Pollen morphology and its taxonomic significance in the genus

Pollen morphology of 52 species (out of c . 79) of the Bomarea subgenus Bomarea was examined using light microscopy and scanning electron microscopy (SEM), or using SEM alone. The studied species of Bomarea were stenopalynous, characterized by large, oblate, monosulcate monads with reticulate exine sculpture in most species. Wide variation was observed in quantitative palynological features. The studied taxa were divided into four major groups based on exine ornamentation observed under SEM: microreticulate, reticulate, coarsely rugulate, and psilate-perforate. The reticulate exine sculpture may be a plesiomorphic character state for the genus Bomarea , and the coarsely rugulate and finely rugulate-perforate or psilate-perforate exine sculptures may have evolved independently more than once. In agglomerative hierarchical clustering (AHC) analyses of the genus Bomarea using quantitative pollen data, the studied species were distributed in either two (similarity-based) or four (dissimilarity-based) major clusters. Neither the recent molecular phylogenetic analyses nor the AHC analyses of Bomarea have recovered clades/clusters that represent traditionally recognized subgeneric taxa for the genus. Therefore, the most reliable infrageneric classification of Bomarea can be achieved by combining morphological, palynological, and molecular data from more extentive sampling of all the species.


Introduction
Bomarea is the most diverse genus of Alstroemeriaceae, and it includes 100-120 species with a Neotropical distribution from Mexico in the north to Argentina/Chile in the south (Neuendorf 1977;Alzate 2005).The genus is largely restricted to the Andean range and its continuation in Central America (Hofreiter & Tillich 2002).Based on morphological features, the genus Bomarea is divided into four subgenera: Baccata (five spp.), Bomarea s. str.(c.79 spp.), Sphaerine (12 spp.), and Wichuraea (18 spp.; Hofreiter & Tillich 2002).The subgenus Bomarea is characterized by a generally twining habit, with flowers pendulous and actinomorphic or horizontally oriented and zygomorphic, tepals deciduous, ovary inferior and dehiscent, and fruit leathery.Baker (1888) divided this subgenus into four main groups according to the structure of the inflorescence and the relation of the inner and outer tepals, and Killip (1935) later named these four groups Multiflorae, Caldasianae, Edules, and Vitellinae.Presently, four sections are recognized within the subgenus Bomarea: Multiflorae, Edules, Goniocaulon, and Pardinae (Harling & Neuendorf 2003; for details see Hofreiter 2008).
As part of a comprehensive survey of pollen morphology in the genus Bomarea, pollen studies of three subgenera, viz.Baccata, Sphaerine, and Wichuraea, have already been published (Sarwar et al. 2015 and references therein).Light microscopy (LM) was mainly employed in previous palynological studies of this genus, and scanning electron microscopy (SEM) was employed in a few cases (for details see Sarwar et al. 2015).We describe here, using both LM and SEM, the pollen morphological features of the subgenus Bomarea, and evaluate their usefulness for the infrageneric classification of this genus.

Materials and Methods
Pollen morphology of 52 species (out of c. 79) of the Bomarea subgenus Bomarea and of one species each of the subgenera Baccata and Wichuraea was examined by light microscopy (LM) and scanning electron microscopy (SEM), or by SEM alone (Tab.1).Polliniferous materials used in this investigation were taken from dried specimens from the herbaria K, MO, MOL, NY, and USM.Herbarium abbreviations follow the Index Herbariorum (Thiers 2007).The pollen parameters studied and the LM and SEM preparation methods used follow Sarwar et al. (2010).Measurements were taken from at least 30 randomly selected grains from each specimen (Tab.2).To visualize the relationships among the studied species of the subgenera Baccata, Bomarea, Sphaerine, and Wichuraea, agglomerative hierarchical clustering (AHC) analyses were conducted using the XLSTAT 2009.3 program, based on the quantitative characters polar length (P), equatorial diameter (E), P/E ratio, and exine thickness.The palynological data for the subgenera Baccata, Sphaerine, and Wichuraea were taken from Sarwar et al. (2015), and dendrograms were built using AHC.Slides of all specimens have been deposited in the Hokkaido University Museum, Sapporo, Japan.Pollen size and shape classes were defined using the criteria of Erdtman (1952)

Results
Based on LM and SEM observations, the Bomarea pollen grains studied were monad, large, ellipsoid (boat-shaped), heteropolar; monosulcate, sulcus on the convex part of the grain, distinct, long, straight, wide at the equator, narrow near the poles, sometimes extended to the proximal pole (Fig. 1A-D  Granula were sometimes visible at the bottom of the lumina; these may correspond to "free standing columellae" (e.g.Fig. 1G; Hesse et al. 2009).The exine sculpture along with sulci was similar to that appearing at the equatorial position, but had relatively smaller lumina.
In agglomerative hierarchical clustering (AHC) analyses of the genus Bomarea using quantitative pollen data, the species studied were distributed in either two (similarity-based) or four (dissimilarity-based) major clusters (Fig. 5).Among these clusters, three include at least one species from each subgenus; cluster 1 (blue) comprises most of the taxa studied (41), cluster 2 (pink) comprises 12 taxa, cluster 3 (brown) comprises 16 taxa, and cluster 4 (black) comprises the single species B. ampayesana from the subgenus Wichuraea.

Discussion
The Bomarea species studied were stenopalynous and characterized by monad, monosulcate, large pollen grains (Figs.1-4 et al. 2015).However, there were significant differences in the values of the quantitative palynological characters, which may to some extent be related to differences in the mounting media (Meltsov et al. 2008) and the methods of preparation of the pollen grains (Schols et al. 2004), as well as the species' geographical distribution, floral size, etc. (AKM Golam Sarwar pers.obs.).For example, there were     significant differences between two B. andreana specimens, one of which had smaller pollen grains with a thinner exine (Davidson 7239, collected from the warmer region of Costa Rica), while the other had larger pollen grains with a thicker exine (Croats 34872, collected from the cooler region of Panama) (Tabs.1, 2).
A wide and generally continuous pattern of variation in P, E, P/E ratio, and exine thickness was observed at both the infra-and inter-species level (Tab.2).Among the sections of Bomarea subgenus Bomarea, section Goniocaulon produces pollen grains with relatively consistent P/E ratios (0.53-0.58) (Tab.2).Pollen grain size within a genus is influenced by internal (chromosome number and floral character) and external (temperature, mineral nutrition, and water conditions) factors (Stanley & Linskens 1974).However, no correlation between ploidy level and palynological features was observed in Bomarea.The only tetraploid species of Bomarea, B. hirtella, produced pollen grains similar in size to or slightly smaller than the diploid species (Tab.2; Cave 1967).
Auricula-like structures in pollen grains have been previously reported from only four Bomarea species, namely, B. brachysepala, B. bracteolata, B. glaucescens, and B. huanuco (Sarwar et al. 2015).In the present study, pollen with auricula-like structures was observed in 19 taxa (Fig. 1E; Tab. 2), including in B. ceratophora (Neuendorf 1977) in the subgenus Bomarea.These auricula-like structures may have some taxonomic importance for the genus Bomarea since they are relatively common in the subgenus Bomarea, but are completely absent in members of the southern group of the subgenus Wichuraea (Tab.2; Sarwar et al. 2015).Auriculate pollen grains are not common among extant plant taxa, and diverse Cretaceous pollen-bearing auriculate appendages have been variously described as gymnospermous and monocotyledonous (Elsik 1974).As the term "auriculae" is only applicable to the spores' structure (Punt et al. 2007), the term "apex" has sometimes been used for these auriculalike pollen structures (see Martín et al. 2012 for detailed terminological discussion).
A recent molecular phylogenetic analysis of Bomarea (Alzate et al. 2008) identified three major clades, but none of them correspond to traditionally recognized subgeneric taxa (Hofreiter & Tillich 2002).Only seven of the species we studied were included in that molecular analysis, and these seven are positioned in two different clades (Fig. 2    Among the quantitative pollen characters analyzed, equatorial diameter had the greatest influence on the position of taxa in the AHC cladogram (Fig. 5; Tab.2; Sarwar et al. 2015).The members of cluster 1 commonly produced pollen grains 52.67-62.93μm in size; cluster 2 produced relatively larger pollen grains (63.93-69.30μm); cluster 3 produced the smallest pollen grains (44.46-55.30 μm).The large pollen grains of B. ampayesana (subgenus Wichuraea) might be one of the reasons for its very distinct position in cluster 4 (black) of the AHC (Fig. 5; Tab.2; Sarwar et al. 2015).The greatest variation in pollen morphological features was observed in the subgenus Wichuraea (Fig. 5; Hofreiter & Tillich 2002), which also exhibited the greatest variation in other morphological features.
There were significant differences in the species composition of the different subclades/clusters recovered from both the molecular phylogenetic analysis (based on a few species; Alzate et al. 2008) and the AHC (based on quantitative pollen features; Fig. 5).Moreover, analyses based on different genomes (nuclear vs. plastid) not only lead to disagreements in the evolutionary relationships of taxa, but also greatly affected other phylogeny-based inferences and interpretations relating to taxonomy, morphological evolution, historical biogeography, and phylogenetic diversity (Zhang et al. 2015).Therefore, the most reliable infrageneric classification of Bomarea will be achieved through combined analyses of morphological, palynological, and molecular data from more extentive sampling-of all the species in the genus.
in Alzate et al. 2008).Exine sculpture was found to be the most important palynological character possessing phylogenetic importance.In the molecular phylogenetic analysis, four members of section Multiflorae (B.vestita syn.B. multiflora; Hofreiter 2008) were included in clade C; this was also supported and confirmed by palynological characters, especially exine sculpture Type II (Figs.2I, O, 3C-F; Tab. 2).The other species of section Multiflorae (B.setacea) are sister to clade B and are characterized by exine sculpture Type IV (Fig. 4K; Tab. 2), although wide variation was observed in the exine sculpture of two specimens of B. setacea (Type I vs. Type IV; Tab. 2), which may be due to polymorphism.Bomarea setacea is polymorphous taxon, especially in its general stature and in the shape and size of the leaves and inflorescence (Harling & Neuendorf 2003).Of the two other species of the subgenus Bomarea, B. edulis was included in clade A, and B. salsilla was sister to B. straminea (Fig. 2 in Alzate et al. 2008).

Figure 5 .
Figure 5. Dendrogram obtained from quantitative data by agglomerative hierarchical clustering analysis.Names of species are abbreviated to the first four to six letters of the specific epithets.

Table 1 .
List of Bomarea taxa used in this study along with their voucher specimens.* New palynological data.

Table 2 .
Variation in pollen characters of Bomarea subg.Bomarea showing mean value in micrometer and standard deviation.Minimum -maximum values in micrometer in parenthesis.Taxa are arranged alphabetically within the group.(A) Pollen grains with auriculae-like structures; n.d.Not discern.
; Tab. 2; Sarwar et al. 2015).The pollen morphology of 36 of these species, indicated by asterisks in Tab. 1, was studied for the first time using either LM or SEM.