Pollen morphology of the tribe Phyllodoceae (Ericoideae, Ericaceae) and its taxonomic significance

Pollen morphology of 13 taxa belonging to 5 genera of the tribe Phyllodoceae (Ericaceae) was examined by means of light and scanning electron microscopy (LM and SEM, respectively), or SEM alone. In Phyllodoceae, 3-colpor(oid)ate, minute to medium, oblate pollen grains are united usually in tetrahedral tetrads. Pollen tetrads are generally characterized by the presence of viscin threads except Elliottia pyroliflora, Epigaea asiatica and Phyllodoce caerulea. The absence of viscin threads might indicate to a secondary loss, since these are present at least in some species within all the genera of Phyllodoceae. The pollen morphological data confirm the infraand inter-generic relationships as identified by molecular phylogeny of Phyllodoceae and/or vice-versa. Although various palynological characters were found to be taxonomically important at different taxonomic levels, the apocolpial exine sculpture is emerged as one of the most important palynological features of systematic importance. The rugulate apocolpial exine with striate secondary sculpture and a reduced colpus might be apomorphic palynological character states for this tribe and Ericaceae as well. Hitherto, it is the first SEM study of Rhodothamnus pollen. Introduction The Phyllodoceae Drude is one of the most morphologically heterogeneous group that apparently has no morphological synapomorphy (Kron, 1997). Moreover, the inclusion of Epigaea in the tribe Phyllodoceae might increase the morphological heterogeneity of this group and the monophyly of this group is not well supported by morphology. Nearly all descriptions of the tribe have diagnosed the group based upon suites of characters that are homoplasious within the broader Ericoideae, rather than recognizing any particular potentially synapomorphic character (Kron et al., 2002). Gillespie and Kron (2010) used molecular data to clarify tribe-level relationships within the Ericoideae and to propose a new classification that includes five tribes, namely Rhodoreae, Empetreae, Ericeae, Bryantheae and Phyllodoceae; the Phyllodoceae is sister to a clade comprised of the other four tribes. Most members of Phyllodoceae are native to temperate-boreal regions of the Northern Hemisphere. The flowers are variable in shape, size and colour. Some species have pollen dispersed by curved stamens that spring out from the flower centre, explosively. Many species in this group are also poisonous, e.g., Kalmia, both to livestock and people. The molecular analyses indicated two strongly supported clades: Kalmia s.l. (including Leiophyllum and Loiseleuria) and a Phyllodoce clade (including Epigaea, Kalmiopsis and Rhodothamnus), and Elliottia is sister to Kalmia + Phyllodoce clade (Kron et al., 2002; Gillespie and Kron, 2013). In addition, at least one taxon from all currently recognized tribes within the Ericoideae have at some point been classified within the Phyllodoceae, illustrating the difficulty in determining relationships of these taxa based on morphological or anatomical evidence alone (Gillespie and Kron, 2013). A part of Ph.D. Thesis Present address: Department of Crop Botany, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. E-mail: drsarwar@bau.edu.bd 130 SARWAR AND TAKAHASHI Pollen of the family Ericaceae is very diverse – monads vs. tetrads, or as polyads of indefinite number of tetrads; and its phylogenetic importance have been well documented (Kron et al., 2002; Sarwar, 2007). Among the genera of Phyllodoceae, the pollen morphology of Kalmia has already been reported (Sarwar and Takahashi, 2012), but fragmentary palynological information is only available for other genera of this tribe (Sladkov, 1954; Oldfield, 1959; Stevens, 1971; Bohm et al., 1978; Comtois and Larouche, 1981; Clements, 1995; Kron et al., 2002; Radcliffe et al., 2010). Light microscopy (LM) was mainly employed in these studies; and scanning electron microscopy (SEM) was employed in a few cases. The aims of the present study are to provide the palynological data of the genera of Phyllodoceae in addition to Kalmia, by using both LM and SEM, and to discuss its systematic significance in light of the recent molecular phylogenetic relationship of this tribe. Materials and Methods Pollen morphology of 13 taxa belonging to 5 (out of 7) genera of the tribe Phyllodoceae, was examined by means of light microscopy (LM) and scanning electron microscopy (SEM), or SEM alone (Table 1). Pollen morphology of the genus Kalmia has been studied in details (Sarwar and Takahashi, 2012) and the data has been included in discussion. The remaining genus Kalmiopsis is endemic to Oregon State, USA and could not be included due to unavailability of pollen samples. Polliniferous materials used in this investigation were taken from the dried specimens of the herbaria GB, S and SAPS. Abbreviations of names of the herbaria are according to the Index Herbariorum (Holmgren et al., 1990). Table 1. List of Phyllodoceae taxa used in this study along with their voucher specimens. No. Name of the taxa Voucher specimens 1. Bejaria aestuans Mutis ex L. Venezuela: Edo, Tachira Hwy. 9, 37 km south of Delicias, 28.01.1978, J.L. Luteyn et al. 5296 (S) 2. B. racemosa Vent. USA: Florida, Dade Co., west of Fulford, 10.02.1930, H.N. Moldenke 601 (S) 3. B. resinosa Mutis ex L. f. Colombia: District Cauca, El Tambo, 08.11.1936, K. von Sneidern 1069 (S) 4. B. subsessilis Benth. Ecuador: Prov. Loja-Zamora road, 17.06.1979, B. Lǿjtnant & U. Molau 15010 (GB) 5. Elliottia bracteata (Maxim.) Benth. et Hook. f. Japan: Hokkaido, Prov. Teshio, Masike-gun, Masike-cho, Mt. Shokanbetsu, 28.07.1983, H. Takahashi 4500 (SAPS) 6. E. paniculata (Sieb. et Zucc.) Benth. et Hook. f. Japan: Hokkaido, Hiyama-sicho, Kudoo-gun, Taisei-cho, Mt. Ohta-san, 06.08.1987, H. Takahashi 7802 (SAPS) 7. E. pyroliflora (Bong.) Brim & Stevens USA: Alaska, Juneau Quadrangle, Mt. Roberts behind, August 1967, L.A. Viereck 8624 (S) 8. Epigaea asiatica Maxim. Japan: Hokkaido, Hiyama-shicho, Kaminokuni-cho, 18.04.1982, M. Hara 5212 (SAPS) 9. E. repens L. USA: New York, Tomplins Co., slopes on east side of valley of Cayuga Intel, 13.04.1935, R.I. Clausen 19207 (S) 10. Phyllodoce aleutica (Spreng.) A. Heller Japan: Hokkaido, Jyozankei, Mt. Yoici-dake, 02.09.1982, H. Takahashi et al. 3666 (SAPS) 11. P. caerulea (L.) Bab. Japan: Hokkaido, the Hidaka range, Mt. Poroshiri – Mt. Tottabetsu, 01.08.1983, H. Takahashi 4569 (SAPS) 12. P. nipponica Makino var. oblong-ovata (Tatew.) Toyokuni Japan: Hokkaido, Hidaka range, Mt. Poroshiri – Mt. Tottabetsu, 01.08.1983, H. Takahashi 4568 (SAPS) 13. Rhodothamnus chamaecistus (L.) Rchb. Austria: Kärnten, Loibl-pass, 28.05.1960, I. Segelberg s.n. (S) POLLEN MORPHOLOGY OF THE TRIBE PHYLLODOCEAE 131 The preparation of pollen grains for LM and SEM, and pollen parameter studied follow Sarwar and Takahashi (2012). Pollen slides of all collection are deposited at the Hokkaido University Museum, Sapporo, Japan. Pollen size and shape classes were made following Erdtman (1986) and descriptive terminology follows Punt et al. (2007). The infrageneric classifications of Bejaria and Phyllodoce follow Clements (1995) and Good (1926 cf. Gillespie and Kron, 2013), respectively. Results and Discussion Pollen grains are united usually in tetrahedral tetrads, sometimes compact tetrahedral tetrads and/or with other configurations (Table 2). Viscin threads are usually present, sometime swelling at base in Bejaria aestuans, or absent in Elliottia pyroliflora, Epigaea asiatica, and Phyllodoce caerulea. The shape of the pollen grains varies from oblate to suboblate. Costae are usually distinct, but indistinct in Bejaria, 3-colpor(oid)ate (Fig. 1B), rarely 4-colporate in Rhodothamnus chamaecistus (Fig. 2K), ectoaperture (colpus) margins are distinct in all species. Endoapertures are usually distinct, but indistinct or absent in Bejaria subsessilis and Phyllodoce aleutica, lalongate, sometimes H-shaped in Epigaea repens; and endocracks are usually indistinct or absent in Phyllodoce and Rhodothamnus. Septum perforations are only observed in Phyllodoce nipponica var. oblong-ovata. The Phyllodoceae pollen ranges in mean values: D 30.0-53.3 μm, P 15.8-26.9 μm, E 22.0-38.7 μm, D/d 1.31-1.39, P/E 0.66-0.76, 2f 11.5-21.5 μm, W 0.9-2.3 μm, 2f/D 0.260.56, endoaperture length 0.8-2.0 μm, width 7.4-13.4 μm, apocolpial exine 1.7-3.0 μm thick, and septum 0.9-2.8 μm thick; tectate, apocolpial exine sculpture from verrucate to rugulate (Table 2). In SEM, apocolpial exine sculptures mainly constitute two distinct groups, viz. i) primary exine sculpture indistinct, secondary sculpture gemmate-pilate (Figs 1C, E, G–H, L) or intermediate type (Fig. 1M); ii) primary exine sculpture moderately to coarsely rugulate or rugulate-psilate or intermediate types (Figs 2B, C, D, G-I, L); colpus membrane largely granulate or granuloid. The genera of the Phyllodoceae employed in the present study reveal variation in SEM. In Bejaria, pollen surface is somewhat flat, primary apocolpial exine sculpture indistinct, secondary sculpture finely (diam. < 0.5 μm) gemmate-pilate (Type FG; Figs 1C, E, G, H); colpus membrane largely granulate or granuloid. In Elliottia, pollen surface is uneven to somewhat flat, primary apocolpial exine sculpture indistinct, secondary sculpture unit moderately (diam. > 0.5 μm) gemmate-pilate (Type MG; Fig. 1L), or intermediate type (MG/R; Fig. 1M); colpus sometimes narrow and elongate, membrane granulate. The pollen surface of Epigaea is somewhat flat, apocolpial exine sculpture coarsely rugulate, the rugulae transversely striate and intermediate type (RS/R; Fig. 2B), or coarsely rugulate-psilate and intermediate type (R/P; Fig. 2C); colpus membrane granulate. Pollen surface in Phyllodoce is uneven and rugged, apocolpial exine sculpture intermediate type (R/FG; Fig. 2G); or the surface is somewhat flat, exine sculpture moderately to coarsely rugulate (Type R; Figs 2HI); colpus membrane granulate or smooth. In Rhodotha


Introduction
The Phyllodoceae Drude is one of the most morphologically heterogeneous group that apparently has no morphological synapomorphy (Kron, 1997).Moreover, the inclusion of Epigaea in the tribe Phyllodoceae might increase the morphological heterogeneity of this group and the monophyly of this group is not well supported by morphology.Nearly all descriptions of the tribe have diagnosed the group based upon suites of characters that are homoplasious within the broader Ericoideae, rather than recognizing any particular potentially synapomorphic character (Kron et al., 2002).Gillespie and Kron (2010) used molecular data to clarify tribe-level relationships within the Ericoideae and to propose a new classification that includes five tribes, namely Rhodoreae, Empetreae, Ericeae, Bryantheae and Phyllodoceae; the Phyllodoceae is sister to a clade comprised of the other four tribes.Most members of Phyllodoceae are native to temperate-boreal regions of the Northern Hemisphere.The flowers are variable in shape, size and colour.Some species have pollen dispersed by curved stamens that spring out from the flower centre, explosively.Many species in this group are also poisonous, e.g., Kalmia, both to livestock and people.The molecular analyses indicated two strongly supported clades: Kalmia s.l.(including Leiophyllum and Loiseleuria) and a Phyllodoce clade (including Epigaea, Kalmiopsis and Rhodothamnus), and Elliottia is sister to Kalmia + Phyllodoce clade (Kron et al., 2002;Gillespie and Kron, 2013).In addition, at least one taxon from all currently recognized tribes within the Ericoideae have at some point been classified within the Phyllodoceae, illustrating the difficulty in determining relationships of these taxa based on morphological or anatomical evidence alone (Gillespie and Kron, 2013).
Pollen of the family Ericaceae is very diverse -monads vs. tetrads, or as polyads of indefinite number of tetrads; and its phylogenetic importance have been well documented (Kron et al., 2002;Sarwar, 2007).Among the genera of Phyllodoceae, the pollen morphology of Kalmia has already been reported (Sarwar and Takahashi, 2012), but fragmentary palynological information is only available for other genera of this tribe (Sladkov, 1954;Oldfield, 1959;Stevens, 1971;Bohm et al., 1978;Comtois and Larouche, 1981;Clements, 1995;Kron et al., 2002;Radcliffe et al., 2010).Light microscopy (LM) was mainly employed in these studies; and scanning electron microscopy (SEM) was employed in a few cases.The aims of the present study are to provide the palynological data of the genera of Phyllodoceae in addition to Kalmia, by using both LM and SEM, and to discuss its systematic significance in light of the recent molecular phylogenetic relationship of this tribe.

Materials and Methods
Pollen morphology of 13 taxa belonging to 5 (out of 7) genera of the tribe Phyllodoceae, was examined by means of light microscopy (LM) and scanning electron microscopy (SEM), or SEM alone (Table 1).Pollen morphology of the genus Kalmia has been studied in details (Sarwar and Takahashi, 2012) and the data has been included in discussion.The remaining genus Kalmiopsis is endemic to Oregon State, USA and could not be included due to unavailability of pollen samples.Polliniferous materials used in this investigation were taken from the dried specimens of the herbaria GB, S and SAPS.Abbreviations of names of the herbaria are according to the Index Herbariorum (Holmgren et al., 1990).
Table 1.List of Phyllodoceae taxa used in this study along with their voucher specimens.

No. Name of the taxa
Voucher specimens The preparation of pollen grains for LM and SEM, and pollen parameter studied follow Sarwar and Takahashi (2012).Pollen slides of all collection are deposited at the Hokkaido University Museum, Sapporo, Japan.Pollen size and shape classes were made following Erdtman (1986) and descriptive terminology follows Punt et al. (2007).The infrageneric classifications of Bejaria and Phyllodoce follow Clements (1995) and Good (1926 cf. Gillespie andKron, 2013), respectively.
The present study reveals that in Phyllodoceae, both medium or minute and oblate pollen grains are united usually in tetrahedral tetrads (Table 2; Sarwar and Takahashi, 2012).Pollen tetrads of this tribe are generally characterized by the presence of viscin threads except in some species (Table 2; Sarwar and Takahashi, 2012).Results of the present study, in general, support previous palynological observations, however, in several instances, marked differences among the observations are apparent e.g., viscin threads on pollen tetrads of Epigaea.We did not observe any  viscin threads on the pollen tetrads of E. asiatica (Table 2), although it was reported that all three species of Epigaea have viscin threads with their pollen (Stevens, 1971).
Pollen tetrads with viscin threads are often regarded as indicating to entomophilous mode of pollination in the Ericaceae (Waha, 1984;Gillespie and Kron, 2013); however, there are many entomophilous taxa in Ericaceae without viscin threads on their pollen tetrads (Buchmann, 1983).High seed-set success was reported for Phyllodoce aleutica whose pollen tetrads have viscin threads, on the other hand, co-occurring P. caerulea whose pollen tetrads do not have viscin threads shown the lower seed-set success (Kasagi and Kudo, 2003).The presence or absence of viscin threads may possibly affect the crossing and selfing ability of these two Phyllodoce species.Viscin threads might be anticipated in all species with open-campanulate inflorescence, since these are associated with insect pollination.The absence of viscin threads in P. caerulea might indicate to a secondary loss (Table 2), since viscin threads are present at least in some species within some genera of the Phyllodoceae (Gillespie and Kron, 2013).
The genera Epigaea, Rhodothamnus, Phyllodoce and Kalmiopsis, are consistently made a clade in all molecular analyses (Kron et al., 2002;Gillespie andKron, 2010, 2013); the close relationship within these four genera also represented in their palynological features.For example, all these genera possess apocolpial exine sculptures rugulate and/or its derivatives (Table 2; Figs 2B, C, G-I, L).The exine sculpture has also been proven as a useful taxonomic tool at the infrageneric level (Sarwar andTakahashi, 2006, 2012), although various palynological characters have been found to be important at different taxonomic levels.For example, Elliottia bracteata and E. paniculata are sister taxa in all molecular analyses (Gillespie and Kron, 2013) and are characterised by the similar value (0.72) of P/E ratio (Table 2).
The exine sculpture of Rhodothamnus chamaecistus, with clearly striate secondary sculpture on the rugulae (Type RS; Fig. 2L), is significantly different than that of other members of this tribe except Epigaea asiatica (RS/R; Fig. 2B), and very much similar to exine sculpture of members of the tribe Vaccinieae of subfamily Vaccinioideae (Sarwar and Takahashi, 2007).Hitherto, it is the first SEM study of Rhodothamnus pollen.In combined analyses of morphological and molecular data, Epigaea repens is strongly supported as sister to a clade of both Rhodothamnus species (Gillespie and Kron, 2013).The similarity in exine sculpture may be one of the indications of closeness of these two genera (Sarwar, 2007).The apocolpial exine with striate secondary sculpture may be an apomorphic palynological character state for this tribe, and Ericaceae as well (Kron et al., 2002;Sarwar and Takahashi, 2007).
The apocolpial exine sculpture types vary often within a single genus.For example, the exine sculptures in Epigaea asiatica and E. repens, are distinctly different (Type RS/R vs. R/P; Figs 2B,  D).The other (external) morphological features of E. asiatica also differ considerably from those of E. repens (Stevens, 1969), which might be due to disjunct geographic distribution.The infrageneric variation in palynological features due to geographic distribution has also been reported for some other genera of Ericaceae e.g., Pyrola (Takahashi, 1986) and Enkianthus (Sarwar and Takahashi, 2006).
The generic circumscription of the tribe Bejarieae (Kron et al., 2002) and the systematic position of Bejaria was a matter of debate for long time (Gillespie and Kron, 2010).Based on palynological, both quantitative and qualitative features it was reported that the monotypic genus Bryanthus is clearly distinguished from other members of the tribe Bejarieae -Bejaria and Ledothamnus (Sarwar, 2007).The molecular phylogenetic studies later confirmed the above finding and transferred the genus Bejaria to the tribe Phyllodoceae and construct a new tribe Bryantheae (includes Bryanthus and Ledothamnus) (Bush and Kron, 2008;Gillespie andKron, 2010, 2013).Between two sections, Bejaria sec.Bejaria is characterized by larger values of P, D/d, P/E and thicker apocolpial exine compared to those of Bejaria sec.Racemosae (Table 2).The close relationship between B. aestuans and B. subsessilis is supported by values of D/d, ecto-and endo-aperture length and apocolpial exine thickness (Table 2; Bush and Kron, 2008).Moreover, the monophyly of Bejaria may be supported by apocolpial exine sculpture (Table 2; Figs 1C, E, G−H).The pollen tetrads of Bejaria are similar to those of some Rhododendron species of the tribe Rhodoreae.But these similarities may be due to homoplasy.Indistinct costae and lower 2f/D in Bejaria are stable and distinct palynological characters within the Ericoideae except for some species of Rhododendron and Therorhodion (Sarwar, 2007).These palynological features might imply an evolutionary tendency to a reduced (smaller) ectoaperture (Warner and Chinnappa, 1986), and might be an apomorphic pollen character state for this genus as well as the family Ericaceae (Sarwar, 2007).Among the other genera of the tribe Phyllodoceae, the systematic significances of pollen morphological features of Kalmia have previously been discussed in details (Sarwar and Takahashi, 2012).
The pollen morphological data confirm the infra-and inter-generic relationships as identified by molecular phylogeny of Phyllodoceae (Gillespie and Kron, 2013) and/or vice-versa.Although various palynological characters were found to be taxonomically important at different taxonomic levels, the apocolpial exine sculpture is emerged as one of the most important palynological features of systematic importance.The rugulate apocolpial exine with striate secondary sculpture, and a reduced colpus might be apomorphic palynological character states for the tribe Phyllodoceae and the family Ericaceae.