Although nudibranchs are common and attractive animals, our understanding of these marine gastropods in Vietnam remains limited. Prior research has suggested that combining morphological examination with molecular analysis results in more accurate identification of nudibranchs. However, previous studies in Vietnam have typically relied solely on morphological methods for nudibranch identification. In this study, the nudibranch species Halgerda batangas was recorded in Vietnam for the first time based on both morphological and molecular approaches. Halgerda batangas was characterized by a network pattern consisting of orange lines, relatively low dorsal tubercles with red-orange caps and white basal rings, and an orange line along the foot margin. Molecular analysis corroborated the morphological findings. These results suggest that integrating morphological and molecular methods is an effective approach for identifying nudibranchs.

Jellyfishes have ecological and societal value, but our understanding of taxonomic identity of many jellyfish species remains limited. Here, an approach integrating morphological and molecular (16S ribosomal RNA and cytochrome oxidase I) data enables taxonomic assessment of the blubber jellyfish found in the Philippines. In this study, we aimed to resolve doubt on the taxonomy of Acromitoides purpurus, a valid binomen at the time of our research. Our morphological findings confirm that this jellyfish belongs to the genus Catostylus, and is distinct from known species of the genus inhabiting the Western Pacific, such as Catostylus ouwensi, Catostylus townsendi, and Catostylus mosaicus. Detailed morphological and molecular analyses of the type specimens from the Philippines with the other Catostylus species revive the binomen Catostylus purpurus and invalidate A. purpurus. Genetic analysis also distinguishes this Philippine jellyfish from C. townsendi and C. mosaicus. Through this study, we arranged several Catostylidae taxa into species inquirendae (Catostylus tripterus, Catostylus turgescens, and Acromitoides stiphropterus) and one genus inquirenda (Acromitoides) and provided an identification key for species of Catostylus. This comprehensive study confirms the blubber jellyfish as C. purpurus, enriching our understanding of jellyfish biodiversity. The integration of morphological and genetic analyses proves vital in resolving taxonomic ambiguities within the Catostylidae family and in the accurate identification of scyphozoan jellyfishes.

Species of Baylisascaris (Nematoda: Ascarididae) are of great veterinary and zoonotic significance, owing to cause Baylisascariosis or Baylisascariasis in wildlife, captive animals and humans. However, the phylogenetic relationships of the current 10 Baylisascaris species remain unclear. Moreover, our current knowledge of the detailed morphology and morphometrics of the important zoonotic species B. procyonis is still insufficient. The taxonomical status of B. procyonis and B. columnaris remains under debate. In the present study, the detailed morphology of B. procyonis was studied using light and scanning electron microscopy based on newly collected specimens from the raccoon Procyon lotor (Linnaeus) in China. The results of the ASAP analysis and Bayesian inference (BI) using the 28S, ITS, cox1 and cox2 genetic markers did not support that B. procyonis and B. columnaris represent two distinct species. Integrative morphological and molecular assessment challenged the validity of B. procyonis, and suggested that B. procyonis seems to represent a synonym of B. columnaris. Molecular phylogenetic results indicated that the species of Baylisascaris were grouped into 4 clades according to their host specificity. The present study provided new insights into the taxonomic status of B. procyonis and preliminarily clarified the phylogenetic relationships of Baylisascaris species.

In recent years, the genus Bryoria (Parmeliaceae, Lecanoromycetes) has been the subject of considerable phylogenetic scrutiny. Here we used information on six gene regions, three nuclear protein-coding markers (Mcm7, GAPDH and Tsr1), two nuclear ribosomal markers (ITS and IGS) and a partial mitochondrial small subunit (mtSSU), to examine infrageneric relationships in the genus and to assess species delimitation in the Bryoria bicolor/B. tenuis group in section Divaricatae. For this purpose, phylogenetic analyses and several of the available algorithms for species delimitation (ASAP, GMYC single, GMYC multiple and bPTP) were employed. We also estimated divergence times for the genus using *BEAST. Our phylogenetic analyses based on the combined data set of six gene loci support the monophyly of sections Americanae, Divaricatae and Implexae, while section Bryoria is polyphyletic and groups in two clades. Species from Bryoria clade 1 are placed in an emended section Americanae. Our study reveals that section Divaricatae is young (c. 5 My) and is undergoing diversification, especially in South-East Asia and western North America. Separate phylogenetic analyses of section Divaricatae using ITS produced a topology congruent with the current species concepts. However, the remaining gene regions produced poorly resolved phylogenetic trees and the different species delimitation methods also generated highly inconsistent results, congruent with other studies that highlight the difficulty of species delimitation in groups with recent and rapid radiation. Based on our results, we describe the new species B. ahtiana sp. nov., characterized by its bicolorous, caespitose, widely divergent thallus, conspicuously thickening main stems, well-developed secondary branches, and rather sparse third-order branchlets. Another new lineage, referred to here as B. tenuis s. lat., is restricted to western North America and may represent a new species recently diverged from B. tenuis s. str., though further work is needed.

There is still a high diversity of lichen-forming fungi that remains undescribed, especially cryptic lineages at the species level. Integrating morphological, chemical, and DNA sequence data has proved useful in corroborating species descriptions and delimitations. Here we reviewed morphological features, secondary metabolites and the DNA sequences of ITS, mtSSU and nuLSU markers to study the diversity of Xanthoparmelia in southern Africa. A total of 37 species were recorded. Three of these appear undescribed, and we therefore describe them here as new: Xanthoparmelia nimisii, with a sorediate thallus and broad lobes, is well supported as a clade separate from X. annexa; X. pseudochalybaeizans with a white medulla is phylogenetically distinct from the otherwise similar X. chalybaeizans; and X. sipmaniana, well supported as a separate clade from the similar X. hypoprocetrarica. In addition, the separation of Xanthoparmelia capensis and X. tinctina requires further studies.

Species of lichen-forming fungi (LFF) display an array of geographical distribution patterns. Among the broadly distributed lichen-forming fungal species, the degree of reproductive isolation and genetic substructure among populations varies widely, in some cases masking unrecognized diversity or meaningful biogeographical patterns. Lecidea atrobrunnea (Raymond ex Lam. & DC.) Schaer. s. lat. (Lecideaceae) is a widespread species complex that has been studied for over two centuries since its initial description. The diversity of the L. atrobrunnea group is highest in western North America, where a dizzying array of morphologies and chemistry can occur at local scales. Here we investigate whether the assumed cosmopolitan distribution of L. atrobrunnea s. lat. is an artifact of taxonomic limitations and masks biogeographical patterns in this species complex. To address these questions, we compiled sequence data from the standard fungal barcoding marker (ITS) for over 100 specimens within this complex, in addition to genome-scale data from a subset of these representing over 1600 single-copy nuclear genes spanning over 3 Mb of the genome. Our study corroborates the perspective that the morphologically and chemically variable Lecidea atrobrunnea group reflects a complex of distinct species-level lineages, with 42–83 candidate species inferred from the ITS region and high levels of diversity inferred from a subset of specimens using genome-scale data. However, both phenotype- and molecular-based species boundaries remained unsettled, with the most common nominal taxa recovered as highly polyphyletic and with conflict among different molecular species delimitation approaches. Our study also highlights the potential for geographically restricted species, with fascinating biogeographical patterns, challenging, in part, the assumed cosmopolitan distribution of L. atrobrunnea s. lat. This study provides valuable direction for future research that will be crucial in understanding diversification and establishing a robust taxonomy for this well-known species complex.

The assassin snail genus Anentome is widespread in Southeast Asia, and is distributed all over the world via the aquarium trade. One species of genus Anentome, Anentome helena, is known to act as intermediate host of parasitic trematodes. This study investigates the taxonomic diversity of larval trematodes infecting A. helena and Anentome wykoffi in Thailand. Larval trematodes were identified by combining morphological and DNA sequence data (cytochrome c oxidase I and internal transcribed spacer 2). Species delimitation methods were used to explore larval trematode species boundaries. A total of 1107 specimens of Anentome sp. were collected from 25 localities in Thailand. Sixty-two specimens of A. helena (n = 33) and A. wykoffi (n = 29) were infected by zoogonid cercariae, heterophyid metacercariae and echinostome metacercariae, with an overall prevalence of 5.6% (62/1107) and population-level prevalences in the range of 0.0–22.3%. DNA sequence data confirmed that the larval trematodes belong to the families Echinostomatidae, Heterophyidae and Zoogonidae. As such, this is the first report of zoogonid cercariae and heterophyid metacercariae in A. helena, and echinostome metacercariae in A. wykoffi. Moreover, this study provides evidence of tentative species-level differentiation between Thai Echinostoma sp. and Cambodian Echinostoma mekongi, as well as within Echinostoma caproni, Echinostoma trivolvis and Echinostoma revolutum.

The widespread species Parmotrema crinitum (Ach.) M. Choisy and Parmotrema perlatum (Huds.) M. Choisy are mainly distinguished by their reproductive strategies. While P. crinitum propagates by isidia, P. perlatum produces soredia. In this study, we aim to evaluate the phylogenetic relationship between both species and to critically examine their species boundaries. To this purpose, 46 samples belonging to P. crinitum and P. perlatum were used in our analysis, including 22 for which we studied the morphology and chemistry, before extracting their DNA. We used 35 sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS) of Parmotrema perlatum from Europe and Africa (20 of which were newly generated), and 11 of Parmotrema crinitum from Europe, North America and North Africa (two newly generated). Additionally, 28 sequences of several species from Parmotrema were included in the ITS dataset. The ITS data matrix was analyzed using different approaches, such as traditional phylogeny (maximum likelihood and Bayesian analyses), genetic distances, automatic barcode gap discovery (ABGD) and the coalescent-based method poisson tree processes (PTP), in order to test congruence among results. Our results indicate that all samples referred to P. crinitum and P. perlatum nested in a well-supported monophyletic clade, but phylogenetic relationships among them remain unresolved. Delimitations inferred from PTP, ABGD and genetic distance analyses were comparable and suggested that P. crinitum and P. perlatum belong to the same lineage. Interestingly, two samples of P. perlatum separate in a different monophyletic clade, which is supported as a different lineage by all the analyses.

In recent years, several species that have long been considered to belong in Bacidia s. lat. have been transferred to other genera such as Bellicidia, Bibbya, Scutula, and also to Toniniopsis, accommodating species previously placed in Bacidia and Toninia. One of its widespread species, Toniniopsis subincompta, can be recognized by its thinly granular thallus, dark brown to black apothecia, green epithecium, red-brown hypothecium, and bacilliform ascospores. However, it shows considerable variation in thallus structure, and coloration of apothecia, hypothecium and exciple. We sequenced 20 specimens of T. subincompta to investigate whether there is phylogenetic support for the delimitation of species in accordance with the variability of the observed characters. For phylogenetic analyses, we used newly generated sequence data from the nuclear internal transcribed spacer (nrITS), mitochondrial small subunit (mtSSU) and DNA-directed RNA polymerase II subunit (RPB2). Maximum likelihood and Bayesian inference analyses, as well as three species delimitation programs, provided consistent evidence that T. subincompta forms two separate lineages, to be recognized at the species level. The complex nomenclature of T. subincompta (basionym Lecidea subincompta) shows it to be a synonym of Bellicidia incompta. For the most common taxon previously called Bacidia (Toniniopsis) subincompta, the new combination T. separabilis is made, rather than proposing a conserved type for Lecidea subincompta. Toniniopsis dissimilis is newly described to accommodate the less common taxon. Toniniopsis dissimilis is characterized by a predominantly wrinkled to warted to subsquamulose thallus; generally grey-brown to dark brown apothecia, often with a lighter margin; a dark brown hypothecium, frequently gradually merging into the coloration of the exciple below and the lateral part of the exciple attached to the hymenium; a mostly colourless rim and lateral part of the exciple. The closely related T. separabilis is characterized by a thallus of mostly single or contiguous ±loose granules, often forming short, coralloid, isidium-like bulges; darker apothecia, with a margin mostly of the same colour or darker than the disc; a comparatively thinner hypothecium easily separated from the exciple below. The rim and lateral part of the exciple often contain either a blue, brown or mixed blue-brown colour in the upper part or along the whole margin. Lectotypes of Bacidia vegeta, Lecidea bacillifera f. melanotica and Secoliga atrosanguinea var. affinis (the synonyms of T. separabilis) are selected. Cyanotrophy and the occurrence of albino morphs in T. separabilis are discussed.

The epicontinental fauna of the Iberian Peninsula is strongly influenced by its geographical history. As the possibilities for dispersion of organisms into and from this region were (and still are) limited, the local fauna consists almost exclusively of endemic species. Almost all Iberian freshwater fishes of the families Leuciscidae and Cyprinidae are endemic and on-going research on these taxa continually uncovers new species. Nevertheless, information on their host-specific parasites remains scarce. In this study, we investigate the diversity and phylogenetic relationships in monogeneans of the genus Dactylogyrus (gill ectoparasites specific to cyprinoid fish) in the Iberian Peninsula. Twenty-two species were collected and identified from 19 host species belonging to Cyprinidae and Leuciscidae. A high degree of endemism was observed, with 21 Dactylogyrus species reported from Iberia only and a single species, D. borealis, also reported from other European regions. Phylogenetic analysis split the endemic Iberian Dactylogyrus into two well-supported clades, the first encompassing Dactylogyrus parasitizing endemic Luciobarbus spp. only, and the second including all Dactylogyrus species of endemic leuciscids and four species of endemic cyprinids. Species delimitation analysis suggests a remarkable diversity and existence of a multitude of cryptic Dactylogyrus species parasitizing endemic leuciscids (Squalius spp. and representatives of Chondrostoma s.l.). These results suggest a rapid adaptive radiation of Dactylogyrus in this geographically isolated region, closely associated with their cyprinoid hosts. Moreover, phylogenetic analysis supports that Dactylogyrus parasites colonized the Iberian Peninsula through multiple dispersion events.

Since the advent of molecular taxonomy, numerous lichen-forming fungi with homoiomerous thalli initially classified in the family Collemataceae Zenker have been transferred to other families, highlighting the extent of morphological convergence within Lecanoromycetes O. E. Erikss. & Winka. While the higher level classification of these fungi might be clarified by such transfers, numerous specific and generic classifications remain to be addressed. We examined the relationships within the broadly circumscribed genus Arctomia Th. Fr., which has been the recipient of several transfers from Collemataceae. We demonstrated that Arctomia insignis (P. M. Jørg. & Tønsberg) Ertz does not belong to Arctomia s. str. but forms a strong monophyletic group with Gabura fascicularis (L.) P. M. Jørg. We also confirmed that Arctomia borbonica Magain & Sérus. and the closely related Arctomia insignis represent two species. We formally transferred A. insignis and A. borbonica to the genus Gabura Adans. and introduced two new combinations: Gabura insignis and Gabura borbonica. We reported Gabura insignis from Europe (Scotland and Ireland) for the first time. While material from Europe and North America is genetically almost identical, specimens from Madagascar, South Africa and Reunion Island belong to three distinct phylogenetic lineages, all of which are present in the latter area and may represent distinct species. In its current circumscription, the genus Gabura may contain up to six species, whereas Arctomia s. str. includes only two species (A. delicatula Th. Fr. and A. teretiuscula P. M. Jørg.). The Gabura insignis group is shown to have an unexpectedly large, subcosmopolitan distribution. With the extended sampling from Arctomiaceae Th. Fr., the placement of Steinera sorediata P. James & Henssen in the genus Steinera Zahlbr. is confirmed and the presence of a new Steinera species from Chile is highlighted.

Species identification in the tropical lichen genus Phyllopsora is generally challenging and is based on ascospore morphology, vegetative dispersal units, thallus structure and secondary chemistry. As several type specimens are in poor condition and difficult to interpret, it is often unclear how these old names fit with the currently used taxonomy. In the present study, we aim to identify species boundaries in Phyllopsora s. str. supported by an integrative approach using multiple sources of evidence. We investigated a substantial amount of herbarium as well as freshly collected material and generated mtSSU and ITS sequence data from most of the described species, including several types. Species delimitation analyses are applied on the gene trees using mPTP and we construct a species tree of both markers with *BEAST, facilitating discussion of species delimitation and sister-relationships. Comparing morphology, chemistry and molecular data, we found that the mPTP analyses split established species repeatedly. Based on our integrative results, we exclude nine species from the genus, resurrect one (P. melanoglauca Zahlbr.), reduce two into synonymy with other Phyllopsora species and describe five as new to science: Phyllopsora amazonica Kistenich & Timdal (which shares the secondary chemistry (atranorin and terpenoid pattern) with P. halei chemotype 1, but differs, e.g., in having smaller areolae that are attached to a thinner, white prothallus, and in having more persistently marginate and less convex apothecia), Phyllopsora concinna Kistenich & Timdal (which shares the secondary chemistry (atranorin and parvifoliellin) with P. parvifoliella and P. rappiana, but differs from both in forming larger isidia, having a white prothallus, apothecial margin paler than the disc, and longer and broader ascospores), Phyllopsora furfurella Kistenich & Timdal (which is here segregated from P. furfuracea based on having a white prothallus and in containing skyrin in the hypothecium (K+ red)), Phyllopsora isidosa Kistenich & Timdal (which differs from P. byssiseda in forming a more crustose thallus with more delicate isidia, and from P. isidiotyla in forming somewhat coarser, less branched isidia) and Phyllopsora neotinica Kistenich & Timdal (a neotropical species here segregated from the now exclusively paleotropical P. chodatinica, differing in containing an unknown xanthone (not chodatin)). Lectotypes are designated for Biatora pyrrhomelaena Tuck., Lecidea leucophyllina Nyl., L. pertexta Nyl., and P. brachyspora Müll. Arg. In total, we accept 54 species in the genus Phyllopsora.

All more than 3000 species of Agrilus beetles are phytophagous and some cause economically significant damage to trees and shrubs. Facilitated by international trade, Agrilus species regularly invade new countries and continents. This necessitates a rapid identification of Agrilus species, as the first step for subsequent protective measures. This study provides the first DNA reference library for ~100 Agrilus species from the Northern Hemisphere based on three mitochondrial markers: cox1–5′ (DNA barcode fragment), cox1–3′, and rrnL. All 329 Agrilus records available in the Barcode of Life Database format, including specimen images and geo data, are released through a public dataset ‘Agrilus1 329’ available at: dx.doi.org/10.5883/DS-AGRILUS1. All Agrilus species were identified using adult morphology and by using molecular phylogenetic trees, as well as distance- and tree-based algorithms. Most DNA-based species limits agree well with the morphology-based identification. Our results include cases of high intraspecific variability and multiple species para- and polyphyly. DNA barcoding is a powerful species identification tool in Agrilus, although it frequently fails to recover morphologically-delimited Agrilus species-group. Even though the current three-gene database covers only ~3% of the known Agrilus diversity, it contains representatives of all principal lineages from the Northern Hemisphere and represents the most extensive dataset built for DNA-delimited species identification within this genus so far. Molecular data analyses can rapidly and cost-effectively identify an unknown sample, including immature stages and/or non-native taxa, or species not yet formally named.

The lichen species Cladonia angustiloba is characterized by a well-developed primary thallus and narrow squamules which show deep incisions, and the presence of usnic and fumarprotocetraric acids. Morphologically it is similar to C. foliacea and C. convoluta, from which it can be distiguished by the squamule size and morphology. Since similar characters were used to distinguish C. foliacea from C. convoluta which do not represent different lineages, it is necessary to examine the taxonomic status of C. angustiloba by means of DNA sequences. In this study, the species delimitation within the C. foliacea complex was studied by sequencing three loci, ITS rDNA, cox1 and RPB2. The data were analyzed by means of phylogenetic and species delimitation methods (GMYC, PTP, ABGD and BPP). Our results show that none of the three species is monophyletic. Most of the species delimitation methods did not support the current species as evolutionary lineages. Only some of the BPP analyses supported C. angustiloba as a species distinct from C. foliacea and C. convoluta. However, the hypothesis that considers the C. foliacea complex as constituted by a unique species obtained the best Bayes Factor value. Therefore, C. angustiloba and C. convoluta are synonymized with C. foliacea. A new, thoroughly checked synonymy with typifications of the whole C. foliacea complex is presented. An updated survey of the world distribution data is compiled.

Specimen-level phylogenetic approaches are widely used in molecular biology for taxonomic and systematic purposes. However, they have been largely ignored in analyses based on morphological traits, where phylogeneticists mostly resort to species-level analyses. Recently, a number of specimen-level studies have been published in vertebrate palaeontology. These studies indicate that specimen-level phylogeny may be a very useful tool for systematic reassessments at low taxonomic levels. Herein, we review the challenges when working with individual organisms as operational taxonomic units in a palaeontological context, and propose guidelines of how best to perform a specimen-level phylogenetic analysis using the maximum parsimony criterion. Given that no single methodology appears to be perfectly suited to resolve relationships among individuals, and that different taxa probably require different approaches to assess their systematics, we advocate the use of a number of methodologies. In particular, we recommend the inclusion of as many specimens and characters as feasible, and the analysis of relationships using an extended implied weighting approach with different downweighting functions. Resulting polytomies should be explored using a posteriori pruning of unstable specimens, and conflicting tree topologies between different iterations of the analysis should be evaluated by a combination of support values such as jackknifing and symmetric resampling. Species delimitation should be consistent among the ingroup and based on a reproducible approach. Although time-consuming and methodologically challenging, specimen-level phylogenetic analysis is a highly useful tool to assess intraspecific variability and provide the basis for a more informed and accurate creation of species-level operational taxonomic units in large-scale systematic studies. It also has the potential to inform us about past speciation processes, morphological trait evolution, and their potential intrinsic and extrinsic drivers in pre-eminent detail.

Ramalina europaea Gasparyan, Sipman & Lücking and R. labiosorediata Gasparyan, Sipman & Lücking, two species of the R. pollinaria group, are described here as new to science. Ramalina europaea, widely distributed in Europe, can be distinguished by small, punctiform, often terminal soralia starting out on small, spine-like branchlets, whereas R. labiosorediata from North America differs from R. pollinaria s. str. and R. europaea in the almost exclusively terminal soralia formed on the tips of normal lobes, originating from the underside and becoming irregularly labriform. Morphological characters, chemistry, ecology and geographical distribution are discussed and a key to the species of the Ramalina pollinaria group is provided. The topology of a maximum likelihood tree based on ITS shows the presence of three well-supported clades, corresponding to the morphological differences of the three species. The status of several historical names variously placed in synonymy with or described as infraspecific entities of R. pollinaria is reassessed and a new neotype and an epitype are designated for Lichen pollinarius, a neotype for L. squarrosus, making it a synonym of R. farinacea, and lectotypes for R. pollinaria var. elatior, making it a synonym of R. pollinaria s. str., and for var. humilis, a taxon of yet unknown affinity.

The Splanchnotrophidae is a family of highly modified endoparasitic copepods known to infest nudibranch or sacoglossan sea slug hosts. Most splanchnotrophid species appear to be specific to a single host, but some were reported from up to nine different host species. However, splanchnotrophid taxonomy thus far is based on external morphology, and taxonomic descriptions are, mostly, old and lack detail. They are usually based on few specimens, with intraspecific variability rarely reported. The present study used molecular data for the first time to test (1) the current taxonomic hypotheses, (2) the apparently strict host specificity of the genus Ismaila and (3) the low host specificity of the genus Splanchnotrophus with regard to the potential presence of cryptic species. Phylogenetic analyses herein used sequences of the barcoding region of the cytochrome oxidase I (COI) gene from 40 specimens representing 13 species of five genera. Species delimitation approaches include distance and barcoding gap analyses, haplotype networks and diagnostic nucleotides. Molecular results are largely compatible with the commonly accepted, morphology-based taxonomy of the Splanchnotrophidae. Strict host specificity could be confirmed for two Ismaila species. COI analyses also supported the idea that Splanchnotrophus angulatus is host-promiscuous. In Ismaila, morphology seems more suitable than barcoding to display speciation events via host switches in a recent Chilean radiation. In Splanchnotrophus, some genetic structure suggests ongoing diversification, which should be investigated further given the inadequate morphology-based taxonomy. The present study thus supports the presence of two different life history strategies in splanchnotrophids, which should be explored integratively.

The recent development of genetic methods allows the delineation of species boundaries, especially in organisms where morphological characters are not reliable to differentiate species. However, few empirical studies have used these tools to delineate species among parasitic metazoans. Here we investigate the species boundaries of Clinostomum, a cosmopolitan trematode genus with complex life cycle. We sequenced a mitochondrial [cytochrome c oxidase subunit I (COI)] gene for multiple individuals (adults and metacercariae) from Middle-America. Bayesian phylogenetic analysis of the COI uncovered five reciprocally monophyletic clades. COI sequences were then explored using the Automatic Barcode Gap Discovery to identify putative species; this species delimitation method recognized six species. A subsample was sequenced for a nuclear gene (ITS1, 5·8S, ITS2), and a concatenated phylogenetic analysis was performed through Bayesian inference. The species delimitation of Middle-American Clinostomum was finally validated using a multispecies coalescent analysis (species tree). In total, five putative species are recognized among our samples. Mapping the second intermediate hosts (fish) onto the species tree suggests that metacercariae of these five species exhibit some level of host specificity towards their fish intermediate host (at the family level), irrespective of geographical distribution.

The diversity of the Trypethelium eluteriae group in Thailand was studied using molecular (ITS and mtSSU rDNA sequences), morphological, and chemical data. Three species were recognized, T. eluteriae, T. platystomum, and T. subeluteriae, with the latter two being new records for Thailand. The separation of the three species, which have sometimes been regarded as synonymous, is supported by molecular and phenotypic characters. The chemical profiles of the three species are distinct, while ascospore size, often used to distinguish species in the group, shows some overlap.