Biogeographic patterns in Mediterranean and Macaronesian species of Saxifraga (Saxifragaceae) inferred from phylogenetic analyses of ITS sequences

A biogeographic study of Saxifraga section Saxifraga was performed based on phylogenetic analyses of ITS (internal transcribed spacer) sequences of nuclear ribosomal DNA. ITS sequences from 21 species and 31 populations were examined to identify colonization patterns for the two species of Saxifraga occurring in Macaronesia and for S. globulifera in the west Mediterranean basin. Phylogenetic analysis of the sequence data yield a single most parsimonious tree with many of the major clades well supported by bootstrap and decay values. The ITS tree provided resolution at specific and populational levels that points to two biogeographic patterns within the genus. In contrast to the molecular evidence provided by other authors for a Mediterranean origin of several Macaronesian genera of angiosperms, our results indicate that the Madeiran archipelago was colonized a single time by a species of Saxifraga originating from the Eurosiberian region. On the other hand, the molecular evidence also suggests that populations of S. globulifera from North Africa have been isolated for a long time from populations occurring in the Iberian Peninsula, and that the endemic S. reuteriana has evolved from the Iberian populations of S. globulifera. The Mediterranean Sea has probably been an effective isolating barrier for some plant groups that occur in Europe and North Africa.     

A phylogenetic study ofSaxifraga sect.Saxifraga (Saxifragaceae) based on nrDNA ITS sequences

Parsimony analyses of 54 nrDNA ITS (Internal Transcribed Spacer) sequences ofSaxifraga sect.Saxifraga were performed. In addition to some unresolved clades, there is strong disagreement between the ITS phylogeny and previous classifications based primarily on morphology. The extensive cytological instability of sect.Saxifraga prevents previous cytotaxonomical results from resolving the incongruence between molecular and morphological data. Dissimilar topologies between chloroplast (matK) and nuclear (ITS) trees for eight species of sect.Saxifraga suggest that gene trees and the true species tree are not coincident. Recent and mid-term reticulation is proposed as an explanation for the incongruence between morphological, cytological, organellar, and nuclear data. Homogenization in multigene families, such as the ITS region, via concerted evolution may be the key to the interpretation of results based on ITS sequences within sect.Saxifraga. The use of organellar genes in a larger sample should help to determine whether extensive reticulation occurs in sect.Saxifraga, as has been documented in various genera of Saxifragaceae.     

Phylogeny of Saxifraga section Saxifraga subsection Arachnoideae (Saxifragaceae) and the origin of low elevation shade-dwelling species

Saxifraga section Saxifraga subsection Arachnoideae is a lineage of 12 species distributed mainly in the European Alps. It is unusual in terms of ecological diversification by containing both high elevation species from exposed alpine habitats and low elevation species from shady habitats such as overhanging rocks and cave entrances. Our aims are to explore which of these habitat types is ancestral, and to identify the possible drivers of this remarkable ecological diversification. Using a Hybseq DNA-sequencing approach and a complete species sample we reconstructed and dated the phylogeny of subsection Arachnoideae. Using Landolt indicator values, this phylogenetic tree was used for the reconstruction of the evolution of temperature, light and soil pH requirements in this lineage. Diversification of subsection Arachnoideae started in the late Pliocene and continued through the Pleistocene. Both diversification among and within clades was largely allopatric, and species from shady habitats with low light requirements are distributed in well-known refugia. We hypothesize that low light requirements evolved when species persisting in cold-stage refugia were forced into marginal habitats by more competitive warm-stage vegetation. While we do not claim that such competition resulted in speciation, it very likely resulted in adaptive evolution.     

matK and rbcL gene sequence data indicate that Saxifraga (Saxifragaceae) is polyphyletic

The large genus Saxifraga, which consists of ≈400 morphologically and cytologically diverse species, has long been considered taxonomically complex. Phylogenetic analysis of over 2500 bp of chloroplast sequence data derived from matK and rbcL was employed to examine relationships among sections of Saxifraga, the segregate genera Zahlbrucknera, Saxifragopsis, and Cascadia, and the relationships of these taxa to other Saxifragaceae sensu stricto. Phylogenetic trees resulting from separate analyses of the matK and rbcL sequences were highly congruent; phylogenetic analysis of a combined matK–rbcL data matrix was therefore also conducted. Our analyses indicate that Saxifraga is polyphyletic, comprising two well-differentiated clades. One clade, Saxifraga sensu stricto, is the sister to the remainder of the family and consists of Saxifraga sections Irregulares, Heterisia, Trachyphyllum, Cymbalaria, Mesogyne, Xanthizoon, Porphyrion, Ciliatae, Cotylea, Ligulatae, Saxifraga, and Gymnopera. With the exception of Gymnopera, the species-rich sections of this clade are monophyletic. Also part of this clade is the problematic Zahlbrucknera paradoxa, which is allied with members of section Saxifraga. A second major clade of Saxifraga species, Micranthes sensu lato, comprises the large section Micranthes, as well as the segregate genus Cascadia, and S. tolmiei of section Merkianae. This clade is allied with the Heuchera, Darmera, and Chrysosplenium-Peltoboykinia groups of genera. The segregate genus Saxifragopsis is only distantly related to species of Saxifraga, and is instead the sister to Astilbe. The monotypic Oresitrophe is confirmed as a member of the Darmera group of genera. These results suggest that the floral features used to define Saxifraga may simply be symplesiomorphic in these well-separated Saxifraga lineages. Furthermore, the enormous cytological diversity encompassed by Saxifraga likely represents two independent instances of extensive aneuploidy and polyploidy in Saxifragaceae.     

Molecular phylogenetics,character evolution and systematics of the genus Micranthes (Saxifragaceae)

With c. 85 species, the genus Micranthes is among the larger genera of the Saxifragaceae. It is only distantly related to the morphologically similar genus Saxifraga, in which it has frequently been included as Saxifraga section Micranthes. To study the molecular evolution of Micranthes, we analysed nuclear ribosomal (internal transcribed spacer, ITS) and plastid (trnL–trnF) DNA sequences in a comprehensive set of taxa comprising c. 75% of the species. The molecular phylogenetic tree from the combined dataset revealed eight well‐supported clades of Micranthes. These clades agree in part with previously acknowledged subsections or series of Saxifraga section Micranthes. As these eight groups can also be delineated morphologically, we suggest that they should be recognized as sections of Micranthes. New relationships were also detected for some species and species groups, e.g. section Davuricae sister to sections Intermediae and Merkianae, and M. micranthidifolia as a member of section Micranthes. Species proposed to be excluded from the genus Micranthes for morphological reasons were resolved in the molecular tree in Saxifraga. Many morphological characters surveyed were homoplasious to varying extents. Micromorphological characters support comparatively well the clades in the phylogenetic tree. An updated nomenclature and a taxonomic conspectus of sections and species of Micranthes are provided. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 47–66.     

Phylogeny of korean rubus (rosaceae) based on its (nrDNA) and trnL/F intergenic region (cpdna)

We examined the phylogeny of the genusRubus in Korea using an internal transcribed spacer (ITS) of the nuclear ribosomal DNA and a trnL-trnF (trnL/F) intergenic region of the chloroplast DNA. In all, 21 ingroup species (1.2 kb for each species) were analyzed using parsimony, resulting in 672 aligned sequences from ITS, and 502 bases with trnL/F. Individual and combined analysis of ITS and trnL/F data proved that the genusRubus is a monophyletic group. This phylogeny also substantiated a previous sectional classification scheme rather than a subgerius classification scheme. However, our results did not support the earlier sectional classification by Focke (sect.Corchorifolii), but did support the sectional classification of Nakai: sect.Crataegifolii (R. crataegifolius, R. takesimensis andR. trifidus), and sect.Villosii (R. corchorifolius). Most of these species, which are found in Korea and belong to subg.Idaeobatus, appeared in two different groups in all data sets. This suggests that this subgenus is a polyphyletic group that has gone through at least two independent evolutionary processes. The taxa, when mapped onto the combined tree, showed that the occurrence of their morphological characters of simple and compound leaves was concurrent in KoreanRubus. ITS sequence data were consistent overall with the geographical distribution of each species. Furthermore, the trnL/F sequence data provided phylogenetic information within closely related species.     

Chloroplast phylogenomics and the taxonomy of Saxifraga section Ciliatae (Saxifragaceae)

Comprising ca. 200 species, Saxifraga sect. Ciliatae is the most species-rich section of Saxifraga s.str., whose center of diversity is in the Tibeto-Himalayan region. The infra-sectional classification of sect. Ciliatae is still in debate due to the high level of species richness, as well as remarkable variations of habitat, morphology, physiology and life cycles. Subdivisions of this section proposed in various taxonomic systems have not been adequately tested in previous phylogenetic studies, partly due to low taxonomic sampling density, but also to the use of few DNA markers. In order to achieve a more robust infra-sectional classification of sect. Ciliatae, complete chloroplast genomes of 94 taxa from this section were analyzed, of which 93 were newly sequenced, assembled and annotated. The length of the 94 plastomes of sect. Ciliatae taxa range from 143,479 to 159,938 bp, encoding 75 to 79 unique protein-coding genes (PCGs). Analyses of the 94 plastomes revealed high conservation in structural organization, gene arrangement, and gene content. Gene loss and changes of IR boundaries were detected but in extremely low frequency. The molecular phylogenetic tree from concatenated PCGs and complete chloroplast genome sequences exhibited high resolution and support values and confirms that sect. Ciliatae is monophyletic. Three well-supported clades were revealed within the section that agree relatively well with the subsectional taxonomy of Gornall (1987), but some minor modifications should be made. Firstly, the monotypic subsection Cinerascentes should be abandoned and its constituent species, S. cinerascens, assigned to subsect. Gemmiparae. Secondly, subsections Rosulares and Serpyllifoliae should be merged and become subsect. Rosulares. Section Ciliatae thus comprises: subsect. Hirculoideae Engl. & Irmsch.; subsect. Rosulares Gornall; subsect. Gemmiparae Engl. & Irmsch.; subsect. Flagellares (C. B. Clarke) Engl. & Irmsch. and subsect. Hemisphaericae (Engl. & Irmsch.) Gornall.     

Systematic significance of pollen nucleus number in the genus Saxifraga (Saxifragaceae)

Pollen cell number was studied in 124 species of Saxifraga and six species of Micranthes. Binucleate pollen is present in all the six species of Micranthes studied and in 96 species of Saxifraga. Trinucleate pollen occurs in 28 species of Saxifraga and is confined to section Ciliatae subsection Hirculoideae, where it is a putative synapomorphy, and to two species of subsection Serpyllifoliae. Trinucleate pollen does not characterise the whole of section Ciliatae as has been thought. The state has probably evolved at least twice, and possibly three times, in the genus. One new nomenclatural combination is made in Micranthes.     

A bridge too far in naming species: a total evidence approach does not support recognition of four species in Desertifilum (Cyanobacteria)

A population of Desertifilum (Cyanobacteria, Oscillatoriales) from an oligotrophic desertic biotope was isolated and characterized using a polyphasic approach including molecular, morphological, and ecological information. The population was initially assumed to be a new species based on ecological and biogeographic separation from other existing species, however, phylogenetic analyses based on sequences of the 16S rRNA gene and 16S–23S ITS region, placed this strain clearly within the type species, Desertifilum tharense. Comparative analysis of morphology, 16S rRNA gene similarity, 16S–23S ITS secondary structure, and percent dissimilarity of the ITS regions for all characterized strains supports placing the six Desertifilum strains (designated as PD2001/TDC17, UAM‐C/S02, CHAB7200, NapGTcm17, IPPAS B‐1220, and PMC 872.14) into D. tharense. The recognition of Desertifilum salkalinema and Desertifilum dzianense is not supported, although our analysis does support continued recognition of Desertifilum fontinale. Pragmatic criteria for recognition of closely related species are proposed based on this study and others, and more rigorous review of future taxonomic papers is recommended.     

Scorzonera ahmet‐duranii sp. nov. (Asteraceae) from southwest Anatolia,and its phylogenetic position

Scorzonera ahmet‐duranii S. Makbul & Coskuncelebi sp. nov. (Asteraceae) is described and illustrated as a new species endemic to Turkey. It is morphologically similar to S. semicana DC., but differs by its rootstock, deeply undulating basal leaves and glabrous achenes. Additionally, the size and micro‐morphological properties of achenes and some anatomical traits of the leaf and stem are helpful in distinguishing these closely related taxa. A phylogenetic analysis based on nrDNA ITS sequence data showed that S. semicana is the sister species of the new taxon presented in this study.     

INTRAGENOMIC NRDNA ITS POLYMORPHISM IN THE STEPHANODISCUS NIAGARAE COMPLEX (BACILLARIOPHYCEAE)

Intragenomic nrDNA ITS variability was examined in representative species of the Stephanodiscus niagarae complex. PCR was performed on DNA extracted from monoclonal cultures, and sequence variability determined using standard cloning techniques. Preliminary data from all species reveal intragenomic polymorphism. ITS sequence data is being used to delineate closely related species of algae and polymorphisms are potentially problematic for phylogenetic reconstruction and identity. We are exploring the extent of polymorphism in other freshwater Thalassiorsiraceae, including Stephanodiscus hantzschii fo. tenuis and Cyclostephanos invisitatus.     

Pachybrachis sassii, a new species from the Mediterranean Giglio Island (Italy) (Coleoptera, Chrysomelidae, Cryptocephalinae)

Pachybrachis sassii, new species is described from Giglio Island, of the Tuscan Archipelago (Italy). The new species belongs to the nominotypical subgenus and is closely related to Pachybrachis salfii Burlini, 1957, from which it differs in the shape of the median lobe of the aedeagus and in the pattern of the yellow raised spots on the elytra and pronotum. Ecological observations are made. The neotype of Pachybrachis salfii from Colloreto, Monte Pollino (Italy) is designated.     

Phylogenetics and seed morphology of African Nymphoides (Menyanthaceae)

We evaluated the boundaries among Nymphoides (Menyanthaceae) species in Africa, a region where the genus has received relatively little attention. We gathered morphological data from seeds using light and scanning electron microscopy, and we conducted molecular phylogenetic analyses using nuclear sequence data from the internal transcribed spacer (ITS) region. Morphological and molecular features distinguished six species and affirmed their respective geographic ranges. No African specimens were attributable to Nymphoides indica, even though this paleotropical species previously had been understood to grow in Africa. We establish the new combination Nymphoides senegalensis (G. Don) Tippery, based on an established African basionym, to accommodate the specimens formerly identified as N. indica. Phylogenetic analyses resolved two distinct clades containing African species. The majority of African species are closely related to neotropical species, with which they share similar petal ornamentation. The morphologically distinct N. ezannoi shares floral and phylogenetic similarity with species from North America and Asia. Results presented here support prior hypotheses that allopolyploid species in the Americas may have originated from one or more parental lineages in Africa. Seed morphological characters remain some of the most reliable features for identifying species, particularly for herbarium specimens lacking observable floral characters.     

A phylogenetic study ofPolygonum sect.Tovara (Polygonaceae) based on ITS sequences of nuclear ribosomal DNA

Polygonum sect.Tovara comprises three morphologically very similar species;P. virginianum,P. filiforme, andP. neofiliforme. Sequences of internal transcribed spacers (ITSs) of nuclear ribosomal DNA of these were determined to examine phylogenetic relationships and the levels of differentiation among them. The size of ITS 1 was 241 bp inP. filiforme andP. neofiliforme, and 242 bp inP. virginianum. The size of ITS 2 was 243 bp, and that of the 5.8S rRNA coding region was 163 bp. The ITS sequences clearly separate North AmericanP. virginianum from the eastern Asian species. Nucleotide divergence between them ranges from 3.3% to 3.8% for ITS 1 and from 9.3% to 10.7% for ITS 2. The molecular data also revealed that two eastern Asian species are closely related but should be treated as distinct species.     

Molecular phylogeny and reticulate origins of the polyploid Bromus species from section Genea (Poaceae)

The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.     

Phylogenetic Relationships of the Silver Saxifrages (Saxifraga, Sect. Ligulatae Haworth): Implications for the Evolution of Substrate Specificity, Life Histories, and Biogeography

The silver saxifrages (Saxifraga sect. Ligulatae Haworth; Saxifragaceae) exhibit remarkable variation of substrate specialization, with strictly calcicole to calcifuge species, as well as life histories which range from semelparity to iteroparity. They occur almost exclusively in the European mountain ranges and display high levels of endemism. Sequences from chloroplast and nuclear ribosomal DNA were obtained to resolve phylogenetic relationships among the silver saxifrages and related taxa and to gain insight into the evolution of substrate specificity, life history, and biogeography. The resulting phylogenies suggested that (1) Saxifraga sect. Ligulatae, as traditionally defined, does not constitute a monophyletic group; (2) lime-secreting hydathodes in calcifuge species apparently represent a secondary nonaptation; (3) semelparity evolved independently two or three times in the silver saxifrages and allied sections, possibly in response to climatic changes that occured during the Pleistocene; and (4) narrow endemics, for example S. cochlearis, likely evolved from the fragmentation of the widespread S. paniculata into refugial populations that became isolated during the glacial maxima of the Pleistocene.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

Diversity in the genus Rhabdias (Nematoda,Rhabdiasidae): Evidence for cryptic speciation

Lungworms from the genus Rhabdias are common parasites of amphibians and reptiles distributed worldwide. To assess the diversity of Rhabdias spp., we performed molecular analyses of 35 specimens sampled in different regions of Brazil. Molecular analyses were based on the internal transcribed spacer (ITS), large subunit (28S) ribosomal and the cytochrome oxidase I (COI) mitochondrial genes. DNA sequence divergence was compared among ribosomal and mitochondrial genes, analyses using the general mixed Yule‐coalescent (GMYC) method based on the COI gene were used to identify possible cryptic diversity, and phylogenetic analyses using concatenated ITS and 28S ribosomal genes were used to test the monophyly of Rhabdiasidae. We revealed five morphospecies: R. cf. stenocephala, R. breviensis, R. pseudosphaerocephala and two new species, Rhabdias sp.4 and Rhabdias sp.5. DNA sequence levels of divergence among genes ITS, 28S and COI were compared, and the efficiency of the molecular markers to identify species (ITS and COI) and lineages (COI) was tested. GMYC was assigned to 17 well‐supported clades (i.e., 17 species), and cryptic diversity was detected in the Neotropical region as evidenced by the multiple lineages in R. breviensis and R. pseudosphaerocephala. In addition, our results suggest evidence for host–parasite cophylogeny in the R. pseudosphaerocephala complex and dispersal events among their populations. Phylogenetic analyses supported the monophyly of Rhabdiasidae and improved the resolution of main clades. Rhabdias breviensis is closely related to Rhabdias cf. africanus, Rhabdias cf. stenocephala, R. pseudosphaerocephala, Rhabdias sp.4 and Rhabdias sp.5 grouping together in a main clade with Neotropical‐related species. The large geographical distribution appeared to be a phylogenetic pattern among the species of Rhabdias from the neotropics.