Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit, 1910
TL;DR: The incompleteness of the morphological data and relatively low host specificity provides the space for large synonymy within this taxon, so a complex approach combining microscopic analyses together with molecular-genetic methods should represent the basic standard for all taxonomic studies.
Abstract: The uniform morphology of the developmental stages of Haemogregarina species and the insufficient information supplied by the simplistic descriptions of previous authors complicates their differential diagnosis and proper species identification. In this study, we detected Haemogregarina spp. in 6 out of 22 (27·2%) examined turtles originating from Southeast Asia, Malayemys subtrijuga (n = 4), Sacalia quadriocellata (n = 1) and Platysternon megacephalum (n = 1), and compared them with the available literature data. Microscopic analysis of our isolates distinguished 2 morphological species, Haemogregarina pellegrini and one new species, being described in this paper as Haemogregarina sacaliae sp. n. Phylogenetic analyses based on 1210 bp long fragment of 18S rDNA sequences placed both haemogregarines firmly within the monophyletic Haemogregarina clade. Isolates of H. pellegrini from 2 distantly related turtle hosts, M. subtrijuga and P. megacephalum, were genetically identical. Despite the fact that numerous Haemogregarina species of turtles have been described, the incompleteness of the morphological data and relatively low host specificity provides the space for large synonymy within this taxon. Therefore, a complex approach combining microscopic analyses together with molecular-genetic methods should represent the basic standard for all taxonomic studies.
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74 citations
TL;DR: An overview of all published 18S rRNA sequence data for haemogregarines suggests that any taxonomic changes now would be premature, and type species need to be assessed, sampling across hosts improved, and multiple genes employed prior to taxonomic alterations.
Abstract: Haemogregarines are a group of apicomplexan parasites composed of 3 families that infect a wide range of hosts. Many species within these families have been subjected to reclassifications and reassignments, especially because the use of molecular tools to estimate their phylogenetic relationships became more widespread. The 18S rRNA gene has been the only widely used gene for studying the diversity of haemogregarines and recent phylogenetic analyses of this gene have indicated incongruences with the current taxonomy, such that a new genus Bartazoon has recently been proposed. To investigate the current taxonomic situation further, we conducted an overview of all published 18S rRNA sequence data for haemogregarines. We highlight that our understanding of the real diversity and phylogenetic relationships of haemogregarines is still limited, which undermines the proposed systematic revision. Notably all the molecular evidence comes from a single gene, and many studies have shown that single-gene trees often do not reflect species trees. Combined with doubts over the relationships of Hemolivia, the recent identification of a new lineage that could also warrant creation of a new genus, and issues with the type species for Hepatozoon, we suggest that any taxonomic changes now would be premature. In our opinion, type species need to be assessed, sampling across hosts improved, and multiple genes employed prior to taxonomic alterations. Otherwise taxonomic instability will be likely.
35 citations
TL;DR: This is the first morphological and molecular characterisation of a haemogregarine within the African Varanidae, and the first report of a species of Karyolysus infecting the monitor lizard family.
Abstract: Within the African monitor lizard family Varanidae, two haemogregarine genera have been reported. These comprise five species of Hepatozoon Miller, 1908 and a species of Haemogregarina Danilewsky, 1885. Even though other haemogregarine genera such as Hemolivia Petit, Landau, Baccam & Lainson, 1990 and Karyolysus Labbe, 1894 have been reported parasitising other lizard families, these have not been found infecting the Varanidae. The genus Karyolysus has to date been formally described and named only from lizards of the family Lacertidae and to the authors’ knowledge, this includes only nine species. Molecular characterisation using fragments of the 18S gene has only recently been completed for but two of these species. To date, three Hepatozoon species are known from southern African varanids, one of these Hepatozoon paradoxa (Dias, 1954) shares morphological characteristics alike to species of the family Karyolysidae. Thus, this study aimed to morphologically redescribe and characterise H. paradoxa molecularly, so as to determine its taxonomic placement. Specimens of Varanus albigularis albigularis Daudin, 1802 (Rock monitor) and Varanus niloticus (Linnaeus in Hasselquist, 1762) (Nile monitor) were collected from the Ndumo Game Reserve, South Africa. Upon capture animals were examined for haematophagous arthropods. Blood was collected, thin blood smears prepared, stained with Giemsa, screened and micrographs of parasites captured. Haemogregarine morphometric data were compared with the data for named haemogregarines of African varanids. Primer set HepF300 and HepR900 was employed to target a fragment of the 18S rRNA gene and resulting sequences compared with other known haemogregarine sequences selected from the GenBank database. Hepatozoon paradoxa was identified infecting two out of eight (25 %) V. a. albigularis and a single (100 %) V. niloticus examined. Phylogenetic analyses revealed that H. paradoxa clustered with the ‘Karyolysus’ clade, and not with those of reptilian Hepatozoon spp. In addition to this being the first morphological and molecular characterisation of a haemogregarine within the African Varanidae, it is the first report of a species of Karyolysus infecting the monitor lizard family. Furthermore, this constitutes now only the third described and named Karyolysus species for which there is a nucleotide sequence available.
30 citations
TL;DR: Maximum likelihood and maximum parsimony analyses of 18S rDNA sequences showed that Haemogregarina parvula formed a monophyletic clade with species of Hemolivia within the Hepatozoon clade, and it is recommended that this haemogRegarine be reassigned to the genus Hemol Bolivia, rendering Hemoliva parvulas the first species of the genus recorded from southern African tortoises.
Abstract: To date, only a single species of Hemolivia, Hemolivia mauritanica (Sergent & Sergent, 1904), has been described from African terrestrial tortoises. Although various haemogregarines have been described from southern African terrapins and tortoises, including species from the genus Haemogregarina and one from the genus Hepatozoon, no species of Hemolivia have been identified previously from southern Africa. Since its morphological redescription, the taxonomic placement of one of these species, Haemogregarina parvula Dias, 1953, was in doubt. Hence, research was undertaken to resolve the true taxonomic position of this haemogregarine. Blood smears from nine wild tortoises of two species, Stigmochelys pardalis and Kinixys zombensis, from South Africa were screened, with the focus on H. parvula. Parasite DNA was extracted from ethanol-preserved blood samples, and fragments of the 18S rDNA gene were amplified by PCR using the primer sets HepF300/HepR900 and 4558/2733. Maximum likelihood and maximum parsimony a...
26 citations
Cites methods from "Haemogregarines of freshwater turtl..."
...DQ096835) was chosen as the outgroup as in Dvořáková et al. (2015) (see Table 2)....
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TL;DR: The observed morphology and the sequencing of nuclear 18S rDNA identified the parasite as Haemogregarina stepanowi Danilewsky, 1885, and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.
Abstract: Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 μm in length and 6.4 ± 0.52 μm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.
24 citations
Cites background from "Haemogregarines of freshwater turtl..."
...The target fragment is considered sufficiently variable and informative at both the generic and specific levels in Apicomplexa (Barta et al. 2012, Dvořáková et al. 2014, 2015, Özvegy et al. 2015)....
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...Conversely, the distribution of haemogregarines seems to be determined by the distri- bution of their definitive host, usually a glossiphoniid leech of the genus Placobdella (Blanchard) (see Dvořáková et al. 2015)....
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References
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TL;DR: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved and modifications are incorporated into a new program, CLUSTAL W, which is freely available.
Abstract: The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order to down-weight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new program, CLUSTAL W which is freely available.
63,427 citations
"Haemogregarines of freshwater turtl..." refers methods in this paper
...Alignment was created using BioEdit (Hall, 1999) with the Clustal W algorithm (Thompson et al. 1994)....
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TL;DR: The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models, inferring ancestral states and sequences, and estimating evolutionary rates site-by-site.
Abstract: Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
39,110 citations
"Haemogregarines of freshwater turtl..." refers methods in this paper
...Genetic distances based on the 18S rDNA sequences of different species were generated by the Mega 5.0 (Tamura et al. 2011)....
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35,309 citations
TL;DR: MrBayes 3 performs Bayesian phylogenetic analysis combining information from different data partitions or subsets evolving under different stochastic evolutionary models to analyze heterogeneous data sets and explore a wide variety of structured models mixing partition-unique and shared parameters.
Abstract: Summary: MrBayes 3 performs Bayesian phylogenetic analysis combining information from different data partitions or subsets evolving under different stochastic evolutionary models. This allows the user to analyze heterogeneous data sets consisting of different data types—e.g. morphological, nucleotide, and protein— and to explore a wide variety of structured models mixing partition-unique and shared parameters. The program employs MPI to parallelize Metropolis coupling on Macintosh or UNIX clusters.
25,931 citations
"Haemogregarines of freshwater turtl..." refers methods in this paper
...(Ronquist and Huelsenbeck, 2003) with a GTR+Γ+I model for 10 million generations....
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TL;DR: This work has used extensive and realistic computer simulations to show that the topological accuracy of this new method is at least as high as that of the existing maximum-likelihood programs and much higher than the performance of distance-based and parsimony approaches.
Abstract: The increase in the number of large data sets and the complexity of current probabilistic sequence evolution models necessitates fast and reliable phylogeny reconstruction methods. We describe a new approach, based on the maximum- likelihood principle, which clearly satisfies these requirements. The core of this method is a simple hill-climbing algorithm that adjusts tree topology and branch lengths simultaneously. This algorithm starts from an initial tree built by a fast distance-based method and modifies this tree to improve its likelihood at each iteration. Due to this simultaneous adjustment of the topology and branch lengths, only a few iterations are sufficient to reach an optimum. We used extensive and realistic computer simulations to show that the topological accuracy of this new method is at least as high as that of the existing maximum-likelihood programs and much higher than the performance of distance-based and parsimony approaches. The reduction of computing time is dramatic in comparison with other maximum-likelihood packages, while the likelihood maximization ability tends to be higher. For example, only 12 min were required on a standard personal computer to analyze a data set consisting of 500 rbcL sequences with 1,428 base pairs from plant plastids, thus reaching a speed of the same order as some popular distance-based and parsimony algorithms. This new method is implemented in the PHYML program, which is freely available on our web page: http://www.lirmm.fr/w3ifa/MAAS/. (Algorithm; computer simulations; maximum likelihood; phylogeny; rbcL; RDPII project.) The size of homologous sequence data sets has in- creased dramatically in recent years, and many of these data sets now involve several hundreds of taxa. More- over, current probabilistic sequence evolution models (Swofford et al., 1996 ; Page and Holmes, 1998 ), notably those including rate variation among sites (Uzzell and Corbin, 1971 ; Jin and Nei, 1990 ; Yang, 1996 ), require an increasing number of calculations. Therefore, the speed of phylogeny reconstruction methods is becoming a sig- nificant requirement and good compromises between speed and accuracy must be found. The maximum likelihood (ML) approach is especially accurate for building molecular phylogenies. Felsenstein (1981) brought this framework to nucleotide-based phy- logenetic inference, and it was later also applied to amino acid sequences (Kishino et al., 1990). Several vari- ants were proposed, most notably the Bayesian meth- ods (Rannala and Yang 1996; and see below), and the discrete Fourier analysis of Hendy et al. (1994), for ex- ample. Numerous computer studies (Huelsenbeck and Hillis, 1993; Kuhner and Felsenstein, 1994; Huelsenbeck, 1995; Rosenberg and Kumar, 2001; Ranwez and Gascuel, 2002) have shown that ML programs can recover the cor- rect tree from simulated data sets more frequently than other methods can. Another important advantage of the ML approach is the ability to compare different trees and evolutionary models within a statistical framework (see Whelan et al., 2001, for a review). However, like all optimality criterion-based phylogenetic reconstruction approaches, ML is hampered by computational difficul- ties, making it impossible to obtain the optimal tree with certainty from even moderate data sets (Swofford et al., 1996). Therefore, all practical methods rely on heuristics that obtain near-optimal trees in reasonable computing time. Moreover, the computation problem is especially difficult with ML, because the tree likelihood not only depends on the tree topology but also on numerical pa- rameters, including branch lengths. Even computing the optimal values of these parameters on a single tree is not an easy task, particularly because of possible local optima (Chor et al., 2000). The usual heuristic method, implemented in the pop- ular PHYLIP (Felsenstein, 1993 ) and PAUP ∗ (Swofford, 1999 ) packages, is based on hill climbing. It combines stepwise insertion of taxa in a growing tree and topolog- ical rearrangement. For each possible insertion position and rearrangement, the branch lengths of the resulting tree are optimized and the tree likelihood is computed. When the rearrangement improves the current tree or when the position insertion is the best among all pos- sible positions, the corresponding tree becomes the new current tree. Simple rearrangements are used during tree growing, namely "nearest neighbor interchanges" (see below), while more intense rearrangements can be used once all taxa have been inserted. The procedure stops when no rearrangement improves the current best tree. Despite significant decreases in computing times, no- tably in fastDNAml (Olsen et al., 1994 ), this heuristic becomes impracticable with several hundreds of taxa. This is mainly due to the two-level strategy, which sepa- rates branch lengths and tree topology optimization. In- deed, most calculations are done to optimize the branch lengths and evaluate the likelihood of trees that are finally rejected. New methods have thus been proposed. Strimmer and von Haeseler (1996) and others have assembled four- taxon (quartet) trees inferred by ML, in order to recon- struct a complete tree. However, the results of this ap- proach have not been very satisfactory to date (Ranwez and Gascuel, 2001 ). Ota and Li (2000, 2001) described
16,261 citations
"Haemogregarines of freshwater turtl..." refers methods in this paper
...(Guindon and Gascuel, 2003) was employed to carry out the ML analysis under the GTR+Г+I model; bootstrap support was calculated for 1000 replicates....
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