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Ted McCloskey and the Hi-Fi's
Ted McCloskey has been telling compelling stories for a long time. He is a singer/songwriter, a multi-instrumentalist, a producer and film composer. His award-winning music has been featured on AMC's The Walking Dead and Mad Men, NBC's, The Voice, PBS, MTV, Discovery, and the History Channel, among others. In March 2013 Honda Motors picked up a track from his 2012 album, The Last of the Pin-Up Girls for use in a national television ad campaign. When you see Ted and his band, The Hi-Fi's, you know you're seeing something special. It's pure energy—a snarling kind of rock 'n' roll that's hard to find these days.
Ted's 9th release, Last Flower Standing, is a complex and haunting exposé on those toxic flowers and beautiful weeds that exist all around us. It's about unshakable shadows that can't just be buried and forgotten. It's about discarded souls that have been uprooted yet survive. Familiarly riff-driven and doused in exposed nerves and slide guitars—Last Flower Standing is both heartbreaking and beautiful; a survival story and a past exhumed.
Location: Allen Street Stage
Address: State College, PA (map)
Website: http://arts-festival.com/event/ted-mccloskey-and-the-hi-fis/
Occurs: July 12th 2019 at 5:30 pm

redpajama_set_name: RedPajamaCommonCrawl


Event marketing is one of the fastest growing fields in marketing today. The competition for mindshare is fierce and going back to the basics of one–on-one marketing can build awareness and establish brand loyalty. Events can take many forms including a tech day, a lunch and learn, roadshow, or holding a cocktail party or breakfast event concurrent with a tradeshow.
Everywhere you look in today's culture a promotional event is happening, although you probably don't easily recognize many of the event marketing programs when you see them. Perhaps it's a national webinar or an all day seminar, or simply the coupon an attractive model hands you from a logo wrapped vehicle at a local market.
You may not be ready to hire a customized tractor/trailer rig to hit the road, but you may be ready to co-market with complementary companies to hold a lunch and learn at a local facility. Whether its national, regional or local outreach, events bring a personal side and intimacy to your marketing efforts.
Antarra and our event experts will work with you to plan, promote and implement an event to fit your budget and achieve your marcom goals.


What is the European supplement to the degree (SET), how and when it is requested?
The expedition of the European supplement to the title (SET) is one of the measures set out with the introduction of the European area of higher education.
The SET can apply for students who completed official university studies (Degrees, diplomas, Bachelor's degree, etc.). Can be requested along with the issuance of the title in the Secretariat of the Centre (school/college) where studies have been completed.
The SET is the document that accompanies each of the degrees of official status and validity throughout the national territory, with information unified, personalized for each University, entitled on the education, the results obtained, acquired professional skills and the level of their qualification in the national system of higher education.
The SET aims to increase the transparency of the various higher education qualifications taught in European countries and facilitate their academic and professional recognition.
(c) information on the level of the qualification.
(d) information on the content and the results obtained.
(e) information on the role of the qualification.
(g) certification of the supplement.
(h) information on the national system of higher education.
The paper support of the European supplements that are issued shall be of identical size for all standardized in format UNE A - 4 (security role model).
SET document issued by the University of Alicante will include the coat of arms of Spain, the logo of the European Union (EU) and Europass, and the coat of arms of the University of Alicante.
The University of Alicante shall issue a document SET, expressed in the two official languages of the autonomous community of Valencia (Spanish and Valencian) and English language. It will be, therefore, a trilingual document in all respects.
Criteria for issuing the European supplement to the title at the University of Alicante.


most people by this level have heard of Makybe Diva; she has had the maximum Melbourne Cup wins by using a horse, with three wins. As a end result, she is the very best stakes-earner in Australasian history, worth $14 million at the time of her retirement in 2005. another Cup winner is so infamous and steeped in Australian records, that his name was even featured in the Australian citizenship test – the enduring Phar lap. Phar Lap ran the race 3 instances; however, notwithstanding heavy favouritism, he simplest controlled one win, which got here in 1930. In 1929, he placed third. As for his very last run within the 1931 Melbourne Cup, he best managed 8th, however, he turned into heavily handicapped during this run, with an additional 10 stone 10 pounds (sixty eight kg). He died the following yr, beneath suspicious situations, thought to be due to both an contamination, or a large dose of arsenic.
As for human winners, "Melbourne Cup King" Bart Cummings, with his record of 12 Melbourne Cup wins is the mainteacher. Astonishingly, Cummings skilled both the primary and second area winners within the Melbourne Cup for five of his 12 winning years. Lee Freedman with 5 wins is his closest rival. Etienne de Mestre (1832-1916) additionally had fivewins, in his time.
betting on the race is a national past time and is sort of considered a ceremony of passage in Australian way of life. it's also one of the less difficult form publications, because the horses walking have had a number of past runs to decidethem with the aid of, including the world famous Cox Plate, wherein triumphing horses can cross without delay into the big one. The Cox Plate consequences also supply the triumphing horse a high-quality position within the Melbourne Cup odds, normally coming in at favored, or at the least an area.
This entry was posted in Uncategorized on October 2, 2018 by stre.


Cybersecurity knowledge is lacking among consumers, according to survey results recently published by the Pew Research Center.
Of the 1,055 adults asked to respond to 13 cybersecurity-related questions, the average person answered about five questions correctly. Approximately 20 percent answered more than eight correctly and only 1 percent received a perfect score.
The results showed that 75 percent of respondents could identify the strongest password from a list of options, and 73 percent knew that public Wi-Fi networks were unsecured and that transmitting sensitive information over public networks could increase the risk of fraud. However, only 54 percent could accurately identify phishing schemes.
Consumer knowledge was lowest with regard to multi-factor authentication processes with only 10 percent of respondents able to correctly identify such methods. Consumer knowledge regarding encrypted websites and emails, private browsing, botnets and virtual private networks was also limited, the survey revealed.
"Internet users' understanding of the remaining cybersecurity issues measured in the survey is lower – in some cases dramatically so," Pew reported. "For instance, 39 [percent] of internet users are aware that internet service providers (ISPs) are able to see the sites their customers are visiting while utilizing the 'private browsing' mode on their internet browsers. Private browsing mode only prevents the browser itself, and in some cases the user's computer or smartphone, from saving this information – it is still visible to the ISP. And one-third (33 percent) are aware that the letter 's' in a URL beginning with 'https://' indicates that the traffic on that site is encrypted.
The survey indicates that age and education levels seem to have the greatest correlation with the results. Younger respondents with a bachelor's degree or higher tended to perform the best.
This article has been viewed 1336 times.


ACC positions its members at the forefront of airport policy, practices, standards and education/training through its committees. ACC committees provide invaluable opportunities for members to interface directly with federal and state government representatives and elected officials, to network with one another and to develop relationships. ACC committee members are kept abreast of the latest technical and policy guidelines and regulations.
All employees of ACC member firms may serve on ACC committees by downloading the committee sign-up form or contacting ACC Headquarters. Sign-up for an ACC committee HERE. Committees meet during ACC-sponsored events, via conference calls, e-mail, and some form LinkedIn groups for further collaboration and discussion.


BAN THE BLOCKS! | JUST WRITE IT!
← You're Not Done Yet!
It's all about the writing. Excuses don't cut it if you are serious about your writing goals. Whining won't get the words onto the paper. Taking your dog for a walk doesn't take all day, and still leaves time for writing. Have an appointment midday? Instead of seeing that as breaking up your day and therefore an excuse for not writing, look at it as a gift – an opportunity to write early, refresh your creative juices, and go back to it with renewed writing spirit.
"I don't have time to meet my writing goals." MAKE TIME! HOW COMMITTED ARE YOU?
"I keep getting interrupted." SET GROUND RULES. LOCK YOURSELF IN A ROOM. GO SOMEPLACE (LIKE A LIBRARY) WHERE THE INTERRUPTIONS ARE ELIMINATED. JUST DO IT!
"I need to be inspired to write." FIND WHAT INSPIRES YOU. MUSIC? ESSENTIAL OILS? CONNECTING PEN TO PAPER INSTEAD OF SITTING AT A COMPUTER SCREEN? IF YOU STILL AREN'T INSPIRED, JUST WRITE. ANYTHING! IT'S LIKE EXERCISING YOUR BODY – ONCE YOU GET PAST THAT CRITICAL FIRST 10-15 MINUTES, IT FLOWS.
"I don't like what I've written." SO SET IT ASIDE AND JUST KEEP WRITING. YOU CAN FIX OR DISCARD IT LATER! THE IMPORTANT THING IS TO MAINTAIN THE HABIT OF WRITING EVERY DAY. EVERY DAY. EVEN IF IT'S ONLY FOR A HALF HOUR. EVEN IF YOU PRODUCE GARBAGE. AGAIN WITH THE WORKOUT ANALOGY, YOU HAVE TO WORK THOSE MUSCLES TO SEE ANY RESULTS. YOU HAVE TO WORK THAT WRITING MUSCLE TO PRODUCE A STORY, A NOVEL, ANY WRITING.
Are you really, truly, not-just-an-excuse busy? EVEN THE BUSIEST PEOPLE CAN CARVE OUT TIME TO WRITE. THE BLOCKS OF TIME MAY BE SHORT. YOU NEED TO TRAIN YOURSELF TO GET RIGHT INTO IT AT THE APPOINTED TIME AND WRITE, FOR A HALF HOUR, FOR AN HOUR, WHATEVER YOU CAN MANAGE.
2:30 am – rise and shine, throw on exercise clothes, drive 1 ½ hours to the gym while listening to books on tape, run 7 miles on the treadmill and spend a half hour on weight work, shower, drive to work.
As you can imagine, by the end of dinner I was done in for the day. Good thing, because I had to get to bed early so I could get up and repeat this 5 days per week. Weekends allowed a little more time for writing, but even writers need a life – so weekends included socializing, doing some kind of workout or hike with my dog and husband, and catching up on TV shows taped throughout the week.
My point is clear. Making time for writing is in your hands.
This entry was posted in Blogs, Books, Goals, Personal Discipline, Reading, Writing and tagged Books, goals, Personal Discipline, Time, Time management, Work, writing, Writing Blocks. Bookmark the permalink.
3 Responses to BAN THE BLOCKS!
I tend to be an early riser even when I don't have to be – just not quite that early any more! Getting up so early was hard the first week or two. But I found that once I established the habit, it wasn't so bad.Good luck with your 3 or 3:30 goal!
Just do it because you love it. It always makes me feel happy and so I notice it when I'm not committing. Life will always get busy but there's always a way. Great post! Thanks.


Rochester, MN (KROC AM News) - The National Weather Service has finally kicked out its prediction about the impact an approaching major winter storm will have on the region.
A blizzard warning will take effect Thursday evening west of Rochester where heavy snow is expected to begin falling. Cities under the warning include Albert Lea and Owatonna. By the time the storm winds down Friday night, the blizzard warning zone could have snowfall totals of 12-15 inches. Strong winds are also expected, which means whiteout conditions will develop.
A winter storm warning will be in effect for the rest of southeast Minnesota, up to the Twin Cities and into western Wisconsin where snowfall totals could be in the 8 to 14 inch range.
Rain and freezing rain could also affect travel Thursday evening, along with some possible thunder.
By the way: the record temperatures that had been talked about for the Rochester area earlier this week never materialized. Rochester's official high Tuesday was 56 degrees. The record for the date is 60. Wednesday's official high was also 56, missing the record for the date by three degrees.


A United Nations committee has added a number of North Korean individuals and firms to a sanctions blacklist.
Five individuals, five firms and two weapons-related items are subject to the new sanctions regime.
A UN resolution in June toughened sanctions against North Korea after it conducted nuclear and missile tests.
The last time the UN imposed sanctions on Pyongyang, it responded by carrying out a nuclear test, says the BBC's Laura Trevelyan in New York.
According to the UN Security Council sanctions committee, nations are now banned from doing business with five firms involved in North Korea's nuclear programme, and five individuals are to have their financial assets frozen and face a travel ban.
The UN resolution in June called for inspections of ships to or from North Korea believed to be carrying goods connected to weapons of mass destruction.
It also broadened the arms embargo and further cut the North's access to the international financial system, but did not authorise the use of force.
Ties between North Korea and the outside world have grown extremely tense since it walked away from six-nation talks aimed at ending its nuclear programme.
It subsequently said it would "weaponise" its plutonium stocks and start enriching uranium, prompting fears that it is working to produce nuclear warheads small enough to put on missiles - though analysts say it could take a long time to do so.


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Colocation Data Centers See Surge in Demand
Colocation data centers have been an option for more than two decades. But the surge in the demand for data processing, the high cost of in-house data centers, and the appearance of cloud providers has dramatically changed the market. Understanding the new dynamics of the market is important for both colocation providers and facility managers in enterprises that may be considering a move to a colo.
Today, colocation data centers remain an attractive choice for many enterprises. But finding the right colocation data center with the right services and price remains a complex task.
Businesses in sectors such as banking and finance have realized that running their own data centers is "not a good use of capital, because they aren't data-center companies," says John Hatem, executive vice president of design, construction, and operations at CyrusOne, which owns more than 40 data centers worldwide. The joke in his industry, he says, used to be that "our biggest competitor was the CIO" at companies where data storage was not the core business.
That's good news for colos, which are growing rapidly. Hatem says his company is spending $1 billion this year on land and construction, "and there's no end in sight."
Another major colocation provider, RagingWire, currently manages 1.5 million square feet of space and is developing 3 million more square feet. "We're building as fast as we can," says Joe Goldsmith, chief revenue officer and senior vice president for RagingWire.
The post Colocation Data Centers See Surge in Demand appeared first on Website Hosting Review.
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Rights to light: are injunctions a thing of the past?
Much has been written about the Supreme Court's decision in the case of Coventry v Lawrence and the impact this will have on the award of injunctions in rights to light cases. There have been a handful of decisions in different areas which have shed some light on the courts' approach to injunctions post-Coventry v Lawrence but until recently nothing on rights to light. On 18 February 2015 Mr Recorder Edward Cole handed down his Judgment in Scott v Aimiuwu  which, although only in the County Court, is the decision the development industry has been waiting for.
The position prior to Coventry v Lawrence was that an injunction would be awarded for a breach of a neighbouring owner's right to light in all but the most exceptional cases. The Supreme Court Justices in that case undertook a review of the case law and concluded that the past tendency towards injunctions had resulted from an overly rigid approach. The indication was that injunctions would be less likely in the future but the Supreme Court failed to provide clear guidance as to how the courts should approach the question. It is for this reason that the first decision in a rights to light case was so eagerly awaited.
The case involved a dispute between the owners of two substantial residential properties in Potters Bar, Hertfordshire. As is often the case in neighbour disputes the facts were complex and neither party emerged from the case unscathed. There had been discussions between the Scotts and the Aimiuwus over a period of years about the possible extension of their properties. The lengthy Judgment records a history of unsuccessful planning applications and appeals none of which are directly relevant to the injunction question. The important paragraphs of the Judgment are relatively brief and record the Judge's approach to the all-important question of whether to award an injunction or damages following his finding that the extension of Mr & Mrs Aimiuwus' house had infringed Mr & Mrs Scotts' right to light.
The conduct of the parties was not without question on both sides.
Ultimately the Judge must carry out a balancing exercise taking into account all of the factors to reach a decision. In this case his main focus was on the fact that the award of an injunction would leave Mr & Mrs Aimiuwu having to pull down part of their extension for what he saw as a relatively minor breach of Mr & Mrs Scotts' rights.
Having found that there was an actual interference with Mr & Mrs Scotts' right to light, the Judge awarded damages rather than an injunction. This comes as something of a surprise considering the pre-Coventry v Lawrence cases. However, when you look more closely at the facts, it is easier to understand how the Judge reached this decision. He went on to assess Mr & Mrs Scotts' damages at £30,000 and in so doing made some interesting comments about the approach to damages. The Judge considered that a share of profits approach might lead to an award of £65,000 which he felt was too high whereas the reduction in the value of Mr & Mrs Scotts' house resulted in a figure which was too low. He opted for something in the middle ground which was the result of an application of the negotiating damages principle drawn from the Wrotham Park  case.
Although the headline is attention grabbing, and no injunction was granted in this case, when you look at the detail it is simply not possible to support a proposition that injunctions are a thing of the past. They remain a very real threat but the decision does provide further encouragement for developers as well as a useful insight into how similar cases might be dealt with in the post-Coventry v Lawrence world.
This article was written by James Souter.


enter signs Free PicturesAlthough currently it is not as common a few years ago websites were an enter button and presentation of the web, a form of introduction.
Keep at your computer all gifs enter signs of signs for websites compressed into a ZIP file.
Do you like this gif of enter signs?
Free enter signs Animated gifs to download. If you are looking for enter signs animated images to a website, a job, a PowerPoint presentation, you're writing an entry for your blog about enter signs, here are all the images you want. Illustrations, charts, photographs and animated videos of the subject. Use your imagination to use these animated gifs in the funniest way enter signs to express your emotions in a WhatsApp conversation, email, or in the social network you use, that way your texts will be more enriched. The word enter is used as an invitation to the user to enter a site visit. These moving words show us this expression in different languages. They are animated text with the word go you can easily insert in the design of your website or blog. You must place it on the first page that appears, and then you link to content. It's a good way that your users are attracted and want to discover what lies behind that button.


text:
Q: json exception no value for f1 I have made an android application and trying to select the data from database  into table-row and textview.
But when I am trying to handle the json response ,getting error, here's the code

 public void selectfromDb(){

        try{
            HttpClient httpclient = new DefaultHttpClient();
            HttpPost httppost = new HttpPost("http://xxxxxxxx/selectall.php");
            HttpResponse response = httpclient.execute(httppost);
            HttpEntity entity = response.getEntity();
            is = entity.getContent();

            Log.e("log_tag", "connection success ");
            //   Toast.makeText(getApplicationContext(), "pass", Toast.LENGTH_SHORT).show();
        }
        catch(Exception e)
        {
            Log.e("log_tag", "Error in http connection "+e.toString());
            Toast.makeText(getActivity(), "Connection fail", Toast.LENGTH_SHORT).show();

        }
        //convert response to string
        try
        {
            BufferedReader reader = new BufferedReader(new InputStreamReader(is,"iso-8859-1"),8);
            StringBuilder sb = new StringBuilder();
            String line = null;
            while ((line = reader.readLine()) != null)
            {
                sb.append(line + "\n");
                //  Toast.makeText(getApplicationContext(), "Input Reading pass", Toast.LENGTH_SHORT).show();
            }
            is.close();

            result=sb.toString();
        }
        catch(Exception e)
        {
            Log.e("log_tag", "Error converting result "+e.toString());
            Toast.makeText(getActivity(), " Input reading fail", Toast.LENGTH_SHORT).show();

        }

        //parse json data
        try
        {

            JSONArray jArray = new JSONArray(result);

            String re=jArray.getString(jArray.length()-1);

            TableLayout tv=(TableLayout)getView().findViewById(R.id.table);
            tv.removeAllViewsInLayout();

            int flag=1;

            for(int i=-1;i<jArray.length()-1;i++)

            {
               TableRow tr=new TableRow(getActivity());

                tr.setLayoutParams(new LayoutParams(
                        LayoutParams.FILL_PARENT,
                        LayoutParams.WRAP_CONTENT));

                if(flag==1)
                {

                    TextView b6=new TextView(getActivity());
                    b6.setText("ID");
                    b6.setTextColor(Color.BLUE);
                    b6.setTextSize(15);
                    tr.addView(b6);

                    TextView b19=new TextView(getActivity());
                    b19.setPadding(10, 0, 0, 0);
                    b19.setTextSize(15);
                    b19.setText("Matier");
                    b19.setTextColor(Color.BLUE);
                    tr.addView(b19);

                    tv.addView(tr);

                    final View vline = new View(getActivity());
                    vline.setLayoutParams(new TableRow.LayoutParams(TableRow.LayoutParams.FILL_PARENT, 2));
                    vline.setBackgroundColor(Color.BLUE);

                    tv.addView(vline);
                    flag=0;

                }else
                {
                    JSONObject json_data = jArray.getJSONObject(i);

                    Log.i("log_tag","id: "+json_data.getInt("f1")+
                                    ", matier: "+json_data.getString("f2")
                    );

                    TextView b=new TextView(getActivity());
              //      String stime=String.valueOf(json_data.getInt("f1"));
                    b.setText(json_data.optString("f1"));
                    b.setTextColor(Color.RED);
                    b.setTextSize(15);
                    tr.addView(b);

                    TextView b1=new TextView(getActivity());
                    b1.setPadding(10, 0, 0, 0);
                    b1.setTextSize(15);
                    String stime1=json_data.optString("f2");
                    b1.setText(stime1);
                    b1.setTextColor(Color.WHITE);
                    tr.addView(b1);

                    tv.addView(tr);

                    final View vline1 = new View(getActivity());
                    vline1.setLayoutParams(new TableRow.LayoutParams(TableRow.LayoutParams.FILL_PARENT, 1));
                    vline1.setBackgroundColor(Color.WHITE);
                    tv.addView(vline1);

                }

            }

        }
        catch(JSONException e)
        {
            Log.e("log_tag", "Error parsing data "+e.toString());
            Toast.makeText(getActivity(), e.toString(), Toast.LENGTH_SHORT).show();
        }

    }

  }

and this is code php

    <?php

         $con = mysql_connect("","","");
         if (!$con)
           {
             die('Could not connect: ' . mysql_error());
           }

           mysql_select_db("databas", $con);

           $i=mysql_query("select * from test",$con);

           $num_rows = mysql_num_rows($i);
           $check='';
           while($row = mysql_fetch_array($i))
             {
                  $r[]=$row;
                  $check=$row['f1'];
             }

         if($check==NULL)
           {
                  $r[$num_rows]="Record is not available";
                  echo json_encode($r);
           }
            else
             {
                $r[$num_rows]="success";
                 echo json_encode($r);
             }

 mysql_close($con);
?>

Error:
JSON exception no value for 'f1'

A: ths is the out put of the php file

[{"0":"66","id":"66","1":"","matier":""},{"0":"65","id":"65","1":"","matier":""},{"0":"64","id":"64","1":"","matier":""},{"0":"63","id":"63","1":"","matier":""},{"0":"62","id":"62","1":"","matier":""},{"0":"61","id":"61","1":"","matier":""},{"0":"60","id":"60","1":"","matier":""},{"0":"59","id":"59","1":"","matier":""},{"0":"58","id":"58","1":"","matier":""},{"0":"57","id":"57","1":"","matier":""},{"0":"56","id":"56","1":"","matier":""},{"0":"55","id":"55","1":"","matier":""},{"0":"54","id":"54","1":"test","matier":"test"},{"0":"53","id":"53","1":"jfhhf","matier":"jfhhf"},{"0":"52","id":"52","1":"jfhhf","matier":"jfhhf"},{"0":"42","id":"42","1":"ykk","matier":"ykk"},{"0":"51","id":"51","1":"jfhhf","matier":"jfhhf"},{"0":"50","id":"50","1":"jfhhf","matier":"jfhhf"},{"0":"49","id":"49","1":"opp","matier":"opp"},{"0":"48","id":"48","1":"opp","matier":"opp"},{"0":"47","id":"47","1":"opp","matier":"opp"},{"0":"46","id":"46","1":"opp","matier":"opp"},{"0":"45","id":"45","1":"opp","matier":"opp"},{"0":"44","id":"44","1":"ykfvjf","matier":"ykfvjf"},{"0":"43","id":"43","1":"ykfvjf","matier":"ykfvjf"},"Record is not available"]

redpajama_set_name: RedPajamaStackExchange


text:
Grimoire of Zero [Review]
by Brittany Vincent September 15, 2017
Grimoire of Zero is the result of what might happen if you combined anthropomorphic animals with The Familiar of Zero and Berserk. With that said, it's not exactly my cup of tea, nor the type of series I'd normally seek out (even though Berserk is one of my favorite series of all time). However, I'm glad I went ahead and took the plunge, because I was rewarded handsomely for my time.
Based on a light novel series of the same name, Grimoire of Zero follows a half-human mercenary that just goes by "Mercenary" from the first episode onward as he meets up with a young witch named Zero who's on the lookout for a magical tome she penned that can educate its readers on how to summon powerful magic.
Though both beastfallen (half-human and half-animal creatures) and witches are looked down upon in the kingdom of Wenias, the two team up with Zero promising Mercenary a way to become fully human if he'll protect her on her search. It's a simple setup, but one that leaves plenty of room for character building, which runs rampant throughout the show. Right after the first episode ended, I was hungry for more, even ravenous. I wanted to see what was coming next like I was engrossed in a classic old storybook.
The dynamic between Zero and Mercenary is cemented quickly in the first episode, as is the way of the world. It's full of cruelty and hatred, even between Zero and Mercenary's people and the rest of the kingdom. It's refreshing to see the two characters come together, and despite not trusting each other at first, grow together and depend on each other like old friends. There's a kindness in Zero that could just as easily have been transplanted with boring, one-dimensional tsundere character traits, but instead you can see a caring individual within her.
Mercenary has an outwardly austere side, but he's also an interesting and multifaceted character to watch. The pain of losing his family and being hunted by humans is evident in his every move. But despite all of that, he's strong, careful, and even shy. He does his best to protect Zero in every situation, and this always makes for engaging interactions between the two.
The series itself plays out a little like shows you've probably already seen before, but it doesn't really matter when you begin to like the characters so much. There's also an intriguing distinction between two types of magic: sorcery and magic, which most people in the kingdom aren't so well versed in. I enjoyed seeing what a witch could do with powers others couldn't.
It's easy to write off Grimoire of Zero as just another magic user and sidekick story, especially because of its art, which resembles some rather childlike fan art and moe tsundere girl, but if what I've written here intrigues you, you should give it a try. It's got quite a few surprising moments that you won't see coming, and the relationship between its two leads is satisfying in itself.
studio/company: Anime Strike (Amazon)
available: Now (Streaming)
This story appears in the October 2017 issue of Otaku USA Magazine. Click here to get a print copy.
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text:
Home News & Analysis Revcap reaches first close on Europe opportunity fund
Revcap reaches first close on Europe opportunity fund
The London-based firm started by former Lehman Brothers bankers has provided a rare bright spot in the European opportunistic fundraising market by reaching £60 million for its first fund.
Revcap Advisors has provided a rare bright spot in the European fundraising scene for opportunistic funds – and first-time funds in particular – by reaching a first close for its higher-return vehicle, which is targeting £150 million (€186 million; $233 million) in commitments.
The London-based firm – started in 2004 by former Lehman Brothers bankers Andrew Pettit, William Killick, François Guiraud and Stefan Lanefelt, plus former Catalyst Capital partner Stephen Pettit – has raised £60 million for Kitty Hawk Capital Partners II, its first commingled fund. Investors in the fund are thought to include existing separate account clients, as well as institutional investors that have not invested with Revcap previously. The firm is aiming for a final close later this year.
The feat will be noted by a clutch of firms that either have launched European opportunistic funds or are considering doing so this year. Indeed, the first close comes at a time when experts say it has become extremely challenging for first-time funds to achieve success, given that many investors appear opposed to backing firms without a long track record.
Revcap, which is being advised by SJ Berwin, Bedell Cristin, Jersey Trust and Godwin Procter, targets real estate co-investment opportunities in smaller market transactions, often below the typical minimum investment size of the larger opportunity funds. It is known to have completed two deals recently, one being in the Paris office sector and the other being a senior loan position in Scotland. It also has been taking strategic positions in the London residential market. However, the firm is perhaps best known for a £60 million recapitalisation of Insite Poster Properties, the largest private owner of billboard properties in the UK, last March.
LOOK AHEAD 2022: Real estate secondaries are 'at an inflection point'
Brookfield finishes 2021 with the year's largest real estate debt fundraising


text:
Q: How to undelete dynamic partition? I've tried to remove mirror from software raid 1 but windows removed the whole array. How to restore it? It was standard windows mirroring on dynamic discs.

A: Have you tried Testdisk ? This software is designed to, among other things, locate missing partitions.


The bruchid beetles Acanthoscelides obtectus, A. obvelatus, A. argillaceus and Zabrotes subfasciatus develop on various bean species in Mexico. Whereas A. obtectus and A. obvelatus develop on Phaseolus vulgaris in the Mexican Altiplano, A. argillaceus feeds on P. lunatus in the Pacific coast. The generalist Z. subfasciatus feeds on both bean species, and is sympatric with A. obtectus and A. obvelatus in the Mexican Altiplano, and with A. argillaceus in the Pacific coast. In order to assess the phylogenetic position of these four species, we amplified and sequenced one nuclear (28S rRNA) and two mitochondrial (cytb, COI) genes.
Whereas species were well segregated in topologies obtained for COI and 28S rRNA, an unexpected pattern was obtained in the cytb phylogenetic tree. In this tree, individuals from A. obtectus and A. obvelatus, as well as Z. subfasciatus individuals from the Mexican Altiplano, clustered together in a unique little variable monophyletic unit. In contrast, A. argillaceus and Z. subfasciatus individuals from the Pacific coast clustered in two separated clades, identically to the pattern obtained for COI and 28S rRNA. An additional analysis showed that Z. subfasciatus individuals from the Mexican Altiplano also possessed the cytb gene present in individuals of this species from the Pacific coast. Zabrotes subfasciatus individuals from the Mexican Altiplano thus demonstrated two cytb genes, an "original" one and an "infectious" one, showing 25% of nucleotide divergence. The "infectious" cytb gene seems to be under purifying selection and to be expressed in mitochondria.
The high degree of incongruence of the cytb tree with patterns for other genes is discussed in the light of three hypotheses: experimental contamination, hybridization, and pseudogenisation. However, none of these seem able to explain the patterns observed. A fourth hypothesis, involving recent horizontal gene transfer (HGT) between A. obtectus and A. obvelatus, and from one of these species to Z. subfasciatus in the Mexican Altiplano, seems the only plausible explanation. The HGT between our study species seems to have occurred recently, and only in a zone where the three beetles are sympatric and share common host plants. This suggests that transfer could have been effected by some external vector such as a eukaryotic or viral parasite, which might still host the transferred fragment.
This article was reviewed by Eric Bapteste, Adam Eyre-Walker and Alexey Kondrashov.
Reviewed by Eric Bapteste, Adam Eyre-Walker and Alexey Kondrashov. For the full reviews, please go to the Reviewers' comments section.
The traditional view of evolution supports that DNA is transferred vertically from parent to offspring. Hybridization and genetic transfer between different species is usually strongly limited. However, exceptions to this rule, i.e. the horizontal transmission of genetic material between distantly related organisms (HGT), are increasingly recognized as an important process of evolution in prokaryotes [1–4]. In eukaryotes, however, reported cases of between-species genetic exchanges not involving hybridization have been essentially limited to noncoding or parasitic transposons or virus sequences. Exceptions to this are a few instances of horizontal transfer of selected genes between prokaryotes and eukaryotes (e. g., [5–7]) and two reports suggesting lateral transfer of mitochondrial genes between genetically distant land plant groups [8, 9]. No instance of HGT in animals has been reported up to now.
In a recent paper, Martin  discusses the relevance of the HGT hypothesis in eukaryotes, focusing on land plants, and stresses the need for corroborating studies in other eukaryote groups. However, he addresses the necessity of careful interpretation of HGT data, and points to the requirement of first discussing such data in the light of standard theories of vertical inheritance of genes.
Our study investigates the phylogenetic relationships between four Neotropical bruchid beetles (Coleoptera, Bruchidae), namely Acanthoscelides obtectus Say, A. obvelatus Bridwell, A. argillaceus Sharp, and Zabrotes subfasciatus Boheman. These beetles develop on seeds of several bean species (genus Phaseolus): Acanthoscelides obtectus and A. obvelatus feed on the common bean (P. vulgaris L. group), A. argillaceus on the Lima bean (P. lunatus L. group), and Z. subfasciatus on both. As A. obtectus,A. obvelatus and Z. subfasciatus can develop on beans of the same species, they are often sympatric in wild or cultivated common beanpopulations. We sampled individuals from these three species in the southern part of the Mexican Altiplano, one such zone of sympatric occurrence of A. obtectus,A. obvelatus and Z. subfasciatus (Figure 1). Although the geographic range of A. argillaceus overlaps with those of its two congeners, it is rarely found in the same habitat, because of its distinct host plant. Acanthoscelides argillaceus and the generalist Z. subfasciatus, however, commonly co-occur in wild Lima bean populations . This is the case in the Pacific coast of Mexico (Figure 1), where individuals from these two species were sampled.
Geographic origin of Acanthoscelides obtectus ( ), A. obvelatus (▽), A. argillaceus ( ) and Zabrotes subfasciatus ( ) individuals sampled. (a) Pacific coast populations; only one cytb gene is present in Z. subfasciatus populations (SJB, ELA). (b) Altiplano populations; two cytb genes are present in the same individual in some Z. subfasciatus (OCM, YAU, TLA); (c) population of origin of the Z. subfasciatus laboratory colony.
The genera Acanthoscelides and Zabrotes are morphologically very different , and are thought to have diverged during the Palaeocene (65 to 54.8 mya) or the Eocene (54.8 to 33.7 mya) , whereas the three Acanthoscelides species studied here belong to the same morphologically defined group within the genus and seem to have diverged during the Miocene (23.8 to 5.3 mya) [14, 15]. To confirm the phylogenetic position of these four species in the context of host plant adaptation (see ), we performed amplification and sequencing of two mitochondrial genes and one nuclear gene.
For each of the three studied genes, different models of evolution were selected by likelihood ratio tests: for COI, the Tamura-Nei model with a proportion of invariable sites and a gamma distribution was selected; for cytb, the Hasegawa-Kishino-Yano model with a gamma distribution was selected; for 28s rRNA, the Kimura 2-parameters model was selected. Topologies of phylogenetic trees were congruent for 28S rRNA and COI, and showed each species as a monophyletic group, the three Acanthoscelides species forming a distinct clade from Z. subfasciatus (see Figure 2a) (28S rRNA [accession numbers AY881176–AY881195, DQ152235–DQ152238]; COI [accession numbers AY881196–AY881214, AY881232, DQ152239–DQ152242]). COI genetic distances ranged from 0.273 to 0.324 substitutions per site between Acanthoscelides spp. and Zabrotes subfasciatus, and from 0.172 to 0.212 substitutions per site among Acanthoscelides species. According to a recent calibration of a beetle mitochondrial molecular clock , such genetic distances correspond to divergence times of 35–40 My between Acanthoscelides spp. and Zabrotes subfasciatus, and 20–25 My among Acanthoscelides species. These divergence times were confirmed by estimates obtained from mitochondrial 12S rRNA , and are fairly similar to the fossil record .
Phylogenetic patterns. a. Phylogenetic relationships between different populations of Acanthoscelides obtectus ( ), A. obvelatus (▽), A. argillaceus ( ) and Zabrotes subfasciatus ( ) for the mitochondrial COI gene. An identical topology is supported by 28S rRNA (not shown); b. Phylogenetic relationships between different populations of Acanthoscelides obtectus ( ), A. obvelatus (▽), A. argillaceus ( ) and Zabrotes subfasciatus ( ) for the cytb gene. Underlined individuals correspond to Z. subfasciatus populations from the Mexican Altiplano – some carry two cytb haplotypes. Stars (*) indicate Z. subfasciatus haplotypes identical to haplotypes from Acanthoscelides. "Z. subfasciatus CLNY" corresponds to an individual of the laboratory colony affected by HGT.
The analysis of cytb sequences, in contrast, revealed an unexpected pattern (see Figure 2b) (accession numbers AY422474–AY422479, AY422485–AY422488, AY881215–AY881232). First, individuals of A. obtectus and A. obvelatus were not separated in two distinct clades, but grouped into a single, little variable monophyletic unit, as would be expected for individuals belonging to the same species. A core clade containing 46 of the 50 sequenced individuals from the two species included 14 closely related haplotypes (maximal distance: 0.0084), some of them shared by the two species, others private to A. obtectus or A. obvelatus. In contrast, A. argillaceus individuals clustered in a separate clade, whose mean distance to the A. obtectus – A. obvelatus core clade was 0.143. Secondly, cytb data revealed a surprising discrepancy among the individuals of Z. subfasciatus. Whereas an expected 0.25 nucleotide divergence from Acanthoscelides spp. was found for the Z. subfasciatus individuals from the Pacific coast of Mexico, three Z. subfasciatus individuals from the Altiplano (coming from three different populations) showed a cytb sequence identical to the most common A. obtectus – A. obvelatus core clade haplotype.
PCR with specific primers and subsequent sequencing revealed that the three Altiplano Z. subfasciatus individuals surprisingly carried both cytb genes (Figure 2b), their own plus the one demonstrating an Acanthoscelides origin. The specific primers were then applied to other Z. subfasciatus individuals from the Altiplano and the Pacific coast. In the Altiplano, 20 individuals out of 70 were revealed to carry both cytb genes – while the remaining 50 only carried their own specific cytb – whereas in the Pacific coast, all the 40 sampled individuals were revealed to carry only their own cytb haplotype. The specific primers were then also applied to 25 individuals of a laboratory colony of Z. subfasciatus that had been maintained for 10 months in the Laboratory of Evolutionary Entomology at the University of Neuchâtel (Switzerland). The colony was founded using individuals from the Mexican Altiplano, i.e., potentially affected by the HGT. These individuals had not been in contact with any Acanthoscelides for about 5–10 generations. Three individuals out of the 25 were revealed to carry both cytb genes, while 22 showed only the Z. subfasciatus haplotype. The number of individuals sampled in each site and the number of individuals fitting the core clade haplotype are summarized in Table 2.
An analysis of cytb sequence variation within the A. obtectus – A. obvelatus core clade revealed no frameshift, and a low (0.106) ratio of non-synonymous to synonymous changes, significantly lower than 1 (likelihood ratio test: 2(ln [Likelihood(M1)]-ln [Likelihood(M2)]) = 47.5; χ2 [1 df]: P < 10-4), indicating that this gene is under selection. Furthermore, these sequences included no mitochondrial stop codon, but typically five nuclear stop codons, strongly suggesting that this gene is replicated by a mitochondrion, and neither by a nuclear nor a bacterial machinery.
The cytb pattern shown by A. obtectus, A. obvelatus and three Mexican Altiplano Z. subfasciatus individuals is highly incongruent with traditional taxonomy, and with the COI and 28S rRNA pattern. This is even more astonishing, considering that cytb and COI are supposedly genetically linked in the non-recombining mitochondrial genome.
Four hypotheses can be addressed to explain this unexpected result: (i) experimental contamination, (ii) hybridization, (iii) pseudogenisation and (iv) horizontal gene transfer.
All experiments were simultaneously conducted on many (>200) bruchid samples, including museum specimens with low amounts of DNA, by the same researcher in the same laboratory. Only the cytb PCR for Mexican Altiplano individuals yielded an additional, unexpected band. In Acanthoscelides, no individual from the Pacific Coast A. argillaceus carried the additional gene, although they were analyzed simultaneously with the A. obtectus and A. obvelatus samples. A putative laboratory contaminant would not only have affected the samples coming from the Mexican Altiplano but also individuals from other sites and particularly museum specimens, which contain very small amounts of DNA, given the fact that samples were randomized on the 96-well plates, both during DNA extraction and PCR reactions. Another strong argument against the contamination hypothesis is that several individuals of the core clade from both A. obtectus and A. obvelatus demonstrated private cytb haplotypes. These haplotypes cannot be the consequence of a contamination, since they were found only once (we sequenced all the amplified cytb fragments). In the case of a contamination, a single haplotype would be expected to occur in a large number of random samples, whereas our data show many related but distinct – and sometimes private – haplotypes found in a non-random subset of the samples. Sampling contamination (e.g., the presence of Acanthoscelides tissues in Z. subfasciatus samples) can also be excluded, since three Z. subfasciatus individuals sampled from the experimental colony showed the additional cytb PCR band. The experiments on colony individuals (DNA extraction, PCR) were conducted several weeks after the analysis of the wild samples, making any cross-experiment contamination impossible.
Hybridization between Acanthoscelides species and Zabrotes subfasciatus is very unlikely. First, the split between the two genera dates back to around 40 mya (molecular clock data) to 60 mya (fossil record) ago. Moreover, they demonstrate a very high level of nucleotide divergence (25%). Usually, the degree of genetic divergence in documented cases of interspecific hybridization in insects never exceeds 5%-10% of nucleotide divergence for protein-encoding mitochondrial genes [18–20], which is much lower than the 25% divergence found between Acanthoscelides and Zabrotes. Secondly, hybridization appears functionally impossible, given the highly differentiated morphology, especially in the male genitalia. The lateral lobes of the male genitalia – which play the decisive role of pushing aside parts of the female genitalia to allow penetration by the median lobe, and which are known to be part of a specific lock/key system – are spectacularly different between the two genera. Borowiec  points out that the structure of the aedeagus is very different in Zabrotes and Acanthoscelides and highly specialized in both. The lateral lobes are particularly distinct, short and almost entirely fused in Zabrotes, but elongate and divided by a deep cleft almost to the base in Acanthoscelides. In the bruchid family, in which many cryptic species remain to be described, male genitalia are often the only character to distinguish between otherwise morphologically identical (but never hybridizing) sibling species. This clearly explains why any attempt to produce hybrid offspring between Acanthoscelides and Zabrotes failed (N. Alvarez, unpublished observations). Moreover, even in the two morphologically similar Acanthoscelides obtectus and A. obvelatus, no evidence of interspecific hybridization has been detected in natura, despite the genetic analysis (by means of diagnostic microsatellite loci) of hundreds of individuals from the two species, among which dozens originated from sympatric populations .
An additional surprise in our data is that the putatively transferred fragment appears to evolve under purifying selection. The rate of non-synonymous substitutions is significantly lower than expected under a model involving a non-selected gene, and there is no insertion or deletion in any cytb sequenced fragment. Moreover, the fragment seems to be expressed in a mitochondrion. Given that the gene is under purifying selection, it could hardly have been translated using the nuclear genetic code, since in that case, five codons would have been translated in stop-codons. These properties argue for an active gene, rather than a pseudogene. A pseudogene – which by definition can hardly ever be under purifying selection – would not show a bias to synonymous over non-synonymous mutations, and its sequence would most likely show nucleotide insertion or deletion. Furthermore, in the exceptional context of a selected pseudogene , the gene must not contain any stop codon when translated with the nuclear genetic code. The combined evidence suggests that genes from the core clade are (i) under purifying selection and are therefore not pseudogenes, and (ii) are translated in a mitochondrion.
Our data clearly argue against any standard explanation for the genetic pattern we observed. A horizontal transfer of the cytb gene between A. obtectus and A. obvelatus, and from one of these species to Z. subfasciatus, seems the most plausible hypothesis, even though the probability of such a mitochondrial genetic exchange between distinct eukaryote species is very low (but is invoked in a recent study in plants [8, 9]). We assume that species polyphyly in the cytb core clade results from a relatively ancient HGT process having started at the time of the core clade ancestor or earlier. Alternatively, one may interpret this clade as resulting from a series of numerous, very recent HGT events. Under the latter hypothesis, the functional constraints we outlined in the last paragraph would have pre-dated HGT's, and would not guarantee that the transferred gene is active and expressed in a mitochondrion.
The data available do not allow formal rejection of this latter hypothesis, under which the transferred pieces of DNA could even be nuclear pseudogenes. This hypothesis requires, however, a sudden, very recent increase of the HGT rate from zero to a very high value. This could be conceivable in the context of a rapid, infectious process (if, for instance, one specific strain of a parasite acquires the ability to switch species and invade the new niche), but in this case one would expect to see a single transferred haplotype (or a small number of them), whereas the intricate phylogeny of the core clade implies several events of HGT involving several distinct haplotypes.
Under either of these two hypotheses (one ancient HGT or several recent ones), the transfer from one species to another could possibly have been carried out by an external vector. Candidate vectors include viruses, prokaryotes such as Wolbachia or other endosymbiotic bacteria (a case of horizontal transfer of genes from Wolbachia to the nuclear genome of its host was recently reported in bruchids ) or even eukaryotes (e.g., intra- or extra-cellular parasites, endoparasitoïds). The location of the transferred cytb in the recipient species remains to be investigated. A parsimonious hypothesis is that the transferred cytb is still hosted by its vector. A eukaryotic parasite might have "domesticated" a bruchid mitochondrion (or at least some bruchid mitochondrial gene), carrying it on when switching to a new species. Such an hypothesis could explain the presence of two cytb genes in Z. subfasciatus individuals from the Altiplano, suggesting that one gene could be carried by a parasite, and the other could belong to the mitochondria of Z. subfasciatus itself. This could also be the case in A. obtectus or A. obvelatus, even though we could not yet demonstrate the existence of a second cytb gene in individuals belonging to the core clade.
If we consider that the vector may belong to a eukaryotic group (i) capable of mitochondrial recombination and (ii) showing a genetic code that uses the same mitochondrial stop-codons as those used by the mitochondria of invertebrates – these two conditions are for example filled by most unicellular eukaryotes – then the mitochondria carried by the parasite may have "included" the "bruchid"-cytb in its genome after recombination. Another interesting point is that the transferred gene replicates in Z. subfasciatus, since it survived ~5–10 generations in the laboratory.
As this HGT event has been detected only between individuals from the Altiplano where the three species co-occur and share the same host plant, it gives clues to the ecological context which may favor such a gene exchange. Indeed, the ecologically distinct A. argillaceus does not demonstrate a HGT pattern for cytb, despite its phylogenetic proximity to A. obtectus and A. obvelatus. This suggests that the probability of the occurrence of lateral gene transfer is partly controlled by the environment. The occurrence of HGT between bruchid species (i) presenting a high degree of phylogenetic divergence but (ii) sharing some dimensions of their ecological niche (e.g., their host plant) could indicate that ecology, as well as phylogeny, might play a role in the distribution of genetic variation across taxa.
Recent horizontal gene transfer (HGT) between A. obtectus and A. obvelatus, and from one of these species to Z. subfasciatus in the Mexican Altiplano, seems the most plausible hypothesis to explain the pattern we observed in our data for cytb. The transfer could have been effected by some external vector such as a eukaryotic parasite, which might still host the transferred fragment.
Individuals of A. obtectus, A. obvelatus, A. argillaceus, and Z. subfasciatus were sampled in the southern Mexican Altiplano. Coordinates of sampled sites are given in Table 1. We amplified and sequenced bi-directionally two mitochondrial genes – cytochrome oxidase I (COI) and cytochrome b (cytb) – and one nuclear gene – 28S ribosomal rRNA (28S rRNA) – from 6 to 25 individuals in each species, using universal primers (28S rRNA: 28ee and 28 mm; COI: C1-J-2183 and TL2-N-3014; cytb: CB-J-10933 and CB-N-11367 ). Total genomic DNA was extracted using DNeasy™ 96-well kit (QIAGEN). PCR amplifications were performed in a final volume of 10 μL, which contained 1 μL of extracted DNA, 1 μL of 25 mM MgCl2, 0.1 μL of 10 mM dNTPs, 1 μL of PCR buffer (Eurogentec), 1 unit of Taq DNA polymerase (Eurogentec Red Goldstar™), 0.5 μL of forward primer, and 0.5 μL of reverse primer. PCRs were performed separately for each primer pair on a PTC-200™ thermocycler using the following cycling conditions: initial denaturation at 92°C (1 min 30 s); 30 cycles of 92°C (30 s), annealing at 55°C (45 s), 72°C (1 min 30 s); final elongation at 72°C (10 min). Sequencing reaction was carried out using Applied Biosystems BygDye™ protocol. Products of the sequencing reactions were then analyzed on an ABI Prism 310 sequencer. Chromatograms were manually corrected using Chromas 2.23 (Technelysium Pty. Ltd., Helensvale, Australia) and further aligned using ClustalW 1.83 . The best-fit substitution model was determined using Modeltest 3.06  through hierarchical likelihood ratio tests. Phylogenetic trees were reconstructed by maximum likelihood using Treefinder . Distances between groups were determined by distance methods using Mega 2.1  with standard parameters (Kimura 2-parameters; gamma shape parameter = 0.5). Primers specific for cytb consensus haplotypes of A. obtectus and A. obvelatus, and for cytb haplotypes of Pacific coast Z. subfasciatus, respectively, were designed. Sequences of specific primers to the most common haplotype shared by A. obtectus and A. obvelatus are: TTGATAACGCAACCTTAACC (forward primer) and GATTAGCAGGAATGAAGTTG (reverse primer). Sequences of specific primers to Pacific-coast Z. subfasciatus cytb are: GAGATAATGCAACATTAACA (forward primer) and GGTTTGCGGGCGTAAAATTA (reverse primer). To test whether the sequenced cytb genes were under purifying selection (i.e., they were not the result of a nuclear mitochondrial pseudogene), we compared rates of synonymous and non-synonymous substitutions. Two models of codon evolution  were tested using PAML 3.14b . In model M1, the ratio of non-synonymous to synonymous evolutionary rate (ω) was set to 1, as expected for a pseudogene. In model M2, the ratio was free to vary. A likelihood-ratio test was performed to compare the two models.
This paper presents potentially interesting data and a possibly compelling result – lateral gene transfers of genes between animal species-, that if confirmed, would certainly deserve to be reported to a broad audience of readers. Yet, a great deal of caution is required before accepting the conclusion proposed here. In my view the present study is still too weak to support the conclusion.
Certainly, the authors reject several alternative hypotheses to explain the bizarre distribution of cytb genes in bruchids. For instance, the presence of two divergent copies of the cytb gene in Z. subfasciatus, one of them being very conserved in sequence and shared by a set of traditionally distantly related taxa, deserves some explanation. In that respect, the authors are right to attract our attention of this result. They exclude the possibility of contamination, hybridization events and the possibility that one of the copies is in fact a pseudogene. Importantly enough though, I feel that more work is needed to test their final proposition and to conclude in favour of the lateral transfer of the gene between animals, based on the sole evidence presented here. Some more in depth analyses should be performed in order to test their claim and to propose a more precise interpretation of the data. Typically, questions about the mechanism of acquisition of this second copy of cytb should be at the heart of such future studies. Nevertheless, some quick adjustments could be incorporated into a moderately revised version of this manuscript.
Number of individuals collected in each site for each species and number of individuals fitting the "core clade" haplotype in each population. In Zabrotes subfasciatus populations, all individuals also fitted their own original cytb haplotype.
It might be of some help in deciding if it is premature or legitimate to quote this study as a case of lateral gene transfer in animals if the authors could answer some of the questions below.
1. Is it really impossible that an endoparasite is a source of a contamination, that the relatively conserved cytb gene shared by the three species is not of bruchid origin and that the observations may be partly artefactual?
If these species hosted a population of closely related endoparasite, could not two cytb sequences be amplified from these organisms? In this case, we would get the sequences of the population of endoparasites (presenting thus some distinct but related haplotypes) and the one for the bruchid? What does a blast of this additional copy teach us: who are the closest completely sequenced relatives having homologues to this additional sequence? Including these data in a broader phylogenetic tree via a broader cytb alignment would certainly be a very important additional result to present to the readers. Are these bruchid sequences still monophyletic when a larger taxonomic sample is investigated and, if not, where do these gene copies branch in the tree? How much evidence support that these two sequences are carried in the genome of the bruchid (i.e. in the mitochondrial DNA of the species as the result of an actual transfer)?
The hypothesis of an endoparasite carrying the unusual cytb sequences deserves some attention. These sequences, however, are undoubtedly of bruchid origin, since a blast search of Genbank using the additional copy shows that the closest sequences outside Acanthoscelides or Zabrotes are carried by four bruchids, two Bruchus ( B. signaticornis [AY390728.1], B. tristiculus [AY390729.1]) and two Bruchidius ( B. saudicus [AY625444.1], B. rubicundus [AY625443.1]), all of them found exclusively in the Old World, and therefore unikely to have been in contact with Acanthoscelides and/or Zabrotes since 90 Mya. Moreover, the first 50 blast hits are all distributed amongst the bruchid family, both in New World genera (i.e. Acanthoscelides and Zabrotes) and in Old World genera (i.e. Bruchus, Bruchidius, Tuberculobruchus, Conicobruchus, Palaeoacanthoscelides and Callosobruchus). More distant sequences are found in species within the superfamily Chrysomeloidea, which comprises bruchids (e.g. Anophophora), or within more distantly related beetles (e.g. Blackburnia) or crickets (e.g. Gryllus). We therefore believe that presenting a broader phylogenetic tree including sequences from these more distantly-related species would not add useful information, since the additional cytb copy is typical of bruchids, and since the root of the tree is obviously in the Zabrotes lineage. Nevertheless, we certainly admit that we cannot be sure by which genome the additional gene is carried; It might be hosted by a eukaryote endoparasite, or by a prokaryotic or viral genome, although we present some evidence for mitochondrial translation. We hope to be able to addressthis question in a middle-term perspective.
2. Could insect species of the outgroup carry as well more than one cytb copy?
If we admit that the bruchid carries two copies and that this is not contamination, and if it carries them within its mitochondria, the scenario of a transfer is appealing, except if the presence of two copies is a "normal" condition for those insects. Without the study of the genetic composition of the outgroup, it is not clear to me if the apparently original situation of the Z. subfasciatus individuals (with two copies of cytb present in the Altiplano) is ancestral (vertically inherited) or derived (laterally acquired).
Could it be the case that the outgroup of these species actually already contained two copies of this gene? Are there precedents of related organisms with two cytb genes? Could it be tested by studying the composition of some completely sequenced mitochondrial genomes? Could then the present result be explained either (i) by a much more ancient lateral gene transfer (somewhere at the base of the bruchids, in which case the scenarios should try to explain this ancient phenomenon) or if not, eventually (ii) by some complex scenarios of independent genes losses to explain the patchy distribution of cytb in current bruchid populations?
In our opinion, the polymorphic nature of this character (i.e. both the ecological/phylogeographic pattern of the distribution of the additional copy and the rare status of Zabrotes individuals carrying the two copies within sites) argues for a relatively recent event (i.e., more recent than the basal divergence of all the beetles studied here, as suggested by the reviewer). Even admitting that the outgroup carried two independent copies – that would, for instance, have two distinct functions and be indispensable to these bruchids – a vertical scenario could not have led to such a low (0–1%) divergence between the original Acanthoscelides copy and one of those carried by some Zabrotes subfasciatus indivudals, knowing that the two species have diverged during more than 50 Mya [13, 30]. Alternatively, one would have to invoke a very bizarre pattern of gene conversion. We believe that sequence identity in this relatively rapidly-evolving mtDNA gene tends to favour the hypothesis of a relatively recent horizontal transfer over the hypothesis of an ancient divergence of the two copies, followed by vertical double inheritance of both copies.
3. Can one deduce anything about the mechanisms of recombination that inserted the new copy, if they are all within the mitochondrial bruchid genome?
If there is only one type of mitochondria in the bruchids, and if this mitochondria carries the two gene copies, it seems at first sight that the presence of two cytb copies in Z. subfasciatus rules out the hypothesis of a legitimate recombination to transfer this gene. Such a mechanism would likely have led to the replacement of one former cytb copy by another one, resulting in one copy per species. Could the opposite observation suggest then that the additional cytb was transferred in kind of a hitch-hiking process, due to the insertion of one (or more) other markers in the mitochondrial DNA, which could have themselves happened by homologous recombination? In this case the HGT event would be even larger, and would concern several markers. By contrast, for the species presenting only one copy of cytb, could we study the location of this single copy with more precision? A (partial) study of the genetic composition of the mitchondrial genome of these bruchids, typically of the genes bordering the cytb gene, could be very informative here. They may or may not have retained evidence of recombination or synteny. Would it be possible to characterise the genetic context of the cytb genes and test their conservation in later studies to decide if there was one or several instance of LGT and what was their mechanism? Alternatively, could the cytb copy be jumping using transposases? Do we have any evidence for/against the implication of transposons in this story?
We agree that sequencing the full mitochondrial genome(s) of bruchid individuals carrying two cytochrome b copies would help understanding further what happened and how. Unfortunately these data are lacking, and are not so easy to collect, given the relatively high level of sequence similarity between the two copies, the lack of knowledge of the genomic structure, and the scarcity of living individuals with two copies – a new round of field sampling is probably required. Sequencing the full mitochondrial genome(s) would also help us in understanding why a "double-pattern" is lacking for other cytb -linked genes such as COI, which seem to be consistent with the scenario supported by nuclear genes and classical taxonomy.
4. About the nature of the most likely vector: virus, endoparasite or... even an host reservoir?
I was interested by the different hypotheses presented in this paper regarding the vector of the possible lateral transfer. Viruses and endoparasites might be good candidates indeed, although I was wondering if a third possibility, inspired by the "you are what you eat theory" (see. W.F. Doolittle, 1998), could not be raised as well. Since these bruchid species share a host, could not their host have provided them the additional cytb copy?
See our response to question 1.
5. Ecological scenarios and the interest of harbouring two cytb copies to survive in the Altiplano.
I may have misunderstood, but I believe that the authors suggest that the persistence of two copies (or at least the swapping of cytb genes) could serve the purpose of an endoparasite. My naïve question is: could not it serve the purpose of the bruchids themselves? Could not they benefit in the Altiplano from such a genomic make up? Do we have any way to investigate the biochemistry/activity of these two divergent copies in more depth that would suggest to us that even if they are related their function is only partially overlapping?
This is an interesting hypothesis. Developing the capacity for research on functional mito-genomics in these non-model animals can only be a very long-term perspective, however.
This is an interesting paper which describes what I believe is one of those observations which almost defies rational explanation, although the observation does appear to be real.
We fully agree with this statement.
In this paper the authors describe a phylogenetic analysis of bruchid beetles from Mexico. They show that for one nuclear and one mitochondrial gene, the species are moderately divergent and phylogenetically well resolved – i.e. each species is monophyletic with high bootstrap support. However, for another mitochondrial gene the pattern is very different; two of the species have an almost identical cytb gene which is also shared by some members of the most divergent of the beetle species. All individuals which share this cytb sequence come from the same geographical area and live on the same host plant. Intriguingly, those individuals of the species Z. subfasciatus, which have the shared cytb gene, also have a cytb gene which is similar to Z. subfasciatus individuals from elsewhere in Mexico; this other cytb sequence is as divergent as one might expect given how distantly related this species is to the others.
So how do we explain this bizarre pattern? The authors consider a number of alternatives including contamination, hybridisation and pseudogenisation, none of which seems likely. This leaves horizontal gene transfer as possibly the only other explanation. They suggest that maybe some eukaryotic vector has transferred the cytb gene between different species. However, two questions remain. Why has only a part of the mitochondrial DNA been transferred? Individuals from different species share similar cytb sequences but have very different mitochondrial COI sequences.
See our response to Reviewer 1 (question 3).
And why don't all species which have this particular cytb sequence also have their own original cytb sequence, as individuals of Z. subfasciatus appear to have. One can explain these patterns but the explanations are not simple.
This question highlights one limitation of the PCR-only experimental strategy we had in this work. PCR can miss existing gene copies, so that we cannot make sure whether Acanthoscelides individuals from the "core clade" carrying the apparently mobile cytb sequence do or do not carry an additional, "private" copy (especially knowing that the mobile copy is probably of Acanthoscelides origin). Given the phylogenetic proximity between these two species, we did not succeed in designing specific primers. Again, full-genome data would clarify these questions but are lacking.
The authors claim that they discovered a case of rather recent lateral gene transfer between mitochondrial genomes of moderately related beetles. While the claim is striking, I see no obvious holes in the data and reasoning and, thus, tend to believe it.
A very interesting observation is that Zabrotes subfasciatus is polymorphic by presence/absence of laterally acquired cytb gene. When this gene is present, it does not replace the original one and, instead, the two coexist within the same mitochondrial genome. This system opens a rare opportunity to study microevolutionary aspects of lateral gene transfer. It would be worthwhile to sequence complete mitochondrial genomes possessing two cytb genes (native and foreign).
Also, standard selective sweep analysis may tell us whether positive selection favors genomes with the extra, foreign cytb. A great system for further analysis of eukaryotic LGT.
This is an interesting suggestion. We analysed separately the sample of "core clade" haplotypes and found significantly ( p <0.05) negative Tajima's D and Fu & Li's F*, which is indicative of a star-like genealogy compatible with the selective sweep (or founder event) hypothesis. Single-locus arguments for selective sweeps are generally weak, however, given the large number of factors potentially influencing the shape of genealogies.
We thank P. Jarne for his support during this study; E. Desmarais, P. Sourrouille and C. Debain for technical assistance; J. Romero, T. Shani and Y. Borcard for providing several Z. subfasciatus individuals; F. Kjellberg and P. David for critical reading of the manuscript; and A. Delgado-Salinas, J. Moreau and A. Aebi for discussion. Financial support was provided by the Swiss National Science Foundation (Project 3100.064821.01) and the Swiss Center of Competence in Research "Plant Survival".
NA carried out most of the molecular genetic studies and drafted the manuscript. BB and AG participated in the writing of the manuscript and revised the final version. MHM and DM contributed in the interpretation of the data and in the writing of the manuscript. Finally, NG supervised data analysis and participated substantially in interpretation of the data and writing of the manuscript. All authors read and approved the final manuscript.


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