Within the family Solanaceae, Withania is a small genus belonging to the Solanoideae subfamily. Here, we report the de novo assembled chloroplast genome sequences of W. coagulans, W. adpressa, and W. riebeckii. The length of these genomes ranged from 154,162 to 154,364 base pairs (bp). These genomes contained a pair of inverted repeats (IRa and IRb) ranging from 25,029 to 25,071 bp that were separated by a large single-copy (LSC) region of 85,635–85,765 bp and a small single-copy (SSC) region of 18,457–18,469 bp. We analyzed the structural organization, gene content and order, guanine-cytosine content, codon usage, RNA-editing sites, microsatellites, oligonucleotide and tandem repeats, and substitutions of Withania plastomes, which revealed high similarities among the species. Comparative analysis among the Withania species also highlighted 10 divergent hotspots that could potentially be used for molecular marker development, phylogenetic analysis, and species identification. Furthermore, our analyses showed that even three mutational hotspots (rps4-trnT, trnM-atpE, and rps15) were sufficient to discriminate the Withania species included in current study.

Comparative Plastomics of Ashwagandha (Withania, Solanaceae) and Identification of Mutational Hotspots for Barcoding Medicinal Plants.

The study was aimed to explore the systematic potential of anatomical characters for identification and delimitation among Euphorbia species. Eight species of leafy spurges of genus Euphorbia L. (Euphorbiaceae) were evaluated for variations in micro morphological characters of foliar epidermal anatomy. While anatomical observations are of importance in the assessments and appraisals, use of these characters as an effective tool in interpreting phyletic evaluations and systematic delineations has its limitations too. The epidermal cell wall in majority of species was wavy to undulate on both adaxial and abaxial surfaces. The observations made in this study indicate that there is not a single type of stomata which appears as characteristic of the genus Euphorbia. Also their distribution whether epistomatic or hypostomatic is not a genuscharacteristic. The trichomes found were simple, unicellular or multicellular, uniseriate. Present investigation revealed the utility of both qualitative and quantitative characters in systematic studies; also the potential influence in the delimitation of species cannot be ignored. Our results show that the micro-morphology of anatomical characters play an important role in definition of taxa at species and sectional levels.

Systematic significance of anatomical characterization in some euphorbiaceous species

The efficacy of genetic material for use as DNA barcodes is under constant evaluation and improvement as new barcodes offering better resolution and efficiency of amplification for specific species groups are identified. In this study, the chloroplast intergenic spacer psbK-psbI was evaluated for the first time as a DNA barcode for distinguishing date palm cultivars. Nucleotide sequences were aligned using MEGA 6.0 to calculate pairwise divergence among the cultivars. The analyzed data illustrated a considerable level of variability in the genetic pool of the selected cultivars (0.009). In fact, five haplotypes were detected among 30 cultivars examined, yielding a haplotype diversity of 0.685. An unweighted pair group method with arithmetic mean phylogenetic tree was constructed and shows a well-defined relationship among date palm cultivar varieties. On the other hand, selective neutrality investigations using Tajima test and Fu and Li tests were negative, providing evidence that date palm has been undergoing rapid expansion and recent population growth. Thus, we suggest that the psbK-psbI spacer can be successfully used to construct reliable phylogenetic trees for P. dactylifera.

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Cultivar-level phylogeny using chloroplast DNA barcode psbK-psbI spacers for identification of Emirati date palm (Phoenix dactylifera L.) varieties.

In the present study the phylogeny of the various selected genera of Acanthaceae was investigated based on macromorphological characters, chloroplast DNA (cp-DNA) genes rbcL, matK and intergenic spacer trnH-psbA sequences. About 34 macro-morphological characters of taxonomic importance were selected from the different floras of the world which were used to construct a phylogeny based on morphology. For morphological analysis selected species belonging to five different genera of Acanthaceae including Ruellia, Justicia, Barleria, Dicliptera and Strobilanthes were studied. The phylogenetic tree was constructed using Multivariate Statistical Package (MVSP 3.1) by the method of UPGMA (Unweighted Pair Group Method Analysis). Our results showed that Ruellia brittoniana seemed to be the closest relative of Justicia and Barleria species based on morphological analysis. The plastid gene regions ribulose-1,5bisphosphatecarboxylase/oxygenase (rbcL), maturase kinase (matK) and trnH–psbA intergenic spacer region were selected for taxonomic study on molecular level. These genes rbcL (1400 bp), matK (800 bp) and trnH–psbA spacer (400 bp) were sequenced and the sequence data was aligned and used for the phylogenetic tree in combination with the other sequences retrieved from GenBank, National Centre of Biotechnology Information (NCBI). Molecular data based on these matK, rbcL and trnH–psbA sequences and the BLAST result shows that genera Barleria, Strobilanthes and Ruellia are monophyletic but genus Justicia is non-monophyletic. Our results conclude that rbcL is more effective barcode; while combination of matK and trnH–psbA can be used for identification and phylogenetic study of plants.

DNA barcoding and molecular systematics of selected species of family Acanthaceae

Anisodus acutangulus (Solanaceae),  an important folk medicinal herb in China, produces up to 1.2% alkaloids more than that in other Solanaceae plants such as Hyoscyamus niger, while its evolutionary position in Hyoscyameae is not very clear. Objective: To explain the evolutionary position of A. acutangulus in the Solanaceae via complete chloroplast genome(cp) sequence. Methods: Complete chloroplast genome of A. acutangulus was obtained and characterized using the Illumina PE150 pair-end sequencing data. Structure of the genome, codon usage, nucleotide variability (Pi) value, distribution of repeats and SSRs between A. acutangulus and other seven Solanaceae species were analyzed. Previously published 22 Solanaceae cp genomes were used to construct phylogenetic tree. Results: The complete cp genome of A. acutangulus is 156082 bp in length, showed the typical quadripartite structure. The complete cp genome of A. acutangulus was highly conserved. A total of 112 unique genes were found in cp genome of A. acutangulus, among which 17 were duplicated. Further, we found eight hotspot regions for genome divergence could be explored as new DNA barcodes for the identification of the Solanaceae species. Phylogenetic analysis showed that A. acutangulus formed a clade with H. niger. Conclusion: A. acutangulus belongs to Hyoscyameae subfamily and the complete cp genome provides valuable information for phylogenetic reconstruction or comparative genomics of A. acutangulus.

The Complete Chloroplast Genome Sequences of Anisodus Acutangulus and a Comparison with Other Solanaceae Species

A phylogenetic analysis of family Solanaceae was conducted using sequence data from the chloroplast intergenic atpβrbcL spacer. Sequence data was generated from 17 species representing 09 out of 14 genera of Solanaceae from Pakistan. Cladogram was constructed using maximum parsimony method and results indicate that Solanaceae is mainly divided into two subfamilies; Solanoideae and Cestroideae. Four major clades within Solanoideae represent tribes; Physaleae, Capsiceae, Datureae and Solaneae are supported by high bootstrap value and the relationships among them are not corroborating with the previous studies. The findings established that subfamily Cestroideae comprised of three genera; Cestrum, Lycium and Nicotiana with high bootstrap support. Position of Nicotiana inferred with atpβ-rbcL sequence is congruent with traditional classification, which placed the taxa in Cestroideae. In the current study Lycium unexpectedly nested with Nicotiana with 100% bootstrap support and identified as a member of tribe Nicotianeae. Expanded sampling of other genera from Pakistan could be valuable towards improving our understanding of intrafamilial relationships within Solanaceae.

Syed Irtifaq Ali

Papers

SUBFAMILIAL RELATIONSHIPS WITHIN SOLANACEAE AS INFERRED FROM atpβ-rbcL INTERGENIC SPACER