Bayesian Phylogenetic Analyses Research Articles

Paramerlinius shamimi n. sp. (Nematoda: Merliniidae) from India with a tabular key for the genus Paramerlinius

Summary The population of the genus Paramerlinius was discovered from potato rhizosphere in a government potato seed farm, located in Tiksen, Munsiari, Pithoragarh, Uttarakhand, India. It is here described as Paramerlinius shamimi n. sp. based on morphological, morphometric and molecular data. The new species is characterised by having a body length of 841.8-1208.8 μm, continuous head bearing 7-8 annuli, a prominent stylet (28.4-32.3 μm) with well-developed posteriorly sloping basal knobs, six lateral incisures at the mid-body, a well-developed median bulb, a secretory-excretory (S-E) pore (123.5-160.7 μm) almost at the level of basal bulb, hemizonid covering 2-3 annuli that are located 3-4 annuli above the S-E pore, phasmids located in the front half of the tail, tail subcylindrical, having a hemispherical smooth terminus (62.7-94.1 μm) with 38-48 tail annuli, males common, spicule arcuate (27.4-31.4 μm), gubernaculum curved ventrally (8.8-9.8 μm) and peloderan bursa. Molecular characterisation of the population was performed by sequencing nuclear ribosomal DNA (rRNA) genes, viz. ITS-1, 18S (partial) and D2-D3 region of 28S rRNA. Maximum likelihood and Bayesian phylogenetic analyses of P. shamimi n.sp. using the D2-D3 region show it forms a distinct clade with other unidentified Paramerlinius spp. and P. neohexagrammus and, using 18S rRNA, groups it in the same clade as P. hexagrammus. A tabular key for 13 species including P. shami1mi n.sp. under the genus Paramerlinius is given.

Oldest record of a crown anomaluroid rodent from sub-Saharan Africa: a new genus and species from the early Oligocene Topernawi formation of northern Kenya

ABSTRACT Anomaluroid rodents (i.e. Anomaluridae + Zenkerellidae) have a long evolutionary history in Africa dating back to at least the middle Eocene, but Palaeogene fossil members of the anomaluroid crown group have never been found south of the Sahara. Here, we describe the oldest crown anomaluroid from sub-Saharan Africa, Eliwourus topernawiensis (new genus and species) from the early Oligocene Topernawi Formation of northern Kenya. Bayesian phylogenetic tip-dating analysis of a combined molecular-morphological dataset places ~29.5 Ma Eliwourus as an advanced stem anomalurid, and suggests that crown Anomaluridae originated ~28.2 Ma. The gliding behaviour seen in all crown anomalurids had, therefore, likely evolved before the close of the early Oligocene. Bayesian geocoordinates analysis places the origin of crown Anomaluroidea (~47.2 Ma) in northern Africa, and identifies a stem anomalurid dispersal into equatorial eastern Africa in the latest Eocene or earliest Oligocene. We present the first quantitative analysis of the pace of anomaluroid dental evolution and confirm a remarkably slow or bradytelic rate of phenotypic change, despite significant transformations in postcranial morphology related to the evolution of gliding behaviour during the same interval. The Topernawi area was evidently sufficiently forested during the early Oligocene to support both arboreal rodents and primates.

Bayesian phylogenetic analysis of Philippine languages supports a rapid migration of Malayo-Polynesian languages

The Philippines are central to understanding the expansion of the Austronesian language family from its homeland in Taiwan. It remains unknown to what extent the distribution of Malayo-Polynesian languages has been shaped by back migrations and language leveling events following the initial Out-of-Taiwan expansion. Other aspects of language history, including the effect of language switching from non-Austronesian languages, also remain poorly understood. Here we apply Bayesian phylogenetic methods to a core-vocabulary dataset of Philippine languages. Our analysis strongly supports a sister group relationship between the Sangiric and Minahasan groups of northern Sulawesi on one hand, and the rest of the Philippine languages on the other, which is incompatible with a simple North-to-South dispersal from Taiwan. We find a pervasive geographical signal in our results, suggesting a dominant role for cultural diffusion in the evolution of Philippine languages. However, we do find some support for a later migration of Gorontalo-Mongondow languages to northern Sulawesi from the Philippines. Subsequent diffusion processes between languages in Sulawesi appear to have led to conflicting data and a highly unstable phylogenetic position for Gorontalo-Mongondow. In the Philippines, language switching to Austronesian in ‘Negrito’ groups appears to have occurred at different time-points throughout the Philippines, and based on our analysis, there is no discernible effect of language switching on the basic vocabulary.

Comparative analysis of the mitochondrial genomes of Smoothhound sharks provide insight into the phylogenetic relationships within the family Triakidae

The genus Mustelus comprises demersal smoothhound sharks, including M. canis and M. norrisi. Despite their economic value and known conservation concerns, only a few genetic and no genomic resources exist for these two species. In this study, we assembled and described the complete mitochondrial genomes of Mustelus canis and M. norrisi. The mitochondrial genomes of M. canis and M. norrisi are 16,758 bp and 16,769 bp in length, respectively. Both mitogenomes are A + T rich and contain 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (CR) 1122 bp and 1121 bp in length, respectively. Mitochondrial synteny observed in M. canis and M. norrisi is identical to that previously reported for congeneric species. In the studied mitochondrial genomes, all PCGs experienced purifying selection. In both species, 21 of the 22 tRNA genes exhibited a typical ‘cloverleaf’ secondary structure, except trnS1 that lacked a complete D-arm. The CR of the studied species lacked tandem repeats, but abundant A + T rich dinucleotide microsatellites were detected. Maximum likelihood and Bayesian inference phylogenetic analyses based on translated PCGs supported the monophyly of the family Triakidae. The same analyses indicated that the genus Mustelus was not monophyletic considering that all representatives of the genus Mustelus clustered together with Triakis megalopterus in a fully supported clade. The assembled mitochondrial genomes will aid with the accurate identification of specimens belonging to the genus Mustelus and support biomonitoring programs based on environmental DNA (eDNA).

Importations of SARS-CoV-2 lineages decline after nonpharmaceutical interventions in phylogeographic analyses

During the early stages of the SARS-CoV-2 pandemic, before vaccines were available, nonpharmaceutical interventions (NPIs) such as reducing contacts or antigenic testing were used to control viral spread. Quantifying their success is therefore key for future pandemic preparedness. Using 1.8 million SARS-CoV-2 genomes from systematic surveillance, we study viral lineage importations into Germany for the third pandemic wave from late 2020 to early 2021, using large-scale Bayesian phylogenetic and phylogeographic analysis with a longitudinal assessment of lineage importation dynamics over multiple sampling strategies. All major nationwide NPIs were followed by fewer importations, with the strongest decreases seen for free rapid tests, the strengthening of regulations on mask-wearing in public transport and stores, as well as on internal movements and gatherings. Most SARS-CoV-2 lineages first appeared in the three most populous states with most cases, and spread from there within the country. Importations rose before and peaked shortly after the Christmas holidays. The substantial effects of free rapid tests and obligatory medical/surgical mask-wearing suggests these as key for pandemic preparedness, given their relatively few negative socioeconomic effects. The approach relates environmental factors at the host population level to viral lineage dissemination, facilitating similar analyses of rapidly evolving pathogens in the future.

Morphology and systematics of a new fossil snake from the early Rupelian (Oligocene) White River Formation, Wyoming

Abstract Extinct snake taxa are recognized primarily from isolated vertebrae. A new specimen from the early Oligocene of Wyoming provides a rare opportunity to examine four nearly complete and articulated fossil snakes. Informally assigned previously to the ‘erycine’ vertebral form taxa Ogmophis and Calamagras, a detailed comparison reveals that this fossil snake exhibits vertebral differences from both taxa and is, furthermore, a new taxon, Hibernophis breithaupti gen. et sp. nov., based on a combination of apomorphies such as absence of basal tubera, low subrectangular prootic, low parasphenoid wings obscuring the anterior opening of the Vidian canal, and foramen for the mandibular branch of the trigeminal nerve posteriorly displaced inside the adductor fossa of the compound. Parsimony and Bayesian phylogenetic analysis of combined morphological and molecular data from a broad selection of snakes places Hibernophis breithaupti as sister taxon to all other booids, distant from both Old and New World ‘erycines’. However, an alternative position close to New World ‘erycines’ and ungaliophiines cannot be rejected.

A late-Ediacaran crown-group sponge animal.

Sponges are the most basal metazoan phylum1 and may have played important roles in modulating the redox architecture of Neoproterozoic oceans2. Although molecular clocks predict that sponges diverged in the Neoproterozoic era3,4, their fossils have not been unequivocally demonstrated before the Cambrian period5-8, possibly because Precambrian sponges were aspiculate and non-biomineralized9. Here we describe a late-Ediacaran fossil, Helicolocellus cantori gen. et sp. nov., from the Dengying Formation (around 551-539 million years ago) of South China. This fossil is reconstructed as a large, stemmed benthic organism with a goblet-shaped body more than 0.4 m in height, with a body wall consisting of at least three orders of nested grids defined by quadrate fields, resembling a Cantor dust fractal pattern. The resulting lattice is interpreted as an organic skeleton comprising orthogonally arranged cruciform elements, architecturally similar to some hexactinellid sponges, although the latter are built with biomineralized spicules. A Bayesian phylogenetic analysis resolves H. cantori as a crown-group sponge related to the Hexactinellida. H. cantori confirms that sponges diverged and existed in the Precambrian as non-biomineralizing animals with an organic skeleton. Considering that siliceous biomineralization may have evolved independently among sponge classes10-13, we question the validity of biomineralized spicules as a necessary criterion for the identification of Precambrian sponge fossils.

The complete mitochondrial genome of Membranipora villosa Hincks, 1880 (Bryozoa: Gymnolaemata: Cheilostomatida): phylogenetic relationship of two kelp-encrusting bryozoans within the suborder Membraniporina

The two commonest kelp-encrusting bryozoans, Membranipora villosa and M. membranacea, are difficult to distinguish morphologically. Molecular studies of M. villosa should thus be helpful for the identification of both species because the mitogenome of M. membranacea was already sequenced. The complete mitogenome of M. villosa collected from Sinjido was determined in this study through Illumina NovaSeq sequencing. Maximum-likelihood (ML) analysis was based on concatenated 13 protein-coding genes dataset from nine bryozoan species. The mitogenome length was 15,407 bp, and its gene arrangement was similar to those of the mitogenome of other membraniporids, having 13 PCGs, two ribosomal RNAs, and 22 tRNAs. It had an overall A + T content of 63.7% (29.7% A, 16.7% C, 19.6% G, and 34.0% T). M. villosa and M. membranacea showed sequence differences of 20% for the total length of mitogenome and 16.1.% for 13 PCGs. Molecular data definitely consider them to be separate species. Phylogenetic analyses based on the amino acids of 13 PCGs indicated that M. villosa has the closest relationship with another kelp-encrusting bryozoan, M. membranacea of membraniporids. The phylogenetic position of genera and families within the suborder Membraniporina coincides with the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions.

The cranial and postcranial morphology of Hutchemys rememdium and its impact on the phylogenetic relationships of Plastomenidae (Testudinata, Trionychidae)

Hutchemys rememdium is a poorly understood softshell turtle (Trionychidae) from the mid Paleocene of the Williston Basin of North America previously known only from postcranial remains. A particularly rich collection of previously undescribed material from the Tiffanian 4 North American Land Mammal Age (NALMA) of North Dakota is here presented consisting of numerous shells that document new variation, some non-shell postcrania, and cranial remains, which are described based on 3D models extracted from micro-CT data. Although the observed shell variation weakens previously noted differences with the younger species Hutchemys arctochelys from the Clarkforkian NALMA, the two taxa are still recognized as distinct. Parsimony and Bayesian phylogenetic analyses reaffirm the previously challenged placement of Hutchemys rememdium within the clade Plastomenidae, mostly based on novel observations of cranial characters made possible by the new material and the micro-CT data. The new topology supports the notion that the well-ossified plastron of plastomenids originated twice in parallel near the Cretaceous/Paleogene boundary, once in the Hutchemys lineage and once in the Gilmoremys/Plastomenus lineage. Hutchemys rememdium is notable for being the only documented species of trionychid in the mid Paleocene of the Williston Basin. The presence of multiple individuals in a carbonaceous claystone indicates this taxon lived in swamps and lakes and its expanded triturating surface suggests it had a durophagous diet.

Phylogenetic Analysis of Rare and Endangered Tulipa Species (Liliaceae) of Kazakhstan Based on Universal Barcoding Markers.

In Kazakhstan, the genus Tulipa is represented by 35 species, 18 of which are listed in the Red Data Book of Kazakhstan and protected by the state. Recent studies of tulip specimens from regions bordering Kazakhstan emphasize the significance of species inventory and report the discovery of several hybrids. In this study, eight tulip species were identified based on morphological characteristics and using DNA barcoding methods. Molecular genetic markers, including nrDNA (ITS) and cpDNA markers (rbcL, matK), of the studied species were sequenced and analyzed using the Bayesian inference and maximum likelihood phylogenetic analysis methods. Our work demonstrates that DNA barcodes based on the ITS, rbcL, and matK marker regions have successful practical applicability, with ITS being the most informative at the intragenic level. However, for distinguishing closely related taxa, the most effective approach would be to use a combined dataset of sequences from multiple DNA markers. The results showed discrepancies in the placement of several taxa (T. kaufmanniana, T. patens), likely due to introgression and natural spontaneous hybridization. The molecular phylogenetic analysis suggests the existence of a previously undescribed hybrid between T. patens and T. alberti. Further detailed population studies are needed to validate this hypothesis.

Rectifying a century of misidentifications: a taxonomic re-evaluation of sipo snakes (Colubridae: Chironius) on Trinidad

The island of Trinidad has several endemic reptile species that in some cases are morphologically indistinguishable, or almost so, from their mainland counterparts. In particular, many snakes from the island have not been examined thoroughly with modern scientific methods and may therefore be misidentified. At least two species of sipo snakes (Chironius carinatus, C. septentrionalis) are reported to inhabit Trinidad, both being considered species already inhabiting the mainland, though their identities are based solely on morphology. Here, we evaluate the molecular distinctiveness of these Trinidadian snakes and assess their relationships to other members of the genus. We constructed a multi-locus data set (12S rRNA, 16S rRNA, cyt b, ND4, cmos, NT3, Rag-1, Rag-2) including novel sequences and those available on GenBank to perform Bayesian and maximum likelihood phylogenetic analyses. Our phylogenetic reconstruction elucidates the identity of both Trinidad species, which have been misidentified since their discovery, and we provide a literature review of their taxonomic history. More specifically, our results suggest that the Trinidad population of Chironius carinatus is an undescribed species, that we describe herein as Chironius nigelnoriegai sp. nov., and is the sister to C. flavopictus. Our results also demonstrate that the Trinidad population previously identified as C. septentrionalis is instead the mainland species C. cochranae, a member of a clade with C. foveatus, C. laurenti, and C. multiventris. Finally, we generated a time tree and inferred that our new species separated from C. flavopictus approximately 4 million years ago in the Pliocene, a time when the island of Trinidad detached from Northern Venezuela. This work contributes to a better understanding of species diversity of Trinidad and we hope that it assists in conservation efforts towards this important endemic region. These findings support the prospect of rapid speciation on Trinidad and implies that more extensive surveys of island squamates will reveal additional cryptic diversity. http://zoobank.org/urn:lsid:zoobank.org:pub:500DB91B-62CD-4051-9831-0BEEAC64C8E2

DNA barcoding reveals cryptic species in the sea slater Ligia italica (Crustacea, Isopoda) from Tunisia

Barcoding studies have provided significant insights into phylogenetic relationships among species belonging to the genus Ligia (Crustacea, Isopoda). Herein the diversity of the Italian sea slater Ligia italica from Tunisia is studied for the first time. Samples were collected from 18 localities in Tunisia, and the analysis included previously published sequences from Italy and Greece available in GenBank. Bayesian and Maximum Likelihood phylogenetic analyses were carried out using a fragment of the mitochondrial COI gene. Putative cryptic species were explored using the ‘barcode gap’ approach in the software ASAP. A genetic landscape shape analysis was carried out using the program Alleles in Space. The analyses revealed highly divergent and well-supported clades of L. italica dispersed across Tunisia (Clades A1 and A2), Greece (Clade B) and Italy (Clades C1 and C2). High genetic dissimilarity among clades suggested that L. italica constitute a cryptic species complex. Divergence among different L. italica lineages (Clades A, B and C) occurred around 7–4.5 Ma. The detected high genetic distances among clades did not result from atypical mitochondrial DNAs or intracellular infection by Wolbachia bacteria. The complex history of the Mediterranean Sea appears to have played a significant role in shaping the phylogeographic pattern of Ligia italica. Additional morphological and molecular studies are needed to confirm the existence of cryptic species in Ligia italica in Mediterranean.

Characterization of the Complete Mitochondrial Genome of Salassa thespis (Lepidoptera: Saturniidae) and Comparison with Other Lepidoptera Species

There are many species of Lepidoptera, but few complete mitochondrial genomes of Lepidoptera have been included in databases. Here, the complete mitochondrial genome sequence of Salassa thespis was isolated and characterized. It was 15,302 bp in length and contained 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. Among the 13 PCGs, the initiation codon of cytochrome c oxidase subunit 1 (cox1) was CGA, and the rest were ATN. The cox1 and cox2 genes had an incomplete stop codon T, while the rest terminated with TAA. Codon usage analysis showed that Phe, Ile, Leu and Asn were the most frequent amino acids, while Trp was the least. Like other Lepidopterans, some conserved motifs were found in the A + T-rich region, including a 17 bp poly-T guided by ATAGA, the AT-rich area and a poly-A element. Bayesian inference and maximum likelihood phylogenetic tree analysis based on 13 PCGs of S. thespis confirmed that it belonged to the Saturniidae family and showed the following relationship: (S. thespis + (Rhodinia fugax + Samia canningi)). The enrichment of mitochondrial DNA provides reference information for the study of the evolution and diversity of Lepidoptera insects.

Distinct evolutionary lineages of Schistocephalus parasites infecting co-occurring sculpin and stickleback fishes in Alaska.

Sculpins (coastrange and slimy) and sticklebacks (ninespine and threespine) are widely distributed fishes cohabiting 2 south-central Alaskan lakes (Aleknagik and Iliamna), and all these species are parasitized by cryptic diphyllobothriidean cestodes in the genus Schistocephalus. The goal of this investigation was to test for host-specific parasitic relationships between sculpins and sticklebacks based upon morphological traits (segment counts) and sequence variation across the NADH1 gene. A total of 446 plerocercoids was examined. Large, significant differences in mean segment counts were found between cestodes in sculpin (mean = 112; standard deviation [s.d.] = 15) and stickleback (mean = 86; s.d. = 9) hosts within and between lakes. Nucleotide sequence divergence between parasites from sculpin and stickleback hosts was 20.5%, and Bayesian phylogenetic analysis recovered 2 well-supported clades of cestodes reflecting intermediate host family (i.e. sculpin, Cottidae vs stickleback, Gasterosteidae). Our findings point to the presence of a distinct lineage of cryptic Schistocephalus in sculpins from Aleknagik and Iliamna lakes that warrants further investigation to determine appropriate evolutionary and taxonomic recognition.

HIV-1 transmission dynamics among people who inject drugs on the US/Mexico border during the COVID-19 pandemic: a prospective cohort study

We examined HIV prevalence and transmission dynamics among people who inject drugs in the U.S./Mexico border region during the COVID-19 pandemic. People who inject drugs aged ≥18 years from 3 groups were recruited: people who inject drugs who live in San Diego (SD) and engaged in cross-border drug use in Tijuana, Mexico (SD CBDUs), and people who inject drugs in SD and Tijuana (TJ) who did not engage in cross-border drug use (NCBDUs). We computed HIV prevalence at baseline and bivariate incidence-density rates (IR) at 18-month follow-up. Bayesian phylogenetic analysis was used to identify local transmission clusters, estimate their age, and effective reproductive number (Re) over time within the clusters. At baseline (n=612), 26% of participants were female, 9% engaged in sex work, and HIVprevalencewas 8% (4% SD CBDU, 4% SD NCBDU, 16% TJ NCBDU). Nine HIV seroconversions occurred over 18 months, IR: 1.357 per 100 person-years (95% CI: 0.470, 2.243); 7 in TJ NCBDU and 2 in SD CBDU. Out of 16 identified phylogenetic clusters, 9 (56%) had sequences from both the U.S. and Mexico (mixed-country). The age of three youngest mixed-country dyads (2018-2021) overlapped with the COVID-related US-Mexico border closure in 2020. One large mixed-country cluster (N=15) continued to grow during the border closure (Re=4.8, 95% Highest Posterior Density (HPD) 1.5-9.1) with 47% engaging in sex work. Amidst the COVID-19 pandemic and the border closure, cross-border HIV clusters grew. Efforts to end the HIV epidemic in the U.S. should take into account cross-border HIV-1 transmission from Tijuana. Mobile harm reduction services and coordination with municipal HIV programs to initiate anti-retroviral therapy and pre-exposure prophylaxisis are needed to reduce transmission. This research was supported by the James B. Pendleton Charitable Trust and the San Diego Center for AIDS Research.

Molecular identification and phylogenetic analysis of the mitogenome in endangered giant nuthatch Sitta magna (Passeriformes, Sittidae)

The Giant Nuthatch Sitta magna (family Sittidae) is a passerine bird, the quantification of the number of habitats and species on a global scale remains low. Most species are restricted to low elevations in southwest China, eastern Myanmar, and northern Thailand. To characterize the mitochondrial genome sequence of S. magna and its phylogenetic relationships with other members within the genus Sitta, the mitochondrial genome of S. magna was sequenced using the whole genome shotgun method. The sequencing results showed that the mitochondrial genome was 16,829 bp long and consisted of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and one control region (D-loop). All tRNAs were predicted to form a typical clover secondary structure. Among the 13 PCGs, only the start codon in COI was ATC, the start codon by the remaining 12 PCGs was ATG, and the stop codons were TAG, TAA, AGG, AGA, and TA. Bayesian inference and maximum likelihood phylogenetic analysis of the sequences of 17 species generated consistent well-supported phylogenies. The family Polioptilidae and the family Troglodytidae were closely related, and the family Sittidae was confined to a single branch. The genus Sitta in the family Sittidae was mainly clustered into three branches. Our findings provide new mitochondrial genomic data that could be used for phylogenetic and taxonomic studies; our results also certificate into the phylogenetic relationships within the genus Sitta ((S. himalayensi+(S. nagaensis + S. europaea))+(S. villosa + S. yunnanensis))+(S. carolinensis + S. magna).

Post-mortem detection of hemoplasmas (hemotropic Mycoplasma spp.) in South American fur seal (Arctocephalus australis) sampled in Rio Grande do Sul State, southern Brazil

Hemotropic mycoplasmas are bacteria that attaches to erythrocytes surface, which some species presents zoonotic concerns. In the suborder Pinnipedia, genera Otaria and Arctocephalus are prominent in Brazil. This study investigated the occurrence of hemoplasmas in Arctocephalus sp. and Otaria flavescens found dead along the coast of a Southern Brazilian State. DNA from 135 spleen samples were extracted and subjected to conventional PCR protocols, targeting the 16 S rRNA and 23 S rRNA gene. Three (2.22 %) Arctocephalus australis were positive in the 16 S rRNA gene, and no samples amplified in the 23 S rRNA gene. Samples from this study clustered with Zalophus californianus and Arctocephalus tropicalis mycoplasmas on a Bayesian phylogenetic analysis. Genetic diversity analysis suggested distinct genotypes, indicating A. australis as a new host for hemoplasma, and also a potential putative novel hemoplasma genotype. These findings raises future awareness for pinnipeds conservation, and adds Mycoplasma spp. to be taken into consideration when clinically evaluating rescued animals.

First Byzantinia from Afro-Arabia and the evolutionary history of extinct cricetodontine rodents investigated through Bayesian phylogenetic inference

The Cricetodontinae is a subfamily of extinct rodents from Europe, northern Africa, and Asia. Byzantinia is a cricetodontine genus that currently includes 10 species. This genus originated in Anatolia in the late Middle Miocene and became extinct at the end of the Miocene. Until now, Byzantinia was known only from Anatolia and Greece. Here, we report isolated molars of Byzantinia from a new locality close to Zahleh, Lebanon. These represent the first remains of Byzantinia from the Arabian area. All these teeth but one, which is very close to Byzantinia dardanellensis, belong to the new taxon Byzantinia rosamariae sp. nov. The evolutionary position of the two Lebanese Byzantinia species allows us to infer an age for this site of approximately 10.9–10 Ma. Bayesian tip-dating phylogenetic analyses, using morphological data for a large variety of cricetodontines, revealed that Anatolia served as the primary centre for speciation and dispersal of this rodent subfamily. This approach allowed us to infer the biogeographical history and time of divergence of the main cricetodontine lineages and sheds light on the evolutionary history of this group. The evolutionary position of the new Lebanese species is intermediate between Byzantinia sp. from Direcik I (Turkey) and B. nikosi from Biodrak (Greece), from which it diverged c. 11.85 Ma. These new Lebanese fossils provide the first physical evidence of the dispersal of these rodents into Afro-Arabia. http://zoobank.org/urn:lsid:zoobank.org:pub:2D8FD3C8-26E1-4C62-8D65-9D58666D70A3

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Discrepancy of flowering time between genetically close sublineages of Aegilops umbellulata Zhuk.

Aegilops umbellulata Zhuk., a wild diploid wheat-related species, has been used as a genetic resource for several important agronomic traits. However, its genetic variations have not been comprehensively studied. We sequenced RNA from 114 accessions of Ae. umbellulata to evaluate DNA polymorphisms and phenotypic variations. Bayesian clustering and phylogenetic analysis based on SNPs detected by RNA sequencing revealed two divergent lineages, UmbL1 and UmbL2. The main differences between them were in the sizes of spikes and spikelets, and culm diameter. UmbL1 is divided into two sublineages, UmbL1e and UmbL1w. These genetic differences corresponded to geographic distributions. UmbL1e, UmbL1w, and UmbL2 are found in Turkey, Iran/Iraq, and Greece, respectively. Although UmbL1e and UmbL1w were genetically similar, flowering time and other morphological traits were more distinct between these sublineages than those between the lineages. This discrepancy can be explained by the latitudinal and longitudinal differences in habitats. Specifically, latitudinal clines of flowering time were clearly observed in Ae. umbellulata, strongly correlated with solar radiation in the winter season. This observation implies that latitudinal differences are a factor in differences in the flowering times of Ae. umbellulata. Differences in flowering time could influence other morphological differences and promote genetic divergence between sublineages.