Mean Genetic Distance Research Articles

Enhanced data on Stolephorus grandis and Stolephorus waitei (Teleostei: Clupeiformes: Engraulidae), two species endemic to the Sahul Shelf, western Pacific Ocean

Eighteen and 23 specimens of two species of the poorly known anchovies Stolephorus grandis Hata & Motomura, 2021b and Stolephorus waitei Jordan & Seale, 1926 (Clupeiformes: Engraulidae), respectively, are examined. This study presents their detailed morphological characters including fresh colorations along with their precise distributional ranges within the Sahul Shelf coastal region. The “barcode” regions of the mitochondrial cytochrome c oxidase subunit I (COI) gene were examined in four specimens of S. waitei and one specimen of S. grandis. Both species diverged by 10.6% and > 10.3% mean (uncorrected) genetic distances from each other and from any other species of Stolephorus, respectively. Stolephorus waitei, S. grandis, and Stolephorus nelsoni Wongratana, 1987 form a monophyletic group of species endemic to the Sahul Shelf region. Additionally, the role and evolution of double lines on the dorsum observed in several species of Stolephorus, including S. grandis, are discussed.

Genetic diversity and structure of Tunisian and Indian date palm (Phoenix dactylifera and sylvestris) cultivars and genotypes revealed by AFLP markers

The date palm breeding programs need to discover valid genetic fingerprints to characterize cultivars and assess their genetic diversity. This study assessed the genetic diversity among thirty-nine date palm cultivars from Tunisia (Phoenix dactylifera) and India (Phoenix sylvestris) by using six AFLP (Amplified Fragment Length Polymorphism) markers. 360 loci were amplified, with 127 loci polymorphic (34.35 %). The Jaccard's similarity coefficient ranged from 0.161 to 0.931, with the mean genetic distances of 0.568. AFLP's average marker index value was 7.28, with a resolving power of 10.91. The analysis of population structure showed two main clusters with a clear separation between Tunisian and Indian cultivars.Furthermore, the heatmap analysis allowed the identification of 10 bands specific to the Indian accessions, which were not detected in Tunisian genotypes. These loci could be linked to genes involved in adapting the species in Indian lands, which allowed the study of the genetic diversity of date palm resources of different origins, confirming the existence of at least two origins of domestication. Additionally, identifying AFLP loci specific to P. dactylifera and P. sylvestris will significantly contribute to breeding programs by exploiting species-specific polymorphisms.

Revealing the Genetic Differentiation of Rattus norvegicus (Berkenhout 1769) Populations by Analyzing Two Mitochondrial Markers

Rattus norvegicus (Brown rat) has a great importance for public health and economy because it lives in close association with human populations. However, molecular systematic studies on global populations of R. norvegicus are very few. In this study, sequences obtained from Europe, Asia, Africa, and America regions were analyzed using mitochondrial Cytochrome-b and Cytochrome oxidase-I gene regions and genetic differentiation levels between these populations were revealed. Accordingly, samples belonging to the studied populations did not split in Bayesian Inference trees and Median-joining networks; these samples also formed common haplotypes, and mean genetic distance and fixation index values were generally low. The results of the study showed that gene flow between these populations may be continuing due to the transportation activity by humans.

Molecular Evolutionary Analyses of the Fusion Genes in Human Parainfluenza Virus Type 4.

The human parainfluenza virus type 4 (HPIV4) can be classified into two distinct subtypes, 4a and 4b. The full lengths of the fusion gene (F gene) of 48 HPIV4 strains collected during the period of 1966-2022 were analyzed. Based on these gene sequences, the time-scaled evolutionary tree was constructed using Bayesian Markov chain Monte Carlo methods. A phylogenetic tree showed that the first division of the two subtypes occurred around 1823, and the most recent common ancestors of each type, 4a and 4b, existed until about 1940 and 1939, respectively. Although the mean genetic distances of all strains were relatively wide, the distances in each subtype were not wide, indicating that this gene was conserved in each subtype. The evolutionary rates of the genes were relatively low (4.41 × 10-4 substitutions/site/year). Moreover, conformational B-cell epitopes were predicted in the apex of the trimer fusion protein. These results suggest that HPIV4 subtypes diverged 200 years ago and the progenies further diverged and evolved.

Studies on Genetic Divergence in Cabbage Genotypes in the Mid Hills of Solan District, Himachal Pradesh, India

Cabbage (Brassica oleracea var. capitata L.) is one of the most important vegetables of the family Brassicaceae and is grown under temperate to tropical climatic conditions for its head in more than ninety countries throughout the world. The diversity in cabbage is immense due to the presence of variation in shape, size, weight and maturity of heads. The greater the variability in the available germplasm, more would be the chances for selecting superior genotypes for use in breeding programmes or direct use as cultivars. Twenty three genotypes of cabbage including check variety, Golden Acre were evaluated in a Randomized Complete Block Design (RCBD) with three replications. The results concluded that twenty-three diverse genotypes of cabbage were grouped into four clusters. The resultant four clusters showed genetic diversity and maximum number of genotypes were accommodated in cluster I (12) followed by II (7), IV (3) and III (1). The maximum inter cluster distance was recorded between cluster II and III, therefore, the hybridization between the genotypes of cluster II and III can be performed to get superior hybrids or recombinants in segregating populations for further breeding programmes. Based on the mean performance, genetic distance and clustering pattern, inter-varietal crosses can be made which may be advantageous in creating wider variability for better heterotic combinations and transgressive segregants in cabbage.

Genetic relationships between and within some Malawian cichlid genera.

Genetic relationships were examined for a total of 90 individuals of 90 species or species var. from 13 genera of Malawian cichlids based on the sequences of an amplified 991-bp fragment of the mtDNA control region (mtDNA-CR). In the network analysis, no exclusive clades were made by all the members of any genera in this study. However, congeneric clades were observed by genera Buccochromis, Copadichromis, Protomelas, and Sciaenochromis, whereas no congeneric clades were observed by genera Mylochromis, Nimbochromis, and Otopharynx. In non-mbuna, an Aulonocara-Lethrinops group was divided into two groups, and the mean genetic distance of the larger group was much lower from mbuna than from other non-mbuna. Overall mean genetic distance within a genus was generally low in mbuna, whereas it was relatively high in non-mbuna. In the genetic tree of each genus, two or more large clades were observed, and some clades, such as those of Aulonocara hansbaenschi and Aulonocara nyassae in genus Aulonocara, Lethrinops micrentodon and Lethrinops sp. "gold harbor" in Lethrinops, and Otopharynx ovatus and Otopharynx brooksi in Otopharynx, were very deeply differentiated. Besides, a mbuna species, Pseudotropheus crabro, was extremely deeply differentiated from other members of this genus. These results suggest a widespread morphological convergence across the taxa in parallel with deep genetic differentiation in the long evolutionary story and some possibility of generation of the species of Aulonocara-Lethrinops group by hybridization of small non-mbuna and mbuna species. Furthermore, taxonomical reexamination is necessary based on a strong support by genetic connection.

Characteristics of sheep in the Near Arctic region by microsatellites and reproductive abilities

The microsatellites polymorphism was analyzed in ewe lambs obtained from intra-breed mating of the Pechora semifine-wool sheep and their littermates from crossing with the Kuibyshev rams. Genotyping of the population by STR loci was performed using the COrDIS Sheep reagent kit recommended by the International Society of Animal Genetics (ISAG). The age of the beginning of reproduction, fertility, milk yield, safety and growth intensity of the litter up to three months of age were taken into account in the ewe lambs. In the group of the Pechora sheep sows 74 alleles were detected, and 81 alleles were detected in the crossbred ones. INRA005 and INRA023 loci were the most polymorphic in both groups, while ETH152 was the least polymorphic. The mean genetic distance between the groups for the 12 STR loci was 0.019 ± 0.003. Pechora sheep (P) and their litters with the Kuibyshev rams (1/2 P + 1/2 K) differed significantly in the frequencies of some STR loci genes. Thus, the INRA023212 allele was found with a frequency of 0.174 in the group of P ewes and was absent in the 1/2 P + 1/2 K group, while the INRA172126 allele was found with a frequency of 0.326 and 0.122, respectively. The mean Fis values in both groups were close to zero. The groups did not differ significantly in the main genetic-population parameters. However, some advantage of the Pechora ewe sows in comparison with the crossbred sheep 1/2 P + 1/2 K on selective traits, apparently, is due to a higher genetic contribution to their heredity of the indigenous northern short-tailed sheep. According to the results of the study, it can be assumed that the age of onset of reproduction of the ewe lambs is related to the level of their heterozygosity for STR loci.

Performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase.

HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.

Description of a new species of Phoxinus from the Ergene River (Aegean Sea Basin) in Türkiye (Actinopterygii, Leuciscidae)

Phoxinus radeki, a new species, is described from the Ergene River (Aegean Sea Basin). It is distinguished from Phoxinus species in Türkiye and the adjacent area by having the scales of the breast, scaled but separated unscaled area anteriorly, short dark rectangular blotches along the lateral line between the lateral line and belly yellowish in both males and females, body depth dorsal fin origin 16–21% SL, caudal peduncle depth 8–10% SL. Additionally, molecular results demonstrated that the new species differed from its closest congeners with a mean genetic distance value of 3.08% (min. 2.82–max. 3.29) and moderately support values in cytochrome b (Cyt b) gene partial sequences (1064 bp.). Further, the species delimitation analysis identified the new species as a single MOTU independent of other Phoxinus species.

Variability Study Analysis for Half-sib Families under Control Environmental Conditions in Neolamarckia cadamba (Kadamb)

Neolamarckia cadamba is a widespread medium-sized deciduous forest tree over most of India, Bangladesh, and Sri Lanka. Twenty-half-sib progenies of kadamb (Neolamarckia cadamba) from various regions in Bihar, Rajasthan, and Uttar Pradesh were sown in a glasshouse in 2022 in order to assess the genetic diversity using non-hierarchical Euclidean cluster analysis. Significant variation was seen in the biomass and morphological traits of a few plus trees. Under greenhouse conditions, significant differences were seen in the growth parameters of plus tree progenies. Six clusters were formed from the twenty NPTs, according to the genetic divergence analysis. Clusters V and VI were found to have the greatest inter-cluster distance (5.530). The cluster (4.258) displayed the highest intra-cluster distance. As a result, there was a genetic divergence between clusters. Therefore, in order to obtain high-yielding genotypes in Neolamarckia cadamba, hybridization involving trees of clusters VI and V is advised. Collar diameter was a major factor in the creation of genetic diversity and contributed the most to the overall divergence. The superior plus trees of cluster V (NPT4, NPT5, NPT11, NPT12, NPT13, NPT17) and cluster VI (NPT18, NPT19, NPT20) may be taken into consideration as possible parents for a future tree improvement program in Neolamarckia cadamba due to their high cluster mean and large genetic distance.

Phylogenetic characterization of two common sand flies Phlebotomus major and P. kandelakii in the West Black Sea Region of Turkey based on mitochondrial genes sequence analysis

Research on the taxonomy and phylogenetic characterization of sand flies (Diptera, Psychodidae, Phlebotominae) has received increasing attention due to their potential role in the transmission of medically significant diseases, including leishmaniasis, bartonellosis, and sand fly fever. Phlebotomus major and Phlebotomus kandelakii are the members of Larroussius subgenus that includes important vector sand fly species. P. major, P. neglectus and P. syriacus from a well-known sand fly “Major” species group are present widely while P. kandelakii has constricted distribution in Turkey. All have the capability to transmit Leishmania infantum, the causative agent of visceral leishmaniasis. Here, we investigated the genetic diversity within each species collected from the West Black Sea Region of Turkey using mitochondrial DNA markers, specifically cytochrome oxidase I (COI) and cytochrome b gene sequences (Cytb). A total of 1889 sand flies specimens were collected from the study area in June 2021 and August 2022, 1596 (84.49%) were identified as P. major s.l., and 253 (13.40%) were identified as P. kandelakii. Nine and four haplotypes of P. major were determined in the study area based on COI and Cytb sequences, respectively. Analysis of the phylogenetic data sets generated from our isolates and published isolates in Genbank revealed high haplotype diversities within P. major (COI = 0.933, Cytb = 0.714). For P. kandelakii, we detected four and three haplotypes within the COI and Cytb sequences, and the haplotype diversities were also high in the datasets including our isolates and published isolates in Genbank (COI = 0.978, Cytb = 1.000). Pairwise mean genetic distance calculated from the COI and Cytb datasets were 0.4% and 1.4% for P. major, and 1.0% and 0.2% for P. kandelakii, respectively, suggesting the absence of cryptic species. Phylogenetic analyses revealed three and two major clusters of Larroussius subgenus in the COI and Cytb datasets, respectively.

Berry texture QTL and candidate gene analysis in grape (Vitis vinifera L.).

Berry texture is a noteworthy economic trait for grape; however, the genetic bases and the complex gene expression and regulatory mechanism for the diverse changes in berry texture are still poorly understood. In this study, the results suggest that it is difficult to obtain high-mesocarp firmness (MesF) and high-pericarp puncture hardness (PPH) grape cultivars with high pericarp brittleness (PerB). The high-density linkage map was constructed using whole-genome resequencing based on 151 F1 individuals originating from intraspecific hybridization between the firm-flesh cultivar 'Red Globe' and soft-flesh cultivar 'Muscat Hamburg'. The total length of the consensus map was 1613.17cM, with a mean genetic distance between adjacent bin markers of 0.59cM. Twenty-seven quantitative trait loci (QTLs) for berry MesF, PPH, and PerB were identified in linkage groups (LGs) 1, 3, 4, 6, 8, 9, 10, 11, 14, 16, and 17, including twelve QTLs that were firstly detected in LGs 6, 11, and 14. Fourteen promising candidate genes were identified from the stable QTL regions in LGs 10, 11, 14, and 17. In particular, VvWARK2 and VvWARK8 refer to chromosome 17 and are two promising candidate genes for MesF and PPH, as the VvWARK8 gene may increase pectin residue binding with WARK for high berry firmness maintenance and the allele for VvWARK2 carrying the 'CC' and 'GA' genotypes at Chr17:1836764 and Chr17:1836770 may be associated with non-hard texture grape cultivars. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) verification revealed that the promising candidate transcription factor genes VvMYB4-like, VvERF113, VvWRKY31, VvWRKY1, and VvNAC83 may regulate cell wall metabolism candidate gene expression for grape berry texture changes.

DNA barcoding of reef-associated fishes of Saint Martin's Island, Northern Bay of Bengal, Bangladesh.

This study employs the DNA barcoding approach to make a molecular taxonomic catalog of reef fishes of Saint Martin's Island (SMI), an ecologically critical area (ECA), and Marine Protected Area (MPA) in Bangladesh. DNA barcoding, along with morphological analysis, confirmed 84 reef-associated fish species in SMI belonging to 16 orders, 39 families, and 67 genera. A total of 184 sequences were obtained in this study where 151 sequences (534-604 bp) of 81 species were identified from the COI barcode gene and 33 sequences (609 bp) of 19 species from the 16S rRNA gene region which were submitted to the GenBank and Barcode of Life Data System (BOLD). Among these sequences, 70 sequences of the COI gene and 16 sequences of 16S rRNA gene region from 41 species were submitted for the first time into the GenBank from Bangladesh. For molecular characterization analysis, another 37 sequences of 15 reef fish species of SMI were added from previous studies, making a total of 221 DNA sequences which comprised 179 sequences of 96 species for the COI gene and 42 sequences of 26 species for the 16S rRNA gene region. The COI sequences contain 145 haplotypes with 337 polymorphic sites, and the mean genetic distances within species, genera, and families were calculated as 0.34%, 12.26%, and 19.03%, respectively. On the contrary, 16S rRNA sequences comprised 31 haplotypes with 241 polymorphic sites, and the mean genetic divergences within species, genera, and families were 0.94%, 4.72%, and 12.43%, respectively. This study is a significant contribution to the marine biodiversity of Bangladesh which would facilitate the assessment of species diversity for strategizing management action. It is also an important input to the DNA barcode library of reef fishes of the northern Bay of Bengal.

Molecular Identification of Acrossocheilus jishouensis (Teleostei: Cyprinidae) and Its Complete Mitochondrial Genome.

Molecular identification, such as DNA barcoding, is a useful tool that is widely applied in distinguishing species. To identify the cyprinid Acrossocheilus jishouensis, which was previously known to be restricted to only its type locality, we conducted molecular identification of this species based on 23 samples in five localities. Molecular identification based on the mitochondrial COI gene sequence showed that the morphologically similar samples from the five populations were all A. jishouensis, as the mean genetic distances between populations were very small (0.1-1.6%); thus, the distribution of this species was substantially expanded. The whole mitochondrial genome of one sample was also assembled, which was 16,594bp in length and consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and one control region. All PCGs began with ATG except the COI gene, which started with GTG; seven PCGs used the complete stop codon TAA, while four terminated in T(AA) and two ended with TAG. The overall base composition reflected a higher proportion of A+T than G+C and a positive AT-skew and negative GC-skew pattern except for the opposite in ND6. Phylogenetic relationships inferred using BI and ML methods revealed that both Acrossocheilus and Onychostoma were nonmonophyletic, which indicated that the traditional diagnoses between these two genera need to be assessed further. The results of this study not only expanded the known distribution ranges of A. jishouensis, but also provided a valuable data resource for future molecular and evolutionary studies of Acrossocheilus and other cyprinids in Barbinae.

GENETIC VARIATION OF LEPTOTROMBIDIUM (ACARI: TROMBICULIDAE) MITES CARRYING ORIENTIA TSUTSUGAMUSHI, THE BACTERIAL PATHOGEN CAUSING SCRUB TYPHUS.

Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626-0.1732, whereas the distances within each subcluster were 0.003-0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010-0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.

DNA Barcoding of Cardinalfish (Apogonidae) in Gilimanuk Bay, Bali, Indonesia

Highlight Research Eight species of cardinalfish in Gilimanuk Bay were delineated using the barcoding method. This study provides the first nucleotide sequence for Ostorhinchus hartzfeldii in GenBank online database. The phylogenetic tree showed that each sample resided in a distinct cluster, indicating that the barcoding method efficiently differentiated at the species level. The mean genetic distance between genera within the family was 31.8-fold higher than the mean genetic distance within species. Abstract The Apogonidae is estimated to consist of nearly 300 fish species, most of which inhabit coral reef areas. The lack of distinctive body markings and overlapping species distribution makes species assignment challenging. Therefore, this study aimed to delineate species and establish barcoding reference databases of Apogonidae in Gilimanuk Bay (Bali, Indonesia) using the Cytochrome Oxidase I (COI) gene of the mitochondrial DNA. A total of 22 fish tissue samples were extracted with 10% Chelex solution. BLAST analysis was performed and genetic differentiation between species was calculated. The phylogenetic tree was constructed using the Maximum Likelihood method and tree visualization was generated using iTOL V5. The morphology and genetic identification results based on the mitochondrial COI gene revealed eight species of seven genera, and one species was new to GenBank online database. This study was the first-ever addition of COI sequence for Ostorhinchus hartzfeldii into the GenBank database. The average K2P genetic distance within species and K2P distance between genera within the family were 0.60% and 19.10%, respectively. The mean genetic distance between genera within the family was 31.8-fold higher than the mean genetic distance within species. The phylogenetic tree showed that each sample resided in a distinct cluster, which indicates that DNA barcoding is a reliable and effective approach for species delimitation in Apogonidae fishes.

MtCO1 Analysis of the Butterfly Genus <i>Papilio</i>, from Indonesia

The genus Papilio is a butterfly that has many subspecies with similar phenotypes. So until now, researchers are still studying the genus Papilio based on morphology and genetics. Partial sequences of mitochondrial cytochrome oxidase-I (mtCOI) genes from individuals from 13 species of Papilionidae from North Sulawesi: Indonesia, Papua New Guinea, Malaysia: Negeri Sembilan, India: Assam, South Korea, China, Taiwan: Nantou, and Thailand: Fang, Chiang Mai have been compared in this study. Nucleotide divergences showed that the mean genetic distance between species was 0.023 (2.3%) to 0.723 (72.32%). Phylogenetic analysis revealed that groups of Papilionidae from North Sulawesi: Indonesia formed their own group; Species from Papua New Guinea are closely related to other Papilionidae species from other regions. There are 50 distinct sites or single nucleotide polymorphism (SNP) in the mtCO1 sequence of eleven species. These mtCO1 sequences can reveal the genetic differentiation of closely related species in the genus Papilio.

Phylogenetic analysis of the critically endangered Karpathos water frog (Anura, Amphibia): conservation insights from complete mitochondrial genome sequencing

Abstract The Karpathos water frog (Pelophylax cerigensis) is classified as Critically Endangered according to the IUCN Red List and its distribution is restricted on the island of Karpathos, South Aegean Sea. Utilizing a next generation sequencing approach, we obtained whole mitochondrial genomes of P. cerigensis to determine the species phylogenetic position within the genus Pelophylax and to clarify the phylogenetic relationship between the species’ population on Karpathos Island and the population of P. cf. bedriagae from the neighboring Rhodes Island. High-throughput sequencing generated mean ± SD = 227 945 ± 18 306 reads averaged per sample. High quality reads were assembled resulting in the complete mitogenome of P. cerigensis of a total size of 17 922 bp. Mitogenome organization was similar to other Pelophylax species, comprising of 13 Protein Coding Genes (PCGs), 22 tRNAs, two rRNAs and one Control Region. Overall mean genetic distance for the 13 PCGs, within the Ranidae family, ranged from 8.01% (COIII) to 11.6% (ATP8), while ratios of synonymous and non-synonymous substitutions were <1 in all the 13 PCGs, indicating purifying selection. Maximum Likelihood and Bayesian Inference analyses based on the concatenated sequences of 13 PCGs and two rRNAs (12S and 16S), showed that individuals from Karpathos and Rhodes form a well-supported monophyletic group with low genetic distance between them (p-distance = 0.1%). Hence, the Rhodes populations seem to belong to P. cerigensis and not to P. bedriagae as formerly thought, highlighting the need for revision of P. cerigensis current conservation status as well as further examination of the P. bedriagae species group.

The taxonomic separateness of the species Aporophyla lueneburgensis (Freyer, 1848) and Aporophyla lutulenta ([Denis & Schiffermüller], 1775) occurring in Poland (Lepidoptera: Noctuidae)

Morphological and genetic comparisons were carried out of specimens of two sister species Aporophyla lueneburgensis (Freyer, 1848) and Aporophyla lutulenta ([Denis & Schiffermüller], 1775), which are distributed allopatrically in Poland. The specimens from different populations of these two species hardly differ morphologically but do differ genetically. The mean genetic distance between the groups of A. lueneburgensis and A. lutulenta examined here indicates that their geographic populations can be treated as belonging to separate species. Hence, A. lueneburgensis and A. lutulenta occurring in Poland can be regarded as two separate but sister species.

DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana.

Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes at different sizes. Among the three barcodes, rbcL was the least efficient as a DNA barcode compared to ITS1 and ITS2. The ITS1 nucleotide analysis revealed that the ITS1 barcode had more variations compared to ITS2. The mean genetic distance (K2P) between them was higher for interspecies compared to intraspecies. The results showed that the DNA barcoding gap existed among Nepenthes species, and differences in the secondary structure distinguish the Nepenthes. The secondary structure generated in this study was found to successfully discriminate between the Nepenthes species, leading to enhanced resolutions.