Species discrimination of novel chloroplast DNA barcodes and their application for identification of Panax (Aralioideae, Araliaceae)

Certain species within the genus Panax L. (Araliaceae) contain pharmacological precious ginsenosides, also known as ginseng saponins. Species containing these compounds are of high commercial value and are thus of particular urgency for conservation. However, within this genus, identifying the particular species that contain these compounds by morphological means is challenging. DNA barcoding is one method that is considered promising for species level identification. However, in an evolutionarily complex genus such as Panax, commonly used DNA barcodes such as nrITS, matK, psbA-trnH, rbcL do not provide species-level resolution. A recent in silico study proposed a set of novel chloroplast markers, trnQ-rps16, trnS-trnG, petB, and trnE-trnT for species level identification within Panax. In the current study, the discriminatory efficiency of these molecular markers is assessed and validated using 91 reference barcoding sequences and 38 complete chloroplast genomes for seven species, one unidentified species and one sub-species of Panax, and two outgroup species of Aralia L. along with empirical data of Panax taxa present in Vietnam via both distance-based and tree-based methods. The obtained results show that trnQ-rps16 can classify with species level resolution every clade tested here, including the highly valuable Panax vietnamensis Ha et Grushv. We thus propose that this molecular marker to be used for identification of the species within Panax to support both its conservation and commercial trade.

Pachytene karyotypes of 17 species of birds

Background: To date less than 10% of bird species have been karyotyped. They are rather conservative with diploid chromosome numbers about 78-80 in most species examined. Immunostaining of meiotic chromosomes at pachytene stage enables more precise estimates of the number, morphology and variability of macro- and microchromosomes than conventional analysis of mitotic metaphase chromosomes does. Analysis of pachytene chromosomes led to discovery of germline-restricted chromosome (GRC) that was present in germline cells and absent in somatic cells in all 16 species of passerine birds examined. GRC has not been found in any non-passerine bird. Results: In this study, using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex (SC) and centromere proteins we examined male pachytene karyotypes of sixteen passerine species and one outgroup species the Common cuckoo Cuculus canorus and provided their idiograms and precise estimates of their diploid chromosome numbers and the numbers of chromosome arms. We provided the first description of the karyotypes of three species, corrected the published data on the karyotypes of ten species and confirmed them for four species. The pachytene cells of the Gouldian finch, Brambling and Common linnet contained heteromorphic bivalents indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, Gouldian finch and Domestic canary have extended centromeres in several macro- and microchromosomes. GRCs of various sizes and shapes were detected in all passerine species examined. Their chromatin was heavily labeled by anticentromere antibodies. The lateral elements of the GRC SC varied in their size from the largest to the smallest element of the pachytene karyotype. They also varied in shape from continuous to fragmented. Conclusions: All songbirds examined, except the Eurasian skylark, have highly conservative karyotypes, 2n=80-82+GRC with seven pairs of macrochromosomes and 33-34 pairs of microchromosomes. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes and sizes of the centromeres. GRC is present in all songbird species examined, varying in size, morphology and SC structure even between closely related species. This indicates its fast evolution.

Systematic review of the firefly genus Emeia Fu, Ballantyne & Lambkin, 2012 (Coleoptera: Lampyridae) from China

The Luciolinae genus Emeia Fu, Ballantyne & Lambkin, 2012 is reviewed. Firefly species tree is reconstructed based upon phylogenetic analysis of CO1 barcoding sequences from 42 fireflies and 2 outgroup species, including three main Lampyridae sub-families: Luciolinae, Photurinae and Lampyrinae. The genus Emeia belongs to Luciolinae based on morphological and DNA sequence level evidences, the diagnostic features for male adults include pink-red pronotum with a black median stripe, subparallel-sided pronotum, un-developed elytral humeral carina. In this study, a new Emeia species,Emeia pulchra sp. nov. is described and illustrated from the wetland of Lishui, Zhejiang, China. The new species is sister to E. pseudosauteri in the Emeia genus which is supported by characteristic morphological features and DNA barcoding data. The two species are separated geographically as shown on the distribution map. A key to the males of the species of Emeia is also provided.

Revised classification of the New World Cylapini (Heteroptera: Miridae: Cylapinae): taxonomic review of the genera Cylapinus, Cylapoides and Peltidocylapus and a morphology-based phylogenetic analysis of tribe Cylapini

Cylapini, as currently circumscribed, is a relatively small group of plant bugs currently comprising 17 genera and 65 species. Most representatives of the tribe are distributed in the New World (10 genera and 47 species) with other members occurring in the Afrotropical, Oriental, and Australian regions. They have primarily tropical and subtropical distributions with only a few members inhabiting temperate regions. This paper provides a taxonomic review of three of the New World Cylapini genera: Cylapinus Carvalho, 1986, Cylapoides Carvalho, 1952, and Peltidocylapus Poppius, 1909. Most species are diagnosed and redescribed. Eight new species are described as new: Cylapinus yasunagai sp. nov., Peltidocylapus calyciformis sp. nov., P. caudatus sp. nov., P. ecuadorensis sp. nov., P. pallidus sp. nov., P. parallelus sp. nov., P. simplex sp. nov., and P. spinosus sp. nov. Cylapus festinabundus Bergroth, 1922 is transferred to Peltidocylapus (comb. nov.). Illustrations of male genitalia, scanning electron micrographs of selected structures of certain species, and an identification key of the genera Cylapinus, Cylapoides and Peltidocylapus are provided. Female genitalia are described and illustrated for the first time for most genera of Cylapini. A cladistic analysis of the tribe based on 81 morphological characters is presented as a contribution to the understanding of the ingroup relationships of Cylapini and its relationships with other groups of Cylapinae. The analysis comprises 30 ingroup species and 15 outgroup species. Both equal- and implied weighting parsimony analyses were used in the phylogenetic reconstruction. This analysis was based solely on morphological characters because an insufficient number of specimens suitable for molecular studies were available for most taxa. The study confirmed a close affinity of the taxa currently included in Cylapini, but the tribe was rendered paraphyletic by inclusion of the tribe Vanniini. The grouping comprising both Cylapini + Vanniini and most of its subordinated clades received low nodal support. Both analyses recovered a decisively supported clade comprising the New World genera Amapacylapus, Cylapus, Peltidocylapus, and Valdasus which accommodate most of the Cylapini species, justifying the recognition of the Cylapus complex suggested by previous authors. The results presented here are discussed and compared with previous phylogenetic hypotheses based on different datasets.

A chelicerate Wnt gene expression atlas: novel insights into the complexity of arthropod Wnt-patterning

The Wnt genes represent a large family of secreted glycoprotein ligands that date back to early animal evolution. Multiple duplication events generated a set of 13 Wnt families of which 12 are preserved in protostomes. Embryonic Wnt expression patterns (Wnt-patterning) are complex, representing the plentitude of functions these genes play during development. Here, we comprehensively investigated the embryonic expression patterns of Wnt genes from three species of spiders covering both main groups of true spiders, Haplogynae and Entelegynae, a mygalomorph species (tarantula), as well as a distantly related chelicerate outgroup species, the harvestman Phalangium opilio. All spiders possess the same ten classes of Wnt genes, but retained partially different sets of duplicated Wnt genes after whole genome duplication, some of which representing impressive examples of sub- and neo-functionalization. The harvestman, however, possesses a more complete set of 11 Wnt genes but with no duplicates. Our comprehensive data-analysis suggests a high degree of complexity and evolutionary flexibility of Wnt-patterning likely providing a firm network of mutational protection. We discuss the new data on Wnt gene expression in terms of their potential function in segmentation, posterior elongation, and appendage development and critically review previous research on these topics. We conclude that earlier research may have suffered from the absence of comprehensive gene expression data leading to partial misconceptions about the roles of Wnt genes in development and evolution.

Comparative transcriptomics provides a strategy for phylogenetic analysis and SSR marker development in Chaenomeles

The genus Chaenomeles has long been considered an important ornamental, herbal and cash crop and is widely cultivated in East Asia. Traditional studies of Chaenomeles mainly focus on evolutionary relationships at the phenotypic level. In this study, we conducted RNA-seq on 10 Chaenomeles germplasms supplemented with one outgroup species, Docynia delavayi (D. delavayi), on the Illumina HiSeq2500 platform. After de novo assemblies, we generated from 40,084 to 49,571 unigenes for each germplasm. After pairwise comparison of the orthologous sequences, 9,659 orthologues within the 11 germplasms were obtained, with 6,154 orthologous genes identified as single-copy genes. The phylogenetic tree was visualized to reveal evolutionary relationships for these 11 germplasms. GO and KEGG analyses were performed for these common single-copy genes to compare their functional similarities and differences. Selective pressure analysis based on 6,154 common single-copy genes revealed that 45 genes were under positive selection. Most of these genes are involved in building the plant disease defence system. A total of 292 genes containing simple sequence repeats (SSRs) were used to develop SSR markers and compare their functions in secondary metabolism pathways. Finally, 10 primers were chosen as SSR marker candidates for Chaenomeles germplasms by comprehensive standards. Our research provides a new methodology and reference for future related research in Chaenomeles and is also useful for improvement, breeding and selection projects in other related species.

Genome sequencing and assembly of Tinospora cordifolia (Giloy) plant

During the ongoing COVID-19 pandemic Tinospora cordifolia also known as Giloy gained immense popularity and use due to its immunity-boosting function and anti-viral properties. T. cordifolia is among the most important medicinal plants that has numerous therapeutic applications in health due to the production of a diverse array of secondary metabolites. Therefore, to gain genomic insights into the medicinal properties of T. cordifolia, the first genome sequencing was carried out using 10x Genomics linked read technology and the draft genome assembly comprised of 1.01 Gbp. This is also the first genome sequenced from the plant family Menispermaceae. We also performed the first genome size estimation for T. cordifolia and was found to be 1.13 Gbp. The deep sequencing of transcriptome from the leaf tissue was also performed followed by transcriptomic analysis to gain insights into the gene expression and functions. The genome and transcriptome assemblies were used to construct the gene set in T. cordifolia that resulted in 19,474 coding gene sequences. Further, the phylogenetic position of T. cordifolia was also determined through the construction of a genome-wide phylogenetic tree using 35 other dicot species and one monocot species as an outgroup species.

Using carrot centromeric repeats to study karyotype relationships in the genus Daucus (Apiaceae)

Background In the course of evolution, chromosomes undergo evolutionary changes; thus, karyotypes may differ considerably among groups of organisms, even within closely related taxa. The genus Daucus seems to be a promising model for exploring the dynamics of karyotype evolution. It comprises some 40 wild species and the cultivated carrot, a crop of great economic significance. However, Daucus species are very diverse morphologically and genetically, and despite extensive research, the taxonomic and phylogenetic relationships between them have still not been fully resolved. Although several molecular cytogenetic studies have been conducted to investigate the chromosomal structure and karyotype evolution of carrot and other Daucus species, detailed karyomorphological research has been limited to carrot and only a few wild species. Therefore, to better understand the karyotype relationships within Daucus, we (1) explored the chromosomal distribution of carrot centromeric repeats (CentDc) in 34 accessions of Daucus and related species by means of fluorescence in situ hybridization (FISH) and (2) performed detailed karyomorphological analysis in 16 of them. Results We determined the genomic organization of CentDc in 26 accessions of Daucus (belonging to both Daucus I and II subclades) and one accession of closely related species. The CentDc repeats were present in the centromeric regions of all chromosomes of 20 accessions (representing 11 taxa). In the other Daucus taxa, the number of chromosome pairs with CentDc signals varied depending on the species, yet their centromeric localization was conserved. In addition, precise chromosome measurements performed in 16 accessions showed the inter- and intraspecific karyological relationships among them. Conclusions The presence of the CentDc repeats in the genomes of taxa belonging to both Daucus subclades and one outgroup species indicated the ancestral status of the repeat. The results of our study provide useful information for further evolutionary, cytotaxonomic, and phylogenetic research on the genus Daucus and may contribute to a better understanding of the dynamic evolution of centromeric satellites in plants.

Diversification Through Gustatorial-Courtship: An X-ray Micro-Computed Tomography Study on Dwarf Spiders

BackgroundSexual selection has been considered to promote diversification and speciation. Sexually dimorphic species have been used to explore the supposed effect, however, with mixed results. In dwarf spiders (Erigoninae), many species are sexually dimorphic - males possess marked prosomal modifications. These male traits vary from moderate elevation to bizarre shapes in various prosomal regions. Previous studies established that male dwarf spiders produce substances in these prosomal modifications that are taken up by the female. Since the transfer of substances increases mating probability of males and oviposition rate in females, the dimorphic traits evolved in the context of sexual selection. Here, we explore the evolutionary lability of the gustatory trait complex by investigating 1) if erigonine modified prosomata are inherently linked to nuptial-gift-producing glands, 2) if gland evolution preceded that of the modified prosomal shapes and by assessing 3) the probability of convergent evolution and cryptic differentiation - with the aim of assessing the role of this trait complex in species divergence.ResultsWe reconstructed the position and extent of the glandular tissue along with the muscular anatomy in the anterior part of the prosoma of 76 erigonine spiders and three outgroup species using X-ray micro-computed tomography. We incorporated the location of glands and muscles into an existing matrix of somatic and genitalic morphological traits of these taxa and reanalyzed their phylogenetic relationship. Our analysis supports that possession of glandular equipment is the ancestral state. The manifold modifications of the prosomal shape have evolved convergently multiple times. We found differences in glandular position between species with both modified and unmodified prosomata, and reported on seven cases of gland loss. ConclusionsOur findings suggest that the occurrence of gustatory gland in sexually monomorphic ancestors has set the stage for the evolution of diverse dimorphic external modifications in dwarf spiders. Variation among congeners indicates that glandular position is highly susceptible to changes. Multiple incidences of gland loss suggest considerable maintenance costs of glandular tissue and nuptial feeding. Our results demonstrate divergent evolutionary patterns of gustatorial-courtship-related traits, and a likely facilitating effect of this type of sexual selection on speciation

A Nation-wide Phylogenetic and Phylogeographic Investigation of the Endemic New Zealand Oyster Borers, Haustrum scobina and Haustrum albomarginatum

The endemic New Zealand sea snails Haustrum scobina and Haustrum albomarginatum are rocky shore intertidal dogwhelks of the Muricidae family. They have direct developing young and are carnivores. Their radula is used to drill into the shells of their prey, and they are commonly referred to as oyster borers. The taxonomic status of these species is still unresolved and therefore the name Haustrum scobina sensu lato is used.<br><br>The overall goal of this thesis research was to investigate the phylogeny and phylogeography of Haustrum scobina sensu lato using mitochondrial DNA sequences. Comparisons made to phylogeographic studies of ecologically similar species such as Cominella spp. provide an opportunity to identify the common environmental determinates of population migration route, genetic differentiation and speciation whenever similar patterns are found.<br><br>A nation-wide collection of samples was used to generate 277 new sequences from a 610 bp portion of the cytochrome c oxidase subunit I (COI) gene. This enabled the formation of a dataset of 654 DNA sequences, which was comprised of the 277 new sequences, 16 retrieved from a published study that deposited them in GenBank, and 361 from a previous unpublished thesis study. An unexpectedly diverse phylogeny of 58 COI haplotypes from 31 sample sites was recovered. These formed three clusters using K-means clustering by pairwise mutational distance. The in-group species did not form reciprocal monophyly groups, and the expected closest outgroup species (Haustrum haustorium) appeared to be as similar to the in-group clusters as they were to each other. A dataset of 27 DNA sequences from an 827 bp portion of the large sub-unit 28S nuclear rRNA gene was produced with the intention of corroborating the findings from the analyses of the COI dataset. This consisted of 26 new sequences and one sequence from a published study that deposited the sequence on GenBank. The expected taxonomic arrangement of Haustrum scobina sensu lato could not be matched by COI sequences due to incongruence with the 28S phylogeny and shell morphology. <br><br>The 28S dataset and shell morphology indicated there are two species in Haustrum scobina sensu lato. These are most likely Haustrum scobina and Haustrum albomarginatum, but they could not clearly be identified in the COI data. As a result, the phylogeographic certainty was limited when using the COI dataset because of the lack of clarity between the haplotypes of the two putative species. Possible reasons for the complicated COI dataset are discussed. Phylogenetic analysis of both the 28S and COI datasets did not support the expected conclusion that members of Haustrum scobina sensu lato are each other’s closest relatives. Haustrum haustorium was the expected immediate outgroup species but formed a polytomy with the in-group. A decrease in COI haplotype diversity was observed in southern samples when they were compared to the samples collected at northern locations. Taranaki sites shared a haplotype with multiple South Island sites that had no haplotype diversity. This suggested post-glacial re-colonisation of southern sites after displacement by ice-age conditions from these locations, a hypothesis consistent with results from studies of the Cominella genus. Association between Purau Bay in Lyttleton Harbour, Titahi Bay, Port Ahuriri and Kawau Island with no associated haplotypes between these locations suggested human-mediated translocation events. A genetic disjunction was also apparent between the south Wellington/Wairarapa coast and the eastern Wairarapa coast. This pattern was consistent with one study of Cominella maculosa and other studies have attributed similar patterns of other species in the region to recent uplift events affecting coastal community composition. The phylogeny of Haustrum scobina sensu lato will require further investigation before it can be used to more confidently resolve the phylogeographic history of the species.