Molecular Phylogenetic Analysis Research Articles

Broad-scale genetic assessment of Southern Ground-Hornbills (Bucorvus leadbeateri) to inform population management

The Southern Ground-hornbill (SGH) (Bucorvus leadbeateri) is considered an umbrella species for biodiversity conservation in savannah biomes since they require large territories and significant protection measures that help to conserve a wide range of biodiversity with similar savanna and grassland requirements. Declines of the species are attributed to low reproductive rates coupled with multiple anthropogenic threats, including secondary poisoning, and persecution. Little is known about connectivity and population structure of SGH populations in Africa, south of the equator. Knowledge of population differentiation is needed to ensure that targeted conservation management plans can be implemented to slow population declines and ensure survival of the species. To inform a long-term conservation strategy, we investigated the broad-scale population structure of Southern Ground-hornbill across their sub-equatorial range. Our study based on 16 microsatellite loci identified moderate variation (average of 5.889 alleles per locus and a mean observed heterozygosity of 0.546) similar to other long-lived avian species. In contrast, mitochondrial DNA sequences analysis identified low diversity (Hd = 0.3313, π = 0.0015). A Bayesian assignment approach, principal component analysis, analysis of molecular variance and phylogenetic analysis identified weak to moderate population structuring across long distances and mitochondrial data showed a shallow phylogeny. Restriction to long-distance dispersal was detected that could not be attributed to isolation by distance, suggesting that other factors, such as their dispersal biology, are shaping the observed genetic differentiation. Although our study does not support the designation of populations as independent conservation units, we advocate that population management should continue to follow the Precautionary Principle (mixing founders from the same range state, rather than allowing mixing of founders from the extremes of the range) until there is scientific certainty. Following further research, if no independent conservation units are detected, then the global captive population can contribute to reintroductions across the range. In the wild, populations at the edge of the species range may need additional management strategies and gene flow should be promoted between neighbouring populations.

Molecular Analysis and Ex Vivo Infectivity of Seronegative Occult Hepatitis C Virus: A Study in Single Haemodialysis Centre.

In occult hepatitis C virus infection (OCI), hepatitis C virus ribonucleic acid (HCV RNA) is detectable in peripheral blood mononuclear cells (PBMCs) but is not evident in serum or plasma. Understanding of OCI in patients with seronegative anti-HCV antibodies is limited. In this study, six HCV isolates from haemodialysis (HD) patients with seronegative OCI were identified by molecular assays and phylogenetic analysis. The virus infectivity was assessed ex vivo using a primary naïve PBMC culture system. HCV isolates obtained from the PBMCs of 10 patients with chronic HCV infection (CCI) were characterised concurrently and used as positive controls in the cell culture. Sequence analysis of the 5' untranslated region (UTR) and non-structural 5B (NS5B) region revealed that HCV genotype 3 was the most prevalent virus type in both the OCI and CCI groups. One of the occult HCV isolates was identified as a mixed type. The mean viral load (log10 RNA copies/106 cells) in the PBMC samples of the OCI group (M = 3.4, SD = 0.7) was lower than that of the CCI group (M = 4.6, SD = 1.7). Upon culture, de novo OCI-HCV replicates were detected in five out of six naïve PBMC cultures. Analysis of the replicates showed a single guanine addition in the domain III of 5'-UTR but the overall molecular structure was retained. Seronegative OCI is an active form of infection that replicates at a low level in PBMCs. Seronegative OCI may share the same route of transmission as CCI. The retained viral competency may have an implication for its persistence.

Protozoan parasites of birds from the Tremembé formation (Oligocene of the Taubaté Basin), São Paulo, Brazil

ObjectiveTo analyze the presence of protozoan parasites in bird coprolites from the Tremembé Formation (Oligocene of the Taubaté Basin). MaterialsTwenty avian coprolites embedded in pyrobituminous shale matrices. MethodsSamples were rehydrated and subjected to spontaneous sedimentation. ResultsPaleoparasitological analyses revealed oocysts compatible with the Eimeriidae family (Apicomplexa) and one single Archamoebae (Amoebozoa) cyst. ConclusionsThe present work increases the amount of information about the spread of infections throughout the Cenozoic Era and reveals that the Brazilian paleoavifauna played an important role in the Apicomplexa and Amoebozoa life cycles. SignificanceThis is the first record of protozoans in avian coprolites from the Oligocene of Brazil. These findings can help in the interpretation of phylogenies of coccidian parasites of modern birds, as certain taxonomic characters observed in the Oligocene Protozoa characterize monophyletic groups in current molecular phylogenetic analyses. LimitationsNone of the oocysts were sporulated; therefore, it is not possible to identify the morphotypes to genus or species. Suggestions for further researchOur results create new perspectives related to biogeographic studies of the parasitic groups described and may improve the understanding of the temporal amplitude of parasitic evolutionary relationships between Protozoans and birds.

Ixeridium malingheense (Asteraceae), a new species from southwestern Guizhou, China

A new Asteraceae species, Ixeridium malingheense Z.Li & Q.Xu sp. nov., from Xingyi City, south-western Guizhou, China, is described and illustrated based on morphological and molecular analyses. Compared with the other species of the genus, the species is most similar to I. yunnanense in a small stature, linear-lanceolate stem leaves, and short phyllaries. However, it is easily distinguished from the latter by its smaller basal leaves 18–34 × 5–13 mm (vs. 10–25 × 5 mm), spatulate or long-spatulate (vs. elliptic, lanceolate, or oblanceolate), petioles ca. 30–55 mm (vs. 10 mm to absent), outer phyllaries 1-seriate (vs. 2-seriate, unequal), achenes smaller 2.0–3.0 × 0.5–1.0 mm (vs. 3.2 × 0.6 mm), and a flowering period of March to May (vs. June). Molecular phylogenetic analyses based on nuclear ITS sequence data determined the systematic position of I. malingheense in Ixeridium.

Vicariance and cryptic diversity revealed by molecular phylogenetic analyses of estuarine Gammarus species (Crustacea: Amphipoda) due to formation of the Labrador Current

The metapopulation of the estuarine species Gammarus tigrinus along the east coast of the United States has been hypothesised to represent two cryptic species divided biogeographically off the coast of North Carolina, USA. This divergence has been attributed to a strong temperature gradient created by the formation of the cold Labrador Current c. 3.0 million years ago. In addition, the northern phylogeographic clade of G. tigrinus has been demonstrated to be invasive in estuarine habitats across a large portion of northern Europe. Recent collections of G. tigrinus from Florida and Maryland, USA, allow for new approaches to test this hypothesis. Using the nuclear 18S and 28S rRNA, and mitochondrial 16S rRNA and cytochrome c oxidase subunit I genes, species delimitation models provide support that the genetic divergence of the northern and southern clades is equivalent to species level. In addition, molecular clock data demonstrate that this phylogeographic divergence coincides with the formation of the Labrador Current. Furthermore, the collections of G. daiberi from Florida, a species with biogeographical and ecological characteristics similar to those of G. tigrinus, provide independent support for the hypothesis. The potential for invasive species to be cryptic highlights the need for accurate identification of taxa to ensure that appropriate biogeographical assessment of potential source populations and mechanisms of dispersal can be made.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Xinjiang, China, from 2020 to 2022.

In recent years, the pig industry in Xinjiang, China, has been severely impacted by outbreaks of porcine epidemic diarrhea (PED), despite vaccination efforts. In this study, we investigated the genetic characteristics of currently prevalent porcine epidemic diarrhea virus (PEDV) strains in the region. We collected 548 samples from animals with suspected PED on large-scale pig farms in Xinjiang. Of these, 258 tested positive for PEDV by RT-PCR, yielding an overall positivity rate of 47.08%. S1 gene sequencing and phylogenetic analysis were conducted on 23 randomly selected RT-PCR-positive samples. Three endemic strains of PEDV (PEDV/CH/XU/2020, PEDV/CH/XK/2020, and PEDV/CH/XA/2020) were isolated, and their complete genome sequences were analyzed for evidence of genetic recombination. Sequence comparison of the S gene indicated significant variations in the S1 gene of the Xinjiang strains compared to the vaccine strains CV777, AJ1102, and LWL, with 90.2%-98.5% nucleotide sequence identity. Notably, both the N-terminal and C-terminal domains of the S protein showed significant variation. Genetic evolutionary analysis identified the GIIa subtype as the dominant genotype among the epidemic strains in Xinjiang. Recombination analysis revealed inter-subtype recombination events in the PEDV/CH/XK/2020 and XJ1904-34 strains. These findings highlight the extensive genetic variation in the predominant GIIa genotype of PEDV in Xinjiang, which does not match the genotype of the currently used vaccine strains. These data may guide further efforts toward the development of effective vaccines for the control of PED.

Unveiling zoonotic threats: molecular identification of Brugia sp. infection in a lion.

Brugia malayi and B. pahangi, potential zoonotic pathogens transmitted by mosquitoes, are believed to primarily infect dogs and cats as reservoir hosts. Although previous studies have indicated nematode infections in lions, particularly in zoo environments where human contact with these reservoirs is possible, limited documentation exists regarding Brugia sp. infections in lions in Thailand. This study aims to investigate a case of Brugia infection in a lion from a zoo in Thailand. The blood sample was collected and examined from a female lion, using staining methods to morphologically identify microfilaria at the genus level. Subsequently, the PCR was employed targeting specific genes, including mitochondrial 12S rDNA, 18S rDNA, cytochrome oxidase I (COI) and Wolbachia surface protein (wsp), to confirm the species of the filarial nematode parasite. The genetic sequencing results revealed a high similarity (99-100%) to B. malayi for the 12S rDNA, 18S rDNA, COI and wsp genes. Phylogenetic analysis based on nucleotide sequences from the 12S rDNA, 18S rDNA, COI and wsp genes showed that the sequences from this study belong to different clusters. This marks the inaugural documentation of molecular identification of Brugia infection in a lion, signifying that lions could function as reservoirs for this parasite and present a potential public health risk in the region. Our research underscores the effectiveness of molecular techniques and phylogenetic analysis in discerning and comprehending the evolution of filarial parasites. Additionally, it emphasizes the significance of these methods in enhancing the diagnosis, control, and prevention of zoonotic filarial nematode infections.

Description of a new marine flatworm of Prosthiostomum (Platyhelminthes, Polycladida, Prosthiostomidae) from the South China Sea

A new species of the polyclad genus Prosthiostomum is described from the intertidal zone of the South China Sea, Huidong, China, based on morphological and molecular analyses. Prosthiostomum huidongensesp. nov. is characterized by i) few marginal eyes scattered between the marginal band and the cerebral eyes; ii) sucker located at two-thirds of the body length, being removed from the female gonopore by twice the distance between the male and female gonopores; iii) shallow male atrium with slightly ruffled inner wall, positioned approximately perpendicular to the body wall. Molecular phylogenetic analyses based on 28S rDNA sequence showed that the new species was nested in a clade composed of Prosthiostomum species. The uncorrected p-distance of COI between P. huidongensesp. nov. and other Prosthiostomum species ranged from 20.3 to 24.3%, and the high genetic divergence further supports P. huidongense as a new species.

Molecular phylogeny and biogeography of the aquatic dance fly subfamily Clinocerinae (Diptera: Empididae)

AbstractThis study presents the first molecular phylogenetic analysis of the Clinocerinae, challenging the traditionally accepted monophyly of this subfamily. DNA was extracted from fresh and museum specimens representing all biogeographical regions. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were performed based on sequences from two mitochondrial genes, cytochrome c oxidase subunit I (COI) and cytochrome β, and three nuclear genes, carbomoylphosphate synthase domain of rudimentary, elongation factor‐1α and isocitrate dehydrogenase. Through molecular data and morphological examination, our results reveal a division within Clinocerinae, distinguishing ‘typical’ or Clinocerinae (s.s.) from several genera, specifically Afroclinocera Sinclair, Asymphyloptera Collin and Proagomyia Collin, possibly lending support for a reclassification of these genera outside Clinocerinae. Bergenstammia Mik is proposed as a junior synonym of Phaeobalia Mik, syn. n., and the following new combinations are recognized: Phaeobalia albanica (Wagner) comb. n., Phaeobalia aurinae (Pusch & Wagner) comb. n., Phaeobalia carniolica (Horvat) comb. n., Phaeobalia frigida (Vaillant) comb. n., Phaeobalia glacialis (Palaczyk & Słowińska) comb. n., Phaeobalia multiseta (Strobl) comb. n., Phaeobalia nudimana (Vaillant) comb. n., Phaeobalia nudipes (Loew) comb. n., Phaeobalia pulla (Vaillant & Wagner) comb. n., Phaeobalia pyrenaica (Vaillant & Vinçon) comb. n., Phaeobalia slovaca (Wagner) comb. n. and Phaeobalia thomasi (Vaillant & Vinçon) comb. n. Re‐evaluation of the genus Roederiodes resulted in the following new combinations: Clinocerella macedonicus (Wagner & Horvat) comb. n. and Clinocerella montenegrinus (Wagner & Horvat) comb. n. The origins of Clinocerinae (s.s.) are traced back to the Holarctic region, Laurasian origin, with a likely complex history of dispersal events into the Southern Hemisphere. Based on current knowledge, the greatest generic and species richness is confined to the Palaearctic Region. These findings provide valuable insights into the evolutionary relationships and distribution patterns of Clinocerinae (s.s.), challenging existing taxonomic classifications and shedding light on their historical biogeography.

Discovery of a new Pseudalomya Telenga, 1930 (Hymenoptera, Ichneumonidae, Ichneumoninae) species from Taiwan and its implications for the systematic position of this genus

The rare genus Pseudalomya Telenga comprises two species, which are found only in the Eastern Palaearctic region and high mountains of the Oriental region. The phylogenetic position of Pseudalomya remains unclear because of its intermediate morphology between two ichneumonine tribes, Alomyini and Phaeogenini. This article reports the discovery of a new species of Pseudalomya: Pseudalomya truncaticornissp. nov. Specimens were collected during a survey of insect fauna in the Dasyueshan area of Shei-Pa National Park, one of the high-altitude regions in Taiwan. The new species can be diagnosed by its body coloration, frontal horn shape, facial punctures, metasomal tergite sculpture, and wing venation. To the best of our knowledge, this is the first record of Pseudalomya in Taiwan. This article also presents a diagnostic key to the global species of Pseudalomya. In this study, molecular phylogenetic analyses were performed using one mitochondrial and two nuclear gene sequences from P. truncaticornissp. nov. and other members of the Ichneumoniformes group. The results indicate that Pseudalomya should be classified within Phaeogenini, distinct from Alomyini, but more comprehensive phylogenomic studies are needed to confirm this placement.

A taxonomic revision of the sponge-associated genus Thoracactis Gravier, 1918 (Anthozoa: Zoantharia) based on an integrated approach

Abstract The integrated approach of molecular phylogenetic and morphological analyses has revolutionized the systematics and our understanding of the evolutionary relationships of marine taxa. One such group is the hexacorallian order Zoantharia Rafinesque, 1815. The monotypic genus Thoracactis Gravier, 1918 has been little investigated since its placement within the order Zoantharia more than 100 years ago. Here, we examined museum specimens collected from the Cape Verde Islands (eastern Atlantic) and newly collected specimens from Brazil (southwestern Atlantic), using a combined molecular and morphological approach. Our results conclusively show Thoracactis to be referable to the family Parazoanthidae. Morphological data show that Thoracactis topsenti Gravier, 1918, the type species of this monotypic genus, has a cyclically transitional arrangement of its sphincter muscle, and this arrangement has previously been reported from the Parazoanthidae. Thoracactis can be distinguished from other hexasterophoran glass-sponge-associated genera (Churabana Kise, Montenegro & Reimer, 2022, Parachurabana Kise, 2023, and Vitrumanthus Kise, Montenegro & Reimer, 2022) by a combination of morphological, ecological and molecular phylogenetic data. In addition, molecular phylogenetic analyses clearly indicate that Thoracactis topsenti is placed within Parazoanthidae. These results are yet another demonstration of the utility of comprehensive combined approaches. From now, research attention should focus on the revision of remaining taxonomic questions within the family Epizoanthidae, with the goal of a comprehensively revised suborder Macrocnemina within reach.

Melanoleuca lalsuhanraensis, a new species from Lal Suhanra National Park, Punjab, Pakistan

A new species of Melanoleuca subg. Urticocystis with urticiform cheilocystidia is proposed from Lal suhanra National Park, Bahawalpur district, Punjab, Pakistan. The identification is based on morphological characters and molecular phylogenetic analyses using ITS sequences. A comparison of morphological features and molecular phylogenetic data with the known taxa of the subgenus confirms the identity of the species as a new taxon. Detailed descriptions, line drawings and phylogenetic results are presented.

Paracapillaria (Ophidiocapillaria) siamensis sp. nov. (Nematoda: Trichuroidea): a new nematode in Naja kaouthia from Thailand.

A comprehensive investigation, incorporating both morphological and molecular analyses, has unveiled the existence of a hitherto unknown nematode species, Paracapillaria (Ophidiocapillaria) siamensis sp. nov., residing in the intestine of the monocled cobra, Naja kaouthia, in the central region of Thailand. This study integrates morphological characteristics, morphometric examination, scanning electron microscopy and molecular phylogenetic analysis (COI, 18S rRNA and ITS1 genes). The findings place the newly described species within the subgenus Ophidiocapillaria, elucidating its distinctive characteristics, including a frame-like proximal spicule shape, approximate lengths of 19 000 and 22 500 μm with approximate widths of 90 and 130 μm for males and females, 39‒45 stichocytes, elevated lips without protrusion, a dorsal bacillary band stripe with an irregular pattern of bacillary cells and evidence of intestinal infection. These features serve to differentiate it from other species within the same subgenus, notably Paracapillaria (Ophidiocapillaria) najae De, , a species coexisting P. siamensis sp. nov. in the monocled cobra from the same locality. This study addresses the co-infection of the novel species and P. najae within the same snake host, marking the second documented instance of a paracapillariid species in the monocled cobra within the family Elapidae. The genetic characterization supports the formal recognition of P. siamensis sp. nov. as a distinct species, thereby underscoring its taxonomic differentiation within the Capillariidae family. This research identifies and characterizes the new nematode species, contributing valuable insights into the taxonomy of this nematode.

Two new species of Sarothrogammarus (Crustacea, Amphipoda) from Swat Valley, Pakistan

Two new species of the genus Sarothrogammarus Martynov, 1935 are described and illustrated from Pakistan. Sarothrogammarus miandamensis sp. nov. is characterized by the oval eyes; pereopod III having filtrative setae on merus, carpus and propodus; uropod III terminal article distinct and inner ramus scale-shaped; and having robust setae and slender setae on urosomites I–III. Sarothrogammarus kalamensis sp. nov. can be distinguished from the above species by pereopod III having filtrative setae on merus, but few on carpus and propodus. Detailed morphological characteristics, as well as comparisons with closely related species are provided. Molecular phylogenetic analyses suggest that S. miandamensis sp. nov. is clustered with S. kalamensis sp. nov., with a genetic distance of 19% for COI between the two species.

Glypturus ferox, a new species of ghost shrimp (Decapoda: Axiidea: Callichiridae) from the Ryukyu Islands, southwestern Japan

The callichirid ghost shrimp genus Glypturus Stimpson, 1866 was represented until now by four extant species world-wide. Only G. armatus A. Milne-Edwards, 1870 is known from the western Pacific. In this study, we describe G. ferox n. sp. based on seven specimens collected from the Ryukyu Islands, southwestern Japan. All specimens of the new species were collected in inner reef lagoons by suction pump. The new species is easily distinguished from all other congeners, including fossil taxa, by having a row of spines on the lower margin of the major cheliped palm. Molecular phylogenetic analysis using the mitochondrial 16S rRNA gene suggests that G. ferox n. sp. is sister to G. armatus; the genetic divergence between the two species is high. An identification key to extant species of Glypturus is provided. A brief overview on Japanese species of Callichiridae is also provided. Glypturus ferox n. sp. is the nineth species of the family known from Japanese waters with certainty.

Molecular phylogeny and taxonomy of Phlomoides (Lamiaceae subfamily Lamioideae) in China: Insights from molecular and morphological data

Phlomoides, with 150–170 species, is the second largest and perhaps most taxonomically challenging genus within the subfamily Lamioideae (Lamiaceae). With about 60 species, China is one of three major biodiversity centers of Phlomoides. Although some Phlomoides species from China have been included in previous molecular phylogenetic studies, a robust and broad phylogeny of this lineage has yet to be completed. Moreover, given the myriad new additions to the genus, the existing infrageneric classification needs to be evaluated and revised. Here, we combine molecular and morphological data to investigate relationships within Phlomoides, with a focus on Chinese species. We observed that plastid DNA sequences can resolve relationships within Phlomoides better than nuclear ribosomal internal and external transcribed spacer regions (nrITS and nrETS). Molecular phylogenetic analyses confirm the monophyly of Phlomoides, but most previously defined infrageneric groups are not monophyletic. In addition, morphological analysis demonstrates the significant taxonomic value of eight characters to the genus. Based on our molecular phylogenetic analyses and morphological data, we establish a novel section Notochaete within Phlomoides, and propose three new combinations as well as three new synonyms. This study presents the first molecular phylogenetic analyses of Phlomoides in which taxa representative of the entire genus are included, and highlights the phylogenetic and taxonomic value of several morphological characters from species of Phlomoides from China. Our study suggests that a taxonomic revision and reclassification for the entire genus is necessary in the future.

Molecular phylogenetic analysis of Echinococcus granulosus sensu lato infecting sheep in Italy.

Cystic Echinococcosis (CE), caused by the larval form of Echinococcus granulosus sensu lato, is a neglected zoonosis still threatening public health worldwide. In Italy different epidemiological scenarios were reported depending on the geographical area and associated socio-economic activities. Although in northern Italy the occurrence of E. granulosus is considered sporadic, in the southern regions and, particularly in Sardinia, CE prevalence reaches high levels. We analysed CE cysts collected from infected sheep from various areas of mainland Italy and the Sardinia island, with the main objective to investigate intergenotypic and intragenotypic variations at national level. CE cysts were collected from slaughtered sheep following post mortem inspection at local abattoirs. Total genomic DNA was extracted and amplification and sequencing of the partial mitochondrial genes nad5 and cox1 were performed. A Bayesian phylogenetic tree was estimated on a nad5 dataset (n=260) composed of E. granulosus samples from this study (n=126) and all the nad5 haplotypes available in GenBank (n=134). In addition, haplotype network, diversity and neutrality analysis were performed on nad5 and cox1 sequences of Italian origin obtained in this study. E. granulosus sensu stricto (s.s.) was found to be the only Echinococcus species infecting sheep in Italy, mainly represented by G1 genotype (76%) and, to a lower extent, by G3 genotype (24%). Phylogenetic analyses revealed 40 nad5 and 33 cox 1 haplotypes, and the presence of two founder haplotypes, belonging to G1 and G3 genotype, showing 100% similarity with DNA sequences from different geographic regions. The lack of geographical segregation, high haplotype and low nucleotide diversity coupled with significant negative values of Tajima's D and Fu's Fs observed in this study indicated high genetic variation and demographic expansion of E. granulosus s.s. in Italy.

Pacific marine gregarines (Apicomplexa) shed light on biogeographic speciation patterns and novel diversity among early apicomplexans

Gregarines are the most biodiverse group of apicomplexan parasites. This group specializes on invertebrate hosts (e.g., ascidians, crustaceans, and polychaetes). Marine gregarines are of particular interest because they are considered to be the earliest evolving apicomplexan lineage, having subsequently speciated (and radiated) through virtually all existing animal groups. Still, mechanisms governing the broad (global) distribution and speciation patterns of apicomplexans are not well understood. The present study examines Pacific lecudinids, one of the most species-rich and diverse groups of marine gregarines. Here, marine polychaetes were collected from intertidal zones. Single trophozoite cells were isolated for light and electron microscopy, as well as molecular phylogenetic analyses using the partial 18S rRNA gene. The cytochrome c oxidase subunit 1 gene was used to confirm morphology-based host identification. This study introduces Undularius glycerae n. gen., n. sp. and Lecudina kitase n. sp. (Hokkaido, Japan), as well as Difficilina fasoliformis n. sp. (California, USA). Occurrences of Lecudina cf. longissima and Lecudina cf. tuzetae (California, USA) are also reported. Phylogenetic analysis revealed a close relationship between L. pellucida, L. tuzetae, and L. kitase n. sp. Additionally, clustering among North Atlantic and Pacific L. tuzetae formed a species complex, likely influenced by biogeography.

Diversity of Cryptophyte Cultures from the Canary Islands

ABSTRACT Several excursions have been undertaken to sample marine, freshwater and terrestrial habitats for the presence of microscopic algae on and around the Canary Islands. Sampling and isolation efforts resulted in several newly established cryptophyte cultures. To preliminarily determine their identity, nuclear SSU rDNA was sequenced and subjected to molecular phylogenetic analyses. All cryptophyte cultures belong to currently known major clades, five out of six freshwater lineages likely represent new Cryptomonas species.

Morphology, phylogeny and pathogenicity of fungal species associated with leaf blight and stem canker of Theobroma cacao in Malaysia

AbstractMalaysia stands prominently among Asia's key cocoa‐producing countries. In the cocoa season of 2022–2023, Malaysia demonstrated its contribution to the industry, with an estimated production of around 364,000 tonnes of cacao bean grindings. Nonetheless, fungal diseases pose undeniable challenges to the cocoa sector. Extensive sampling conducted between September 2018 and March 2019 across multiple states in Malaysia revealed concerning symptoms of leaf blight and stem canker affecting Theobroma cacao plants. The aim of this study was to identify and characterize fungal species associated with leaf blight and stem canker of T. cacao in Malaysia through morphological, molecular and pathogenicity analyses. Morphological and molecular phylogenetic analyses using multiple DNA regions (rDNA internal transcribed spacer [ITS], TEF1 and TUB2) were performed and identified 40 fungal isolates found in this study as Diaporthe tulliensis (17 isolates), Fusarium solani (seven isolates), Fusarium proliferatum (six isolates) and Neopestalotiopsis clavispora (10 isolates). Pathogenicity tests with mycelial plugs and wound treatments showed that D. tulliensis and N. clavispora were responsible for causing leaf blight whereas D. tulliensis, F. solani and F. proliferatum caused stem canker of T. cacao. The present study provides insights into disease aetiology and symptomatology that may be useful in planning effective disease management for the host plant.