Analysis of honey environmental DNA indicates that the honey bee (Apis mellifera L.) trypanosome parasite Lotmaria passim is widespread in the apiaries of the North of Italy.

Lotmaria passim is a trypanosomatid that infects honey bees. In this study, we established an axenic culture of L. passim from Italian isolates and then used its DNA as a control in subsequent analyses that investigated environmental DNA (eDNA) to detect this trypasonosomatid. The source of eDNA was honey, which has been already demonstrated to be useful to detect honey bee parasites. DNA from a total of 164 honey samples collected in the North of Italy was amplified with three L. passim specific PCR primers and 78% of the analysed samples gave positive results. These results indicated a high prevalence rate of this trypanosomatid in the North of Italy, where it might be considered another threat to honey bee health.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Lotmaria passim (Kinetoplastea: Trypanosomatidae) in honey bees from Argentina.

Lotmaria passim (Kinetoplastea) is considered the most prevalent as well as the most virulent trypanosomatid associated to the European honey bee Apis mellifera. We used qPCR to screen for the presence of this parasite in 57 samples from ten Argentinian provinces, and were able to detect its presence throughout most of the country with 41% of the samples testing positive. In a retrospective analysis, we detected L. passim in 73% of honey bee samples from 2006 showing that this flagellate has been widely present in Argentina for at least ~15 years. Additionally, three primer sets for L. passim detection were compared, with the pair that produced smallest PCR product having the best detection capability. Finally, we also found L. passim DNA in 100% (n = 6) of samples of the mite Varroa destructor. The role of this ectoparasite in the lifecycle of Lotmaria, if any, remains unrevealed.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Noninvasive Biological Samples to Detect and Diagnose Infections due to Trypanosomatidae Parasites: A Systematic Review and Meta-Analysis.

Unicellular eukaryotes of the Trypanosomatidae family include human and animal pathogens that belong to the Trypanosoma and Leishmania genera. Diagnosis of the diseases they cause requires the sampling of body fluids (e.g., blood, lymph, peritoneal fluid, cerebrospinal fluid) or organ biopsies (e.g., bone marrow, spleen), which are mostly obtained through invasive methods. Body fluids or appendages can be alternatives to these invasive biopsies but appropriateness remains poorly studied. To further address this question, we perform a systematic review on clues evidencing the presence of parasites, genetic material, antibodies, and antigens in body secretions, appendages, or the organs or proximal tissues that produce these materials. Paper selection was based on searches in PubMed, Web of Science, WorldWideScience, SciELO, Embase, and Google. The information of each selected article (n = 333) was classified into different sections and data were extracted from 77 papers. The presence of Trypanosomatidae parasites has been tracked in most of organs or proximal tissues that produce body secretions or appendages, in naturally or experimentally infected hosts. The meta-analysis highlights the paucity of studies on human African trypanosomiasis and an absence on animal trypanosomiasis. Among the collected data high heterogeneity in terms of the I2 statistic (100%) is recorded. A high positivity is recorded for antibody and genetic material detection in urine of patients and dogs suffering leishmaniasis, and of antigens for leishmaniasis and Chagas disease. Data on conjunctival swabs can be analyzed with molecular methods solely for dogs suffering canine visceral leishmaniasis. Saliva and hair/bristles showed a pretty good positivity that support their potential to be used for leishmaniasis diagnosis. In conclusion, our study pinpoints significant gaps that need to be filled in order to properly address the interest of body secretion and hair or bristles for the diagnosis of infections caused by Leishmania and by other Trypanosomatidae parasites.

Field Isolation and Cultivation of Trypanosomatids from Insects.

Monoxenous (one host) trypanosomatids from insects and other invertebrates can be introduced into axenic culture relatively easily and efficiently, allowing for their transfer from the field into the laboratory. Here we describe simple methods and alternative cultivation protocols, the wider application of which will allow substantial expansion of trypanosomatids available for research.

Canine vector-borne protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. in dogs from Northern Algeria.

Dogs are competent reservoirs/hosts of several protozoan pathogens transmitted by blood-feeding arthropods. Throughout their long history of domestication, they have served as a link for the exchange of parasites among livestock, wildlife, and humans and therefore remain an important source of emerging and re-emerging diseases. In Algeria, while canine leishmaniosis (CanL) is well known to be endemic, no data are available on other vector-borne protozoans. Here, we investigate the occurrence and diversity of trypanosomes, piroplasms and Hepatozoon spp. and update the epidemiological status of CanL in dogs from Kabylia, northern Algeria. A total of 227 dogs from three regions of Kabylia were enrolled, including 77 dogs with clinical signs. Dogs were clinically examined and were tested for L. infantum antibodies using a Rapid Immuno-Migration (RIM™) and a quantitative indirect Immunofluorescence Antibody Test (IFAT). PCR screening and sequencing were performed for vector-borne protozoa. Sixty two percent (141/227) of dogs presented at least one infection, whereas 26% (59/227) were co-infected. L. infantum antibodies were detected in 35.7% (81/227) of dogs including 88.7% (68/77) of sick dogs. Molecular investigation revealed prevalence of: 6.6% (15/227), 13.2% (30/227), 41% (93/227) for Trypanosoma spp., B. vogeli and H. canis, respectively. T. evansi (3.1%) and potential new subspecies of T. congolense had been identified. Dog''s clinical status correlates positively with L. infantum antibody titers and the presence of co-infections. Susceptibility to CanL varied according to the dog's aptitude and guard dogs were more infected (51%) (P-value = .001). B. vogeli infection was more frequent in juveniles than adults (32% vs 9%, P-value < .001) and in females than males (21% vs 10%, P-value = .02). To the authors' knowledge, this is the first report on vector-borne protozoa infected dogs in Algeria. Current results are important not only for animal health, but also to avoid serious public health and livestock problems.

High resolution melting based method for rapid discriminatory diagnosis of co-infecting Leptomonas seymouri in Leishmania donovani-induced leishmaniasis.

Leishmania donovani, a protozoan parasite of family Trypanosomatidae, causes fatal visceral leishmaniasis (VL) in the Indian subcontinent and Africa and cutaneous leishmaniasis (CL) in Sri Lanka. Another member of Trypanosomatidae, Leptomonas seymouri, resembling Leishmania was discovered recently to co-exist with L. donovani in the clinical samples from India and Sri Lanka and therefore, interfere with its investigations. We earlier described a method for selective elimination of such co-existing L. seymouri from clinical samples of VL exploiting the differential growth of the parasites at 37 °C in vitro. Here, we explored ways for a rapid discriminatory diagnosis using high resolution melting (HRM) curves to detect co-occurring L. seymouri with L. donovani in clinical samples. Initial attempt with kDNA-minicircle (mitochondrial DNA) based HRM did not display different Tm values between L. donovani and L. seymouri. Surprisingly, all of their minicircle sequences co-existed in similar clades in the dendrogram analysis, although the kDNA sequences are known for its species and strain specific variations among the Trypanosomatids. However, an HRM analysis that targets the HSP70 gene successfully recognized the presence of L. seymouri in the clinical isolates. This discovery will facilitate rapid diagnosis of L. seymouri and further investigations in to this elusive organism, including the clinico-pathological implications of its co-existence with L. donovani in patients.

Insect trypanosomatids in Papua New Guinea: high endemism and diversity.

The extreme biological diversity of Oceanian archipelagos has long stimulated research in ecology and evolution. However, parasitic protists in this geographic area remained neglected and no molecular analyses have been carried out to understand the evolutionary patterns and relationships with their hosts. Papua New Guinea (PNG) is a biodiversity hotspot containing over 5% of the world's biodiversity in less than 0.5% of the total land area. In the current work, we examined insect heteropteran hosts collected in PNG for the presence of trypanosomatid parasites. The diversity of insect flagellates was analysed, to our knowledge for the first time, east of Wallace's Line, one of the most distinct biogeographic boundaries of the world. Out of 907 investigated specimens from 138 species and 23 families of the true bugs collected in eight localities, 135 (15%) were infected by at least one trypanosomatid species. High species diversity of captured hosts correlated with high diversity of detected trypanosomatids. Of 46 trypanosomatid Typing Units documented in PNG, only eight were known from other geographic locations, while 38 TUs (~83%) have not been previously encountered. The widespread trypanosomatid TUs were found in both widely distributed and endemic/sub-endemic insects. Approximately one-third of the endemic trypanosomatid TUs were found in widely distributed hosts, while the remaining species were confined to endemic and sub-endemic insects. The TUs from PNG form clades with conspicuous host-parasite coevolutionary patterns, as well as those with a remarkable lack of this trait. In addition, our analysis revealed new members of the subfamilies Leishmaniinae and Strigomonadinae, potentially representing new genera of trypanosomatids.

Correlates of blood parasitism in a threatened marshland passerine: infection by kinetoplastids of the genus Trypanosoma is related to landscape metrics of habitat edge.

In birds, vector-borne parasites invading the bloodstream are important agents of disease, affect fitness and shape population viability, thus being of conservation interest. Here, we molecularly identified protozoan blood parasites in two populations of the threatened Aquatic Warbler Acrocephalus paludicola, a migratory passerine nesting in open marsh. We explored whether prevalence and lineage diversity of the parasites vary by population and whether infection status is explained by landscape metrics of habitat edge and individual traits (body mass, fat score, wing length and sex). Aquatic Warblers were infected by genera Plasmodium, Leucocytozoon and Trypanosoma, with seven, one and four lineages, and 29.9, 0.7 and 12.5% prevalence, respectively. No Haemoproteus infections were detected. Prevalence did not vary between the populations, but lineage diversity was higher in Polesie than in Biebrza for all the lineages pooled and for Plasmodium. Infection by Trypanosoma decreased with patch core area and increased with density of habitat edge. Infection status was not predicted by the individual traits. Our study is the first to show an association between edge-related landscape features and blood parasitism in an open habitat bird. This finding will support informed conservation measures for avian species of the globally shrinking marshland and other treeless habitats.

A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species.

Leishmaniasis and Chagas disease are endemic in many countries, and re-emerging in the developed countries. A rapid and accurate diagnosis is important for early treatment for reducing the duration of infection as well as for preventing further potential health complications. In this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of Leishmania major and Trypanosoma cruzi. The assay consists of a singleplex PCR step that amplifies a highly homologous DNA sequence which encodes for the RNA component of the large ribosome subunit. The amplicons of the two different parasites differ between them by single nucleotide variations, known as "Single Nucleotide Fingerprint" (SNF) markers. The SNF markers can be easily identified by naked eye using a novel micro Spin-Tube device "Spin-Tube", as each of them creates a specific spot pattern. Moreover, the direct use of ribosomal RNA without requiring the PCR pre-amplification step is also feasible, further increasing the simplicity of the assay. The molecular assay delivers sensitivity capable of identifying up to 8.7 copies per µL with single mismatch specificity. The Spin-Tube thus represents an innovative solution providing benefits in terms of time, cost, and simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries.

High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island.

We describe the monoxenous trypanosomatids parasitizing true bugs and flies on the island of Curaçao. Out of 248 examined true bugs belonging to 17 species, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 flies, six were infected. All detected trypanosomatids were compared based on their 18S rRNA sequences with TUs parasitizing bugs and flies described from mainland South America, allowing us to assess their diversity and distribution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two known species of the subfamily Leishmaniinae, our analysis revealed six new TUs falling into the groups 'jaculum', Blastocrithidia and Herpetomonas. Moreover, two new members of the genus Phytomonas and three new TUs belonging to the monophyletic group designated as 'new clade II' sensu Mol. Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids were characterized by moderate diversity (13 TUs) species richness. Out of nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 TUs have not been encountered before. Although a sampling bias may partially affect the comparison between trypanosomatid communities on Curaçao and the mainland, the high proportion of unique TUs from the former location suggests that the prominent role of islands in increasing the global diversity of macroscopic organisms may also extend to their protistan parasites.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

BACKGROUND: Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. OBJECTIVES: We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. METHODS: The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. FINDINGS: In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. CONCLUSIONS: This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Triplex real-time PCR for detection of Crithidia mellificae and Lotmaria passim in honey bees.

Currently, light microscopic examination of cell morphology cannot discriminate Crithidia mellificae and Lotmaria passim with 100% certainty. Here, a minor groove-binding (MGB) probe-based multiplex real-time PCR assay was developed for the simultaneous and quantitative detection of C. mellificae and L. passim in honey bees. A conserved Hymenoptera 18S rRNA gene was built in as an internal control that allows accurate detection of PCR inhibition and failure of DNA extraction. The newly developed assay was also applied to field samples. Of 21 honey bee colonies (446 bees) sampled from six counties in both central and eastern Massachusetts, 3 colonies (14.29%) and 8 bees (1.79%) were infected with L. passim, and 1 colony (4.76%) and 1 bee (0.22%) with C. mellificae. Our data showed a low rate of trypanosomatid infection, and L. passim was more prevalent than C. mellificae in honey bee samples in Massachusetts.

Prevalence of Trypanosoma cruzi and Other Trypanosomatids in Frequently-Hunted Wild Mammals from the Peruvian Amazon.

To better understand the ecology of Trypanosoma cruzi in the northeastern Peruvian Amazon, we evaluated the prevalence of T. cruzi and other trypanosomatids in four orders of wild mammals hunted and consumed by inhabitants of three remote indigenous communities in the Peruvian Amazon. Of 300 wild mammals sampled, 115 (38.3%) were infected with trypanosomatids and 15 (5.0%) with T. cruzi. The prevalence of T. cruzi within each species was as follows: large rodents (Cuniculus paca, 5.5%; Dasyprocta spp., 2.6%), edentates (Dasypus novemcinctus, 4.2%), and carnivores with higher prevalence (Nasua nasua, 18.8%). The high prevalence of T. cruzi and other trypanosomatids in frequently hunted wild mammals suggests a sizeable T. cruzi sylvatic reservoir in remote Amazonian locations.

Leptomonas seymouri narna-like virus 1 and not leishmaniaviruses detected in kala-azar samples from India.

The great majority of kala-azar/visceral leishmaniasis (VL) cases, which are caused by Leishmania donovani (LD), are reported in Asia. We investigated whether leishmaniaviruses (LRVs) are present in LD isolates. These dsRNA viruses contribute to hyperpathogenicity, as observed in the case of other members of the genus Leishmania. However, LRVs could not be detected in 22 Indian LD isolates tested in the present study, while 70% of these original LD isolates harboured a virus that was not of LD but instead of Leptomonas seymouri (LS) origin. LS is another protozoon that parasitizes the sandfly vector of LD. Historically, LD clinical isolates from India often showed high incidence of LS coinfection. LS was detected in 20 out of the 22 (91%) above-mentioned LD isolates. Leptomonas seymouri narna-like virus 1 (Lepsey NLV1) was identified by whole-genome sequencing in an LD-LS coinfected sample, and its presence was confirmed by PCR and sequencing in 15 (75%) of the 20 LD-LS coinfected samples. The LS-negative LD samples were also virus negative by PCR. That the human host is exposed to an RNA virus in LS, another coinfecting parasite with LD, i.e., the "LD-LS-Lepsey NLV1 triple pathogen" phenomenon, unveils a new paradigm of research towards revisiting the mysteries of Indian leishmaniasis pathogenesis and management.

The evolution of trypanosomatid taxonomy.

Trypanosomatids are protozoan parasites of the class Kinetoplastida predominately restricted to invertebrate hosts (i.e. possess a monoxenous life-cycle). However, several genera are pathogenic to humans, animals and plants, and have an invertebrate vector that facilitates their transmission (i.e. possess a dixenous life-cycle). Phytomonas is one dixenous genus that includes several plant pathogens transmitted by phytophagous insects. Trypanosoma and Leishmania are dixenous genera that infect vertebrates, including humans, and are transmitted by hematophagous invertebrates. Traditionally, monoxenous trypanosomatids such as Leptomonas were distinguished from morphologically similar dixenous species based on their restriction to an invertebrate host. Nonetheless, this criterion is somewhat flawed as exemplified by Leptomonas seymouri which reportedly infects vertebrates opportunistically. Similarly, Novymonas and Zelonia are presumably monoxenous genera yet sit comfortably in the dixenous clade occupied by Leishmania. The isolation of Leishmania macropodum from a biting midge (Forcipomyia spp.) rather than a phlebotomine sand fly calls into question the exclusivity of the Leishmania-sand fly relationship, and its suitability for defining the Leishmania genus. It is now accepted that classic genus-defining characteristics based on parasite morphology and host range are insufficient to form the sole basis of trypanosomatid taxonomy as this has led to several instances of paraphyly. While improvements have been made, resolution of evolutionary relationships within the Trypanosomatidae is confounded by our incomplete knowledge of its true diversity. The known trypanosomatids probably represent a fraction of those that exist and isolation of new species will help resolve relationships in this group with greater accuracy. This review incites a dialogue on how our understanding of the relationships between certain trypanosomatids has shifted, and discusses new knowledge that informs the present taxonomy of these important parasites.

PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries.

The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples.

Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n.

In this study, we surveyed six species of cockroaches, two synanthropic (i.e. ecologically associated with humans) and four wild, for intestinal trypanosomatid infections. Only the wild cockroach species were found to be infected, with flagellates of the genus Herpetomonas. Two distinct genotypes were documented, one of which was described as a new species, Herpetomonas tarakana sp. n. We also propose a revision of the genus Herpetomonas and creation of a new subfamily, Phytomonadinae, to include Herpetomonas, Phytomonas, and a newly described genus Lafontella n. gen. (type species Lafontella mariadeanei comb. n.), which can be distinguished from others by morphological and molecular traits.