Species differentiation in the genus Chara (Charophyceae): considerable phenotypic plasticity occurs within homogenous genetic groups

Charophytes are benthic algae with a complex morphology and high phenotypic plasticity. This has led to ambiguities in species delineation. However, until now genetic studies on Chara have been based on samples collected from a restricted geographic range or only included a restricted number of taxa. This may have hindered a general interpretation of the results. We applied barcoding of matK, a rapidly evolving coding section of the plastid genome, in 324 Chara samples collected from 19 countries, in order to test whether the distribution of barcode haplotypes among individuals was consistent with species boundaries as they are currently understood. The phylogenetic tree grouped the 324 Chara individuals, which according to commonly used identification keys represented 29 species, into 12 well-defined groups (i.e. monophyletic morphospecies or groups of morphospecies). Considerable morphological variation occurred within genetically homogeneous groups. This included traits which are commonly used for Chara species determination, such as the length and number of spine cells, the length of stipulodes and bract cells, cortication (tylacanthous, isostichous, aulacanthous and absent cortication), as well as sex differentiation. However, there were also substantial genetic differences among morphologically similar species (e.g. C. virgata – C. globularis – C. connivens). No morphological trait consistently reflected genetic differences. This indicates that morphological traits for specific taxa may serve as diagnostic tools for species delimitation, but that they are not generally suitable for inferring genetic differentiation or phylogenetic relationships. We propose that (i) C. virgata and C. strigosa, (ii) C. liljebladii, C. horrida and C. baltica, and (iii) C. hispida, C. rudis and C. polyacantha are conspecific. Our data also indicate that C. gymnophylla should be divided into tylacanthous forms (which are closely related to C. contraria) and aulacanthous forms (which are related to C. vulgaris).     

Oospore variation in <Emphasis Type="Italic">Nitella gracilis</Emphasis> and <Emphasis Type="Italic">Nitella mucronata</Emphasis> (Charales,Charophyceae) from Poland

The determination of the members of Charales (Charophyceae) is not easy. The main reason of this is a wide range of variation in vegetative characters within most of the species. Many features such as the extent of branchlet cortication, the degree of incrustation, length and number of bract cells, spine cells, stipulodes, diameter of the main axis and oospore size and color do not have universal taxonomic significance. In similar, many Nitella species characters such as: general habitus, branchlets and presence of the top segment (mucro), are helpful in determination, however they are often not developed. In this case, the features of oospore wall ornamentation revealed by light and scanning microscopy can be important taxonomic characters of the Charales. The present study shows interspecific variations of the oospore in two Nitella species from Poland which can be helpful in correct determination of both species. Some of the oospore features like number of striae and fossa width can not be used, as they appeared statistically unimportant, and cannot be used in distinguishing between both species. The presented results are the first detailed report of oospore wall ornamentation pattern from Poland for the species studied. Oospore features are presented in detail on the background of some additional tips and among them particularly: length and width of the oospore, ISI idex, width of fossa, width of striae and number of striae.     

DNA barcoding the genus Chara: molecular evidence recovers fewer taxa than the classical morphological approach

Charophytes (Charales) are benthic algae with a complex morphology. They are vulnerable to ecosystem changes, such as eutrophication, and are red‐listed in many countries. Accurate identification of Chara species is critical for understanding their diversity and for documenting changes in species distribution. Species delineation is, however, complicated, because of high phenotypic plasticity. We used barcodes of the ITS2, matK and rbcL regions to test if the distribution of barcode haplotypes among individuals is consistent with species boundaries as they are currently understood. The study included freshly collected and herbarium material of 91 specimens from 10 European countries, Canada and Argentina. Results showed that herbarium specimens are useful as a source of material for genetic analyses for aquatic plants like Chara. rbcL and matK had highest sequence recoverability, but rbcL had a somewhat lower discriminatory power than ITS2 and matK. The tree resulting from the concatenated data matrix grouped the samples into six main groups contrary to a traditional morphological approach that consisted of 14 different taxa. A large unresolved group consisted of C. intermedia, C. hispida, C. horrida, C. baltica, C. polyacantha, C. rudis, C. aculeolata, and C. corfuensis. A second unresolved group consisted of C. virgata and C. strigosa. The taxa within each of the unresolved groups shared identical barcode sequences on the 977 positions of the concatenated data matrix. The morphological differences of taxa within both unresolved groups include the number and length of spine cells, stipulodes, and bract cells. We suggest that these morphological traits have less taxonomic relevance than hitherto assumed.     

Morphological and molecular differentiation of smooth‐hound sharks (Genus Mustelus,Family Triakidae) from the Gulf of California

The genus Mustelus is the most species‐rich of the widespread family Triakidae whereby its taxonomy and systematics have been historically challenging. They represent a significant fraction of the shark catches of small‐scale fisheries in the Gulf of California. In order to provide information useful for their management and conservation, the morphological and genetic distinction of the four species found in the northern Gulf of California (M. albipinnis, M. californicus, M. henlei and M. lunulatus) were analyzed. Discriminant analysis of 10 morphometric variables placed each species in a distinct and significantly different region of multivariate morpho‐space. The variables contributing most to their distinction were inter‐nostril width, mouth length, upper and lower labial furrow length, and mouth width. Restriction fragment length polymorphisms (PCR‐RFLP) of the nuclear ITS2 ribosomal DNA (rDNA) confirmed that each species represents a genetically cohesive and independent evolutionary lineage. In spite of the difficulty in differentiating these Mustelus species, a few cephalic measurements are useful to separate them. A PCR‐RFLP assay (using RsaI and MspI on ITS2 rDNA amplicons) is also proposed for the molecular differentiation of these commercially harvested smooth‐hound sharks, constituting the first molecular marker available for their identification. These data provide morphological and genetic tools that can be used to improve their management and conservation.     

Morphological assessment of the Octopus vulgaris species complex evaluated in light of molecular‐based phylogenetic inferences

Cryptic species are common in the ocean, particularly among marine invertebrates such as octopuses. Delineating cryptic species is particularly problematic in octopus taxonomy where the plasticity recorded among taxonomic characters often results in low resolution at the species level. This study investigated the morphological relationships among seven phylogenetic clades (identified using cytochrome c oxidase subunit I) of the broadly distributed Octopus vulgaris species complex and close relatives. Morphological analyses in this study were successful in delimiting O. sinensis, Brazilian O. vulgaris and O. vulgaris sensu stricto, which was congruent with the molecular findings of this study. Analyses based on male morphology were successful in distinguishing 14 of 15 total pairwise comparisons and proved to be a more reliable indicator of species‐level relationships in comparison with female morphology. The majority of characters with the greatest discriminatory power were male sexual traits. Significant morphological differences were also recorded among sampling localities of conspecifics, with phenotype showing correlation with local environmental data. The findings of this study support the hypothesis that multiple O. vulgaris‐like species are currently being incorrectly treated under a single species name, O. vulgaris. Octopuses being exported globally under the name O. vulgaris are of extremely high fisheries market value and profile. Our findings have potentially significant implications for the naming and conservation of commercially harvested members of this species complex throughout their ranges.     

A POPULATION STUDY OF CHARA ZEYLANICA IN TEXAS,OKLAHOMA, AND NEW MEXICO

Examination of 140 collections of Chara zeylanica from 3 southwestern states shows that a strong dichotomy exists in the collections. Some produce 4-plated antheridia while others produce 8-plated antheridia. Absolute separation of the study material is possible on the basis of plate number. In addition, 3 other characters (length/width ratio of oospores; fertility of first branchlet node; and number of distal ecorticate branchlet internodes) show strong correlation with the antheridial type. Preliminary chromosomal work indicates that a segregation at this level may also be possible. In 4-plated collections examined, n = 28; 8-plated collections are usually n = 56; however, a few 8-plated collections are n = 42. No conclusion has been reached regarding the status of the latter collections.     

PHYSIOLOGICAL VARIABILITY WITHIN TEN STRAINS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

The effects of temperature, ionic composition, and conductivity on growth rates of ten strains of Chaetoceros muelleri Lemmerman (mostly var. subsalsum Johan. & Rushf.) were studied. Lipid content of stressed and unstressed cells and fatty acid composition were also determined. Considerable physiological variability was observed in the ten strains, although principal components analysis of physiological data indicated that all strains fell into one of two major groups: C. muelleri (var. muelleri and var. subsalsum) and an undescribed Chaetoceros species morphologically close to C. muelleri var. subsalsum. A high degree of agreement was found among morphological, physiological, and biochemical data sets, indicating that physiological and biochemical data may be helpful in making taxonomic decisions in diatoms, particularly in taxa with few morphological characters. We also conclude that nonmorphological characters such as those employed in the present study can be used to test phylogenetic hypotheses formulated from traditional morphological data.     

Molecular phylogeny and ecogeography of Onobrychis viciifolia Scop. (Fabaceae) based on nrDNA ITS sequences and genomic ISSR fingerprinting

In this study 40 specimens from 8 representative Iranian populations of Onobrychis viciifolia SCOP. were collected from their natural habitats. The specimens were biometrically assessed using 43 quantitative and 15 qualitative morphological characters. At each sample site we recorded ecogeographical variables. In order to evaluate the difference between the variation due to phenotypic responses and genetic adaptation, we generated nucleotide sequence data from the internal transcribed spacer of the nuclear rDNA genes (ITS) and carried out genomic fingerprints using inter‐simple sequence repeat PCR (ISSR). Cluster analysis of morphological characters divided the eight populations into three major groups. Leaf length, leaflet length and width, calyx length, plant height, and stem length were introduced as diagnostic characters for three different morphological groups. Furthermore, canonical correspondence analysis (CCA) of ecogeographical data showed correlations between morphological variations and ecogeographical factors. Clay%, saturation percentage (SP), total neutralizing value (TNV%), texture, minimum temperature, and geographic separation were the main environmental variables associated with morphological groups of O. viciifolia. ISSR analysis revealed three main groups which are in correspondence with morphological groups, indicating that the three morphological groups have been shaped by genetic and phenotypic responses to environmental conditions. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A multivariate morphological analysis of the Cardamine concatenata alliance (Brassicaceae)

Principal coordinate analysis using Gower’s similarity coefficient and canonical discriminant analysis were performed using morphological data to assess species distinctness in theCardamine concatenata alliance, comprising the eastern North American dentarias. The results of an overall principal coordinate analysis of the alliance using quantitative and qualitative characters revealed distinct and separate clusters that correspond to five previously recognized species (C. angustata, C. concatenata, C. diphylla, C. dissecta, C. maxima). One additional taxon,C. incisa, was found to group closely with theC. diphylla cluster. In a second principal coordinate analysis including only individuals fromC. diphylla andC. incisa, both are supported as distinct morphological entities. Canonical discriminant analysis of only above-ground quantitative characters was less effective in distinguishing previously recognized taxa, in keeping with the emphasis on qualitative characters in previous taxonomic treatments of the group.Cardamine incisa (Eames) Schumann (pro hybr.) is raised to the rank of species. A key to the species of theCardamine concatenata alliance is provided.     

Cryptic diversity and species delimitation in the Xiphinema americanum‐group complex (Nematoda: Longidoridae) as inferred from morphometrics and molecular markers

The Xiphinema americanum‐group constitutes a complex of about 55 species of polyphagous plant‐ectoparasitic nematodes with a worldwide distribution. This group of plant‐parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X. americanum‐group, five of which were tentatively identified as belonging to three new species and are described herein as X iphinema plesiopachtaicum sp. nov., X iphinema vallense sp. nov. , and X iphinema astaregiense sp. nov. , and 11 populations belonging to nine known species: Xiphinema brevisicum, Xiphinema duriense, Xiphinema incertum, Xiphinema luci, Xiphinema madeirense, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema parapachydermum, and Xiphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length (V), stylet length, oral aperture‐guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X. pachtaicum‐subgroup that includes X. plesiopachtaicum sp. nov. and X. vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X. pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral aperture‐guiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [D2‐D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 (ITS1)] and the protein‐coding mitochondrial gene, cytochrome c oxidase subunit 1 (coxI) were congruent, showing two main clades separating most of the species of X. americanum‐subgroup ‘sensu stricto’ from the X. pachtaicum‐subgroup. Agreement between phylogenetic trees and some morphological characters (viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA‐based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X. americanum‐group species. © 2015 The Linnean Society of London     

Numerical analysis of morphology of natural hybrids between Carex hostiana and the members of Carex flava agg. (Cyperaceae)

Uni‐variate and multi‐variate statistical methods, based on data taken from dried specimens, were used to determine the morphological variance of Carex hostiana 3 Carex flava agg. hybrids and to establish their parents among members of the C. flava complex. The following hybrids were found: C. demissa 3 hostiana [C. 3 fulva], C. hostiana 3 lepidocarpa [C. ×leutzii] and C. flava 3 hostiana [C. 3 xanthocarpa]. The least variable traits, namely beak length, utricle length, ratio of beak length to the overall utricle length, female spike width, and width of the lowest bract, proved to be the most useful in delimiting the hybrids. Carex flava 3 hostiana specimens usually have long utricles and beaks, wide male and female spikes, as well as wide bracts and leaf blades. Carex hostiana 3 lepidocarpa specimens are characterized by relatively short beaks (with low ratio of beak length to the overall utricle length) and narrow bracts. Common features of C. demissa 3 hostiana specimens, on the other hand, are male spikes with long peduncles, usually longly parted female spikes and a long beak compared to the overall utricle length.     

Foliar micro‐morphology of Carex sect. Phacocystis in northern Europe

A micro‐morphological analysis of leaf epidermis (adaxial and abaxial sides) of fifteen taxa of Carex section Phacocystis was carried out using light microscopy and scanning electron microscopy (SEM). Three taxa were epistomatic (C. nigra var. nigra, C.nigra var. juncea and C. subspathacea), seven taxa were hypostomatic (C. acuta, C. bigelowii ssp. bigelowii, C. bigelowii ssp. rigida, C. cespitosa, C. elata, C. lyngbyei and C. paleacea) and five amphistomatic (C. aquatilis, C.×halophila, C. rufina, C. stans and C. trinervis). Epidermal modifications such as prickles were present in many species. The micro‐morphological leaf characters of the investigated species were found to be important for distinguishing individual taxa but not for subsectional classification.     

A Phylogenetic Analysis of Ilyocryptus Sars, 1862 (Cladocera: Ilyocryptidae)

Kotov and Štifter (2006) revised the taxonomy of the genus Ilyocryptus Sars, 1862 (Branchiopoda: Anomopoda: Ilyocryptidae) and concluded that 28 species can be recognized as valid for the world fauna. In order to test phylogenetic relationships between the species and to contribute to a better understanding of the genus, a cladistic (using branch‐and‐bound search) analysis was conducted for 25 Ilyocryptus species (two of them with two subspecies). In total, 32 morphological characters were used. An evolutionary‐morphological interpretation of the results was also made. Phylogenetic analysis revealed that Ilyocryptus elegans is an earlier derived member of the genus, while other species are separated into two main branches: agilis ‐ and sordidus ‐lines (without reference to type of moulting). Species with incomplete moulting and species with complete moulting do not form separate clusters, suggesting an independent origin of incomplete moulting within different species groups. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Variation in Potentilla sect. Aureae (Rosaceae) in the Baltic states

The morphological variation of four Baltic representatives of the genus Potentilla section Aurea–P. neumanniana, P. arenaria, P. subarenaria and P. crantzii was studied with multivariate methods. Altogether 41 characters were used. The most important characters for the phenetical classification are those of the epidermis: characters of stellate and glandular hairs and numbers of cells. Macromorphological characters are less important, the most useful of these being the length of sepals and stipules and the number of teeth of the central leaflet. All four species are significantly distinct. Even P. subarenaria, a putative hybrid of P. arenaria and P. neumanniana, is clearly separated. At the same time, the species are morphologically quite variable, and it is possible to distinguish subclusters (morphotypes) within P. neumanniana, P. subarenaria and P. crantzii, which are also statistically distinct. The varieties described by Wolf (1908) under the name P. verna (P. neumanniana) do not agree well with the morphs in our material. However, it can be admitted that var. typica and var. neumanniana axe prevalent, var. pseudo-incis? and var. incis? occur occasionally; only var. longifoli? can be quite clearly delimited.     

Bupleurum shanianum sp. nov. (Apiaceae) from China

Bupleurum shanianum X. G. Ma & X. J. He, previously recognized in China as B. gracillimum, is proposed here as a new species. This new species and B. gracillimum differ in many morphological characters (flowering stem position, bract size, number of flowers per umbellule) and have non‐overlapping distribution ranges. Bupleurum shanianum is most similar to B. yunnanense Franch., but the new species has shorter cauline leaves, and umbellules usually with 4 bracteoles and 3–6(–9) flowers. Bupleurum shanianum is known from eastern Himalayas to western Sichuan.     

BIODIVERSITY OF CORALLINE ALGAE IN THE NORTHEASTERN ATLANTIC INCLUDING CORALLINA CAESPITOSA SP. NOV. (CORALLINOIDEAE,RHODOPHYTA)1

The Corallinoideae (Corallinaceae) is represented in the northeastern Atlantic by Corallina officinalis L.; Corallina elongata J. Ellis et Sol.; Haliptilon squamatum (L.) H. W. Johans., L. M. Irvine et A. M. Webster; and Jania rubens (L.) J. V. Lamour. The delimitation of these geniculate coralline red algae is based primarily on morphological characters. Molecular analysis based on cox1 and 18S rRNA gene phylogenies supported the division of the Corallinoideae into the tribes Janieae and Corallineae. Within the Janieae, a sequence difference of 46–48 bp (8.6%–8.9%) between specimens of H. squamatum and J. rubens in the cox1 phylogeny leads us to conclude that they are congeneric. J. rubens var. rubens and J. rubens var. corniculata (L.) Yendo clustered together in both phylogenies, suggesting that for those genes, there was no genetic basis for the morphological variation. Within the Corallineae, it appears that in some regions, the name C. elongata has been misapplied. C. officinalis samples formed two clusters that differed by 45–54 bp (8.4%–10.0%), indicating species‐level divergence, and morphological differences were sufficient to define two species. One of these clusters was consistent with the morphology of the type specimen of C. officinalis (LINN 1293.9). The other species cluster is therefore described here as Corallina caespitosa sp. nov. This study has demonstrated that there is a clear need for a revision of the genus Corallina to determine the extent of “pseudocryptic” diversity in this group of red algae.     

Which Morphological Characteristics Are Most Influenced by the Host Matrix in Downy Mildews? A Case Study in Pseudoperonospora cubensis

Before the advent of molecular phylogenetics, species concepts in the downy mildews, an economically important group of obligate biotrophic oomycete pathogens, have mostly been based upon host range and morphology. While molecular phylogenetic studies have confirmed a narrow host range for many downy mildew species, others, like Pseudoperonospora cubensis affect even different genera. Although often morphological differences were found for new, phylogenetically distinct species, uncertainty prevails regarding their host ranges, especially regarding related plants that have been reported as downy mildew hosts, but were not included in the phylogenetic studies. In these cases, the basis for deciding if the divergence in some morphological characters can be deemed sufficient for designation as separate species is uncertain, as observed morphological divergence could be due to different host matrices colonised. The broad host range of P. cubensis (ca. 60 host species) renders this pathogen an ideal model organism for the investigation of morphological variations in relation to the host matrix and to evaluate which characteristics are best indicators for conspecificity or distinctiveness. On the basis of twelve morphological characterisitcs and a set of twelve cucurbits from five different Cucurbitaceae tribes, including the two species, Cyclanthera pedata and Thladiantha dubia, hitherto not reported as hosts of P. cubensis, a significant influence of the host matrix on pathogen morphology was found. Given the high intraspecific variation of some characteristics, also their plasticity has to be taken into account. The implications for morphological species determination and the confidence limits of morphological characteristics are discussed. For species delimitations in Pseudoperonospora it is shown that the ratio of the height of the first ramification to the sporangiophore length, ratio of the longer to the shorter ultimate branchlet, and especially the length and width of sporangia, as well as, with some reservations, their ratio, are the most suitable characteristics for species delimitation.     

Differential sensitivity of green algae to allelopathic substances from Chara

Three short-term laboratory experiments were conducted to investigate allelopathic effects of a mixture of Chara globularis var. globularis Thuillier and Chara contraria var. contraria A. Braun ex Kützing on three different green algae. Single phytoplankton species were exposed to filtered water originating from charophyte cultures. Phytoplankton growth was monitored by determination of chlorophyll concentrations in batch cultures. The change in chlorophyll concentration during the experiments was analysed with a logistic growth model, resulting in an estimate of the exponential growth rate and the duration of the lag phase of the single green algae. The results indicate allelopathic effects of Chara on the growth of the green algae Selenastrum capricornutum Printz and Chlorella minutissima Fott et Nováková, whereas Scenedesmus obliquus (Turpin) Kützing did not seem to be affected. The exponential growth rate of S. capricornutum decreased 7% in the presence of water from a charophyte culture, while the growth rate of C. minutissima decreased with 3%. The allelopathic effect of Chara did not increase when the green alga C. minutissima was P-limited. The effect of Chara was different when young sprouts were used. With young sprouts the duration of the lag phase of C. minutissima was extended (25%), whilst for old plants the growth rate of this green alga decreased. Although the inhibiting effect of charophytes on specific phytoplankton species is rather small, the differential sensitivity of the species to Chara might influence the composition and biomass of phytoplankton communities in the field.     

Micromorphological studies of the leaf epidermis of Iranian Cotoneaster Medik

In this study, micromorphological features of the leaf epidermal cells of 17 species of Iranian Cotoneaster were determined. The studied species were compared with each other and with phylogenetic relationships established by previous studies. Three populations of C. integerrimus and C. multiflorus were studied as representatives of C. subgen. Cotoneaster and Chaenopetalum, respectively. To evaluate quantitative and qualitative characters, multiple correspondence analysis (MCA) and factor analysis of mixed data (FAMD) were performed. Finally, relationships among the taxa were illustrated by hierarchical clustering analysis. Three stomatal types were identified, i.e. anomocytic, stephanocytic and actino‐stephanocytic. The wax and cuticle density was higher in species from semi‐moist habitats than in those from dry and semi‐dry habitats. The patterns formed by the anticlinal walls of the epidermal cells were sinuous, repand and straight‐curved. Interestingly, there was a significant relationship between the length of leaves, the density of the indumentum and the pattern formed by the anticlinal cell walls. The leaves of species with large leaves also have lower hair density and more curved anticlinal cell walls. It appears probable that climatic conditions affected the evolution of leaf micro‐morphological characters during the early diversification of the genus, but that these characters have subsequently been permanently fixed by phylogenetic constraints and thus appear diagnostic for extant species.