A new species of Enchytraeus (Enchytraeidae, Clitellata) from sewage sludge of a plum processing plant in Japan.

Enchytraeus ohtakai sp. nov. (Enchytraeidae, Clitellata, Oligochaeta) was discovered in the organic matter of a wastewater treatment facility of a plums processing plant in Honshu, Japan. The wastewater is characterized by high organic matter content and low salt concentration. Morphological analysis and DNA-sequencing of a fragment of the COI barcoding gene show that the new species belongs to the E. albidus species group. Within this group it differs in: vasa deferentia restricted to XII, preclitellar bundles with mostly three chaetae, postclitellar bundles with two or three, dorsal blood vessel from XII or XIII, spermathecal ectal duct completely glandular. spermatheca with a large diverticulum, accessory sexual glands present in XII, clitellum ventrally almost absent. The individual gene trees of COI analysis recovered this new species as a monophyletic group within the genus Enchytraeus, closely related to E. albidus species group.

Origin and diversification of pheretimoid megascolecid earthworms in the Japanese Archipelago as revealed by mitogenomic phylogenetics.

Megascolecid earthworms of the pheretimoid group are dominant detritivores of soil ecosystems in the Japanese Archipelago and East Asia. However, their diversity and phylogenetic relationships are poorly understood. We assembled whole mitogenome sequences for 197 megascolecid earthworms collected throughout Japan to study the phylogenetic relationships, phylogeography, divergence times, and diversification of important morphological characteristics among pheretimoid earthworms. Using 197 mitogenome sequences and 24 published mitogenome sequences from the East Asian mainland (221 sequences in total), we constructed a maximum likelihood tree and found that the pheretimoid earthworms currently assigned to Amynthas, Metaphire, Duplodicodrilus, and Manus are involved in the most senior genus Amynthas; thus, Amynthas can be treated as the sole genus encompassing all of the above genera. Within the Amynthas group, we identified three major lineages that led to four groups of endemic species in Japan. These lineages originated from different lineages on the East Asian mainland and Taiwan Island, indicating multiple colonization events from the East Asian mainland by different ancestral lineages, possibly after the Miocene. We also assembled nuclear ribosomal DNA sequences encompassing the 18S to 28S rRNA genes. The nuclear gene tree showed major groups consistent with the mitogenome tree except for different (and not well-resolved) relationships among major clades. Our molecular data covered 115-158 native and 7 non-native Amynthas group species in Japan in terms of DNA-based species delimitation. Our findings provide a basis for understanding the evolutionary relationships among diversified megascolecid earthworms in the Amynthas group in Japan and adjacent regions.

Chameleon-Inspired Brilliant and Sensitive Mechano-Chromic Photonic Skins for Self-Reporting the Strains of Earthworms.

The skins of chameleons have attracted growing interest because they have sensitive mechano-chromic properties and bright colors due to the large surface-to-surface distances (Ds-s) between neighboring particles and contrast of the refractive index (Δn), respectively. Inspired by these, artificial mechano-chromic photonic skins (MPSs) mimicking those of chameleons were fabricated by the large Δn and Ds-s. The fabrication is considerably simple and efficient based on the self-assembly strategy using commercial chemicals and materials. The reflectance of MPSs depends on the value of Δn, which can be greatly increased to 70% with a Δn of 0.035, leading to their brilliant colors. Because of the large Ds-s, the MPSs possess outstanding mechano-chromic performances, including a large maximal (Δλ = 205 nm) and effective (Δλe = 184 nm) tuning range of the reflection wavelength, high sensitivity (368), fast responsiveness (2.2 nm/ms), good stabilities (>1 year), and reversibility (>100 times). Based on these advantages, MPSs have been used for self-reporting the strain of earthworms by outputting diverse colors during the peristaltic process, indicating the great potential of the MPSs as visual sensors and optical coatings.

DNA Barcoding: Molecular Identification and Phylogenetic Analysis of Pheretimoid Earthworm (Metaphire sp. and Amynthas sp.) Based on Mitochondrial Partial COI Gene from Sialkot, Pakistan.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Authentication of three main commercial Pheretima based on amino acids analysis.

Pheretima has been used as an animal-derived traditional Chinese medicine for thousands of years in Asian countries due to its multi-activities. However, more than half of the commercial Pheretima are adulterants according to the previous research. Besides, the standards of Pheretima are still inadequate in the identification of Pheretima species. In this study, an amino acids (AAs) analytical method established based on the ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QqQ-MS) in multiple reaction monitoring (MRM) mode through derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for qualitative and quantitative analysis of the total AAs of three main commercial Pheretima (two major Pheretima species, Amynthas aspergillum, Metaphire vulgaris, and one main counterfeit, M. magna). As a result, 16 AAs were detected and quantitated in their hydrolyzed samples. Then, multivariate statistical analysis was applied to distinguish the three commercial Pheretima based on their AAs level. Finally, four AAs (Thr, Glu, Asp, and Arg) were screened as species-differential AAs, which may be used as chemical markers to distinguish the three commercial Pheretima. This study deeply described the outline of AAs in Pheretima and offered a good reference for its species authentication.

ESIDE: A computationally intelligent method to identify earthworm species (E. fetida) from digital images: Application in taxonomy.

Earthworms (Crassiclitellata) being ecosystem engineers significantly affect the physical, chemical, and biological properties of the soil by recycling organic material, increasing nutrient availability, and improving soil structure. The efficiency of earthworms in ecology varies along with species. Therefore, the role of taxonomy in earthworm study is significant. The taxonomy of earthworms cannot reliably be established through morphological characteristics because the small and simple body plan of the earthworm does not have anatomical complex and highly specialized structures. Recently, molecular techniques have been adopted to accurately classify the earthworm species but these techniques are time-consuming and costly. To combat this issue, in this study, we propose a machine learning-based earthworm species identification model that uses digital images of earthworms. We performed a stringent performance evaluation not only through 10-fold cross-validation and on an external validation dataset but also in real settings by involving an experienced taxonomist. In all the evaluation settings, our proposed model has given state-of-the-art performance and justified its use to aid earthworm taxonomy studies. We made this model openly accessible through a cloud-based webserver and python code available at https://sites.google.com/view/wajidarshad/software and https://github.com/wajidarshad/ESIDE.

Distribution of epi-endogeic and endogeic earthworm species (Oligochaeta: Lumbricidae) in the forest belt of the Northwest Caucasus.

The distribution of the often dominant in density and biomass epi-endogeic and endogeic earthworm species in forests of the Northwest Caucasus was estimated based on a large array of field data and GIS modeling of modern potential areas. Quantitative accountings of earthworms were conducted annually from 2014 to 2019 in different types of forests: sticky alder forest, small-leaved forests, broadleaf forests (hornbeam and beech forests), coniferous-deciduous forests, dark coniferous forests, and pine forests (792 geographic locations). It is shown that the native species of the Caucasian fauna dominate in the number and biomass from piedmont to high mountain forests. The most widespread species that is found in all types of forests from low to high mountains and makes the main contribution to the biomass of earthworms is the Crimean-Caucasian subendemic Dendrobaena schmidti. Peregrine species of epi-endogeic and endogeic earthworms inhabit sticky alder forest, small-leaved, broadleaf and pine forests in which they inhabit together with native species, while not dominating either in numbers or in biomass among other species. Dark coniferous forests are inhabited mainly by the native endogeic species D. schmidti and A. jassyensis. Maxent modeling shows that the species of the endogeic earthworm group have wider potential areas than epi-endogeic species. The most significant bioclimatic factors that make the greatest contribution to the distribution: amount of precipitation in the driest month, altitude above the sea level, isothermality.

Global data on earthworm abundance, biomass, diversity and corresponding environmental properties.

Earthworms are an important soil taxon as ecosystem engineers, providing a variety of crucial ecosystem functions and services. Little is known about their diversity and distribution at large spatial scales, despite the availability of considerable amounts of local-scale data. Earthworm diversity data, obtained from the primary literature or provided directly by authors, were collated with information on site locations, including coordinates, habitat cover, and soil properties. Datasets were required, at a minimum, to include abundance or biomass of earthworms at a site. Where possible, site-level species lists were included, as well as the abundance and biomass of individual species and ecological groups. This global dataset contains 10,840 sites, with 184 species, from 60 countries and all continents except Antarctica. The data were obtained from 182 published articles, published between 1973 and 2017, and 17 unpublished datasets. Amalgamating data into a single global database will assist researchers in investigating and answering a wide variety of pressing questions, for example, jointly assessing aboveground and belowground biodiversity distributions and drivers of biodiversity change.

Occurrence of bioluminescent and nonbioluminescent species in the littoral earthworm genus Pontodrilus.

Pontodrilus litoralis is a cosmopolitan littoral earthworm known to exhibit bioluminescence. Recently, a congeneric species, Pontodrilus longissimus, from Thailand was described. These species are sympatric, but their burrowing depths on Thai beaches are different. In this study, we examined the in vivo and in vitro bioluminescent properties of P. longissimus and P. litoralis. Mechanical stimulation induced in vivo luminescence in P. litoralis, as reported previously, but not in P. longissimus. In vitro cross-reaction tests between these species revealed the absence of luciferin and luciferase activities in P. longissimus. The coelomic fluid of P. litoralis had strong fluorescence that matched the spectral maximum of its bioluminescence, but the same result was not observed for P. longissimus. These results suggest that P. litoralis has luminescence abilities due to the creation of bioluminescent components (i.e., luciferin, luciferase, and light emitters). The presence of both luminous and nonluminous species in a single genus is likely widespread, but only a few examples have been confirmed. Our findings provide insight into the possible functions of bioluminescence in earthworms, such as avoiding predation by littoral earwigs.

Three new species and four new species records of earthworms of the genus emMoniligaster/em Perrier, 1872 (Clitellata: Moniligastridae) from Kerala region of the Western Ghats Biodiversity Hotspot, India.

Three new species of Moniligaster Perrier, 1872, namely M. bahli Narayanan Julka, sp. nov., M. blakemorei Narayanan Julka, sp. nov. and M. keralensis Narayanan Julka, sp. nov. are described from materials collected from the Indian state of Kerala. Moniligaster cernosvitovi Gates, 1962, Moniligaster horsti Gates, 1940, Moniligaster michaelseni Gates, 1940 and Moniligaster stephensoni Gates, 1940 are recorded for the first time from the state. With the new findings, a total of 10 Moniligaster species are known from Kerala. Moniligaster species are restricted to southern peninsular India, except Moniligaster ivaniosi Manazhy, 2011, decribed from the Andaman Islands, outside the currently known distributional range of the genus. Hence we critically reviewed the original description and reinvestigated the holotype. As a result, Moniligaster ivaniosi is considered a junior synonym of Drawida nepalensis Michaelsen, 1907.

Taxonomical study of Japanese emAulodrilus/em Bretscher (Annelida, Clitellata, Tubificinae) with descriptions of two new species.

Seven species of the genus Aulodrilus (Annelida, Clitellata, Tubificinae) are studied, based on new material from Japan. Aulodrilus dentosus sp. nov. is characterized as having tubular atrium, forked or bifid distal ends of dorsal crotchets, but no genital chaetae, and A. aestivus sp. nov. is characterized as having crescent-shaped atrium, median male bursa, and bifid chaetae in the dorsal bundles. Five other congeners are redescribed: A. limnobius Bretscher, A. pluriseta (Piguet), A. pigueti Kowalewski, A. japonicus Yamaguchi, and A. americanus Brinkhurst Cook. Comparison of taxonomic characters among the 14 species recognized in the genus to date shows that three species differ from other congeners in several features: A. paucichaeta Brinkhurst Barbour, A. adetus (du Bois-Reymond Marcus), and A. apeniatus Cui Wang. They are provisionally maintained in the genus.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

First reported quantitative microbiota in different livestock manures used as organic fertilizers in the Northeast of Thailand.

Northeastern Thailand relies on agriculture as a major economic activity, and has used high levels of agrochemicals due to low facility, and salty sandy soil. To support soil recovery and sustainable agriculture, local farmers have used organic fertilizers from farmed animal feces. However, knowledge about these animal fecal manures remains minimal restricting their optimal use. Specifically, while bacteria are important for soil and plant growth, an abundance and a diversity of bacterial composition in these animal fecal manures have not been reported to allow selection and adjustment for a more effective organic fertilizer. This study thereby utilized metagenomics combined with 16S rRNA gene quantitative PCR (qPCR) and sequencing to analyze quantitative microbiota profiles in association with nutrients (N, P, K), organic matters, and the other physiochemical properties, of the commonly used earthworm manure and other manures from livestock animals (including breed and feeding diet variations) in the region. Unlike the other manures, the earthworm manure demonstrated more favorable nutrient profiles and physiochemical properties for forming fertile soil. Despite low total microbial biomass, the microbiota were enriched with maximal OTUs and Chao richness, and no plant pathogenic bacteria were found based on the VFDB database. The microbial metabolic potentials supported functions to promote crop growth, such as C, N and P cyclings, xenobiotic degradation, and synthesis of bioactive compounds. Pearson's correlation analyses indicated that the quantitative microbiota of the earthworm manure were clustered in the same direction as N, and conductivity, salinity, and water content were essential to control the microbiota of animal manures.

Cryptic speciation and blurred species boundaries of the earthworm: A challenge for soil-based toxicological risk assessments.

The toxicological risk assessment of chemicals is largely based on the Organization for Economic Co-operation and Development (OECD) guidelines. These internationally approved methodologies help shape policy and political strategy of environment and human health issues. Risk assessments which pertain to soil biota 'recruit' sentinel organisms, including the earthworm Eisenia fetida. Despite E. fetida being morphologically similar to Dendrobaena veneta, they are characterized by a several-fold difference in sensitivity to xenobiotics. Worms, sold as either as pure E. fetida stocks or E. fetida/D. veneta mixed cultures, were obtained from five commercial suppliers. The species identity of 25 earthworms was determined by sequencing the cytochrome c oxidase subunit 2 (COII). We revealed that only one of 25 worms was E. fetida, the remaining worms were all identified as D. veneta. This underlines the notion that E. fetida and D. veneta are easily mis-identified. The occurrence of cryptic speciation combined with the well-documented species-specific variation in toxicological responses highlights the pressing need to accurately classify earthworms to species level prior to any toxicological research. Only this will ensure the validity and reliability of risk assessments.

A New Species of Monocystis (Apicomplexa: Gregarina: Monocystidae) from the Asian Invasive Earthworm Amynthas agrestis (Megascolecidae), with an Improved Standard for Monocystis Species Descriptions.

Monocystis perplexa n. sp., a parasite of an important invasive Japanese earthworm in North America, Amynthas agrestis, is described from a site in Vermont. An improved standard for Monocystis species descriptions is proposed including a standard nomenclature to reduce synonymies, a standard set of biometrics and shape descriptions for living cells, and a DNA genomic sequence for the 18S rRNA (∼1,700 base pairs). Comparing morphologies of Monocystis parasites in sympatric earthworm species indicates that M. perplexa is specific to A. agrestis in the study region. Also, polymerase chain reaction primers specific to M. perplexa amplified samples of A. agrestis earthworms taken from several sites in Japan. This suggests the parasite entered North America from Japan, the origin of the invasive Amynthas earthworm, and thus M. perplexa would be the first Monocystis described from the diverse Japanese Amynthas earthworms and the first from East Asia. Monocystis perplexa was found in every population of A. agrestis surveyed in Vermont, always reaching 100% prevalence by late summer (the host has an annual life cycle in Vermont). The 18S gene sequence differed from that of Monocystis agilis from the sympatric earthworm Lumbricus terrestris (the only other sequence available for Monocystis), and a genetic similarity tree places them closest among other gregarines. Many of the 95 described species of Monocystis are very similar in morphology (based on species descriptions), so the 18S gene can act as a barcode for Monocystis species and thus will help to eliminate both synonymies and reveal cryptic species.

Exploring new records of Eutyphoeus sp. (haplotaxida: Octochaetidae) from garo hills, Meghalaya, North Eastern state of India with use of DNA barcodes.

The work was aimed to investigate earthworms species particularly Eutyphoeus endemic to India with the use of DNA barcodes and usual morpho-anatomical standards of earthworm taxonomy from protected areas of Garo Hills, Meghalaya, the north-east region (NER) of India. The study revealed two new records Eutyphoeus kempi Stephenson, E. nepalensis Michaelsen and confirms availability of three known sp. namely E. callosus Gates, E. gammiei Beddard and E. turaensis Stephenson. The neighbor-joining tree was constructed using the K2P substitution model and the genomic signature of each species using COI-1 gene was generated for the first time and were used to reconfirm the identification of species.

Testing species hypotheses for Fridericia magna, an enchytraeid worm (Annelida: Clitellata) with great mitochondrial variation.

BACKGROUND: Deep mitochondrial divergences were observed in Scandinavian populations of the terrestrial to semi-aquatic annelid Fridericia magna (Clitellata: Enchytraeidae). This raised the need for testing whether the taxon is a single species or a complex of cryptic species. RESULTS: A total of 62 specimens from 38 localities were included in the study, 44 of which were used for species delimitation. First, the 44 specimens were divided into clusters using ABGD (Automatic Barcode Gap Discovery) on two datasets, consisting of sequences of the mitochondrial markers COI and 16S. For each dataset, the worms were divided into six not completely congruent clusters. When they were combined, a maximum of seven clusters, or species hypotheses, were obtained, and the seven clusters were used as input in downstream analyses. We tested these hypotheses by constructing haplowebs for two nuclear markers, H3 and ITS, and in both haplowebs the specimens appeared as a single species. Multi-locus species delimitation analyses performed with the Bayesian BPP program also mainly supported a single species. Furthermore, no apparent morphological differences were found between the clusters. Two of the clusters were partially separated from each other and the other clusters, but not strongly enough to consider them as separate species. All 62 specimens were used to visualise the Scandinavian distribution, of the species, and to compare with published COI data from other Fridericia species. CONCLUSION: We show that the morphospecies Fridericia magna is a single species, harbouring several distinct mitochondrial clusters. There is partial genetic separation between some of them, which may be interpreted as incipient speciation. The study shows the importance of rigorous species delimitation using several independent markers when deep mitochondrial divergences might give the false impression of cryptic speciation.

Blind spots in global soil biodiversity and ecosystem function research.

Soils harbor a substantial fraction of the world's biodiversity, contributing to many crucial ecosystem functions. It is thus essential to identify general macroecological patterns related to the distribution and functioning of soil organisms to support their conservation and consideration by governance. These macroecological analyses need to represent the diversity of environmental conditions that can be found worldwide. Here we identify and characterize existing environmental gaps in soil taxa and ecosystem functioning data across soil macroecological studies and 17,186 sampling sites across the globe. These data gaps include important spatial, environmental, taxonomic, and functional gaps, and an almost complete absence of temporally explicit data. We also identify the limitations of soil macroecological studies to explore general patterns in soil biodiversity-ecosystem functioning relationships, with only 0.3% of all sampling sites having both information about biodiversity and function, although with different taxonomic groups and functions at each site. Based on this information, we provide clear priorities to support and expand soil macroecological research.

Characterization of the complete mitochondrial genome of Drawida gisti (Metagynophora, Moniligastridae) and comparison with other Metagynophora species.

Here, the complete mitochondrial genome (mitogenome) of Drawida gisti was sequenced and compared with the mitogenomes of other Metagynophora species. The circular mitogenome was 14,648 bp in length and contained two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and 22 transfer RNA genes (tRNAs). The types of constitutive genes and the direction of the coding strand that appeared in Drawida mitogenome were identical to those observed in other Metagynophora species, except for a missing lengthy non-coding region. The conservative relationships between Drawida species were supported by the overall analyses of 13 PCGs, two rRNAs, and 22 tRNAs. A comparison of the Metagynophora mitogenomes revealed that the ATP8 gene possessed the highest polymorphism among the 13 PCGs and two rRNAs. Phylogenetic analysis suggested that the Moniligastridae contained Drawida, which is a primitive Metagynophora group. Our study provides a step forward toward elucidating the evolutionary linkages within Drawida and even Metagynophora.

Delimitation of five astome ciliate species isolated from the digestive tube of three ecologically different groups of lumbricid earthworms, using the internal transcribed spacer region and the hypervariable D1/D2 region of the 28S rRNA gene.

BACKGROUND: Various ecological groups of earthworms very likely constitute sharply isolated niches that might permit speciation of their symbiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different host groups. The nuclear highly variable ITS1-5.8S-ITS2 region and the hypervariable D1/D2 region of the 28S rRNA gene have proven to be useful tools for the delimitation of species boundaries in closely related free-living ciliate taxa. In the present study, the power of these molecular markers as well as of the secondary structure of the ITS2 molecule were tested for the first time in order to discriminate the species of endosymbiotic ciliates that were isolated from the gastrointestinal tract of three ecologically different groups of lumbricid earthworms. RESULTS: Nineteen new ITS1-5.8S-ITS2 region and D1/D2-28S rRNA gene sequences were obtained from five astome species (Anoplophrya lumbrici, A. vulgaris, Metaradiophrya lumbrici, M. varians, and Subanoplophrya nodulata comb. n.), which were living in the digestive tube of three ecological groups of earthworms. Phylogenetic analyses of the rRNA locus and secondary structure analyses of the ITS2 molecule robustly resolved their phylogenetic relationships and supported the distinctness of all five species, although previous multivariate morphometric analyses were not able to separate congeners in the genera Anoplophrya and Metaradiophrya. The occurrence of all five taxa, as delimited by molecular analyses, was perfectly correlated with the ecological groups of their host earthworms. CONCLUSIONS: The present study indicates that morphology-based taxonomy of astome ciliates needs to be tested in the light of molecular and ecological data as well. The use of morphological identification alone is likely to miss species that are well delimited based on molecular markers and ecological traits and can lead to the underestimation of diversity and overestimation of host range. An integrative approach along with distinctly increased taxon sampling would be helpful to assess the consistency of the eco-evolutionary trend in astome ciliates.