A combined morphological and molecular approach to Nitzschia varelae sp. nov., with discussion of symmetry in Bacillariaceae

A previously unknown member of the Bacillariaceae was discovered almost simultaneously in four different brackish coastal wetlands on the Atlantic and Mediterranean coasts of the Iberian Peninsula. It appears to tolerate a wide range of salinities but was never common in samples where it occurred. The frustules were consistently hantzschioid (i.e. with the raphe systems always on the same side of the frustule) and the valve outline was asymmetrical about the apical plane, two features that have until recently been considered characteristic of Hantzschia. Molecular phylogenies based on rbcL and LSU rDNA indicated, however, that the new species does not belong in Hantzschia but among the several disparate lineages that comprise the paraphyletic genus Nitzschia. This finding, coupled with the recent discovery of other diatoms with constant hantzschioid symmetry but with a morphology very similar to the type species of Nitzschia, is discussed in relation to the status and characterization of Hantzschia as an independent genus. It is concluded that, while a core of hantzschioid species may exist that can be classified together, corresponding to the traditional understanding of the genus Hantzschia, there is no single morphological feature common to all of them that can be used to diagnose the group and differentiate it from the various hantzschioid lineages that are separate from true Hantzschia and currently placed in e.g. Nitzschia or Cymbellonitzschia. Testing whether a hantzschioid species does or does not belong to Hantzschia will in many cases require molecular evidence. Although the new coastal species does not belong to the same lineage as the type species of Nitzschia, N. sigmoidea, it is described for the moment as N. varelae Carballeira, D.G. Mann & Trobajo, sp. nov., until there is a better understanding of generic limits in the Bacillariaceae following a wider molecular and morphological survey of that family.     

Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences

Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.     

杉科、柏科的系统发生分析——来自28S rDNA序列分析的证据

对杉科（Taxodiaceae）与柏科（Cupressaceae s.s.）的28S rRNA基因的部分序列（约630 bp）进行PCR扩增、序列测定和系统发生关系分析,用简约法和邻接法构建的系统发生树基本一致。结果表明,杉科与柏科构成一个单系群,支持将杉科、柏科（Sciadopitys除外）合并为一个科——广义柏科（Cupressaceae sensu lato）的观点。在广义柏科中,Taiwania、Athrotaxis分别形成一支系;Metasequoia、Sequoia、Sequoiadendron关系较近,聚成一支系; Taxodium、Glyptostrobus、Cryptomeria聚成一支系;柏科聚成一支系。这一分析结果与叶绿体基因序列的分析结果相吻合,但是由于28S rRNA基因的进化速率较慢,尚不能分辨上述各个支系之间的系统演化关系。 Abstract:DNA sequences from 28S rDNA were used to assess relationships between and within traditional Taxodiaceae and Cupressaceae s.s.The MP tree and NJ tree generally are similar to one another.The results show that Taxodiaceae and Cupressaceae s.s.form a monophyletic conifer lineage excluding Sciadopitys.In the Taxodiaceae-Cupressaceae s.s.monophyletic group,the Taxodiaceae is paraphyletic.Taxodium,Glyptostrobus and Cryptomeria forming a clade(Taxodioideae),in which Glyptostrobus and Taxodium are closely related and sister to Cryptomeria;Sequoia,Sequoiadendron and Metasequoia are closely related to each other,forming another clade (Sequoioideae),in which Sequoia and Sequoiadendron are closely related and sister to Metasequoia;the seven genera of Cupressaceae s.s.are found to be closely related to form a monophyletic lineage (Cupressoideae).These results are basically similar to analyses from chloroplast gene data.But the relationships among Taiwania,Sequoioideae,Taxodioideae,and Cupressoideae remain unclear because of the slow evolution rate of 28S rDNA,which might best be answered by sequencing more rapidly evolving nuclear genes.     

The matK gene: sequence variation and application in plant systematics

Although the matK gene has been used in addressing systematic questions in four families, its potential application to plant systematics above the family level has not been investigated. This paper examines the rates, patterns, and types of nucleotide substitutions in the gene and addresses its utility in constructing phylogenies above the family level. Eleven complete sequences from the GenBank representing seed plants and liverworts and nine partial sequences generated for genera representing the monocot families Poaceae, Joinvilleaceae, Cyperaceae, and Smilacaceae were analyzed. The study underscored the high rate of substitution in the gene and the presence of mutationally conserved sectors. The use of different sectors of the gene and the cumulative inclusion of informative sites showed that the 3' region was most useful in resolving phylogeny, and that the topology and robustness of the tree reached a plateau after the inclusion of 100 informative sites from that region for the taxa used. The impact of using partial sequencing on sample size is addressed. The presence of a relatively conserved 3' region and the less conserved 5' region provides two sets of characters that can be used at different taxonomic levels from the tribal to the division levels.     

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.     

Phylogenetic relationships within the tribe Janieae (Corallinales,Rhodophyta) based on molecular and morphological data: a reappraisal of Jania1

Generic boundaries among the genera Cheilosporum, Haliptilon, and Jania—currently referred to the tribe Janieae (Corallinaceae, Corallinales, Rhodophyta)—were reassessed. Phylogenetic relationships among 42 corallinoidean taxa were determined based on 26 anatomical characters and nuclear SSU rDNA sequence data for 11 species (with two duplicate plants) referred to the tribe Corallineae and 15 species referred to the tribe Janieae (two species of Cheilosporum, seven of Haliptilon, and six of Jania, with five duplicate plants). Results from our approach were consistent with the hypothesis that the tribe Janieae is monophyletic. Our data indicate, however, that Jania and Haliptilon as currently delimited are not monophyletic, and that Cheilosporum should not be recognized as an independent genus within the Janieae. Our data resolved two well‐supported biogeographic clades for the included Janieae, an Indian‐Pacific clade and a temperate North Atlantic clade. Among anatomical characters, reproductive structures reflected the evolution of the Janieae. Based on our results, three genera, Cheilosporum, Haliptilon, and Jania, should be merged into a single genus, with Jania having nomenclatural priority. We therefore propose new combinations where necessary of some species previously included in Cheilosporum and Haliptilon.     

Cryptic goldfields: a molecular phylogenetic reinvestigation of Lasthenia californica sensu lato and close relatives (Compositae: Heliantheae sensu lato)

Maximum parsimony analysis of DNA sequence data from the internal and external transcribed spacer (ITS and ETS) regions of 18S-26S nuclear ribosomal DNA and the 3' trnK intron of chloroplast DNA from over 60 populations of Lasthenia sect. Amphiachaenia yielded a well-supported tree showing that the most common species of Lasthenia, L. californica sensu lato (s.l.), is not monophyletic. Members of Lasthenia californica s.l. belong to two well-supported but morphologically cryptic clades. One clade includes members of L. macrantha; the other represents a basally divergent lineage in L. sect. Amphiachaenia. Members of each clade can be diagnosed by pappus morphology and by geographic distribution, except for epappose plants that occur in a broad region of sympatry in central California. Overall diversification in the clade corresponding to L. sect. Amphiachaenia has been accompanied by minimal morphological divergence, which has resulted in previously underappreciated cryptic diversity.     

The interrelationships of the echinoderm classes: morphological and molecular evidence

The relationship between the five echinoderm classes has perplexed phylogeneticists for some time. Although each of the crinoids (C), asteroids (A), ophiuroids (O), echinoids (E) and holothuroids (H) are morphologically distinct, evidence from larval and adult morphology, molecular biology, and stratigraphy have failed to provide a single consensus solution. We have reviewed all available morphological and molecular data, added new data and reanalysed independent data sets individually and in combination, in order to resolve echinoderm class relationships. In total, we present 21 larval and 50 adult morphological characters, partial 28S-like large subunit rRNA gene data for 39 taxa and complete 18S-like small subunit rRNA gene data for 37 taxa. For a 5 taxon problem there are 105 possible rooted tree topologies, and yet we were consistently presented with three competing hypotheses when data sets were analysed both individually and in combination. The total evidence solution favoured (outgroup(C(A(O(E, H))))) although the alternative tree topology, (outgroup(C(O(A(E, H))))) was only one step longer and (outgroup(C((A, O),(E, H)))) was two steps longer. Only these three trees are serious contenders and the distribution of morphological characters suggests we should discount the solution placing ophiuroids as sister group to an asteroid+echinoid + holothurian clade. Thus we are left with (outgroup(C(A(O(E, H))))) and (outgroup(C((A, O),(E, H)))) as the two most plausible phylogenetic hypotheses. Our data showed high levels of phylogenetic signal and these trees best fit the available data.     

Out of Africa: molecular phylogenetics and biogeography of Wolffiella (Lemnaceae)

The monophyletic genus Wolffiella (Lemnaceae) comprises 10 species divided taxonomically into three sections. Relative to other genera of Lemnaceae, Wolffiella has a restricted range, with species distributed in warm temperate to tropical areas of Africa and the Americas, with only one species occurring in both areas. Sequence data from coding (rbcL and matK) and non‐coding (trnK and rpl16 introns) regions of cpDNA were analyzed phylogenetically to resolve relationships within Wolffiella, and these results were compared to earlier allozyme and morphological studies. Allozymes, cpDNA and morphology all supported the recognition of three sections. Relationships among species were similar in most respects between the allozyme and cpDNA trees, as well as among the different plastid partitions. In Wolffiella, both non‐synonymous and synonymous substitutions were greater in matK than in rbcL, as observed in other taxa. The synonymous substitution rate in matK was similar to the substitution rate of the non‐coding regions. All partitions, including coding regions, exhibited some homoplasy. Biogeographical reconstructions from a combination of cpDNA partitions indicated that Wolffiella originated in Africa with early movement to and radiation in the Americas. The one species found in both Africa and the Americas, W. welwitschii, likely originated in the Americas and subsequently dispersed to Africa. Using the SOWH test, the cpDNA data could reject two alternative biogeographical hypotheses suggested from analyses of morphological and allozyme data. The present distribution of Wolffiella can be explained by two major dispersal events and this contrasts with the more complex species distributions in other Lemnaceae genera. Limited dispersal in Wolffiella relative to other Lemnaceae genera may be due to more recent origins of species, lower dispersibility and poorer colonizing ability. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79 , 565–576.     

A multidisciplinary approach to the fine-systematics within Tisbe - an evaluation of morphological and molecular methods

Six species from the species-rich taxon Tisbe (Copepoda, Harpacticoida) were selected that could be reared in the laboratory as mass cultures. Phylogenetic relationships among these species were assessed by morphological studies of adults and larvae, DNA restriction site polymorphisms, allozymic, immunological distance, and lipid composition. Limits of scope and practicability of these analyses became apparent, as well as their potential and importance for future work in zoological systematics.     

French Polynesian Scytosiphonaceae (Ectocarpales,Phaeophyceae): A combined molecular and morphological approach to their diversity and systematics

This study revisited the taxonomy and diversity of brown macroalgae within the Scytosiphonaceae family in French Polynesia, which had previously been recognized as encompassing only six species. Using the chloroplast and mitochondrial genes rbcL, psbA, and cox3 as molecular markers in conjunction with morpho-anatomical observations, we unveiled the presence of 11 species spanning six genera: Chnoospora minima, Colpomenia claytoniae, Co. sinuosa [groups IIIa and IIIb], Hydroclathrus rapanuii, H. tenuis, H. tilesii, Manzaea minuta, Pseudochnoospora implexa, Rosenvingea australis, and the newly described species R. polynesiensis sp. nov. and R. tahitiensis sp. nov. This encompasses the recognition of two previously unreported genera in this region: Manzaea and Pseudochnoospora. Sequences were successfully acquired for four taxa that had been documented previously, while the absence of sequences for H. clathratus and H. tumulis in French Polynesia raises queries about their presence in this region. With these additions, the total species count now stands at 13 (including H. clathratus and H. tumulis), one being an endemic species. The molecular-assisted alpha taxonomic approach used here allowed for a critical revision of the Scytosiphonaceae species checklist for French Polynesia. The diversity revealed in this region accounts for a substantial 20% of the family's global diversity. Additionally, our study presents an updated species-level phylogeny for the Scytosiphonaceae.     

Phylogeny of drepanosiphine aphids sensu lato (Hemiptera,Aphidoidea) inferred from molecular and morphological data

As the second largest and most diverse group in the superfamily Aphidoidea, the phylogeny of drepanosiphine aphids sensu lato (s.l.) is critical for discussing the evolution of aphids. However, the taxa composition and phylogenetic relationships of drepanosiphine aphids s.l. have not been fully elucidated to date. In this study, based on total-evidence analyses combining 4 molecular genes (3 mitochondrial, COI, tRNA-Leu/COII, and CytB; 1 nuclear, EF-1ɑ) and 64 morphological and biological characteristics, the phylogeny of this group was reconstructed for the first time at the subfamily level using different datasets, parsimonies and model-based methods. All of our phylogenetic inferences clearly indicated that the drepanosiphine aphids s.l. was not a monophyletic group and seemed to support the division of the drepanosiphine aphids s.l. into different groups classified at the subfamily level. Calaphidinae was also not a monophyletic group, and Saltusaphidinae was nested within this subfamily. Drepanosiphinae was not clustered with Chaitophorinae, which was inconsistent with the previous hypothesis of a close relationship between them, illustrating that their phylogeny remains controversial. Overall, some groups of drepanosiphine aphids s.l., including Phyllaphidinae, Macropodaphidinae, Pterastheniinae, Lizeriinae, Drepanosiphinae, Spicaphidinae, Saltusaphidinae, and Calaphidinae, clustered together and might constitute the actual drepanosiphine aphids s.l. To a certain extent, our results clarified the phylogenetic relationships among drepanosiphine aphids s.l. and confirmed their taxonomic status as subfamilies.     

Adaptive radiation in African weakly electric fish (Teleostei: Mormyridae: Campylomormyrus): a combined molecular and morphological approach

We combined multiple molecular markers and geometric morphometrics to revise the current taxonomy and to build a phylogenetic hypothesis for the African weakly electric fish genus Campylomormyrus. Genetic data (2039 bp DNA sequence of mitochondrial cytochrome b and nuclear S7 genes) on 106 specimens support the existence of at least six species occurring in sympatry. We were able to further confirm these species by microsatellite analysis at 16 unlinked nuclear loci and landmark-based morphometrics. We assigned them to nominal taxa by comparisons to type specimens of all Campylomormyrus species recognized so far. Additionally, we showed that the shape of the elongated trunk-like snout is the major source of morphological differentiation among them. This finding suggests that the radiation of this speciose genus might have been driven by adaptation to different food sources.     

A morphological and molecular assessment of the genus Sinotubimorpha (Halymeniaceae, Rhodophyta)

A critical reassessment of the morphological features of two closely related red algal genera,Grateloupia C.Agardh and Sinotubimorpha W.X.Li & Z.F.Ding (Halymeniaceae),pointed out that members of the t...     

Quantitative analyses of the genetic structure of closely related conifers with disparate distributions and demographics: the Cupressus arizonica (Cupressaceae) complex

Seedling populations of seven Cupressus taxa were compared in common gardens to assess relationships among taxa and to elucidate the genetic structure of three taxa with different distributions and demographics. Discriminant analyses of 15 quantitative traits separated C. sargentii and C. forbesii from taxa within the C. arizonica complex (C. arizonica, C. glabra, C. stephensonii, C. nevadensis, and C. montana); aligned a population of C. arizonica from Sierra de Arteaga, Mexico, with families of the same taxon from southwestern United States; and described a close relationship between C. nevadensis and C. glabra. but nevertheless separated taxa within the C. arizonica complex. Analyses of wind-pollinated progenies of single trees showed the narrow endemic, C. glabra, to have a greater degree of population differentiation than the more broadly dispersed C. arizonica. In addition, genetic variances within the numerically small populations of C. arizonica were about the same as in the much larger populations of C. glabra. The results also suggested that genetic variability in C. stephensonii, a taxon occurring in a single population, may occur primarily among inbred lines. While the genetic structures of C. arizonica and C. glabra have counterparts among broadly dispersed conifers, that of C. stephensonii appears to be unique. Together, the results support the view that genetic structure primarily reflects the uncertain, chance events that are interspersed throughout evolutionary history.     

Andalucia (n. gen.)--the deepest branch within jakobids (Jakobida; Excavata), based on morphological and molecular study of a new flagellate from soil

A new heterotrophic flagellate (Andalucia godoyi n. gen. n. sp.) is described from soil. Earlier preliminary 18S rRNA analyses had indicated a relationship with the phylogenetically difficult-to-place jakobid Jakoba incarcerata. Andalucia godoyi is a small (3-5 mum) biflagellated cell with a ventral feeding groove. It has tubular mitochondrial cristae. There are two major microtubular roots (R1, R2) and a singlet root associated with basal body 1 (posterior). The microtubular root R1 is associated with non-microtubular fibres "I,"B," and "A," and divides in two parts, while R2 is associated with a "C" fibre. These structures support the anterior portion of the groove. Several features of A. godoyi are characteristic of jakobids: (i) there is a single dorsal vane on flagellum 2; (ii) the C fibre has the jakobid multilaminate substructure; (iii) the dorsal fan of microtubules originates in very close association with basal body 2; and (iv) there is no "R4" microtubular root associated with basal body 2. Morphological analyses incorporating the A. godoyi data strongly support the monophyly of all jakobids. Our 18S rRNA phylogenies place A. godoyi and J. incarcerata as a strong clade, which falls separately from other jakobids. Statistical tests do not reject jakobid monophyly, but a specific relationship between Jakoba libera and J. incarcerata and/or A. godoyi is rejected. Therefore, we have established a new genus Andalucia n. gen. with the type species Andalucia godoyi n. sp., and transfer Jakoba incarcerata to Andalucia as Andalucia incarcerata n. comb.     

Delimitating cryptic species in the Gracilaria domingensis complex (Gracilariaceae,Rhodophyta) using molecular and morphological data

Species in the genus Gracilaria that display conspicuously flattened vegetative morphologies are a taxonomically challenging group of marine benthic red algae. This is a result of their species richness, morphological similarity, and broad phenotypic plasticity. Within this group, the Gracilaria domingensis complex is one of the most common, conspicuous, and morphologically variable species along the tropical western Atlantic Ocean. Previous research has identified that members of this complex belong to two distantly related clades. However, despite this increased phylogentic resolution, species delimitations within each of these clades remain unclear. Our study assessed the species diversity within this difficult complex using morphological and molecular data from three genetic markers (cox1, UPA, and rbcL). We additionally applied six single‐marker species delimitation methods (SDM: ABGD, GMYCs, GMYCm, SPN, bPTP, and PTP) to rbcL, which were largely in agreement regarding species delimitation. These results, combined with our analysis of morphology, indicate that the G. domingensis complex includes seven distinct species, each of which are not all most closely related: G. cervicornis; a ressurected G. ferox; G. apiculata subsp. apiculata; a new species, Gracilaria baiana sp. nov.; G. intermedia subsp. intermedia; G. venezuelensis; and G. domingensis sensu stricto, which includes the later heterotypic synonym, G. yoneshigueana. Our study demonstrates the value of multipronged strategies, including the use of both molecular and morphological approaches, to decipher cryptic species of red algae.     

Evolutionary genetics and biogeographic structure of Rhizobium gallicum sensu lato, a widely distributed bacterial symbiont of diverse legumes

We used phylogenetic and population genetics approaches to evaluate the importance of the evolutionary forces on shaping the genetic structure of Rhizobium gallicum and related species. We analysed 54 strains from several populations distributed in the Northern Hemisphere, using nucleotide sequences of three 'core' chromosomal genes (rrs, glnII and atpD) and two 'auxiliary' symbiotic genes (nifH and nodB) to elucidate the biogeographic history of the species and symbiotic ecotypes (biovarieties) within species. The analyses revealed that strains classified as Rhizobium mongolense and Rhizobium yanglingense belong to the chromosomal evolutionary lineage of R. gallicum and harbour symbiotic genes corresponding to a new biovar; we propose their reclassification as R. gallicum bv. orientale. The comparison of the chromosomal and symbiotic genes revealed evidence of lateral transfer of symbiotic information within and across species. Genetic differentiation analyses based on the chromosomal protein-coding genes revealed a biogeographic pattern with three main populations, whereas the 16S rDNA sequences did not resolve that biogeographic pattern. Both the phylogenetic and population genetic analyses showed evidence of recombination at the rrs locus. We discuss our results in the light of the contrasting views of bacterial species expressed by microbial taxonomist and evolutionary biologists.