Genome size and base composition of Bromeliaceae species assessed by flow cytometry

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of Bromeliaceae species, which constitutes relevant information for studies of taxonomy, evolution, genetic diversity, and reproductive biology in bromeliads. Nevertheless, C values have only been estimated for 58 out of the 3,140 existing Bromeliaceae species. Aiming to contribute to the genome database of Bromeliaceae, the current study was carried out to measure the nuclear DNA content and base composition of Bromelioideae and Tillandsioideae species occurring in the Atlantic Rainforest. The most adequate FCM procedure provided histograms exhibiting G₀/G₁ peaks with coefficients of variation below 5%, so that these histograms were used to measure the mean 2C and AT% values for all collected Bromelioideae and Tillandsioideae species. These values were statistically compared, and dendrograms were plotted. A second comparison was performed among all mean 2C values reported for Pitcairnioideae, Tillandsioideae, and Bromelioideae species. In accordance with previous statistical comparisons, two groups were formed: cluster 1, composed by Tillandsia loliacea, Tillandsia usneoides, and Tillandsia cyanea, and cluster 2, gathering other 69 species. Based on these results, we concluded that FCM was a rapid, accurate, and reliable technique to assess genome size and base composition. Furthermore, the FCM data reported here will contribute to the Monocot and Bromeliaceae database, which still displays several ongoing gaps, especially for endemic species.     

Ovule ontogeny in Billbergia nutans in the evolutionary context of Bromeliaceae (Poales)

The infrasubfamilial relations within Bromeliaceae are still not fully understood, especially in Bromelioideae. Billbergia nutans (Bromelioideae) is one of the most widely cultivated bromeliads. This study aims to characterise ovule ontogeny in B. nutans and to review the literature concerning ovules at a family level, to verify useful data for taxonomy and identify the evolutionary scenario of embryological characters within the family. Novel data are recorded for the genus. The ovules of Bromeliaceae are anatropous, bitegmic and crassinucellate, with trizonate ovule primordia, a monosporic development, a linear type of tetrad, callosic cell walls during meiosis, a functional chalazal gynospore, a Polygonum-type gametophyte, the presence of a hypostase, and a dermal origin of the integuments. In B. nutans, the chalazal appendage morphology shows intraspecific variability and might thus not be an appropriate character for taxonomic use. This is the first report on the three-dimensional reconstruction of the female gametophyte of a monocotyledon. Many useful characters are suggested for the taxonomy of Bromeliaceae. The evolutionary scenario of the ovule in the family indicates that the earliest-divergent subfamily, Brocchinioideae, possesses a reduced state of several characters. Among the family, some characters appear homoplasious and might be relevant when applied to lower taxonomic levels. The two-layered integuments and the small number of ovules per carpel are probably plesiomorphic states in Poales.     

The systematic implications of the complexity of leaf flavonoids in the bromeliaceae

In a leaf survey of 61 species of the Bromeliaceae, an unexpectedly wide spectrum of flavonoid constituents was encountered. The family is unique amongst the monocotyledons in the frequency and variety of flavonoids with extra hydroxylation or methoxylation at the 6-position. More common flavonols (in 43% of species) and flavones (in 13%) are distributed throughout the family whereas the rarer flavonoid classes are restricted to one or two of the three subfamilies. Thus 6-hydroxyflavones were found in both the Pitcairnioideae (in 50%) and the Tillandsioideae (in 14%) but patuletin (in 19%), gossypetin (in 1 species) and methylated 6-hydroxymyricetin derivatives (in 24%) were detected only in the Tillandsioideae. A new flavonol, 6,3′,5′-trimethoxy-3,5,7-4′-tetrahydroxyflavone, was identified as the 3-glucoside in Tillandsia usneoides and a new glycoside, patuletin 3-rhamnoside, in Vriesea regina. Myricetin glycosides were found only in the Bromelioideae and their presence here and the concomitant absence of 6-hydroxyflavonoids could indicate the primitive condition of this subfamily. The flavonoid results, in toto, confirm the view based on morphology, that the Bromeliaceae occupies an isolated position in relation to other monocot families.     

Chromosome numbers and DNA content in Bromeliaceae: additional data and critical review

For the large Neotropical plant family Bromeliaceae, we provide new data on chromosome numbers, cytological features and genome size estimations, and combine them with data available in the literature. Root‐tip chromosome counts for 46 species representing four subfamilies and a literature review of previously published data were carried out. Propidium iodide staining and flow cytometry were used to estimate absolute genome sizes in five subfamilies of Bromeliaceae, sampling 28 species. Most species were diploid with 2n = 50 in Bromelioideae, Puyoideae and Pitcairnioideae, followed by 2n = 48 observed mainly in Tillandsioideae. Individual chromosome sizes varied more than tenfold, with the largest chromosomes observed in Tillandsioideae and the smallest in Bromelioideae. Genome sizes (2C‐values) varied from 0.85 to 2.23 pg, with the largest genomes in Tillandsioideae. Genome evolution in Bromeliaceae relies on two main mechanisms: polyploidy and dysploidy. With the exception of Tillandsioideae, polyploidy is positively correlated with genome size. Dysploidy is suggested as the mechanism responsible for the generation of the derived chromosome numbers, such as 2n = 32/34 or 2n = 48. The occurrence of B chromosomes in the dysploid genus Cryptanthus suggests ongoing speciation processes closely associated with chromosome rearrangements. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 349–368.     

Leaf epidermal characteristics and genetic variability in Central European populations of broad-leaved Festuca L. taxa

Leaf epidermal (micromorphological and micromorphometric) features of the leaf blade were examined with light and scanning electron microscopy in 49 Central European natural populations of species belonging to subgenera Schedonorus and Drymanthele within the genus Festuca L. In addition, molecular biological (random amplified polymorphic DNA) studies were conducted on selected populations. The goal of the study was to reveal the degree of anatomical and genetic variability at the taxon and population level, as well as to determine the most important characters that allow differentiation of the taxa in these two subgenera. The anatomical variation was compared with the genetic distance between taxa. Investigated taxa exhibited great anatomic variability in terms of both qualitative (presence or absence, shape and appearance of various cell types) and quantitative characters (frequency and size of cells). Strong correlation was found between the genetic distance of populations and the frequency and size of silica cells in the costal zone of the epidermis, which indicates that silica cells are largely determined by the genetic background, regardless of varying environmental conditions. Our studies highlight that despite the high level of variability in anatomical features and genetic polymorphism, these fescue populations clearly segregate both in terms of epidermal structure and genetic constitution.     

Alcantarea (Bromeliaceae) leaf anatomical characterization and its systematic implications

Alcantarea (Bromeliaceae) has 26 species that are endemic to eastern Brazil, occurring mainly on gneiss–granitic rock outcrops (‘inselbergs’). Alcantarea has great ornamental potential and several species are cultivated in gardens. Limited data is available in the literature regarding the leaf anatomical features of the genus, though it has been shown that it may provide valuable information for characterizing of Bromeliaceae taxa. In the present work, we employed leaf anatomy to better characterize the genus and understand its radiation into harsh environments, such as inselbergs. We also searched for characteristics potentially useful in phylogenetic analyses and in delimiting Alcantarea and Vriesea. The anatomical features of the leaves, observed for various Alcantarea species, are in accordance with the general pattern shown by other Bromeliaceae members. However, some features are notable for their importance for sustaining life on rock outcrops, such as: small epidermal thick‐walled cells, uneven sinuous epidermal walls, hypodermis often differentiated into lignified layers with thick‐walled cells, aquiferous hypodermis bearing collapsible cells, and the presence of well developed epicuticular stratum. Alcantarea leaves tend to show different shapes in the spongy parenchyma, and have chlorenchymatous palisade parenchyma arranged in more well‐defined arches, when compared to Vriesea species from the same habitat.     

Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil)

This work describes the leaf anatomy of 16 species belonging to three genera of the Malpighiaceae family found in the Cerrado (Minas Gerais State, Brazil). The scope of this study was to support the generic delimitation by contributing to the identification of the species and constructing a dichotomous identification key that includes anatomical characters. The taxonomic characters that were considered to be the most important and used in the identification key for the studied Malpighiaceae species were as follows: the presence and location of glands; presence of phloem in the medullary region of the midrib; mesophyll type; presence and type of trichomes; and presence, quantity, and disposition of accessory bundles in the petiole. It was also possible to indicate promising characters for future taxonomic and phylogenetic studies in the Malpighiaceae family, especially for the Banisteriopsis, Byrsonima, and Heteropterys genera.     

Phylogenetics of Puya (Bromeliaceae): Placement, major lineages, and evolution of Chilean species

Puya (Bromeliaceae), a large genus of terrestrial bromeliads found throughout a range of elevations in the Andes and central Chile, is of great systematic, evolutionary, and biogeographical interest. This first molecular phylogenetic study of Puya and related bromeliads employs matK, trnS-trnG, rps16, and PHYC sequences. Chloroplast DNA, nuclear DNA, and combined DNA data all place Puya closest to subfamily Bromelioideae. Nuclear and combined data support Puya as monophyletic, and the two subgenera are nonmonophyletic. All data indicate that the Chilean species of Puya are early diverging within the genus, consistent with Chilean genera as the first-diverging members of subfamily Bromelioideae. Central Chile is identified as a key region for understanding the biogeographical history of Bromeliaceae, as is true with other South American plant groups. A complicated history involving early chloroplast capture and later secondary hybridization and/or introgression is seen in Chilean lineages. These events help explain the occurrence of sterile inflorescence tips, floral color and shape, and leaf indument. The ecological radiation of Puya appears coincident with the final, recent rise of the Andes and subsequent high-elevation habitat diversification. Additionally, geographical distribution, rather than moisture or elevational adaptations, correlates to species relationships. Evolution of CAM photosynthesis has occurred multiple times.     

Leaf anatomical characteristics of Ugandan species of Festuca L. (Poaceae)

The importance of leaf anatomical characters in the taxonomy of Ugandan Festuca species was investigated. In particular, leaf cross sections were studied for variation in disposition of the sclerenchyma tissue to determine whether this is as taxonomically useful as is implied in much of the literature. Also the leaf epidermis was investigated for the presence of diagnostic features. A lot of variation in disposition of sclerenchyma tissue was found in F. abyssinica, which did not support the recognition of F. elgonensis and F. richardii as good species, yet they were described mainly based on leaf anatomy. The ring of sclerenchyma in F. pilgeri and the presence of papillae on the epidermis of F. claytonii supported them to be good species. The presence of silica bodies on the epidermis of Ugandan species of Festuca was investigated for the first time and is confirmed using energy dispersive X-ray analysis. In conclusion, leaf anatomy should be used together with other non-anatomical characters before recognizing species.     

Comparative vegetative anatomy and classification of Diseae (Orchidaceae)

The leaf, stem, root, tuber and dropper anatomy of the orchid tribe Diseae (including the subtribes Satyriinae, Disinae, Brownlecinac, Huttonaeinae and Coryciinae) is reviewed. The study is largely based on investigations of 123 species, and data from several previous publications have also been incorporated. Two characters were identified as being taxonomically valuable: (1) the presence of sclerenchyma caps associated with leaf vascular bundles, and (2) the degree of dissection of the siphonostele of the tuber (‘polystelic’ or ‘monostelic’). The phylogenetic analysis shows that anatomical characters do not change the basic structure of a cladogram that is based on morphological characters. The taxa of Diseae are discussed on the basis of anatomical data. Subtribes Satyriinae (excluding the anatomically unusual genus Pachites), Brownleeinae, Huttonaeinae, and Coryciinae are uniform in. critical anatomical characters. However, subtribe Disinae is rather diverse in vegetative anatomy. Disa sect. Micranthae differs from the rest of the genus in its leaf anatomy. The occurrence of foliar sclerenchyma bundle caps and ‘polystelic’ tubers supports the incorporation of Herschelianthe in Disa sect. Stenocarpa.     

Comparative leaf anatomy and morphology of some neotropical Rutaceae: Pilocarpus Vahl and related genera

Previous anatomical studies have been restricted to the foliar aspects of Pilocarpus. However, no anatomical studies analyzing the foliar aspects of Pilocarpus in relation to related genera have been carried out. Therefore, the aim of this study was to identify characters for future taxonomic and phylogenetic studies in Rutaceae, particularly in Pilocarpus, and to discuss the characteristics associated with the simple or compound leaf condition for the group. The petiole and the leaf blade of 14 neotropical Rutaceae species were analyzed, and the following characteristics were observed in all leaves studied: stomata on both surfaces; secretory cavities, including mesophyll type; camptodromous?Cbrochidodromous venation pattern; and free vascular cylinder in the basal region of the petiole. Additional promising characters were identified for future taxonomic and phylogenetic studies in the Rutaceae family, especially for the Pilocarpus genera.     

Floral anatomy of Bromeliaceae, with particular reference to the evolution of epigyny and septal nectaries in commelinid monocots

Floral anatomy is described in ten genera of Bromeliaceae, including three members of subfamily Bromelioideae, three Tillandsioideae, and four genera of the polyphyletic subfamily Pitcairnioideae (including Brocchinia, the putatively basal genus of Bromeliaceae). Bromeliaceae are probably unique in the order Poales in possessing septal nectaries and epigynous or semi-epigynous flowers. Evidence presented here from floral ontogeny, vasculature, and the relative positions of nectary and ovules indicates that there could have been one or more reversals to apparent hypogyny in Bromeliaceae, although this hypothesis requires a better-resolved phylogeny. Such evolutionary reversals probably evolved in response to specialist pollinators, and in conjunction with other aspects of floral morphology of Bromeliaceae, such as the petal appendages of some species. The ovary is initiated in an inferior position even in semi-epigynous or hypogynous species. The ovary of all so-called hypogynous Bromeliaceae is actually semi-inferior, because the septal nectary is infralocular; in these species the nectaries have a labyrinthine surface and many vascular bundles. Brocchinia differs from most other fully epigynous species in that each carpel is secretory at the apex and reproductive, rather than secretory, at the base.     

Do leaves in Cyperoideae (Cyperaceae) have a multiple epidermis or a hypodermis?

In Cyperaceae, leaf anatomical characters, in particular the presence of a hypodermis or of a multiple epidermis, have contributed in taxonomic and phylogenetic studies. In this family, the leaf epidermis is often described as uniseriate, and the cells of the subepidermal layers having no chloroplasts are treated as hypodermis. Both tissues have a different ontogenetic origin and hence are not homologous. The aim of the present work was to verify the origin of the subepidermal layers in eight species belonging to Cyperoideae. All species studied presented multiple epidermal layers that were confirmed by leaf ontogeny. In Fimbristylis complanata, F. dichotoma, Pycreus flavescens and P. polystachyos the mature leaves present multiple epidermal layers with cells of the distinct layers similar in shape and size; in the other species studied these cells are different. Especially in the latter case, a multiple epidermis is easily interpreted erroneously as a hypodermis, possibly leading to erroneous evolutionary conclusions. Making correctly distinction between a hypodermis and a multiple epidermis, and hence in case of doubt investigating the origin of the questioned tissue, is compulsory in order to use both characters in a phylogenetic context. Though in the past often called ‘hypodermis’, our leaf ontogenetical observations show that in all species studied, the subepidermical layers constitute a multiple epidermis, originating from the protodermis.     

Comparative ecophysiology of CAM and C3 bromeliads. I. The ecology of the Bromeliaceae in Trinidad

Abstract This article deals with the physiological ecology of the Bromeliaceae, a large neotropical family containing both terrestrial and epiphytic forms, as well as many species with crassulacean acid metabolism (CAM). The article is in two parts. In the first, we review what is known of the occurrence of CAM and C₃ species in the Bromeliaceae. The photosynthetic pathways are discussed in the context of the major taxonomic divisions within the family and the great diversity of bromeliad life-forms. Of the three subfamilies, the Pitcairnioideae contain both C₃ and CAM species and are essentially all terrestrial. In contrast, the Tillandsioideae are entirely epiphytic or saxicolous, with CAM species being restricted to the genus Tillandsia, And in the Bromelioideae all species show CAM, but terrestrial and epiphytic forms are found in about equal numbers. The evidence suggests that both CAM and the epiphytic habit arose more than once in the family's evolutionary history. In the second part we consider the photosynthetic ecology of the various bromeliad life-forms in more detail using the specific example of Trinidad (West Indies). CAM bromeliads tend to be centred on the drier regions of the island and C₃ forms on the wetter areas. However, at any one site there is a marked vertical stratification of species within the forest profile. Based on the known habitat preferences of the bromeliads, six contrasting sites were selected for field studies in Trinidad. These ranged from arid coastal scrub to montane rain forest, the vegetational and climatic characteristics of which are described here. The constancy of δ¹³C values (carbon-isotope ratios) for individual CAM species in these markedly different habitats emphasized the need for ecophysiological studies to characterize environmental effects on CO₂ assimilation and transpiration. The following papers in this series present the results of a comparative investigation of gas exchange and leaf water relations of CAM and C₃ bromeliads in situ at the various sites.     

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.