Adaptive Evolution of Digestive RNASE1 Genes in Leaf-Eating Monkeys Revisited: New Insights from Ten Additional Colobines

Pancreatic RNase genes implicated in the adaptation of the colobine monkeys to leaf eating have long intrigued evolutionary biologists since the identification of a duplicated RNASE1 gene with enhanced digestive efficiencies in Pygathrix nemaeus. The recent emergence of two contrasting hypotheses, that is, independent duplication and one-duplication event hypotheses, make it into focus again. Current understanding of Colobine RNASE1 gene evolution of colobine monkeys largely depends on the analyses of few colobine species. The present study with more intensive taxonomic and character sampling not only provides a clearer picture of Colobine RNASE1 gene evolution but also allows to have a more thorough understanding about the molecular basis underlying the adaptation of Colobinae to the unique leaf-feeding lifestyle. The present broader and detailed phylogenetic analyses yielded two important findings: 1) All trees based on the analyses of coding, noncoding, and both regions provided consistent evidence, indicating RNASE1 duplication occurred after Asian and African colobines speciation, that is, independent duplication hypothesis; 2) No obvious evidence of gene conversion in RNASE1 gene was found, favoring independent evolution of Colobine RNASE1 gene duplicates. The conclusion drawn from previous studies that gene conversion has played a significant role in the evolution of Colobine RNASE1 was not supported. Our selective constraint analyses also provided interesting insights, with significant evidence of positive selection detected on ancestor lineages leading to duplicated gene copies. The identification of a handful of new adaptive sites and amino acid changes that have not been characterized previously also provide a necessary foundation for further experimental investigations of RNASE1 functional evolution in Colobinae.

Stepwise loss of motilin and its specific receptor genes in rodents

Specific interactions among biomolecules drive virtually all cellular functions and underlie phenotypic complexity and diversity. Biomolecules are not isolated particles, but are elements of integrated interaction networks, and play their roles through specific interactions. Simultaneous emergence or loss of multiple interacting partners is unlikely. If one of the interacting partners is lost, then what are the evolutionary consequences for the retained partner? Taking advantages of the availability of the large number of mammalian genome sequences and knowledge of phylogenetic relationships of the species, we examined the evolutionary fate of the motilin (MLN) hormone gene, after the pseudogenization of its specific receptor, MLN receptor (MLNR), on the rodent lineage. We speculate that the MLNR gene became a pseudogene before the divergence of the squirrel and other rodents about 75 mya. The evolutionary consequences for the MLN gene were diverse. While an intact open reading frame for the MLN gene, which appears functional, was preserved in the kangaroo rat, the MLN gene became inactivated independently on the lineages leading to the guinea pig and the common ancestor of the mouse and rat. Gain and loss of specific interactions among biomolecules through the birth and death of genes for biomolecules point to a general evolutionary dynamic: gene birth and death are widespread phenomena in genome evolution, at the genetic level; thus, once mutations arise, a stepwise process of elaboration and optimization ensues, which gradually integrates and orders mutations into a coherent pattern.

Rapid and parallel chromosomal number reductions in muntjac deer inferred from mitochondrial DNA phylogeny

Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chromosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of karyotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46-->14/13-->8/9-->6/7. For example, Muntiacus muntjak (2n = 6/7) shares a common ancestor with Muntiacus feae (2n = 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n greater than or equal to 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another pa;allel reduction lineage from the ancestral 2n = 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus. Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr.

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta -bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.

The genetic structure and phylogenetics of pikeperch (Sander lucioperca) Anzali and Amirkolaye wetlands and perch (Perca fluviatilis) populations in Aras Dam and south-west of the Caspian Sea

The genetic structure of pikeperch (Sander lucioperca) and perch (Perca fluviatilis) populations was studied using microsatellite technique. A total of 207 specimens of adult pikeperch were collected from Aras dam (57 specimens), Anzali wetland (50 specimens), Talesh (50 specimens) and Chaboksar (50 specimens) coasts. Also a total of 158 specimens of adult perch were collected from Anzali (Abkenar (50 specimens)and Hendekhale(48 specimens)) and Amirkolaye(60 specimens) wetlands. About 2 g of each specimen's dorsal fin was removed, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using ammonium-acetate method. The quality and quantity of DNA was assessed using 1% agarose gel electrophoresis. Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 pairs of microsatellite primers. PCR products were electrophoresed on poly acryl amide gels (6%) that were stained that were stained using silver nitrate. DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected and observed heterozygosity , allele number and the effective allele number, genetic similarity and genetic distance, Fst, Rst, Hardy Weinberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendogram for genetic distances and identities were calculated using TFPGA program for any level of hierarchy. The results for P. fluviatilis showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 4.1±1.1 and mean observed and expected heterozygosity was 0.56±0.12 and 0.58±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.095) with Nm=2.37 was observed between Hendekhale and Amirkolaye and the lowest Fst (0.004) with Nm=59.31 was observed between Abkenar and Hendekhale. According to AMOVA Significant difference (P<0.05) was observed between recorded Rst in the studied regions in Anzali and Amirkolaye lagoons. In another words there are two distinct populations of this species in Anzali and Amirkolaye lagoons. The highest genetic distance (0.181) and lowest genetic resemblance (0.834) were observed between specimens from Hendekhale and Amirkolaye and the lowest genetic distance (0.099) and highest genetic 176 resemblance (0.981) were observed between specimens from Abkenar and Hendekhale. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Anzali and Amirkolaye wetlands have the same ancestor. On the other hand there is no noticeable genetic distance between the specimens of these two regions. Also the results for S. lucioperca showed that from 15 pair of primers that were examined 6 polymorphic and 7 monomorphic loci were produced, while 2 loci didn't produce any DNA bands. Mean allele number was 3.0±0.6 and mean observed and expected heterozygosity was 0.52±0.21 and 0.50±0.14 respectively. It was also seen that specimens from all regions were not in Hardy Weinberg Equilibrium in some of loci (P<0.001). Highest Fst (0.093) with Nm=2.43 was observed between Aras dam and Anzali wetland and the lowest Fst (0.022) with Nm=11.27 was observed between Talesh and Chaboksar coasts. Significant differences (P<0.05) were observed between recorded Rst in the studied regions exept for Talesh and Chaboksar Coasts. In another words there are three distinct populations of this species in Caspian sea, Anzali wetland and Aras dam. Highest genetic distance (0.110) and lowest genetic resemblance (0.896) were observed between specimens from Aras dam and Anzali wetland and the lowest genetic distance (0.034) and highest genetic resemblance (0.966) were observed between specimens from Talesh and Chaboksar coasts. Based on the genetic dendogram tree derived by applying UPGMA algorithm, specimens from Talesh and Chaboksar coasts have the lowest genetic distance. On the other hand the main population of this species belongs to Anzali wetland. Phylogenetic relationship of these two species was inferred using mitochondrial cytochrome b gene sequencing. For this purpose 2 specimens of P. fluviatilis from Anzali wetland, 2 specimens of S. lucioperca from Aras dam and 2 specimens of S. lucioperca from Anzali wetland were sequenced and submitted in Gene Bank. These sequences were aligned with Clustal W. The phylogenic relationships were assessed with Mega 4. The results of evolutionary history studies of these species using Neighbor-Joining and Maximum Parsimony methods showed that the evolutionary origin of pikeperch in Aras Dam and Anzali wetland is common. On the other hand these two species had common ancestor in about 4 million years ago. Also different sequences of any region specimens are supposed as different haplotypes. 177 As a conclusion the results of this study showed that microsatellite and mtDNA sequencing methods respectively are effective in genetic structure and phylogenic studies of P. fluviatilis and S. lucioperca.

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene, GlTop 2 was identified. It is a single copy gene with a 4476 by long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a similar to 100 as longer central domain; a similar to 200 as shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate" G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

Sox genes evolution in closely related young tetraploid cyprinid fishes and their diploid relative

Previous study and analysis of cytochrome b suggested that polyploidization event in the genus Tor occurred about 10 Mya ago. In order to understand evolutionary fates of Sox gene in the early stage of genome duplication at the nucleotide level, PCR surveys for Sox genes in three closely related cyprinid fishes T douronensis (2n = 100), T qiaojiensis (2n = ?), T sinensis (2n = 100) and their relative T brevifilis (2n = 50) were performed. Totally, 52 distinct Sox genes were obtained in these four species, representing SoxB, SoxC, and SoxE group. As expected, isoforms of some Sox genes correspond with the ploidy of species, such as two copies of Sox9a exist in tetraploid species. Analysis indicated that duplicated Sox gene pairs caused by polyploidization evolved independently of each other within polyploid species. Results of substitution rate showed nearly equal rate of nonsynonymous substitution of duplicated Sox orthologs among different polyploid species and their diploid relative orthologs, suggesting at the early stage of genome duplicated Sox orthologs are under similar selective constraints in different polyploidy species and their diploid relative at the amino acid level. All PCR fragments of Sox genes obtained in this study are not accompanied by obvious increase in mutations and pseudogene formation which means that they are under strong purifying selection, suggesting that they are functional at the DNA level. Cenealogical analysis revealed that T qiaojiensis was tetraploid, and T douronensis, T qiaojiensis as well as T sinensis had an allotetraploid ancestor. (C) 2009 Elsevier B.V. All rights reserved.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Multiple invasions of europe by east asian cobitid loaches (Teleostei : Cobitidae)

While it has been widely suggested that freshwater fishes from East Asia invaded the western Palaearctic, details about this process are largely unknown. Here, using the cytochrome b gene, we evaluated the phylogenetic relationships of a small group of Eurasian primary freshwater fishes (Cobitidae), which are widely distributed and species rich in East Asia and Europe, with the purpose of inferring their invasion process of Europe from East Asia. Though phylogenetic relationships of cobitids were not well resolved, our analysis could identify three sister groups formed by the European and East Asian cobitids, which brought new insights into the biogeography of the genera Cobitis, Misgurnus, and Sabanejewia. The present results support the view that Asian cobitid fishes may have invaded Europe at least five times independently, and once reverse colonization of European cobitids to East Asia could also be found. Ancestral Sabanejewia might have been the first cobitids to cross Siberia and invade the EMZS (Euro-Mediterranean zoogeographic subregion) about 33.54 million years ago (MYA). One lineage of Cobitis and the ancestor of Misgurnus fossilis (Linnaeus) almost in the same time invaded the Europe, responding to 16.71 MYA and 16.59 MYA, respectively. Three different lineages of Cobitis were found to have invaded the EMZS from East Asia, and once reverse invasion to East Asia occurred to one subclade of European Cobitis. And our data also suggest that the diversity of East Asian cobitid fishes, especially of the genus Cobitis, is greatly underestimated.

Origin of Gymnocypris przewalskii and phylogenetic history of Gymnocypris eckloni (Teleostei : Cyprinidae)

The origins and phylogenetic patterns were assessed for G. przewalskii and G. eckloni by analyzing the complete mtDNA cytochrome b gene sequence (1140bp). Phylogenetic analyses further supported that there were three mtDNA lineages (A-C) identified in G. przewalskii and G. eckloni, demonstrating that outer rakers of the first gill have little significance in the phylogeny of the Gymnocypris fishes. The network established showed that G. eckloni of the Yellow River specific haplotype A1 was a founder and it radiated all haplotypes of G. przewalskii which suggested G. przewalskii might only originate from one of two maternals of G. eckloni from the Yellow River. Fs test and mismatch analysis showed at least two expansion events in the population of G. przewalskii about 0.2734 Ma and 0.0658 Ma, while G. eckloni from Qaidam Basin could have experienced severe bottleneck effect about 0.0693 Ma. The population expansion was detected in subclades A1 and A21 with the most recent common ancestor (TMRCA) about 0.2308 +/- 0.01 Ma and 0.1319 +/- 0.015 Ma, respectively, which were within the geological age range of "Gonghe Movement" event that caused the separation of Lake Qinghai from the upper Yellow River. These results suggested the effect of the fish diversification by rapid uplift of the Qinghai-Tibetan Plateau in the Late Pleistocene.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Identification of the Angel-related elements in cyprinid fishes and their phylogenetic implications

Angel-related element belongs to the family of miniature inverted-repeat transposable elements (MITEs). In this paper we report the identification of an Angel-related element in the series Leuciscini of cyprinid fishes, which is located in the second intron of the growth hormone (GH) gene. We have also found that this element is absent in orthologous locus in the series Barbini of cyprinid fishes, that provides new evidence for the monophyly of the series Leuciscini. The insertion of Angel-related element into the GH gene might take place in the common ancestor of the series Leuciscini after its divergence from the series Barbini. The high sequence divergence and relatively broad species distribution of Angel-related elements implies that they might be ancient transposons which appeared about 26 million years ago.

Molecular phylogenetics of Gymnocypris (Teleostei : Cyprinidae) in Lake Qinghai and adjacent drainages

149 complete mitochondrial DNA (mtDNA) cytochrome b (Cyt b) genes (1140 bp) of Gymnocypris przewalskii, Gymnocypris eckloni and Gymnocyptis scolistomus from the Lake Qinghai, Yellow River and Qaidam Basin were sequenced and analyzed. Consistent dendrogram indicated that the samples collected from the same species do not constitute a separate monophyletic group and all the samples were grouped into three highly divergent lineages (A, B and C). Among them, Lineage A contained all samples of G. przewalskii from the Lake Qinghai and partial samples of the G. eckloni from the Yellow River. Lineage B contained the remaining samples of G. eckloni from the Yellow River. Lineage C was composed of a monophyletic group by G. eckloni from the Qaidam Basin. Analysis of molecular variance (AMOVA) indicated that most of genetic variations were detected within these three mtDNA lineages (93.12%), suggesting that there are three different lineages of Gymnocypris in this region. Our Cyt b sequence data showed that G. przewalskii was not a polytypic species, and G. scolistomus was neither an independent species nor a subspecies of G. eckloni. The divergent mtDNA lineages of G. eckloni from the Yellow River suggested that gene flow between the different populations was restricted to a certain extent by several gorges on the upper reach of the Yellow River. Lineage B of G. eckloni might be the genetic effect from the ancestor which was incorporated with the endemic schizothoracine fishes when the headward erosion of the Yellow River reached to its current headwaters of late. The G. eckloni from Basin Qaidam was a monophyletic group (lineage C) and F-st values within G. eckloni from the Yellow River were higher than 0.98, suggesting that the gene flow has been interrupted for a long time and the G. eckloni from Basin Qaidam might have been evolved into different species by ecology segregation. The correlation between the rakers number of Gymnocypris and population genetic variation was not significant. All Gymnocypris populations exhibited a low nucleotide diversity (pi = 0.00096-0.00485). Therefore the Gymnocyptis populations from Basin Qaidam could have experienced severe bottleneck effect in history. Our result suggested Gym-nocypris populations of Basin Qaidam should give a high priority in conservation programs.

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i.e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E, galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E, hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.