Rhododendron kuomeianum (Ericaceae), a new species from northeastern Yunnan (China), based on morphological and genomic data

Rhododendron kuomeianum Y.H. Chang, J. Nielsen & Y.P. Ma, a new species of Rhododendron (Ericaceae) within subsect. Maddenia in sect. Rhododendron from Yiliang County, NE Yunnan, China, is described and illustrated. The new species is similar to R. valentinianum, but it can be easily distinguished by its sparse scales on the abaxial surface of the leaf blade, fewer flowers per inflorescence and white corolla with pale red margins. There are also differences in the widths of calyx lobes, leaf blade shape and indumentum characteristics of the petiole between the new species and Rhododendron linearilobum. We confirmed that R. kuomeianum is a new species closely related to R. valentinianum and R. changii with phylogenomic studies of 10 species within this subsection based on restriction site-associated DNA sequencing (RAD-seq) data. These phylogenomic analyses also clarified additional taxonomic problems in this subsection previously raised by morphological analysis. Our findings make a strong case for using next-generation sequencing to explore phylogenetic relationships and identify new species, especially in plants groups with complicated taxonomic problems.     

Taxonomic notes on Gaudinia (Poaceae) based upon morphological characters and the first record of G. maroccana for Portugal

Gaudinia maroccana is considered a Moroccan endemism. After a qualitative and quantitative study of 13 morphological characters of Gaudinia based upon 43 specimens from western Iberian Peninsula, northwestern Morocco and Azores Islands, with statistical analyses (correspondence analysis, box-plots and cluster), we can conclude that this species also occurs on the coast of Portugal. Some taxonomic remarks on G. fragilis, G. coarctata, G. hispanica and G. valdesii and a key for determination are presented.     

Phylogenetic Species Identification in Rattus Highlights Rapid Radiation and Morphological Similarity of New Guinean Species

The genus Rattus is highly speciose, the taxonomy is complex, and individuals are often difficult to identify to the species level. Previous studies have demonstrated the usefulness of phylogenetic approaches to identification in Rattus but some species, especially among the endemics of the New Guinean region, showed poor resolution. Possible reasons for this are simple misidentification, incomplete gene lineage sorting, hybridization, and phylogenetically distinct lineages that are unrecognised taxonomically. To assess these explanations we analysed 217 samples, representing nominally 25 Rattus species, collected in New Guinea, Asia, Australia and the Pacific. To reduce misidentification problems we sequenced museum specimens from earlier morphological studies and recently collected tissues from samples with associated voucher specimens. We also reassessed vouchers from previously sequenced specimens. We inferred combined and separate phylogenies from two mitochondrial DNA regions comprising 550 base pair D-loop sequences and both long (655 base pair) and short (150 base pair) cytochrome oxidase I sequences. Our phylogenetic species identification for 17 species was consistent with morphological designations and current taxonomy thus reinforcing the usefulness of this approach. We reduced misidentifications and consequently the number of polyphyletic species in our phylogenies but the New Guinean Rattus clades still exhibited considerable complexity. Only three of our eight New Guinean species were monophyletic. We found good evidence for either incomplete mitochondrial lineage sorting or hybridization between species within two pairs, R. leucopus/R. cf. verecundus and R. steini/R. praetor. Additionally, our results showed that R. praetor, R. niobe and R. verecundus each likely encompass more than one species. Our study clearly points to the need for a revised taxonomy of the rats of New Guinea, based on broader sampling and informed by both morphology and phylogenetics. The remaining taxonomic complexity highlights the recent and rapid radiation of Rattus in the Australo-Papuan region.     

Morphological and molecular data support recognition of a new rupicolous species of Pinguicula (Lentibulariaceae) from the Iberian Peninsula

A new rupicolous species, Pinguicula saetabensis, belonging to P. sect. Pinguicula is described from calcareous cliffs of central-southern Valencia province, in the eastern Iberian Peninsula. It has previously been confused with P. mundi and P. vallisneriifolia, two close allies endemic to southern Spain which share some morphological traits and a similar habitat. However, some peculiarities allow recognition of those Valencian plants at the specific rank. Data on morphology, ecology, biogeography and conservation are reported for the new species, and its taxonomic affinities are discussed on the basis of phylogenetic analyses of the internal transcribed spacer region of nuclear ribosomal DNA. Furthermore, the presence of one population of P. vallisneriifolia is confirmed in that province, also based on morphological and molecular data.     

Morphological Characters Useful for the Delimitation of Taxa Within Viola Subsect. Viola (Violaceae): A Morphometric Study from the West Carpathians

Forty-nine morphological characters were scored or measured on 44 populations (376 individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech Republic, Austria and Hungary). The presence of six species, namely V. alba (represented by subsp. alba), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility, cytological and morphometric analyses. The morphological characters traditionally used to delimit taxa within the subsection and those revealed by our study as most reliable are widely discussed. A key for identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is presented. Chromosome counting and flow cytometry were used to determine the ploidy levels of the populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.     

Molecular evidence for the hybridity ofIlex × wandoensis and the phylogenetic study of KoreanIlex based on ITS sequence data

In this study, internal transcribed spacer (ITS) regions within the nuclear ribosomal DNA of KoreanIlex were analyzed in order to investigate any molecular evidence thatI. × wandoensis could serve as a putative hybrid betweenI. cornuta andI. integra. We also sought to clearly identify taxonomic relationships and problems caused by consecutive external morphological characters among taxa in the genus. We sequenced 20 clones fromI. × wandoensis and found these individuals displayed intra-genomic polymorphisms within ITS regions. The analysis of the clones clearly demonstrated the presence of discrete sequences from bothI. cornuta andI. integra, thereby confirmingI. × wandoensis is a species that was formed by crossingI. cornuta andI. integra. Lastly, the subgenusIlex, which contains the evergreen species, failed to form a monophyletic group in a strict consensus tree that was prepared based upon ITS regions.I. crenata var.microphylla in the subgenus KoreaIlex, which has presented taxonomic problems previously, formed an independent clade within the consensus tree, thereby showing distinction fromI. crenata.) Genetic discontinuity ofI. macropoda andI. Macropoda for.pseudo-macropoda individuals couldn't be confirmed.     

The Model Organism Hermissenda crassicornis (Gastropoda: Heterobranchia) Is a Species Complex

Hermissenda crassicornis is a model organism used in various fields of research including neurology, ecology, pharmacology, and toxicology. In order to investigate the systematics of this species and the presence of cryptic species in H. crassicornis, we conducted a comprehensive molecular and morphological analysis of this species covering its entire range across the North Pacific Ocean. We determined that H. crassicornis constitutes a species complex of three distinct species. The name Hermissensa crassicornis is retained for the northeast Pacific species, occurring from Alaska to Northern California. The name H. opalescens is reinstated for a species occurring from the Sea of Cortez to Northern California. Finally, the name H. emurai is maintained for the northwestern species, found in Japan and in the Russian Far East. These three species have consistent morphological and color pattern differences that can be used for identification in the field.     

DNA barcoding of genus Metapenaeopsis (Decapoda: Penaeidae) and molecular phylogeny inferred from mitochondrial and nuclear DNA sequences

Metapenaeopsis Bouvier, 1905 is the most diverse genus within Penaeidae. Metapenaeopsis shrimps exhibited subtle morphological differences, which make identification a difficult task based on taxonomic keys alone. In this study, we carried out DNA barcoding and phylogenetic analyses to examine taxonomy and phylogeny of genus Metapenaeopsis based on mitochondrial (COI) and nuclear (PEPCK and NaK) genes. Despite limited performance of DNA barcoding in delineating Metapenaeopsis shrimps, it questioned the taxonomic status of the two subspecies, Metapenaeopsis mogiensis intermedia and Metapenaeopsis mogiensis mogiensis, as well as three separate species: Metapenaeopsis provocatoria longirostris, Metapenaeopsis quinquedentata and Metapenaeopsis velutina. The major pattern of relationships between all studied taxa of Metapenaeopsis was similar across all analytical methods in which species with one-valved petasma were genetically distinct from those with two-valved petasma. As expected from morphology, the remaining species with stridulating organ constituted a strongly supported clade. In contrast, a paraphyletic clade was resolved for species without stridulating organ which contradicts Crosnier's morphological classification scheme for Metapenaeopsis. Overall, the present molecular data indicated that the shape of petasma and stridulating organ were both phylogenetically significant morphological characters for this genus, adding further evidence for the Crosnier's proposal.     

Integrative taxonomy of the Rhinolophus macrotis complex (Chiroptera,Rhinolophidae) in Vietnam and nearby regions

The taxonomic status of Rhinolophus macrotis sensu lato (s.l.) in Vietnam and adjacent territories remains problematic. To address this issue, we performed an integrated study of morphological, acoustic, and genetic characters of R. macrotis s.l. specimens and compared these with sympatric species within the philippinensis group (R. marshalli, R. paradoxolophus, and R. rex). Our results reveal that in addition to a cryptic species of R. macrotis previously found in Jiangxi and Jingmen, China, R. macrotis s.l. in continental Asia includes three further species, namely R. cf. siamensis, R. cf. macrotis, and R. cf. macrotis “Phia Oac.” These four taxa are distinguished from genuine R. macrotis in Nepal and R. siamensis in Thailand by their morphological and/or genetic features. Further taxonomic evaluation of the subspecies of R. macrotis s.l. is needed to determine their affinities with recently recognized cryptic species and to possibly describe new taxa. Our results also show that interspecific divergences in mitochondrial DNA sequences (Cytb and COI genes) among taxa within the philippinensis group (particularly between R. cf. siamensis/R. cf. macrotis and R. rex/R. paradoxolophus) are significantly lower than those of other morphological groups in the genus. These phylogenetic patterns might be explained by recent allopatric speciation or ancient introgression events among ancestors of the taxa during the Pleistocene. However, further investigations including genetic analyses of nuclear genes are needed to test the latter hypothesis.     

Phylogenetic affiliation of <Emphasis Type="Italic">Choiromyces magnusii</Emphasis> and <Emphasis Type="Italic">C. venosus Tuberaceae Ascomycota</Emphasis>) from Spain

The only two species of Choiromyces collected up to now in Spain, C. venosus and C. magnusii, were studied with both morphological and molecular methods. Phylogenetic inference of nrDNA ITS and LSU sequences showed that they should be considered independent species in the genus Choiromyces. Choiromyces venosus is a hypogeous fungus, infrequent but naturally present in the Iberian Peninsula, that is imported into Spain from central Europe without any indication of its origin or proper taxonomic identification. It is usually sold as highly-prized edible species of the genus Tuber such as T. magnatum. Choiromyces magnusii is a more frequent hypogeous fungus in Spain that is commercialized in spring on a local scale together with different species in the genus Terfezia. Additionally, the Australian species Reddellomyces westraliensis, which was introduced with Eucalyptus camaldulensis plantations, is cited here for the first time in Spain and Europe.     

Species delimitation of three species within the Russula subgenus Compacta in Korea: R. eccentrica,R. nigricans,and R. subnigricans

Distinguishing individual Russula species can be very difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. In this study, we use the internal transcribed spacer (ITS) and 28S nuclear ribosomal large subunit (LSU) markers to identify and study the genetic diversity of species in the Russula subgenus Compacta in Korea. We focus on two morphologically similar species that are often misidentified for each other: R. nigricans and R. subnigricans. Based on molecular phylogenetic analyses, we identify three subgroups of R. nigricans, with two from Asia and one from Europe/North America. Surprisingly, we find Korean R. subnigricans are more closely related to R. eccentrica from North America than the type specimen of R. subnigricans from Japan. These molecular data, along with habitat data, reveal that Korean R. subnigricans had previously been misclassified and should now be recognized as R. eccentrica. Both ITS and LSU exhibit high interspecific and low intraspecific variation for R. eccentrica, R. nigricans, and R. subnigricans. These markers provide enough resolutional power to differentiate these species and uncover phylogeographic structure, and will be powerful tools for future ecological studies of Russula.     

Flow cytometric and karyological analyses of Calendula species from Iberian Peninsula

Calendula L. (Asteraceae) is a taxonomically and cytologically complex genus due to its high morphological and karyological variation. To gather consistent cytological information aiming to consolidate the existing knowledge, sustain the taxonomic revision of the genus and explore the evolutionary relationships among species, the genome size and chromosome number of the Iberian Peninsula representatives of this genus were assessed. The study included 11 taxa that occur in the Iberian Peninsula, one in Madeira and two from Morocco. Chromosome counts were made using the squash technique in root tips and flower buds, while nuclear DNA contents were assessed using propidium iodide flow cytometry. The following chromosome numbers are reported: 2n = 44 for C. arvensis, 2n = 30 for C. tripterocarpa, and 2n = 32 for the remaining Iberian taxa. The genome size of Calendula species was assessed for the first time and ranged from 1.75 pg/2C in C. maroccana to 5.41 pg/2C in C. arvensis. Within the complex formed by C. incana and C. suffruticosa, a gradient of genome size values was obtained. Intraspecific variation in genome size was detected in some taxa. The obtained genome size values and their variation are discussed in the light of the theories proposed for the speciation of the genus, with events of hybridization, genome duplication and dysploidy being hypothesized to play a major role in the evolution of this genus.     

Tree-based delimitation of morphologically ambiguous taxa: a study of the lizard malaria parasites on the Caribbean island of Hispaniola

Malaria parasites in the genus Plasmodium have been classified primarily on the basis of differences in morphology. These single-celled organisms often lack distinguishing morphological features, and this can encumber both species delimitation and identification. Six saurian malaria parasites have been described from the Caribbean island of Hispaniola. All six infect lizards in the genus Anolis, but only two of these parasites can be distinguished using morphology. The remaining four species overlap in morphology and geography, and cannot be consistently identified using traditional methods. We compared a morphological approach with a molecular phylogenetic approach for assessing the taxonomy of these parasites. We surveyed for blood parasites from 677 Anolis lizards, representing 26 Anolis spp. from a total of 52 sites across Hispaniola. Fifty-five of these lizards were infected with Plasmodium spp., representing several new host records, but only 24 of these infections could be matched to previously described species using traditional morphological criteria. We then estimated the phylogeny of these parasites using both mitochondrial (cytb and coxI) and nuclear (EF2) genes, and included carefully selected GenBank sequences to confirm identities for certain species. Our molecular results unambiguously corroborated our morphology-based species identifications for only the two species previously judged to be morphologically distinctive. The remaining infections fell into two well-supported and reciprocally monophyletic clades, which contained the morphological variation previously reported for all four of the morphologically ambiguous species. One of these clades was identified as Plasmodium floridense and the other as Plasmodium fairchildi hispaniolae. We elevate the latter to Plasmodium hispaniolae comb. nov. because it is polyphyletic with the mainland species Plasmodium fairchildifairchildi and we contribute additional morphological and molecular characters for future species delimitation. Our phylogenetic hypotheses indicate that two currently recognised taxa, Plasmodium minasense anolisi and Plasmodium tropiduri caribbense, are not valid on Hispaniola. These results illustrate that molecular data can improve taxonomic hypotheses in Plasmodium when reliable morphological characters are lacking.     

First identification of Decennatherium Crusafont, 1952 (Mammalia,Ruminantia, Pecora) in the Siwaliks of Pakistan

Previously undescribed remains of a new large giraffid have been identified from the late Miocene Siwaliks Hills of the Potwar Plateau in Pakistan. This taxon is very intimately related to the late Miocene giraffid genus Decennatherium, previously only certainly identified in the Iberian Peninsula, and with some possible remains assigned to Decennatherium crusafonti from Aliäbäd (Iran). The new material collected in the Siwaliks Hills shows a high morphological similarity with the Spanish remains of the genus, especially the early Vallesian Decennatherium pachecoi. Two previously undescribed ossicone fragments from the same area are also described and identified as cf. Decennatherium. These findings represent the easternmost occurrence of the genus Decennatherium and show a late Miocene migration of the genus throughout the Iberian Peninsula and Southern Asia.     

Notocotylus ikutai n. sp. (Digenea: Notocotylidae) from lymnaeid snails and anatid birds in Hokkaido,Japan

An unknown species of the genus Notocotylus (Digenea: Notocotylidae) was found as the larval stage from the lymnaeid snail, Radix auricularia, in a static water area of the Chubetsu River, Hokkaido, the northernmost island of Japan. A DNA barcoding identification system was applied to detect the adult stage. Through the inspection of anatid game birds in Hokkaido, Anas crecca, Anas platyrhynchos, Anas zonorhyncha, and Mareca penelope were demonstrated to serve as the definitive hosts. The detailed morphological features of the species were characterized using adults raised experimentally in immunosuppressed mice and naturally developed larvae in R. auricularia. Although the species is morphologically similar to Notocotylus attenuatus and Notocotylus magniovatus in both adult and larval stages, its taxonomic independence was confirmed by a comprehensive study based on molecular phylogeny, morphology, and ecology. Here we propose Notocotylus ikutai n. sp. for this species. The migratory behavior of the anatid hosts and the North-Eurasian distribution of R. auricularia suggest that the new species is widely distributed in the northern Far East.