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' shape was ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to the pyramid (Fig. 1). As the out-groups, the shape of Asperugo procumbense was semicircular, and the Myosotis sylvatica one was an ellipse. These two genera belong to Asperugeae and Myosotideae tribes, respectively.
Nine qualitative characters including shape, the centerline of the raphe, base surface of prickles on the desk, the arrangement of glochids, the type of lamellae, tubercles on the desk, appendages on nutlet desk, Number of glochid rows on nutlet edge, and emergence type were selected for 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 between studied taxa based on nutlet ultrastructure characters as follow:
Type I: Heterocaryum and Pseudolappula (Syn: L. siniaca)
There is no glochid or appendage on the nutlet disk, but there is a row of glochid (Heterocaryum) or glochid-like (Pseudolappula) on the nutlet edge. The glochids are distributed in very low numbers in the edges of the nutlets of Pseudolappula. The nutlet disk ornament of Heterocaryum is “papilla verrucose with verrucae minutely muricate” (called complex papilla) while it is “papilla with aggregate verrucose in the center” in Pseudolappula. It appears that each of the microcapillaries found in Psudolappula has become complex in the Heterocaryum, and each has formed warts (verrucose) that have become more complex and denser.
Type II: Lappula (L. barbata, L. microcarpa and L. semiglabra)
Glochids in different sizes and rows can be seen in the nutlet edge and sometimes on the nutlet disk surface. Glochids have an anchor with 2–4 branches at the apex, and the surface of the glochids is smooth. The ultrastructure of the nutlet emergencies is stellar-aculeate, and sometimes the appendages are prickles or tubercles are seen (scattered or collected) on the surface of the nutlet disk and edge. The glochid stem is composed of fusiform cells, and there are tubercles with 2 to 5 mineralized spines on the stem. These tubercles are also present on the entire surface of the nutlet with a different distribution.
Type III: Rochelia (R. disperma, R. sessiflora = L. sessiflora)
The prickles are stellate and are scattered throughout the surface of the nutlet. The surface of the prickles is not glossy and has verrucose. The tubercles often have more than 2 spines, and the emergencies are stellar-aculeate (similar to type II). Although the nutlet surface of R. sessiflora is similar to type II (presence of glochid on the nutlet edge). Moreover, the prickles and verrucose on it and the accumulation of tubercles with more than 5 spines around each prickle show more similarity to type III.
Type IV: L. ceratophora and L. spinocarpus
There is not any glochid, tubercle, or prickles on the nutlet surface. The nutlet surface of L. ceratophora is not smooth, and the papilla appears as a verrucose-like. While the ultrastructure of the nutlet in L. spinocarpus seems papilla with flowerlike verrucose. Also, the tubercles appear as verrucose and lack any spines.
Type V: Asperugo
The nutlet surface lacks any glochid and prickles. Papilla appears as dome-shaped in different sizes, and it is verrucose at the base of them.
Type VI: Myosotis
The surface of the nutlet is smooth, and there is not any ornamentation (nonexpressiate).
a–h = Type I; (a-d) Heterocaryum rigidum; a, b: An overview photograph of nutlet with stereomicroscope and SEM. c, d: The close up views of nutlet disk with “papilla verrucose with verrucae minutely muricate”. (e-h) Pseudolappula siniaca; e, f: An overview photograph of nutlet with stereomicroscope and SEM. g, h: The close up views of nutlet disk with “papilla with aggregate verrucose in center”.
i-o Type II. (i, j) Lappula. barbata; (k, l) Lappula. Microcarpa; (The more detail of these two species were described between different specimens in Fig. 3). Lappula semiglabra m, n: An overview photograph of nutlet with stereomicroscope and SEM. o: The close up views of nutlet disk.
p-v = Type III; (p-r) Rochelia sessiflora; p, q: An overview photograph of nutlet with stereomicroscope and SEM. r: The close up views of nutlet disk with prickles and verrucose on it. (s-v) Rochelia disperma s, t: An overview photograph of nutlet with stereomicroscope and SEM. u, v: The close up views of stellare-aculeate emergencies in nutlet disk.
w-ac = Type IV; (w-y) Lappula spinocarpus; w, x: An overview photograph of nutlet with stereomicroscope and SEM. y: The close up views of nutlet disk with papilla with flowerlike verrucose on it. (z-ab) Lappula ceratophora z, aa: An overview photograph of nutlet with stereomicroscope and SEM. ab: The close up views of nutlet disk with papilla appears as a verrucose-like on it.
ac-af = Type V; Asperugo procumbense; ac-ad: the overview photograph of nutlet with stereomicroscope and SEM. af: The close up views of nutlet disk with Papilla appears as dome-shape on it.
ag-aj = Type VI; Myosotis sylvatica; ag-ai: the overview photograph of nutlet with stereomicroscope and SEM. aj: The close up views of nutlet disk with smooth surface.
3.2. Evolution of microstructural characters of nutlet
The resulting ancestral state reconstruction and the proportional likelihoods for character states are shown in Fig. 2. The out-group species Asperugo and Myosotis were unique regarding the bilaterally flattened and ellipse with a smooth surface, respectively. Tracing the evolution of nutlet micromorphology indicated that the glochids were not an ancestral character.
Arrangement of glochid character:
The status of ancestral taxa (with or without glochids) was unclear, and the proportional likelihoods of any three characters were almost equal (node A). Transition to the glochids character occurs 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 likelihoods 1) were differentiated these two tribes from each other and Rocheliea tribe. The tubercle and prickles on the disk were ancestral characters (the proportional likelihoods 0.43). While the ancestor of these characters is unclear in node A, the status “tubercle and prickles on disk” had more proportional likelihoods in A group and then C, D, F. 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.
Tracing of surface emergence character was unclear in node A. However, 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.
3.3. Close boundary of nutlet microstructures between L. barbata and L. microcarpa
Both L. microcarpa and L.barbata had high micro-morphological similarities (Fig. 3). Different clustering and ordination methods produced similar results; therefore, only WARD tree of micro-morphological characters is presented here (Fig. 4). In general, plant samples of each species did not group and formed a separate group. This result shows that the micro-morphological characters studied could not delimit these two species.