Comparative analysis of the two suborders of Thysanoptera and characterization of the complete mitochondrial genome of Thrips parvispinus.

Thrips parvispinus is a serious sucking pest on a number of economically important crops in the oriental region. It has gained importance recently for its drastic range extension distribution as an invasive pest. Here, the complete mitochondrial genome (15,067 bp) of Thrips parvispinus was sequenced and characterized. It possesses 37 genes and the putative noncoding region is duplicated. Comparative analyses of nucleotide diversity, skewness, codon usage bias, and selection pressure in mitochondrial protein-coding genes of the available 31 thrips mitogenomes (24 Terebrantia + 7 Tubulifera) were performed. Phylogenetic analysis showed a sister relationship of T. parvispinus to the clade (T. florum + T. hawaiiensis). Phylogenetic analyses formed the monophyly of subfamilies Phlaeothripinae and Idolothripinae within the family Phlaeothripidae (Suborder Tubulifera). Low nucleotide diversity was indicative of reversal of strand asymmetry in the Tubulifera. Neutrality analysis showed that directional mutation plays a major role in shaping codon usage bias in both suborders. Principal component analysis indicated distinct codon usage patterns in each suborder. Our data suggested weaker selection constrains on Terebrantia than in the Tubulifera. More tubuliferan mitogenomes are required to resolve previous classification hypotheses and elucidate genome evolution in these two suborders.

Gene arrangement, phylogeny and divergence time estimation of mitogenomes in Thrips.

BACKGROUND: The metazoan mitogenomes usually display conserved gene arrangement while thrips are known for their extensive gene rearrangement, and duplication of the control region. METHODS AND RESULT: We sequenced complete mitogenomes of eight species of thrips to determine the gene arrangement, phylogeny and divergence time estimation. All contain 37 genes and one control region, (CR) except four species with two CRs. Duplicated tRNAs were detected in Mycterothrips nilgiriensis and Thrips florum. nad4-nad4L were not found adjacent to each other in Phibalothrips peringueyi and Plicothrips apicalis. Both Bayesian and likelihood phylogenetic analyses of thrips mitogenomes supported the monophyly of two suborders (Terebrantia and Tubulifera) and the two largest families (Phlaeothripidae and Thripidae). Out of seven earlier proposed ancestral gene blocks, six are conserved in Panchaetothripinae, three in Thripinae and two in Phlaeothripidae. Additionally, eight Thrips Gene Blocks were identified, of which, three conserved in Tubulifera, four in Terebrantia, and one only in Aeolothripidae. Forty-two gene boundaries (15 from previous study + 27 new) were identified. The molecular divergence time is estimated for the order Thysanoptera and suggested that these insects may have been diversified from hemipterans in the late Permian period. The most recent ancestors belong to family Thripidae and Phlaeothripidae, which were diversified in upper Cretaceous period and showed higher rates of rearrangement from the ancestral gene order. CONCLUSIONS: The current study is the first largest effort to provide the new insights into the mitogenomic features, gene arrangement, phylogeny and divergence time estimation of thrips belonging to the order Thysanoptera.

Gene rearrangement in the mitogenome of whiteflies (Hemiptera: Aleyrodinae) along with their phylogeny and characterization of complete mitogenome of Aleurodicus rugioperculatus.

BACKGROUND: Mitochondrial genome rearrangements have been used for defining evolutionary relationships, but there have been incidences of convergences at different taxonomic levels. To gain new insights into whitefly mitogenome evolution, gene arrangement and phylogeny, we sequenced the complete mitogenome of Aleurodicus rugioperculatus (Aleyrodidae: Aleurodicinae) and conducted a comparative analysis with the previously published mitogenomes across the family Aleyrodidae. METHODS AND RESULTS: The complete mitogenome of Aleurodicus rugioperculatus was generated by Next generation sequencing method. It is 15,060 bp circular molecule with 86.5 A + T and 5.5% G + C content. It contains 37 genes (13 PCGs, two rRNAs, 22 tRNAs) and a non-coding control region (CR). Comparative analysis of codon usage indicated that the subfamily Aleyrodinae have weaker bias than Aleurodicinae. Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses yielded similar topologies supporting the monophyly of Aleyrodinae and Aleurodicinae. The gene order of 13 whiteflies was compared with ancestor to examine the plesiomorphies, synapomorphies and autapomorphies. We identified five gene blocks (I-V) in the whitefly ancestor that are shared plesiomorphies retained in different whitefly lineages. Gene block I is conserved in all whiteflies except three species. Conversely, we detected 83 derived gene boundaries within the family. Mapping these gene boundaries onto a phylogenetic tree revealed that 16 were symplesiomorphies for two subfamilies; 9 were synapomorphies between the species, and 28 autapomorphies for individual species. CONCLUSIONS: Comparative analyses of gene order of whiteflies revealed the derived gene boundaries which can be further investigated with more mitogenome data to examine the genome evolution in whiteflies.

Complete mitogenome of the endangered and endemic Nicobar treeshrew (Tupaia nicobarica) and comparison with other Scandentians.

The Nicobar treeshrew (Tupaia nicobarica) is an endangered small mammal endemic to the Nicobar Island of the Andaman Sea, India regarded as an alternative experimental animal model in biomedical research. The present study aimed to assemble the first mitochondrial genome of T. nicobarica to elucidate its phylogenetic position with respect to other Scandentians. The structure and variation of the novel mitochondrial genome were analyzed and compared with other Scandentians. The complete mitogenome (17,164 bp) encodes 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs), two ribosomal RNA (rRNAs), and one control region (CR). Most of the genes were encoded on majority strand, except nad6 and eight tRNAs. The nonsynonymous/synonymous ratio in all PCGs indicates strong negative selection among all Tupaiidae species. The comparative study of CRs revealed the occurrence of tandem repeats (CGTACA) found in T. nicobarica. The phylogenetic analyses (Maximum Likelihood and Bayesian Inference) showed distinct clustering of T. nicobarica with high branch supports and depict a substantial divergence time (12-19 MYA) from the ancestor lineage of Tupaiidae. The 16S rRNA dataset corroborates the taxonomic rank of two subspecies of T. nicobarica from the Great and Little Nicobar Islands. In the future, whole nuclear genome sequencing is necessary to further improve our understanding of evolutionary relationships among treeshrews, and will have implications for biomedical research.

The complete mitochondrial genome of Aeolothrips indicus Bhatti, 1964 (Thysanoptera: Thripidae).

Here, we have generated the complete mitochondrial sequence of Aeolothrips indicus Bhatti, 1964. So far, this is the first largest mitogenome with 17,042 bp length in order Thysanoptera. It includes 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes along with three non-coding regions. AT composition of A. indicus is 72.5% (37.7% A and 34.8% T) and GC 27.5% (15.6% C and 11.9% G). The constructed phylogeny revealed the monophyly of family Aeolothripidae in the order Thysanoptera. The data would provide further insight into the evolution and phylogeny of the order Thysanoptera.

The complete mitochondrial genome of Taeniothrips tigris Bhatti, 1995 (Thysanoptera: Thripidae).

In this study, we sequenced complete mitogenome of Taeniothrips tigris Bhatti 1995. It was 15,501 bp in length containing 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes along with two non-coding regions. The overall base composition of Ta. tigris is 43.66% A, 35.20% T, 11.46% C, and 9.68% G, with a high AT bias of 78.86%. The constructed phylogeny using 19 mitogenomes revealed that the genus Taeniothrips is in close relationship with genus Thrips. This mitogenome data would help in deducing phylogenetic relationships studies in the order Thysanoptera.

A new species of Apelaunothrips (Thysanoptera: Phlaeothripidae) from India with new synonym of Podothrips sasacola Kurosawa.

The first objective of this paper is to describe a new fungus-feeding species in the Phlaeothripinae genus Apelaunothrips, and to provide a key to the six species of this genus recorded from India. The second objective is to establish a new synonym in the Poaceae-associated genus, Podothrips, based on structural variation observed among recently collected samples from bamboo. The slide-mounted specimens were studied with a Leica Trinocular Microscope (Leica DM-1000) and a Leica software application suite (LAS EZ 2.1.0) was used to make line drawing and photographs.

Tryphactothrips rutherfordi (Bagnall) (Thysanoptera, Panchaetothripinae): first description of the male.

The subfamily Panchaetothripinae (family Thripidae) is represented by 140 species under 40 genera (Thrips Wiki 2019). In India, 36 species under 16 genera are recorded (Tyagi Kumar 2016, Tyagi et al. 2017, Rachana and Varatharajan, 2018, Johnson et al. 2019). The members of this subfamily are leaf-feeders and usually dark brown in colour with strongly reticulate sculpture on body, terminal antennal segments needle-shaped, tarsi 1- or 2-segmented, fore wing upper vein fused with costa. The genus Tryphactothrips was established by Bagnall (1919), and this genus remains monobasic with only Dinurothrips rutherfordi Bagnall from Sri Lanka as the type species. The genus Tryphactothrips can be distinguished from related genera by the presence of sculptured round areolae on abdominal segments. It is closely related to Anisopilothrips Stannard Mitri but can be identified by paired sigmoidal setae on abdominal tergites (absent in Anisopilothrips), mesonotum without complete median longitudinal split (complete median longitudinal in Anisopilothrips). Recently, a series of both sexes of Tryphactothrips rutherfordi with banded fore wings was collected on fern from Kerala state of India. Females were identified using published keys (Wilson 1975), and the male is here described for the first time. DNA was isolated from the studied specimens and partial fragment of mtCOI gene was amplified and sequenced (Tyagi et al. 2017). Four sequences were submitted in the GenBank (Accession No. MN627201 to MN627204). Photographs and illustrations were taken through a Leica Trinocular Microscope (Leica DM-1000) using Leica software application suite (LAS EZ 2.1.0). The studied specimens were deposited in the National Zoological Collections (NZC), Zoological Survey of India, Kolkata, India.

Identification of Indian Spiders through DNA barcoding: Cryptic species and species complex.

Spiders are mega diverse arthropods and play an important role in the ecosystem. Identification of this group is challenging due to their cryptic behavior, sexual dimorphism, and unavailability of taxonomic keys for juveniles. To overcome these obstacles, DNA barcoding plays a pivotal role in spider identification throughout the globe. This study is the first large scale attempt on DNA barcoding of spiders from India with 101 morphospecies of 72 genera under 21 families, including five endemic species and holotypes of three species. A total of 489 barcodes was generated and analyzed, among them 85 novel barcodes of 22 morphospecies were contributed to the global database. The estimated delimitation threshold of the Indian spiders was 2.6% to 3.7% K2P corrected pairwise distance. The multiple species delimitation methods (BIN, ABGD, GMYC and PTP) revealed a total of 107 molecular operational taxonomic units (MOTUs) for 101 morphospecies. We detected more than one MOTU in 11 morphospecies with discrepancies in genetic distances and tree topologies. Cryptic diversity was detected in Pardosa pusiola, Cyclosa spirifera, and Heteropoda venatoria. The intraspecies distances which were as large as our proposed delimitation threshold were observed in Pardosa sumatrana, Thiania bhamoensis, and Cheiracanthium triviale. Further, shallow genetic distances were detected in Cyrtophora cicatrosa, Hersilia savignyi, Argiope versicolor, Phintella vittata, and Oxyopes birmanicus. Two morphologically distinguished species (Plexippus paykulli and Plexippus petersi) showed intra-individual variation within their DNA barcode data. Additionally, we reinstate the original combination for Linyphia sikkimensis based on both morphology and DNA barcoding. These data show that DNA barcoding is a valuable tool for specimen identification and species discovery of Indian spiders.

DNA Barcoding Reveals Association of Glossiphoniidae Species on Endangered Freshwater Turtles in Northeast India.

BACKGROUND: The identification of Glossiphoniidae species is often confusing due to the lack of both morphological and molecular data worldwide. PURPOSE: The study aimed to identify the collected leech specimens from two endangered freshwater turtles, Chitra indica and Pangshura sylhetensis from northeast India. METHODS: We generated DNA barcode sequences and estimated the genetic distances and phylogenetic relationship of the studied taxa with another 14 Glossiphoniidae genera (114 barcode sequences). RESULTS: The high genetic distinctiveness (17.9 to 26.3%) and distinct clustering in Neighbor-Joining (NJ) phylogeny, we identified the studied specimens as Glossiphoniidae sp. We assumed that the studied specimens might be a new member of Glossiphoniidae or an extant species without DNA information. CONCLUSION: Hence, the study recommends further sampling of leeches from the similar host as well as from same and various localities and also generates the molecular data to perceive the exact diversity. The aimed study is also helpful to encourage the awareness and conservation management of freshwater turtles and other threatened animals.

DNA barcoding of freshwater fishes from Brahmaputra River in Eastern Himalaya biodiversity hotspot.

The genetic diversity of freshwater fishes is still anonymous in several drainage systems in northeast India. Moreover, the comparative genetic analysis is largely sporadic to judge their actual diversity and true status. We generated 89 DNA barcodes of 40 morphologically identified fishes collected from two major tributaries of Brahmaputra River. The comparative study revealed that most of the species were clearly discriminated by their estimated genetic distances and monophyletic clustering in Bayesian (BA) tree. Considering the genetic divergence (2%) for species discrimination boundary, the high genetic diversity (2.36-10.73%) was detected in 11 species (Macrognathus pancalus, Channa punctata, Puntius terio, Bangana ariza, Garra arupi, Badis badis, Mystus vittatus, Rita rita, Gagata cenia, Mastacembelus armatus, and Danio dangila), which signified the occurrence of concealed genetic diversity in this ecozone. However, the insignificant genetic distances were also noticed in few reportedly valid species: Channa stiktos and C. ornatipinnis (1.43%); Mystus ngasep, M. rufescens, and M. carcio (0.4%); Glyptothorax trilineatus, G. churamanii, and G. verrucosus (0.4%); Botia almorhae, B. histrionica, B. lohachata, and B. rostrata (0-0.4%); Barilius barilia and B. vagra (0.4%); Batasio merianiensis and B. tengana (1.2%); Puntius chola and P. fraseri (0%), Schistura beavani and S. paucireticulata (0%); hence to validate this species, generation of more barcode data was required from their types or topotypes. The present study would help to develop conservation schemes for the native species and collegiate ecosystem, which associated with the livelihoods of millions of ethnic communities in this region.

Mitochondrial DNA identified bat species in northeast India: electrocution mortality and biodiversity loss.

Northeast India with two biodiversity hotspots is recognized as a biodiversity-rich region. However, several extant animals including chiropterans are currently at jeopardy due to habitat loss, electrocution mortality, and other anthropogenic threats. This study examines the efficacy of mitochondrial Cytochrome b (mtCytb) sequences for species-level identification of five electrocuted bat specimens from Manipur state. The similarity search results in the global database, Kimura 2 parameter (K2P) genetic distances, and neighbor-joining (NJ) tree identified all bat specimens into two species, Cynopterus sphinx and Megaerops niphanae. The detection of M. niphanae is the first record of this mammal from the state. In comparison with other Pteropodidae species, the genetic distances clearly discriminate both C. sphinx (7.9-30.2%) and M. niphanae (12.2-25.7%). In addition, the combined tree analysis of present and earlier genetic information of C. sphinx suggested the presence of cryptic lineages and sympatric population in India. This similar approach with more sampling from a wide distribution area could assist the future genetics research on chiropterans and their precise conservation.

DNA barcoding of freshwater fishes from the transboundary river of Indo-Bhutan: multiple clades and cryptic diversity.

The species diversity of freshwater fishes from the transboundary river, Jaldhaka is still unknown to the scientific communities. We generated 40 DNA barcode sequences of 16 morphologically identified freshwater fishes and compared genetically with the database sequences. Ten species (Acanthocobitis botia, Barilius bendelisis, Crossocheilus latius, Channa punctata, Channa quinquefasciata, Garra gotyla, Garra kempi, Opsarius barna, Psilorhynchus balitora, and Pseudecheneis sulcata) showed unique haplotypes in the studied riverine system. Further, the estimated genetic divergences, BA tree topology, and ABGD species delimitation methods revealed the presence of cryptic diversity in Badis badis, Garra annandalei, G. gotyla, G. kempi, P. balitora, Rasbora daniconius, and Pethia ticto. The study suggested more exhaustive sampling and generation of more molecular data to strengthen the fact. The aimed integrated approach will be helpful to detect the extant species diversity, helps to reevaluate the checklist and promote sustainable conservation management to protect this unparalleled ecosystem.

DNA barcoding of selected Scirtothrips species (Thysanoptera) from India.

The members of the genus Scirtothrips are highly polyphagous, including major pest and vector species. We applied both morphology and molecular approaches to delimit the selected Scirtothrips species from India. Out of 43 generated barcode sequences, six sequences of three species (S. hitam, S. mangiferae, and S. malayensis) are the novel contribution in global database. The Bayesian (BA) phylogeny clearly distinguishes all the studied species with reciprocal monophyletic criteria and represents multiple clades in S. dorsalis and S. oligochaetus. The high Kimura-2-Parameter (K2P) genetic divergences were observed between the multiple clades of S. dorsalis (4.5-8.8%) and S. oligochaetus (6.4%), which indicating possible existence of cryptic diversity. The current study also provided the morphological keys for six Scirtothrips species including S. hitam as a new record to India.

Complete mitochondrial genome of Black Soft-shell Turtle (Nilssonia nigricans) and comparative analysis with other Trionychidae.

The characterization of mitochondrial genome has been evidenced as an efficient field of study for phylogenetic and evolutionary analysis in vertebrates including turtles. The aim of this study was to distinguish the structure and variability of the Trionychidae species mitogenomes through comparative analysis. The complete mitogenome (16796 bp) of an endangered freshwater turtle, Nilssonia nigricans was sequenced and annotated. The mitogenome encoded for 37 genes and a major non-coding control region (CR). The mitogenome was A + T biased (62.16%) and included six overlapping and 19 intergenic spacer regions. The Relative synonymous codon usage (RSCU) value was consistent among all the Trionychidae species; with the exception of significant reduction of Serine (TCG) frequency in N. nigricans, N. formosa, and R. swinhoei. In N. nigricans, most of the transfer RNAs (tRNAs) were folded into classic clover-leaf secondary structures with Watson-Crick base pairing except for trnS1 (GCT). The comparative analysis revealed that most of the tRNAs were structurally different, except for trnE (TTC), trnQ (TTG), and trnM (CAT). The structural features of tRNAs resulted ≥ 10 mismatched or wobble base pairings in 12 tRNAs, which reflects the nucleotide composition in both H- and L-strands. The mitogenome of N. nigricans also revealed two unique tandem repeats (ATTAT)8, and (TATTA)20 in the CR. Further, the conserved motif 5'-GACATA-3' and stable stem-loop structure was detected in the CRs of all Trionychidae species, which play an significant role in regulating transcription and replication in the mitochondrial genome. Further, the comparative analysis of Ka/Ks indicated negative selection in most of the protein coding genes (PCGs). The constructed Maximum Likelihood (ML) phylogeny using all PCGs showed clustering of N. nigricans with N. formosa. The resulting phylogeny illustrated the similar topology as described previously and consistent with the taxonomic classification. However, more sampling from different taxonomic groups of Testudines and studies on their mitogenomics are desirable for better understanding of the phylogenetic and evolutionary relationships.

DNA barcoding of Cloridopsis immaculata: genetic distance and phylogeny of stomatopods.

The changes of coastal topography might have genetically altered the extant species diversity in Chilika Lake. The genetic assessment of stomatopods has never been attempted from this ecosystem. The study generate the first genetic information (mtCOI) of Cloridopsis immaculata. DNA sequences of C. immaculata shows 12.9% genetic divergence with Harpiosquilla harpax and clade as sister species in NJ tree. Alima, Harpiosquilla, and Oratosquilla shows high congeneric/conspecific genetic divergence (20.9%, 15.7%, and 7.2%) and cladded separately in the phylogeny; correlate to their diverse populations. We recommend more extensive survey of stomatopods and generation of molecular data to resolve the taxonomic uncertainty.