Taxonomic re-evaluation of the Azolla genus in Portugal

ABSTRACT

The Portuguese and the European Flora refer to the presence of two or three Azolla species in Portugal: A. filiculoides Lam., A. caroliniana Willd. and/or Azolla mexicana Presl., the latter included in the last edition of Flora Europaea. In the present work, the taxonomy of Azolla species is reviewed using the two most important characters that can distinguish between these two/three species: papillae in the dorsal leaf lobe and perine architecture of the megaspore apparatus. Other characteristics, such as the hyaline border cells of the dorsal leaf lobes and the number of glochidia septa in microsporangium massulae, are also used. All the Azolla specimens, collected from several locations in Portugal, were identified as Azolla filiculoides Lam. This identification disagrees with previous published reports on Azolla taxonomy in Portugal as well as with herbarium identification.     

Leaf architecture of the genus Didymaea Hook. f. (Rubiaceae)

We examined 532 specimens representing eight species of Didymaea (Rubiaceae) to describe their leaf architecture. Characters observed in the herbarium specimens and cleared leaves were leaf base, apex, and margin; trichome type and distribution; vein orders from primary to higher order veins, veinlets, and ultimate marginal vein. Six morphometric variables were analyzed using variance and principal component analysis to identify which characters are useful to distinguish species. Most of the species present brochidodromous venation pattern, except D. hispidula, which has eucamptodromous venation pattern; Didymaea floribunda is unique in having cylindrical and bicellular trichomes. Principal component analysis revealed that 78% of the total variation is mainly related to leaf maximum width. Variance analysis showed significant differences between D. microflosculosa and the other species. Architectural features were useful for differentiating all the species, and an identification key for the species of Didymaea based only on leaf characters is presented.     

Rhus coriaria var. zebaria (Anacaridaceae), a new variety from Iraq

Rhus coriaria var. zebaria from Duhok/Aqrae, Kurdistan, Iraq is described as a variety new to science. Field expeditions started in spring 2011, continued during 2012 and 2013 with the aim of collecting sumac specimens from different physiographic regions of Kurdistan, Iraq. A total of ten plants for each variety were sampled. An equal number of tagged herbarium specimens were prepared. The holotype was designated and deposited together with other herbarium specimens in the herbarium of the Faculty of Agriculture and Forestry, Univ. of Duhok. Morphological characters of the leaf, inflorescence, flowers, infructescence, and fruit were studied. The pH value of the pericarp in addition to the hilum length was measured. Results were compared with Rhus coriaria L. var. coriaria. The most distinctive feature of the new variety is the dull whitish brown color of the drupe pericarp of var. zebaria as compared to the reddish brown color of the drupe pericarp of var. coriaria. The whitish color of the drupes of var. zebaria, in combination with their larger size, represents diagnostic characters for distinguishing this variety. In contrast, most leaf characters are of little or no taxonomic value in this species. Only shape, apex and marginal teeths of the leaflets show minor differences for separating the two varieties. Female and male inflorescences and flowers characters show no significant differences. Both hilum length and pH value characters may be useful for taxonomic application if mean values are taken in consideration.     

SPORE MORPHOLOGY IN THE DICKSONIACEAE. I. THE GENERA CYSTODIUM,THYRSOPTERIS, AND CULCITA

The most mature spores available in herbarium specimens of the dicksoniaceous genera Cystodium, Thyrsopteris, and Culcita were studied by scanning electron microscopy and light microscopy, and representative specimens were analyzed to determine the number of spores produced per sporangium. Thyrsopteris and Culcita feature 64-spored sporangia, but Cystodium is consistently 32-spored. Spores were analyzed both in their native state as found on the specimens and in a perine-free state achieved by treatment with sodium hydroxide or acetolysis mixture. The sodium hydroxide assay demonstrated the presence of a perine in Cystodium, Thyrsopteris, and Culcita subgenus Culcita, but no evidence of a perine so defined was found in Culcita subgenus Calochlaena. Spores of Cystodium feature a nearly psilate exine overlain by a striate inner perine and a granular outer perine and are in several respects similar to those of Metaxya in the cyatheoid-dicksonioid complex and to those of Saccoloma in the dennstaedtioids. The most mature Thyrsopteris spores available may not have been fully mature. They feature a sparsely distributed, granular perinous layer over a microverrucate sculptine. The latter topography is taken as largely perinous since treatment with sodium hydroxide left a nearly psilate exine. The spore morphologies of Cystodium and Thyrsopteris reinforce the taxonomic distinctness of these monotypic genera indicated by their other characters. The two subgenera of Culcita are very dissimilar in their spore morphologies. The exine in subgenus Culcita ranges from psilate to slightly microverrucate proximally and distally, with varying margo development. Spores of subgenus Calochlaena are strongly differentiated from those of subgenus Culcita by their exine of broad spinules which vary in their degree of lateral fusion to each other and in the granular appearance of their distal surfaces. Spore morphology in Culcita strongly supports the argument of those who would raise its subgenera to generic rank.     

Morphological analysis of three equivocal sibling species of Deprea (Solanaceae)

Taxonomic confusion among closely related and morphologically similar Deprea species has persisted in the literature and in the identification of species. Morphological variation among three closely related, monophyletic Deprea species was studied to determine if and how they can be distinguished. Their sympatric occurrence in Venezuela afforded an opportunity to couple field study with analysis of herbarium specimens representing their entire geographic range. An analysis of 94 morphological characters resulted in five vegetative and 13 reproductive taxonomically informative traits. Canonical variates analysis clearly separated the three species using six quantitative traits. We conclude that these taxa, although quite variable and similar morphologically, are taxonomically distinct. Results of character analysis indicated that D. orinocensis is morphologically more similar to D. bitteriana than either are to D. paneroi. In D. paneroi, small, sterile anthers on fruit-bearing plants and the absence of fruits on plants possessing large, plllen-bearing anthers, suggest cryptic dioecy. Based on these data, D. granulosa is considered to be a synonym of D. orinocensis: Athenaea bitteriana, a misapplied synonym, is the correct basionym and is applicable to many specimens identified as D. granulosa. We submit a new combination, D. bitteriana (Werderm.) Sawyer & Benítez, and designate a lectotype to accommodate these findings.     

Changes in plant collection practices from the 16th to 21st centuries: implications for the use of herbarium specimens in global change research

Background and AimsHerbaria were recently advertised as reliable sources of information regarding historical changes in plant traits and biotic interactions. To justify the use of herbaria in global change research, we asked whether the characteristics of herbarium specimens have changed during the past centuries and whether these changes were due to shifts in plant collection practices.MethodsWe measured nine characteristics from 515 herbarium specimens of common European trees and large shrubs collected from 1558 to 2016. We asked botanists to rank these specimens by their scientific quality, and asked artists to rank these specimens by their beauty.Key ResultsEight of 11 assessed characteristics of herbarium specimens changed significantly during the study period. The average number of leaves in plant specimens increased 3-fold, whereas the quality of specimen preparation decreased. Leaf size negatively correlated with leaf number in specimens in both among-species and within-species analyses. The proportion of herbarium sheets containing plant reproductive structures peaked in the 1850s. The scientific value of herbarium specimens increased until the 1700s, but then did not change, whereas their aesthetic value showed no systematic trends.ConclusionsOur findings strongly support the hypothesis that many characteristics of herbarium specimens have changed systematically and substantially from the 16th to 21st centuries due to changes in plant collection and preservation practices. These changes may both create patterns which could be erroneously attributed to environmental changes and obscure historical trends in plant traits. The utmost care ought to be taken to guard against the possibility of misinterpretation of data obtained from herbarium specimens. We recommend that directional changes in characters of herbarium specimens which occurred during the past 150‒200 years, primarily in specimen size and in the presence of reproductive structures, are accounted for when searching for the effects of past environmental changes on plant traits.     

Comparative anatomy of leaves and stems in some species of the South American genus Chrysolaena (Vernonieae,Asteraceae) and taxonomic implications

Vegetative anatomical features are poorly known in the South American genus Chrysolaena. In this study, leaves and stems of six Chrysolaena species were described and compared morphologically and anatomically using diaphanization, microtome serial sectioning and scanning electron microscopy. The species differed in leaf epidermis, type of stomata, shape of anticlinal walls of epidermal cells, trichome density, and presence or absence in stems of small air spaces in the cortical parenchyma and of druse‐shaped oxalate crystals. Furthermore, glandular trichomes and three types of non‐glandular trichomes with different number of basal cells were identified on leaves and stems. Collectively, these features proved instrumental to discriminate among the six studied species, suggesting that leaves and stems of Chrysolaena can represent a source for taxonomically useful characters. We also discuss anatomical features in relation to the environmental conditions in the species’ habitats.     

Combining traditional morphometrics and molecular markers in cryptic taxa: towards an updated integrative taxonomic treatment for Veronica subgenus Pentasepalae (Plantaginaceae sensu APG II) in the western Mediterranean

The aim of this study was to quantify the phenotypic differences amongst the taxa included in Veronica subgenus Pentasepalae, represented in the western Mediterranean area. In combination with previously obtained data on genotypic differences, this enabled the recognition of eight taxonomic units. The delimitation of the boundaries among species and subspecies was mainly based on molecular data, whereas the best characters to discriminate taxa were selected after an accurate study of herbarium material and living populations. Such a selection was also supported by a detailed morphometric study of 30 quantitative leaf traits in 88 populations. Several morphological characters, including the length of the trichomes, length of the petiole and leaf division, were found to be robust, and could be useful in the identification of the taxa involved. As a conclusion, a key to the taxa is proposed, as well as a complete nomenclatural treatment for the group, which includes the typification of several names. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 68–87.     

SPORE MORPHOLOGY IN THE CYATHEACEAE. III. THE GENUS TRICHIPTERIS

Scanning electron microscopy supported by light microscopic L-O analysis is used in a palynological study of the genus Trichipteris of the tree fern family Cyatheaceae. The spores of all but two of the 55 species are investigated and the results are related to previous findings for Trichipteris species in the literature. A perine layer consisting of a network of slender, smooth- or rough-textured interlocking strands with free ends uniformly characterizes the genus and is the same as the perine type previously reported in neotropical species of Sphaeropteris excluding the S. horrida group. The exine is analyzed both in spores which have not yet developed the perine layer and in spores whose perine has been removed by chemical treatment. Exine sculpturing shows great variation in the genus, ranging from essentially psilate to variously pitted (foveolate, foraminate), with topography ranging from plane to verrucate-tuberculate, and with the verrucae-tubercles themselves often variously pitted. In many instances, exine sculpturing types correlate with the infrageneric species groupings or affinities hypothesized by the recent monographers. In other cases, palynological characters offer new insight into species relationships that were previously unclear to monographers or that were interpreted differently on the basis of other characters. The exine sculpturing types reinforce the relationship evident between Trichipteris and neotropical Sphaeropteris on the basis of perine morphology.     

HIGH-RESOLUTION LEAF X-RADIOGRAPHY IN SYSTEMATICS AND PALEOBOTANY

The foliar vein nets of many seed plants and ferns display systematically informative characters. These venation characters traditionally have been observed by chemically clearing and staining leaves, a process that is slow, involves toxic chemicals, and yields delicate, glass-mounted specimens that require long-term maintenance to prevent or correct bubbling or crystallization of the mounting medium. A technique that uses X-rays and photographic film to produce images of leaf venation consistently shows veins that are 50–100 μm thick. Although the X-ray images are slightly less detailed than the best cleared and stained leaves, the images can be made much more quickly, are more easily stored and reproduced, and do not require permanent alteration of the original herbarium specimen. The technique should facilitate the use of vein characters in systematics, and the identification and systematic analysis of fossil leaves.     

Taxonomic implications of foliar epidermis in Impatiens (Balsaminaceae): Investigating 12 Pakistani taxa as an example

Impatiens L. is taxonomically a complicated genus that necessitates fresh characterization to resolve its taxonomy. This study therefore aimed at ascertaining micro-morphological characters in the foliar epidermis of Impatiens. For this purpose, we gathered twelve taxa (10 species and 2 subspecies) mostly from the northern regions of Pakistan and studied qualitative and quantitative characters depicting variation in epidermal cells, stomata, and trichomes on both surfaces. Multicellular, uniseriate trichomes were observed only in I. balsamina L., and raphides in I. balfourii Hook. f. and I. thomsonii Hook. f. while stomata were exclusively anomocytic in all species. Morphological variation in the foliar epidermal characters and their diagnostic value has been depicted by constructing a taxonomic key to all taxa. The study revealed that although foliar epidermal characters in Impatiens did not desirably assort taxa on the basis of structural topographies, yet demonstrated adequate variation distinguishing the Pakistani taxa.     

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.     

Leaf anatomy ofDombeya andNesogordonia (Sterculiaceae), emphasizing epidermal and internal idioblasts

We studied leaf anatomy, using clearings, resin sections, and scanning electron microscopy, from mostly herbarium specimens of 123 species ofDombeya and 11 species ofNesogordonia (Sterculiaceae). Species were placed in seven idioblast categories, ranging from those without any to single and bicelled epidermal forms to multicelled nodules and single mesophyll idioblasts. Idioblast contents are possibly mucilaginous, but were not identified. In these two genera the range of foliar idioblast morphology surpasses that known previously for the entire family. Leaves are dorsiventral with mostly abaxial anomocytic stomata and typical palisade and spongy layers; paraveinal mesophyll is lacking. Miniature glandular (clavates, capitates) and nonglandular (mostly stellate) trichomes occur. Prismatic crystals predominate; epidermal prismatics and mesophyll druses are rare.     

国产柿属(Diospyros)的叶表皮形态研究及其分类学意义

为了解柿属(Diospyros)植物的叶表皮特征,在光学显微镜下观察了46种2变种植物的叶表皮微形态特征,并进行了主成分分析。结果表明,柿属植物的叶表皮微形态可分为2组：A组的表皮细胞为不规则形,垂周壁浅波状或深波状;B组的表皮细胞为多边形,垂周壁平直或弓形。气孔全部位于下表皮,以无规则型为主,但同时有辐射型、环列型和十字型气孔存在。大多数种具有表皮毛,表皮毛有非腺毛和头状腺毛的区别。柿属植物的叶表皮微形态特征类型多样,能够为部分类群的分类提供较好的形态学证据。     

DNA extraction from processed wood: A case study for the identification of an endangered timber species (Gonystylus bancanus)

We applied human forensic techniques to the extraction of whole genomic DNA from processed wood samples to explore the possibility of identifying an endangered tropical timber species by using DNA sequencing technology. High-yield and high-quality DNA samples were obtained from 2 commercial wood and 3 herbarium samples. Large PCR fragments ranging from 500–800 bp were successfully amplified from 2 chloroplast and 1 mitochondrial regions in all 5 samples, indicating limited degradation of the cytoplasmic genomes. DNA extraction from stem wood taken from herbarium specimens appeared superior to that from stem wood with bark intact or from leaf samples. DNA sequences from thetrn regions allowed for easy identification of the focal species based on GenBank Blast search. Little sequence variation was observed in the 3 regions, with the mitochondrialcox3 region completely conserved. Extraction of high-quality and large intact DNA fragments makes dry wood materials amenable to various DNA marker-based applications, including fingerprinting and historical approaches. By sampling stemwood, the wealth of historical information housed in international herbaria can be explored with minimal damage to taxonomically important features.     

Phytochemical data parallel morpho-colorimetric variation in Polygala flavescens DC.

Abstract

Phytochemical data, integrated with other sources of information, represent a valuable tool helping to solve different kinds of taxonomic problems in plant systematics. In the present study, a comparative investigation, in order to clarify the systematic relationships of the three subspecies currently recognized within the Italian endemic Polygala flavescens, was carried out. Preliminarily, a morphometric and colorimetric analysis, in order to test the degree of morphological distinctiveness among the taxa, was performed. Then, a phytochemical analysis based both on volatile and non-volatile compounds was obtained. Concerning the morpho-colorimetric analysis, our results confirm most of the characters as useful to discriminate the three subspecies. In addition, some volatile and non-volatile compounds are good taxonomic markers. Morpho-colorimetric variation is clearly paralleled by phytochemical results, confirming the value of this kind of data to infer relationships in plant systematics. Based on these results, we support a taxonomic treatment at subspecific level for the involved taxa. Finally, based on the most significant morphological characters, a revision of herbarium specimens allowed to redefine the distribution pattern of the three subspecies. Accordingly, the range of P. flavescens subsp. maremmana is limited to Mt. Argentario (southern Tuscany) only. A key is also reported for the identification of the three subspecies.     

The micromorphologies of seed coats and petal trichomes of the taxa of Viola subsect. Boreali-Americanae (Violaceae) and their utility in discerning orthospecies from hybrids

Viola subsect.Boreali-Americanae, confined to North America including northern México, has long been considered one of the most taxonomically difficult temperate groups of the angiosperms. Hybridization, followed in some localities by introgression, has made it difficult for systematists to discern the “core” species by using only standard macromorphological characters. Analyses that employed scanning electron microscopy (SEM) of seed coats and petal trichomes generated new characters. SEM of the surface of seeds of each of the orthospecies revealed a suite of primary and secondary sculpture characters unique to each species that are correlated with a suite of macromorphological characters. In a number of species, SEM of petal trichomes provided additional unique characters. SEM of the surface of seed coats enabled, also, the identification of instances of hybridization by revealing blends of structures of the primary and secondary sculpture typical of different orthospecies and instances of putative introgression by revealing deviations in surface structures from those typical of orthospecies. A procedure based on iterative examinations of macromorphology and micromorphology, which was developed in this research, enable the discernment of hybrids and putative introgressants. Combining the micromorphological and macromorphological data with ecological and phytogeographical data enabled the delimitation of sixteen orthospecies.     

Systematics ofPrimula veris (Primulaceae)

Primarily, 50 characters taken from herbarium specimens, cultivated plants, and whole populations ofPrimula veris L. were critically evaluated. Only 24 characters were found to be suitable for taxonomic use. The geographical and multivariate analysis of about 1000 herbarium specimens from the whole area lead to a reduction of the number of subspecies. Only a less restricted subsp.veris (incl. subsp.canescens, subsp.inflata and probably including subsp.macrocalyx) and subsp.columnae (incl. subsp.suaveolens) can be distinguished considering the variability of characters within an individuum, under different culture conditions, and during the growth period. The status of the taxon macrocalyx cannot be cleared presently.