Protein-coding Gene Sequences Research Articles

Complete mitochondrial DNA genome of Thryssa dussumieri (Valenciennes, 1848)

In this study, the complete mitochondrial genome (mitogenome) sequence of Thryssa dussumieri was determined by long polymerase chain reaction and primer walking methods. The mitogenome was a circular molecule of 16,883 base pairs (bp) in length and contained 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and a control region similar to those found in other bony fishes. The overall nucleotide composition was estimated to be 32.1% A, 26.1% C, 23.7% T, and 18.1% G. The neighbor-joining tree was reconstructed based on the 13 PCGs sequences of T. dussumieri and other 10 Engraulidae species. The mitogenome data will provide useful information for further studies on the phylogenetic analysis and population genetics.

The complete mitochondrial genome of Triplophysa sellaefer (Cypriniformes: Balitoridae)

The complete mitochondrial genome of Triplophysa sellaefer is a circular molecule of 16,571 bp in length with the A + T contents of 56.84%, containing 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and a putative control region. Six gene overlaps and 10 intergenic spacers were observed, with the total length of 26 bp and 34 bp, respectively. The phylogenetic analysis based on the concatenated PCG sequences revealed that T. sellaefer is closely related to T. alticeps. This study would provide useful information on phylogenetic and evolutionary studies in Triplophysa.

Overview of inactivating mutations in the protein-coding genome of the mouse reference strain C57BL/6J.

Mice are extremely important as the premier model organism in human biomedical and mammalian genetic research. The genomes of several tens of mouse inbred strains have been sequenced. They have been compared to the genome of C57BL/6J, considered by convention as the reference genome. Based on a comparison of this reference genome with 36 other sequenced mouse strains, we generated an overview of all protein-coding genes that are deviant in this reference genome, compared with consensus protein-coding mouse gene sequences. We provide PROVEAN scores, reflecting the likelihood that these C57BL/6J proteins have lost function. We thus identified numerous abnormal proteins, and biological pathways, specifically present in C57BL/6J, suggesting the important caveats of this reference mouse strain, and linking candidate genes to some of the best-known phenotypes of this strain.

Codon Usage Pattern of Genes Involved in Central Nervous System.

Codon usage bias (CUB) is the non-uniform usage of synonymous codons in which some codons are more preferred to others in the transcript. Analysis of codon usage bias has applications in understanding the basics of molecular biology, genetics, gene expression, and molecular evolution. To understand the patterns of codon usage in genes involved in the central nervous system (CNS), we used bioinformatic approaches to analyze the protein-coding sequences of genes involved in the CNS. The improved effective number of codons (ENC) suggested that the overall codon usage bias was low. The relative synonymous codon usage (RSCU) revealed that the most frequently occurring codons had a G or C at the third codon position. The codons namely TCC, AGC, CTG, CAG, CGC, ATC, ACC, GTG, GCC, GGC, and CGG (average RSCU > 1.6) were over-represented. Both mutation pressure and natural selection might affect the codon usage pattern as evident from correspondence and parity plot analyses. The overall GC content (59.93) was higher than AT content, i.e., genes were GC-rich. The correlation of GC12 with GC3 suggested that mutation pressure might affect the codon usage pattern.

Global analysis of threonine metabolism genes unravel key players in rice to improve the abiotic stress tolerance

The diversity in plant metabolites with improved phytonutrients is essential to achieve global food security and sustainable crop yield. Our study using computational metabolomics genome wide association study (cmGWAS) reports on a comprehensive profiling of threonine (Thr) metabolite in rice. Sixteen abiotic stress responsive (AbSR) – Thr metabolite producing genes (ThrMPG), modulate metabolite levels and play a significant role determining both physiological and nutritional importance of rice. These AbSR-ThrMPG were computationally analysed for their protein properties using OryzaCyc through plant metabolic network analyser. A total of 1373 and 1028 SNPs were involved in complex traits and genomic variations. Comparative mapping of AbSR-ThrMPG revealed the chromosomal colinearity with C4 grass species. Further, computational expression pattern of these genes predicted a differential expression profiling in diverse developmental tissues. Protein interaction of protein coding gene sequences revealed that the abiotic stresses (AbS) are multigenic in nature. In silico expression of AbSR-ThrMPG determined the putative involvement in response to individual AbS. This is the first comprehensive genome wide study reporting on AbSR –ThrMPG analysis in rice. The results of this study provide a pivotal resource for further functional investigation of these key genes in the vital areas of manipulating AbS signaling in rice improvement.

Actively transcribed and expressed atp8 gene in Mytilus edulis mussels.

BackgroundAnimal mitochondrial genomes typically encode 37 genes: 13 proteins, 22 tRNAs and two rRNAs. However, many species represent exceptions to that rule. Bivalvia along with Nematoda and Platyhelminthes are often suspected to fully or partially lack the ATP synthase subunit 8 (atp8) gene. This raises the question as to whether they are really lacking this gene or is this maybe an annotation problem? Among bivalves, Mytilus edulis has been inferred to lack an ATP8 gene since the characterization of its mitochondrial genome in 1992. Even though recent bioinformatic analyses suggested that atp8 is present in Mytilus spp., due to high divergence in predicted amino acid sequences, the existence of a functional atp8 gene in this group remains controversial.ResultsHere we demonstrate that M. edulis mitochondrial open reading frames suggested to be atp8 (in male and female mtDNAs) are actively translated proteins. We also provide evidence that both proteins are an integral part of the ATP synthase complex based on in-gel detection of ATP synthase activity and two-dimensional Blue-Native and SDS polyacrylamide electrophoresis.ConclusionMany organisms (e.g., Bivalvia along with Nematoda and Platyhelminthes) are considered to be lacking certain mitochondrial genes often only based on poor similarity between protein coding gene sequences in genetically closed species. In some situations, this may lead to the inference that the ATP8 gene is absent, when it is in fact present, but highly divergent. This shows how important complementary role protein-based approaches, such as those in the present study, can provide to bioinformatic, genomic studies (i.e., ability to confirm the presence of a gene).

According to mitochondrial DNA evidence, Parascaris equorum and Parascaris univalens may represent the same species.

Parascarosis is caused mainly by parasitic infections with Parascaris equorum and Parascaris univalens, the most common ascarid nematodes, in the small intestine of equines. Parascarosis often causes severe illness and even death in foals and yearlings. In this study, we obtained the complete sequence of the P. equorum mitochondrial (mt) genome and compared its organization and structure with that of P. equorum Japan isolate (nearly complete), and the complete mtDNA sequences of P. univalens Switzerland and USA isolates. The complete mtDNA genome of P. equorum China isolate is 13,899 base pairs (bp), making it the smallest of the four genomes. All four Parascaris mt genomes are circular, and all genes are transcribed in the same direction. The P. equorum mtDNA genome consists of 12 protein-coding genes, two ribosomal RNA genes, 22 transfer (t) RNA genes and one non-coding region, which is consistent with P. equorum Japan isolate and P. univalens Switzerland isolate but distinct from P. univalens USA isolate, which has 20 tRNA genes. Differences in nucleotide sequences of the four entire mt genomes range from 0.1-0.9%, and differences in total amino acid sequences of protein-coding genes are 0.2-2.1%. Phylogenetic analyses showed that the four Parascaris species clustered in a clade, indicating that P. equorum and P. univalens are very closely related. These mt genome datasets provide genetic evidence that P. equorum and P. univalens may represent the same species, which will be of use in further studies of the taxonomy, systematics and population genetics of ascarids and other nematodes.

Assessing the functional association of intronic miRNAs with their host genes.

In human, nearly half of the known microRNAs (miRNAs) are encoded within the introns of protein-coding genes. The embedment of these miRNA genes within the sequences of protein-coding genes alludes to a possible functional relationship between intronic miRNAs and their hosting genes. Several studies, using predicted targets, suggested that intronic miRNAs influence their hosts’ function either antagonistically or synergistically. New experimental data of miRNA expression patterns and targets enable exploring this putative association by relying on actual data rather than on predictions. Here, our analysis based on currently available experimental data implies that the potential functional association between intronic miRNAs and their hosting genes is limited. For host-miRNA examples where functional associations were detected, it was manifested by either autoregulation, common targets of the miRNA and hosting gene, or through the targeting of transcripts participating in pathways in which the host gene is involved. This low prevalence of functional association is consistent with our observation that many intronic miRNAs have independent transcription start sites and are not coexpressed with the hosting gene. Yet, the intronic miRNAs that do show functional association with their hosts were found to be more evolutionarily conserved compared to other intronic miRNAs. This might suggest a selective pressure to maintain this architecture when it has a functional consequence.

Extensive gene rearrangements in the mitochondrial genomes of two egg parasitoids, Trichogramma japonicum and Trichogramma ostriniae (Hymenoptera: Chalcidoidea: Trichogrammatidae)

Animal mitochondrial genomes usually exhibit conserved gene arrangement across major lineages, while those in the Hymenoptera are known to possess frequent rearrangements, as are those of several other orders of insects. Here, we sequenced two complete mitochondrial genomes of Trichogramma japonicum and Trichogramma ostriniae (Hymenoptera: Chalcidoidea: Trichogrammatidae). In total, 37 mitochondrial genes were identified in both species. The same gene arrangement pattern was found in the two species, with extensive gene rearrangement compared with the ancestral insect mitochondrial genome. Most tRNA genes and all protein-coding genes were encoded on the minority strand. In total, 15 tRNA genes and seven protein-coding genes were rearranged. The rearrangements of cox1 and nad2 as well as most tRNA genes were novel. Phylogenetic analysis based on nucleotide sequences of protein-coding genes and on gene arrangement patterns produced identical topologies that support the relationship of (Agaonidae + Pteromalidae) + Trichogrammatidae in Chalcidoidea. CREx analysis revealed eight rearrangement operations occurred from presumed ancestral gene order of Chalcidoidea to form the derived gene order of Trichogramma. Our study shows that gene rearrangement information in Chalcidoidea can potentially contribute to the phylogeny of Chalcidoidea when more mitochondrial genome sequences are available.

Complete mitochondrial genome of the Northern Long-eared Owl (Asio otus Linnaeus, 1758) determined using next-generation sequencing

In this study, the mitogenome of Asio otus, the Northern Long-eared Owl, was analysed using Illumina next-generation sequencing. The mitogenome was found to be a circular molecule, 17,735 bp long with a slight AT bias (53.0%). The gene arrangement pattern was the same as that of a typical vertebrate, containing 37 genes (13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a non-coding control region). In the putative control region (1984 bp), there were two types of tandem repeats at the end of the region. A phylogenetic tree was constructed using the 13 PCG sequences discovered in this study and those of that have been previously published of other Strigidae species and revealed a close relationship between A. otus and A. flammeus. The newly generated mitogenome from this study enriches the genomic resources available for future evolutionary studies and promotes conservation genetics of this species.

Organellar phylogenomics inform systematics in the green algal family Hydrodictyaceae (Chlorophyceae) and provide clues to the complex evolutionary history of plastid genomes in the green algal tree of life.

Phylogenomic analyses across the green algae are resolving relationships at the class, order, and family levels and highlighting dynamic patterns of evolution in organellar genomes. Here we present a within-family phylogenomic study to resolve genera and species relationships in the family Hydrodictyaceae (Chlorophyceae), for which poor resolution in previous phylogenetic studies, along with divergent morphological traits, have precluded taxonomic revisions. Complete plastome sequences and mitochondrial protein-coding gene sequences were acquired from representatives of the Hydrodictyaceae using next-generation sequencing methods. Plastomes were characterized, and gene order and content were compared with plastomes spanning the Sphaeropleales. Single-gene and concatenated-gene phylogenetic analyses of plastid and mitochondrial genes were performed. The Hydrodictyaceae contain the largest sphaeroplealean plastomes thus far fully sequenced. Conservation of plastome gene order within Hydrodictyaceae is striking compared with more dynamic patterns revealed across Sphaeropleales. Phylogenetic analyses resolve Hydrodictyon sister to a monophyletic Pediastrum, though the morphologically distinct P. angulosum and P. duplex continue to be polyphyletic. Analyses of plastid data supported the neochloridacean genus Chlorotetraëdron as sister to Hydrodictyaceae, while conflicting signal was found in the mitochondrial data. A phylogenomic approach resolved within-family relationships not obtainable with previous phylogenetic analyses. Denser taxon sampling across Sphaeropleales is necessary to capture patterns in plastome evolution, and further taxa and studies are needed to fully resolve the sister lineage to Hydrodictyaceae and polyphyly of Pediastrum angulosum and P. duplex.

Complete chloroplast genome of the medicinal plant Amomum compactum: gene organization, comparative analysis, and phylogenetic relationships within Zingiberales

BackgroundAmomum compactum is one of the basic species of the traditional herbal medicine amomi fructus rotundus, with great pharmacology effect. The system position of A. compactum is not clear yet, and the introduction of this plant has been hindered by many plant diseases. However, the correlational molecular studies are relatively scarce.MethodsThe total chloroplast (cp) DNA was extracted according to previous studies, and then sequenced by 454 GS FLX Titanium platform. Sequence assembly was complished by Newbler. Genome annotation was preformed by CPGAVAS and tRNA-SCAN. Then, general characteristics of the A. compactum cp genome and genome comparsion with three Zingiberaceae species was analyzed by corresponding softwares. Additionally, phylogenetical trees were reconstructed, based on the shared protein-coding gene sequences among 15 plant taxa by maximum parsimony (MP) and maximum likelihood (ML) methods.ResultsThe A. compactum cp genome with a classic quadripartite structure, consisting of a pair of reverse complement repeat regions (IRa/IRb) of 29,824 bp, a large single copy (LSC, 88,535 bp) region as well as a small single copy (SSC, 15,370 bp) region, is 163,553 bp in total size. The total GC content of this cp genome is 36.0%. The A. compactum cp genome owns 135 functional genes, that 113 genes are unique, containing eighty protein-coding genes, twenty-nine tRNA (transfer RNA) genes and four rRNA (ribosomal RNA) genes. Codon usage of the A. compactum cp genome is biased toward codons ending with A/T. Total 58 SSR loci and 24 large repeats are detected in the A. compactum cp genome. Relative to three other Zingiberaceae cp genomes, the A. compactum cp genome exhibits an obvious expansion in the IR regions. In A. compactum cp genome, the ycf1 pseudogene is 2969 bp away from the IRa/SSC border, whereas in other Zingiberaceae species, it is only 4–5 bp away from the IRa/SSC border. Comparative cp genome sequences analysis of A. compactum with other Zingiberaceae reveals that the gene order and gene content differ slightly among Zingiberaceae species. The phylogenetic analysis based on 67 protein-coding gene sequences supports the phylogenetic position of A. compactum.ConclusionsThe study has identified unique features of the A. compactum cp genome which would be helpful for us to understand the cp genome evolution and offer useful information for phylogenetics and further studies of this traditional medicinal plant.

The complete mitochondrial genome of Thryssa hamiltonii (Engraulinae, Engranlidae, Clupeoidei) and phylogenetic studies of Clupeoidei

The complete mitochondrial genome of Thryssa hamiltonii has been determined. The whole sequence was 16,894 bp in length and included 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region (D-loop). The overall base composition is A 30.77%, C 27.97%, G 16.25%, T 25.01%, with a slightly A + T bias of 55.78%. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Three tandem repeat sequences were observed in the control region. Phylogenetic tree was constructed based on 13 protein-coding genes sequences of 21 clupeoidei species, and the result showed that T. hamiltonii is most closely related to T. dussumieri. These mitogenome sequence data will be useful for population genetics and phylogenetic analysis of the Clupeoidei.

The complete mitochondrial genome of Gadus chalcogramma and phylogenetic analysis

Here, we report the complete mitochondrial genome sequence of Alaska Pollock, Gadus chalcogramma. The genome sequence was obtained via long PCR reactions using universal primer sets for the fish mitochondrial genome. The total length of mitochondrial genome was 16,571 bp, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and one control region (D-loop). Except for ND6 and eight tRNA genes, all of the other mitochondrial genes were encoded on the heavy strand. The NJ tree of the combined 13 protein-coding gene sequences of Gadus chalcogramma possesses a relatively closer relationship with the Okhotsk Sea Pollock (Gadus chalcogrammus). Our complete mitochondrial genome will be valuable resources for phylogeny, genetics, and conservation of the Gadus chalcogramma in Korea.

The complete mitochondrial genome of Thryssa setirostris (Engraulinae, Engranlidae, Clupeoidei) and phylogenetic studies of Clupeoidei

The complete mitochondrial genome of Thryssa setirostris has been determined. The whole sequence was 16,926 bp in length and included 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A 29.88%, C 29.23%, G 16.77%, T 24.02%, with a slightly A + T bias of 54%. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Phylogenetic tree was constructed based on 13 protein-coding genes sequences of 21 clupeoidei species, and the result showed that T. setirostris is most closely related to T. dussumieri. These mitogenome sequence data will be useful for population genetics and phylogenetic analysis of the Clupeoidei.

The complete mitochondrial genome of Epinephelus Chlorostigma (Serranidae; Epinephelus) with phylogenetic consideration of Epinephelus

The complete mitochondrial genome of Epinephelus chlorostigma has been determined. It contains a typically conserved structure including 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region, and the whole sequence was 16,894 bp in length. The overall base composition is A 28.70%, C 27.99%, G 16.28%, T 27.03%. Except ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Phylogenetic tree was constructed based on 12 protein-coding genes sequences of 18 Epinephelus species, four species belong to Cephalopholis as outgroup, the result showed that E. chlorostigma is most closely related to Epinephelus areolatus. We sequenced the complete mitochondrial genome of E. chlorostigma to enrich the resource of molecular markers for examination of phylogenetic relationships in Epinephelus.

Light in the darkness: New perspective on lanternfish relationships and classification using genomic and morphological data

Massive parallel sequencing allows scientists to gather DNA sequences composed of millions of base pairs that can be combined into large datasets and analyzed to infer organismal relationships at a genome-wide scale in non-model organisms. Although the use of these large datasets is becoming more widespread, little to no work has been done in estimating phylogenetic relationships using UCEs in deep-sea fishes. Among deep-sea animals, the 257 species of lanternfishes (Myctophiformes) are among the most important open-ocean lineages, representing half of all mesopelagic vertebrate biomass. With this relative abundance, they are key members of the midwater food web where they feed on smaller invertebrates and fishes in addition to being a primary prey item for other open-ocean animals. Understanding the evolution and relationships of midwater organisms generally, and this dominant group of fishes in particular, is necessary for understanding and preserving the underexplored deep-sea ecosystem. Despite substantial congruence in the evolutionary relationships among deep-sea lanternfishes at higher classification levels in previous studies, the relationships among tribes, genera, and species within Myctophidae often conflict across phylogenetic studies or lack resolution and support. Herein we provide the first genome-scale phylogenetic analysis of lanternfishes, and we integrate these data from across the nuclear genome with additional protein-coding gene sequences and morphological data to further test evolutionary relationships among lanternfishes. Our phylogenetic hypotheses of relationships among lanternfishes are entirely congruent across a diversity of analyses that vary in methods, taxonomic sampling, and data analyzed. Within the Myctophiformes, the Neoscopelidae is inferred to be monophyletic and sister to a monophyletic Myctophidae. The current classification of lanternfishes is incongruent with our phylogenetic tree, so we recommend revisions that retain much of the traditional tribal structure and recognize five subfamilies instead of the traditional two subfamilies. The revised monophyletic taxonomy of myctophids includes the elevation of three former lampanyctine tribes to subfamilies. A restricted Lampanyctinae was recovered sister to Notolychninae. These two clades together were recovered as the sister group to the Gymnoscopelinae. Combined, these three subfamilies were recovered as the sister group to a clade composed of a monophyletic Diaphinae sister to the traditional Myctophinae. Our results corroborate recent multilocus molecular studies that infer a polyphyletic Myctophum in Myctophinae, and a para- or polyphyletic Lampanyctus and Nannobrachium within Lampanyctinae. We resurrect Dasyscopelus and Ctenoscopelus for the independent clades traditionally classified as species of Myctophum, and we place Nannobrachium into the synonymy of Lampanyctus.

The complete mitochondrial genome sequence and phylogenetic analysis of white-lipped deer (Cervus albirostris)

The white-lipped deer (Cervus albirostris) is a rare and endangered species endemic to the Qinghai–Tibet Plateau in China. In this study, we determined the complete mitochondrial genome sequence of the white-lipped deer. The total length of the mitogenome is 16473 bp. It has the typical structure of a vertebrate mitochondrial genome including 13 protein-coding genes(PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a control region (D-loop). tRNASer lacks the stem of the dihydrouridine (DHU) arm and fails to form a typical cloverleaf secondary structure. A phylogenetic tree based on the 13 PCG sequences of nine species closely related to white-lipped deer shows the classification status of the species within the family Cervidae. These results provide new molecular biology information to further understand the diversity of the white-lipped deer, which will help to protect this population.

The complete mitochondrial genome of the alvinocaridid shrimp Shinkaicaris leurokolos (Decapoda, Caridea): Insight into the mitochondrial genetic basis of deep-sea hydrothermal vent adaptation in the shrimp

Deep-sea hydrothermal vent is one of the most extreme environments on Earth with low oxygen and high levels of toxins. Decapod species from the family Alvinocarididae have colonized and successfully adapted to this extremely harsh environment. Mitochondria plays a vital role in oxygen usage and energy metabolism, thus it may be under selection in the adaptive evolution of the hydrothermal vent shrimps. In this study, the mitochondrial genome (mitogenome) of alvinocaridid shrimp Shinkaicaris leurokolos (Kikuchi & Hashimoto, 2000) was determined through Illumina sequencing. The mitogenome of S. leurokolos was 15,903bp in length, containing 13 protein-coding genes, 2 rRNAs, and 22 tRNAs. The gene order and orientation were identical to those of sequenced alvinocaridids. It has the longest concatenated sequences of protein-coding genes, tRNAs and shortest pooled rRNAs among the alvinocaridids. The control regions (CRs) of alvinocaridid were significantly longer (p<0.01) than those of the other caridaen. The alignment of the alvinocaridid CRs revealed two conserved sequence blocks (CSBs), and each of the CSBs included a noncanonical open reading frame (ORF), which may be involved in adjusting mitochondrial energy metabolism to adapt to the hydrothermal environment. Phylogenetic analysis supported that the deep-sea hydrothermal vent shrimps may have originated from those living in shallow area. Positive selection analysis reveals the evidence of adaptive change in the mitogenome of Alvinocarididae. Thirty potentially important adaptive residues were identified, which were located in atp6, cox1, cox3, cytb and nad1–5. This study explores the mitochondrial genetic basis of hydrothermal vent adaptation in alvinocaridid for the first time, and provides valuable clues regarding the adaptation.

Molecular phylogeny of Bradyrhizobium bacteria isolated from root nodules of tribe Genisteae plants growing in southeast Poland

The phylogeny of 16 isolates from root nodules of Genista germanica, Genista tinctoria, Cytisus ratisbonensis, and Cytisus scoparius growing in southeast Poland was estimated by comparative sequence analysis of core (16S rDNA, atpD, glnII, recA) and symbiosis-related (nodC, nodZ, nifH) genes. All the sequences analyzed placed the studied rhizobia in the genus Bradyrhizobium. Phylogenetic analysis of individual and concatenated housekeeping genes showed that the Genisteae microsymbionts form a homogeneous group with Bradyrhizobium japonicum strains. The phylogeny of nodulation and nitrogen fixation genes indicated a close relationship of the examined rhizobia with B. japonicum, Bradyrhizobium canariense, Bradyrhizobium cytisi, Bradyrhizobium rifense and Bradyrhizobium lupini strains infecting other plants of the tribe Genisteae. For the first time, the taxonomic position of G. germanica and C. ratisbonensis rhizobia, inferred from multigenic analysis, is described. The results of the phylogenetic analysis based on the protein-coding gene sequences presented in this study also indicate potential pitfalls concerning the choice of marker and reference strains, which may lead to conflicting conclusions in species delineation.