Molecular evidence for glacial expansion and interglacial retreat during Quaternary climatic changes in a montane temperate pine (Pinus kwangtungensis Chun ex Tsiang) in southern China

It has been suggested that the eastern Asian temperate flora have responded to Quaternary climatic changes in a different way compared with temperate plants in Europe and Northern America. However, knowledge about their phylogeographic structure and evolutionary history is still limited. In this study we investigated mitochondrial DNA variation in 17 populations of Pinus kwangtungensis, a five-needled pine inhabiting in isolated mountains of southern China and northern Vietnam. A total of ten mitochondrial haplotypes (mitotypes) were characterized by the polymorphisms of two fragments (cox-1-2 and nad7 intron1), and total genetic diversity was high (h _T = 0.847). The construction of phylogenetic relationships of the ten mitotypes detected three major, well-distinct clades, largely corresponding to four population groups identified by SAMOVA analysis. SAMOVA also indicated that most genetic variance should be attributed to among-group differentiation (F _CT = 0.868), consistent with the substantial genetic structure found within P. kwangtungensis (G _ST = 0.751). The genetic distances of P. kwangtungensis weakly but significantly correlated with geographical distances (R = 0.228, P = 0.03), revealing a pattern of isolation-by-distance. Demographic analysis did not detect any bottleneck events in the recent history of P. kwangtungensis. These results clearly suggested that there were three major refugia for this montane temperate conifer during warm stages (along with other minor refugia), and that the species would have tracked Quaternary climatic changes by expanding to nearby lowlands in the glacial periods, and by retreating to mountaintops (the refugia) during interglacial warmer stages, experiencing fragmentation and isolation among refugia.     

Clear genetic structure of Pinus kwangtungensis (Pinaceae) revealed by a plastid DNA fragment with a novel minisatellite

Background and Aims

Pinus kwangtungensis is a five-needled pine, inhabiting isolated mountain tops, cliffs or slopes in the montane areas of southern China and northern Vietnam. Global warming and long-term deforestation in southern China threaten its existence and genetic integrity, and this species is listed as vulnerable in the China Species Red List. However, the level and distribution of genetic diversity in this vulnerable species are completely unknown. In this paper, the genetic diversity and structure are examined using paternally inherited plastid markers to shed light on its evolutionary history and to provide a genetic perspective for its conservation.

Methods

By means of direct sequencing, a new polymorphic fragment containing a minisatellite site was identified within the plastid genome of P. kwangtungensis. Using the minisatellite site along with five SNPs (one indel and four substitutions) within the same fragment, the population genetic structure and pollen flow were analysed in 17 populations of P. kwangtungensis in southern China.

Key Results

Analysis of 227 individuals from 17 populations revealed ten haplotypes at the minisatellite site. The haplotype diversity at species level was relatively high (0·629). Genetic diversity of each population ranged from 0 to 0·779, and the western populations harboured more genetic variation than the eastern and Hainan populations, although the former appeared to have experienced a bottleneck in recent history. Population subdivision based on this site was high (F_ST = 0·540 under IAM; R_ST = 0·677 under SMM). Three major clusters (eastern, western and Hainan) were identified based on a neighbor-joining dendrogram generated from genetic distances among the populations. The genetic structures inferred from all the polymorphic sites and the SNPs were in concordance with that from the minisatellite site.

Conclusions

The results suggest that there are at least three refugia for P. kwangtungensis and that populations in these refugia should be treated as separate evolutionarily significant units or conservation units. The high diversities in the western populations suggest that these were much larger in the past (e.g. glacial stages) and that the shrinking population size might have been caused by recent events (e.g. deforestation, global warming, etc.). The western populations should be given priority for conservation due to their higher genetic diversity and limited population sizes. It is concluded that the newly found minisatellite may serve as a novel and applicable molecular marker for unravelling evolutionary processes in P. kwangtungensis.Key words: Pinus kwangtungensis, minisatellite, population genetics, conservation     

Pestalotiopsis yunnanensis sp. nov., an endophyte from Podocarpus macrophyllus (Podocarpaceae) based on morphology and ITS sequence data

Pestalotiopsis is a common and important plant-associated pathogen and endophyte with wide geographical and host distribution. In an investigation of endophytic Pestalotiopsis species associated with Podocarpaceae in China, a new species Pestalotiopsis yunnanensis was isolated from Podocarpus macrophyllus in Kunming, southwestern China. This new species produced pycnidium-like conidiamata in culture, distinct from its morphologically similar species, P. funereoides, P. funerea and P. thujae, which produce acervuli in manual media and hosts. P. yunnanensis also possesses a greater conidium length/width ratio, and longer apical and basal appendages as its distinguishing morphology characters. Phylogenetic analysis of internal transcribed spacer (ITS) sequences showed that P. yunnanensis is a member of Pestalotiopsis, and is distinct from morphologically similar P. funereoides, P. funerea, and P. thujae, as well as other Pestalotiopsis species. A dichotomous key to 26 Pestalotiopsis species occurring on Podocarpus plants is also presented.     

A New Species of Tiger Pleco Panaqolus (Siluriformes: Loricariidae) from the Xingu Basin,Brazil

Panaqolus tankei is described from the Xingu River, Brazil. The new species is diagnosed from P. albomaculatus, P. dentex, P. nix, P. nocturnus, and P. koko by its color pattern consisting of dark and light diagonal bars on the body and bands on the fins (vs. body and fins without bars or bands); from P. albivermis, P. maccus, and P. purusiensis by the width of the dark bars being more or less the same of the light bars (vs. dark bars at least two or three times wider than light bars) and from P. changae by the absence of vermiculation on the head (vs. vermiculation present on head). The new species differs from P. gnomus by the orientation of the bars from posterodorsal to anteroventral direction (vs. anterodorsal to posteroventral direction), and from P. claustellifer by the orientation of the bands in the dorsal fin that are not parallel to the margin (vs. parallel to the margin). The barcoding region (COI) was sequenced for the new species, sequences were deposited in GenBank and were compared with congeners from other drainages. With regard to the current construction of a hydroelectric power plant (a so-called mega dam) in the Xingu River, herewith we increase knowledge of the river Xingu’s ichthyofauna and, thus improve the assessment of the impacts of that construction on the river.     

Transcriptome Analysis of Poplar during Leaf Spot Infection with Sphaerulina spp.

Diseases of poplar caused by the native fungal pathogen Sphaerulina musiva and related species are of growing concern, particularly with the increasing interest in intensive poplar plantations to meet growing energy demands. Sphaerulina musiva is able to cause infection on leaves, resulting in defoliation and canker formation on stems. To gain a greater understanding of the different responses of poplar species to infection caused by the naturally co-evolved Sphaerulina species, RNA-seq was conducted on leaves of Populus deltoides, P. balsamifera and P. tremuloides infected with S. musiva, S. populicola and a new undescribed species, Ston1, respectively. The experiment was designed to contain the pathogen in a laboratory environment, while replicating disease development in commercial plantations. Following inoculation, trees were monitored for disease symptoms, pathogen growth and host responses. Genes involved in phenylpropanoid, terpenoid and flavonoid biosynthesis were generally upregulated in P. balsamifera and P. tremuloides, while cell wall modification appears to play an important role in the defense of P. deltoides. Poplar defensive genes were expressed early in P. balsamifera and P. tremuloides, but their expression was delayed in P. deltoides, which correlated with the rate of disease symptoms development. Also, severe infection in P. balsamifera led to leaf abscission. This data gives an insight into the large differences in timing and expression of genes between poplar species being attacked by their associated Sphaerulina pathogen.     

Phytophthora species isolated from alpine and sub-alpine regions of Australia,including the description of two new species; Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov

Plant deaths had been observed in the sub-alpine and alpine areas of Australia. Although no detailed aetiology was established, patches of dying vegetation and progressive thinning of canopy suggested the involvement of root pathogens. Baiting of roots and associated rhizosphere soil from surveys conducted in mountainous regions New South Wales and Tasmania resulted in the isolation of eight Phytophthora species; Phytophthora cactorum, Phytophthora cryptogea, Phytophthora fallax, Phytophthora gonapodyides, Phytophthora gregata, Phytophthora pseudocryptogea, and two new species, Phytophthora cacuminis sp. nov and Phytophthora oreophila sp. nov, described here. P. cacuminis sp. nov is closely related to P. fallax, and was isolated from asymptomatic Eucalyptus coccifera and species from the family Proteaceae in Mount Field NP in Tasmania. P. oreophila sp. nov, was isolated from a disturbed alpine herbfield in Kosciuzsko National Park. The low cardinal temperature for growth of the new species suggest they are well adapted to survive under these conditions, and should be regarded as potential threats to the diverse flora of sub-alpine/alpine ecosystems. P. gregata and P. cryptogea have already been implicated in poor plant health. Tests on a range of alpine/subalpine plant species are now needed to determine their pathogenicity, host range and invasive potential.     

Variation in cuticular lipid profiles of black butterflies of the genus Papilio in Japan

Insect (epi)cuticular lipids characterize sex and species and often play an important role in mating behavior. We previously revealed that two black-colored swallowtail butterflies, Papilio polytes and Papilio protenor, show sexual dimorphism and species specificity in cuticular lipid composition and that P. polytes males use specific monoene components for mate discrimination. These findings suggest that their closely related species may have different profiles of cuticular lipids. We examined the cuticular lipid compositions of five Papilio species (P. bianor, P. maackii, P. helenus, P. macilentus, and P. memnon), closely related and sympatric to P. polytes and P. protenor, and discussed whether it is possible to discriminate between sexes, and between species based on their chemical profiles. The cuticular lipids consist mainly of C23–C31 aliphatic hydrocarbons, in which n-tricosane, n-heptacosane, and n-nonacosane are predominant. Several aliphatic ketones, aliphatic acids, and oxygenated terpenoids were also identified as major components shared by several species. There were no components exclusive to a particular species. Conspecific males and females shared most of the components but were mostly distinguishable based on their composition. Moreover, P. helenus males, P. polytes females, and P. protenor females had two different phenotypes of lipid composition. Although unrelated to the genetic lineage, the seven species were classified into four clusters based on their lipid profiles. The first cluster was composed of only P. memnon. The other six species were broadly classified into three clusters consisting of subclusters for each species: 1) P. polytes, P. helenus, P. macilentus, and several P. protenor females; 2) P. bianor and P. protenor; and 3) P. maackii and several P. helenus males. These results indicate that cuticular lipid profiles characterize the species and sex of the Papilio species and may be responsible for mate discrimination among them.     

Two new Pythium species from China based on the morphology and DNA sequence data

In an investigation of Pythium species in lawn grassland of south China, two new species, Pythium breve and P. baisense, were identified based on morphological characteristics and DNA sequence data. These two new species differ morphologically from the other Pythium species by the oogonia encompassed by many antheridia and with 1?C3 antheridia on the wavy and curved stalks. Furthermore, P. baisense with complexly lobed antheridial stalks differs from P. breve with antheridial stalks entwining the hyphae nearby the oogonia with several turns. Results of phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species based on the internal transcribed spacer region (ITS1, 5.8S, ITS2) and cytochrome c oxidase subunit I (COXI) gene sequences using maximum parsimony and Bayesian methods. These two new species are described and illustrated in detail.     

Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.     

Two new species,Pythium agreste and P. wuhanense,based on morphological characteristics and DNA sequence data

In an investigation of Pythium species in China, two new species, P. agreste and P. wuhanense, were identified based on morphological characteristics and DNA sequence data. Pythium agreste has slightly inflated sporangia, oogonia encompassed by antheridia and antheridial stalks forming a very complicated knot, and plerotic oospores. It differs from the morphologically similar P. volutum which has inflated sporangia, bigger oogonia, and aplerotic oospores; from P. kashmirense which has contiguous inflated sporangia, catenulate oogonia and coiled or bent oogonial stalks; and from P. pectinolyticum which has catenulate oogonia and bigger oogonia and oospores. Pythium wuhanense can be differentiated from morphologically similar P. emineosum and P. paroecandrum by its possession of intercalary cylindrical to elongated sporangia and intercalary oogonia catenulate with sporangia and antheridia. Phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species, based on the internal transcribed spacer (ITS) region and cytochrome C oxidase subunit I (COI) gene sequences using maximum parsimony and Bayesian methods. The two new species are described and illustrated in detail.     

Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.     

Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation,including Two New Species,Phytophthora crassamura and P. ornamentata sp. nov.

The Mediterranean basin is recognized as a global biodiversity hotspot accounting for more than 25,000 plant species that represent almost 10% of the world’s vascular flora. In particular, the maquis vegetation on Mediterranean islands and archipelagos constitutes an important resource of the Mediterranean plant diversity due to its high rate of endemism. Since 2009, a severe and widespread dieback and mortality of Quercus ilex trees and several other plant species of the Mediterranean maquis has been observed in the National Park of La Maddalena archipelago (northeast Sardinia, Italy). Infected plants showed severe decline symptoms and a significant reduction of natural regeneration. First studies revealed the involvement of the highly invasive wide-host range pathogen Phytophthora cinnamomi and several fungal pathogens. Subsequent detailed research led to a better understanding of these epidemics showing that multiple Phytophthora spp. were involved, some of them unknown to science. In total, nine Phytophthora species were isolated from rhizosphere soil samples collected from around symptomatic trees and shrubs including Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus. Based on morphological characters, growth-temperature relations and sequence analysis of the ITS and cox1 gene regions, the isolates were identified as Phytophthora asparagi, P. bilorbang, P. cinnamomi, P. cryptogea, P. gonapodyides, P. melonis, P. syringae and two new Clade 6 taxa which are here described as P. crassamura sp. nov. and P. ornamentata sp. nov. Pathogenicity tests supported their possible involvement in the severe decline that is currently threatening the Mediterranean maquis vegetation in the La Maddalena archipelago.     

Repeated Reticulate Evolution in North American Papilio machaon Group Swallowtail Butterflies

Hybridization between distinct populations or species is increasingly recognized as an important process for generating biodiversity. However, the interaction between hybridization and speciation is complex, and the diverse evolutionary outcomes of hybridization are difficult to differentiate. Here we characterize potential hybridization in a species group of swallowtail butterflies using microsatellites, DNA sequences, and morphology, and assess whether adaptive introgression or homoploid hybrid speciation was the primary process leading to each putative hybrid lineage. Four geographically separated hybrid populations were identified in the Papilio machaon species group. One distinct mitochondrial DNA clade from P. machaon was fixed in three hybrid taxa (P. brevicauda, P. joanae, and P. m. kahli), while one hybrid swarm (P. zelicaon x machaon) exhibited this hybrid mtDNA clade as well as widespread parental mtDNA haplotypes from both parental species. Microsatellite markers and morphology showed variable admixture and intermediacy, ranging from signatures of prolonged differential introgression from the paternal species (P. polyxenes/P. zelicaon) to current gene flow with both parental species. Divergences of the hybrid lineages dated to early- to mid-Pleistocene, suggesting that repeated glaciations and subsequent range shifts of parental species, particularly P. machaon hudsonianus, facilitated initial hybridization. Although each lineage is distinct, P. joanae is the only taxon with sufficient evidence (ecological separation from parental species) to define it as a homoploid hybrid species. The repetition of hybridization in this group provides a valuable foundation for future research on hybridization, and these results emphasize the potential for hybridization to drive speciation in diverse ways.     

Selective bactericidal activity of nanopatterned superhydrophobic cicada Psaltoda claripennis wing surfaces

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on its physical surface structure. As such, they provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. Their effectiveness against a wide spectrum of bacteria, however, is yet to be established. Here, the bactericidal properties of the wings were tested against several bacterial species, possessing a range of combinations of morphology and cell wall type. The tested species were primarily pathogens, and included Bacillus subtilis, Branhamella catarrhalis, Escherichia coli, Planococcus maritimus, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Staphylococcus aureus. The wings were found to consistently kill Gram-negative cells (i.e., B. catarrhalis, E. coli, P. aeruginosa, and P. fluorescens), while Gram-positive cells (B. subtilis, P. maritimus, and S. aureus) remained resistant. The morphology of the cells did not appear to play any role in determining cell susceptibility. The bactericidal activity of the wing was also found to be quite efficient; 6.1?±?1.5?×?10⁶ P. aeruginosa cells in suspension were inactivated per square centimeter of wing surface after 30-min incubation. These findings demonstrate the potential for the development of selective bactericidal surfaces incorporating cicada wing nanopatterns into the design.     

The genus Eodasycladus (Lower Jurassic dasycladalean green alga) and its relationship with Palaeodasycladus

The Lower Jurassic genus Eodasycladus is discussed according to the characters of type species and compared with the other species known in the literature. The architecture of two species attributed to the genus Palaeodasycladus, P. alanensis Soka?, and P. benceki Soka?, is examined and an alternative organization of the thallus is prospected. P. alanensis is considered a valid species, but its characters require to transfer the taxon to the genus Eodasycladus. P. benceki is considered synonymous with P. alanensis.     

New Pseudomonas spp. Are Pathogenic to Citrus

Five putative novel Pseudomonas species shown to be pathogenic to citrus have been characterized in a screening of 126 Pseudomonas strains isolated from diseased citrus leaves and stems in northern Iran. The 126 strains were studied using a polyphasic approach that included phenotypic characterizations and phylogenetic multilocus sequence analysis. The pathogenicity of these strains against 3 cultivars of citrus is demonstrated in greenhouse and field studies. The strains were initially grouped phenotypically and by their partial rpoD gene sequences into 11 coherent groups in the Pseudomonas fluorescens phylogenetic lineage. Fifty-three strains that are representatives of the 11 groups were selected and analyzed by partial sequencing of their 16S rRNA and gyrB genes. The individual and concatenated partial sequences of the three genes were used to construct the corresponding phylogenetic trees. The majority of the strains were identified at the species level: P. lurida (5 strains), P. monteilii (2 strains), P. moraviensis (1 strain), P. orientalis (16 strains), P. simiae (7 strains), P. syringae (46 strains, distributed phylogenetically in at least 5 pathovars), and P. viridiflava (2 strains). This is the first report of pathogenicity on citrus of P. orientalis, P. simiae, P. lurida, P. moraviensis and P. monteilii strains. The remaining 47 strains that could not be identified at the species level are considered representatives of at least 5 putative novel Pseudomonas species that are not yet described.     

Ecological Niche Conservatism in Doucs (Genus Pygathrix)

The impressive diversity and unique distribution of primate taxa in Southeast Asia creates a region of interesting biogeography and evolutionary history that remains poorly understood. The three species in the Asian colobine genus Pygathrix (P. cinerea, P. nemaeus, and P. nigripes) appear to follow the unique distribution pattern, replacing one another along the north–south axis. However, the evolutionary history and taxonomic status within Pygathrix is currently debated. We constructed climate-based ecological niche models (ENMs) for the three species, using six environmental variables, to confirm that the bioclimate envelopes of Pygathrix cinerea, P. nemaeus, and P. nigripes follow the north–south gradient. We also used the ENMs to test whether the three species lack ecological exchangeability, meaning differentiation among factors that define the fundamental niche, and whether they exhibit ecological niche conservatism, or the tendency for related species to differ less ecologically than might be expected by the climate available to each species. Our ENMs for Pygathrix followed a north–south gradient as expected, with P. nemaeus extending from ca. 14°N to 21°N, P. cinerea from 14°N to 16°N, and P. nigripes from 11°N to 13.5°N. This study presents the first use of ENMs for doucs that yield significant predictive ability based only on climatic variables. The models are significantly different for all three species, supporting a lack of ecological exchangeability between them, as well as the recent elevation of Pygathrix cinerea to species status. Importantly, Pygathrix cinerea and P. nemaeus show evidence of ecological niche conservatism, which lends support to the occurrence of an allopatric speciation event. This result also suggests that the current overlapping distribution of Pygathrix cinerea and P. nemaeus is the result of secondary contact. The north–south distribution gradient, which exists in similar form among other groups of sister taxa in the region, may be attributed to a zoogeographic barrier, vicariance, or current or historical climatic shifts.     

Novel palmicolous taxa within Pleosporales: multigene phylogeny and taxonomic circumscription

Palm fungi are highly diverse in the tropical regions of Asia. Recent investigations on these palmicolous fungi have led to the collection of astrosphaeriella-like taxa from Calamus, Caryota, and Licuala species in Thailand (Chiang Rai and Narathiwat provinces) and southwest China (Yunnan Province). This study characterizes fungal taxa, which are new to science, based on morphological examination and concatenated DNA sequence data, to infer their familial relationships. Morphological comparisons reveal six new species, viz. Astrosphaeriellopsis caryotae, Fissuroma calami, F. caryotae, Neoastrosphaeriella sribooniensis, Pithomyces caryotae, and P. licualae.Their similarities and differences to other extant species are discussed. The phylogenetic results indicate that all of these new taxa belong to Aigialaceae and Astrosphaeriellaceae (Pleosporales) and support their establishment. Astrosphaeriellopsis is assigned to Astrosphaeriellaceae and the family is amended in order to accommodate both coelomycetous and hyphomycetous asexual morphs. A generic key is presented for Astrosphaeriellaceae to delimit Astrosphaeriella, Astrosphaeriellopsis, Pteridiospora, and Pithomyces. Asexual morph connections of Pithomyces caryotae and P. licualae are established from axenic cultures derived from single ascospores. DNA-based sequence data supports the establishment of our new species; however, the affinities of Astrosphaeriella tornata to other Astrosphaeriella and Pithomyces species are unclear and warrant further investigations with increased taxon sampling.