Mitochondrial 16S rRNA Gene Sequences Research Articles

Assessment of Fish Species in Wanlv Lake, the Largest Drinking Water Source in South China, by Environmental DNA Metabarcoding Technology

As the largest lake in South China, Wanlv Lake is also an important drinking water source for Guangdong and Hong Kong and is responsible for water supplying of more than 40 million people in the Guangdong-Hong Kong-Macao Greater Bay Area. The study of fish diversity in Wanlv Lake can help us to effectively understand the changes and states of its aquatic ecosystem and provide scientific basis for natural ecological protection, biodiversity, sustainable use and scientific management of fishery resources. However, research on the lake’s fish is scarce. The high throughput environmental DNA metabarcoding technology (eDNA technology) of the mitochondrial 12S rRNA gene sequence was herein used to conduct a preliminary exploration of Wanlv Lake fish diversity. A total of 10 sampling sites were set up to monitor fish diversity and analyze the composition and richness of the species. The results revealed a total of 83 genera in 42 families and 17 orders of fish in the lake, with the highest proportion found in Cypriniformes (38 species), accounting for 45.24% of the total. Five alien species and eight rare and endangered species were also detected. The proportion of invasive species was 5.95%, and the combined proportion of rare and endangered species was 9.52%. The results indicated that Wanlv Lake serves as an important drinking water source, and overall, the condition of fish is satisfactory. However, proactive measures should be implemented to control the overpopulation of invasive species, as their unchecked proliferation may lead to a decline in species abundance, particularly among endangered species. This was the first overall assessment report on fish of the Wanlv Lake via 12S rRNA; the results herein lay a foundation for water quality assessment of Wanlv Lake as a drinking water source.

Clarification of the identity of deep-sea crab Tymolus uncifer (Ortmann, 1892) and validation of Tymolus melanommus (Doflein, 1904) (Decapoda: Brachyura: Cyclodorippidae).

Examination of material from Japanese waters has disclosed that two species have been confounded under Tymolus uncifer (Ortmann, 1892) (Decapoda: Brachyura: Cyclodorippidae) in previous literature. The identity of Tymolus uncifer sensu stricto is clarified in reference to the lectotype, and the second taxon is herein attributed to T. melanommus (Doflein, 1904), which was originally recognised as a forma of T. uncifer with one specimen from off Kenya and one specimen from Japan. A lectotype is herein selected for T. melanommus in the interest of nomenclatural stability. Differentiating characters between the two species are assessed in detail, and those two species are compared also with T. glaucommus (Alcock, 1894), T. similis (Grant, 1905), T. brucei Tavares, 1991, T. daviei Tavares, 1997, T. hirtipes Tan & Huang, 2000 and T. globosus Spiridonov & Trkay, 2007, all of them are considered as valid. Sexual dimorphism and ontogenetic variation are seen in the relative length and stoutness of the pereopods 2 and 3 in T. uncifer and T. melanommus, but the morphometric characters derived from the two pereopods are useful as diagnostic at comparison between same sex in adults. Comparison of partial fragments of the mitochondrial COI and 16S rRNA gene sequences strongly supports that the two taxa are distinct. Previous records of T. uncifer, including fossil records, are reviewed based on the present findings. Tymolus uncifer and T. melanommus are restricted to Japanese waters at present.

Distribution and molecular identification of ixodid ticks infesting cattle in Kilombero and Iringa Districts, Tanzania

BackgroundHard ticks infesting cattle are vectors of Tick-borne diseases that causes major public health problems and considerable socioeconomic losses to the livestock industry in tropical and subtropical countries. A repeated cross-sectional study was carried out by collecting ticks on cattle during the wet and dry seasons from January to August 2021 in order to determine hard tick prevalence, distribution, and abundance on cattle in Kilombero and Iringa Districts of Tanzania. The collected ticks were identified morphologically using published morphological keys under a stereomicroscope and confirmed by polymerase chain reaction (PCR) and sequencing.ResultsOut of 740 examined cattle, 304, (41.08%) were infested with ticks. In total, 1,780 ticks were counted on one side of the animal’s body and doubled, whereby resulting in a total of 3,560 ticks were recorded. Individual tick burden ranged from n = 2 to 82 ticks per animal, with a mean tick burden of n = 11.7 ± 0.68 per animal. A total of 1,889 ticks were collected from infected cattle including 109 additional ticks observed while collecting ticks based on the animal’s posture when restrained to the ground. Out of 1,889 ticks, nine species from three genera were identified morphologically: 1,377 fit in the genus Rhipicephalus, 459 to the genus of Amblyomma, and 53 to the genus Hyalomma. Rhipicephalus microplus was the most prevalent (n = 909, 48.1%), followed by Rhipicephalus evertsi (n = 310, 16.4%), Amblyomma lepidum (n = 310, 16.4%), Rhipicephalus appendiculatus (n = 140, 7.4%), Amblyomma gemma (n = 120, 6.4%), Hyalomma rufipes (n = 50, 2.6%), Amblyomma variegatum (n = 29, 1.5%), Rhipicephalus decoloratus (n = 18, 1.0%), while the least common was Hyalomma albiparmatum (n = 3, 0.2%). Tick diversity was higher in Iringa compared to Kilombero District. Tick prevalence was higher in wet season (n = 148, 41.11%). Ticks were widely distributed in different parts of the host body, with the highest distribution on zone 4 (n = 1,060, 56.11%), which includes groin, flank, abdomen, and around inner thigh of the hind legs and the lowest distribution on zone 2 (n = 14, 0.74%), which includes the back surface of the body. Both Amblyomma lepidum and Rhipicephalus microplus species were found in all the five body zones, and with the highest proportions recorded on zone 4: A. lepidum (n = 209, 67.42%) and R. microplus (n = 714, 78.55%). The nine tick species identified morphologically were also confirmed using molecular methods. However, during sequencing, two species (Rhipicephalus appendiculatus and R. decoloratus) had poor quality sequences and were excluded from the sequence analysis. Sequencing results indicated high nucleotide identity (96–100%) with sequences available in GenBank and Barcode of Life Database (BOLD). The phylogenetic analysis of partial mitochondrial COI and 16S rRNA gene sequences of ticks were used to confirm the morphological identification.ConclusionThe results showed a high burden of tick infestation on cattle, which could reduce animal production and potentially increase the risk of tick-borne diseases. Therefore, it is necessary to explore the epidemiological and molecular aspects of various tick species in other regions of Tanzania.

Occurrence of an unidentified harrimaniid acorn worm in the genus Stereobalanus (Hemichordata: Enteropneusta) from the Japan Trench hadal zone

A number of soft fragments of uncertain identity, mostly taking a form of a disk or button, were found among trawled material from the hadal zone (7426–7654 m in depth) in the Japan Trench during the cruise KH-22-8 of the R/V Hakuho-maru in October 2022. These enigmatic ‘disks’ subsequently turned out to be the proboscides of enteropneusts severed from the collar. A molecular phylogenetic analysis based on the mitochondrial 16S rRNA and nuclear 18S rRNA gene sequences determined from one of the fragments along with others available in public databases indicated that the species in question was sister to the harrimaniid enteropneust Stereobalanus canadensis (Spengel, 1893). Some morphological features of the fragments—the proboscis longitudinal musculature arranged in radial plates, the four tubular ovaries, and the absence of synapticula on the gill bars—support that the Japan Trench species would belong to Stereobalanus Spengel, 1902. This study represents the deepest record of Stereobalanus and the first report of enteropneusts from the Japan Trench hadal zone, albeit identified to the genus level. While the species is putatively undescribed, a formal taxonomic description should await intact specimens in future studies.

Analysis of Partial Mitochondrial COI and 16S rRNA Gene Sequences Variation of Pagurus brachiomastus and Pagurus proximus (Decapoda: Paguridae) Populations from the South of Russian Far East

Mitochondrial COI and 16S rRNA gene partial sequences were generated for Pagurus brachiomastus from two locations and P. proximus from single location of the south of Russian Far East. Genetic variation and differentiation of P. brachiomastus Russian and South Korean (GenBank data) populations were estimated and compared on the basis of sequence data on two mitochondrial genes separately. Additionally, P. simulans from South Korea was analyzed. Results show P. brachiomastus from two locations of the south of Russian Far East and South Korea and Korean P. simulans differs at intraspecific level based on molecular markers. Median-Joining analysis indicate presence of two main groups of haplotypes, representing P. proximus and P. brachiomastus + P. simulans, respectively. These data support that P. brachiomastus and P. simulans are conspecific. Mismatch distribution analysis showed population growth for P. brachiomastus at the south of Russian Far East. Sequence pairwise variations of both molecular markers appear differently in two far eastern samples of P. brachiomastus, that can be partially explained by active gene flow through hermit crab migrations between two localities after their isolation.

A new Asian leaf litter toad of the genus Leptobrachella (Amphibia, Anura, Megophryidae) from central south China.

A new species of the Asian leaf litter toad genus Leptobrachella from central south China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and nuclear RAG1 gene sequences indicated the new species as an independent clade in the genus. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 29.2-34.2 mm in 15 adult males and 34.4-43.1 mm in seven adult females); distinct black spots present on flanks; toes rudimentary webbed, with wide lateral fringes; ventral belly white with distinct nebulous brown speckling on ventrolateral flanks; skin on dorsum shagreened with fine tiny granules or short ridges; iris copper above, silver below; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaches the middle eye; dorsal surface of tadpole semi-transparent light brown, spots on tail absent, keratodont row formula I: 3+3/2+2: I; call series basically consist of repeated long calls, at dominant frequency (5093 ± 412 Hz).

Diversity, Distribution, and Habitat Association of Anuran Species from Keffa, Southwest Ethiopia

The aim of the current study was to provide a description of the species composition and habitat association of anuran species found in the Keffa area of southwest Ethiopia, among the least studied natural areas in Ethiopia. Data were gathered in both the rainy and dry seasons. Opportunistic observations, audio, and visual encounters were used to perform the survey. To capture different frog species, drift fence and pitfall trap techniques were used. Three habitat types—wetlands, agricultural land, and riverine forests—were explored for the frog species. A total of 3672-person field hours were spent and 5678 individuals of 26 different anuran species from eight different families were recorded. Of these, six species were new records for the Keffa area. Species determination was assessed using morphology and validated with genetic barcoding. The mitochondrial 16S rRNA and COI gene sequence analysis have been used in molecular systematic analyses of the frog species. The Leptopelis ragazzii that was discovered to be present in the earlier study was actually Leptopelis vannutellii, as evidenced by DNA sequencing. The Hyperolius individuals were initially categorized as H. nasutus, but after conducting DNA barcoding, they were found to be H. microps. At least three potential new candidate species (Leptopelis sp. 1, Leptopelis sp. 2, and Phrynobatrachus sp. 1) were discovered. Hyperoliidae was the most abundant family from the recorded anurans, followed by Phrynobatrachidae, while Conrauidae was the least abundant. Most species were found in the wetland habitat, followed by riverine forest, while agricultural land had the least. Wetlands had the highest values for the Shannon diversity index (2.131), followed by agricultural land (1.58) and riverine forests (1.459). The environmental variables revealed that temperature had a considerable negative impact, while precipitation had a non-significantly positive relationship with anuran abundance. The Keffa area is rich in frog species, accounting for 35% of the country’s batrachofauna. However, the habitats have been fragmented and ruined by agricultural expansion, water drainage from the wetlands, and the conversion of horticulture to field crops. We recommend that non-governmental organizations, districts, regional officials, and local people in and around Keffa actively engage in wetland and forest conservation and protection.

Molecular phylogenetic investigation of toads based on sequencing of the mitochondrial 16S ribosomal RNA gene

Amphibians inhabit a wide variety of habitats and environments, such as arboreal, terrestrial and freshwater aquatic ecosystems, and are considered among the vertebrate classes to exhibit one of the most diverse distributions. Their life histories, behaviour and diversity play vital roles in assessing environmental and evolutionary theories. The mitochondrial 16S rRNA gene sequence analysis has been widely used in molecular taxonomy and is frequently used in systematic analyses of families and genera. This study examines taxonomic relationships among 18 specimens of the Arabian toad (Sclerophrys arabica) and the Dhofar toad (Bufo dhufarensis) from Al Madinah Province, Saudi Arabia by estimating phylogenetic relationships using the mitochondrial 16S ribosomal RNA gene (16S rRNA). Results indicate the presence of four new lineages of toads among the samples in this study. The generated phylogenetic tree grouped all samples of Arabian toad in one clade, while the Dhofar toad samples were divided into three subclades. This is the first study to be reported the genetic diversity of Al Madinah Province toads.

Phylogenetic position of the enigmatic genus Atherospio and description of Atherospio aestuarii sp. nov. (Annelida: Spionidae) from Japan.

BackgroundThere are currently two species within the small enigmatic genus Atherospio Mackie & Duff, 1986, which belongs to the Pygospiopsis-Atherospio group in the family Spionidae Grube, 1850. The taxonomic relationship of the genus Atherospio with other spionid or spioniform genera is currently not well understood due to its unusual morphological characteristics.MethodsHere, we describe a new Atherospio species, Atherospio aestuarii sp. nov., based on materials collected from three localities in Japan: Hirota Bay (Iwate Prefecture), Ago Bay (Mie Prefecture), and Yakushima Island (Kagoshima Prefecture). We have also evaluated the possible systematic position of this new species by conducting molecular phylogenetic analyses using the nuclear 18S, 28S, and mitochondrial 16S rRNA gene sequences.ResultsThe morphology of A. aestuarii sp. nov. resembles that of A. disticha Mackie & Duff, 1986 and A. guillei (Laubier & Ramos, 1974) in having branchiae fused to the notopodial lamellae on a restricted number of segments from chaetiger 7, modified neurochaetae on chaetiger 5, and at least some bidentate neuropodial hooks with the secondary tooth below the main fang. The form and arrangement of the modified aristate neurochaetae in double vertical rows closely resemble those found on chaetigers 4 and 5 of A. disticha. The new species lacks the occipital antenna present in A. disticha. In this respect it resembles A. guillei, however, that species differs in having robust neuropodial spines on chaetiger 5 and peristomial papillae, and a preponderance of unidentate neurochaetae. Both A. guillei and the new species have slender needle-like notochaetae in their posteriormost chaetigers. Atherospio aestuarii sp. nov. is distinguished from both congeneric species by its branchial and neuropodial hook distributions. The new species is also unique in that it was recorded at relatively shallow depths, which included intertidal zones. The results of our molecular phylogenetic analysis indicate that the new species was included in a clade that included the genera of the Polydora complex, Pygospio Claparède, 1863, Glandulospio Meißner, Bick, Guggolz, Götting, 2014, Spio Fabricius, 1785, Microspio Mesnil, 1896, Marenzelleria Mesnil, 1896, Rhynchospio Hartman, 1936, Scolelepis Blainville, 1828, Dispio Hartman, 1951, and Malacoceros Quatrefages, 1843 with robust statistical support. The new species formed a clade with Dispio and Scolelepis, however, statistical support for the node was not significant.

Description of Alvaniawangi Xu, Qi & Kong, sp. nov. (Mollusca, Gastropoda, Littorinimorpha, Rissoidae) from the East China Sea.

Alvaniawangi Xu, Qi & Kong, sp. nov. (Mollusca, Gastropoda, Littorinimorpha, Rissoidae) was discovered within the intertidal zone in the Nanji Islands and Zhoushan Islands, Zhejiang Province, China. It has a radula characteristic of Alvania Risso, 1826, a protoconch sculptured with micro pits and lamellae between spiral lirae, and a teleoconch with growth lines and subobsolete cords. Specimens were examined using an integrative taxonomic approach incorporating morphological observations and phylogenetic analyses of concatenated mitochondrial 16S rRNA and nuclear 28S rRNA gene sequences. The findings suggest that the new species is sister to Alvaniacircinata A. Adams, 1861 and is probably endemic to the shallow waters of the East China Sea.

Molecular analysis of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) from the Mediterranean and near Atlantic.

In the present paper we used a molecular data set (including mitochondrial partial 16S rRNA and COI gene sequences) to examine the genetic structure of Lepidopleuruscajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) - a distinctive shallow water chiton and member of the basal branching Lepidopleurida, which is widespread in and adjacent to the Mediterranean. The analyses of the two mt-standard marker fragments resolved two main discrete clusters reported as L.cajetanus s.s. and L.aff.cajetanus, respectively. Lepidopleuruscajetanus s.s. is widespread throughout the area under study, while the second distinct lineage apparently co-occurs on the eastern Spanish mainland coast of the Balearic Sea. This result is discussed comparing our data with those reported, in 2014, by Fernández and colleagues who described L.cajetanus as exhibiting "a 'chaotic patchiness' pattern defined by a high genetic variability with locality-exclusive haplotypes, high genetic divergence, and a lack of geographic structure". Although genetic data alone are not sufficient to draw any definitive conclusions, nevertheless we believe that present results shed new light on L.cajetanus which apparently shows more geographically patterned genetic structure than supposed so far.

Molecular characterisation of Rhipicephalus sanguineus sensu lato ticks from domestic dogs in Nigeria.

Rhipicephalus sanguineus is the most widely reported tick in the world. Molecular characterisation is important to verify its taxonomic status in the different parts of the world. In this study, we provide information on the molecular characterisation of R. sanguineus tick of dogs collected from Nigeria. Ticks were collected from 62 of 93 sampled dogs. The collected ticks were subjected to morphological identification with the aid of appropriate entomological keys. Deoxyribonucleic acid (DNA) was extracted from the most prevalent tick species (R. sanguineus) and was subjected to further molecular characterisation protocols. The partial mitochondrial 16S rRNA gene sequences (∼300 bp) were obtained from representative specimens. Data were statistically analysed using the chi‐square (χ 2) test. Phylogenetic analysis was performed including different lineages of R. sanguineus (sl) from Africa, Asia, Europe and America, and other species belonging to the R. sanguineus ‘tropical lineage’ (R. linnaei) as well as Rhipicephalus turanicus and Ixodes ricinus. Results of this study showed that R. sanguineus was the most abundant ticks of dogs with a prevalence of 61.8% (68/110; 95% CI = 52.5–70.54), followed by Amblyomma variegatum (20.0%) and Haemaphysalis leachi (18.2%). The molecular analysis shows that they are genetically different from the temperate strains but closely related to those from other West African countries. There is a need to establish the vector competence of this common Nigerian dog tick.

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific.

Due to the lack of visible barriers to gene flow, it was a long-standing assumption that marine coastal species are widely distributed, until molecular studies revealed geographically structured intraspecific genetic differentiation in many taxa. Historical events of sea level changes during glacial periods are known to have triggered sequential disjunctions and genetic divergences among populations, especially of coastal organisms. The Parasesarma bidens species complex so far includes three named plus potentially cryptic species of estuarine brachyuran crabs, distributed along East to Southeast Asia. The aim of the present study is to address phylogeography and uncover real and hidden biological diversity within this complex, by revealing the underlying genetic structure of populations and species throughout their distribution ranges from Japan to West Papua, with a comparison of mitochondrial COX1 and 16S rRNA gene sequences. Our results reveal that the P. bidens species complex consists of at least five distinct clades, resulting from four main cladogenesis events during the mid to late Pleistocene. Among those clades, P. cricotum and P. sanguimanus are recovered as monophyletic taxa. Geographically restricted endemic clades are encountered in southeastern Indonesia, Japan and China respectively, whereas the Philippines and Taiwan share two clades. As individuals of the Japanese clade can also be found in Taiwan, we provide evidence of a third lineage and the occurrence of a potential cryptic species on this island. Ocean level retreats during Pleistocene ice ages and present oceanic currents appear to be the main triggers for the divergences of the five clades that are here addressed as the P. bidens complex. Secondary range expansions converted Taiwan into the point of maximal overlap, sharing populations with Japan and the Philippines, but not with mainland China.

New record of blunthead pufferfish, Sphoeroides pachygaster (Muller & Troschel, 1848 (Tetraodontiformes: Tetraodontidae) from Indian water along with DNA barcode and some biological aspects

Blunthead pufferfish, Sphoeroides pachygaster (Muller and Troschel 1848) was recorded for the first time in Indian seas. A single female specimen of S. pachygaster was caught in bottom trawl operation at 263 – 310 m depth of the E astern Arabian S ea. Morphometric and meristic description are presented in detail and compared with the pertinent records available elsewhere. The specimen collected was fully matured with Gonado Somatic Index (GSI) of 17.9 %. Gonad occupied two-third of the abdominal cavity, ova diameters ranged between 0.45 and 0.6 mm and the absolute fecundity estimated was 0.238 million eggs. Histological studies revealed that, this species performs single spawning strategy and it is the first information on the reproduction of S. pachygaster in India. Available reports on the occurrence of this species indicated distribution in the Mediterranean Sea, Atlantic, Indian and Pacific Oceans ; however , the present record confirms its distribution in the Arabian Sea. Wide variations in the meristic counts among the specimens of this species collected from various locations are also discussed. Molecular analysis of the present specimen using Mitochondrial 16S rRNA gene sequences, confirmed the identity as S. pachygaster with intra specific divergence of 00.0 – 0.04 %. This report is the first well documented, confirmed record and re-description of S. pachygaster from the Indian Ocean, which documents a new addition to the family, Tetraodontidae of the Indian ichthyofauna.

A new species of Odorrana (Anura, Ranidae) from Hunan Province, China.

A new species, Odorrana sangzhiensissp. nov., is described, based on five specimens from Sangzhi County, Zhangjiajie City, Hunan Province, China. Molecular phylogenetic analyses, based on mitochondrial 12S rRNA and 16S rRNA gene sequences, strongly support the new species as a monophyletic group nested into the O. schmackeri species complex. The new species can be distinguished from its congeners by a combination of the following characters: (1) body size medium (SVL: 42.1–45.1 mm in males, 83.3–92.7 mm in females); (2) dorsolateral folds absent; (3) tympanum diameter 1.53 times as long as the width of the disc of finger III in females; 2.3 times in males; (4) dorsal skin green with dense granules and sparse irregular brown spots; males with several large warts on dorsum; (5) two metacarpal tubercles; (6) relative finger lengths: I ≤ II < IV < III; (7) tibiotarsal articulation beyond the tip of the snout; (8) ventral surface smooth in females; throat and chest having pale spinules in adult males; (9) dorsal limbs green or yellow green with brown transverse bands; and (10) paired external vocal sacs located at corners of the throat, finger I with light yellow nuptial pad in males. This discovery increases the number of Odorrana species to 59 and those known from China to 37.

Molecular Identification, Transcriptome Sequencing and Functional Annotation of Pulex irritans.

Pulex irritans are vectors of various zoonotic pathogens. However, molecular studies on P. irritans and flea-borne diseases are limited due to the lack of molecular data. This study aimed to conduct transcriptome sequencing, functional annotation, and pathogen analysis of P. irritans. Fleas collected from a dog were identified morphologically and molecularly. RNA was extracted for transcriptome sequencing and functional annotation. Open reading frames (ORFs) of unigenes were confirmed by employing bioinformatics strategies, and maximum likelihood (ML) trees were reconstructed based on the highly expressed genes of ejaculation globulin-specific 3-like protein, salivary protein, and actin for phylogenetic relationship analysis. The obtained mitochondrial 16S rRNA gene sequences showed 99.71% of similarity with P. irritans obtained from GenBank database. Transcriptome sequencing generated 74,412 unigenes, of which 53,211 were functionally annotated. A total of 195 unigenes were assigned to fleas, of which 69 contained complete ORFs. Phylogenetic trees of both ejaculatory globulin and salivary protein genes demonstrated that P. irritans first clustered with Pulicidae sp., indicating the reliability of transcriptome data. It is noteworthy that 1070 unigenes were assigned to Hymenolepis microstoma and Dipylidium caninum, of which 62 contained complete ORFs. The phylogenetic tree of the actin gene showed that the unigenes had closer relationships with Echinococcus sp., suggesting the role of P. irritans as intermediate hosts of tapeworms. The results of this study provide the possibility for functional exploration of important genes and lay foundations for the prevention and control of P. irritans and flea-borne diseases.

Caridina sinanensis, a new species of stygobiotic atyid shrimp (Decapoda, Caridea, Atyidae) from a karst cave in the Guizhou Province, southwestern China.

From a biodiversity survey of a subterranean habitat near Sinan County, Guizhou Province, southwestern China, a new atyid shrimp of the genus Caridina H. Milne Edwards, 1837, C. sinanensissp. nov. was discovered. The new species can be separated from other congeners based on a combination of characters including depigmentation in body and reduction of eyes, small pigment spot at the centre of the cornea, the shape of rostrum and the endopod of the 1st male pleopod, and the relatively longer appendix interna on the appendix masculina of the 2nd pleopod. Mitochondrial COI and 16S rRNA gene sequences also support the establishment of the new species. Information on the habitat, and the levels of threat are discussed to guide the conservation of C. sinanensissp. nov.

The phylogenetic position of Arhaphe deviatica within Hemipteran insects: A potential model species for eco-devo studies of symbiosis.

Insecta is known to be the most diverse group of species, exhibiting numerous forms of endosymbiotic associations. Molecular techniques have provided significant indicators for insect-microbe interactions. The present study aimed to register one of the true bugs of pentatomomorpha and clarify its taxonomic position through phylogenetic analysis of the partial 16S rRNA gene region. A maximum likelihood analysis retrieved a generally well-supported phylogeny based on Tamura 3-parameter model. Based on the partial mitochondrial 16S rRNA gene sequences, a phylogenetic study of suborder Heteroptera relationships within Hemipteras' order was constructed. Sequences of 221 bases of the 3' end of the gene from 28 species within 16 families were analyzed. This analysis and bootstrap confidence revealed two major clades comprising four suborders within Hemiptera, with a close relationship between Heteroptera + (Sternorrhyncha + (Auchenorrhycha + Coleorrhyncha)). Infraorder Pentatomomorpha is forming a sister group with a substantial bootstrap value to Cimicomorpha. Pyrrhocoroidea forms a sister relationship with Lygaeoidea + Coreoidea. There is a close relationship between Largidae and Pyrrhocoridae within Pyrrhocoroidea. The results show that the present species is firmly embedded in the genus Arhaphe with 94.35% sequence resemblance to its congeners. Besides, the recovered hemipteran species considered a potential model group for studying different symbionts. We propose both phylogenetic and ecological evolutionary developmental biology viewpoints for a more synthetic understanding of insect populations' molecular evolution.

A NEW CRYPTIC SPECIES OF BUSH FROG (AMPHIBIA: ANURA: Raorchestes) FROM THE SOUTHERN EASTERN GHATS, INDIA

A new cryptic lineage of bush frogs of the genus Raorchestes from the understudied Eastern Ghats of India is described. Molecular phylogenetic reconstructions using mitochondrial 16S rRNA gene sequences suggest that the new species belongs to the Raorchestes charius clade, is sister to the allopatric R. charius of the Western Ghats and support the species-level distinctiveness of the lineage. The new species morphologically resembles R. charius from which it can be distinguished using the following characters: narrower head (HW/SVL 0.31 vs 0.34–0.36), wider inter upper eyelid (IUE/SVL 0.14–0.16 vs 0.11–0.13), greater maximum upper eyelid width (UEW/SVL 0.10–0.12 vs 0.06–0.09) and shorter thigh (TL/SVL 0.32–0.43 vs 0.44–0.51). Acoustic observations from the breeding males further corroborate the distinct specific status of the new species. The discovery of this new species of bush frog from an understudied landscape suggests that additional extensive surveys in the Eastern Ghats would yield several more amphibian lineages yet unknown to science.

Importance of genetic data in resolving cryptic species: A century old problem of understanding the distribution of Minervarya syhadrensis Annandale 1919, (Anura: Dicroglossidae).

Frogs of the genus Minervarya are cryptic and widely distributed in South Asia. However, many of them lack information about the precise type locality, genetic data, and distribution range. The present study aimed to examine the genetic affinities of a widely distributed species Minervarya syhadrensis around its type locality in the northern Western Ghats (Pune, Maharashtra). We studied the type specimen of M. syhadrensis and collected similar sized Minervarya frogs from Pune district. In the field, we observed two different calls from morphologically similar (M. syhadrensis like) males suggesting the sympatric occurrence of two cryptic species (that we initially named Minervarya species A and Minervarya species B). We analyzed morphology, call pattern, and mitochondrial 16S rRNA gene sequence of both species. Minervarya species A has a long call with a low pulse repetition rate and higher dominant frequency compared to that of the Minervarya species B. These species cannot be differentiated based on morphometric data. However, they can be sorted out using morphological characters such as the presence of longitudinal skin folds on the dorsal side (Minervarya species A) and differences in foot webbing. DNA sequences of Minervarya species A and Minervarya species B are matching with those of M. caperata and M. agricola respectively. After studying the type specimens of M. syhadrensis and M. caperata, we found morphological similarities (longitudinal skin folds) with the samples of Minervarya species A collected during the present study. Based on the results of our study (morphology and genetic) and available literature, we propose to redefine M. syhadrensis as applying to the lineage initially named Minervarya species A, and to treat the species M. caperata as a junior synonym of M. syhadrensis. Our study will be helpful in further taxonomic revision of the genus, and provides natural history information for M. syhadrensis and M. agricola.