Nuclear ITS Region Research Articles

Spatially distinct and regionally endemic Symbiodinium assemblages in the threatened Caribbean reef-building coral Orbicella faveolata

Recently, the Caribbean reef-building coral Orbicella faveolata was listed as “threatened” under the U.S. Endangered Species Act. Despite attention to this species’ conservation, the extent of geographic variation within O. faveolata warrants further investigation. O. faveolata is unusual in that it can simultaneously harbor multiple genetically distinct and co-dominant species of endosymbiotic dinoflagellates in the genus Symbiodinium. Here, we investigate the geographic and within-colony complexity of Symbiodinium-O. faveolata associations from Florida Keys, USA; Exuma Cays, Bahamas; Puerto Morelos, Mexico; and Carrie Bow Cay, Belize. We collected coral samples along intracolony axes, and Symbiodinium within O. faveolata samples was analyzed using the nuclear ITS2 region and chloroplast 23S rDNA genotyping. O. faveolata associated with species of Symbiodinium in clades A (type A3), B (B1 and B17), C (C3, C7, and C7a), and D (D1a/Symbiodinium trenchii). Within-colony distributions of Symbiodinium species correlated with light availability, cardinal direction, and depth, resulting in distinct zonation patterns of endosymbionts within a host. Symbiodinium species from clades A and B occurred predominantly in the light-exposed tops, while species of clade C generally occurred in the shaded sides of colonies or in deeper-water habitats. Furthermore, geographic comparisons of host–symbiont associations revealed regional differences in Symbiodinium associations. Symbiodinium A3 was detected in Mesoamerican coral colonies, but not in colonies from the Florida Keys or Bahamas. Likewise, Symbiodinium B17 was unique to Mesoamerican O. faveolata, whereas Symbiodinium B1 was found at all localities sampled. However, using cp23S genotyping paired with ITS2 analysis revealed geographically endemic haplotypes among Symbiodinium clades A, B, and C. Since Symbiodinium spatial heterogeneity among this coral species is greater than most corals, a question arises as to whether all western Atlantic populations of O. faveolata should be considered equally “threatened”? Alternatively, geographically and spatially distinct coral–symbiont associations may benefit from specialized management protocols.

A PCR-RFLP method to detect hybridization between the invasive Eurasian watermilfoil (Myriophyllum spicatum) and the native northern watermilfoil (Myriophyllum sibiricum), and its application in Ontario lakes

The discovery of hybridization between the invasive Eurasian watermilfoil (Myriophyllum spicatum L.) and native northern watermilfoil (Myriophyllum sibiricum Kom.) has generated interest in establishing the hybrid’s distribution and invasiveness. Identification of hybrid M. spicatum × M. sibiricum requires molecular genetic analysis, however, as the hybrid’s morphology overlaps with both parent species. Using plants collected from 10 lakes in Ontario, Canada, we compared a previous method of identification (sequencing the nuclear ITS region) with a simpler screening method (PCR-RFLP of the ITS region). Both methods agreed on the identification of hybrid M. spicatum × M. sibiricum and both parent species, supporting the suitability of PCR-RFLP to screen for the hybrid. Four of 29 samples were identified as hybrid M. spicatum × M. sibiricum, which were all found in three adjacent lakes associated with the Rideau Canal Waterway. The PCR-RFLP method should enable greater sampling effort to screen for hybrid M. spicatum × M. sibiricum and establish its geographic distribution across connected waterways.

DNA based revised geographic circumscription of species of Physcomitrella s.l. (Funariaceae): P. patens new to East Asia and P. magdalenae new to East Africa

Physcomitrella, as traditionally defined, accommodates certain Funariaceae with immersed and cleistocarpous capsules. Recent molecular inferences revealed that the three or four taxa typically recognized within Physcomitrella did not arise from a unique common ancestor, and hence that their morphological similarities likely resulted from convergence, while one potential taxon, P. patens subsp. californica (=Physcomitridium readeri s.l.), is currently regarded as a polyphyletic entity, making Physcomitridium polyphyletic. Following recent discoveries that would greatly expand the known range of two taxa, we sampled populations of Physcomitrella s.l. from all main geographic regions, and sequenced the nuclear ITS regions and four plastid loci to assess the geographic circumscription of each clade. We recovered three unambiguous monophyletic entities matching the three morphotypes recognized by Fife, each with a distinct geographic range: Physcomitrella patens s.str. with a range in Europe and North America extended to East Asia (China); P. magdalenae with a range from West and Central Africa extended eastwards to Ethiopia; and Physcomitridium (Physcomitrella) readeri from western Europe and western North America to Japan and Australia, plus a doubtful occurrence in China. Although the distinction of P. californica from P. readeri remains doubtful, we reject the hypothesis that Physcomitridium is polyphyletic, arguing that this resulted from a misidentification of the sequenced voucher.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described.

First Report of Phytophthora hedraiandra Causing Rhododendron Dieback and Root Rot of Common Beech in the Czech Republic.

From 2010 to 2012, Phytophthora isolates were obtained from brownish diffusion leaf lesions usually up to 2 to 3 cm in diameter of Rhododendron caucasicum 'Cheer,' from withered twigs of Rhododendron sp. with blackish elongated lesions up to ~5 cm in length, and from rotten feeder roots of 2-year-old, chlorotic, wilting seedlings of Fagus sylvatica collected from ornamental and forest nurseries in three areas (central and eastern Bohemia and northern Moravia) in the Czech Republic. Isolates formed chrysanthemum-like to slightly stellate, appressed colonies with sparse aerial mycelium on V8 agar (V8A) plates at 20°C after 5 days, whereas on carrot agar (CA) plates the pattern was vague with no aerial mycelium. The cardinal growth temperatures were: min. 3°C, optimum 23 to 27°C, and max. 31°C. Radial growth was 5.7 to 6.6 mm/day at 20°C on V8A. The isolates were homothallic and produced colorless, smooth-walled, spherical oogonia with an average diameter 29.9 to 33.8 μm on CA. Oospores were aplerotic (avg. 26.4 to 29.3 μm), oospore wall was hyaline and averaged 1.3 to 1.7 μm thick, oospore wall index was 0.26 to 0.32. Paragynous or occasionally amphigynous antheridia averaged 13.4 to 15.0 × 10.9 to 12.5 μm (height × width). Sporangia were caducous, papillate, globose, spherical to ovoid, with short pedicels (avg. 2.1 to 2.6 μm) and averaged 30.9 to 41.5 × 25.5 to 30.6 μm, L:B ratio was 1.2 to 1.4. Chlamydospores were not observed. Morphological characters resembled those described for P. hedraiandra (1). The isolates were deposited in the collection of phytopathogenic oomycetes of RILOG Pruhonice and given accession nos. 450.11, 531.11, and 578.12. The isolates were sequenced for nuclear rDNA ITS region and partial Cox I gene. Obtained sequences were compared with sequences present in GenBank database using BLAST. The ITS sequences of all isolates (GenBank Accession Nos. KJ567081, 82, and 83) of overall length of 792 bp were identical to that of P. hedraiandra AY707987 (1). The Cox I sequences of overall length of 880 bp (KJ567084, 85, and 86) showed 99% homology (1 bp substitution) with AY769115 (1) and 100% identity with other Cox I sequences of P. hedraiandra, i.e., JN376067 (4) and EF174432 (3). Koch's postulates were confirmed by wound-inoculating of 3-year-old rhododendron and common beech plants (10 host plants per corresponding isolate). Rhododendron leaves were gently abraded near the midrib, whereas 5-mm-diameter bark plugs were removed from the beech collars. The inoculum (5-mm-diameter V8A plug with actively growing mycelium) was placed over wounds and sealed with Parafilm. Control plants were treated in the same manner with sterile agar plugs. Plants were maintained in a greenhouse at 22°C. All inoculated plants showed disease symptoms after 10 days of incubation; the lesions were up to 2 cm in rhododendron leaves and ~1 cm in beech collars. Control plants remained healthy. The pathogen was re-isolated from all infected plants. To our knowledge, this is the first report of P. hedraiandra in the Czech Republic. Besides it, the pathogen was found in southern and western Europe (Italy, Slovenia, Spain, the Netherlands) and in the United States (2).

Evaluación de la posición filogenética de la familia monotípica Halophytaceae (Portulacinae, Caryophyllales) basada en conjuntos de datos moleculares nucleares y plástidos

A pesar de numerosos estudios filogenéticos para determinar las relaciones de las familias del orden Caryophyllales y particularmente del suborden Portulacinae, no se ha establecido aún la posición de Halophytaceae. Halophytum ameghinoi es el único representante de esta familia de hierbas suculentas, endémico de la ecoregión Monte Argentino, creciendo en vegetación arbustiva árida o semi-árida. Algunos autores han sugerido una relación con Chenopodiaceae y otros con Basellaceae y/o Portulacaceae y Montiaceae. Para determinar la posición de Halophytum en el suborden Portulacinae se llevaron a cabo análisis filogenéticos utilizando regiones nucleares (18S, ITS, 26S) y regiones de cloroplasto ( atpB , trnK/matK , ndhF , rbcL y rpl16 ), de secuencias de ADN previas y secuenciadas en este proyecto. El análisis filogenético basado en la matriz de evidencia total confirmó una cercana relación entre Halophytum y Basellaceae. Estos dos grupos resultaron cercanamente emparentados con Didiereaceae. La región de ADN con mayor número de sitios variables fue la región de cloroplasto trnk/matK seguida por ndhF , aunque la región nuclear de ITS resultó con más sitios variables si se toma en cuenta el porcentaje de sitios variables/sitios informativos. Sin embargo, si esta región nuclear es eliminada, los árboles filogenéticos muestran valores de soporte de ramas más altos.

Radiation of the red algal parasite Congracilaria babae onto a secondary host species, Hydropuntia sp. (Gracilariaceae, Rhodophyta).

Congracilaria babae was first reported as a red alga parasitic on the thallus of Gracilaria salicornia based on Japanese materials. It was circumscribed to have deep spermatangial cavities, coloration similar to its host and the absence of rhizoids. We observed a parasitic red alga with morphological and anatomical features suggestive of C. babae on a Hydropuntia species collected from Sabah, East Malaysia. We addressed the taxonomic affinities of the parasite growing on Hydropuntia sp. based on the DNA sequence of molecular markers from the nuclear, mitochondrial and plastid genomes (nuclear ITS region, mitochondrial cox1 gene and plastid rbcL gene). Phylogenetic analyses based on all genetic markers also implied the monophyly of the parasite from Hydropuntia sp. and C. babae, suggesting their conspecificity. The parasite from Hydropuntia sp. has a DNA signature characteristic to C. babae in having plastid rbcL gene sequence identical to G. salicornia. C. babae is likely to have evolved directly from G. salicornia and subsequently radiated onto a secondary host Hydropuntia sp. We also recommend the transfer of C. babae to the genus Gracilaria and propose a new combination, G. babae, based on the anatomical observations and molecular data.

Taxonomic reinvestigation of Petalonia (Phaeophyceae, Ectocarpales) in southeast of Honshu, Japan, with a description of Petalonia tenuis sp. nov.

:We conducted a phylogenetic and morphological reinvestigation of Petalonia species from the Pacific coast southeast of Honshu, Japan. Phylogenetic analyses based on plastid rbcL and the nuclear ITS regions resulted in three genetically distinct species: P. binghamiae, P. fascia, and Petalonia sp. Petalonia sp. was characterized by the medullary structure showing intermediate characteristics between those of P. binghamiae and P. fascia as well as by the thinner thallus. In addition, the habitat and the seasonality of Petalonia sp. were slightly different from those of the two species. Sexual fusion of plurizoids was observed on Petalonia sp., and the morphology of the prostrate sporophytes agreed with the previous reports of P. binghamiae and P. fascia. According to the characteristics, Petalonia sp. is described as a new species, P. tenuis sp. nov.

Molecular phylogeny of Cotoneaster (Rosaceae) inferred from nuclear ITS and multiple chloroplast sequences

Cotoneaster Medik. (Rosaceae, Maloideae) is distributed in Europe, North Africa, and temperate areas of Asia except Japan. Members of the genus exhibit considerable morphological variation. The infrageneric classification is also obscured by polyploidy, hybridization, and apomixis. In this study, phylogenetic analyses were conducted to test infrageneric classifications of this genus using DNA sequence data from the nuclear ITS (nrITS) region and three chloroplast intergenic spacer regions. Maximum parsimony and Bayesian inference analyses of both datasets agreed with the two sections/subgenera of Koehne’s classification system, and suggested that four subsections (Microphylli, Chaenopetalum, Adpressi, and Cotoneaster) and the series of Koehne’s classification system were non-monophyletic. The incongruence length difference test indicated that the nrITS and cpDNA datasets were significantly incongruent (P = 0.001), and the placement of 14 species was discordant in phylogenetic trees derived from the two datasets. Within Cotoneaster, hybridization was indicated to be an important factor contributing to the incongruence between the nrITS and cpDNA data. By mapping nine morphological characters onto the combined nrITS–cpDNA phylogenetic tree, we inferred that a deciduous habit, glabrous fruit, white anthers, erect and light pink petals, and white filaments are plesiomorphic character states in Cotoneaster.

DNA barcodes and phylogenetic affinities of the terrestrial slugs Arion gilvus and A. ponsi (Gastropoda, Pulmonata, Arionidae)

The Iberian Peninsula is a region with a high endemicity of species of the terrestrial slug subgenus Mesarion. Many of these species have been described mainly on subtle differences in their proximal genitalia. It therefore remains to be investigated 1) whether these locally diverged taxa also represent different species under a phylogenetic species concept as has been shown for other Mesarion species outside the Iberian Peninsula, and 2) how these taxa are phylogenetically related. Here, we analysed DNA sequence data of two mitochondrial (COI and 16S) genes, and of the nuclear ITS1 region, to explore the phylogenetic affinities of two of these endemic taxa, viz. Arion gilvus Torres Mínguez, 1925 and A. ponsi Quintana Cardona, 2007. We also evaluated the use of these DNA sequence data as DNA barcodes for both species. Our results showed that ITS did not allow to differentiate among most of the Mesarion molecular operational taxonomic units (MOTUs) / morphospecies in Mesarion. Yet, the overall mean p-distance among the Mesarion MOTUs / morphospecies for both mtDNA fragments (16.7% for COI, 13% for 16S) was comparable to that between A. ponsi and its closest relative A. molinae (COI: 14.2%; 16S: 16.2%) and to that between A. gilvus and its closest relative A. urbiae (COI: 14.4%; 16S: 13.4%). Hence, with respect to mtDNA divergence, both A. ponsi and A. gilvus, behave as other Mesarion species or putative species-level MOTUs and thus are confirmed as distinct ‘species’.

Molecular phylogeny of the Lactuca alliance (Cichorieae subtribe Lactucinae, Asteraceae) with focus on their Chinese centre of diversity detects potential events of reticulation and chloroplast capture.

The first comprehensive molecular phylogenetic reconstruction of the Cichorieae subtribe Lactucinae is provided. Sequences for two datasets, one of the nuclear rDNA ITS region, the other of five concatenated non-coding chloroplast DNA markers including the petD region and the psbA-trnH, 5′trnL(UAA)-trnF, rpl32-trnL(UAG) and trnQ(UUG)-5′rps16 spacers, were, with few exceptions, newly generated for 130 samples of 78 species. The sampling spans the entire subtribe Lactucinae while focusing on its Chinese centre of diversity; more than 3/4 of the Chinese Lactucinae species are represented. The nuclear and plastid phylogenies inferred from the two independent datasets show various hard topological incongruences. They concern the internal topology of major lineages, in one case the placement of taxa in major lineages, the relationships between major lineages and even the circumscription of the subtribe, indicating potential events of ancient as well as of more recent reticulation and chloroplast capture in the evolution of the subtribe. The core of the subtribe is clearly monophyletic, consisting of the six lineages, Cicerbita, Cicerbita II, Lactuca, Melanoseris, Notoseris and Paraprenanthes. The Faberia lineage and the monospecific Prenanthes purpurea lineage are part of a monophyletic subtribe Lactucinae only in the nuclear or plastid phylogeny, respectively. Morphological and karyological support for their placement is considered. In the light of the molecular phylogenetic reconstruction and of additional morphological data, the conflicting taxonomies of the Chinese Lactuca alliance are discussed and it is concluded that the major lineages revealed are best treated at generic rank. An improved species level taxonomy of the Chinese Lactucinae is outlined; new synonymies and some new combinations are provided.

Molecular evidence confirms the parasite Congracilaria babae (Gracilariaceae, Rhodophyta) from Malaysia

A phylogenetic study of two parasites found on the common host species Gracilaria salicornia from Japan and Malaysia based on nuclear, plastid, and mitochondrial molecular markers was conducted. The Japanese and Malaysian parasites were placed in the same cluster in the nuclear phylogenies inferred from the LSU rRNA gene and ITS region, supporting the monophyly of these parasites despite the slight anatomical variations observed. In addition to the molecular analyses, shared morphological and reproductive features including the lack of rhizoids, similar coloration to hosts, and the presence of deep spermatangial conceptacles supported the inclusion of both the Japanese and Malaysian parasites in Congracilaria babae. Phylogenetic analyses based on ITS sequences indicated that C. babae most likely evolved directly from its current host species, G. salicornia. C. babae was shown to be a member of Gracilaria sensu stricto along with its host. C. babae growing on G. salicornia is characterized by having plastid rbcL and mitochondrial cox1 gene sequences identical to those of its host, whereas the nuclear LSU rRNA gene and ITS region sequences are different from those of its host.

Oligochaeta (Annelida) of the profundal of Lake Hazar (Turkey), with description of Potamothrix alatus hazaricus n. ssp..

Lake Hazar is an alkaline oligotrophic lake of tectonic origin, located in the Eastern Anatolia region in Turkey, 1248 a a.s.l. Its surface area is 80 km2, the average depth 93 m and maximum depth 205 m. The lake and its surroundings an under protection as a region of historical value. During the present study (2007-2012), samples were taken from 15 stations located at a depth of 2-200 m. Oligochaeta comprised 69% of the total invertebrate abundance. The profundal olgochaete fauna was found to consist of only three tubificid taxa, all of the subfamily Tubificinae. Potamothrix alatus hazaricus Timm & Arslan, n. ssp. was dominating anywhere down to maximum depths while Psammoryctides barbatus (Grube) and Ilyodrilus(?) sp. occurred seldom. All three are new records for Lake Hazar. Potamothrix alatus hazaricus shares the "winged" body shape in its genital region with the nominal, brackish-water subspecies P. a. alatus Finogenova, 1972, and the lateral position of the spermathecal pores and the shape of the ventral chaetae with the freshwater subspecies P. a. paravanicus Poddubnaja & Pataridze, 1989 known from Transcaucasian lakes. The mitochondrial COI barcoding gene suggests long separation between the two taxa, but the nuclear ITS region shows no variation. The generic position of Ilyodrilus (?) sp. remains obscure since its internal genitalia could not be studied.

Primulina cardaminifolia (Gesneriaceae), a rare new species from limestone areas in Guangxi, China.

BackgroundPrimulina cardaminifolia Yan Liu & W.B. Xu (Gesneriaceae), a distinct new species with imparipinnate leaves, is described and illustrated from a limestone valley in Guangxi Zhuangzu Autonomous Region, China. To assure its generic placement and phylogenetic affinity, phylogenetic analyses were performed using DNA sequences of nuclear ITS and chloroplast trnL-F intron spacer region. Additionally, somatic chromosome number was counted and pollen stainability was tested.ResultsPhylogenetic analyses support its placement in Primulina; however, two phylogenetically distinct ITS sequence types were detected, suggesting a probable hybrid origin. Its pollen stainability is 100% and its chromosome number, 2n = 36, is congruent with all known counts of diploid species of the genus.ConclusionAll available data support the recognition of the new species Primulina cardaminifolia and suggest that it could have derived from homoploid hybrid speciation. Color plates, line drawings and a distribution map are provided to aid in identification.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-19) contains supplementary material, which is available to authorized users.

Barcoding Poplars (Populus L.) from Western China

Background Populus is an ecologically and economically important genus of trees, but distinguishing between wild species is relatively difficult due to extensive interspecific hybridization and introgression, and the high level of intraspecific morphological variation. The DNA barcoding approach is a potential solution to this problem.Methodology/Principal FindingsHere, we tested the discrimination power of five chloroplast barcodes and one nuclear barcode (ITS) among 95 trees that represent 21 Populus species from western China. Among all single barcode candidates, the discrimination power is highest for the nuclear ITS, progressively lower for chloroplast barcodes matK (M), trnG-psbK (G) and psbK-psbI (P), and trnH-psbA (H) and rbcL (R); the discrimination efficiency of the nuclear ITS (I) is also higher than any two-, three-, or even the five-locus combination of chloroplast barcodes. Among the five combinations of a single chloroplast barcode plus the nuclear ITS, H+I and P+I differentiated the highest and lowest portion of species, respectively. The highest discrimination rate for the barcodes or barcode combinations examined here is 55.0% (H+I), and usually discrimination failures occurred among species from sympatric or parapatric areas.Conclusions/SignificanceIn this case study, we showed that when discriminating Populus species from western China, the nuclear ITS region represents a more promising barcode than any maternally inherited chloroplast region or combination of chloroplast regions. Meanwhile, combining the ITS region with chloroplast regions may improve the barcoding success rate and assist in detecting recent interspecific hybridizations. Failure to discriminate among several groups of Populus species from sympatric or parapatric areas may have been the result of incomplete lineage sorting, frequent interspecific hybridizations and introgressions. We agree with a previous proposal for constructing a tiered barcoding system in plants, especially for taxonomic groups that have complex evolutionary histories (e.g. Populus).

Comprehensive phylogenetic analyses of the Ruppia maritima complex focusing on taxa from the Mediterranean

Recent molecular phylogenetic studies reported high diversity of Ruppia species in the Mediterranean. Multiple taxa, including apparent endemics, are known from that region, however, they have thus far not been exposed to phylogenetic analyses aimed at studying their relationships to taxa from other parts of the world. Here we present a comprehensive phylogenetic analyses of the R. maritima complex using data sets composed of DNA sequences of the plastid genome, the multi-copy nuclear ITS region, and the low-copy nuclear phyB gene with a primary focus on the Mediterranean representatives of the complex. As a result, a new lineage, "Drepanensis", was identified as the seventh entity of the complex. This lineage is endemic to the Mediterranean. The accessions included in the former "Tetraploid" entity were reclassified into two entities: an Asia-Australia-Europe disjunct "Tetraploid_α" with a paternal "Diploid" origin, and a European "Tetraploid_γ" originating from a maternal "Drepanensis" lineage. Another entity, "Tetraploid_β", is likely to have been originated as a result of chloroplast capture through backcrossing hybridization between paternal "Tetraploid_α" and maternal "Tetraploid_γ". Additional discovery of multiple tetraploidizations as well as hybridization and chloroplast capture at the tetraploid level indicated that hybridization has been a significant factor in the diversification of Ruppia.

Evolutionary relationships in Gorteria: A re‐evaluation

AbstractA molecular phylogeny of the mainly South African genus Gorteria is presented for the first time, based on Bayesian inference and parsimony analyses using DNA sequences from plastid trnL‐F and from nuclear ETS and ITS regions. It is shown that the genus is strongly paraphyletic in relation to one subgroup of Hirpicium, a finding that changes the prevailing generic concept and circumscription of Gorteria and Hirpicium. Furthermore, Gorteria diffusa and G. personata both seem to be polyphyletic assemblages, comprised of some more or less well diagnosed monophyletic groups with different distributions.

Relationships of <I>Asanthus</I> (Asteraceae, Eupatorieae)

Abstract Analysis of DNA sequence data was used to clarify the circumscription and relationships of the small Mexican genus Asanthus. Results from non-coding regions, including the nuclear ITS and the chloroplast psbA-trnH spacer showed that Asanthus is clearly monophyletic and distinct from Steviopsis and Brickellia, two genera with which it is sometimes lumped, as well as from the closely related Brickelliastrum and Carminatia. The DNA sequence data also agree with morphological observations in showing that Asanthus is composed of three species, A. thyrsiflorus from the Sierra Madre Oriental and A. squamulosus and A. solidaginifolius from the Sierra Madre Occidental. Samples from the southern Sierra Madre Occidental that have been identified as A. thyrsiflorus are suggested by the presence of an additive pattern of polymorphisms in the nuclear ITS region to be of interspecific hybrid origin between A. thyrsiflorus and A. solidaginifolius, which is consistent with reports of their morphological intergrad...

An approach to identify putative hybrids in the ‘coalescent stochasticity zone’, as exemplified in the African plant genus Streptocarpus (Gesneriaceae)

The inference of phylogenetic relationships is often complicated by differing evolutionary histories of independently-inherited markers. The causes of the resulting gene tree incongruence can be challenging to identify, often relying on coalescent simulations dependent on unverifiable assumptions. We investigated alternative techniques using the South African rosulate species of Streptocarpus as a study group. Two independent gene trees - from the nuclear ITS region and from three concatenated plastid regions (trnL-F, rpl20-rps12 and trnC-D) - displayed widespread, strongly supported incongruence. We investigated the causes by detecting genetic exchange across morphological borders using morphological optimizations and genetic exchange across species boundaries using the genealogical sorting index. Incongruence between gene trees was associated with ancestral shifts in growth form (in four species) but not in pollination syndrome, suggesting introgression limited by reproductive barriers. Genealogical sorting index calculations showed polyphyly of two additional species, while individuals of all others were significantly associated. In one case the association was stronger according to the internal transcribed spacer data than according to the plastid data, which, given the smaller effective population size of the plastid, may also indicate introgression. These approaches offer alternative ways to identify potential hybridization events where incomplete lineage sorting cannot be rejected using simulations.

Restricted variation in plant barcoding markers limits identification in closely related bryophyte species

Species-level identification and delimitation of bryophytes using the proposed general barcode markers for land plants has been challenging. Bryophyta (mosses) is the second most species-rich group of land plants after angiosperms, and it is thus of great importance to find useful barcoding regions also for this group of plants. We investigated how the plastid regions atpF-atpH, rbcL and trnH-psbA and the nuclear ITS2 region performed as barcode markers on closely related bryophyte taxa of selected moss (Bartramia, Distichium, Fissidens, Meesia and Syntrichia) and liverwort (Blepharostoma) genera from boreal and arctic regions. We also evaluated how sequencing success of herbarium specimens is related to length of the sequenced fragment, specimen age and taxonomic group. Sequencing success was higher for shorter fragments and younger herbarium specimens, but was lower than expected in the genera Distichium and Fissidens, indicating imperfect universality of the primers used. None of the studied DNA barcode regions showed a consistent barcode gap across the studied genera. As a single locus, the region atpF-atpH performed slightly better than rbcL and ITS2 and much better than trnH-psbA in terms of grouping conspecific sequences in monophyletic groups. This marker also gave a higher percentage of correct hits when conducting blast searches on a local database of identified sequences. Concatenated data sets of two and three markers grouped more conspecific sequences in monophyletic groups, but the improvement was not great compared with atpF-atpH alone. A discussion of recent studies testing barcode regions for bryophytes is given. We conclude that atpF-atpH, rbcL and ITS2 are to be the most promising barcode markers for mosses.