Mitochondrial COI-NC-COII sequences in talitrid amphipods (Crustacea)

Mitochondrial (mt) sequences from cytochrome oxidase subunit I to the subunit II gene (COI, COII) were analysed in crustacean talitrid amphipods. Species of the genera Orchestia, Talitrus and Talorchestia from the Mediterranean-East Atlantic area were examined. The expected tRNALeu-UUR gene was not revealed between COI and COII. Instead, a short (35-48 bp) noncoding (NC) AT-rich (ca. 90%) region with putative stem loops was found. Here, we discuss briefly the NC region and explore its potential involvement in generating this novel rearrangement. The COI-NC-COII organization, as well as preliminary phylogenetic results, based on both COI-COII nucleotide and amino-acid sequence indicate monophyly of these talitrid taxa.     

Phylogenetic relationships within the genus Jesogammarus (Crustacea, Amphipoda, Anisogammaridae) deduced from mitochondrial COI and 12S sequences

The genus Jesogammarus contains 16 species in two subgenera, Jesogammarus and Annanogammarus. To examine relationships among species in the genus, a molecular phylogenetic study including eight species of the former subgenus and four of the latter was conducted using partial DNA sequences of the mitochondrial COI and 12S rRNA genes. MP, NJ, and ML trees based on the combined COI and 12S data indicated monophyly of the subgenus Annanogammarus, though the monophyly of Jesogammarus was left unresolved. Consistent with few morphological differences, Jesogammarus (A.) naritai and J. (A.) suwaensis showed low genetic differentiation and did not show reciprocal monophyly, which suggests a close affinity of these taxa.     

基于COI基因序列的九种真蝽属昆虫的分子系统学研究(半翅目:蝽科)

通过对真蝽属Pentatoma 9种昆虫线粒体COI基因约798bp的序列进行分子进化分析,并以同蝽科宽铗同蝽Acanthosoma labiduroides为外群,采用最大简约法、最大似然法和邻接法构建了分子系统树,来探讨真蝽属的系统发育关系.研究结果支持褐真蝽群P. semiannulata-group的划分,绿角真蝽Pentatoma viridicornuta应划归到褐真蝽群P. Semiannulata-group;红足真蝽群中的角肩真蝽P. angulata与红足真蝽P. rufipes遗传距离较小,它们是否为1个物种值得关注;真蝽属各群之间的系统发育关系以及是否可分为3个属值得进一步研究.     

Molecular characterization of Gonatocerus tuberculifemur (Ogloblin) (Hymenoptera: Mymaridae), a prospective Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) biological control candidate agent from South America: divergent clades

We genetically characterized the prospective South American egg parasitoid candidate, Gonatocerus tuberculifemur, of the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, for a neoclassical biological control program in California. Two molecular methods, inter-simple sequence repeat-polymerase chain reaction DNA fingerprinting and a phylogeographic approach inferred from the mitochondrial cytochrome oxidase subunit I gene (COI), were utilized. Five geographic populations from South America were analyzed; in addition, a phylogenetic analysis was performed with several named and one unnamed Gonatocerus species using the COI gene. DNA fingerprinting demonstrated a fixed geographic banding pattern difference in the population from San Rafael, Mendoza Province, Argentina. The COI analysis uncovered haplotype or geographic structure in G. tuberculifemur. A neighbour-joining distance (NJ) and a single most parsimonious tree (MP) clustered the populations into two well-supported distinct clades with strong bootstrap values (97-99% and 92-99%, respectively) with populations from San Rafael clustering into clade 2 and the rest of the populations clustering into clade 1. No haplotype sharing was observed between individuals from the two clades. Phylogenetic analyses performed by NJ and MP methods with 15 Gonatocerus species confirmed species boundaries and again uncovered two distinct clades in G. tuberculifemur with strong bootstrap support (95-100% and 68-100%, respectively). However, the NJ tree supported the morphologically defined relationships better than the MP tree. The molecular evidence in the present study is suggestive of a species level divergence. Because G. tuberculifemur is under consideration as a potential biological control agent for GWSS in California, understanding cryptic variation in this species is critical.     

A new method for species identification via protein-coding and non-coding DNA barcodes by combining machine learning with bioinformatic methods

Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.     

Relative efficiencies of the maximum-parsimony and distance-matrix methods of phylogeny construction for restriction data.

The relative efficiencies of the maximum-parsimony (MP), UPGMA, and neighbor-joining (NJ) methods in obtaining the correct tree (topology) for restriction-site and restriction-fragment data were studied by computer simulation. In this simulation, six DNA sequences of 16,000 nucleotides were assumed to evolve following a given model tree. The recognition sequences of 20 different six-base restriction enzymes were used to identify the restriction sites of the DNA sequences generated. The restriction-site data and restriction-fragment data thus obtained were used to reconstruct a phylogenetic tree, and the tree obtained was compared with the model tree. This process was repeated 300 times. The results obtained indicate that when the rate of nucleotide substitution is constant the probability of obtaining the correct tree (Pc) is generally higher in the NJ method than in the MP method. However, if we use the average topological deviation from the model tree (dT) as the criterion of comparison, the NJ and MP methods are nearly equally efficient. When the rate of nucleotide substitution varies with evolutionary lineage, the NJ method is better than the MP method, whether Pc or dT is used as the criterion of comparison. With 500 nucleotides and when the number of nucleotide substitutions per site was very small, restriction-site data were, contrary to our expectation, more useful than sequence data. Restriction-fragment data were less useful than restriction-site data, except when the sequence divergence was very small. UPGMA seems to be useful only when the rate of nucleotide substitution is constant and sequence divergence is high.     

线蛱蝶亚科蝶类部分类群线粒体COI基因的分子系统发生分析

以线粒体细胞色素氧化酶I（COI）基因作分子标记,对线蛱蝶亚科蝴蝶进行序列测定．序列分析的结果表明．经比对和处理后的序列总长度是645bp,其中有199个变异位点,147个简约信息位点;所编码的氨基酸序列中有18个变异位点,7个信息位点．A＋T平均含量为69．6％,G＋C平均含量为30．4％,碱基组成出现AT偏斜．以蛱蝶亚科及秀蛱蝶亚科物种为外类群,用NJ、MP及贝叶斯法重建了该亚科的系统发生树,探讨了它们主要类群间的系统发生关系．分子系统树显示,线蛱蝶亚科由以下3大支系：环蛱蝶族＋翠蛱蝶族、线蛱蝶族、丽蛱蝶族构成;其中,环蛱蝶族为单系群（NJ树也支持线蛱蝶族的单系性）;翠蛱蝶族与环蛱蝶族亲缘关系较近：丽蛱蝶族可能是该亚科较早分化出的一支．     

利用肠道菌群的分布和16S DNA序列研究鲤科肠道菌系统演化关系

肠道微生物与寄主具有复杂的、多方面的相互依存效应,这种依存效应所产生的共生关系或协同进化关系既可反映寄主间的系统演化关系,也可显示肠道微生物间的系统演化关系,共生关系或协同进化关系是由于寄主与肠道微生物两者之间存在着相互自然选择作用所形成的,在长期的进化历程中逐步发生的共生关系信息很可能被记录在DNA序列中。本文通过检测鱼鲤鱼科8种鱼中9种肠道菌群的分布含量对这9种菌群进行分析,且利用从GenBank调取这9种肠道细菌菌属的43个种或亚种的16S DNA序列的构建NJ树和MP树,将这6个科9个属43个种或亚种分为革兰氏阴性和革兰氏阳性两大类群（一级分枝）。在这两类群中,又以科为单位分为6个亚类群（二级分枝）,而肠杆菌科中则以属为单位分为4个小类群（三级分枝）,此外球状菌与杆状菌也能截然分开。将16S DNA的NJ树隐去所有的种,以属为单位所得到的以分枝形式的无根树在拓扑结构上与菌群分布含量（寄主范围）所构建的无根树相近,但芽孢杆菌在两种无根树的位置中有较大的差异。如果提高检测水平,扩大所检测的寄主对象,这种差异有可能消除。     

Phylogenetic relationship of Turkish Apis mellifera subspecies based on sequencing of mitochondrial cytochrome C oxidase I region

Mitochondrial DNA sequence variation can be used to infer honey bee evolutionary relationships. We examined DNA sequence diversity in the cytochrome C oxidase I (COI or Cox1) gene segment of the mitochondrial genome in 112 samples of Apis mellifera from 15 different populations in Turkey. Six novel haplotypes were found for the COI gene segment. There were eight variable sites in the COI gene, although only three were parsimony-informative sites. The mean pairwise genetic distance was 0.3% for the COI gene segment. Neighbor-joining (NJ) trees of the COI gene segment were constructed with the published sequences of A. mellifera haplotypes that are available in GenBank; the genetic variation was compared among the different honeybee haplotypes. The NJ dendogram based on the COI sequences available in GenBank showed that Eastern European races were clustered together, whereas the Mellifera and Iberian haplotypes were clustered far apart. The haplotypes found in this study were clustered together with A. mellifera ligustica and some of the Greek honey bees (accession Nos. GU056169 and GU056170) found in NCBI GenBank database. This study expands the knowledge about the mitochondrial COI region and presents the first comprehensive sequence analysis of this region in Turkish honeybees.     

基于线粒体ND1和CO1基因序列探讨锯眼蝶-亚科主要类群的系统发生关系

研究测定了锯眼蝶亚科4族、10属共20个国产代表种的线粒体ND1和COI基因的部分序列,结合从GenBank中获得的4个国外产种类的同源序列,以凤蝶科的迪洛尔娟凤蝶、丝带凤蝶,以及娟蝶科的西猛娟蝶为外类群,通过邻接法、最大简约法、最大似然法和贝叶斯法重建了分子系统树,分析了该亚科及其主要类群的系统发生关系。分析结果表明：帻眼蝶族和锯眼蝶族具有较近的亲缘关系;黛眼蝶族不是单系群,该族中的黛眼蝶属、荫眼蝶属与眉眼蝶族具有较近的亲缘关系,带眼蝶属、藏眼蝶属、毛眼蝶属和帕眼蝶属聚合为一个独立的支系,其中带眼蝶属和藏眼蝶属在所有的分析方法中均以100%的置信度（BP=100%, PP=1.00）相聚合,笔者倾向于将它们合并为一属。     

Reanalysis of Murphy et al.'s data gives various mammalian phylogenies and suggests overcredibility of Bayesian trees

Murphy and colleagues reported that the mammalian phylogeny was resolved by Bayesian phylogenetics. However, the DNA sequences they used had many alignment gaps and undetermined nucleotide sites. We therefore reanalyzed their data by minimizing unshared nucleotide sites and retaining as many species as possible (13 species). In constructing phylogenetic trees, we used the Bayesian, maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ) methods with different substitution models. These trees were constructed by using both protein and DNA sequences. The results showed that the posterior probabilities for Bayesian trees were generally much higher than the bootstrap values for ML, MP, and NJ trees. Two different Bayesian topologies for the same set of species were sometimes supported by high posterior probabilities, implying that two different topologies can be judged to be correct by Bayesian phylogenetics. This suggests that the posterior probability in Bayesian analysis can be excessively high as an indication of statistical confidence and therefore Murphy et al.'s tree, which largely depends on Bayesian posterior probability, may not be correct.     

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. urceol     

Phylogenetic relationships between the six superoxide dismutase proteins (FeSOD) of Trichomonas vaginalis and FeSOD6 genetic diversity

The parasitic protozoan Trichomonas vaginalis is known to contain several types of Fe-containing superoxide dismutase proteins (FeSOD). Using three different methods of phylogenetic analysis, maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods, we examined the phylogenetic relationships among the six FeSOD (FeSOD1-FeSOD6) based on their amino acid sequences. All the analyses consistently suggested that the six proteins formed a monophyletic group implying that they probably be originated from an ancestral protein form through repeated duplication events. Although MP tree was totally unresolved, the NJ and ML trees revealed that FeSOD6 placed the most basal position and thus emerged earlier than the other five gene types during the evolution of T. vaginalis. Phylogenetic relationships among the five remaining proteins were (FeSOD2, FeSOD3), (FeSOD4, (FeSOD1, FeSOD5)) although weakly supported in terms of bootstrapping values. In addition to this, we newly designed two PCR primer specifically amplifying full-length FeSOD6 gene and examined its genetic diversity among 12 T. vaginalis isolates from five countries and three continents. They had the same nucleotide sequences except those of three Korean isolates which showed one to three different nucleotides.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

从细胞色素b基因全序列探讨大额牛的分子系统发生

大额牛是一种半野生半家养的珍稀牛种, 有关其起源和系统地位一直存在争议。通过PCR扩增、测序等步骤共获得了11头大额牛细胞色素b(Cyt b)基因全序列(1 140 bp)。应用分析软件, 对大额牛11条Cyt b序列进行了分析, 并结合GenBank中牛属动物6个近缘种的同源序列, 以亚洲水牛(Bubalus bubalis)为外群, 分别采用邻接法(NJ)和最大简约法(MP)构建了牛属动物分子系统发育树。序列分析结果表明, 11条大额牛Cyt b序列1 140位点中, 共发现95个变异位点(占分析位点总数的8.33 %), 定义了6种单倍型, 表明大额牛群体的Cyt b基因遗传多态性比较丰富。构建的NJ和MP分子系统树均显示, 大额牛研究群体明显分为3支, 第1支与普通牛(Bos taurus)相聚, 第2支与瘤牛(Bos indicus)相聚, 第3支与印度野牛(Bos gaurus)相聚。系统发育分析表明, 大额牛很可能是印度野牛的家养型或驯化种, 我国大额牛群体可能曾受到其他牛种血缘的入侵。     

New aspects of an old discussion - phylogenetic relationships of Ammotragus and Pseudois within the subfamily Caprinae based on comparison of the 12S rDNA sequences

A mitochondrial 12S rDNA fragment was amplified by PCR and directly sequenced from eight members of the Caprinae ( Ammotragus lervia, Capra aegagrus, Ovis ammon, O. musimon, O. nivicola, O. orientalis, O. vignei and Pseudois nayaur ). Multiple alignment of the sequences and genetic distance calculation with the'distance of Taijama and Nei and the algorithms of Saitou and Nei (neighbour-joining - NJ) showed that Pseudois is grouped together with Capra and Ammotragus . All members of the genus Oxis formed a second cluster. Phylogenetic analyses supported these results. NJ-analyses and maximum parsimony (MP) trees did not support the present phylogenetic classification. Bootstrap values of 95% (NJ) and 96% (MP) confirms the grouping of P. nayaur and C. aegagrus . Another clade, O. ammon and O. orientalis supported weakly by bootstrap values of 65% (MP) and 70% (NJ) and the clade O. musimonand O. vignei had bootstrap values of 50% (MP) and 60% (NJ). So the phylogenetic relationship within the genus Oxis is still unsafe. But the parsimony analyses lead to the conclusion that Ammotragus represents an old lineage that diverged from ancestral caprines together with true sheep a long time ago and that both true goats and blue sheep forms represent one evolutionary lineage.     

中国绿水螅分子系统发生地位的初步探讨

目的:初步探讨中国绿水螅(Hydra sinensis)分子系统发生地位以及水螅属内部各类群系统发生关系。方法:采用酚-氯仿法提取中国绿水螅总DNA,扩增线粒体COI和16S r RNA基因片段并进行DNA序列测定,再利用Clustal及MEGA等生物信息学分析软件进行系统发生分析。结果:在本研究重建的所有系统发生树中,中国绿水螅始终与绿水螅Hydra viridissima的不同种群一起构成绿水螅单系群。同时,棕色水螅群的单系性被基于COI基因的NJ树以及基于16S r RNA基因的NJ树和ML树支持,唯独基于COI基因的ML树不支持棕色水螅群的单系发生。在基于COI基因的ML树中纤弱水螅族在系统树的基部独立为一支系,而绿水螅群和其他棕色水螅群水螅一起组成另一支系,提示纤弱水螅族水螅的系统发生地位值得进一步探讨。值得注意的是,根据本文的结果,棕色水螅群内3族的划分仍然有一定疑问。基于COI基因的NJ树和ML树支持普通水螅族、寡水螅族和纤弱水螅族各自族内的单系发生,但16S r RNA基因的NJ树和ML树中仅普通水螅族水螅聚为单系群,而寡水螅族和纤弱水螅族水螅各自并非单系发生。结论:把水螅属划分为绿水螅群及棕色水螅群有一定的合理性,但棕色水螅群内寡水螅族、普通水螅族和纤弱水螅族3族的划分还有待商榷。     

Statistical properties of bootstrap estimation of phylogenetic variability from nucleotide sequences: II. Four taxa without a molecular clock

Summary The statistical properties of sample estimation and bootstrap estimation of phylogenetic variability from a sample of nucleotide sequences were studied by considering model trees of three taxa with an outgroup. The cases of constant and varying rates of nucleotide substitution were compared. From sequences obtained by simulation, phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor joining (NJ) methods. The effectiveness and consistency of the MP method were studied in terms of proportions of informative sites. The results of simulation showed that bootstrap estimation of the confidence level for an inferred phylogeny can be used even under unequal rates of evolution if the rate differences are not large so that the MP method is not misleading. The condition under which the MP method becomes misleading (inconsistent) is more stringent for slowly evolving sequences than for rapidly evolving ones, and it also depends on the length of the internal branch. If the rate differences are large so that the MP method becomes consistently misleading, then bootstrap estimation will reinforce an erroneous conclusion on topology. Similar conclusions apply to the NJ method with uncorrected distances. The NJ method with corrected distances performs poorly when the sequence length is short but can avoid the inconsistency problem if the sequence length is long and if the distances can be estimated accurately.Offprint requests to: W.-H. Li     

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm

Three new members of the cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The amino acid alignment showed conservation of the cysteine, histidine, and asparagine residue that form the catalytic triad. With 57 cysteine proteinases including PwCP1-5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1-5 were clustered with cruzipain-like or cathepsin L-like cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22 cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported cruzipain-like cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to cathepsin L-like cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six cruzipain signature sequences known previously, whereas PwCP5 do not have any cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175--human cathepsin L numbering) for cathepsin L-like group are conserved in PwCP5, which are conserved within cathepsin L-like group and also different from those of cruzipain and other cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser-Tyr-Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr-Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1-4 (cruzipain) and PwCP5 (cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1-4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the cruzipain and cathepsin L-like cysteine proteinases. In addition, the fact that most of cysteine proteinases from P. westermani are cruzipain-like type implies strongly that a new powerful drug for paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani cruzipain-like cysteine proteinases.