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Blood Drive is the poignant story of the men and women who drive fast cars and kill zombies for sport. There's an announcer, occasional verbal sparring and a bunch of climactic cut-scenes, but let's be honest, you're reading this review because it says "blood" in the title and below that is a picture of a car smashing a zombie so does this game let us smash zombies or what?
Yes. The sole purpose of Blood Drive is to smash zombies. Unfortunately, that's all it does and it doesn't even do that very well.
Wait, a bland zombie game? How did anyone whiff in the game genre equivalent of the tee-ball league? To be fair, Blood Drive provides the occasional glimpse of what could have been. But as it stands, the game is incomplete -- mind-numbingly incomplete. It's as if a gentle, loving hand crafted the game halfway, then the art teacher walked by, snatched it up and said, "Sorry students, we're all out of time."
Okay, let's grade this funky smash of unfinished clay.


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Black God, White Devil ( Deus e o Diabo na Terra do Sol ) ( God and the Devil in the Land of the Sun ) [ NON-USA FORMAT, PAL, Reg.0 Import Review
Black God, White Devil ( Deus e o Diabo na Terra do Sol ) ( God and the Devil in the Land of the Sun ) [ NON-USA FORMAT, PAL, Reg.0 Import Feature
United Kingdom released, PAL/Region 0 DVD: it WILL NOT play on standard US DVD player. You need multi-region PAL/NTSC DVD player to view it in USA/Canada: LANGUAGES: Portuguese ( Dolby Digital 2.0 ), English ( Subtitles ), French ( Subtitles ), Portuguese ( Subtitles ), Spanish ( Subtitles ), SPECIAL FEATURES: Interactive Menu, Remastered, Scene Access, SYNOPSIS: Widely considered to be the greatest Brazilian film of all time, Black God, White Devil (Deus e o Diabo na Terra do Sol) influenced such major filmmakers as Martin Scorsese and Sergio Leone and is Glauber Rocha s finest and most moving work. This masterpiece of cinema makes its in its UK theatrical debut, courtesy of Mr Bongo Films An epic and moving work, Black God, White Devil blends mysticism, religion, and popular culture, into a powerful, bleak and cruel epic, that unashamedly expresses its frustration with many of the major institutions of Brazilian life government, Church and corrupt landowners. Set in the 1940s, the picture follows Manuel, who kills his employer after he tries to cheat him of his wages. From then on Manuel and his wife, Rosa, move around the country, robbing and killing as they go, listening to the words of religious mystics and self-proclaimed saints as try to find their place in a ruthless land of corruption, anguish and violence. What makes this film all the more remarkable is the fact that director Glauber Rocha was only 25 years old when he wrote and began filming this masterwork, a film which cries out for man to determine his own path. The picture was nominated for the 1964 Golden Palm award at the Cannes Film Festival and is considered to be a seminal piece of the Brazilian Cinema Novo movement. SCREENED/AWARDED AT: Cannes Film Festival,

subset: commoncrawl


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Sponsored By
2 Whales Found Beached On East Hampton ShoreTwo whales were found beached Sunday on separate stretches on the shores of East Hampton.
Dolphin Dies Hours After Beaching Itself In Far RockawayThe 5-foot female dolphin was found around 9:30 a.m. Friday, but died several hours later.
Beached Dolphin Dies On South Jersey ShoreAn officer and a volunteer had poured buckets of water on the stranded dolphin in a failed effort to keep it alive.


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We now know that actor Stacy Keach had a heart attack on stage at Goodman Theatre on May 30 during the opening night of his show, "Pamplona." I was in the audience that night and it was clear that Keach was having trouble recalling parts of the script and missing light cues. He appeared to be confused and finally after an hour of struggling through, someone over a loud speaker said, "Hold, please. Mr. Keach, would you exit the stage?" Keach exited and director Bob Falls came on stage and explained that Keach had been ill all day, but insisted on performing that night. Falls added that the performance had not gone as planned and they were canceling it for the night. Goodman continued to cancel performances for another three shows before canceling the run altogether. The good news is that Keach is expected to make a recovery and Goodman plans to reschedule "Pamplona" for 2018. Keach is expected to be released from the hospital today and remain in Chicago during his recover.
The hit 1988 film "Working Girl" starring Melanie Griffith, Harrison Ford and Sigourney Weaver is headed to a Broadway stage. Singer/song-writer Cyndi Lauper will write the music for the show. Lauper previously won a Tony for the score of "Kinky Boots," which had its pre-Broadway run right here in Chicago. The film which was nominated for six Academy Awards including Best Picture focuses on a Staten Island secretary trying to make in a man's world. When her female boss takes a brief leave, she fills in and trouble ensues. No date yet on that Broadway bow.
So while a sequel has not been officially announced, almost everyone in the film industry expects it. "Wonder Woman" grossed more than $200 million worldwide in its opening week. Both star Gal Gadot and director Patty Jenkins are contractually obligated to a sequel should Warner Bros. green light it. Jenkins has also said she knows what she wants in a sequel including setting it in the US. Critics and fans are giving "Wonder Woman" great reviews and I agree. Definitely worth your money. Check it out.
The film that gave starring roles to both Tupac Shakur and Omar Epps, "Juice," is out on Blu-ray for the first time and celebrating the film's 25th anniversary. The Blu-ray also includes an alternate ending to "Juice." "Juice" is available from Paramount Home Entertainment on DVD and Blu-ray in stores everywhere now.
franklyHANK: Stacy Keach, Working Girl, Wonder Woman
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Small business in the 21st century cannot survive without an internet presence. Tens of millions of people start the shopping process online, and if most consumers cannot find your business on Google they will not shop at that business. But gaining traction on search engines can be a daunting process. It can be hard to distinguish your company from the hundreds or thousands of other companies around the world who sell a similar product or service, especially if a company is relatively new. Search Engine Optimization (SEO) can help push consumers away from those competitors and towards your company.
SEO is an approach where companies try to figure out how to reach a high area of Google search results surrounding their products or services. The first organic page of those search results is the overall goal. According to KatRank, over 90% of all Google searches do not make it to the second page. Therefore, SEO attempts to bring users as close as possible to that first page by shifting the marketing formula of the company to fit the search engine. The company may reduce barriers in its website to better allow for Google's web crawler to analyze it. A company may also fill its marketing posts with a certain mix of keywords that will help them show up in more searches. More searches means greater visibility and a better change of appearing on that first Google page.
SEO can be helpful for nearly any type of business, large or small. These techniques can help bring a company's products to an audience of millions. SEO is also much less costly than other forms of advertising and can be scaled easily for multiple advertising campaigns. When it comes to internet visibility and racking up sales in an internet-driven world, SEO is much more of a requirement than an option.


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About Us
The Grossman Burn Foundation's mission is to promote effective, sustainable partnership solutions for the comprehensive treatment, care, and support of burn survivors and their families in the United States and around the world.
GBF's focus is on building in-country and local-level capacity through its global partnerships. Established in 2007, the GBF was created as the philanthropic arm of the Grossman Burn Centers, a pioneer in international burn treatment facilities. Founded by Dr. Peter H. Grossman (Director of the Grossman Burn Centers, Inc.), and his wife Rebecca Grossman, the GBF is headquartered in Los Angeles County and works to provide burn prevention education and self-sustainable burn treatment communities in the United States and around the world.
Global Healthcare & Education Initiatives for Burn Injuries
Senior Burn Prevention Education
Senior citizens are at particularly high risk of scalds, burn injury and death from accidental fires.
Changes in vision, comprehension, and mobility that accompany aging, combined with
medication side effects and attitudinal risks, put seniors at higher risk than younger adults
of burns and scalds.
The Fifth Annual SoCal Burn Ride will be held on Sunday, October 7, 2018 beginning at a Los Angeles Fire Station in Woodland Hills and end at the prestigious Exile Cycles, "Rider Round Up at the Ranch" charity event and show at Paramount Ranch along with LA Calendar Show's Custom Bike Building Championship.
So many of us at ABC News know about the work that the Grossman Burn Foundation has done, to provide so much care, new life, for women, children, people around the US and the world, in need of urgent medical care.
Diane Sawyer – ABC News Anchor


text: Two months in to my Parisian adventure and I now see Paris for what it truly is.. a beautiful and enchanting, not simply the Eiffel Tower and Le Louvre. The best thing about being out here, is being able to show my favourite spots in the city to my friends and family. Usually their visits … Continue reading Travel | How to spend one day à Paris


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Nothing really solidifies the passage of time like having children and dogs. Children grow up so fast leaving you wondering where the time went and dogs…well, you swear yesterday they were just a puppy and today they look at you with those sweet grey muzzles and eyebrows and you realize they are growing older right under your nose.
London as a puppy was as different from Lexi as night and day. She was wildly happy all the time with a tail that never quite wagging, and a tongue that never quit licking. Lexi was much more reserved, quietly loyal with a strong sense of right and wrong. With London everything was a party. Somehow these two were the perfect counterpoint for each other. Lexi used to worry about riding in the vehicle and would get sick if the trip lasted more than five minutes, having London along was exactly what she needed and she stopped the drooling almost immediately. Lexi taught London that you must come immediately when you're called, and showed her through good behavior how to be a well behaved dog. Together they are the perfect team.
London is a group placing champion and has her CGN. She also has one leg towards her CD and who knows..maybe one day we'll get the other two. She is the dam of five champions as well as many more much loved companions.
So today, Happy 8th Birthday to you my London Bridges….here's to many more birthdays to come!


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Guidance has been launched by UNICEF to increase awareness among UK clinicians of the marketing practices of infant formula companies.
It notes that while most people are aware of aggressive marketing techniques to sell infant formula in the developing world, there is a lack of understanding that similar practices exist in the UK.
The new document – A guide for health workers to working within the International Code of Marketing of Breastmilk Substitutes – sets out ways formula manufacturers may seek to influence health professionals to endorse their product, for example, through using branded pens or attending sponsored study days.
Baby Friendly director Sue Ashmore, director of the UNICEF UK Baby Friendly Initiative, said: "Health professionals, particularly midwives and health visitors, are extremely valuable to formula companies because of their trusted status and constant access to new parents."
Sign our Speak Out Safely petition to support a transparent and open NHS. We are calling on the government to implement recommendations from the Francis report that will increase protection for staff who raise concerns about patient care.
Readers' comments (2)
I find it rather offensive to assume that by attending a study day arranged (and subsidised) by a formula company I am compromising my integrity in any way. I would not display or use any article that promoted formula feeding, when I believe wholeheartedly that breast feeding is best. However when offered a place on a valuable learning session that is within my financial abilities to pay for, because it is subsidised, I see no reason not to. I do not doubt the companies have ulterior motives for what they do - that is not my problem. We are all 'professionals' shouldn't we be treated as such and given a little trust?
It is not new practice for companies to sponsor training days, it happens in all professions, I attended training days organised by hardware manufacturers, it didn't mean that I was compromising my professional judgement to use the best hardware for the job. Drug companies visit GP surgeries with their version of a statin, for example, which is why one GP may prescribe Atorvastatin and another Simvastatin, does this mean that either GP has compromised their integrity?
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This teacher training is designed to guide you through an intense yoga program. Work will be hard but it will be fun, challenging, life changing and you will leave Malta ready to teach yoga, which means not only asanas and technique, but being able to truly connect deeply within yourself finding the profound joy and wisdom that comes only from being able to be mindful, aware and present. This is one investment not only for professional growth but for personal development you would not want to miss for the world.
Learn to incorporate the elements of yoga into purposeful sequencing. Practice teaching and receive feedback. Expand your expertise and confidence. Discover your individual potential and unique voice as a teacher. Our creative approach in offering the technical, inspirational and business aspects of yoga will prepare you to shine your extraordinary light - on and off the mat.
A luxury accommodation awaits aspiring yoga teachers for this retreat.
Freemyme concentrated a lot of things in 4 weeks, so you can definitely expect to be entirely dedicated to learning and improving in all aspects of your yoga practice: spiritual, mental, physical. Your schedule, even if quite full, will anyway allow you to take your rest times and refresh your mind, with whatever you feel would be best for you. You can use your free time for studying or having a nice, refreshing swim in the beautiful waters of the Maltese sea, visit some of the stunning natural or cultural amenities of this island, the choices are really infinite. This is how your typical day will look like during the course:
You will have one day and a half (half Saturday and all Sunday) of free time, and even though Freemyme encourages you to find the time to review and study in preparation of your final exam, taking some time just for yourself and to rest is also very important in order to get the maximum out of this course. Freemyme hopes you will find your balance between hard work and well deserved rest, being able to find peace and tranquility even when it seems that you have too many things to accomplish in a short time, this is also part of your training as a yogi.
Sailing trip around the Malta, Comino and Gozo
This training will take place in the beautiful island of Malta.
Healthy vegetarian meals will be served daily with snacks throughout the day.
Experience beach yoga while you enjoy the sunset and sunrise. Go sunbathing, horse riding and SUP yoga.
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For information on travel options, please send your inquiries to Freemyme, they will be happy to assist you on your travel queries.
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text: My Uncle Bill died February 19th after suffering a heart attack while driving, causing his car to crash. I believe my uncle was 81, I believe. We share the same birthday and so does my cousin John. He and my mother were very close, so here are some pictures in honour of him. Graduation with my mother, they went through school together as they were only 17 months apart.


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- Research article
- Open Access
The evolutionary history of holometabolous insects inferred from transcriptome-based phylogeny and comprehensive morphological data
- Ralph S Peters†1,
- Karen Meusemann†2, 3,
- Malte Petersen2,
- Christoph Mayer2,
- Jeanne Wilbrandt2,
- Tanja Ziesmann2,
- Alexander Donath2,
- Karl M Kjer4,
- Ulrike Aspöck5, 6,
- Horst Aspöck7,
- Andre Aberer8,
- Alexandros Stamatakis8, 9,
- Frank Friedrich10,
- Frank Hünefeld11,
- Oliver Niehuis2,
- Rolf G Beutel11 and
- Bernhard Misof2Email author
© Peters et al.; licensee BioMed Central Ltd. 2014
- Received: 8 October 2013
- Accepted: 4 March 2014
- Published: 20 March 2014
Despite considerable progress in systematics, a comprehensive scenario of the evolution of phenotypic characters in the mega-diverse Holometabola based on a solid phylogenetic hypothesis was still missing. We addressed this issue by de novo sequencing transcriptome libraries of representatives of all orders of holometabolan insects (13 species in total) and by using a previously published extensive morphological dataset. We tested competing phylogenetic hypotheses by analyzing various specifically designed sets of amino acid sequence data, using maximum likelihood (ML) based tree inference and Four-cluster Likelihood Mapping (FcLM). By maximum parsimony-based mapping of the morphological data on the phylogenetic relationships we traced evolutionary transformations at the phenotypic level and reconstructed the groundplan of Holometabola and of selected subgroups.
In our analysis of the amino acid sequence data of 1,343 single-copy orthologous genes, Hymenoptera are placed as sister group to all remaining holometabolan orders, i.e., to a clade Aparaglossata, comprising two monophyletic subunits Mecopterida (Amphiesmenoptera + Antliophora) and Neuropteroidea (Neuropterida + Coleopterida). The monophyly of Coleopterida (Coleoptera and Strepsiptera) remains ambiguous in the analyses of the transcriptome data, but appears likely based on the morphological data. Highly supported relationships within Neuropterida and Antliophora are Raphidioptera + (Neuroptera + monophyletic Megaloptera), and Diptera + (Siphonaptera + Mecoptera). ML tree inference and FcLM yielded largely congruent results. However, FcLM, which was applied here for the first time to large phylogenomic supermatrices, displayed additional signal in the datasets that was not identified in the ML trees.
Our phylogenetic results imply that an orthognathous larva belongs to the groundplan of Holometabola, with compound eyes and well-developed thoracic legs, externally feeding on plants or fungi. Ancestral larvae of Aparaglossata were prognathous, equipped with single larval eyes (stemmata), and possibly agile and predacious. Ancestral holometabolan adults likely resembled in their morphology the groundplan of adult neopteran insects. Within Aparaglossata, the adult's flight apparatus and ovipositor underwent strong modifications. We show that the combination of well-resolved phylogenies obtained by phylogenomic analyses and well-documented extensive morphological datasets is an appropriate basis for reconstructing complex morphological transformations and for the inference of evolutionary histories.
- Rate Heterogeneity
- Tree Reconstruction
- Reference Species
- Sister Group Relationship
- Gamma Model
Holometabola (or Endopterygota) are, given their evolutionary age, by far the most species-rich subgroup of insects (Hexapoda) and comprise more than 60% of all described metazoan species [1]. Within the Holometabola, the mega-diverse orders Coleoptera (beetles), Diptera (midges, mosquitos, and flies), Lepidoptera (moths and butterflies), and Hymenoptera (sawflies, bees, wasps, and ants) comprise together almost 800,000 species [2] and therefore more than 95% of the total species diversity of the entire lineage. The smaller orders are Neuroptera (lacewings), Megaloptera (alderflies and dobsonflies), Raphidioptera (snakeflies), Trichoptera (caddisflies), Mecoptera (scorpionflies and relatives), and Siphonaptera (fleas). Complete metamorphosis, which is characterized by the presence of a more or less inactive and non-feeding pupal stage between a feeding larva and a reproducing adult, is the most striking difference between Holometabola and other hexapods. Whereas the monophyly of Holometabola and of all its orders (with few exceptions, see below) has been consistently recovered (e.g., [1, 3]), the interordinal relationships are still insufficiently resolved. This impedes our understanding of the ancestral holometabolan morphology and life history and the modifications that occurred during the subsequent diversification of this highly successful lineage.
A reliable reconstruction of evolutionary transformations within Holometabola requires a well-founded hypothesis of the phylogenetic relationships of the major included groups. The first comprehensive reconstruction of holometabolan phylogenetic relationships was presented by Hennig [4], although a substantial contribution had already been made earlier by Hinton [5]. Alternative concepts to Hennig's proposal were presented by Rasnitsyn and Quicke [6] and Kukalová-Peck and Lawrence [7], with the main difference that Hymenoptera were not placed as sister group of Mecopterida (Diptera, Siphonaptera, and Mecoptera (= Antliophora), and Lepidoptera and Trichoptera (= Amphiesmenoptera)) (as in [4] and, e.g., [1, 8, 9]), but as the first diverging extant holometabolan insect order. A distinctly different view was presented by Wheeler and colleagues [10] (see also [11, 12]): they discussed a sister group relationship between Hymenoptera and Mecopterida (as in Hennig's concept), a sister group relationship between Strepsiptera and Diptera (Halteria), and paraphyletic Mecoptera, with the mecopteran Boreidae as sister group of Siphonaptera. Based on entirely new molecular and morphological datasets, Wiegmann et al. [13], McKenna and Farrell [14], and Beutel et al. [15] (see also [16]) congruently revived the view that Hymenoptera are sistergroup of all remaining Holometabola; Strepsiptera were recovered as closely related to Coleoptera, and Mecoptera were found monophyletic. Recently, these hypotheses gained additional support by a phylogenetic analysis of nucleotide sequence data from whole genome sequencing projects [17]. However, several interordinal relationships within Holometabola remained elusive. Despite remarkable progress, the genomic depth of published molecular sequence data, which potentially offers a plethora of phylogenetically informative characters, is still very low: large-scale transcriptome or genome data have been only available for representatives of less than half of all recognized holometabolan orders, with most studies so far dealing with model species. Consequently, the aim of our study was to present the first reconstruction of holometabolan relationships based on transcriptomic data of representatives of all currently recognized orders.
- 1.
Are Hymenoptera the sister group of Mecopterida (Antliophora and Amphiesmenoptera) or of all other holometabolan insect lineages (e.g., [4] versus [13])?
- 2.
Are Neuropteroidea (Neuropterida, Coleoptera, and Strepsiptera) monophyletic? Neuropteroidea were found monophyletic by Wiegmann et al. [13] but not found by Wheeler et al. [10], Kukalová-Peck and Lawrence [7], and Beutel et al. [15].
- 3.
Are Megaloptera monophyletic? and 4. Are Neuroptera and Megaloptera sister groups? Proposed relationships of the groups of Neuropterida (Megaloptera, Neuroptera, and Raphidioptera) are incongruent, and nearly all possible topological arrangements concerning this problem have been published over the last years (see, e.g., [1, 3, 15, 18–21]).
- 5.
Are Coleopterida (Coleoptera and Strepsiptera) monophyletic? The whole genome-based analyses by Niehuis et al. [17] inferred Strepsiptera as sister group of Coleoptera, but did not include representatives of Neuropterida.
- 6.
Are Mecopterida monophyletic? This group was neither found monophyletic by Kukalová-Peck and Lawrence [7] nor by some of the analyses in Beutel et al. [15], but was monophyletic in Wiegmann et al. [13], though not well supported.
- 7.
What are the phylogenetic relationships within Antliophora? Contradicting phylogenetic relationships among Diptera, Mecoptera, and Siphonaptera have been published, and the monophyly of Mecoptera has been questioned (see above, and [8, 10, 13, 15]).
In order to address the above questions, we generated transcriptomic data of at least one representative of each holometabolan order. For transcriptome sequencing, we selected species mostly characterized by plesiomorphic morphological character conditions and representing taxa that presumably diverged early in the evolutionary history of each group (see [15]). In our molecular phylogenetic analyses, we used specific decisive datasets for each of our phylogenetic questions. Following the arguments put forth by Dell'Ampio et al. [22], a dataset is deemed "decisive" if information of each gene is available from each taxonomic group of interest and thus can contribute to resolving the relationships among these groups. In addition to maximum likelihood (ML) based tree inference, we applied Four-cluster Likelihood Mapping (FcLM) [23] to study potential incongruent signal in our datasets that might not be revealed by a phylogenetic multi-species tree.
We mapped a comprehensive set of morphological data [15] on the transcriptome-based phylogeny, and addressed the following issues regarding the evolutionary history of Holometabola:
Major morphological features of the ancestral larva and the ancestral adult of Holometabola (groundplan) (e.g., larval eyes, legs, prognathous versus orthognathous head; adult prognathous versus orthognathous head, size of pterothoracic segments, eyes)
Ancestral larval and adult life habits of Holometabola (e.g., diet, phytophagy/fungivory versus carnivory)
Major transformations of larval and adult characters within Holometabola (e.g., flight apparatus transformations: shift of segment and wing size, wing coupling mechanisms; modifications of oviposition strategy)
Ancestral mode of ontogenetic development of Holometabola (e.g., pupal characters)
In summary, we aimed to trace evolutionary changes of phenotypic features and to reconstruct groundplans for Holometabola and well-established clades within the Holometabola tree. An evolutionary history based on a solid phylogenetic background represents an important step toward a better understanding of the unparalleled diversification of this exceptional group of organisms.
Results and discussion
The phylogeny of Holometabola
Holometabola species, for which data were newly sequenced
No. of contigs
No. of OGs
Xyela alpigena (Strobl, 1895)
Raphidia ariadne Aspöck & Aspöck, 1964
Nevrorthus apatelios Aspöck, Aspöck & Hölzel, 1977
Sialis lutaria (Linnaeus, 1758)
Corydalinae sp.
Priacma serrata (Leconte, 1861)
Carabus granulatus (Linnaeus, 1758)
Mengenilla moldrzyki Pohl et al., 2012
Micropterix calthella (Linné, 1761)
Philopotamus ludificatus McLachlan, 1878
Tipula maxima Poda, 1761
Archaeopsylla erinacei (Bouché, 1835)
Nannochorista philpotti (Tillyard, 1917)
The seven datasets, designed to address seven phylogenetic questions
Addressed phylogenetic question
Covered subgroups/FcLM clusters (4 clusters per analysis)
No. of species
No. of OGs
Alignment length (aa)
Coverage [%] all species
Coverage [%] addressed groups
Dataset 1 (complete dataset)
Position of Hymenoptera?
1) Hymenoptera
2) outgroup taxa
3) Mecopterida
4) Neuropteroidea
Dataset 2
Are Neuropteroidea monophyletic?
1) Neuropterida
2) Mecopterida
3) Coleopterida
4) Hymenoptera
Dataset 3
Are Megaloptera monophyletic?
1) Raphidioptera
2) Corydalidae
3) Sialidae
4) Neuroptera
Dataset 4
Are Neuroptera and Megaloptera sister groups?
1) Raphidioptera
2) Megaloptera
3) Neuroptera
4) remaining holometabolans
Dataset 5
Are Coleopterida monophyletic?
1) Neuropterida
2) Strepsiptera
3) Coleoptera
4) remaining holometabolans
Dataset 6a
a) Are Mecopterida monophyletic? or
a) 1) Antliophora
Dataset 6b
b) Are Antliophora + Coleopterida monophyletic?
2) Amphiesmenoptera
3) Neuropteroidea
4) remaining holometabolans
b) 1) Antliophora
2) Amphiesmenoptera
3) Coleopterida
4) remaining holometabolans
Dataset 7
Relationships within Antliophora?
1) Diptera
2) Siphonaptera
3) Mecoptera
4) remaining holometabolans
FcLM Results
Possible unambiguous topologies
No. of drawn quartets
Support T1 [%] 1,2 | 3,4
Support T2 [%] 1,3 | 2,4
Support T3 [%] 1,4 | 2,3
Dataset 1 (complete dataset)
T1: Hymenoptera,outgroup taxa | Mecopterida, Neuropteroidea
T2: Hymenoptera, Mecopterida | outgroup taxa, Neuropteroidea
T3: Hymenoptera, Neuropteroidea | outgroup taxa, Mecopterida
Dataset 2
T1: Neuropterida, Mecopterida | Coleopterida, Hymenoptera
T2: Neuropterida, Coleopterida | Mecopterida, Hymenoptera
T3: Neuropterida, Hymenoptera | Mecopterida, Coleopterida
Dataset 3
T1: Raphidioptera, Corydalidae | Sialidae, Neuroptera
T2: Raphidioptera, Sialidae | Corydalidae, Neuroptera
T3: Raphidioptera, Neuroptera | Corydalidae, Sialidae
Dataset 4
T1: Raphidioptera, Megaloptera | Neuroptera, remaining holometabolans
T2: Raphidioptera, Neuroptera | Megaloptera, remaining holometabolans
T3: Raphidioptera, remaining holometabolans | Megaloptera, Neuroptera
Dataset 5
T1: Neuropterida,Strepsiptera | Coleoptera,remaining holometabolans
6 (8)
55 (53)
38 (38)
T2: Neuropterida, Coleoptera | Strepsiptera,remaining holometabolans
T3: Neuropterida,remaining holometabolans | Strepsiptera, Coleoptera
Dataset 6a
T1: Antliophora, Amphiesmenoptera | Coleopterida, remaining holometabolans
T2: Antliophora, Coleopterida | Amphiesmenoptera, remaining holometabolans
T3: Antliophora, remaining holometabolans | Amphiesmenoptera, Coleopterida
Dataset 6b
T1: Antliophora, Amphiesmenoptera | Coleopterida, remaining holometabolans
T2: Antliophora, Coleopterida | Amphiesmenoptera, remaining holometabolans
T3: Antliophora, remaining holometabolans | Amphiesmenoptera, Coleopterida
Dataset 7
T1: Diptera, Siphonaptera | Mecoptera, remaining holometabolans
T2: Diptera, Mecoptera | Siphonaptera, remaining holometabolans
T3: Diptera, remaining holometabolans | Siphonaptera, Mecoptera
The analysis of dataset 1 yielded Hymenoptera as sister group to all remaining holometabolan orders in both ML tree reconstruction and FcLM (Table 3, Figure 1). This relationship had already been recovered in several multiple gene studies (e.g., [13, 14]), and based on whole genome data but a limited taxon sampling [17]. Previously published analyses of morphological data yielded contradictory results, such as for instance Hymenoptera + Mecopterida in Beutel and Gorb [9]versus Hymenoptera + remaining holometabolan orders in Beutel et al. [15]. Potential problems of topological artifacts in these analyses that are caused by convergent reductions in many morphological character systems were discussed in detail by Friedrich and Beutel [24] and Beutel et al. [15]. The placement of Hymenoptera as sister group to all remaining holometabolan orders implies that presumptive synapomorphies of Hymenoptera and Mecopterida (e.g., single claw of larvae, sclerotized sitophore plate of adults; see [1]) are in fact homoplasies.
Our analyses of dataset 2 yielded monophyletic Neuropteroidea (i.e., a clade comprising Neuropterida, Coleoptera, and Strepsiptera) with maximal support in the ML tree reconstruction and strong support in the FcLM (Table 3, Figure 1). Neuropteroidea was not supported as a clade in Beutel et al. [15], but was found monophyletic in many previous studies [1, 8, 9, 13, 14, 25], even though in most cases with weak or without support.
We did not find any signal for paraphyletic Megaloptera as discussed by Beutel et al. [15] and Winterton et al. [26] (dataset 3, Table 3, Figure 1). Within Neuropterida, our ML analyses maximally supported a sister group relationship between Raphidioptera and Neuroptera + Megaloptera, which was also supported by more than 2/3 of all quartets in the FcLM (dataset 4, Table 3, Figure 1). Phylogenetic relationships among neuropterid orders have been discussed controversially with two alternative hypotheses: Raphidioptera + Megaloptera being monophyletic (e.g., [8, 9, 13–15, 27]) or Neuroptera + Megaloptera being monophyletic (e.g., [18, 19, 25, 28, 29]). Our results strongly support the latter hypothesis.
Analysis of dataset 5 yielded ambiguous results with respect to a possible clade comprising Coleoptera and Strepsiptera (Coleopterida) (Table 3, Figure 1). Resolving this longstanding problem is difficult due to the extremely modified morphology (e.g., [30]) and the distinctly derived genomic features [17, 31] of the endoparasitic Strepsiptera ("the Strepsiptera problem", [1]; "insects from outer space", [32]). In most recent contributions, evidence was found for monophyletic Coleopterida (e.g., [13–15, 17]). However, the studies based on molecular data remained ambiguous in their results. Coleopterida were not supported by all datasets analyzed by McKenna and Farrell [14]. The results of Wiegmann et al. [13] were based on a relatively small set of genes and showed only weak support for this clade. Niehuis et al. [17] analyzed whole genome nucleotide sequences of holometabolous insects and found well-supported Coleopterida but the taxon sampling did not include any neuropterid orders. In our study, Coleopterida is supported in the ML tree (with maximal bootstrap support), but not in the FcLM analyses (Table 3, Figure 1). In the ML tree, Strepsiptera are placed within Coleoptera (like in some of the trees of McKenna and Farrell [14]), however, with poorly supported relationships (Additional file 2: Figure S12). We further analyzed whether the incongruence between ML tree reconstruction and FcLM analyses vanished considering partitioned ML and FcLM analyses using different models on different partitions. Partitioned analyses might reduce potential model misspecifications and might yield congruent topologies. However, the incongruence between ML and FcLM analyses did not disappear (Table 3, Additional files 2 and 3). This implies that model misspecifications due to unpartitioned analyses are not the source of incongruence (see also [22] and discussion therein). Apparently, the data and analytical procedures of our study did not yield an unambiguous solution of the question whether or not Coleopterida is a monophyletic group. However, evidence from morphology clearly suggests monophyletic Coleopterida (see also [17]) as the most plausible result.
In order to test the monophyly of Mecopterida, a clade comprising Amphiesmenoptera (Lepidoptera + Trichoptera) and Antliophora (Diptera + Siphonaptera + Mecoptera), we analyzed two versions of dataset 6 to account for two possible hypotheses (dataset 6a, b; Tables 2 and 3). Both analyses recovered monophyletic Mecopterida with strong support (Table 3, Figure 1). Monophyletic Mecopterida, as proposed by Hinton [5] under the name Panorpoidea (or panorpoid complex), was not well supported in Kjer et al. [25] and Wiegmann et al. [13], and only supported in the Bayesian analyses of morphological characters in Beutel et al. [15]. Niehuis et al. [17] found tentative support for this clade based on whole genome data but the incomplete taxon sampling – genomes of Neuropterida, Trichoptera, Siphonaptera, and Mecoptera have not been sequenced yet – diminished the decisiveness of this dataset concerning the question of monophyletic Mecopterida.
Our analyses clearly corroborated the monophyly of Amphiesmenoptera (Trichoptera + Lepidoptera) (Figure 1). However, we did not test this hypothesis with a specifically designed dataset because it has never been seriously disputed [1].
Within Antliophora, which showed maximal bootstrap support in the ML tree, we found a sister group relationship of Mecoptera and Siphonaptera, also with maximal bootstrap support and with maximal support in the FcLM (dataset 7, Table 2, Figure 1). This result corroborates views put forward by Beutel and Gorb [8], McKenna and Farrell [14], and Wiegmann et al. [13], though the clade Mecoptera + Siphonaptera was not well supported in the latter study. A sister group relationship between Diptera and Siphonaptera as retrieved in Beutel et al. ([15], see discussion therein) is highly unlikely based on our analyses.
With this study, we do not contribute to the question whether Mecoptera are a monophyletic group as only one species, Nannochorista philpotti, was part of our taxon sampling. However, morphological data [15] and analyses of nine nuclear genes [14] strongly suggest that Mecoptera indeed form a monophyletic group.
Selection of groundplan characters and apomorphies of Holometabola and of those holometabolan subgroups whose phylogenetic relationships were addressed in this study and whose monophyly was confirmed
•* Larval head orthognathous
•* Larval compound eyes simplified but present
•* Ocelli absent in larvae
•* Larval tentorium X-shaped
•* Retractile larval abdominal prolegs absent
• Larval cerci absent (possible reversal in Strepsiptera [homology uncertain])
•* Adult head orthognathous
• Meso- and metasternum invaginated
• Meso- and metacoxae closely adjacent medially
• Appearance of fully developed compound eyes including external apparatus in the pupal stage (reversal in Strepsiptera)
• External wing buds absent in larval stages (partial reversal in Strepsiptera)
Aparaglossata (Holometabola excluding Hymenoptera)
• Larval head prognathous
• Well-developed larval stemmata
• Larval tentorium H-shaped
• Paraglossae vestigial or absent, without muscles
• Ventral sclerites of segment VIII (gonocoxae and gonapophyses) indistinct (reversals within Neuropterida)
Neuropteroidea § (Neuropterida and Coleopterida)
• Adult head prognathous or slightly inclined (reversal in Neuroptera)
Megaloptera §
• Sensorium on antepenultimate larval antennomere
• Larval salivary duct strongly narrowed, without recognizable lumen
• Setiferous lateral abdominal gills present in larvae
Neuroptera + Megaloptera
• Mesothoracic prealare present (also in Amphiesmenoptera)
• Muscular connection between metafurcal arm and epimeral apophysis
• Aquatic larvae (with reversal)
Coleopterida (Coleoptera and Strepsiptera)
• Antenna with 9 flagellomeres or less
• Pronotum and propleuron partly or completely connected (also in Diptera)
• Metathorax enlarged, hind wings used as flight organs (posteromotorism)
• Membranous area between mesoscutellum and mesopostnotum present
Mecopterida (Antliophora and Amphiesmenoptera)
• Larval dorsal tentorial arm strongly reduced or absent
• Less than 3 larval antennomeres (reversal to 3 in some groups)
• Larval galea and lacinia extensively or completely fused (also missing as separate structures in Neuroptera and Strepsiptera)
• Larval Musculus craniodististipitalis present
Siphonaptera + Mecoptera §
• Muscle connecting profurcal arms (Musculus profurca-spinalis) present
• Acanthae of proventriculus close-set, prominently elongated
Our compilation of molecular sequence datasets and our design of the phylogenetic analysis exhibit some major differences compared to earlier studies on the phylogeny of Holometabola. Specifically, i) we used a massive amount of data generated with Illumina Next Generation Sequencing (Table 1). ii) We ensured decisiveness of our datasets by specifically designing datasets for each of our seven research questions (Table 2) (see [22]). Decisiveness means that all genes included in a dataset are covered by at least one representative of all taxonomic groups that are relevant for the specific phylogenetic relationship under study. Accordingly, each dataset has a coverage of 100% in terms of presence of genes, with respect to the relevant taxonomic groups. By ensuring decisiveness, we alleviate the potentially misleading effects of missing data. Missing data can lead to inference of highly supported but wrong topologies (see [22]). iii) We performed FcLM [23] for each of our seven datasets (Table 3). We re-implemented FcLM in RAxML to cope with these large-scale data matrices and complemented the method by newly-written scripts that map respective results into 2D simplex graphs. Bootstrap support in phylogenetic trees alone is of limited conclusiveness in analyses of very large datasets [22, 34]. FcLM is a method to identify possible support for alternative topologies in a dataset, i.e., a method to display incongruent signal that might not be observable in phylogenetic trees. This study is the first to apply FcLM to large phylogenomic supermatrices. Finally, iv) we checked all datasets for rogue taxa. Rogue taxa are taxa that assume multiple phylogenetic positions in a set of bootstrap trees. They decrease resolution and/or support, for example, when building bootstrap consensus trees. Removing rogues may produce a more informative bootstrap consensus tree [35, 36] (see Additional file 4, Chapter 4). All our datasets were free of rogues.
With a compilation of datasets as presented here (i.e., by extracting the maximum number of genes that can contribute to resolving the phylogenetic relationship in question) we also ensured that inferred topologies were not based on an arbitrary selection of genes with respect to their inherent phylogenetic signal. Dell'Ampio et al. [22] showed that the selection of genes – if not driven by considerations concerning decisiveness of a dataset – can generate topologically different trees that may nonetheless all exhibit high support. Furthermore, Simon et al. [37, 38] showed that genes involved in different biological pathways can support different topologies for a specific phylogenetic relationship. It can therefore be concluded that phylogenetic trees inferred from studying only a set of few to several genes are easily biased and thus might not reflect the correct species tree. While the currently best approach to address this problem is to include the maximum feasible amount of potentially informative data, we will have to further disentangle the contributing factors of topological incongruences in datasets (see also [22]).
Phylogenetic studies exclusively based on morphology (e.g., [15, 24]) also yielded problematic groupings in some cases. The authors addressed and discussed apparent artifacts that were mainly caused by parallel reductions in character complexes (e.g., the flight apparatus). However, the problems turned out as intractable given the data and analytical procedures at hand [15]. With our molecular datasets we were able to provide reliable solutions for most interordinal phylogenetic relationships within Holometabola (Figure 1, and above). For tracing evolutionary changes on the phenotypic level we used the most extensive morphological dataset presently available, including 356 characters of representatives of all holometabolan orders and of carefully selected outgroup taxa [15]. The characters were mapped onto the transcriptome-based phylogeny in a formal approach (see Methods section for details). This allowed us to trace and re-interpret evolutionary changes of numerous characters and to conduct parsimony-based groundplan reconstructions for all clades of the tree (see "The evolution within Holometabola" below).
The evolution within Holometabola
Larvae and development
The ancestral aparaglossatan larva was likely prognathous and equipped with stemmata. Whether these larvae were of the agile campodeid type, like the larvae of many beetles (e.g., Adephaga, Myxophaga [partim], Staphylinoidea), Strepsiptera (first instar), Neuropterida, and some groups of Trichoptera (e.g., Rhyacophilidae), remains unclear. It is conceivable that this larval type is an apomorphic condition characterizing Neuropteroidea, with parallel evolution in Trichoptera. Prognathism is often linked with carnivorous feeding habits (Neuropterida, Adephaga, and some polyphagan subgroups), but can also be related with penetrating narrow crevices or burrowing in substrates, as it is the case in the wood-associated larvae of Archostemata (Coleoptera), but also in early lepidopteran lineages (e.g., [1]). Thus, it is unclear whether or not the ancestral aparaglossatan larvae were predaceous. Larvae of Mecopterida display some simplifications (tentorium and antennal segments), and a distinct trend towards reductions characterizes antliophoran larvae, especially those of Siphonaptera and Diptera. Both have entirely lost their thoracic legs (distinctly shortened in Mecoptera) and are characterized by simplifications of cephalic structures, especially of the muscle system [39]. This reflects the widespread larval life history in Antliophora, with larvae living in the upper soil layer, leaf litter, moist substrates, or different water bodies, feeding mainly on soft substrates or small particles. The important question whether ancestral antliophoran larvae were terrestrial (Lepidoptera, Mecoptera, Siphonaptera, Mecoptera excl. Nannochoristidae, Diptera partim) or aquatic (Trichoptera, Nannochoristidae, Diptera partim) remains ambiguous.
Our phylogenetic results clearly indicate that a typical holometabolous development with larvae completely lacking external wing buds ("endopterygote insects") and also lacking cerci belongs to the groundplan of Holometabola (see also [1, 17]). The conditions characterizing strepsipteran primary larvae (abdominal segment XI and cerci present) and secondary larvae (external wing buds recognizable as external convexities) are apparently the result of reversals, like the early appearance of the prospective compound eyes (see [17]). Largely immobilized pupae with immobilized mandibles (pupa adectica) have almost certainly evolved several times independently. It appears likely that a mobile pupa with movable mandibles as it is characteristic for Raphidioptera is ancestral for Holometabola even though this is not confirmed by a formal character analysis.
Adults and egg deposition
The ancestral holometabolan adult apparently differed only slightly from the neopteran groundplan (Neoptera: all winged insects except Odonata and Ephemeroptera). Cephalic structures, the entire muscle system, the flight apparatus, and abdominal structures appear largely unmodified [15, 23, 39, 40]. The most profound apomorphies in adult holometabolan insects are related to the invagination of the pterothoracic sternites (e.g., closely adjacent meso- and metacoxae) [24]. Our data do not lead to a reliable assessment of ancestral feeding habits of holometabolan adults, but it is apparent that feeding in the adult stage played a minor role compared to feeding in the larval stages. Exceptions to this rule are for instance predaceous beetles (e.g., Dytiscidae and Carabidae) with a very rapid postembryonic development and long-lived adults.
Distinct morphological character transformations characterize the rise of Aparaglossata: the reduction of the labial endite lobes (paraglossae), including muscles, the distinct modification of the orthopteroid ovipositor, and possibly the reduced number of Malpighian tubules (also in Acercaria (true bugs, psocopterans, lice, and relatives)) [15, 41]. Our results do not allow for an unambiguous reconstruction of the ancestral condition of the flight apparatus for Holometabola and Aparaglossata. It appears plausible that approximately equally sized pterothoracic segments (as in Neuropterida, early lepidopteran lineages, and Mecoptera) are plesiomorphic for Aparaglossata, but the reconstruction of the ancestral state of this character in the formal analysis remained ambiguous. As pointed out above, the question whether or not Coleopterida is a monophylic group is not completely settled. However, it appears plausible to assume that posteromotorism evolved only once in a common ancestor of Strepsiptera and Coleoptera, with a suite of related features, such as the size reduction of the mesothorax, a distinct reduction of the mesothoracic muscle system [42], and an increased size of the metathorax. A distinct anteromotorism as it is present in Hymenoptera, Trichoptera, "higher" Lepidoptera, and Diptera is possibly ancestral in Holometabola, but it is conceivable that this condition has evolved (secondarily?, see above) several times independently (e.g., almost equally sized pterothoracic segments in non-glossatan Lepidoptera).
Wing coupling mechanisms have apparently evolved independently in Hymenoptera (hamuli as an autapomorphy of the order, see Additional file 4, Chapter 5), Trichoptera, Lepidoptera, and some families of Neuroptera (different mechanisms occur in these orders).
The primary mode of egg deposition in Holometabola was very likely endophytic, as it can be assumed for the groundplan of Hymenoptera ("Symphyta"). This mode of egg deposition is arguably maintained in the groundplan of Neuropteroidea. Raphidioptera have a modified, elongated ovipositor which they use to deposit eggs under bark or into ground litter. This resembles egg deposition as assumed for the groundplan of Holometabola and Hymenoptera; however, it might also be a derived character. The complete or nearly complete reduction of elements of the primary ovipositor is a characteristic of Mecopterida and obviously related with superficial egg-deposition or oviposition in soft substrates. Our results mostly confirm an evolutionary scenario for the female postabdomen and egg-deposition as outlined in detail in Hünefeld et al. [41].
For the first time in insect systematics a scenario for transformations on the phenotypic level is based on a strictly formal procedure, using a well-documented comprehensive morphological data-set in combination with analyses of phylogenomic data. Our combined approach may lead to a new level of reciprocal enlightenment between researchers with a main focus on morphology and molecular data, respectively, and eventually to new and well-founded insights into the evolution of Hexapoda and other groups of organisms.
Data acquisition
Our study included a total of 88 species: 71 holometabolan species, and 17 species belonging to different hemimetabolous lineages for outgroup comparison. Of these, we generated transcriptomic data de novo for 13 holometabolan species. From all remaining species, we used published transcriptomic data or the transcripts of the official gene set (OGS) if the genome of a species is already sequenced (see below).
The 13 holometabolan species (at least one representative of each order) with newly generated transcriptomic data are listed in Table 1 (for details see Additional file 6, Table S1). Extraction of RNA, cDNA library construction, library normalization, sequencing of 12.5 million paired end reads (~ 2.5 Gigabases raw reads per species) using the Illumina Technique (Hiseq 1000), and sequence processing (vector-clipping, trimming and soft-masking of raw reads, and assembly into contigs) were done by LGC Genomics, Berlin, Germany (see Additional file 4, Chapter 1, and Additional files 6 and 7: Tables S1 and S2 for details). All raw nucleotide sequence reads are deposited at the NCBI Sequence Read Archive (SRA). The corresponding nucleotide assemblies have been deposited at the NCBI's Transcriptome Sequences Database (TSA) (Umbrella project ID PRJNA176423). For further details and accession numbers, please refer to Additional file 4, Chapter 1, and Additional file 7: Table S2.
Nucleotide sequence assemblies of published transcriptome data were obtained from the Deep Metazoan Phylogeny (DMP) database (http://www.deep-phylogeny.org/), NCBI's Transcriptome Sequences Database (TSA) and from various web sources of species whose official gene set was available. We only used species with more than 3,000 available contigs (status: November 2012) (Additional file 8: Table S3).
Orthology assignment
We mapped the transcripts to a set of 1,343 ortholog groups (OGs), i.e., a set of genes that have been identified as single-copy orthologs in 14 reference species (13 insects, 1 crustacean) in OrthoDB 4 (http://cegg.unige.ch/orthodb4/) (see Additional file 9: Table S4 for reference species, and Additional file 10: Table S5 for included orthologs; for details on the design of the ortholog reference set see Additional file 4, Chapter 2). Orthology of transcripts was assigned using HaMStRad, a modified version of HaMStR v.8 [43] (see Additional file 4, Chapter 2 for details on modifications). The modified program files are available at https://github.com/mptrsen/HaMStRad (Status: March 2013). HaMStRad maps transcripts to a set of OGs using hidden Markov models and the best reciprocal hit criterion. We ran HaMStRad with the following settings: (i) the E-value cut-off for the pHMM search was 1e-5, (ii) the reciprocity criterion was considered fulfilled if the candidate OG was found as best hit in at least one of the 14 reference species during the reciprocal best hit search (RBH) (−relaxed option), (iii) in case of multiple transcripts being assigned to a given OG, the best set of non-overlapping transcripts was chosen while non-overlapping transcripts are automatically concatenated (−representative option). Transcripts that were assigned to more than one OG were removed from the dataset using Perl scripts (available upon request) (redundancy check). Furthermore, we removed terminal stop codons and masked internal stop codons with 'X'.
Multiple amino acid sequence alignment, refinement, and masking
We aligned all OGs separately at the amino acid level using MAFFT L-INS-i [44] v6.951. Then we checked for misaligned sequences (henceforth called "outliers") in multiple amino acid sequence alignments (MSAs) of all OGs. This check was done with Perl scripts (available upon request) applying the following procedure: first, the maximal alignment length of a given multiple amino acid sequence alignment was recorded. Then, mean, median, and quartiles of BLOSUM62 distances of the amino acid sequences of all reference species were calculated. After that, the BLOSUM62 distance of each transcript to the sequence of its closest reference taxon (i.e., the reference taxon found as best reciprocal hit) was calculated. Subsequently, it was checked whether this distance was below or above a cut-off value of 2.25 times the distance of the upper quartile to the mean of the BLOSUM62 distances among the reference species. Transcripts with a minimal BLOSUM62 distance to a reference species above the cut-off were classified as outliers, and also sequences with less than 20 overlapping sites to the corresponding sequence of the reference species. All outliers were extracted from the respective MSAs. Each outlier amino acid sequence was separately aligned to only the aligned orthologous sequences of the reference species, using the "--add" option in MAFFT L-INS-i. The refined outlier amino acid sequences were reintegrated into the respective MSA using the alignment of the reference species as a backbone. The outlier check procedure as described above was repeated for each MSA. Sequences that were still classified as outliers were finally removed from the respective MSA (see Additional file 8: Table S3). Gap-only sites were also removed from the MSAs.
Ambiguously aligned sections were identified with a modified version of ALISCORE [45–47]; for modifications, see [47]). We applied the default sliding window size, the maximal number of pairwise comparisons (−r option) and a special EST data scoring (−e option). Identified ambiguously aligned sections were removed ("masked") from the MSAs with ALICUT v.2.0 ([48], http://www.museumkoenig.de/web/ZFMK_Mitarbeiter/KckPatrick/Software/AliCUT/Download/index.de.html) (see Additional file 11: Table S6).
Design of seven specific decisive datasets addressing particular phylogenetic relationships
We call a dataset phylogenetically decisive if all included OGs contain at least one sequence of a representative of each taxonomic group of interest. To compile decisive datasets, we selected four taxonomic groups of interest for each of our seven phylogenetic questions (Table 2 and Table 3). All species relevant for a specific question were assigned to one of the four groups (also called "clusters", see below; see also Additional file 12). The monophyly of each group of species is assumed. All OGs that contained at least one sequence of a representative of each group were extracted with Perl scripts (available upon request) and concatenated into seven supermatrices that constitute the seven decisive datasets. The taxa that are not relevant for answering the respective question were removed (see also Additional file 13: Table S7). The amount and distribution of missing data in each dataset was visualized with mare v. 0.1.2-rc ([49], http://mare.zfmk.de) (Additional file 1: Figures S1-S7).
Phylogenetic analyses
For each of the seven datasets, we performed phylogenetic tree reconstruction with the maximum likelihood (ML) optimality criterion and Four-cluster Likelihood Mapping (FcLM) at the amino acid level. We refrained from calculating the Relative Composition Variability (RCV, see [50]) among the sequences in a dataset to select an optimal data subset (e.g., first, second, and third codon positions of nucleotide sequence dataset, and amino acid sequence dataset) because the statistics is not independent of sequence length, number of sequences, and frequency of symbols. This renders a comparison of RCV between datasets with a different number of symbols and different lengths inappropriate.
For maximum likelihood tree inference, the smaller and larger datasets were treated in slightly different ways because of RAM limitations. For analyzing our small datasets (datasets 3, 4, 5, 7), we conducted one tree-search per dataset to determine the best fitting model, using the −AUTO function implemented in RAxML-Light [51] v. 1.0.9., under the GAMMA model of rate heterogeneity [52] using the median for the discrete GAMMA approximation. Then, ML trees for the small datasets were inferred applying the −f a command line option in RAxML [53], v.7.3.1, HYBRID [54, 55] with the CAT model of rate heterogeneity [53], the best-scoring amino acid substitution matrix, and empirical amino acid frequencies (PROTCAT, bestMODEL, F option). The final tree-searches were conducted under the GAMMA model of rate heterogeneity, again using the median for the discrete approximation. For analyzing our larger datasets (1, 2, and 6), we used RAxML-Light v. 1.0.9 to determine the best-scoring protein substitution model and for subsequent tree inferences. Based on randomized topologies of starting trees, we conducted 50 tree-searches with the CAT model of rate heterogeneity (PROTCATAUTOF) and estimated the best-scoring model using empirical frequencies (+ F) for each tree-search. We subsequently estimated the best final GAMMA likelihood and additional parameters under the GAMMA model using the median for the discrete approximation. For all datasets, the best-scoring amino acid model was the LG model [56].
We assessed statistical support for each node from bootstrap replicates. Bootstrap analyses were performed with the rapid bootstrap algorithm [53], using bootstopping criteria ([57], command line option: −# autoMRE -B 0.01). For analyzing the small datasets, the search for the best tree and the bootstrap analyses were performed in one single step (−f a option). For analyzing the large datasets, bootstrap analyses were performed separately and the bootstrap support was plotted on the respective best tree.
All ML analyses were conducted on Linux clusters at the Cologne High Efficient Operating Platform for Science (CHEOPS), Regionales Rechenzentrum Köln (RRZK) (http://rrzk.uni-koeln.de/cheops.html).
After tree inference, we scrutinized our trees for rogue taxa ([36, 58], see Additional file 4, Chapter 4).
Trees were edited with Treegraph 2.0 [59], and rooted with respective outgroups (see Additional file 2: Figures S8-S15). Supermatrices (i.e., datasets) are deposited at labarchives repository, DOI10.6070/H4G73BMJ, https://mynotebook.labarchives.com/share/ubulin/MC4wfDIzNDAzLzAvVHJlZU5vZGUvMjA0NzAzNzkzMHwwLjA.
Four-cluster Likelihood Mapping (FcLM)
We used FcLM proposed by Strimmer and von Haeseler [23] as an alternative method for analyzing single phylogenetic splits. In each decisive dataset, all included species were binned into four clusters that correspond to the taxonomic groups that are relevant for the respective phylogenetic relationship (see above, Table 2, and Additional file 12). The phylogenetic relationships between these four clusters represent the phylogenetic question of interest. In one case (dataset 6), we defined two different sets of clusters because two phylogenetic hypotheses had to be tested. For each dataset, we calculated the log-likelihood values of all non-redundant quartets drawn from the predefined species groups ("clusters") (see Additional file 4, Chapter 3). We implemented this in RAxML (as of v. 7.3) to be able to handle large-scale datasets. Calculation of log-likelihood values was performed using the GAMMA model of rate heterogeneity and empirical base frequencies with RAxML 7.3.1 (PTHREADS) on the MESCA System of the HPC Linux Cluster CHEOPS, RRZK, University of Cologne. We developed an additional tool written in Perl to map the support values of the RAxML analyses for each quartet onto 2D simplex graphs (available upon request). Results from the analysis of all seven datasets were plotted on the main tree (Figure 1). For the final phylogenetic inference, we compared support inferred from FcLM with ML bootstrap support.
Additional partitioned ML tree and FcLM analyses of dataset 5
We repeated ML tree reconstruction and FcLM based on partitioned analyses for dataset 5 to identify possible sources for incongruence between results of tree reconstruction and FcLM in this specific case. For the partitioned ML tree reconstruction (with 972 partitions), we followed the procedure applied on the large datasets (see above), but using ExaML (version 4.1 [2013-06-19]) instead of RAxML-Light, with the PSR model of rate heterogeneity (equal to CAT in RAxML-Light). We subsequently estimated the optimal parameters and the log-likelihood using the GAMMA model of rate heterogeneity. We performed 50 tree searches and choose the one with the best log-likelihood as best tree (Additional file 2: Figure S15). For partitioned FcLM analysis, we used the respective best models for each partition, selected during the preceding ML tree search (–AUTO option in RAxML), as input (Additional file 14: Table S8). For calculating the log-likelihood support for each drawn quartet, we used again the GAMMA model of rate heterogeneity and empirical base frequencies in RAxML 7.7.2 (PTHREADS). Results were again mapped onto a 2D simplex graph (Additional file 3: Figure S25).
Reconstruction of character evolution and groundplans
Morphological characters of immatures and adults were mapped onto the reconstructed tree using Mesquite ([33], http://mesquiteproject.org). As input, we used the datamatrix of morphological characters published by Beutel et al. [15] and the interordinal topology of the transcriptome-based phylogeny inferred from dataset 1, which represents the complete molecular datamatrix (Figure 1). The taxon sampling at the species level is not congruent between Beutel et al. [15] and the present study. However, all orders are covered in both studies, and only evolutionary transformations between orders or supraordinal taxa are considered here. To reconstruct the character evolution and groundplan features at each node, we used the "Trace Character History" option and performed maximum parsimony reconstructions of groundplans (select "Parsimony Ancestral States") for categorical characters under unordered states assumption.
Availability of supporting data
The datasets supporting the results of this article are available in the labarchives repository, DOI10.6070/H4G73BMJ, https://mynotebook.labarchives.com/share/ubulin/MC4wfDIzNDAzLzAvVHJlZU5vZGUvMjA0NzAzNzkzMHwwLjA.
We thank Dominique Zimmermann, Michael A. Ivie, Kai Schütte, Ewald Altenhofer, Dieter Stüning, Douglas Craig, Alexander Blanke, Martin Kubiak, Hans Malicky, Hans Pohl, and Eva Buscher and her husband for collecting or help in collecting, and identifying species. We acknowledge Torsten Struck, ZFMK, for kindly providing a Perl script for checking multiple assignments of transcripts to different orthologs. We want to thank LGC Genomics Berlin, especially Berthold Fartmann, Victor Achter at Cologne High Efficient Operating Platform for Science (CHEOPS), Regionales Rechenzentrum Köln (RRZK), Julia Schwarzer (EAWAG, Kastanienbaum), and Claudia Etzbauer (ZFMK), as well as two anonymous reviewers for their helpful comments. RGB was funded by the DFG grant BE 1789/8-1. RSP, KM and BM were funded by the DFG grant MI 649/10.
Authors' Affiliations
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Technology In Red Dot Sights
As you may know, red dot sights are an attachment for guns that gives you a clear view of your target with a red dot. We know what they are for but do we really know how they work? What goes into making one? When were they first invented? All these are questions that I can answer for you in a few short paragraphs. For more information and reviews on red dot sights read here.
What Is Inside A Red Dot Sight?
There really is nothing to these sights actually. The inside is a hollow tube and includes a concave lens with a thin coating that's metallic that reflects red light but other colors are transmitted freely, pretty neat, huh? The dot itself is just a reflection off of a diode that emits light inside of the tube. The first one of this king was created in 1975 and was known as the "Aimpoint Electronic".
Reflex Sight Technology
This is the category that most red dot sights fall into. This is pretty much just a mirror inside of a scope. The aiming reticle is projected forward by a point that comes from behind the objective lens and then is reflected back off of that lens assembly towards your eye. The coating on these lenses are tuned carefully to reflect only light wavelengths that are emitted by the reticle illumination system. Some are fiber-optic and some have a tritium beta lamp used for reticle illumination.
Holographic Sight Technology
These have almost the same technology as the reflex sights. The only difference is that with a holographic sight, the reticle used for sighting is superimposed on your view of the target by a laser transmitted hologram. Basically a picture of the reticle is taken and forms a window in which you can view your target. These sights are also made so that light will not be altered when going through the window. And yes, there is in fact a laser inside of the sight. The beam is bounced back to the shooters eye from a hologram window that creates the reticle image. This image will then project 50 yards in front of you.
Laser Sight Technology
Essentially this is a large laser pointer used for shooting things and can tolerate recoil. The laser needs to travel from the gun, to the target, and then back to the gun and still be visible to the shooter. The visibility can be affected by daylight, reflectivity of the target, fog, rain, smoke, and other environmental factors. The laser needs more power than the other sights for this exact reason. Unlike the other sights, since this is made from lasers, you will be able to see the red laser beam shining on your target and through the fog or snow.
The technology of these sights are really nothing too confusing. They are essentially mirrors and reflections. The fact that they have been around so long means a lot. Back in the day, there wasn't much fancy technology and people made these sights with just the bare essentials. It is surprising that there aren't just sights coming straight out of the gun without it being an attachment, or is there?


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See also: IRC log
<ht> Norm, shall I start the call w/o you?
<ht> http://www.w3.org/XML/XProc/2007/10/18-agenda
<ht> scribenick: ht
HST: Agenda approved
HST: Minutes approved
... Next meeting 25 October
... NW apologies for 25 Oct and 1 Nov, HST to be in the chair _pro tem_
... Charter has been extended for 1 year
PG: F2F agenda?
NW: one day on comment processing, one day on the future, I guess
PG: I find a detailed agenda helps folk to be prepared
NW: Well, item 1 is "Read and be familiar with the details of the issues list"
<scribe> scribenick: Norm
Extended 1 year.
A-86-01: Alex to review XSLT streaming requirements before the face-to-face.
<scribe> Continued
A-86-03: Henry to reply to the commenter (non-string parameters; issue 30)
<scribe> Completed
A-86-04: Henry to craft the prose to cover the defaulted output case
<scribe> Continued
A-87-01: Norm to take a stab at reconsidering the default inputs feature applying it only to ports that are not primary
<scribe> Continued.
A-87-02: Alex to propose some text about imports and circularity
<scribe> Continued
A-87-03: Norm to attempt to incorporate Richard's draft text about step type scope
<scribe> Continued.
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#029
<scribe> Continued pending Henry's action
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#006
<scribe> Continued pending Norm's action
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#018
<scribe> Continued pending Norm's action
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#024
Norm outlines the issue and summarizes Jeni's observed options
Henry: I think we leave it impl. defined.
Some discussion of how defaulted attributes fit in: probably covered by the spirit of the best efforts clause.
Proposal: Leave it implementation-defined.
<scribe> ACTION: Henry will respond to the commenter on comment 24: passing PSVIs between steps. [recorded in http://www.w3.org/2007/10/18-xproc-minutes.html#action01]
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#007
Norm reviews the points in Mike's message.
Point 1: accepted.
Point 2: changed validate-* to validate-with-*; accepted.
<MoZ> http://lists.w3.org/Archives/Public/public-xml-processing-model-comments/2007Sep/0034.html
Point 3-6 are editorial.
Point 7: rejected, also made a separate issue
Point 8: discussion
Henry: I think we're pretty clear
that we're not answering this question.
... We're trying not to be too precise.
Henry: No, that doesn't work. An
XML document isn't an Infoset, the infoset is just a set of
... But maybe it's ok.
... Sure let's try this.
Norm: What about A.3?
Henry: Yes, that looks fine too.
Norm: Anyone unhappy with the editor's resolution of Mike's point 8?
None heard.
Point 9:
Norm: Anyone unhappy with the editor's resolution of Mike's point 9?
None heard.
That leaves some editorial clarifications, but I think we've covered the technical issues
-> http://www.w3.org/XML/XProc/2007/09/lastcall/comments#012
Let's leave the "editorial" and "clarification" points to the editor unless they turn out not to be
Henry: I'm prepared to skip to
... I think he's right.
Norm: I don't think these apply to the things *inside* the elements in the step
Henry: So you meant "children" not "contains"? If so, you'll have to repeat it endlessly.
Norm: Uhm...
Henry: Can't we just say that these rules don't apply inside p:inline?
Norm: Perhaps
Some discussion about what constitutes a step.
Norm: I think children would work.
Henry: What about giving the
fifth and sixth bullets a parallel construction
... If any element in the XProc namespace other than p:inline, or any step, has text node children...
Norm: Sure, that would work for
... I think that resolves point 7.
Point 8:
Henry: I think replacing "within
its container" by "immediately contained by that steps
... Or if we've formally defined subpipline as the immediately contained steps, then "the last step in document order in the subpipeline"
Norm: I'm happy to attempt to clarify that.
Point 11:
Some of this is editorail.
Norm: I think we have clarified that select only selects elements or documents.
Henry: I've always said that
select needs the same namespace fixup we already
... We've already re-worded 4.2 so there isn't a double "each" anymore.
Norm: But that leaves "wrapped"
and an explicit pointer to 2.6.1
... The select question points to 5.2 and aon through to p:input where it's covered.
Henry: No, I don't think we want to make the reference to 2.6.1 explicit.
<scribe> ACTION: Norm to see if the commenter agree's we've addressed his concerns. [recorded in http://www.w3.org/2007/10/18-xproc-minutes.html#action02]
Point 12:
Henry: I think we can replace "aggregated" with "concatentated" here.
Norm: I'm willing to do that and see if it helps.
Henry: The prose is still a bit
... I think we should unpack it and make it more explicit.
Norm: Ok.
Point 13:
Norm: We probably need to make the distinction between match and select more clear.
Henry: Not that you need to make the point about match only matching element or document nodes here. There's no free ride.
Norm: The WG's intent is clear
but the prose needs to be clearer.
... Should we make it an error to select a document node?
Henry: Yes.
Point 14:
Norm: I think it can be a static error.
Henry: Is this just a case where a processor could detect it statically if it wanted to?
Norm: It could, but why not make it static?
Henry: A select option to a step may be computed, so it has to be a dynamic error.
Norm: But for select on our compound steps and for test on when, then they should be static errors.
Henry: Let's try that and see if there's any pushback.
Norm: Any objections?


text:
Call of Duty isn't the most difficult first-person shooter to play by any means, but it can be challenging. That is arguably even more true now with the recent release of Advanced Warfare. This latest title in the series has added several new elements to the game revolving around the use of the Exosuit. Mastering the use of weapons and the new movement capabilities can be a bit daunting. But, we're here to help.
Advanced Warfare Multiplayer Tips
We've complied some of the most helpful tips & tricks that will help you improve your performance and boost your kill-to-death ratio while playing Call of Duty: Advanced Warfare. Use these tips to better your class selections, increase your score in each match, and dominate the competition in online multiplayer.
Loadout Creation is Key
The first, and possibly most important, tip is to simply fine-tune your class setups. Knowing which weapons you perform best with and what perks help you to get more kills and avoid deaths is a key component in becoming a better player.
Not what you're after? Don't forget to read over our Infinite Warfare tips and tricks to learn how to play Infinity Ward's latest title better!
If you feel like you consistently do better playing a run 'n gun role, equip something like an assault rifle, SMG, or shotgun as your primary weapon. If you choose to take a secondary, make it something that is quick to swap to and can pop off a few bullets relatively fast (the Atlas 45 or PDW are excellent choices).
If you think you perform better supporting your teammates with fire from across the map you may want to use something like a sniper rifle or LMG as your primary gun and use a launcher to take down scorestreaks or a pistol to kill enemies that get too close for comfort.
Experiment with different weapons, use a variety of attachments to find out which ones best suit your play style, try out different perks, and sculpt the perfect loadout that'll help you get more kills every match. Pay attention to your overall K/D with each weapon to determine which ones you work best with.
Movement Advantages & Disadvantages
In Advanced Warfare your Exosuit serves as a tool that can help you maneuver around maps faster, conceal yourself from enemies, and more. Learn to use it well. Familiarize yourself with all of the Exo movements and Exo abilities by testing each one thoroughly to learn how each one works.
With your Exosuit you can dodge enemy fire with boost-powered strafing, severely damage unsuspecting opponents with aerial boost slams, and jump to strategic spots. There are advantages and disadvantages to all of this. A key thing to remember is to not be in the air too often. A flying player is an easy target for soldiers firmly planted on the ground. Don't make yourself an easy kill.
Want to really dominate the battlefield? Use the tips, tricks, and strategies found in our Call of Duty Advanced Warfare guide to learn how to beat the competition every match.
Exo abilities can help you do survive firefights longer, sustain flight, conceal your character from enemy vision, and even shield you from oncoming bullets. Play around with the different abilities to figure out which one helps you the most. We personally like Overclock, which allows you to temporarily run faster than normal.
NOTE: Need help in BO3 instead? We've got you covered! Check out our Black Ops 3 tips to learn how to improve your play in that game now!
Cover Up
Don't be afraid to take shelter behind a wall, a crate, or any other object that can block enemy fire. Despite the fact that many players may insult you for "camping", it's a legitimate strategy that can save you from certain death. After all, if this were reality we wouldn't be running out into the open amidst enemy fire, that's suicide.
This is an especially useful technique when you find yourself running low on ammo and need to reload your weapon. Hide behind a solid object, reload, then, once you're prepared for confrontation, move into position to take your shot. There's even a special Exo ability, the Exo shield, that can protect you for a short time.
Eyes on the Bullet
One of the most important things to do that will greatly improve your overall playing ability and your K/D is to focus on accuracy. Make every bullet count. Most guns don't have unlimited ammo so you'll need to be conservative with it. Only fire at targets that are within your weapon's effective range and take good aim to ensure every bullet hits its mark.
Most firefights in Advanced Warfare come down to one of two things:
- Which Player Sees & Reacts to the Other the Fastest
- Which Player Has the Better Aim
Accuracy can mean the difference between a certain death and a satisfying kill. Oftentimes, if your accuracy is spot-on you can still best players that see you before you see them. Not everyone is a deadeye. But if you become one yourself, you'll find yourself getting more kills and dying less.
Practice your aim by playing private matches with bots on medium to high difficulty settings. And don't forget to take weapons into the virtual firing range to learn the recoil plots of each gun (how the gun moves when fired) so you can figure out exactly what to do to ensure every bullet hits its target in multiplayer matches.
Hey! We've got an in-depth guide for Call of Duty Black Ops 3, too! Check it out.