Plant DNA‐barcode library and community phylogeny for a semi‐arid East African savanna

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA‐barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL‐F, trnH–psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra‐ and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack “barcode gaps”, defined as disparities between the maximum intra‐ and minimum interspecific genetic distances. We found barcode gaps for 72%–89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode‐gap presence or absence for all of the markers in combination and for matK, trnL‐F, and trnH–psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations.     

Isolation and characterization of di‐ and tetranucleotide microsatellite loci in the yellow‐spotted night lizard Lepidophyma flavimaculatum (Squamata: Xantusiidae)

We report the development of 17 di‐ and tetranucleotide microsatellite markers in the Yellow‐Spotted Night Lizard Lepidophyma flavimaculatum. Levels of heterozygosity ranged from 0 to 100%. Several loci also amplify in other Lepidophyma species and several species of the sister genus Xantusia. High levels of variation at some loci indicate that they will be useful for parentage assessment and population genetic studies, whereas the 15 loci that amplify across multiple Lepidophyma species suggest these will be useful in determining the origin of parthenogenetic populations.     

Frequency of hybridization between Ostrinia nubilalis E‐and Z‐pheromone races in regions of sympatry within the United States

Female European corn borer, Ostrinia nubilalis, produce and males respond to sex pheromone blends with either E‐ or Z‐Δ11‐tetradecenyl acetate as the major component. E‐ and Z‐race populations are sympatric in the Eastern United States, Southeastern Canada, and the Mediterranean region of Europe. The E‐ and Z‐pheromone races of O. nubilalis are models for incipient species formation, but hybridization frequencies within natural populations remain obscure due to lack of a high‐throughput phenotyping method. Lassance et al. previously identified a pheromone gland‐expressed fatty‐acyl reductase gene (pgfar) that controls the ratio of Δ11‐tetradecenyl acetate stereoisomers. We identified three single nucleotide polymorphism (SNP) markers within pgfar that are differentially fixed between E‐ and Z‐race females, and that are ≥98.2% correlated with female pheromone ratios measured by gas chromatography. Genotypic data from locations in the United States demonstrated that pgfar‐z alleles were fixed within historically allopatric Z‐pheromone race populations in the Midwest, and that hybrid frequency ranged from 0.00 to 0.42 within 11 sympatric sites where the two races co‐occur in the Eastern United States (mean hybridization frequency or heterozygosity (H_O) = 0.226 ± 0.279). Estimates of hybridization between the E‐ and Z‐races are important for understanding the dynamics involved in maintaining race integrity, and are consistent with previous estimates of low levels of genetic divergence between E‐ and Z‐races and the presence of weak prezygotic mating barriers.     

Comparative intra‐ and interspecific sexual organ reciprocity in four distylous Primula species in the Himalaya‐Hengduan Mountains

• Distyly is a mechanism promoting cross‐pollination within a balanced polymorphism. Numerous studies show that the degree of inter‐morph sexual organ reciprocity (SOR) within species relates to its pollen‐mediated gene flow. Similarly, a lower interspecific SOR should promote interspecific isolation when congeners are sympatric, co‐blooming and share pollinators. In this comparative study, we address the significance of SOR at both intra‐ and interspecific levels.
• Seventeen allopatric and eight sympatric populations representing four Primula species (P. anisodora, P. beesiana, P. bulleyana and P. poissonii) native to the Himalaya‐Hengduan Mountains were measured for eight floral traits in both long‐ and short‐styled morphs. GLMM and spatial overlap methods were used to compare intra‐ and interspecific SOR.
• While floral morphology differed among four Primula species, SOR within species was generally higher than between species, but in species pairs P. poissonii/P. anisodora and P. beesiana/P. bulleyana, the SOR was high at both intra‐ and interspecific levels. We did not detect a significant variation in intraspecific SOR or interspecific SOR when comparing allopatric versus sympatric populations for all species studied.
• As intraspecific SOR increased, disassortative mating may be promoted. As interspecific SOR decreased, interspecific isolation between co‐flowering species pairs also may increase. Hybridisation between congeners occurred when interspecific SOR increased in sympatric populations, as confirmed in two species pairs, P. poissonii/P. anisodora and P. beesiana/P. bulleyana.

     

Colonization of disturbed Scots pine trees by bark‐ and wood‐boring beetles

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Effects of size‐ and sex‐selective harvesting: An integral projection model approach

Harvesting is often size‐selective, and in species with sexual size dimorphism, it may also be sex‐selective. A powerful approach to investigate potential consequences of size‐ and/or sex‐selective harvesting is to simulate it in a demographic population model. We developed a population‐based integral projection model for a size‐ and sex‐structured species, the commonly exploited pike (Esox lucius). The model allows reproductive success to be proportional to body size and potentially limited by both sexes. We ran all harvest simulations with both lower size limits and slot limits, and to quantify the effects of selective harvesting, we calculated sex ratios and the long‐term population growth rate (λ). In addition, we quantified to what degree purely size‐selective harvesting was sex‐selective, and determined when λ shifted from being female to male limited under size‐ and sex‐selective harvesting. We found that purely size‐selective harvest can be sex‐selective, and that it depends on the harvest limits and the size distributions of the sexes. For the size‐ and sex‐selective harvest simulations, λ increased with harvest intensity up to a threshold as females limited reproduction. Beyond this threshold, males became the limiting sex, and λ decreased as more males were harvested. The peak in λ, and the corresponding sex ratio in harvest, varied with both the selectivity and the intensity of the harvest simulation. Our model represents a useful extension of size‐structured population models as it includes both sexes, relaxes the assumption of female dominance, and accounts for size‐dependent fecundity. The consequences of selective harvesting presented here are especially relevant for size‐ and sex‐structured exploited species, such as commercial fisheries. Thus, our model provides a useful contribution toward the development of more sustainable harvesting regimes.     

Regional connectivity and coastal expansion: differentiating pre‐border and post‐border vectors for the invasive tunicate Styela clava

The dramatic increase in marine bio‐invasions, particularly of non‐indigenous ascidians, has highlighted the vulnerability of marine ecosystems and the productive sectors that rely on them. A critical issue in managing invasive species is determining the relative roles of ongoing introductions, versus the local movement of propagules from established source populations. Styela clava (Herdman, 1882), the Asian clubbed tunicate, once restricted to the Pacific shores of Asia and Russia, is now abundant throughout the northern and southern hemispheres and has had significant economic impact in at least one site of incursion. In 2005 S. clava was identified in New Zealand. The recent introduction of this species, coupled with its restricted distribution, provided an ideal model to compare and contrast the introduction and expansion process. In this study, the mitochondrial DNA cytochrome oxidase subunit I gene (COI) gene and 11 microsatellite markers were used to test the regional genetic structure and diversity of 318 S. clava individuals from 10 populations within New Zealand. Both markers showed significant differentiation between the northern and southern populations, indicative of minimal pre‐ or post‐border connectivity. Additional statistics further support pre‐ and post‐border differentiation among Port and Harbour populations (i.e. marinas and aquaculture farms). We conclude that New Zealand receives multiple introductions, and that the primary vector for pre‐border incursions and post‐border spread is most likely the extensive influx of recreational vessels that enter northern marinas independent of the Port. This is a timely reminder of the potential for hull‐fouling organisms to expand their range as climates change and open new pathways.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Using RAD‐seq to develop sex‐linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking.     

Assessing the potential of candidate DNA barcodes for identifying non‐flowering seed plants

In plants, matK and rbcL have been selected as core barcodes by the Consortium for the Barcode of Life (CBOL) Plant Working Group (PWG), and ITS/ITS2 and psbA‐trnH were suggested as supplementary loci. Yet, research on DNA barcoding of non‐flowering seed plants has been less extensive, and the evaluation of DNA barcodes in this division has been limited thus far. Here, we evaluated seven markers (psbA‐trnH, matK, rbcL, rpoB, rpoC1, ITS and ITS2) from non‐flowering seed plants. The usefulness of each region was assessed using four criteria: the success rate of PCR amplification, the differential intra‐ and inter‐specific divergences, the DNA barcoding gap and the ability to discriminate species. Among the seven loci tested, ITS2 produced the best results in the barcoding of non‐flowering seed plants. In addition, we compared the abilities of the five most‐recommended markers (psbA‐trnH, matK, rbcL, ITS and ITS2) to identify additional species using a large database of gymnosperms from GenBank. ITS2 remained effective for species identification in a wide range of non‐flowering seed plants: for the 1531 samples from 608 species of 80 diverse genera, ITS2 correctly authenticated 66% of them at the species level. In conclusion, the ITS2 region can serve as a useful barcode to discriminate non‐flowering seed plants, and this study will contribute valuable information for the barcoding of plant species.     

Development of New Oomycete Taxon‐specific Mitochondrial Cytochrome b Region Primers for Use in Phylogenetic and Phylogeographic Studies

Here, we describe the development of an oomycete‐specific primer pair for amplification of the cytochrome b region in plant pathogenic species that span the order Peronosporales (Phytophthora spp., downy mildews). Because of the high number of variable sites at both inter‐ and intra‐specific levels this marker provides a powerful tool for population genetics and phylogenetic studies in this taxa. We also demonstrate its potential compared with other oomycete‐specific mitochondrial markers currently available.     

Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.     

Both morph‐ and species‐dependent asymmetries affect reproductive barriers between heterostylous species

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre‐ versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre‐ and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co‐occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species‐dependent asymmetries, morph‐dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short‐styled flowers to stigmas of long‐styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long‐styled flowers to stigmas of short‐styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long‐styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph‐ and species‐dependent asymmetries shape boundaries between heterostylous species.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.     

Enantiopure 4‐oxazolin‐2‐ones and 4‐methylene‐2‐oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles

Enantiopure 3‐((R)‐ and 3‐((S)‐1‐phenylethyl)‐4‐oxazoline‐2‐ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N‐(R)‐ or N‐(S)‐1‐phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4‐ and 5‐positions of the 4‐oxazolin‐2‐one ring through thermal and MW‐promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six‐membered carbo‐ and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4‐methylene‐2‐oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero‐Diels‐Alder cycloaddition.     

Oleanane‐Type Glycosides from Tremastelma palaestinum (L.) Janchen

Three new oleanane‐type glycosides, 1 – 3 , were isolated from the whole plant of Tremastelma palaestinum (L.) Janchen, along with eight known triterpene glycosides. The structures of the new compounds were established as 3‐O‐[β‐d‐ glucopyranosyl‐(1→3)‐α‐l‐ rhamnopyranosyl‐(1→3)‐β‐d‐ glucopyranosyl‐(1→3)‐α‐l‐ rhamnopyranosyl‐(1→2)‐α‐l‐ arabinopyranosyl]hederagenin ( 1 ), 3‐O‐[β‐d‐ glucopyranosyl‐(1→3)‐α‐l‐ rhamnopyranosyl‐(1→3)‐β‐d‐ glucopyranosyl‐(1→3)‐α‐l‐ rhamnopyranosyl‐(1→2)‐α‐l‐ arabinopyranosyl]hederagenin 28‐O‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranosyl ester ( 2 ), and 3‐O‐[α‐l‐ rhamnopyranosyl‐(1→3)‐β‐d‐ glucopyranosyl‐(1→3)‐α‐l‐ rhamnopyranosyl‐(1→2)‐α‐l‐ arabinopyranosyl]oleanolic acid 28‐O‐β‐d‐ glucopyranosyl‐(1→6)‐β‐d‐ glucopyranosyl ester ( 3 ) by using 1D‐ and 2D‐NMR techniques and mass spectrometry. This is the first report on the phytochemical investigation of a species belonging to Tremastelma genus.     

Isolation and characterization of tri‐ and tetranucleotide microsatellite loci in the Asian elephant,Elephas maximus

Asian elephants (Elephas maximus) are an endangered species. Their future survival depends on intensive conservation and management, based on in‐depth knowledge of particular populations. Molecular genetic methods, especially microsatellite analysis through noninvasive sampling, provides an effective means of obtaining such information. The use of tri‐ and tetranucleotide microsatellite markers is advantageous in noninvasive sampling through dung analysis. Here we describe the isolation and characterization of five tri‐ and tetranucleotide markers in the Asian elephant. All five loci were found to be polymorphic in a sample of 20 Asian elephants from Sri Lanka.