. microcarpa
Both L. microcarpa and L.barbata had high micro-morphological similarities (Fig. 3). The very close relationship was studied by PCA analysis about other studied taxa in the tribe Rochelieae. In total, nine nutlet characteristics of the studied taxa were investigated (see nutlet SEM characters in Table 2). PCA analysis of nutlet characteristics revealed that the first two PCs comprised 75.83% of the total variability of the taxa. In the first PCA axis, characters such as the appendage on nutlet desk, shape of nutlet; surface emergence and prickles surface showed the highest correlation, while in the second PCA axis, other characters, such as seed shape of nutlet, tubercles on the desk and the centerline of nutlet showed the highest correlation. Therefore, these were the most varied micro-morphological characters of the taxa. PCA-biplot of micro-morphological characters (Fig. 4a) separated the taxa into distinct groups. All specimens of L. barbata and L. microcarpa were clustered together and close to L. semiglabra (Fig. 4a), while other taxa were grouped together. Moreover, L. ceratophora and L. spinocarpos overlapped each other.
Different clustering and ordination methods produced similar results; therefore, only the WARD tree of micro-morphological characters is presented here (Fig. 4b). The taxa were separated in the WARD tree of micro-morphological features, subsequently resulted in main branches. One included three genera arranged in two sub-groups: subgroup I, including all specimens, which belonged to L.barbata and L.microcarpa, while L.semiglabra diverged from others and placed in sub-group II. The rest of the taxa were clustered with each other in the same branch, similar to the results of the PCA plot (Fig. 4a). This result implies that the micro-morphological characters could not delimit two species L. barbata and L.microcarpa.