- Research article
- Open Access
Horizontal gene transfer between Wolbachia and the mosquito Aedes aegypti
© Klasson et al; licensee BioMed Central Ltd. 2009
- Received: 22 December 2008
- Accepted: 20 January 2009
- Published: 20 January 2009
The evolutionary importance of horizontal gene transfer (HGT) from Wolbachia endosymbiotic bacteria to their eukaryotic hosts is a topic of considerable interest and debate. Recent transfers of genome fragments from Wolbachia into insect chromosomes have been reported, but it has been argued that these fragments may be on an evolutionary trajectory to degradation and loss.
We have discovered a case of HGT, involving two adjacent genes, between the genomes of Wolbachia and the currently Wolbachia-uninfected mosquito Aedes aegypti, an important human disease vector. The lower level of sequence identity between Wolbachia and insect, the transcription of all the genes involved, and the fact that we have identified homologs of the two genes in another Aedes species (Ae. mascarensis), suggest that these genes are being expressed after an extended evolutionary period since horizontal transfer, and therefore that the transfer has functional significance. The association of these genes with Wolbachia prophage regions also provides a mechanism for the transfer.
The data support the argument that HGT between Wolbachia endosymbiotic bacteria and their hosts has produced evolutionary innovation.
- Horizontal Gene Transfer
- Anopheles Gambiae
- Cytoplasmic Incompatibility
- Horizontal Gene Transfer Event
- Wolbachia Strain
Wolbachia pipientis is an intracellular inherited bacterium found in arthropods, where it manipulates host reproduction using phenotypes such as cytoplasmic incompatibility (CI), male killing, parthenogenesis and feminization, and can spread rapidly through insect populations . It is also an obligate mutualist of a number of filarial nematode species .
Several cases where sections of the Wolbachia genome, sometimes large, have been transferred to the host chromosomes are now known in both insects and nematodes [3–5]. These are either recent events where Wolbachia and host sequences are highly similar or involve extensive pseudogenization . Transcription was reported for 2% of the genes transferred to Drosophila ananassae but the levels were estimated to be 104 to 107 fold lower than for a control gene, act5C [5, 6], and it has been argued that this could represent background transcriptional noise (as occurs for many pseudogenes) rather than functional expression [7, 8] – translation has yet to be demonstrated. It has therefore been suggested that these fragments are on an evolutionary trajectory to degradation by neutral mutation and play no significant part in host evolution . If so they would be analogous to the non-functional nuclear fragments of mitochondrial DNA present in some animal genomes , which are transient and in the process of decay.
The case has therefore been made that if Wolbachia-insect HGT has evolutionary significance, both longevity and integration into host biology would need to be demonstrated ; and furthermore that we would expect to see Wolbachia-like genes in species that do not currently harbour Wolbachia but presumably did in the past. Both phylogenetic analyses and theory suggest that Wolbachia can be lost over time from host species by a variety of mechanisms [10–12]. Aedes aegypti, the most important mosquito vector of human dengue fever and various other arboviruses, is naturally Wolbachia-uninfected but has been shown to be able to support Wolbachia following artificial transinfection – with both high rates of maternal inheritance and the expression of high levels of CI . The examination of its sequenced genome  for any genes that could have originated in Wolbachia was therefore undertaken.
Sequences (5'-3'), optimal annealing temperatures (°C) and amplified fragment sizes (base pairs) for primers used in the study.
optimal annealing temperature (°C)
PCR amplification results from genomic DNA to examine the distribution of Ae. aegypti genes AAEL004181 and AAEL004188, plus presence/absence of Wolbachia, among other species in the Aedes subgenus Stegomyia.
simpsoni, heischi, calceatus, metallicus, soleatus
wsp – Wolbachia
Aeg S7 – control
The w Pip genes are located at the end of a genomic prophage region, providing a putative mechanism for the HGT. Wolbachia have been shown to contain phage particles by EM in several studies; WO prophage have been shown to be highly variable and rapidly evolving regions in the genomes of mosquito Wolbachia, and non-congruent with host phylogeny [19–24], strongly suggesting that lateral transfer of phage between Wolbachia strains has occurred. The two w Mel genes WD0512 and WD0513 are part of an operon that also contains the ankyrin repeat domain (ANK) encoding gene WD0514. This operon is present in mod+ strain variants of w Mel (able to induce CI in males) but not in the related mod- strain w Au (unable to induce CI) . The operon is located in a region of the w Mel genome that was shown to be missing in w Au, WD0506-WD0518 in w Mel, and in fact all these genes have homologs in the prophage regions of the w Pip genome, except for the ANK gene WD0514. Therefore, although not annotated as prophage , these genes in w Mel are likely to be remnants of an old prophage region, the rest of which has been deleted or rearranged.
The Ae. aegypti gene AAEL004181 also shares considerably lower amino acid similarity with a group of genes in the Ae. aegypti, Anopheles gambiae and Culex pipiens genomes. One of these Ae. aegypti genes showed female salivary gland specific expression and was named aaSGS1 (SGS = Salivary Gland Specific). This gene and homologs in Anopheles are candidate Plasmodium sporozoite receptors [26, 27]. It has already been suggested that the SGS-type mosquito genes might have arisen from an ancient transfer between Wolbachia and mosquitoes, but with weak support [26, 27].
The data presented provide a robust case for HGT between Wolbachia and mosquitoes, and we consider Wolbachia-to-host to be the most likely direction of this transfer of the genes AAEL004181/8 in Ae. aegypti and mascarensis. Our results support the argument that HGT between Wolbachia and their insect hosts has led to the acquisition of evolutionary innovation, provide a putative mechanism for transfer via nuclear-phage recombination, and suggest that the previously documented examples of recent/ongoing Wolbachia-host HGT may have considerably more significance than interesting, but transient, phenomena.
Mosquito DNA and RNA extraction, PCR, sequencing, and RT-PCR using a Qiagen one-step RT-PCR kit were carried out as previously described [22, 28]. DNA from Museum specimens was extracted using a Qiagen QIAamp DNA micro kit according to the manufacturers instructions. Primers were designed using Primer3  and previously published primers for gene wsp (81F and 691R) were used to check for presence of Wolbachia, and AegS7F and R primers amplifying the ribosomal S7 gene  as controls for Aedes DNA quality. DNA from Aedes simpsoni, heischi, soleatus, calceatus, metallicus and mascarensis (all considered phylogenetically close to Ae. aegypti) was extracted from preserved Museum specimens; only data from specimens where strong AegS7 amplification was observed were included. DNA from Ae. aegypti and Ae. albopictus was extracted from laboratory specimens.
Gene expression levels were monitored using quantitative RT-PCR (qRT-PCR). Total RNA was extracted with TRIzol™ reagent from groups of ten Aedes aegypti females and cDNA was synthesized from 1 microgram of total RNA using SuperScript II enzyme (Invitrogen) following the manufacturer's protocol. qRT-PCR was performed on a 1 in 20 dilution of the cDNAs using dsDNA dye SYBR Green I. Reactions were run on a DNA Engine thermocycler (MJ Research) with Chromo4 real-time PCR detection system (Bio-Rad) using the following cycling conditions: 95C for 15 minutes, then 45 cycles of 95C for 10s, 59C for 10s, 72C for 20s, with fluorescence acquisition at the end of each cycle, then a melting curve analysis after the final one. The cycle threshold (Ct) values were determined and background fluorescence was subtracted. Gene expression levels of target genes were calculated, relative to the internal reference gene RpS17 (ribosomal protein S17). Primer pairs used to detect target gene transcripts were as follows: AAEL004181 (forward: 5'-GTT TCC GCA GAA GAA TCA GC-3', reverse: 5'-AGT TCG TCT CCA AAG CAG GA-3'); AAEL004188 (forward: 5'-TGA ATT GCT GCT ACG GTT TG-3', reverse: 5'-TGA ATG GGT CTT TGT GTC CA-3'); Actin5C (forward: 5'-ATC GTA CGA ACT TCC CGA TG-3', reverse: 5'-ACA GAT CCT TTC GGA TGT CG-3') and control RpS17 (forward: 5'-CAG GTC CGT GGT ATC TCC AT-3', reverse: 5'-CAG GAC ATC ATC GAA GTC GA-3').
Custom Ae. aegypti microarrays were designed using Agilent eArray software . A probe set, containing two unique 60-mers per annotated Ae. aegypti gene, was designed using the gene expression probe design module. These probes were randomly position across the surface of the array.
Aedes aegypti were reared using standard procedures to either 0–2 days (young) or 14–16 days (old) post-eclosion before collection. Total RNA was extracted from pools of 10 female Ae. aegypti using Trizol reagent (Invitrogen) and following the manufacturer's protocol. Isolated total RNA was quantified on the Nanodrop ND-1000 spectrophotometer. The two-colour low RNA input linear amplification kit PLUS kit (Agilent Technologies) was used to amplify cyanine-3 (Cy3) and cyanine-5 (Cy5) labeled complimentary RNA (cRNA) from 1.5 μg of total RNA from each pool of female mosquitoes. cRNA samples were purified using RNeasy mini kits (Qiagen), then cRNA concentration and dye incorporation (labeling efficiency) where quantified using the Nanodrop. Prepared cRNA samples were hybridized to four 4 × 44K format microarrays using Agilent reagents and protocols. Microarray slides were washed following Agilent protocols to prevent ozone degradation and scanned with an Agilent scanner at 5 μm scan resolution using the extended dynamic range (XDR) function (XDR Hi 100%, XDR Lo 10%). Agilent feature extraction software (version 9.5.3) was run on all array datasets using the GE2-v5_95_Feb07 protocol. This protocol reports processed Cy3 and Cy5 signal intensities and identifies probes significantly expressed above background (2-sided t-test; P = 0.01).
The two genes WP1346 and WP1348 in Wolbachia strain w Pip and the two genes WD0512 and WD0513 from Wolbachia strain w Mel were concatenated. The gene AAEL004181 from Aedes aegypti was extended to the new start codon and the parts annotated as introns were included. All genes identified as putative SGS family members by searching the genomes of Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus with tblastn in VectorBase  were extracted. All gene sequences were, if necessary, extended to the putatively correct start codon based on homology with AAEL004181 and introns were included and translated together with the exon sequences. The gene annotated as CPIJ007816 contained two large open reading frames that were both similar to the SGS genes, they were both included and are called CPIJ007816a and b. Two SGS genes from Anopheles gambiae, agSGS2 and agSGS3 sequenced by Korochkina et al. , were retrieved separately from Genbank, since the corresponding region in the Anopheles gambiae genome contains sequence gaps. The amino acid sequences were aligned using MUSCLE . Phylogenetic reconstruction was performed using MrBayes 3.12 , with the mixed amino acid model. The program was run for 200,000 generations, sampling every 100th generation, using 2 runs with 4 chains in each. A consensus tree was constructed using a burnin of 25% of the sampled trees. A maximum likelihood analysis was conducted using RAxML ver. 7.0.4 . A rapid boostrap analysis using 1000 replicates with a following search for the best scoring ML tree was conducted in two separate runs using the WAGF+GAMMA+I model. For each run, the final ML optimization was conducted for every 5th bootstrapped tree to search for the best scoring ML tree.
Sequences are deposited in GenBank accession numbers FM958472–FM958475.
We thank Leonard Munstermann (Yale) for advice on Aedes subgenus Stegomyia; Theresa Howard (Natural History Museum, London) and George McGavin (University Museum, Oxford) for kindly providing preserved Aedes specimens; Charles Godfray (Oxford) and Julian Parkhill (Wellcome Trust Sanger institute) for their helpful comments on the manuscript; and Peter Atkinson (UCR) for permission to use SGS sequences from the Culex quinquefasciatus unpublished genome database. This work was supported by the Wellcome Trust, and the European Union Marie Curie Fellowship to LK.
- Turelli M, Hoffmann AA: Rapid spread of an inherited incompatibility factor in California Drosophila. Nature. 1991, 353: 440-442. 10.1038/353440a0.View ArticlePubMedGoogle Scholar
- Taylor MJ, Bandi C, Hoerauf A: Wolbachia bacterial endosymbionts of filarial nematodes. Adv Parasitol. 2005, 60: 245-284. 10.1016/S0065-308X(05)60004-8.View ArticlePubMedGoogle Scholar
- Kondo N, Nikoh N, Ijichi N, Shimada M, Fukatsu T: Genome fragment of Wolbachia endosymbiont transferred to X chromosome of host insect. Proceedings of the National Academy of Sciences of the United States of America. 2002, 99: 14280-14285. 10.1073/pnas.222228199.PubMed CentralView ArticlePubMedGoogle Scholar
- Fenn K, Conlon C, Jones M, Quail MA, Holroyd NE, Parkhill J, Blaxter M: Phylogenetic relationships of the Wolbachia of nematodes and arthropods. PLoS Pathog. 2006, 2: e94-10.1371/journal.ppat.0020094.PubMed CentralView ArticlePubMedGoogle Scholar
- Hotopp JC, Clark ME, Oliveira DC, Foster JM, Fischer P, Torres MC, Giebel JD, Kumar N, Ishmael N, Wang S, Ingram J, Nene RV, Shepard J, Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko BE, Tettelin H, Werren JH: Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science. 2007, 317 (5845): 1753-1756. 10.1126/science.1142490.View ArticleGoogle Scholar
- Nikoh N, Tanaka K, Shibata F, Kondo N, Hizume M, Shimada M, Fukatsu T: Wolbachia genome integrated in an insect chromosome: evolution and fate of laterally transferred endosymbiont genes. Genome research. 2008, 18: 272-280. 10.1101/gr.7144908.PubMed CentralView ArticlePubMedGoogle Scholar
- Bordenstein SR: Evolutionary genomics: transdomain gene transfers. Curr Biol. 2007, 17: R935-936. 10.1016/j.cub.2007.09.022.View ArticlePubMedGoogle Scholar
- Blaxter M: Symbiont genes in host genomes: fragments with a future?. Cell host & microbe. 2007, 2: 211-213. 10.1016/j.chom.2007.09.008.View ArticleGoogle Scholar
- Richly E, Leister D: NUMTs in sequenced eukaryotic genomes. Molecular biology and evolution. 2004, 21: 1081-1084. 10.1093/molbev/msh110.View ArticlePubMedGoogle Scholar
- Werren JH, Zhang W, Guo LR: Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods. Proceedings. 1995, 261: 55-63.Google Scholar
- Reuter M, Pedersen JS, Keller L: Loss of Wolbachia infection during colonisation in the invasive Argentine ant Linepithema humile. Heredity. 2005, 94: 364-369. 10.1038/sj.hdy.6800601.View ArticlePubMedGoogle Scholar
- Sinkins SP, Godfray HC: Use of Wolbachia to drive nuclear transgenes through insect populations. Proceedings. 2004, 271: 1421-1426.Google Scholar
- Xi Z, Khoo CC, Dobson SL: Wolbachia establishment and invasion in an Aedes aegypti laboratory population. Science. 2005, 310 (5746): 326-328. 10.1126/science.1117607.View ArticlePubMedGoogle Scholar
- Nene V, Wortman JR, Lawson D, Haas B, Kodira C, Tu ZJ, Loftus B, Xi Z, Megy K, Grabherr M, Ren Q, Zdobnov EM, Lobo NF, Campbell KS, Brown SE, Bonaldo MF, Zhu J, Sinkins SP, Hogenkamp DG, Amedeo P, Arensburger P, Atkinson PW, Bidwell S, Biedler J, Birney E, Bruggner RV, Costas J, Coy MR, Crabtree J, Crawford M, Debruyn B, Decaprio D, Eiglmeier K, Eisenstadt E, El-Dorry H, Gelbart WM, Gomes SL, Hammond M, Hannick LI, Hogan JR, Holmes MH, Jaffe D, Johnston JS, Kennedy RC, Koo H, Kravitz S, Kriventseva EV, Kulp D, Labutti K, Lee E, Li S, Lovin DD, Mao C, Mauceli E, Menck CF, Miller JR, Montgomery P, Mori A, Nascimento AL, Naveira HF, Nusbaum C, O'Leary S, Orvis J, Pertea M, Quesneville H, Reidenbach KR, Rogers YH, Roth CW, Schneider JR, Schatz M, Shumway M, Stanke M, Stinson EO, Tubio JM, Vanzee JP, Verjovski-Almeida S, Werner D, White O, Wyder S, Zeng Q, Zhao Q, Zhao Y, Hill CA, Raikhel AS, Soares MB, Knudson DL, Lee NH, Galagan J, Salzberg SL, Paulsen IT, Dimopoulos G, Collins FH, Birren B, Fraser-Liggett CM, Severson DW: Genome sequence of Aedes aegypti, a major arbovirus vector. Science. 2007, 316 (5832): 1718-1723. 10.1126/science.1138878.View ArticlePubMedGoogle Scholar
- Klasson L, Walker T, Sebaihia M, Sanders MJ, Quail MA, Lord A, Sanders S, Earl J, O'Neill SL, Thomson N, Sinkins SP, Parkhill J: Genome Evolution of Wolbachia Strain wPip from the Culex pipiens Group. Mol Biol Evol. 2008, 25 (9): 1877-1887. 10.1093/molbev/msn133.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, Brownlie JC, McGraw EA, Martin W, Esser C, Ahmadinejad N, Wiegand C, Madupu R, Beanan MJ, Brinkac LM, Daugherty SC, Durkin AS, Kolonay JF, Nelson WC, Mohamoud Y, Lee P, Berry K, Young MB, Utterback T, Weidman J, Nierman WC, Paulsen IT, Nelson KE, Tettelin H, O'Neill SL, Eisen JA: Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS biology. 2004, 2: E69-10.1371/journal.pbio.0020069.PubMed CentralView ArticlePubMedGoogle Scholar
- Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JM, Wides R, Salzberg SL, Loftus B, Yandell M, Majoros WH, Rusch DB, Lai Z, Kraft CL, Abril JF, Anthouard V, Arensburger P, Atkinson PW, Baden H, de Berardinis V, Baldwin D, Benes V, Biedler J, Blass C, Bolanos R, Boscus D, Barnstead M, Cai S, Center A, Chaturverdi K, Christophides GK, Chrystal MA, Clamp M, Cravchik A, Curwen V, Dana A, Delcher A, Dew I, Evans CA, Flanigan M, Grundschober-Freimoser A, Friedli L, Gu Z, Guan P, Guigo R, Hillenmeyer ME, Hladun SL, Hogan JR, Hong YS, Hoover J, Jaillon O, Ke Z, Kodira C, Kokoza E, Koutsos A, Letunic I, Levitsky A, Liang Y, Lin JJ, Lobo NF, Lopez JR, Malek JA, McIntosh TC, Meister S, Miller J, Mobarry C, Mongin E, Murphy SD, O'Brochta DA, Pfannkoch C, Qi R, Regier MA, Remington K, Shao H, Sharakhova MV, Sitter CD, Shetty J, Smith TJ, Strong R, Sun J, Thomasova D, Ton LQ, Topalis P, Tu Z, Unger MF, Walenz B, Wang A, Wang J, Wang M, Wang X, Woodford KJ, Wortman JR, Wu M, Yao A, Zdobnov EM, Zhang H, Zhao Q, Zhao S, Zhu SC, Zhimulev I, Coluzzi M, della Torre A, Roth CW, Louis C, Kalush F, Mural RJ, Myers EW, Adams MD, Smith HO, Broder S, Gardner MJ, Fraser CM, Birney E, Bork P, Brey PT, Venter JC, Weissenbach J, Kafatos FC, Collins FH, Hoffman SL: The genome sequence of the malaria mosquito Anopheles gambiae. Science. 2002, 298 (5591): 129-149. 10.1126/science.1076181.View ArticlePubMedGoogle Scholar
- Hilburn LR, Rai KS: Genetic analysis of abnormal male sexual development in Aedes aegypti and Ae. mascarensis backcross progeny. The Journal of heredity. 1982, 73: 59-63.PubMedGoogle Scholar
- Masui S, Kamoda S, Sasaki T, Ishikawa H: Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods. Journal of molecular evolution. 2000, 51: 491-497.PubMedGoogle Scholar
- Masui S, Kuroiwa H, Sasaki T, Inui M, Kuroiwa T, Ishikawa H: Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods. Biochem Biophys Res Commun. 2001, 283: 1099-1104. 10.1006/bbrc.2001.4906.View ArticlePubMedGoogle Scholar
- Wright JD, Sjostrand FS, Portaro JK, Barr AR: The ultrastructure of the rickettsia-like microorganism Wolbachia pipientis and associated virus-like bodies in the mosquito Culex pipiens. J Ultrastruct Res. 1978, 63: 79-85. 10.1016/S0022-5320(78)80046-X.View ArticlePubMedGoogle Scholar
- Sinkins SP, Walker T, Lynd AR, Steven AR, Makepeace BL, Godfray HC, Parkhill J: Wolbachia variability and host effects on crossing type in Culex mosquitoes. Nature. 2005, 436: 257-260. 10.1038/nature03629.View ArticlePubMedGoogle Scholar
- Sanogo YO, Dobson SL: Molecular discrimination of Wolbachia in the Culex pipiens complex: evidence for variable bacteriophage hyperparasitism. Insect Mol Biol. 2004, 13: 365-369. 10.1111/j.0962-1075.2004.00498.x.View ArticlePubMedGoogle Scholar
- Bordenstein SR, Wernegreen JJ: Bacteriophage flux in endosymbionts (Wolbachia): infection frequency, lateral transfer, and recombination rates. Molecular biology and evolution. 2004, 21: 1981-1991. 10.1093/molbev/msh211.View ArticlePubMedGoogle Scholar
- Iturbe-Ormaetxe I, Burke GR, Riegler M, O'Neill SL: Distribution, expression, and motif variability of ankyrin domain genes in Wolbachia pipientis. Journal of bacteriology. 2005, 187: 5136-5145. 10.1128/JB.187.15.5136-5145.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Arca B, Lombardo F, Valenzuela JG, Francischetti IM, Marinotti O, Coluzzi M, Ribeiro JM: An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae. The Journal of experimental biology. 2005, 208: 3971-3986. 10.1242/jeb.01849.View ArticlePubMedGoogle Scholar
- Korochkina S, Barreau C, Pradel G, Jeffery E, Li J, Natarajan R, Shabanowitz J, Hunt D, Frevert U, Vernick KD: A mosquito-specific protein family includes candidate receptors for malaria sporozoite invasion of salivary glands. Cellular microbiology. 2006, 8: 163-175. 10.1111/j.1462-5822.2005.00611.x.View ArticlePubMedGoogle Scholar
- Walker T, Klasson L, Sebaihia M, Sanders MJ, Thomson NR, Parkhill J, Sinkins SP: Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group. BMC Biol. 2007, 5: 39-10.1186/1741-7007-5-39.PubMed CentralView ArticlePubMedGoogle Scholar
- Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000, 132: 365-386.PubMedGoogle Scholar
- Zhou W, Rousset F, O'Neil S: Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences. Proceedings. 1998, 265: 509-515.Google Scholar
- Dutton TJ, Sinkins SP: Strain-specific quantification of Wolbachia density in Aedes albopictus and effects of larval rearing conditions. Insect Mol Biol. 2004, 13: 317-322. 10.1111/j.0962-1075.2004.00490.x.View ArticlePubMedGoogle Scholar
- Agilent Technologies eArray. [https://earray.chem.agilent.com/earray]
- VectorBase. [http://www.vectorbase.org]
- Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research. 2004, 32: 1792-1797. 10.1093/nar/gkh340.PubMed CentralView ArticlePubMedGoogle Scholar
- Huelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics (Oxford, England). 2001, 17: 754-755. 10.1093/bioinformatics/17.8.754.View ArticleGoogle Scholar
- Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics (Oxford, England). 2006, 22: 2688-2690. 10.1093/bioinformatics/btl446.View ArticleGoogle Scholar
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