To assess the accuracy of species delimitation in the genus Aphaenomurus Yosii, we conducted a comparative micro-morphological study and molecular analysis using two mitochondrial (COI, 16S) and two nuclear genes (18S, 28S) on 118 specimens from 24 localities. The results showed that the morphological characters of A. vicinus and A. interpositus, as presented in the original description, were intermixed in phylogenetic lineages within the genus and did not form independent lineages. Furthermore, there were enough differences among Aphaenomurus individuals to be considered as morphologically distinct species (Th. III is 1+1 or 2+2, Abd. I is 2+2 or 3+3, Abd. II is 2+2 or 3+3), but they do not form an independent lineage. Molecular and morphological analyses have determined that A. vicinus and A. interpositus are the same species, exhibiting morphological variation in dental spines formula, claws, chaetotaxy, and other features. Additionally, the genus Aphaenomurus forms a monophyletic clade, which is further divided into several lineages within the genus. No morphological differences were observed to distinguish these lineages. This cladistic divergence is attributed to heteroplasmy, which is supported by previous studies that have suggested the possibility and problems of heteroplasmy in Collembola, and by the high genetic distances between individuals in the mitochondrial genes of Aphaenomurus.

Food and agricultural production sector, especially livestock production is vital for Mongolia’s economic and social development. Domestic sheep play key roles for Mongolians, providing food (meat, milk) and raw materials (wool, sheepskin), but genetic diversity, origin of sheep populations in Mongolia have not been well studied. Studies of population genetic diversity is important research field in conservation and restoration of animal breeds and genetic resources. Therefore, this study aimed to investigate genetic characteristics and estimate origin through the analysis of mitochondrial DNA control region D-loop and Cytochrome b of Mongolian indigenous sheep (Mongolian native, Orkhon and Altanbulag) and one Europe sheep (Suffolk). As a result of there were found, 220 SNPs (Single nucleotide polymorphism) in the D-loop region, 28 SNPs in the Cytochrome B region, furthermore, 77 Haplotypes. The nucleotide diversity was only found in D-loop region (n = 0.0184). Phylogenetic analysis showed that 3 (A, B, and C) of 5 haplogroups of sheep have been identified in our research. Haplogroup C was only found in Mongolian indigenous sheep. Haplogroup D and E were not observed. As a result of haplogroups, haplogroup A was dominant (n = 46 of 94 sheeps), followed by haplogroup B (n = 36) and haplogroup C (n = 12). Sequence analysis showed that T deletion, insertion and heteroplasmy in D-loop region occurred at a high rate in Mongolian indigenous sheep population (T insertion = 47, T deletion = 83). The heteroplasmy, which has never been found in Mongolian sheep, has been newly discovered in this study. As a result, the Mongolian sheep varieties, which mainly derived from Asia, were in hybridization with European sheep varieties.

A partial sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene is widely used as a molecular marker for species identification in animals, also termed a DNA barcode. However, the presence of more than one sequence type in a single individual, also known as heteroplasmy, is one of the shortcomings of barcode identification. In this study, we examined the extent and divergence of COI heteroplasmy, including nuclear-encoded mitochondrial pseudogenes (NUMTs), at the genomic-DNA level from 13 insect species, including four individuals of orthopteran Anapodisma miramae. Furthermore, a long fragment of mitochondrial DNA (~13.5 kb) and cDNA from A. miramae were used as a template for COI PCR to compare the patterns of heteroplasmy between DNA sources and to investigate a possible way to avoid ambiguity in DNA barcoding. When multiple numbers of clones originated from genomic DNA were sequenced, heteroplasmy was prevalent in all species (3~16 heteroplasmic copies), with a varying degree of maximum sequence divergence (<1% in 7 species, <4% in 3 species, <6% in 2 species and 2.15-8.03% in four A. miramae individuals). In five species, NUMTs also were observed when genomic DNA was used as a template. Long fragment DNA also is a source of heteroplasmic amplification, but the divergent haplotypes and NUMTs obtained in the genomic DNA-based PCR were not detected in A. miramae. On the other hand, cDNA was heteroplasmy-free, without NUMTs when multiple numbers of clones were sequenced. Consistently, one dominant haplotype was always obtained from the genomic DNA-origin clones in all species and also from the long fragment- and cDNA-origin clones of A. miramae. Furthermore, the dominant haplotype was identical in sequence, regardless of the DNA source. Thus, one possible solution to avoid the barcoding problem in relationship to heteroplasmy could be the acquisition of multiple numbers of barcoding sequences to determine a dominant haplotype that can be assigned as barcoding sequence for a given species.