SYSTEMATIC POSITION OF MIYAKEA INTEGRIFOLIA MIYABE & TATEW. IN THE INFRAGENERIC CLASSIFICATION OF PULSATILLA (RANUNCULACEAE): PALYNOLOGICAL CHALLENGES

Miyakea integrifolia Miyabe and Tatew. was considered to be an endemic genus and species to Sakhalin Island, Russia, but the monotypic genus Miyakea was later subsumed into Pulsatilla as P. integrifolia (Miyabe & Tatew.) Vorosch. This taxonomic treatment has been supported by many botanists. At present, there are two opinions on the systematic position of P. integrifolia within the genus Pulsatilla : i) a separate monotypic subgenus Miyakea situated at the last position; ii) a close relative of P. patens and P. vernalis in ser. Patentes within the species-rich subgen. Pulsatilla . Since palynological evidence might be promising morphological traits for clarifying the systematic position of Miyakea , a table summarizing the pollen morphological traits within the genus Pulsatilla was prepared using the previous appropriate palynological reports. Hitherto, the morphology of Miyakea pollen supports the second opinion i.e., a close relative of P. patens and P. vernalis . Moreover, we pointed out the issues that should be focused on in future research on the pollen morphology of Pulsatilla s.l.

Miyakea integrifolia Miyabe and Tatew., a closely related taxon of Pulsatilla, was collected from the mountain range of the palaeozoic formation situated on the Sea of Okhotsk side of Sakhalin Island in 1934 and was first described as a new monotypic and endemic genus in 1935 (Miyabe andTatewaki, 1935, 1937).The presence of coriaceous, simple, entire, and evergreen radical leaves with three prominent parallel veins on the underside was regarded as the most prominent generic feature of this taxon (Miyabe andTatewaki, 1935, 1941).
Although the generic status had been maintained by Sugawara (1939), Czerepanov (1995), Starodubtsev (1995), Smirnov (2002), Barkalov and Taran (2004), Nishikawa (2008) and Eryemin et al. (2019).Voroshilov (1966) considered the genus Miyakea to be Pulsatilla and published the name P. integrifolia (Miyabe & Tatew.)Vorosch.This view was followed by Czerepanov (1973) and Voroshilov (1982); furthermore, Tamura (1991Tamura ( , 1995) ) and Grey-Wilson (2014, 2020) recognized this species as a separate monotypic subgenus Miyakea situated at the last position within the classification system of Pulsatilla.On the other hand, the most recent molecular phylogenetic study by Sramkó et al. (2019) showed that M. integrifolia is subsumed into the genus Pulsatilla but is considered to be a closely related sister species to P. vernalis in ser.Patentes of subgen.Pulsatilla.Thus, ser.Patentes has been composed of three Pulsatilla species: P. integrifolia [≡Miyakea integrifolia], P. patens, and P. vernalis.The inclusion of P. integrifolia and P. vernalis within ser.Patentes is an unexpected result, as no specialist of Pulsatilla has ever considered the species included in this clade to be related (Sramkó et al., 2019).

An overview of pollen morphological traits in the infrageneric taxonomy of Pulsatilla by Sramkó et al. (2019)
The most recent DNA-based classification of Pulsatilla by Sramkó et al. (2019) is characterized by the following three main results; i) the genus was separated into three subgenera, Kostyczewianae, Preonanthus and Pulsatilla, ii) the monotypic subgen.Kostyczewianae consisting of only P. kostyczewii was basally placed in the classification, iii) three species (P.integrifolia [≡Miyakea integrifolia], P. patens s.l. and P. vernalis) formed the single ser.Patentes of sect.Pulsatilla within the species-rich third subgen.Pulsatilla.
The basally positioned subgen.Kostyczewianae in the Sramkó et al.'s system was characterized by 2-to 3-colpate pollen with small grain size below 30 μm in the longest axis (Table 1).The two apertures state is palynologically exceptional within the genus Pulsatilla and supports the subgeneric status, but further studies are needed because 2-colpate pollen was reported in only 40% of pollen grains in Xi (1985).
Within the recent DNA-based classification by Sramkó et al. (2019), most species examined in the second subgen.Preonanthus (including two sects.Preonanthus and Preonanthopsis) were characterized by having commonly pantocolpate pollen with a medium (30 μm or more but below 40 μm) grain size (Table 1).The palynological evidence would support possibly the unity of subgen.Preonanthus.
The irregular and occasional appearance of pantocolpate pollen adding to the usual 3-colpate pollen was observed in P. patens, P. vulgaris, P. halleri, P. chinensis, and P. dahurica within the third subgen.Pulsatilla (Table 1).This palynological phenomenon indicates at least a similarity between the second subgen.Preonanthus and the species-rich third subgen.Pulsatilla.
The majority of the species (eight examined among the twelve species) comprising the last series Albanae within the third subgen.Pulsatilla was characterized by having the most specialized pantoporate pollen within the genus Pulsatilla (Table 1).The evolutionary trend of pollen types; from tricolpate through pantocolpate to pantoporate was postulated in Pulsatilla (Xi, 1985).Series Albanae could be distinguished from other series within the genus Pulsatilla in the pollen aperture traits (Huynh, 1970;Tamura, 1995).Monophyly and the last position of series Albanae in the classification of Sramkó et al. (2019) was well supported by the present palynological evidence, and the section-level separation of Albanae should be possibly considered.
Although Nowicke and Skvarla (1995) pointed out that the pollen morphology of Ranunculaceae has generally limited taxonomic values, palynological traits, in some cases, indicate homogeneity at the infrageneric level in the Pulsatilla classification by Sramkó et al. (2019).

Systematic affiliation of Miyakea based on the palynological traits and future challenges
At present, there are two opinions on the systematic position of Miyakea [P.integrifolia] within the genus Pulsatilla: i) a separate monotypic subgen.Miyakea situated at the last position mainly based on morphological and anatomical studies (Tamura, 1991(Tamura, , 1995;;Grey-Wilson, 2014, 2020), ii) a close relative of P. patens and P. vernalis in ser.Patentes within the species-rich subgen.Pulsatilla mainly based on DNA and chromosome analyses (Sramkó et al., 2019).
Miyakea pollen was first studied by Nowicke and Skvarla (1995) in SEM and they showed that 3-colpate aperture and supratectal microspinulate ornamentation with distinct tectal perforations.A tri-colpate aperture of Miyakea pollen is the same as in many species of subgen.Pulsatilla (excluding series Albanae) within Pulsatilla.Although exine ornamentation such as supratectal microspinules is the same character between Pulsatilla and Miyakea, this feature is common within the Ranunculaceae (44 of the 48 genera examined; Nowicke and Skvarla, 1995).Nowicke and Skvarla (1995) suggested that elongated tectal perforations on the surface view of Miyakea pollen were regarded as characteristic palynological features distinguished from Pulsatilla.But the surface shape of tectal perforations of Miyakea [as P. integrifolia] in their SEM micrograph may not be evidently different from that of P. patens (Xi, 1985;Plate 3, fig. 4) and P. vernalis (Clarke et al., 1991;Plate 47, Fig. 2) within ser.Patentes.Therefore, the exine ornamentation of Miyakea pollen may not support positively the opinion with the separate monotypic subgen.Miyakea (Miyabe and Tatew.)Tamura.
As in Table 1, the presence of distinct tectal perforations in SEM micrographs in most Pulsatilla pollen was different from indistinct perforations on the pollen in some samples of Pulsatilla; P. kostyczewii, P. patens, P. grandis and P. pratensis and P. cernua.However, considering the different preparation methods and micrograph resolution between them, we did not consider the presence/absence (or distinct/indistinct) of tectal perforations as the taxonomic informative palynological character at the current research stage.To compare the exine ornamentation between species within Pulsatilla, it is necessary to thoroughly remove the surface material (perine, pollenkitt, and so on) and observe the surface ornamentation of the swollen pollen grains by SEM.The palynological trait of flat tectum versus undulate tectum (Nowicke and Skvarla, 1995) may be problematical for comparison at the species level.Palynological studies need to be performed on more species that have not yet been observed (Table 1).The pollen morphology of Far East Asian species (e.g., P. magadanensis, P. tatewakii, P. sugawarai, and P. tongkangensis) in particular should be clarified.
Palynological studies by LM should clarify aperture type and pollen grain size within a species or between populations.These palynological traits are related to chromosome number (cf.Table 1).The TEM study on Pulsatilla pollen wall has been conducted only in restricted species; P. chinensis and P. campanella in Xi (1985), and P. grandis in PalDat (2023).Structural diversity within the exine wall in Pulsatilla pollen is not yet known.