Interspecies Genetic Distance Research Articles

Using a DNA mini-barcode within the ITS region to identify toxic Amanita in mushroom poisoning cases

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning.Key points• Development of a DNA mini-barcoding method for Amanita species identification without fresh samples.• The ITS-a primer set was optimized for robust universality in Amanita samples.• The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.Graphical

Molecular Data and Karyotype Revealed Two Distinct Species of Domesticated Water Buffaloes in the Philippines

The six species of the genus Bubalus include the lowland and mountain anoa, Asian wild buffalo, tamaraw, swamp buffalo, and riverine buffalo. Few studies were conducted on the matrilineal phylogeny and genetic diversity of Bubalus species. In addition, there is controversy regarding the identity of the two domesticated water buffaloes in the Philippines – the introduced riverine and the native swamp buffalo. Thus, this study aimed to determine the genetic distance and phylogenetic relationship of Bubalus species using the mitochondrial cytochrome b (cytb) variation and to re-evaluate the taxonomic species designation of Bubalus bubalis. A total of499 Philippine cytb sequences from this study (n = 179), as well as other Asian countries (n = 319)and Syncerus caffer (n = 1), from NCBI were included in the analysis. The highest interspecies genetic distance (d) (0.040) was between the tamaraw and lowland anoa, whereas the lowest genetic distance (0.020) was between mountain and lowland anoa. Moreover, the phylogenetic tree indicated 52 haplotypes that delineated three groups, including lowland anoa, mountain anoa, and tamaraw with two domesticated buffaloes. The latter group further separated into three species – tamaraw, riverine, and swamp buffaloes. The analyses of genetic distance,phylogeny, and median-joining network structure based on cytb confirmed that the swamp buffaloes from Occidental Mindoro and South Cotabato, Philippines belong to maternal lineage B. Karyotype analysis consistently confirms that riverine buffaloes possess 50 chromosomes, whereas swamp buffaloes have 48 chromosomes, aligning with previous findings. Moreover, the swamp and riverine buffaloes appear to be distinct species, with the swamp buffalo having a closer affinity to the tamaraw than to the riverine buffalo. We, therefore, propose to revive the species assignment of the Bubalus kerabau Fitzinger, 1860 as a distinct species to the swamp buffalo. Furthermore, a common maternal ancestry was confirmed between the tamaraw and two domesticated buffaloes – the swamp and riverine. Research findings provided valuable information on understanding and monitoring the matrilineal genetic diversity of the genus Bubalus toward conservation and management.

New species of the genus Pseudocuneopsis Huang, Dai, Chen & Wu, 2022 (Bivalvia, Unionidae) from Guangxi Province, China.

A new species of freshwater mussel belonging to the genus Pseudocuneopsis, namely Pseudocuneopsisyangshuoensissp. nov., is diagnosed and described from Guangxi Province, China. This paper provides a detailed morphological description, photograph of the type specimen, and anatomical characteristics along with partial sequences of mitochondrial COI as DNA barcode data for this novel species. The new species can be distinguished from its congeners (Pseudocuneopsissichuanensis and Pseudocuneopsiscapitata) by shell shape, beak position and surface sculpture. The interspecies genetic distance based on the COI barcode between P.yangshuoensissp. nov. and P.sichuanensis is 8%, while it reaches 9% with P.capitata. Therefore, we provide robust morphological and molecular evidence to support the validity of this new species.

Mitochondrial DNA Corroborates the Genetic Variability of Clarias Catfishes (Siluriformes, Clariidae) from Cameroon.

The airbreathing walking catfish (Clariidae: Clarias) comprises 32 species that are endemic to African freshwater systems. The species-level identification of this group is challenging due to their complex taxonomy and polymorphism. Prior to this study, the biological and ecological studies were restricted to a single species, Clarias gariepinus, resulting in a biased view of their genetic diversity in African waters. Here, we generated the 63-mitochondrial Cytochrome c oxidase subunit 1 (COI) gene sequences of Clarias camerunensis and Clarias gariepinus from the Nyong River in Cameroon. Both C. camerunensis and C. gariepinus species maintained adequate intra-species (2.7% and 2.31%) and inter-species (6.9% to 16.8% and 11.4% to 15.1%) genetic distances with other Clarias congeners distributed in African and Asian/Southeast Asian drainages. The mtCOI sequences revealed 13 and 20 unique haplotypes of C. camerunensis and C. gariepinus, respectively. The TCS networks revealed distinct haplotypes of C. camerunensis and shared haplotypes of C. gariepinus in African waters. The multiple species delimitation approaches (ABGD and PTP) revealed a total of 20 and 22 molecular operational taxonomic units (MOTUs), respectively. Among the two Clarias species examined, we found more than one MOTU in C. camerunensis, which is consistent with population structure and tree topology results. The phylogeny generated through Bayesian Inference analysis clearly separated C. camerunensis and C. gariepinus from other Clarias species with high posterior probability supports. The present study elucidates the occurrence of possible cryptic diversity and allopatric speciation of C. camerunensis in African drainages. Further, the present study confirms the reduced genetic diversity of C. gariepinus across its native and introduced range, which might have been induced by unscientific aquaculture practices. The study recommends a similar approach to the same and related species from different river basins to illuminate the true diversity of Clarias species in Africa and other countries.

Trichotorquatus salinus sp. nov. (Oculatellaceae, Cyanobacteria) from a Saltern of Gomso, Republic of Korea

Six strains of subaerial cyanobacteria were isolated from a Gomso saltern in the Republic of Korea, all of which were studied using morphological and molecular traits. Trichotorquatus salinus sp. nov. was studied using a light microscope (LM), transmission electron microscope (TEM), 16S rRNA, 16S–23S ITS region, and ecological data. T. salinus is a thin and simple filament with a false branch and a firm collar sheath. The phylogenetic analyses of 16S rRNA revealed that Trichotorquatus formed a monophyletic lineage and the strains of T. salinus formed a distinct clade among the species in the genus Trichotorquatus. In the statistical analysis, the inter-species genetic distance of the five species of Trichotorquatus, including T. salinus, is shown to be greater than the distance of the previously reported species of Trichotorquatus. Additionally, 16S–23S ITS gene sequences between T. salinus and four species of Trichotorquatus showed dissimilarities of 55.3–59.4%. In the secondary structure of 16S–23S ITS region (type 2 operon), D1–D1′, Box-B, and V3 helix of T. salinus were different from the other taxa in the genus Trichotorquatus. These results demonstrate that T. salinus sp. nov. has unique morphological, ecological, and molecular traits. Therefore, we propose that T. salinus sp. nov. is a novel species belonging to the genus Trichotorquatus.

Molecular Identification of Brown Algae Sargassum sp. from the Lombok Coastal Waters

Sargassum is a well-known genus of brown algae in Indonesia that has long been investigated due to its economic importance. To support its biological research, it is important to identify and classify the species studied. Morphological identification had greatly contributed to taxonomy, however, it cannot distinguish Sargassum species accurately due to its plasticity. The current study aims to identify Lombok Sargassum molecularly using an ITS2 DNA barcode. Fresh algae were collected from Ekas Bay (EB) and Aan Cape (AC) in Lombok. DNA was first purified, and then its gene product was amplified using ITS2 primers. DNA sequences were examined and traced using the Basic Local Alignment Search Tool (BLAST). DNA sequences were processed in MEGA-X to reconstruct the phylogenetic tree and estimate the genetic distance. Three species were identified based on the BLAST results: Sargassum cf granuliferum, Sargassum polycystum, and Sargassum oligocystum. The base length obtained ranged from 521 to 637 bp, with a similarity percentage of 99.25 to 100%. The phylogenetic tree exhibited each recognized Sargassum species was clustered with the same species from the gene bank. Interspecies genetic distance was 0,000-0,0039, while amongst Sargassum species it's 0,0136-0,2395. The genetic distance between Sargassum and Ulva adherens (outgroup) was >1. Sargassum species found in Lombok were closely related to other similar species in the GenBank.

New species, redescriptions and new records of deep-sea brittle stars (Echinodermata: Ophiuroidea) from the South China Sea, an integrated morphological and molecular approach

Deep-sea ophiuroids were collected by the manned submersible ‘Shenhaiyongshi’ from the South China Sea at depths of 500–3550 m, in 2017 to 2020. A total of 18 species were identified, including three new species and eight new records, increasing the total number of species known from the South China Sea to 304. Most of the ophiuroids recorded from the South China Sea were found in shallow waters (77.9%) and a few of them occurred only in deep water (20.4%). Three new species are described as Ophiacantha aster sp. nov., Ophiomoeris petalis sp. nov. and Ophiopristis shenhaiyongshii sp. nov. We provide comprehensive descriptions of morphological features, including characteristics of the arm skeletons, and a phylogenetic analysis based on COI and 16S sequences. Overall intraspecific and interspecific genetic distance variations among the families found in this study were 0.5% to 2.47% and 1.16% to 44.16%, respectively, along the South Pacific region to the South China Sea. Our phylogenetic analysis suggested that COI partial genes resolved the interspecies complexity in the class Ophiuroidea better than 16S partial genes. The order Euryalida had low interspecies genetic distance variation within the class Ophiuroidea. The present study suggests a high probability that species of Asteroschema and Gorgonocephalus are more widely spread around the Indo-Pacific region than previously expected.

DNA Barcoding of Key Storage Pests Using Mitochondrial Cytochrome Oxidase I

Stored grain insect pests are of economic importance, and they spread rapidly through the grain trade. Hence, it is important to identify these pests accurately. Although several morphological methods exist, it is tedious and time-consuming. DNA barcoding using mitochondrial COI is an alternative approach that aids in precise species identification. In this study, 13 stored grain insect pests belonging to the order Coleoptera and Lepidoptera were collected from different storage structures in Coimbatore. A fragment of mt-COI was amplified and sequenced. Sequence analyses were carried out with BOLD (Barcode of Life Data System) and ABGD (Automatic Barcode Gap Discovery) tools. The barcoding gap analysis revealed that the inter-species genetic distance is greater than the intra-species genetic distance. The ABGD analysis for species delimitation partitioned the Coleopteran and Lepidopteran datasets into 9 and 4 putative species, respectively. The barcode gap was absent in more closely related species. However, analysis of their sequences revealed significant variations. Our results showed the ability of the mt-COI to discriminate between the species, thus provide a complementary technique for the diagnosis of stored grain insect pests.

DNA barcoding of deep-sea shrimp Sicyonia parajaponica Crosnier, 2003 (Crustacea: Decapoda: Sicyoniidae) from southwest coast of India.

The present work reports detailed taxonomic information on deep-sea shrimp Sicyonia parajaponica from southwest Indian waters. The samples were caught in bottom trawls conducted between the depths of 200 and 230 m from Sakthikulangara fishing harbour off Kollam, Kerala along the Arabian Sea during November 2019. DNA barcoding and a phylogenetic analysis was used to explore the relationship of the genus Sicyonia based on mitochondrial cytochrome c oxidase subunit 1 (COI: MN816389, MN816390) with the present specimen and sequences retrieved from NCBI GenBank. There is no difference in the intraspecies genetic distance (0%) while interspecies genetic distance (19.028.1%) revealed divergence between the species (COI) of the genus Sicyonia.

Molecular and Otolith Shape Analyses of Scorpaena spp. in the Turkish Seas

This study aims to investigate the discrimination and the phylogenetic relationship of five Scorpaena species (S. elongata, S. maderensis, S. notata, S. porcus, and S. scrofa), sampled from eight locations in the four seas (Mediterranean Sea, Aegean Sea, Black Sea, and Sea of Marmara) in Turkey, using both molecular and otolith shape analyses. Totally, 1360 samples were examined for otolith shape analyses, and 51 of them were also used in the molecular analysis (COI sequences, 652 bp). Eighteen haplotypes were determined in the 5 species of the genus Scorpaena; 3 haplotypes for S. elongata, S. maderensis and S. scrofa, 4 haplotypes for S. notata, and 5 haplotypes for S. porcus. The highest interspecific genetic distance was observed between S. maderensis and S. notata (0.1569), and the lowest interspecific genetic distance (0.0527) was observed between S. elongata and S. scrofa. The lowest interspecies genetic distance was found to be considerably higher than the highest intraspecies genetic distances for these five Scorpaena species. The barcode range in the study alone provided a high rate of barcoding success in identifying species belonging to the genus Scorpaena. A Principal Component Analysis (PCA) and Canonical Discriminant Analysis (CDA) were conducted for interspecific discrimination of the five Scorpaena species. The CDA produced an overall classification success rate of 96.3% with the highest rate for S. scrofa (98.8%), followed by S. maderensis (97.5%), S. notata 96.6%), S. elongata (94.6%), and with the lowest rate for S. porcus (94.4%). The results indicated that molecular analysis was generally compatible with otolith shape analysis and confirmed that otoliths have phylogenetic signals for Scorpaena species. This is the first study in which five Scorpaena species sampled from eight locations in four seas have been distinguished using both otolith shape and molecular analyses.

Identification and Genetic Relationship Analysis of <i>Torreya</i> Based on nrDNA ITS Sequence

In order to evaluate the identification ability of nrDNA ITS sequences on Torreya , this study used ITS sequence and four different analysis methods (BLAST, K2P genetic distance, SNP analysis, NJ tree) to identify species of Torreya , and established phylogenetic trees to discuss their phylogenetic relationships. The results showed that the length of 48 ITS sequences was 1 095 bp~1 105 bp, the average intraspecies genetic distance was 0.001 6 and the average interspecies genetic distance was 0.015 0. At the species level, BLAST alignment and NJ tree had the highest efficiency in Torreya . The SNP loci analysis could effectively identify T. grandis cv . ‘ Merrillii ’ and T. grandis . The analysis of genetic relationship showed that all the other species of Torreya were single-line branches, but the quince tree of T. yunnanensis and T. fargesii were clustered into a cluster in the ML tree, indicating that the two were closely related. This provided evidence for the nuclear genome sequence proposed the incorporation of T. yunnanensis and T. fargesii . This study showed that ITS sequence could be used as a DNA barcode for the identification of Torreya , and providing reference for the identification and systematic relationship of Torreya.

DNA barcoding of 4 species of cheyletid mites based on COI and 18S rRNA gene sequences

To analyze the sequences of mitochondrial cytochrome C oxidase subunit I gene (COI) and 18S ribosomal RNA gene (18S rRNA), so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites. Cheyletid mite samples were collected from small-scale flour mills in Fuyang, Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019, extracted and morphologically identified. Then, genomic DNA was extracted from a single cheyletid mite, and the COI and 18S rRNA gene sequences were obtained by PCR amplification, cloning and sequencing. The obtained sequences were aligned using the BLAST software. Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites. The genetic distance was calculated using the software MEGA X, and the phylogenetic tree was created using the maximum likelihood method. The DNA barcoding results of Cheyletus malaccensis, C. carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification, while no sequences pertaining to Eucheyletia reticulate were retrieved in the GenBank database. The proportions of A + T were 69.6% and 55.1% in the COI and 18S rRNA sequences of 4 cheyletid mites species, respectively, and the numbers of base substitutions were 137 and 46, respectively. There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, respectively, and the intra-species genetic distance was all 0.020 or less in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114, respectively. Phylogenetic analysis based on COI and 18S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100% supportive rate, which was consistent with the morphological identification. Mitochondrial COI gene is superior to 18S rRNA gene as DNA barcodes for 4 species of cheyletid mites, which is more suitable to be used to investigate the phylogenetic relationship of at genus and species levels.

<p><strong>Morphology and molecular description of <em>Wilmottia koreana</em> <em>sp. nov.</em> (Oscillatoriales, Cyanobacteria) isolated from the Republic of Korea</strong></p>

Two types of Wilmottia (Cyanobacteria) were collected from the tree-bark of a tree on Mt. Gwanggyo in Suwon City, Gyeonggi-do and a rock wall of Haje Port, Geum River in Jeollabuk-do, Republic of Korea. Morphological observations using light microscopy (LM) and transmission electron microscopy (TEM) revealed that one species was Wilmottia murrayi (Coleofasciculaceae, Oscillatoriales) and the other had similar morphology to W. murrayi, with considerable genetic variations, and thus, it was described as a new species named Wilmottia koreana. Phylogenetic analyses of the 16S rRNA revealed that the family Coleofasciculaceae was clearly separated into taxonomic groups of each genus, and in them, Wilmottia forms a monophyletic lineage. W. koreana diverged the earliest, forming a unique clade separate from the others. Statistical analyses showed that the inter-species genetic distances of Wilmottia were significantly different from the intra-species distances (t-test, P < 0.001). Additionally, the secondary structures of D1–D1’, Box-B, and the V3 helices were different among the Wilmottia species. These results demonstrated that the proposed new Wilmottia species was unique in molecular traits. Therefore, we propose this to be a new species belonging to the genus Wilmottia with the name Wilmottia koreana sp. nov.

Morphological identification and molecular validation of anchovies (Engraulidae) in the Persian Gulf and Oman Sea.

Validation of species using independent lines of evidence is sometimes desirable when their identification using only one approach is difficult or questionable. The identification of anchovies (Engraulidae) are often challenging based on morphology because closely related species exhibit only slight morphological differentiation. This study utilized morphological characteristics and DNA barcodes for identification and validation of anchovies in the Persian Gulf and Oman Sea. Based on morphology, we identified eight species: Thryssa hamiltonii, T. setirostris, T. vitrirostris, T. whiteheadi, T. dussumieri, Encrasicholina punctifer, E. pseudoheteroloba and Stolephorus indicus. A 658 bp region of mitochondrial cytochrome oxidase subunit I (COI) was generated for 53 specimens from these eight species. From these sequences, we built a Maximum Likelihood phylogenetic tree. In this tree, each species forms a monophyletic group confirming our initial morphological identification. In addition, we provided (and registered in GenBank) the first barcode sequences for T. whiteheadi, an endemic species of this region. Interspecies genetic distances were comprised between 0.168 to 0.275. The largest genetic distance was found between T. vitrirostris and S. indicus and the smallest between T. dussumieri and T. whiteheadi. This study successfully identified eight species of anchovies in the Persian Gulf and Oman Sea based on both morphological and molecular characters.

Species identification of zooplankton resting eggs based on DNA barcode technology: A case study of Lake Liuye (Changde), Lake Dongting Basin

以洞庭湖流域的典型城市湖泊常德柳叶湖表层沉积物中浮游动物休眠卵为研究对象，采用DNA条形码技术进行种类鉴定，从191个休眠卵中成功获得101条有效序列，鉴定成功率约为53%.根据NCBI数据库比对成功鉴定休眠卵9科12属11种，另有6个样品鉴定到属或科；3个类群的种间遗传距离平均为种内遗传距离的68倍，表明可以利用线粒体细胞色素c氧化酶第一亚基编码基因（COⅠ）对休眠卵进行有效物种鉴定.通过Neighbor-Joining树进行系统发育分析，发现所鉴定的物种与其参比序列聚类为一支，所有物种分别聚为独立的一个支系，不同物种可有效区分.研究结果均表明，COⅠ基因作为DNA条形码可以实现沉积物中浮游动物休眠卵的物种鉴定.;With the rapid development of modern molecular biology tools, especially DNA barcoding technology, a rapid, accurate and efficient identification method for species of zooplankton resting eggs is possible. In this study, zooplankton resting eggs, including copepods,cladocerans and rotifers in the surface sediments of Lake Liuye (Changde), a typical urban lake in Lake Dongting Basin, were studied. Using DNA barcode technology for species identification, 101 valid DNA sequences were successfully obtained from 191 resting eggs, and the identification success rate was 53%. According to the NCBI database comparison, 9 families, 12 genera and 11 species of resting eggs were successfully identified, and 6 samples were only identified to the genus or family level. The inter-species genetic distance of the three taxa was 68 times that of the intra-species genetic distance, indicating that the cytochrome oxidase Ⅰ (COⅠ) gene could be used for effective species identification of zooplankton resting eggs. Through the phylogenetic analysis by Neighbour-Joining tree, it was found that the identified species and their reference sequences were clustered into one branch, and all different species were clustered into independent branches respective. Therefore, different species could be effectively distinguished. The above results all indicate that the mitochondrial COⅠ gene, as a DNA barcode, can be used to identify the species of zooplankton resting eggs in sediment of lakes.

First record of the dotted grouper Epinephelusepistictus (Temminck & Schlegel, 1843) (Perciformes, Serranidae) in Malaysia.

Five specimens of Epinephelusepistictus (Temminck & Schlegel, 1843) were collected from a major landing site located on the west coast of Peninsula Malaysia during a fish faunal survey on 23 August 2017. The present study extends the distribution range of E.epistictus southwards from Andaman Sea to the Strait of Malacca. Species identification was confirmed by colour pattern and DNA barcoding (567 bp of cytochrome C oxidase I) of all E.epistictus specimens and nine closely related Epinephelus species. The interspecies genetic distance ranged from 0.002–0.245. This study also presents, for the first time for Malaysia, data on length-weight relationships and otolith measurements. It contributes to a better understanding of taxonomy, and phylogenetic and genetic diversity of E.epistictus.

DNA barcoding inferred maternal philopatric affinity of ocean maskray (Neotrygon indica) in the Bay of Bengal

The Indian Ocean maskray, Neotrygon indica, is a recently described species from the Bay of Bengal in the eastern coast of India. The conspicuous morphology and less genetic information of this group are often confusing for their identity and phylogeography. In this study, we have collected the specimens from Odisha coast in eastern India and generated the DNA barcode data for comparative analysis with publicly available barcodes of N. indica and other congeners. The morphological characters are similar to the previous record of N. indica. However, the generated barcode data of N. indica showed 2.2–8.0% inter-species genetic distances with the other six species. The studied population of N. indica showed 0.2–1.5% genetic distances with a different known population (Tamil Nadu, Andhra Pradesh, and Bangladesh) in the Bay of Bengal. The BA phylogeny revealed monophyletic clustering of all the studied species. Henceforth, the shallow genetic divergences within the different population of N. indica depicted possible female precise natal philopatric affinity within their range distribution. To corroborate this unique phylogeographic structure, this study evoked more rigorous sampling and detailing with other molecular markers. This study also exhibits the expand range distribution of N. indica beyond its type locality and furnishes a new state record for Odisha. Our data would be a valuable resource for further studies on genetic diversity of N. indica and related species, which will provide new insights for better conservation plans.

Genetic diversity of Northern snakehead (Channa argus) based on complete mitochondrial COI gene sequences

White type Channa argus, which has the common name of the great northern snakehead, has an uncertain classification for many years. In the present study, mitochondrial COI gene has been used to analyze the relationships and genetic diversity of nine kinds of Channa. For the Chnannidae family, the average of the inter-species genetic distance was 0.156 (ranged: 0.000–0.245). And the intra-species genetic distances were ranged from 0.000 to 0.002. For the haplotypes of two-colour morphs of Channa argus, the mean of the pair-wise genetic distances was estimated as 0.0009. This value was within the intra-species genetic distance interval, indicating that two-colour morphs of Channa argus are not subspecies of white type at the molecular level. Moreover, take Lateolabrax maculates and Epinephelus coioides as outgroups, molecular phylogenetic tree showed that all the haploids gather together as a branch and crossing each other. These indicated that the white type Channa argus should be regarded as an albino of bio colour type.

Geometric vs. traditional morphometric methods for exploring morphological variation of tadpoles at early developmental stages

Abstract We conducted a comparative (2D landmark-based geometric and traditional) morphometric analysis on tadpoles at early developmental stages. Two species of brown frog (Rana dalmatina and R. temporaria) and the common toad (Bufo bufo) were involved, all raised in the laboratory from fertilized eggs collected in their natural habitat. Taxonomic identification was confirmed by the DNA barcoding method with the 16S rRNA sequence as the gene marker. Interested to compare the methodologies for quantification and description of morphological differences among tadpoles of mentioned species, we aimed to: 1) calculate interspecies genetic distances as the most relevant measurement for species differentiation, 2) determine and describe size and shape variation, 3) identify relationships among the analyzed species at the morphological level and 4) assess their classification accuracy. Within the framework of the specified aims, both methodologies produced very similar results, i.e., the smallest divergence was between R. dalmatina and R. temporaria, while the most discriminative were B. bufo and R. temporaria. However, we observed subtle shape variation of the distal region of the tail that was detected only by the geometric morphometrics. Our findings support the following. Geometric morphometric method captures more subtle shape differences that were unable to be recovered from linear measurements. It performs slightly better in classification rate. Although it was not quantified, it stands to reason that there is no difference in time investment between the two approaches. Geometric morphometrics provides more information that can be leveraged to answer further questions and it has a clear advantage in visualizing.

Neoprotoparmelia gen. nov. and Maronina (Lecanorales, Protoparmelioideae): species description and generic delimitation using DNA barcodes and phenotypical characters.

Multilocus phylogenetic studies revealed a high level of cryptic diversity within the lichen-forming fungal genus Maronina (Protoparmelioideae, Parmeliaceae). Coalescent-based species delimitation suggested that most of the cryptic molecular lineages warranted recognition as separate species. Here we study the morphology and chemistry of these taxa and formally describe eight new species based on phenotypical and molecular characters. Further, we evaluate the use of ITS rDNA as a DNA barcode for identifying species in this genus. For the first time, we obtained an ITS sequence of Maroninaaustraliensis, the type species of the genus and showed that it is phylogenetically not closely related to species currently placed in Maronina or Protoparmelia. We assembled a dataset of 66 ITS sequences to assess the interspecies genetic distances amongst the twelve Maronina species using ITS as DNA barcode. We found that Maronina and Protoparmelia form a supported monophyletic group whereas M.australiensis is sister to both. We therefore propose a new genus Neoprotoparmelia to accommodate the tropical-subtropical species within Protoparmelioideae, with Neoprotoparmeliacorallifera as the type, N.amerisidiata, N.australisidiata, N.brasilisidiata, N.capensis, N.crassa, N.pauli, N.plurisporibadia and N.siamisidiata as new species and N.capitata, N.isidiata, N.multifera, N.orientalis and N.pulchra as new proposed combinations. We provide a key to Neoprotoparmelia and confirm the use of ITS for accurately identifying species in this group.