Genetic diversity and phylogenetic relationship among the western and the Asian honey bees based on two mitochondrial gene segments (COI and ND5)

The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.     

Two new species of the chrysomelid genus Trichomimastra Weise, 1922 (Coleoptera,Chrysomelidae) from Pakistan

Two new species of the genus Trichomimastra Weise, 1922 from Pakistan, T. pakistanica sp. n. and T. kaganica sp. n., are described; a key to the species of this genus from the Himalayas and neighboring areas (Pakistan, Afghanistan) is proposed.     

An insight into molecular taxonomy of bufonids,microhylids, and dicroglossid frogs: First genetic records from Pakistan

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.     

Description of Tylenchorhynchus qasimii sp. n. with a New Report of T. kegasawai from Pakistan

A new stunt nematode, from soil around the roots of coconut (Cocos nucifera L.) and rice (Oryza sativa L.) from Karachi, Pakistan, is described and illustrated as Tylenchorhynchus qasimii n. sp. This new species is characterized by having females with 3–4 head annules, anteriorly directed stylet knobs, absence of post anal extension, presence of rounded sperm filled spermatheca and conoid to bluntly rounded hemispherical tail terminus. Males are common. Also included is the record of T. kegasawai from around the roots of rice (O. sativa L.), a new report from Sindh, Pakistan.     

First genetic evidence for the presence of the rumen fluke Paramphistomum epiclitum in Pakistan

More than 70 species of the Superfamily Paramphistomoidea, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes within this family have a predilection for the forestomach (rumen) or bile duct of the liver, where they may cause epithelial damage. Identification of adult Paramphistomum, Calicophoron, Gastrothylax and Fischoederius at the species level based on morphology requires specialised expertise, whereas molecular genetic marker analysis is more precise and transferable. In the present study, we performed molecular characterisation of twenty seven adult flukes collected from the forestomachs of buffalo, cattle and goats in the Punjab province of Pakistan. PCR and sequencing of the ITS-2 rDNA region revealed a single haplotype in all cases. Phylogenetic comparison of P. epiclitum ITS2-rDNA sequences with those from other Paramphistomum, Calicophoron, Gastrothylax and Fischoederius species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for P. epiclitum identification. This work provides a validated species-specific marker of P. epiclitum and the first report of this parasite species from Pakistan. The results of this study also have implications for the diagnosis and control of rumen flukes in the region and the need for accurate species identification to understand parasite distribution and population genetics.     

Descriptions of Two New Species of Tylenchorhynchus Cobb, 1913 (Nematoda: Tylenchida), with Details on Morphology and Variation of T. claytoni

Two new species of plant parasitic nematodes (Tylenchorhynchus quaidi n. sp. and T. tritici n. sp.) from Pakistan are described and illustrated. Tylenchorhynchus quaidi n. sp., from soil around roots of potato (Solanum tuberosum) from an experimental field of NNRC, Karachi, Pakistan, is distinguishable from other species by its peculiar sunken dome-shaped head. Although similar to T. goffarti, it differs by head shape, areolation of lateral field, ratios a (23-28 vs. 29-37) and c (11-14 vs. 13-20), and a vagina that is half sclerotized and half unsclerotized. Tylenchorhynchus tritici n. sp., from soil around roots of wheat (Triticum aestivum) from Campbellpur, Pakistan, is similar to T. ventrosignatus and T. nordiensis. It differs from T. ventrosignatus by a continuous lip region, number of head annules (2-3 vs. 4), coarse body annulation, absence of a wave-like structure near the vulva, and by tail shape and number of tail annules (15-23 vs. 28-32). It differs from T. nordiensis by stylet length (12.4-14.6 vs. 11-13 μm), shape of stylet knobs, number of head annules (2-3 vs. 4), non-areolated lateral field in region of phasmids, and not fusing in posterior third of tail. Morphometrics of Tylenchorhynchus claytoni from soil around stunted maize (Zea mays L.), in Muscatine County, Iowa, and several other populations are given. Detailed morphometric data on T. claytoni based on topotypes collected from type locality and several other populations revealed that this species shows variations in the shape of tail in females, number of tail annules (and sometimes annules extending further back on the terminus, almost being an annulated terminus), position of phasmid, and shape of lip region. The subgenus Bitylenchus is proposed as a new synonym of Tylenchorhynchus and its species referred to the latter genus.     

New and little known species of the family Saldidae (heteroptera) from india and adjacent countries

Data on 13 species of the family Saldidae distributed in India, Pakistan, Afghanistan, Nepal, and Vietnam are reported. Two new species, Macrosaldula indica sp. n. (Himachal Pradesh) and Saldula pericarti sp. n. (Jammu and Kashmir, Himachal Pradesh), are described from the Himalayas. Macrosaldula indica is similar to the dark specimens of M. scotica (Curtis, 1835) and M. variabilis variabilis (Herrich-Schaeffer, 1835) of the scotica group, but differs in a shorter body and in the structure of the genitalia. The montane Middle Asian M. tadzhika (Kiritshenko, 1912) is larger and differs from the new species in the color pattern of its fore wing and in a short and arched paramere. An isolated dark spot on the fore tibia and a pale pattern on the corium identify Saldula pericarti sp. n. as a member of the saltatoria group, but the species differs from the other members of this group in the presence of erect hairs on the head, antennae, pronotum, and wings (the character of the orthochila group). New records of Micracanthia minor Hamid et Sultana, 1972, comb. n., are made in Western and Eastern Pakistan. Micracanthia ornatula (Reuter, 1882) is found for the first time in Nepal and Vietnam, Saldula burmanica Lindskog, 1975 and S. palustris (Douglas, 1874), in Pakistan, and S. pilosella (Thomson, 1871), in Afghanistan.     

New Alicornops (Rhinocerotidae) remains from Lower and Middle Siwaliks,Pakistan

Unpublished rhinocerotid remains from the Lower and Middle Siwaliks of Pakistan are described in this paper and recognized as two species of Alicornops. Alicornops complanatum (Heissig) is identified in the Dhok Pathan Formation of the Middle Siwaliks and Alicornops laogouense Deng in the Kamlial Formation of the Lower Siwaliks. The Dhok Pathan Formation levels with A. complanatum are roughly correlated with the late Miocene-Pliocene European mammal zones MN10-15. In turn, levels with A. laogouense of the Kamlial Formation would correlate with the middle–late Aragonian (middle Miocene) European MN5. The recognition of the Chinese species A. laogouense in the Potwar Plateau represents the first discovery of this taxon in Pakistan and increases the geographical and stratigraphic distributions of this species, and adds to the rhinocerotid association from the Siwaliks. In turn, the presence of A. complanatum in the Siwaliks of Potwar Plateau also enlarges its geographic distribution in Pakistan, as it was previously known from the Bugti Hills of Balouchistan. The absence of Alicornops from the Siwaliks in the Chinji and Nagri formations (between late MN5 and MN9 zones) might be due to an inadequate fossil record, as other rhinocerotid species are known from Kamlial to Dhok Pathan formations. However, the two recorded species of Alicornops could also reveal two independent migration waves as supported by the appearance of other taxa in different formations. A summary of fossil Cenozoic rhinocerotids from different areas of Pakistan is also presented.     

Early Paleogene decapod crustaceans from the Sulaiman and Kirthar Ranges,Pakistan

A set of Paleocene and Eocene decapod crustaceans is described from the Sulaiman and Kirthar Ranges of Pakistan. The fossil crabs Proxicarpilius planifrons Collins and Morris, 1978 and Pakicarcinus orientalis (Collins and Morris, 1978), already known in the Eocene of northern Pakistan, are reported for the first time in the Paleocene of southern Pakistan, enlarging the stratigraphic and the palaeobiogeographical ranges of these species. The callianassid genus Calliax de Saint Laurent, 1973 is reported for the first time in the Paleocene of southern Pakistan; this is the oldest record for the genus.     

First record and taxonomic description of the genus Thysanoplusia (Fabricius) (Lepidoptera: Noctuidae: Plusiinae) from Pakistan

Species belonging to genus, Thysanoplusia Fabricius of the subfamily Plusiinae which are polyphagous in nature and pest of vegetables, foods, legumes, fodder, fruits, ornamental plants and cotton crops. Samples were collected from different localities of district Bahawalpur. For collection, comprehensive and comparative surveys were carried out during 2017–18 on taxonomic account of species of the genus Thysanoplusia Fabricius and resulted identified one species Thysanoplusia orichalcea (Fabricius) first time from Pakistan. Morphological characters viz., vertex, frons, labial palpi, antennae, compound eyes, ocelli, proboscis, wing venation, male and female genital characteristics were used for the identification and classification. Dichotomous keys and photographs are also provided. There is hardly any substantial research work on taxonomic studies of subfamily Plusiinae Pakistan. So to fill this gap the present proposal was designed to study the diversity of Noctuid moths from Pakistan and very fruitful results have been obtained.     

DNA Barcoding of Bemisia tabaci Complex (Hemiptera: Aleyrodidae) Reveals Southerly Expansion of the Dominant Whitefly Species on Cotton in Pakistan

Background

Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan.

Methods/Principal Findings

Sequence diversity in the DNA barcode region (mtCOI-5′) was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3′ to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage “Pakistan”. The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and “Pakistan” were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan.

Conclusions

DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.     

Morphology and molecular characterization of Acanthogyrus (Acanthosentis) bilaspurensis Chowhan,Gupta & Khera, 1987 (Acanthocephala: Gyracanthocephala: Quadrigyridae) from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan

Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 is a poorly known acanthocephalan species reported from the reba carp Cirrhinus reba (Hamilton) (Cypriniformes: Cyprinidae) in India. In the present study, the detailed morphology of A. (Acanthosentis) bilaspurensis were studied using light microscopy and, for the first time, scanning electron microscopy (SEM), based on newly collected specimens from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan. The SEM observations revealed the outer shell of eggs with numerous remarkable protuberances and the gonopore of female located at a discoid protrusion surrounded by some very small spines. This is the first time that A. (Acanthosentis) bilaspurensis was reported in Pakistan, and the common carp represents a new host for this species. In addition, the molecular characterization of the 18S and 28S rDNA of A. (Acanthosentis) bilaspurensis is provided for the first time. There was no intraspecific genetic variation detected in the 18S and 28S regions between different individuals of A. (Acanthosentis) bilaspurensis, but high level of interspecific nucleotide divergence was found in the 18S (4.15–16.7%) and 28S regions (3.60–15.4%). Phylogenetic results based on the 18S, 28S and 18S + 28S sequence data, respectively, all revealed the Quadrigyridae, Pallisentinae and the genus Neoechinorhynchus are not monophyletic groups, and the genera Acanthogyrus and Pallisentis have far relationship. The systematic status of the genus Acanthogyrus and some species of Neoechinorhynchus need further clarify based on different mtDNA and nuclear DNA data including broader representatives of the class Eoacanthocephala.     

Contribution to the flora of Pakistan: Hedysarum alii and H. shahjinalense spp. nov. (Fabaceae) and three new records

Two new species of Hedysarum, H. alii Haidar Ali & Qaiser sp. nov. and H. shahjinalense Haidar Ali & Qaiser sp. nov. are described from Chitral in Pakistan. As far as known, both new species are endemic to Chitral and are categorized as ‘Critically Endangered’ (CR). In addition, three other species of Hedysarum are recorded from Pakistan for the first time, viz. H. volkii Rech. f., H. brahuicum Boiss. and H. sericeum M. Bieb. These are also ‘Critically Endangered’ (CR) at a national level. A key to all 12 species of Hedysarum occurring in Pakistan is provided.     

Description of Quinisulcius solani n.sp. (Nematoda: Tylenchorhynchidae) with a Key to the Species and Data on Scutylenchus koreanus from Pakistan

A new species. Quinisulcius solani, is described and illustrated from specimens on Solanum tuberosum from Murree Hills, Pakistan. Q. solani n.sp. differs from its closest relative, Q. acutus (Allen, 1955) Siddiqi, 1971, by its spiral to open ''C'' shaped body and stylet length of 19 μn, vs. 17 μm in Q. acutus. In Q. acutus the stylet knobs project anteriorly but slope posteriorly in Q. solani n.sp. Tail annules number 17 in Q. acutus but 38 in Q. solani n.sp.; also the phasmids in the former species are at mid-tail and in the latter are in anterior half of tail, at about 34%. Q. solani n.sp. is also closely related to Q. capitatus (Allen, 1955) Siddiqi, 1971 but differs in some characters. Head annules number eight and stylet length is 17 μm in Q. capitatus, but head annules are six and stylet length is 19 μm in Q. solani n.sp. In Q. capitatus T/ABW is 3 vs. 2.2 in Q. solani n.sp. Also, phasmids are located at mid-tail on Q. capitatus but at ahout one-third of the tail on Q. solani n.sp. A key to the 10 species of Quinisulcius is also presented. Scutylenchus koreanus (Choi &Geraert, 1971) Siddiqi, 1979 is recorded for the first time in Pakistan and morphometric data and illustrations given.     

Systematic status of true katydids Sathrophyllia (Orthoptera,Tettigonioidea, Pseudophyllinae) from Pakistan,with description of two new species

The genus Sathrophyllia Stål, 1874 from Pakistan is reviewed with four species recorded. The diagnostic characters are given and two new species Sathrophyllia saeedi sp. n. and Sathrophyllia irshadi sp. n. are described. In addition to that Sathrophyllia nr. rugosa (Linnaeus, 1758) and Sathrophyllia femorata (Fabricius, 1787) are re-described. Further information on the distribution and ecology of the species is given and a key to studied species of Sathrophyllia is presented. Sathrophyllia femorata (Fabricius, 1787) and Sathrophyllia rugosa (Linnaeus, 1758) are recorded from Rawalakot (KPK) and Tharparker (Sindh), Pakistan for first the time.     

Contribution to the marine algae from the coast of West Pakistan III. Marine Vaucheria from Karachi

The coast of West Pakistan is represented by three marine Vaucheria, i.e. V. piloboloides Thur., V. longicaulis Hopp., and V. marina Lyngbye. (?). The latter two species are new records from this area. The ecology and distribution of these species have been described.     

Notes on genusPotentilla (Rosaceae) from Pakistan and Kashmir

A new species,P. grandiloba, and a new variety,P. pamirica var.khunjrabensis, from Pakistan are described. New status has been given toP. grisea var.vilijuga. P. aphanes is recorded for the first time from Pakistan.