Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson–Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson–Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB^TM is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5′-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Taxonomic interpretation of chromosomal and mitochondrial DNA variability in the species complex close to Polyommatus (Agrodiaetus) dama (Lepidoptera,Lycaenidae)

In this paper, by using combination of molecular and chromosomal markers, populations of Polyommatus (Agrodiaetus) karindus (Riley, 1921) from north-west and central Iran are analyzed. It has been found that taxon usually identified as Polyommatus (Agrodiaetus) karindus is represented in Iran by two geographically separated groups of individuals, strongly differentiated by their karyotypes and mitochondrial haplotypes. It is demonstrated that populations from NW Iran have the haploid chromosome number n = 68, while the haploid chromosome number of Polyommatus (Agrodiaetus) karindus from central Iran is found to be n = 73. Phylogenetic analysis revealed that these groups also differ by at least eight nucleotide substitutions in a 690 bp fragment of the mitochondrial COI gene and form separated groups of clusters in Bayesian inference tree. Thus, population entities from central Iran are described here as a new subspecies Polyommatus (Agrodiaetus) karindussaravandissp. n. Strong chromosomal and molecular differentiation are confirmed between Polyommatus (Agrodiaetus) karindus and its sister species, Polyommatus (Agrodiaetus) dama (Staudinger, 1892).     

Dobzhansky's rule and reinforcement of pre-zygotic reproductive isolation in zones of secondary contact

It is well known that closely related sympatric species are usually more different in characters involved in species recognition (e.g., in visual and acoustic signals) than allopatric species of the same evolutionary age. In this article I call this phenomenon Dobzhansky's rule in accordance with the name of the scientist who first discovered it. There are two alternative explanations for this pattern. Under hypothesis of reinforcement by Dobzhansky, these species-specific differences evolve in situ, exactly in zone of overlap between two populations. Under hypothesis of differential fusion by Templeton, the differences originate in geographically separated regions, and only those populations that have evolved such differences can persist in secondary sympatry. These evolutionary scenarios are significantly different. The scenario by Dobzhansky is an essentially sympatric model, in which natural selection reinforces pre-zygotic isolation between divergent populations by selecting against unfit hybrids. The scenario by Templeton is based on classic allopatric speciation model that consider the formation of reproductive isolation to be a by-product of divergent evolution. In this work we show that the sympatric distribution of sister taxa of Agrodiaetus butterflies strongly correlates with differences in male wing colour. We also use a new quantitative phylogenetic test to distinguish between the models by Dobzhansky and by Templeton and to demonstrate that the pattern observed is, most likely, the result of reinforcement.     

What gene and chromosomes say about the origin and evolution of insects and other arthropods

At the turn of the 21st century, the use of molecular and molecular cytogenetic methods led to revolutionary advances in systematics of insects and other arthropods. Analysis of nuclear and mitochondrial genes, as well as investigation of structural rearrangements in the mitochondrial chromosome convincingly supported the Pancrustacea hypothesis, according to which insects originated directly from crustaceans, whereas myriapods are not closely related to them. The presence of the specific telomeric motif TTAGG confirmed the monophyletic origin of arthropods (Arthropoda) and the assignment of tongue worms (Pentastomida) to this type. Several different types of telomeric sequences have been found within the class of insects. Investigation of the molecular organization of these sequences may shed light on the relationships between the orders Diptera, Siphonaptera, and Mecoptera and on the origin of such enigmatic groups as the orders Strepsiptera, Zoraptera and suborder Coleorrhyncha.     

Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.

Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation.     

The origin and taxonomic position of the Crimean endemic <Emphasis Type="Italic">Agrodiaetus pjushtchi</Emphasis> (Lepidoptera,Lycaenidae) based on the data on karyology,ecology, and molecular phylogenetics

The geographic ranges, karyotypes, ecological preferences, and mitochondrial DNA polymorphism were analyzed to reveal the origin and taxonomic position of Agrodiaetus pljushtchi and related species of A. damone species group. According to the data obtained, the common ancestor of these species originated in Asia Minor in Late Pliocene, 1.2–1.8 million years ago. In Early Pleistocene (0.8–1.2 million years ago), a representative of this group migrated into the Crimea and gave rise to A. pljushtchi. Another branch originated from the common ancestor, which migrated into Central Asia and Siberia and gave rise to a variety of partly sympatric taxa. Recently, representatives of the Central Asian — Siberian lineage occupied Eastern Europe and approached the area of A. pljushtchi but did not overlap with it. The karyotype n = 67 is an apomorphy of the A. damone species complex. However, this karyotype is not species-specific and cannot be used to differentiate taxa within the complex. The similarity of karyotypes is thus not sufficient to consider A. pljushtchi and A. damone as conspecific taxa. The natural history data support the isolated position of A. pljushtchi, and phylogenetic analysis shows that A. pljushtchi and A. damone are not closely related.     

Species Delimitation and Analysis of Cryptic Species Diversity in the XXI Century

The potentials and limitations of different approaches to revealing species boundaries and describing cryptic species are discussed. Both the traditional methods of species delimitation, mostly based on morphological analysis, and the approaches using molecular markers are considered. Besides, the prospects of species identification using digital image recognition and machine learning are briefly considered. It is concluded that molecular markers provide very important material for species delimitation; the value of these data increases manifold if they can be compared with information on morphology, geographic distribution, and ecological preferences of the studied taxa. In many cases, only a practicing taxonomist who knows his or her group thoroughly can correctly interpret the molecular data and incorporate them into the existing knowledge system in order to make a taxonomic decision.

     

Butterflies (Lepidoptera,Rhopalocera) of West Altai

A total of 176 species of butterflies have been found in West Altai. Their distribution ranges, phenology, biological preferences, and taxonomic structure are analyzed. Numerous Siberian butterfly species from the genera Erebia, Oeneis, Clossiana, Boeberia, and Colias have been found in West Altai which may be accounted for its westernmost position in the mountain system of southern Siberia and very humid climate. At the same time, many western Palaearctic as well as few Middle Asian butterflies have been discovered in West Altai for the first time. West Altai is the region where pairs of sister species which diverged in the Western and Eastern Palaearctic can be found in secondary contact. In this study, a zone of stable secondary sympatry of Melitaea didyma (western element) and M. latonigena (eastern element) as well as that of Maculinea arion (western element) and M. cyanecula (eastern element) has been found. These findings provide the first strong evidence for the distinctness of the species in these sister pairs.