Does the fish-infecting Trypanosoma micropteri belong to Trypanosoma carassii?

Recently, based on a limited morphological characterisation and partial 18S rRNA gene sequence, Jiang et al. (2019) described Trypanosoma micropteri Jiang, Lu, Du, Wang, Hu, Su et Li, 2019 as a new pathogen of farmed fish. Here we provide evidence based on the expanded sequence dataset, morphology and experimental infections that this trypanosome does not warrant the establishment as a new species, because it is conspecific with the long-term known Trypanosoma carassii Mitrophanow, 1883, a common haemoflagellate parasite of freshwater fish. The former taxon thus becomes a new junior synonym of T. carassii.

Nile tilapia (Oreochromis niloticus) can be experimentally infected with both marine and freshwater fish trypanosomes.

Trypanosomes are haemoflagellates found in vertebrate species and many of them can cause death in infected hosts including fish and humans. With the development of high-density farming in marine and freshwater fish aquaculture systems, severe disease or death, caused by trypanosomiasis, has been frequently reported. However, due to the lack of a model system, particularly for marine fish trypanosomes, and a paucity in the understanding of the biology and pathogenesis of these parasites, effective treatment for fish trypanosomiasis is significantly hampered. The goldfish is the common model system for freshwater fish trypanosomes, mainly of the species Trypanosoma carassii, while a similar model for marine fish trypanosomes has not yet been established. To address this issue, we found that Nile tilapia (Oreochromis niloticus) could be easily infected with a marine fish trypanosome, Trypanosoma epinepheli isolated from Lates calcarifer. Obvious clinical symptoms, associated with a high parasitemia (>108/ml), were found in the infected tilapias and more than 70% mortality was recorded in individuals within 20 days of infection. Interestingly, we also found that the Nile tilapia could also be infected with a freshwater fish trypanosome isolated from the largemouth bass (Micropterus salmoides) and caused significant death (more than 13%) in infected fish. This system not only provides an economical and effective laboratory model to study the biology and pathogenesis of marine and freshwater fish trypanosomes, but also provides a useful platform to develop vaccines and screen compounds for the protection and treatment of fish trypanosomiasis.

A new species of mammalian trypanosome, Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis.

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27-37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.

Mucosal immune response of Nile tilapia Oreochromis niloticus during Gyrodactylus cichlidarum infection.

Monogenean Gyrodactylus cichlidarum can cause severe mortality of Nile tilapia (Oreochromis niloticus) fry. To date, reports about mucosal immunity of O. niloticus against this parasite have been rare. In order to explore the mucosal immunity of Nile tilapia against G. cichlidarum infection, the expressions of six adaptive immune-related genes and the contents of specific immunoglobulin IgM and IgT in the skin-associated lymphoid tissues (SALT) were dynamically analyzed after primary and secondary infections. The abundances of G. cichlidarum on the hosts after secondary infection were lower than those after primary parasite infection, which implied that hosts could initiate immune protection against G. cichlidarum reinfection to some degree. The transcription levels of TCR-ß and CD4 genes in the skin tissue were significantly up-regulated after primary G. cichlidarum infection, while genes pIgR and IgT were only detected with significant up-regulations during secondary infection. With the exception of pIgR, which had remarkably higher expression in the fish with low parasite loads, all other genes studied tended to have higher mRNA level in the fish with higher parasite loads. The specific IgM content in the skin mucus increased significantly on the 2nd day after the primary exposure, higher than the corresponding value during the secondary exposure, and had significantly positive correlation with the parasite loads during the first parasite infection. These results manifested that acquired immune responses in the SALT of Nile tilapia participated in the resistance against G. cichlidarum infection, underscoring the involvement of mucosal immunity in fish against monogenean infection, and suggesting potential prophylactic treatment of gyrodactylid disease of tilapia.

Monogenean fauna of alien tilapias (Cichlidae) in south China.

Tilapias are important aquaculture fishes that have been introduced widely all over the world, often carrying their monogenean parasites with them. An extensive investigation on monogeneans of invasive tilapias was conducted in 19 natural water sources in south China between July 2015 and December 2017. We found nine known species of monogeneans, i.e., Enterogyrus coronatus, E. malmbergi, Cichlidogyrus cirratus, C. halli, C. sclerosus, C. thurstonae, C. tilapiae, Scutogyrus longicornis, Gyrodactylus cichlidarum, and one unknown Gyrodactylus species. In addition to reporting ten new hosts and four new geographical records, we observed new morphological characteristics of these species. Observation on living specimens of Enterogyrus spp. demonstrated that these two species have characteristic opisthaptoral retraction capacities, while the opisthaptor glands were not observed in our specimens of E. coronatus and E. malmbergi. The morphological differences of the accessory piece of the male copulatory complex between C. cirratus and C. mbirizei (character for species differentiation) could result from the observation at different perspectives, which indicates that C. mbirizei is likely a synonym of C. cirratus. A more detailed structure of the sclerotized parts of Cichlidogyrus spp. and S. longicornis were revealed by scanning electron microscopy. As was the case for the monogeneans found on alien tilapias from other geographic regions, the present study confirmed the high potential of these monogeneans to establish populations in new habitats.

Expression of immune-related genes of Nile tilapia Oreochromis niloticus after Gyrodactylus cichlidarum and Cichlidogyrus sclerosus infections demonstrating immunosupression in coinfection.

Gyrodactylus cichlidarum and Cichlidogyrus sclerosus, two monogenean ectoparasite species commonly found on the body surface and gills of Nile tilapia (Oreochromis niloticus) respectively, inflicted considerable economic losses in intensive tilapia farming. In order to explore the immune response of tilapia against these two species of monogeneans, expression patterns of five immune-related genes were studied after singular G. cichlidarum or C. sclerosus infection and their coinfection. The transcription levels of IL-1ß were up-regulated in the skin after G. cichlidarum infection, reaching a peak at day 5 PI, and in the gills after C. sclerosus infection (peaking at day 8 PI), with significant elevation only detected in the gills after high-dose C. sclerosus infection. A trend favoring increased gill TNF-α expression at day 8 PI of C. sclerosus infection was statistically significant only in the low-dose infection group. TNF-α expression in the skin did not change significantly after G. cichlidarum infection. TGF-ß had extremely up-regulated expressions in the gills at day 8 PI after both high- and low-dose C. sclerosus infections, but its significantly promoted expression in the skin was observed only after infection of high-dose G. cichlidarum. Significantly increased expressions of HSP70 and COX-2 in the skin were detected after high-dose G. cichlidarum infections. In comparison to singular infection with either G. cichlidarum or C. sclerosus, concurrent infection resulted in significantly advanced expression of TGF-ß in both skin and gills, and lower expressions at day 8 PI, and similar patterns were observed in the expression of IL-1ß and TNF-α in the gills. G. cichlidarum infection on the body surface significantly down-regulated the expressions of TNF-α, TGF-ß and COX-2 in the gills. In addition, the intensity of G. cichlidarum was significantly positively correlated with that of C. sclerosus (correlation index 0.922, p = 0.000) at day 2 PI under concurrent infection. These results contribute to the understanding of mucosal immunity of fish against monogenean infection, particularly when two monogenean species infect concurrently.

Nitric oxide blocks the development of the human parasite Schistosoma japonicum.

Human schistosomiasis, caused by Schistosoma species, is a major public health problem affecting more than 700 million people in 78 countries, with over 40 mammalian host reservoir species complicating the transmission ecosystem. The primary cause of morbidity is considered to be granulomas induced by fertilized eggs of schistosomes in the liver and intestines. Some host species, like rats (Rattus norvegicus), are naturally intolerant to Schistosoma japonicum infection, and do not produce granulomas or pose a threat to transmission, while others, like mice and hamsters, are highly susceptible. The reasons behind these differences are still a mystery. Using inducible nitric oxide synthase knockout (iNOS-/-) Sprague-Dawley rats, we found that inherent high expression levels of iNOS in wild-type (WT) rats play an important role in blocking growth, reproductive organ formation, and egg development in S. japonicum, resulting in production of nonfertilized eggs. Granuloma formation, induced by fertilized eggs in the liver, was considerably exacerbated in the iNOS-/- rats compared with the WT rats. This inhibition by nitric oxide acts by affecting mitochondrial respiration and energy production in the parasite. Our work not only elucidates the innate mechanism that blocks the development and production of fertilized eggs in S. japonicum but also offers insights into a better understanding of host-parasite interactions and drug development strategies against schistosomiasis.

Phylogeography of Angiostrongylus cantonensis (Nematoda: Angiostrongylidae) in southern China and some surrounding areas.

Angiostrongylus cantonensis is of increasing public health importance as the main zoonotic pathogen causing eosinophilic meningitis or meningoencephalitis, which has been documented all over the world. However, there are very limited studies about its phylogeography and spread pattern. In the present study, the phylogeography of A. cantonensis in southern China (including Taiwan) and partial areas of Southeast Asia were studied based on the sequences of complete mitochondrial cytochrome b (Cytb) gene. A total of 520 individuals of A. cantonensis obtained from 13 localities were sequenced for the analyses and grouped into 42 defined haplotypes. The phylogenetic tree (NJ tree and BI tree) revealed a characteristic distribution pattern of the four main lineages, with detectable geographic structure. Genetic differentiation among populations was significant, but demographic expansion could not be detected by either neutrality tests or mismatch distribution analysis, which implied a low gene flow among the local populations in different regions where the samples were collected. Two unique lineages of the A. cantonensis population in Taiwan were detected, which suggests its multiple origin in the island. Populations in Hekou (China) and Laos showed the highest genetic diversities, which were supported by both genetic diversity indices and AMOVA. These results together infer that the area around Thailand or Hekou in Yunnan province, China are the most likely origins of Angiostrongylus cantonensis.

Two new and one redescribed species of Acanthobothrium (Cestoda: Onchoproteocephalidea: Onchobothriidae) from Dasyatis akajei (Myliobatiformes: Dasyatidae) in the China Sea.

Acanthobothrium ningdense n. sp. and Acanthobothrium guanghaiense n. sp. are described from the spiral intestine of the whip stingray, Dasyatis akajei (Müller & Henle). Acanthobothrium ningdense n. sp. is reported based on 38 cestode specimens collected at five locations along the Chinese coast, i.e. Taizhou, Zhejiang Province, Ningde and Xiamen, Fujian Province, Taishan, Guangdong Province and Sanya, Hainan Province between 2012 and 2015. Acanthobothrium ningdense n. sp. belongs to the "species category 4" characterised by Ghoshroy & Caira. Among category 4 Acanthobothrium species, A. ningdense n. sp. most closely resembles A. micracantha Yamaguti, 1952 and A. latum Yamaguti, 1952, both from the same host species. Acanthobothrium ningdense n. sp. differs from A. micracantha by lacking a long robust hook handle that connects the bases of medial and lateral hooks, and from A. latum by having much longer axial prongs than abaxial prongs. Acanthobothrium guanghaiense n. sp., only found in Taishan, Guangdong Province in 2014, is a category 2 Acanthobothrium species. Among category 2, A. guanghaiense n. sp. most closely resembles A. semnovesiculum Verma, 1928, but can be differentiated from it by the arrangement of testes (in two staggered columns rather than in two tandem columns), and the shape of abaxial prong (nearly straight instead of with a conspicuous curve in the proximal portion). Acanthobothrium macrocephalum Wang & Yang, 2001 is redescribed based on new specimens collected from D. akajei in Guanghai and Sanya. With this study, the total number of Acanthobothrium species reported from D. akajei is brought to nine. It is possible that some of the species of Acanthobothrium previously reported from D. akajei not encountered in this study may have geographically restricted distributions, as was observed here for A. guanghaiense n. sp..

Comparative phylogeography of two monogenean species (Mazocraeidae) on the host of chub mackerel, Scomber japonicus, along the coast of China.

In the present paper, the phylogeographies of two monogenean species, Pseudokuhnia minor and Kuhnia scombri, on the same species of host, Scomber japonicus, were studied. Fragments of the mitochondrial cytochrome c oxidase subunit 1 gene were sequenced for 264 individuals of P. minor and 224 individuals of K. scombri collected from 10 localities along the coast of China. Genetic diversity of K. scombri was higher than that of P. minor, which may imply that P. minor has a lower evolution rate and/or is a younger species. The neighbour-joining (NJ) trees of both parasites were comprised of two clades without association to sample sites, which is the signature of remixing populations following past division. Analyses of molecular variance and pairwise fixation index revealed different genetic structures for the populations of these two closely related species along the coast of China: P. minor without significant genetic structure, while K. scombri has some genetic differentiation. Both neutrality tests and mismatch distribution suggested that the populations of these two species of parasites experienced population expansion in the late Pleistocene era due to the glacial-interglacial cycles induced by climatic oscillations.

Phylogeography of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) on the host of mottled spinefoot (Siganus fuscescens) in the South China Sea, inferred from mitochondrial COI and ND2 genes.

To examine the phylogeographical pattern of Tetrancistrum nebulosi (Monogenea, Dactylogyridae) in the South China Sea, fragments of mitochondrial cytochrome c oxidase subunit I and NADH dehydrogenase subunit 2 genes were obtained for 220 individuals collected from 8 localities along the southeast coast of China and 1 locality in Terengganu, Malaysia. Based on these two genes, two and three distinct clades with geographic signals were revealed on the phylogenetic trees respectively. The divergence between these clades was estimated to occur in the late Pleistocene. Analysis of molecular variance and pairwise FST suggested a high rate of gene flow among individuals sampled from the Chinese coast, but with obvious genetic differentiation from the Malaysian population. Mismatch distribution and neutrality tests indicated that the T. nebulosi population experienced expansion in Pleistocene low sea level periods. Vicariance was considered to account for the genetic divergence between Chinese and Malaysian populations, while sea level fluctuations and mainland-island connections during glacial cycles were associated with the slight genetic divergence between the populations along the mainland coast of China and those off Sanya. On the contrary, oceanographic circulations and host migration could lead to genetic homogeneity of populations distributed along the mainland coast of China.

Complete mitogenome of the foxface rabbitfish Siganus vulpinus (Perciformes, Siganidae): indication of potential interbreeding in rabbitfishes.

In the present study, the complete mitochondrial genome of the foxface rabbitfish Siganus vulpinus (Perciformes, Siganidae) is determined. The entire sequence is 16,505 bp in length, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. The genome organization is similar to those found in other rabbitfishes. Apart from ND6 and 8 tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Overall base compositions of mitogenome are 29.3% of A, 29.3% of C, 25.7% of T, and 15.7% of G, showing an obvious anti-G bias which is commonly found in fishes. The high similarity of mitochondrial genome between S. vulpinus and S. unimaculatus indicate that natural interbreeding might exist in breeding season.

Complete mitochondrial genome of the orange-spotted spinefoot Siganus guttatus (Perciformes, Siganidae).

The complete mitochondrial genome of the orange-spotted spinefoot Siganus guttatus (Perciformes, Siganidae) is presented in this paper. The entire sequence, 16,505 bp in length, is comprised of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. Structurally, the composition and order of genes are same as those found in other rabbitfishes. Except ND6 and 8 tRNA, all other mitochondrial genes were encoded on the heavy strand. Overall base compositions of mitogenome are 29.3% of A, 29.3% of C, 25.7% of T, and 15.7% of G, showing an obvious anti-G bias which was commonly found in fishes. Three conserved sequence blocks (CSB-1, CSB-2 and CSB-3) and long series of di- and mono-nucleotide microsatellite repeats in 3' end are observed in the putative control region. The complete mitogenome sequence of S. guttatus could provide useful information for further phylogenetic and population genetic studies.

Complete mitochondrial genome of the giant African snail, Achatina fulica (Mollusca: Achatinidae): a novel location of putative control regions (CR) in the mitogenome within Pulmonate species.

The whole sequence (15,057 bp) of the mitochondrial DNA (mtDNA) of the terrestrial snail Achatina fulica (order Stylommatophora) was determined. The mitogenome, as the typical metazoan mtDNA, contains 13 protein-coding genes (PCG), 2 ribosomal RNA genes (rRNA) and 22 transfer RNA genes (tRNA). The tRNA genes include two trnS without standard secondary structure. Interestingly, among the known mitogenomes of Pulmonata species, we firstly characterized an unassigned lengthy sequence (551 bp) between the cox1 and the trnV which may be the CR for the sake of its AT bases usage bias (65.70%) and potential hairpin structure.

Complete mitochondrial genome of the pelagic stingray Pteroplatytrygon violacea (Myliobatiformes: Dasyatidae).

The pelagic stingray Pteroplatytrygon violacea is the only pelagic species of the Dasyatidae (Chondrichthyes: Elasmobranchii), which is widely distributed in all tropical, subtropical and temperate oceans. In this study, the complete mitochondrial genome of Pteroplatytrygon violacea was determined. It is 17,665 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 control region, with the typical gene order and direction of transcription of vertebrates. The overall nucleotide compositions of the whole mitogenome are 30.8% A, 26.9% C, 28.6% T and 13.7% G. The ND2 and CO1 sequences are highly similar to the corresponding sequences of this species available in NCBI collected from the Atlantic sea.

Two new species of Euryhaliotrema Kritsky et Boeger, 2002 (Monogenea: Dactylogyridae) from Lutjanus russellii (Bleeker) and L. argentimaculatus (Forsskål) (Teleostei: Lutjanidae) in the South China Sea.

Euryhaliotrema russellum sp. n. and Euryhaliotrema tenuiaccessorium sp. n. are described from the gills of Lutjanus russellii (Bleeker) and L. argentimaculatus (Forsskål), respectively, in the South China Sea. Euryhaliotrema russellum sp. n. differs from all other members of Euryhaliotrema Kritsky et Boeger, 2002 by its unique male copulatory organ, a straight tube with a bulbous base and without an accessory piece. Euryhaliotrema tenuiaccessorium sp. n. differs from its congeners by having a copulatory organ with a bulbous base, a meandering copulatory tube with clockwise coil having less than one complete ring, and a slender accessory piece along the middle length of the copulatory tube. All species of Euryhaliotrema are listed and divided to three groups based on morpholgy of the male copulatory organ.

Two new species of Haliotrema Johnston & Tiegs, 1922 (Monogenea: Dactylogyridae) from Acanthurus nigrofuscus (Forsskål) and A. triostegus (Linnaeus) (Teleostei: Acanthuridae) in the South China Sea.

Haliotrema nanhaiense n. sp. and Haliotrema triostegum n. sp. are described respectively from the gills of Acanthurus nigrofuscus (Forsskål) and Acanthurus triostegus (Linnaeus) in the South China Sea. Haliotrema nanhaiense n. sp. differs from other existing congeneric species by its male copulatory complex, comprising a C-shaped copulatory tube, a saucer-shaped base and a sickle-shaped accessory piece. Haliotrema triostegum n. sp. can be differentiated from all other members of Haliotrema by having a unique copulatory complex, a cup-shaped base, inverted L-shaped copulatory tube with a small sclerotised piece arising from its distal portion, and a large accessory piece from the proximal portion of copulatory tube.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen.

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Complete mitochondrial genome of the masked spinefoot Siganus puellus (Perciformes, Siganidae).

In the present study, the complete mitochondrial genome of the masked spinefoot Siganus puellus (Perciformes, Siganidae) is determined. The entire sequence is 16,504 bp in length, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. Gene organization and gene order are similar to those found in other rabbitfishes. Apart from ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Overall base compositions of mitogenome are 28.6% of A, 29.5% of C, 25.6% of T and 16.3% of G. The overall mean genetic distances of 13 protein-coding genes among three Siganus species with known complete mitogenome ranges from 0.076 (COXII) to 0.154 (ATP6), higher than the divergence (0.107) of control region, which implies that ATP6 gene may be more appropriate for population genetic structure analyses of Siganus species.

Studies on Lethrinitrema Lim & Justine, 2011 (Monogenea: Dactylogyridae), with the description of two new species, a key to the genus and a phylogenetic analysis based on rDNA sequences.

Six species of Lethrinitrema Lim & Justine, 2011, including two new taxa, are described from the gills of Lethrinus nebulosus (Forsskål) from the South China Sea. Lethrinitrema nebulosum n. sp. differs from all other members of the genus in possessing a copulatory organ with a short, distally recurved tube, a cup-shaped base and a thin accessory piece, arising from distal end of tube, junction inconspicuous. Lethrinitrema zhanjiangense n. sp. can be distinguished from its congeners by possessing a copulatory organ with a C-shaped tube, a cup-shaped base and without accessory piece. Lethrinitrema grossecurvitubum (Li & Chen, 2005) n. comb. and Lethrinitrema austrosinense (Li & Chen, 2005) n. comb., previously included in Haliotrema Johnston & Tiegs, 1922, are transferred to Lethrinitrema and redescribed with additional details, including the intestinal caeca that unite posterior to gonads and continue posteriorly as two diverticula, and the elongate tubular distal end of each haptoral reservoir bifurcating prior to entering a superficial lateral groove on each side of the ventral anchor. The sclerotised parts of two unidentified species of Lethrinitrema are also described. Lethrinitrema sp. 1 differs from the other Lethrinitrema spp. in possessing a male copulatory organ consisting of a short tapered tube with a robust cup-shaped base and in lacking accessory piece. Lethrinitrema sp. 2 differs from its congeners in possessing a copulatory organ comprised of a short slender tube and without accessory piece, delicate ventral and dorsal bars, and poorly developed outer roots of the anchors. Sequences of partial 28S rDNA (domains D1-D2) and complete 18S rDNA for 27 dactylogyrids including five species of Lethrinitrema, i.e. Lethrinitrema fleti (Young, 1968) Lim & Justine, 2011, L. nebulosum, L. zhanjiangense, L. grossecurvitubum and Lethrinitrema sp. 1 were used to assess the monophyly of Lethrinitrema which was supported by high bootstrap values.