Average Interspecific Distance Research Articles

Challenges in using DNA barcodes for authentication of Sida species

The Indian system of medicine’s “Ayurvedic pharmacopeia of India (API)” recommends the use of Sida cordifolia Linn (root), Sida cordata (Burm.f.) Borss.Waalk. (aerial part), Sida rhombifolia Linn. (root) and Abutilon indicum (Linn.) Sw. (root) in drug preparations of Bala, Nagabala, Mahabala and Atibala respectively. Moreover many Sida sp. are being used in China, South East Asia, Africa and South America in their traditional healthcare systems. It is a taxonomically complex genus often difficult to authenticate from dried/chopped herbal market samples. Many Sida sequences from the NCBI database, including published reports, were highly suspect and were redesignated into species groups during phylogenetic clustering. Among the four loci studied, ITS2 region was identified as the best for the Sida species identification followed by trnH-psbA. The trnH-psbA phylogeny however fails to differentiate between (1) S. beddomei and S. cordata, (2) S. alnifolia and S. scabrida, (3) S. cordifolia and S. fryxellii that formed monophyletic clusters. The average evolutionary divergence over Sequence Pairs within each species group for ITS2 locus ranged from 0.000 to 0.009 (Average=0.0021), while average Interspecific distance between species was 0.1175 making them ideal for authentication of Sida species. The matK and rbcL is recommended as a back-up loci for identifying intergeneric adulterants in case, the ITS2 or trnH-psbA amplification fails. The present study identified two market samples as adulterant species; (1) S. alnifolia and (2) a mixture of S. acuta and S. alnifolia/S.scabrida. The study provides a roadmap for Ayurvedic/herbal industry to utilize DNA barcoding for authentication of Sida species. At the same time the presence of “Unknown Sida group” highlights the need for further research to accurately classify and identify all Sida species at the phylogenetic level, utilizing the DNA barcode sequences to thoroughly understand the diversity and evolution of the Sida genus.

Smittia solominae sp. nov. (Diptera: Chironomidae: Orthocladiinae), living on ice of high mountain glaciers of the Elbrus Region (North Caucasus).

Illustrated morphological description with a study of DNA barcoding and biology of adult male, pupa and larva of Smittia solominae sp. nov., living on the ice surface of glaciers at an altitude of about 3000 m above sea level in the Elbrus region of the North Caucasus is given. DNA barcoding provided support that the new species unique within genus Smittia. The average interspecific distances between S. solominae sp. nov. and other Smittia from BOLD above 12% that correspond to species level.

DNA barcoding of the genus Alburnoides Jeitteles, 1861 (Actinopterygii, Cyprinidae) from Anatolia, Turkey

The present study investigated the ability of DNA barcoding to reliably identify the endemic freshwater species in Turkey, known as biodiversity hotspots. The barcode region (652 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) was used to barcode 153 individuals from 13 morphologically identified species of the genus Alburnoides. Based on the Kimura two-parameter (K2P) evolution model, the average interspecific distance (0.0595) was 31-fold higher than the average intraspecific distance (0.0019). There was a clear-cut barcode gap (0.0158–0.0187) between maximum intraspecific distance (A. tzanevi and A. velioglui) and minimum nearest-neighbour distance (A. freyhofi and A. kurui) for Anatolian Alburnoides species and a common genetic threshold of 0.0158 sequence divergence was defined for species delimitation. The multiple species delimitation methods (ABGD, ASAP, GMYC and bPTP) revealed a total of 11 molecular operational taxonomic units (MOTUs) for 13 morphospecies. Neighbour-joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI) tree analysis indicated that all haplotypes were clustered into two major clades, which corresponded to eleven Alburnoides species clusters, with strong bootstrap support. Furthermore, all the specimens clustered in concurrence with the morpho-taxonomic status of the species, except for two species (A. coskuncelebii and A. emineae) that were morphologically differentiated, but showed overlap in variation for COI-based DNA barcode data with other species. Overall, present results identified that COI-based DNA barcoding is effective for species identification and cataloguing of genus Alburnoides in Turkey.

DNA Barcoding of Fish Species Diversity in Guizhou, China

Guizhou is an important ecological barrier in the upper reaches of the Yangtze River and the Pearl River basins with abundant fish species. However, fish from these regions are threatened by anthropogenic activities, including overfishing and habitat destruction. Here, we assessed the fish diversity including more than half of the species from the region using DNA barcoding (partial sequence of cytochrome c oxidase subunit I (COI) gene). We obtained 800 mitochondrial COI barcode sequences from 82 genera, 18 families and 8 orders of fishes. The average Kimura two-parameter (K2P) distances within species and genera were 0.35% and 5.44%, respectively. The average interspecific distance was 15.54 times higher than the mean intraspecific distance. Moreover, DNA barcodes revealed 175 operational taxonomic units (OTUs) based on consensus demarcation schemes. Barcoding gaps were detected in 94.81% of morphospecies. Three fish species (Schistura fasciolata, Vanmanenia pingchowensis, and Misgurnus dabryanus) have considerable intraspecific variability, and each was divided into multiple molecular operational taxonomic units (MOTUs) using molecular definition methods (Automatic Barcode Gap Discovery, Refined Single Linkage, General Mixed Yule Coalescent, and Poisson Tree Processes), possibly indicating the occurrence of cryptic species. Altogether, our study reveals the complex diversity of fish species in Guizhou Province, serving as a reference for the conservation and monitoring of fish species in this region.

DNA Barcoding Revealing the Parrotfish (Perciformes: Scaridae) Diversity of the Coral Reef Ecosystem of the South China Sea

Parrotfish are an important group in the coral reef ecosystem that maintain ecological stability and have a close relationship with coral reefs. Around the world, parrotfish and coral reefs are being destroyed due to human activities and global climate change. Here, we investigated the diversity of parrotfish in the South China Sea (SCS) using DNA barcoding, and initially established a DNA barcode database of the SCS parrotfish. A total of 1620 parrotfish specimens, belonging to 23 species of 6 genera in the family Scaridae, were collected in the SCS, accounting for 64% of the parrotfish species in the SCS. Genetic distance analysis at each taxonomic level showed that the average genetic distance was 0.23% within species, 8.52% within genus and 13.89% within family. The average inter-specific distance was 37.04-fold the intra-specific distance. Barcode gap analysis showed that 6 of the 21 parrotfish species with multiple samples had no barcode gap, resulting in an overall identification success rate of 74%. The ABGD analysis revealed that there could be 37 potential operational taxonomic units (OTUs) and the BIN analysis showed 32 identifiable taxonomic units, which by far exceed the number of morphologically previously known species. Overall, this study complements the lack of parrotfish DNA barcode sequences, and our findings provide an important stepping-stone to further study the diversity of parrotfish in South China.

Molecular diversity of Uzbekistan\u2019s fishes assessed with DNA barcoding

Uzbekistan is one of two doubly landlocked countries in the world, where all rivers are endorheic basins. Although fish diversity is relatively poor in Uzbekistan, the fish fauna of the region has not yet been fully studied. The aim of this study was to establish a reliable barcoding reference database for fish in Uzbekistan. A total of 666 specimens, belonging to 59 species within 39 genera, 17 families, and 9 orders, were subjected to polymerase chain reaction amplification in the barcode region and sequenced. The length of the 666 barcodes was 682 bp. The average K2P distances within species, genera, and families were 0.22%, 6.33%, and 16.46%, respectively. The average interspecific distance was approximately 28.8 times higher than the mean intraspecific distance. The Barcode Index Number (BIN) discordance report showed that 666 specimens represented 55 BINs, of which five were singletons, 45 were taxonomically concordant, and five were taxonomically discordant. The barcode gap analysis demonstrated that 89.3% of the fish species examined could be discriminated by DNA barcoding. These results provide new insights into fish diversity in the inland waters of Uzbekistan and can provide a basis for the development of further studies on fish fauna.

A new, undescribed species of Melanocharis berrypecker from western New Guinea and the evolutionary history of the family Melanocharitidae

Western New Guinea remains one of the last biologically underexplored regions of the world, and much remains to be learned regarding the diversity and evolutionary history of its fauna and flora. During a recent ornithological expedition to the Kumawa Mountains in West Papua, we encountered an undescribed species of Melanocharis berrypecker (Melanocharitidae) in cloud forest at an elevation of 1200 m asl. Its main characteristics are iridescent blue‐black upperparts, satin‐white underparts washed lemon yellow, and white outer edges to the external rectrices. Initially thought to represent a close relative of the Mid‐mountain Berrypecker Melanocharis longicauda based on elevation and plumage colour traits, a complete phylogenetic analysis of the genus based on full mitogenomes and genome‐wide nuclear data revealed that the new species, which we name Satin Berrypecker Melanocharis citreola sp. nov., is in fact sister to the phenotypically dissimilar Streaked Berrypecker Melanocharis striativentris. Phylogenetic relationships within the family Melanocharitidae, including all presently recognized genera (Toxorhamphus, Oedistoma, Rhamphocharis and Melanocharis), reveal that this family endemic to the island of New Guinea diversified during the main uplift of New Guinea in the Middle and Late Miocene (14.6 Mya), and represents an evolutionary radiation with high disparity in bill morphology and signalling traits across species. Rhamphocharis berrypeckers fall within the Melanocharis clade despite their larger beaks and should be included in the latter genus. Interspecific genetic distances in Melanocharis are pronounced (average interspecific distance: 8.8% in COI, 12.4% in ND2), suggesting a long history of independent evolution of all lineages corresponding to currently recognized species, including the Satin Berrypecker, which shares a most recent common ancestor with its sister species in the early Pleistocene (˜ 2.0 Mya).

DNA Barcoding of Chinese species of the genus Eurydema Laporte, 1833 (Hemiptera: Pentatomidae)

The phenotype, as a significant identification character for the bug species Eurydema Laporte, 1833, possesses many variation patterns, making this species difficult to determine. In this context, DNA barcoding provides an efficient method for species identification. In our study, we used distance-based and tree-based methods to assess the effectiveness of COI gene as an additional taxonomic method. We examined COI sequences of 203 specimens collected in China. Intra- and interspecific genetic distances were calculated, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method. The program TaxonDNA was used to calculate the distribution of distances and investigated the barcode gap. DNA barcodes revealed 100% (ABGD), 99.5% (TaxonDNA) and 97.5% (Barcode Index Numbers) successful identification rates. In the NJ tree, all taxonomic species showed monophyletic clusters that were separated from each other by large genetic distances (> 4.3%). Low divergence (< 0.6%) and non-monophyly clusters that included different phenotype variations were found in E. dominulus and E. oleracea. Compared with other Eurydema species, E. sp. had an average interspecific distance of 7.4% that was congeneric distance, and formed a distinct cluster among Eurydema tree clades. We also confirmed a new combination of Eurydema qinlingensis (Zheng, 1982) comb. nov. based on DNA barcoding and morphology data. Overall, our data showed that, despite the little barcode overlap between intra- and interspecific genetic distances, DNA barcoding is a useful and effective method for enhancing morphological data and resolving taxonomic problems.

<b>DNA barcode regions for differentiating <i>Cattleya walkeriana</i> and <i>C. loddigesii</i>

Growers appreciate Cattleya walkeriana and C. loddigesii due to striking shape and rarity. Thus, this study aimed to evaluate the feasibility of DNA barcode regions, namely ITS1, ITS2 and rpoC1, to discriminate between C. walkeriana and C. loddigesii species. DNA barcode regions were successfully amplified using primers designed to amplify plants. We also included sequences from public databases in order to test if these regions were able to discriminate C. walkeriana and C. loddigesii from other Cattleya species. These regions, and their combinations, demonstrated that the ITS1+ITS2 had the highest average interspecific distance (11.1%), followed by rpoC1 (1.06%). For species discrimination, ITS1+ITS2 provided the best results. The combined data set of ITS1+ITS2+rpoC1 also discriminated both species, but did not result in higher rates of discrimination. These results indicate that ITS region is the best option for molecular identification of these two species and from some other species of this genus.

Molecular identification of the genus Thryssa based on DNA barcoding.

DNA barcoding is an effective method for identifying species by analyzing one or a few short standardized DNA sequences. In this study, we examined the utility of mitochondrial cytochrome oxidase subunit I (COI) sequences as a DNA barcode for the identification of six species belonging to the genus Thryssa: T. dussumieri, T. hamiltonii, T. kammalensis, T. mystax, T. setirostris, and T. vitrirostris. We obtained an intraspecific distance of 0.000 for T. vitrirostris and T. hamiltonii, 0.006 for T. mystax, 0.002 for T. dussumieri, and 0.005 for T. kammalensis. The average intraspecific distance was 0.002, while the average interspecific distance was 0.137. Thus, the interspecific genetic distance was approximately 67-fold larger than the intraspecific genetic distance; the average genetic distance among species was greater than the minimum of 0.020 between species suggested elsewhere. The genetic distance between T. vitrirostris and T. mystax was 0.003. A maximum-likelihood phylogenetic tree constructed using best-fitting tree topology showed distinct clusters corresponding to the species (except for T. vitrirostris and T. mystax). The closest relationship was found between T. vitrirostris and T. mystax. These two species clustered together in the phylogenetic tree. This conclusion contradicts the evolutionary relationship based on morphological classification.

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest.

DNA Barcoding of Fish, Insects, and Shellfish in Korea

DNA barcoding has been widely used in species identification and biodiversity research. A short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence serves as a DNA bio-barcode. We collected DNA barcodes, based on COI sequences from 156 species (529 sequences) of fish, insects, and shellfish. We present results on phylogenetic relationships to assess biodiversity the in the Korean peninsula. Average GC% contents of the 68 fish species (46.9%), the 59 shellfish species (38.0%), and the 29 insect species (33.2%) are reported. Using the Kimura 2 parameter in all possible pairwise comparisons, the average interspecific distances were compared with the average intraspecific distances in fish (3.22 vs. 0.41), insects (2.06 vs. 0.25), and shellfish (3.58 vs. 0.14). Our results confirm that distance-based DNA barcoding provides sufficient information to identify and delineate fish, insect, and shellfish species by means of all possible pairwise comparisons. These results also confirm that the development of an effective molecular barcode identification system is possible. All DNA barcode sequences collected from our study will be useful for the interpretation of species-level identification and community-level patterns in fish, insects, and shellfish in Korea, although at the species level, the rate of correct identification in a diversified environment might be low.