Molecular Phylogenetic Analysis Research Articles

Molecular characteristics and phylogenetic analysis of pigeon paramyxovirus type 1 isolates from pigeon meat farms in Shanghai (2009–2012)

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3′-NP-P-M-F-HN-L-5′. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai’s meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks.

Molecular identification and characterizations of rhizobacterial isolates collected from lentil rhizosphere of Indo-gangetic plains.

Plant growth promoting rhizobacteria (PGPR) are also known to colonize in the soil rhizosphere and prevent the development of other soil borne pathogens residing in the root surface. These microorganisms play a vital role in growth and development of the plant and also enhances the soil fertility by enriching the soil with different beneficial nutrients. This study was aimed at isolation of different rhizobacteria and their molecular characterization in search of efficient bacterial strains with multiple growth regulating activities. A total 36 bacteria were isolated from lentil root nodule as well as soil from different lentil growing fields with a view to screen/evaluate their plant growth promoting potential. Morphological characterization of isolated rhizobacterial candidates were done by observing the colonies on YEMA and nutrient agar media. Determination of CFU, Congo red test and gram staining tests were done to further screen them according to their morphology. All the isolates were then undergone molecular phylogenetic analysis using the partial sequences of the 16S rDNA. Based upon the Gram staining test, all the isolates were negative in gram reaction except six Bacillus isolates, PSB2 and AB3. Results of Ribosomal Database Project (RDP) and Basic Local Alignment Search Tool for Nucleotide Sequences (BLASTn) from 16S rDNA gene sequences showed that these isolates are genetically diverse. A total of 15 isolates of Rhizobium, 6 isolates of Bacillus, 3 isolates of Pseudomonas, 2 isolates of Phosphate Solubilizing Bacteria, 4 isolates of actinomycetes were identified by molecular sequencing of their 16S rDNA region and comparing them with the other isolates enlisted in the database of NCBI for the similarity percentage, query coverage. The purpose of the present study was to select native rhizosphere bacteria from the lentil nodule and soil of Lentil field and to evaluate their plant growth promoting potential as an alternative of chemical fertilizer for sustainable, environment friendly agriculture and assessment of their phylogenetic characterization.

Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria’s ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

Contributions to the Knowledge of Nemognathinae (Coleoptera: Meloidae) from China

Despite being the most widespread blister beetle subfamily, Nemognathinae is unfairly understudied in China. In this study, a new genus and species, Sinostenoria yangi Pan, from northern China is described and illustrated. The antennae, elytra, hind wings, and claws of the new genus form a truly unique set of characteristics never observed in other genera of Nemognathini Laporte de Castelnau, 1840. Three species from China are newly recorded and illustrated: Megatrachelus sibiricus (Tauscher, 1812), Zonitomorpha dollei (Fairmaire 1889), and Stenodera djakonovi Aksentjev, 1978. The genus Oreomeloe Tan, 1981, is transferred from the tribe Meloini Gyllenhal, 1910, to Nemognathini based on an examination of the types. Aiming to test the morphology-based placement of the new genus, we conducted molecular phylogenetic analyses using two mitochondrial (COI, 16S) and three nuclear markers (28S, CAD, ITS2). The results confirm our tribal assignment of the new genus and support a clade that consists of Sinostenoriagen. n., Longizonitis Pan and Bologna, 2018, Stenoria cf. grandiceps, and Ctenopus cf. persicus.

Molecular phylogenetic analysis of the East Asian hemicultrine fishes (Teleostei: Cyprinidae: Xenocypridinae), with suggestions to their generic classification and redescription of the recently described species Hemiculter yungaoi Vasil'eva et al. 2022.

The hemicultrine fishes are a group of small-sized cyprinids, widely distributed but endemic to East Asian rivers and lakes. Till now, the taxonomic boundaries and relationships within this group remain poorly explored. In the present study, we study the phylogeny of this group, providing suggestions for classification of the hemicultrine group. Using two mitochondrial and three nuclear genes, and samples representing all genera, our results showed that the group consists of seven major lineages, of which four (Hemiculterella, Hainania, Pseudolaubuca, and Anabarilius) were monophyletic and three (Hemiculter, Toxabramis, and Pseudohemiculter) were not. Based on the phylogenetic tree, we redefined the genera. We revive the genus Siniichthys, which has three species, Siniichthys bleekeri, Siniichthys lucidus, and S. varpachovskii, that were previously treated as members of the genus Hemiculter but showed distant relationships to the genus Hemiculter in our phylogenetic tree. With the new results, a diagnostic key for clades of the hemicultrine group is provided. Furthermore, we provide more detailed information on diagnostic features of the recently described species Hemiculter yungaoi (Vasil'eva et al., 2022). This work will facilitate future systematic studies, pave the way for evolutionary studies, and provide valuable information for the urgent conservation of hemicultrine fishes.

Two new species of Dictyota (Dictyotales, Phaeophyceae), D. dimorphosa sp. nov. and D. recumbens sp. nov., based on morphological and molecular investigations

ABSTRACT The brown algal genus Dictyota (Dictyotales, Phaeophyceae) is widespread, particularly in warm temperate and tropical waters, and plays a vital role in coastal ecosystems. This study reassessed the taxonomy of Dictyota, focusing on specimens from Myanmar and Japan, using a combination of morphological and molecular analyses. Molecular phylogenetic analyses using concatenated DNA sequences of rbcL, psaA, psbA, cox1 and cox3 genes revealed that Dictyota specimens from Myanmar, Japan, and other regions, belong to nine different clades, each representing a different species. Based on morphological and molecular data, seven out of these nine clades are identified as known species, while the remaining two clades are described as new species D. recumbens sp. nov. (=D. sp. A) and D. dimorphosa sp. nov. (=D. sp. B). Dictyota recumbens is characterized by the recumbent thallus, with transverse yellowish-green iridescent lines, and the formation of solitary spores irregularly distributed over the thallus. Dictyota dimorphosa is characterized by two morphological forms: decumbent and erect thalli, with bluish iridescence mainly in the decumbent thallus, and the formation of spores in oval ring shapes distributed over both thallus surfaces. Dictyota recumbens was typically found in tropical regions, while D. dimorphosa appears to be a cool-warm temperate species, distributed from northeastern Hokkaido to southern Kyushu with an extension along South Korea. Our molecular phylogenetic analyses have revealed the existence of several species whose names appeared in multiple clades, suggesting the potential for future increases in species diversity and the presence of cryptic species within certain groups.

Integrative Taxonomy of Urocleidoides spp. (Monogenoidea, Dactylogyridae) Parasites of Pseudanos trimaculatus (Characiformes: Anostomidae) from Eastern Amazon, Brazil.

Two new species of Urocleidoides are described from the gills of Pseudanos trimaculatus (Characiformes: Anostomidae) from the coastal drainages of the Eastern Amazon, Brazil. Urocleidoides itabocaensis n. sp. is characterized by having a sclerotized, tubular, sigmoid male copulatory organ (MCO), a circular sclerotized tandem brim associated with the base of the MCO; an accessory piece articulated with the MCO, aV-shaped, divided into two subunits, distal subunit spoon-shaped; and a vaginal pore dextroventral with opening marginal. Urocleidoides omphalocleithrum n. sp. is characterized by presenting a C-shaped or sigmoid MCO; anaccessory piece articulated with the MCO, L-shaped, divided into two subunits, distal subunit gutter-shaped; a vaginal pore dextroventral with opening marginal; and aventral bar broadly V-shaped, with anteromedial projection. Molecular phylogenetic analysis based on partial 28S rDNA and COI mtDNA genes indicate that U. itabocaensis n. sp. and U. omphalocleithrum n. sp. are closely related and appear as a sister group to other Urocleidoides species (U. paradoxus, U. digitabulum and U. sinus) parasitizing anostomid fishes. This study represents the first record of monogenoids from the gills of P. trimaculatus for the Eastern Amazon.

Two new species of Platanthera (Orchidaceae, Orchidoideae) from Xizang, China

Two new orchid species, Platanthera linzhiensis, P. deergongensis, are described and illustrated from Motuo, Xizang, China. P. linzhiensis is closely related to P. uniformis, but differs from it by lip yellowish green, spur yellow, nearly as long as ovary, connective obtuse-truncate, not protruding from anther, column lateral appendages ovate to oblong, slightly shorter than the anther. P. deergongensis is morphologically similar to P. contigua, but is distinguished from it by having white lip, obtuse-truncate connective and ovate to oblong column lateral appendages. Molecular phylogenetic analysis based on six markers (nrITS, psaB, matK, psbA-trnH, rbcL, trnL-F) revealed that P. linzhiensis and P. deergongensis formed a clade with high support, and are close to P. superantha.

A new species of Platanthera (Orchidaceae, Orchideae) from southeast of Xizang, China

A new species of orchid, Platanthera medogensis, was described and illustrated from Xizang Autonomous Region, China. P. medogensis is close to P. bakeriana and P. chiloglossa, but is readily distinguished from the latter by having ovate-lanceolate lip concave at base with two calli, linear visidium, two stigmas below the rostellum. Molecular phylogenetic analysis based on five markers (nrITS, matK, psbA-trnH, rbcL, trnL-F) revealed that P. medogensis is sister with P. exelliana by full support.

Molecular phylogeny and secondary structure analysis of hop stunt viroid (HSVd) associated with Mulberry (Morus alba) in India.

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants.

A new name and two new combinations in Leucheria Lag. (Asteraceae; Nassauvieae)

Molecular phylogenetic analysis has demonstrated that Polyachyrus Lag. species must be reclassified in Leucheria Lag. Here, Polyachyrus cinereus Ricardi & Weldt is renamed as Leucheria duraniorum, and Polyachyrus gayi J.Rémy and Polyachyrus poeppigii Kunze ex Less. subsp. multifidis (D.Don) Ricardi & Weldt are recombined in Leucheria.

Impatiensbeipanjiangensis (Balsaminaceae), a new species from Guizhou, China.

Impatiensbeipanjiangensis Jian Xu & H. F. Hu (Balsaminaceae), a new species of Impatienssubg.Clavicarpa discovered in Guizhou, China, is described and illustrated in this study along with its molecular phylogenetic analysis. I.beipanjiangensis is similar to I.liboensis, I.chishuiensis and I.clavigera in morphology, but I.tubulosa has the closest relationship to it. However, there are various ways in which the new species can be easily distinguished from these four species: Inferior nodes swollen rhizoid, pale green and with hooked outer sepals, longer lateral united petals, subovate auricle, deeper lower sepal and shorter spur that is reflexed towards the lower sepal. Furthermore, I.beipanjiangensis is distinguished from other Impatiens species, based on morphological, micromorphological and palynological evidence and molecular data (PP 0.967).

A new species of Habenaria (Orchidaceae, Orchidoideae, Orchideae) from Yunnan, China

A new species of Orchidaceae, Habenaria tiechangensis, from Yunnan province is described and illustrated based on morphological characters and molecular phylogenetic analysis. The results indicated that it belongs to H. josephi group and is similar to H. diplonema but differs from the latter by having short and lanceolate lateral lobes of lip and a pair of distinctive horn-like stigmas.

Description of Pauronemoides siddiqii n. gen., n. sp. (Tylenchomorpha, Paurodontidae), a morphological and molecular phylogenetic study

Summary Three populations of Pauronemoides siddiqii n. gen., n. sp. were recovered from three different locations in northwestern and northern Iran from bark and soil samples. The new genus was described and illustrated based upon morphological and molecular characteristics. Pauronemoides n. gen. belongs to the family Paurodontidae and is characterised by the medium-sized body, asymmetrical knobs in stylet base, stem-like extension into intestine, post-vulval uterine sac (PUS) absent or rudimentary, different tail tip shape between sexes, and bursa enveloping tail or reaching to the tail end. The type species, Pauronemoides siddiqii n. gen., n. sp., is mainly characterised by 752-1281 μm body length in females and 660-1085 μm in males, six equally-sized lips, stylet 9-11 μm long with asymmetrical knobs, six incisures in lateral fields forming five bands, three of which (two outers and one central) are wider, and variably lengthened stem-like extension projecting into the intestine. Vulva situated posteriorly (V = 87.7-91.7%), PUS absent. Conical tail with widely rounded end, having a small mucron at tip in females, males with conical tail ending to a narrower tip and bursa enveloping tail completely. The type species of the new genus was compared with all known species of the genera Prothallonema, Paurodontus, Paurodontoides and Paurodontella. Pauronemoides siddiqii n. gen., n. sp. resembles Prothallonema macrocellum and P. mucronatum by having the bulb extension penetrating into the intestine, dimorphism in tail shape of both sexes and bursa enveloping tail, and Paurodontella iranica by tail shape dimorphism between sexes and bursa ending near tail tip. Therefore, the above-mentioned species are transferred to the new genus as Pauronemoides macrocellus n. gen., n. comb. (= Prothallonema macrocellum), P. mucronatus n. gen., n. comb. (= Prothallonema mucronatum) and P. iranicus n. gen., n. comb. (= Paurodontella iranica). In addition to morphological comparisons, molecular phylogenetic analyses were performed using two sequences of small subunit (SSU), and three partial sequences of D2-D3 expansion segments of the large subunit (LSU) rDNA, and the resulting topologies were discussed.

Meconopsis biluoensis (Papaveraceae), a new species revealed by population-level investigation.

Meconopsis biluoensis, a new species of Papaveraceae in an alpine meadow from Yunnan, Southwest China, is described and illustrated. Morphologically, it resembles Meconopsis georgei, while it is distinct in acaulescent and hispid with clearly expanded bases on the leaves. A genus-level molecular phylogenetic analysis supported the closest relationship between M. biluoensis and M. georgei. In a finer population-level molecular phylogenetic analysis using ribosomal DNA (rDNA) and the chloroplast genome, individuals from M. biluoensis and M. georgei were clearly separated, and the extremely short branch length indicated that the two species had a very short differentiation time. The species has currently been assessed as "endangered" (EN) due to its small-sized population and narrow distribution following the IUCN categories and criteria.

Host–pathogen interaction between the African fig fly, Zaprionus indianus, and its external mycobiome under laboratory conditions

AbstractThe African fig fly, Zaprionus indianus Gupta (Diptera: Drosophilidae), is an ecologically diverse pest species that interacts with and feeds on various microbial pathogens including bacteria and yeast found in decomposing fruits. However, the interaction of Z. indianus with its microbial community and microbial pathogens is obscure. To determine the presence of fungal pathogens, we collected wild Z. indianus from the northwestern part of the Indian subcontinent. Based on molecular identification and phylogenetic analysis, Debaryomyces hansenii (Zopf) Lodder & Kreger‐van Rij (isolate ziha1) was the most commonly observed fungus associated with Z. indianus. Furthermore, we identified two more opportunistic fungal pathogens: Aspergillus flavus Link (isolate zias2), and Pichia kudriavzevii Boidin, Pignal & Besson (isolate zibd3). The interaction of isolated fungi with Z. indianus was evaluated in terms of larval mortality, adult emergence, and fecundity. Debaryomyces hansenii ziha1 resulted in 90% emergence rate for adults, and did not cause significant mortality in the larval stage. In contrast, A. flavus zias2 and P. kudriavzevii zibd3 showed a significant reduction in fecundity and caused 99% and 74% larval mortality of Z. indianus, respectively. In the laboratory oviposition preference assay, mated females of Z. indianus preferred D. hansenii ziha1 supplemented food with a positive oviposition index compared to the uninfected control and compared to food infected with P. kudriavzevii zibd3 or A. flavus zias2. These findings underscore Z. indianus' potential to act as a reservoir for both symbiotic and pathogenic fungal species, some of which may be further harnessed for effective fruit fly pest control strategies.

Molecular detection and phylogenetic analysis of tick-borne pathogens in cattle from southern Malawi.

Tick-borne diseases (TBDs) pose a major hindrance to livestock production in countries with limitedresources. Effective prevention and management of TBDs require a thorough understanding of disease vectorsand pathogens. However, there is limited information on studies of bovine tick-borne pathogens (TBPs) using molecular methods in Malawi. This study aimed to detect TBPs of cattle populations in southern Malawi, which has the largest cattle population in the country. A total of 220 blood samples from apparently healthy cattle were collected in six districts, and werescreened for selected TBPs using polymerase chain reaction (PCR). The overall detection rate of TBPs was 72.3%. Among the detected pathogens, Babesia bigemina hadthe highest detection rate (34.5%), followed by Anaplasma marginale (23.2%), Anaplasma phagocytophilum(22.3%), Theileria taurotragi (22.3%), Theileria parva (15.5%),Anaplasma bovis (9.6%), Babesia bovis (7.3%),Theileria mutans (4.1%), and Babesia naoakii (2.7%). Among the positive samples, 64.2% were found to be co-infected with two or more TBPs, with the highest number of seven pathogens detected in a single sample. Thestudy documents the existence of A. phagocytophilum, B. bovis, and B. naoakii in Malawian cattle for the first time. The findings herein demonstrate a significant burden of TBPs on cattle in Malawi, which gives a challenge in combating TBDs. The high TBP burden, along with the high co-infection frequencies in Malawian cattle necessitates the urgency to implement effective control strategies to enhance cattle production in the country.

Molecular phylogeny confirms the subspecies delineation of the Malayan Siamang (Symphalangussyndactyluscontinentis) and the Sumatran Siamang (Symphalangussyndactylussyndactylus) based on the hypervariable region of mitochondrial DNA.

Siamangs (Symphalangussyndactylus) are native to Peninsular Malaysia, Sumatra and southern Thailand and their taxonomical classification at subspecies level remains unclear. Morphologically, two subspecies were proposed as early as 1908 by Thomas namely Symphalangus s.syndactylus and Symphalanguss.continentis. Thus, this study aims to clarify the Siamang subspecies status, based on mtDNA D-loop sequences. Faecal samples were collected from wild Siamang populations at different localities in Peninsular Malaysia. A 600-bp sequence of the mitochondrial D-loop region was amplified from faecal DNA extracts and analysed along with GenBank sequences representing Symphalangus sp., Nomascus sp., Hylobates sp., Hoolock sp. and outgroups (Pongopygmaeus, Macacafascicularis and Papiopapio). The molecular phylogenetic analysis in this study revealed two distinct clades formed by S.s.syndactylus and S.s.continentis which supports the previous morphological delineation of the existence of two subspecies. Biogeographical analysis indicated that the Sumatran population lineage was split from the Peninsular Malaysian population lineage and a diversification occurrred in the Pliocene era (~ 3.12 MYA) through southward expansion. This postulation was supported by the molecular clock, which illustrated that the Peninsular Malaysian population (~ 1.92 MYA) diverged earlier than the Sumatran population (~ 1.85 MYA). This is the first study to use a molecular approach to validate the subspecies statuses of S.s.syndactylus and S.s.continentis. This finding will be useful for conservation management, for example, during Siamang translocation and investigations into illegal pet trade and forensics involving Malayan and Sumatran Siamangs.

Molecular Identification, Multigenic Characterization and Phylogenetic Analysis of Ctenocephalides Canis Fleas from Humans and Domestic Animals in Iran.

Fleas belonging to the Pulicidae are prevalent ectoparasites infesting mammals and birds in Iran. This study focused on genetically identifying and characterizing Ctenocephalides canis collected both off-host and infesting humans and various domestic animals in the country. A total of 918 adult flea samples were collected from 10 sites in western and northwestern Iran between April 2018 and May 2019. Out of these, 71 specimens were found off-host, while the remaining fleas were collected from humans (121), sheep (126), goats (184), and dogs (416). Morphological identification at the genus level was performed on all fleas, and ten selected specimens selected based on the sampling sites and hosts were subjected to molecular detection at the species level by using partial amplification and sequencing of the internal transcribed spacer 1 and 2, as well as the cytochrome oxidase I (COXI) markers. The morphological identification confirmed all fleas as Ctenocephalides spp. Alignment and phylogenetic analysis of nuclear and mitochondrial partial sequences confirmed the presence of C. canis. However, molecular divergence was observed among the ten isolates based on the ITS1 and ITS2 with diversity rates estimated at 0.15% and 3.36%, respectively. Notably, the analysis of the COXI marker revealed no molecular divergence among the partial sequences representing the ten studied isolates from C. canis. This study explores the diversity of C. canis in the western and northwestern regions of Iran, providing insights into their molecular taxonomy and potential role as disease vectors in these areas.

The phylogeny of the tribe Podonomini (Chironomidae: Podonominae) is clarified by new inclusion of Rheochlus Brundin in molecular analysis

Abstract The immature stages of the patagonian podonomine (Diptera: Chironomidae) genus Rheochlus (Diptera: Chironomidae) have been unknown although the adult stage was described over half a century ago. New pupal associations with described adults belonging to two species of Rheochlus allow better understanding of the genus, its diversity and its phylogeny. The pupa of Rheochlus insignis Brundin is the same as an unreared damaged pupal taxon designated Podochlus sp. “Canteras’‘ by Brundin and is described more completely here. Inclusion of R. insignis in a molecular phylogenetic analysis shows it to be sister to two specimens (one a male, the other pharate female) allocated previously to Podochlus but better transferred to Rheochlus. The male morphology is compatible with Rheochlus prolongatus Brundin but we refrain from asserting identity by using ‘cfr.’ prolongatus to indicate the status. Molecular evidence also provides association of the female adult and pupa that are newly described here. We validate Rheochlus as distinguished on pupa and adult, with the type-species Rheochlus insignis from Patagonian Chile and Argentina, Rheochlus prolongatus Brundin from Southern Patagonia Argentina and possibly also from central Patagonia, Rheochlus latisetus Siri & Brodin from Patagonia, and the eastern Australian Rheochlus wirthi Brundin. The latter two species remain known only from their original adult male descriptions. Strengthened support for our concept of Rheochlus as a valid genus, sister group to Podochlus, derives also from an expanded and reanalysed morphological matrix.