"QDIA Fiduciary Red Flags 401k Plan Sponsors Must Look Out For," (FiduciaryNews.com, June 14, 2016) Do 401k plan sponsors know the fiduciary minefield they're stepping into when they select a QDIA? Why don't they? And what can be done to both make them more aware and to reduce their fiduciary liability?
On one hand, a movement towards acceptance. On the other hand, the resistance to move away from the past remains depressingly resilient.
"IRA rollover under DOL rule: Where to start?" (Financial Planning, June 10, 2016) So it begins, the long hard slog towards adapting to the new compliance regime.
"Alicia Munnell is wrong: Social Security needs a quick fix," (MarketWatch, June 10, 2016) Someone wants to entice Munnell into a verbal contest. Will she take the bait? On the other hand, this is just another example of "Inside the Beltway (Box)" thinking.
John Oliver's show was the talk of the week as he hit the fiduciary issue head on. Too bad the audience was distracted by the usual meme of "high fees" and "index funds" to remember the more important "fiduciary" point. Then again, those other two have been repeated so often they're easy for folks to remember. Maybe a few more pop culture shows highlighting "fiduciary" will lead to the same.
"Fiduciary duty finally hits mainstream — and Main Street," (InvestmentNews, June 13, 2016) While the content may have oversimplified things to the point of missing a few good points, overall the John Oliver retirement piece avoided the biggest mistakes of Frontline's "Retirement Gamble." More importantly, it may have been instructive when it comes to showing how best to educate retirement savers.
"DOL Defines 'Best Interest' More Clearly Than Many Think," (Financial Advisor, June 14, 2016) Harold Evensky does a good job here answering an important question.
"401k Flaws Nothing to Laugh At: John Oliver," (The 401(k) Specialist, June 14, 2016) Based on informal conversations with regular folk, in addressing the three points of this article they remembered the fees, needed to be reminded about the fiduciary, and basically ignored the index vs. passive point.
"No SEC Fiduciary Rule Till After Obama Departs: SEC Chief White," (ThinkAdvisor, June 14, 2016) Surprised, anyone?
"Under new fiduciary rule, DOL has reason to pay attention to reverse churning," (InvestmentNews, June 16, 2016) "Reverse churning" looks a lot like buy and hold, except it occurs in non-RIA arrangements when there is no formal duty to monitor the portfolio continuously.
You know you have a financial literacy problem when retail investors think fees are more important than returns. That's like saying you don't want to win the lottery because then you'd have to pay more taxes.
"What's More Important than 401k Fund Performance?" (The 401(k) Specialist, June 12, 2016) In further testament that propaganda works, a new survey reveals 401k participants prefer lower fees to higher investment returns. This is enough to test the mettle of the true fiduciary. Again, the role of the fiduciary is not to accede to the beneficiaries every demand, but to do what's in the best interests of the beneficiary. Now, ask yourself this question (and to make it easier we'll use a real life scenario): Is it better for retirement savers to invest in almost no-fee (and absolutely no net return) money market fund or a high-fee (and a net return of 8%) equity fund? Too obvious? What if we replace the money market fund with a low-fee fund that consistently yields a net return of 4%? Is your answer still the same? If you're a fiduciary, it should be. Believe it or not, not everyone passes this test.
"Grappling with fiduciary, active-passive debate and more at Morningstar," (Financial Planning, June 14, 2016) Here's a twist, it's not about active or passive, it's about low fee or high fee, and plenty of passive funds have conflict-of-interest fees.
If you're old enough to remember the song by the group "War" then you're probably singing the lyrics now to that catchy chorus. Still, the truth is we may have reach a milestone truce this past week. Time will tell.
"Socially responsible funds can give your 401k a conscience," (USA Today, June 11, 2016) SRIs are a lot like longevity annuities, everybody talks about them but nobody asks for them. Most important, there's a huge fiduciary liability risk to 401k plan sponsors when they try to make (or even accommodate) political points with retirement funds.
"How Retirees Can Protect Themselves From Sequence Risk," (Wall Street Journal, June 12, 2016) A popular concern with a simple solution, although not necessarily the way the article says.
"Active Versus Passive Doesn't Have to Be Either/Or," (Wealth Management, June 13, 2016) This really isn't news (see "3 Reasons Why the 401k Fiduciary Should Use Both Active and Passive Funds to Reduce Fiduciary Liability," FiduciaryNews.com, October 28, 2009), but it does offer a different take for individual investors and that might have an impact on plan sponsors.
I've watched this segment three times now over the past couple of says and I just don't see where John Oliver is advocating for "education".
Oliver said the retirement plan industry is a potential mine field because essentially anyone can call him or herself a "financial adviser", there potentially high fee, and actively managed funds often under perform. So…you need to pay attention because given the current system, the deck is stacked against you.
It was interesting to me that he was so embarrassed by the initial plan with high costs from Hancock that he decided to scrap it and he will "…pay almost all the fees for our employees" (of which there are 35). Apparently no one told Mr. Oliver that as the plan sponsor, he himself has a fiduciary duty to act in the SOLE interest of plan participants/beneficiaries. Did the Hancock rep inform Oliver that as plan sponsor under ERISA he is a fiduciary?
He was embarrassed and so did the right thing…pay for most of the costs of offering a 401(k) plan. My takeaway is that many if not most small employers (under 100 ees for sure, maybe 250) shouldn't sponsor a 401(k) plan as they are complicated and very costly to maintain.
Oliver made no mention of the need for participants to know that bond prices are inversely rated to changes in interest rates (or anything that would qualify as becoming investment literate). He said that you need to pay attention because the potential exists that the retirement plan industry is a minefield.
He ended by basically saying — start saving now (or better yet invest a time machine and start investing 10 years ago; (2) use low cost index funds; (3) if your financial adviser isn't a fiduciary, RUN; (4) as you age, gradually shift from stocks to bonds (although to do that requires knowledge about stocks and bonds that many participants are clueless about); and (5) keep total fees less than 1%.
According to Oliver, "no one in their right mind" wants to sit through an enrollment meeting. And your takeaway from this is that he is somehow advocating for participant education?
Dave, thanks for your comment and you're absolutely correct! The HBO show said nothing about employee education. On the other hand, what it did do was suggest a better way to conduct employee education. Given the number of You Tube viewers, it's clear this segment has gone well beyond the insiders and is being viewed by rank and file investors. Consider this – in order for those rank and file investors to watch this video, they have to actively search it out, find it, and press a button to view it. All those activities involve the use of behavioral techniques that have been shown to increase audience (and student) retention rates. They have made the commitment to watch the segment, and now possess a self-fulfilling vested interest in understanding what that show said. This is the demonstration of the successful education model the show presents – if only by accident.
Chris, My experience is that many/most eligible employees attending enrollment meetings are like Oliver, e.g. no one in their right mind wants to sit through one, (let alone actually learn anything about investing).
Over the past three decades or so, our national "retirement income program" has defaulted quite accidentally into participant directed 401(k) plans that require extremely knowledgeable individuals if it's going to work. Looking objectively at the results (take median account balances for virtually any cohort) and it's clear that our national system is utterly broken.
Investment expertise cant be achieved via a series of 20-minute YouTube posts.
The "default" investment arrangement for 401(k) plans should be that they are employer directed. But, that's not going to happen.
However, getting even 25% of the 401(k) participant population to a satisfactory level of investment expertise via "education" is not going to happen either, in my opinion.
"No one in their right mind wants to sit through an enrollment meeting" let alone taking the time and making the academic effort it would take to actually learn how to be a successful investor.
Is investment expertise the goals? Or is it savings expertise?


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Raxio launches a new data centre in Tanzania
As the sixth data centre in Raxio's portfolio, Raxio Tanzania will play a key part in plugging the East African digital infrastructure gap.
In March, Raxio Group announced it would establish the first carrier-neutral, Tier III data centre in Tanzania. The new facility will be Raxio's sixth data centre on the continent, and will play a vital role in improving connectivity across Tanzania and beyond.
"We have a very clear strategy to build state-of-the-art facilities in what we call secondary markets on the continent," Robert Mullins, CEO of Raxio Group, tells EMEA Finance. "A lot of the attention on data centre infrastructure has been around Kenya, South Africa and Nigeria, but if you look at the continent beyond that you have 20 countries with populations greater than 25 million. Many of these countries are completely underserviced in terms of digital infrastructure."
While the African data centre market is growing fast, it is far from reaching saturation. To date, the continent accounts for less than 1% of the world's co-location data supply, according to Xalam Analytics, despite playing host to 17% of the global population. To put matters into perspective, Africa as a whole has about the same amount of data centre space as Switzerland.
However, the continent is seeing a surge in the number of internet users, with around half its population poised to have internet access by 2030, according to GSMA. This in turn is driving the need for digital infrastructure such as data centres.

redpajama_set_name: RedPajamaCommonCrawl


Every year for Christmas I try to make many of my Christmas decor items. This year I wanted to warm up my decor with a wreath that looked like a warm and cozy blanket. I think this wreath accomplished that and what I love most is that with a simple change of the bow it can carry you through from Fall to Christmas right to the end of Winter.
Straw wreath form 2" diameter x 18"
You can also use foam core or you can even use a pool noodle.The thicker diameter helps to show off the stitches better, a 3" would be even better but I was unable to find it.
This size once crocheted into a chain was the exact size for my wreath form. Sizes will vary depending on your wreath size and diameter. I tried to find a nice thick yarn, colors and sizes are completely your choice.
If you have never crocheted before this can sound intimidating but using only your fingers you will see this project will be very fast and very simple. Let's get started!
You will begin with your yarn to form a loop from which your chain will begin. Begin with about a 6 - 8 inch tail and cross the yarn over to form a teardrop.
Holding the tail, put your thumb and pointer finger into the teardrop and grab the yarn and pull though the loop.
This becomes the first link on your chain.
See I told you this would be easy!
Once you have your first link done, you just keep repeating the steps reaching your two fingers into a loop and drawing the yarn though the chain until you come to the end of the yarn.
Ideally what you are looking for is a loose chain, but you want the links to be close enough that once wrapped around the form you will not see the form underneath it. Don't worry about it being perfect, once your wrap it you will not see any uneven links.
Depending what kind of wreath form you chose, you may or may not need to prepare it. I was unable to find a foam core wreath so I chose a straw wreath. It worked perfectly but is very messy. I chose to use cling wrap to cover my wreath and contain the fibers from messing up my yarn.
Taking the cling wrap, I rolled it out about 10 inches and layered my wreath form on top of it.
Using scissors, cut the cling wrap.
Taking the wrap, and tightly wrap the form to encase all the straw.
Continue this step to completely cover the wreath.
If you do not have cling wrap, you can use packing tape or duct tape for this step.
With your completed chain you now need to wrap it around the wreath.
Take your completed chain and roll it back into a ball so it is easy to slip into the wreath for wrapping.
Split one end of the chains 8" tail into two strands and tie it to the wreath form.
Lay the chain flat on the wreath and slowly start to wrap it around the wreath.
Be sure when you wrap it to keep the chain from twisting, this will give you the knitted look you are going for.
Continue wrapping it around the wreath, either spreading it our squishing it to fit the entire chain on the wreath. You want to make sure that you do not wrap it so tight that you lose the texture of the chain but not so loose that you see the straw behind it.
When you get to the end of the chain, split the ends into two pieces and tie it to the wreath.
In stead of cutting my end I added another knot and used it as my hook for hanging.
The final step to completing the wreath is adding the bow and any additional embellishments as desired. This is always the fun part as you can make it as embellished as you want or keep it simple like mine.
Take any ribbon that you like, the size doesn't matter really except that it will become fuller or flatter depending on the width or weight of the ribbon.
My ribbon width is 2.5" and the width of my finished bow is 15"
Place the end of the ribbon against a ruler at the 7.5" mark, fold the ribbon over at the 0" mark; this becomes the first loop.
Take the ribbon and lay it on top of the first layer till it is at the 15" mark and fold it again on top of itself; you now have the two back loops done.
Lay the ribbon on top of itself till it gets to the 2" mark and fold again on the other side at the 13" mark. Now you have your second layer of loops.
Fold the ribbon again at the 4" and 11" mark.
Cut the ribbon at 7" mark.
Pinch the ribbon at the 7.5" mark and wrap twine around it a couple of times securing it in the back and cutting the ends off.
Start to fluff your bow to make the loops stand out.
Cut another piece of ribbon approximately double the size of your finished bow or to desired length. Mine is 30".
Fold the ribbon in half to find center and place the center over the loops you created, this will become your bow tails.
Pinch the ribbon at the back and twist it, tie with twine.
You now have completed your bow and you can flatten or mold the loops as desired to get the fullness you want for your wreath.
The final step is adding your bow to the wreath. There are many ways to do that, you can tie them on, hot glue or use floral picks. I preferred the floral picks because I like to change the bow up with the season. I didn't have any on hand so I use paper clips. Using your wire cutters cut the paper clip so that each size is the same length. You can reshape the paper clip to use as a pick. Take the pick and insert them into the fabric of your bow and then pushing them into the straw form.
Finally you have completed your wreath, with just a quick tug you can change the bow to suit the season.
I hope you have fun making your own!


The Modelling News: Andrea Miniatures latest - Dont go running around like a headless chicken this Christmas!
Andrea Miniatures latest - Dont go running around like a headless chicken this Christmas!
WY-12 SANTA´S DISGUISE DOESN´T WORK..
It seems someone has rumbled Santa! Our canine best friend isn't acting like it in this Vignette – and it seem the tempting treat that's being offered in exchange isn't working either!!
This set comes with parts for the base, ladder, presents and of course Santa and his best mate. The kit can come in unpainted and a painted form as seen here by Diego Ruina.
This Bare-legged bodyguard seems to not be one to lose his head! Seen in full battle armour ( apart from his pants it seems) he is carrying his long pike/sword and also the head of a vanquished enemy.
The Samurai's helmet and armour look resplendent painted up here and one can only guess why he is still hanging on to that old head – If you would like to paint your own of course the kit comes in Painted and unpainted versions.
This figure in 54mm (1/32scale) shows a scene of the battle for Zama in 202 BC. This is the last battle in the Carthaginian War in which Scipio Africanus beat the forces of the terror of ancient Rome the General Hannibal.
The Romans used the strength of Hannibal's elephants against his own army – the Romans startled them with horns and flame spikes and arrows – some of these large beasts turned and fled into their own ranks – allowing the roman cavalry to attack from the sides and therefore causing the mercenary army of Hannibal to be broken and flee. This ended the Punic Wars and Carthage as a world power.
I would assume most of us would want to get out hands on her and do it ourselves! The lady in question has been "caught" by some prickly flora – this is a great representation of well-done Pin-Up art in the style of Vargas and others - and I for one love it!
On our recent trip to Euro Militaire we were treated to a Masterclass by none other than the legendary Julio Cabos who gathered quite a crowd giving lessons on figure painting – we felt really privileged to be able to watch this master at work – we also loved to see some of Andrea Miniatures figures painted and on display at the event – including some very large versions of figures we know well.. Did I forget to mention Andrea Miniatures have some great new figures coming out this month?
Built in ten parts of lusciously sculpted white metal this figure of a real charmer (punTM.) is pretty neat – Sculpted as usual of many of Andrea Miniatures figures by the very sharp eyed Nelya Vitvitska and in this instance painted by the no less talented Julio Cabos.
The figure comes in painted and unpainted flavours and if this is what you get painted I may be tempted. The pillow and the basket are of course supplied and I had better leave it at that before I say too much more – it is a great looking figure though!
This figure of the soon to be most important man in Rome celebrates his victories over the Gauls in 52BC – this was the year the romans defeated Gauls led by Vercingetorix at the Battle of Alesia, thus breaking the back of the Gallic insurrection. The final pacification of Gaul became complete the following year.
5 white metal casting parts for assembly and painting are the kit's contents – in 54MM(1/32) this tiny figure comes painted or unpainted in it's original form.
Sculpted by Joaquín Palacios and in this case painted by Elías Alonso this figure looks great in the way Joaquin has sculpted the drapes on Caesars body and his elaborate gladius – his short stabbing sword.
Of course you can get this sculpture painted as well and this particular one was brought to life by master painter Julio Cabos. He has done a great job on making the Ronin look lifelike in 1/24th scale, his long spear and Katana look impressive as does his facial expressions and muscle tone especially.
The Firereach Commander is part of the "Dark Nova" series of figures tilted at science fiction and war gaming fans – though most fellas I met like the "figure" without knowing the story behind her.
This figure was painted here by Elias Alonso who has done a great job of rendering the scale effect in colour and shading. This will certainly be a good seller.
Till next month keep an eye on their site for more news or just check back here – well keep you updated!
This figure is sold likewise painted (280 €) and unpainted in raw kit form for Kit 42€.
Sculpted by Ángel Terol in White metal this 75mm figure is painted in this instance by the very talented Julio Cabos who show this warrior with his Nagana and layered extremely tough armour. His armour is less ornate than his master the samurai who's land they probably lived on and worked when not in military service. These soldiers were Japan's first attempt at the foot solider.
Part of the famous - and need I say very popular PYN-UP series from Andrea Miniatures is this the latest of their series number 25 which will be released soon.
PIN-UP 25 is made in 80mm white metal and painted by Nelya Vitvitska - who I must say has a great eye for detail! Julio Cabos has painted this beauty so very lifelike it looks like an old picture from a 50's magazine - excellent ring-tone!


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Home Industry News
A Tiny Amount of Michter's Barrel Strength Bourbon is Hitting Shelves Ahead of the Kentucky Derby
G. Clay Whittaker - April 18, 2019
Limited supplies of Michter's Barrel Strength Bourbon are arriving in shops this week, likely in an effort to stock shelves one last time before the Derby. Bottles of Michter's Barrel Strength Bourbon with 2018 bottling dates quietly began appearing with...
Indian Whisky: Everything You Need to Know
Brad Japhe - January 14, 2019
The meteoric rise of Asian single malt is perhaps the most widely reported whisky story of the past decade. Japan and Taiwan have positioned themselves at the center of the spectacle, birthing liquids that have become feverishly revered and...
Jim Rutledge Talks Cream of Kentucky Pre-Sales, Breaking Ground on a Distillery in 2019
Maggie Kimberl - January 2, 2019
Since retirement, Jim Rutledge has shown no signs of slowing down. He's been contract distilling at Castle & Key while simultaneously launching the J.W. Rutledge Distillery, which includes reviving the heritage brand Cream of Kentucky. The Bourbon Review recently...
Retirement for Jim Rutledge Means Two Distilleries Instead of One
Maggie Kimberl - September 14, 2018
Former Four Roses Master Distiller Jim Rutledge's retirement has doubled the amount of work he's doing in the whiskey world these days, or at least the number of distilleries where his talents are finding a home. In addition to...
The Next Batch of Jim Beam's Booker's Rye Hasn't Been Barreled Yet
G. Clay Whittaker - September 11, 2018
Kentucky distiller Jim Beam has not laid down a single barrel of Booker's Rye whiskey—the award-winning limited edition bottle released in 2016—according to Master Distiller Fred Noe. During an exclusive interview with The Bourbon Review earlier this year, Noe was...
Jefferson's Bourbon Twin Oak Custom Barrel
The Bourbon Review - August 31, 2018
Jefferson's Bourbon has taken maturation technology to the next level with their latest release of Jefferson's Twin Oak Custom Barrel. The brand's newest release marks the result of a six-year project with Independent Stave Company The small-batch bourbon producer, famous for...
Kentucky Bourbon Festival continues to expand events & engagement opportunities
Bob Eidson - August 29, 2018
Story & Interview by Bob Eidson It's that time of year when the mornings start out cool & refreshing, but by noon in the Bluegrass, you know you're still in the humid Ohio Valley. The kids are back in school...
Cleanup at Barton 1792 is Underway after Warehouse Collapse
Caroline Paulus - August 13, 2018
A month after the collapse of Warehouse 30, cleanup at Barton 1792 Distillery is underway in Bardstown, KY. The initial collapse on June 22nd caused 9,000 bourbon barrels to tumble to the ground, with broken barrels spilling into a nearby...


VISITORS: Mayor Renny Perry reported the City Council was waiting for a student to express interest in serving on the Recreation and Facilities and Programs Advisory Committee; we now have four interested candidates. He introduced Sierra Bertrand, Summer Chabot and Anna Rakowski and advised Jacob Kadric is also interested in serving on the committee but was unable to attend this meeting. Each of the students gave a brief statement on why they would like to serve on the committee. Mayor Renny Perry suggested maybe appointing 2 of the students as primary delegates and 2 as alternates. The appointment(s) will be made in accordance with the agenda.
MINUTES: Alderman Lowell Bertrand moved to approve minutes to the City Council meetings held August 28th and September 11th. That motion was seconded by Alderman Mark Koenig. An amendment was made to the August 28th minutes on the last page; under South Water Street Sidewalk the words "throw up the sidewalk" were replaced with "tear up the sidewalk". Minutes to the September 11th meeting were amended on the first page under Visitors. The words "New Haven is contributing $2,000" were corrected to read "New Haven was being asked to contribute $2,000". On the last page of the September 11th minutes, under City Manager's Report, "was told by an Alderman to remove" was replaced with "was told to remove". In the next paragraph "revolving flashing beacons" was replaced with "rapid flashing beacons". Alderman Bertrand moved to accept the minutes as amended, seconded by Alderman Koenig, with all voting in favor.
WARRANTS: The Warrant totaling $770,111.87 was circulated for review and signatures of approval. City Manager Chabot pointed out the Warrant includes the 1st quarter installment for the education tax.
CITIZENS COMMENTS: Robert Trepanier advised he is back to follow-up on a national issue he brought to the City Council a few months ago when he asked how the City Council stood on fair and equitable treatment. He cited the restaurant conversation he shared with the City Council regarding a patron being asked to leave a restaurant due to their political affiliation which is not protected under civil rights law. Unless a municipality has an ordinance prohibiting this right there is no standing in the courts, he stated. Mayor Renny Perry advised he has not been able to research this yet to make sure we can legally do it. Mr. Trepanier stated we, the citizens, want to know what protection we can expect in Vergennes so you looking into that would be much appreciated. Alderman David Austin did not feel that we wanted to enact an ordinance as the answer is complex and costly. We should respond to his concern with perhaps a resolution instead. Alderman Koenig suggested letting Mayor Perry do his part first and see if it is feasible and can we do it. Mayor Renny Perry advised he will work with VLCT and do some research.
STUDENT NOMINEES TO RECREATION COMMITTEE: Because four students were interested in being on the recreation committee, discussion was had on how all students could participate with their busy schedules. Mayor Renny Perry advised the Charge addresses it being a 7-member committee but we could appoint all four students who collectively would have up to two votes at a meeting. Deputy Mayor Jeff Fritz moved to appoint Sierra Bertrand, Summer Chabot, Anna Rakowski and Jacob Kadric to the recreation committee with a limit of two votes. Alderman David Austin seconded the motion with all voting in favor.
PUBLIC HEARING: At 6 p.m. Mayor Renny Perry called the duly warned and posted public hearing to order regarding an amendment to the Land Use Map of the Vergennes zoning and subdivision regulations as proposed by the Vergennes Planning Commission. The Notice of Public Hearing, as required by 24 V.S.A. §4442, was read by the Mayor. As no one wished to give testimony on the proposed amendment, Mayor Perry closed the public hearing at 6:01 p.m.
TRIANGLE BIKE LOOP FUNDING: Carrie Macfarlane, a member of the Walk-Bike Council of Addison County, advised the Town of New Haven has approved their request for $2,000 towards the local match ($3,500) and she hopes Vergennes will support them and approve the remaining $1,500 as was requested. The grant will allow them to hire a consultant who will develop a master plan for a designated bicycle route connecting Addison County's Designated Downtowns (Vergennes, Middlebury, and Bristol) and New Haven's Village Center and it is in agreement with Vergennes' Municipal Plan. City Manager Chabot reported the $1,500 can come from the Water Tower Fund. Alderman Bertrand inquired why Middlebury and Bristol were not contributing to this project. Carrie Macfarlane advised Middlebury cannot be an applicant because they already have a grant and the Town of Bristol does not have a Town Plan so they are not eligible either. Neither one of them is qualified to be applicants of this grant. Alderman Lowell Bertrand moved to approve the request for $1,500 towards the matching funds and the fund to be taken from the Water Tower Fund. The motion was seconded by Alderman Mark Koenig with all voting in favor. A FY19 Municipal Resolution for a Municipal Planning Grant was circulated for signatures from the legislative body.
ACRPC TOWN ENERGY PLANNING ELEMENT PILOT: Shannon Haggett, Chair of the Planning Commission, reported Addison County Regional Planning Commission has State funding to assist three towns within this Region with development of city plan energy elements that are consistent with the provisions of Act 174. These grants require no matching investment, he advised. A letter of intent is needed from him as the Chair and a decision is likely next week. Unanimous City Council support was offered; Mayor Renny Perry will sign Shannon's letter indicating that support.
ALTERNATE TO ACSWMD: City Manager Matt Chabot suggested he take retired City Manager Hawley's position as the alternate delegate on the Addison County Solid Waste Management District's Board of Directors. Alderman Lowell Bertrand moved to appoint City Manager Chabot as an alternate delegate to the ACSWMD, seconded by Deputy Mayor Jeff Fritz, with all voting in favor.
VLCT DRAFT MUNICIPAL POLICY: Mayor Renny Perry advised he sent a copy of the draft municipal policy to the City Council early so they would have time to review it. The VLCT policy is their guidance manual. As he is on the F.A.I.R. Committee he should be voting what the City standing wants and not what he wants himself. The election portion was discussed and the City Council supported no "gap" day as it relates to elections.
DOWNTOWN-BASIN MASTER PLAN TASK FORCE APPOINTMENT: Mayor Renny Perry advised Kathy Rossier was appointed to the Basin Task Force as a community representative but she should be switched to a Vergennes Partnership representative as she sits on that Board. Mike Winslow will remain a community appointee. He advised he would like to recommend David Austin for the Council representative and we will wait for the recommendation from the recreation committee for their representative. Deputy Mayor Jeff Fritz moved to appoint Alderman David Austin to the Basin Task Force as the City Council representative. Alderman Mark Koenig seconded the motion with all voting in favor.
APPOINTMENT OF TRUSTEE TO BIXBY BOARD: Mayor Renny Perry advised the Bixby Memorial Free Library will make their recommendation for a new trustee that will replace Christine Bradford. That appointment to the Bixby Memorial Free Library Board of Trustees will occur at the next regular meeting.
CITY MANAGER'S REPORT: City Manager Chabot reported we will be having our firewall replaced this week and are expecting two short blackout periods on Thursday. The Citizens Bank branch office in Vergennes is closing about November 17th so the Clerk/Treasurer and I are searching out and interviewing alternative banks, he advised. Scott Gaines reported to him a Vergennes Day Little City Race profit of $627 and recommends $500 be donated to Vergennes Area Rescue Squad and the remaining $127 be kept by the City for additional signage and supplies for next year. A budget report was made available to the Board. He informed the City Council that he is watching the overtime line item, supplies and equipment, as well as discretionary uniforms in the Police Department budget. He is establishing monthly meetings with each department.


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communications biology
Phylogenomics illuminates the evolution of bobtail and bottletail squid (order Sepiolida)
Morphology and genome of a snailfish from the Mariana Trench provide insights into deep-sea adaptation
Kun Wang, Yanjun Shen, … Shunping He
Phylogeography, colouration, and cryptic speciation across the Indo-Pacific in the sea urchin genus Echinothrix
Simon E. Coppard, Holly Jessop & Harilaos A. Lessios
Mitogenomics suggests a sister relationship of Relicanthus daphneae (Cnidaria: Anthozoa: Hexacorallia: incerti ordinis) with Actiniaria
Madelyne Xiao, Mercer R. Brugler, … Estefanía Rodríguez
Prolonged morphological expansion of spiny-rayed fishes following the end-Cretaceous
Ava Ghezelayagh, Richard C. Harrington, … Thomas J. Near
Whole-genome sequences of Malawi cichlids reveal multiple radiations interconnected by gene flow
Milan Malinsky, Hannes Svardal, … Richard Durbin
New bobtail squid (Sepiolidae: Sepiolinae) from the Ryukyu islands revealed by molecular and morphological analysis
Gustavo Sanchez, Jeffrey Jolly, … Daniel S. Rokhsar
Mitogenomics provides new insights into the phylogenetic relationships and evolutionary history of deep-sea sea stars (Asteroidea)
Shao'e Sun, Ning Xiao & Zhongli Sha
Genome sequences reveal global dispersal routes and suggest convergent genetic adaptations in seahorse evolution
Chunyan Li, Melisa Olave, … Qiang Lin
The complete mitochondrial genome of the eusocial sponge-dwelling snapping shrimp Synalpheus microneptunus
Solomon T. C. Chak, Phillip Barden & J. Antonio Baeza
Gustavo Sanchez ORCID: orcid.org/0000-0002-6490-28421,2 na1,
Fernando Á. Fernández-Álvarez ORCID: orcid.org/0000-0002-8679-73773 na1,
Morag Taite3,
Chikatoshi Sugimoto2,
Jeffrey Jolly2,
Oleg Simakov ORCID: orcid.org/0000-0002-3585-45114,
Ferdinand Marlétaz5,
Louise Allcock ORCID: orcid.org/0000-0002-4806-00403 &
Daniel S. Rokhsar ORCID: orcid.org/0000-0002-8704-22242,6,7
Communications Biology volume 4, Article number: 819 (2021) Cite this article
Bobtail and bottletail squid are small cephalopods with striking anti-predatory defensive mechanisms, bioluminescence, and complex morphology; that inhabit nektobenthic and pelagic environments around the world's oceans. Yet, the evolution and diversification of these animals remain unclear. Here, we used shallow genome sequencing of thirty-two bobtail and bottletail squids to estimate their evolutionary relationships and divergence time. Our phylogenetic analyses show that each of Sepiadariidae, Sepiolidae, and the three subfamilies of the Sepiolidae are monophyletic. We found that the ancestor of the Sepiolinae very likely possessed a bilobed light organ with bacteriogenic luminescence. Sepiolinae forms a sister group to Rossinae and Heteroteuthinae, and split into Indo-Pacific and Atlantic-Mediterranean lineages. The origin of these lineages coincides with the end of the Tethys Sea and the separation of these regions during the Eocene and the beginning of the Oligocene. We demonstrated that sepiolids radiated after the Late Cretaceous and that major biogeographic events might have shaped their distribution and speciation.
Sepiolids are small round-bodied cephalopods of around 1–8 cm of dorsal mantle length which live in a range of habitats from shallow coastal waters to mesopelagic environments across the globe1. These animals are nektobenthic and pelagic species that have evolved remarkable anti-predatory defense mechanisms. Bobtail squids, for example, use counter-illumination2 or discharge luminous secretions3, while bottletail squids secrete toxic substances through their slime4. Taxonomically, sepiolids comprise the order Sepiolida Keferstein, 1866 which is split into the families Sepiolidae Leach, 1817 and Sepiadariidae Fisher, 1882, commonly known as bobtail and bottletail squid, respectively.
Bobtail and bottletail squids are emerging as model organisms for cephalopods due to their easy culture under laboratory conditions5. Most notably, the Hawaiian bobtail squid Euprymna scolopes Berry, 1913 is a model for host-symbiont interaction with the bacterium Allivibrio fischeri (Beijerinck 1889)6, which interacts with the light organ of the animal2,7,8. The luminous organs of bobtail squids emit light of bacteriogenic or autogenic origin; these organs have either bilobed or rounded forms3,9. Genomic resources to study these animals have also increased through the recent sequencing of the nuclear genome of E. scolopes10 and transcriptomes of several different species11,12,13,14.
Previous phylogenetic analyses have placed the order Sepiolida as sister to the family Idiosepiidae Appellöf, 189815,16. The recent study of Anderson and Lindgren 202017, however, finds Sepiolida to be sister to a clade containing the Oegopsida Orbigny, 1845 (oceanic squid with no cornea), Myopsida Naef, 1916 (neritic squid with cornea), and Sepiida Zittel, 1895 (cuttlefish) adding a controversy about the phylogenetic position of these animals. Sepiolida forms the third largest group of Decapodiformes after cuttlefishes and oegopsids18. Bobtail squids are the most diverse of the order with around 68 valid species grouped within three subfamilies, Sepiolinae Leach, 1817, Rossinae Appellöf, 1898, and Heteroteuthinae Appellöf, 1898. In contrast, their relatives, the bottletail squids have only five species grouped into two genera, Sepiadarium Steenstrup, 1881 and Sepioloidea d'Orbigny 1845. The genus Euprymna Steenstrup, 1887 has been recently redescribed using both molecular and morphological analyses11. The systematics of Sepiolinae has recently been reviewed by Bello19, who proposed subdividing Euprymna and other genera into multiple new genera according to variation in the shape of the hectocotylus and other morphological characters. However, more molecular studies with confident taxonomic identifications are required to clarify the systematics, diversity, and evolutionary relationships of Sepiolida.
Recently, transcriptome sequencing provided several nuclear DNA markers and resolved the evolutionary relationships of Euprymna and other species formerly classified in the genus Sepiola Leach, 1817 (see Bello19) collected in the shallow waters (up to 20 m depth) of the Ryukyu archipelago and mainland Japan11. Other members of the order Sepiolida, however, are found in oceanic waters (the record is for Heteroteuthis nordopacifica Kubodera and Okutani, 2011 collected at 1000 m depth, off Toba district in Japan20), and are often caught as by-catch in which the animal does not survive the collection. While this makes the sampling of fresh tissue of these animals for transcriptome sequencing difficult, genomic DNA suitable for sequencing is readily recovered.
While full genome sequencing is costly, low-coverage shotgun sequencing allows the recovery of phylogenetically useful markers, including complete or nearly complete mitochondrial genomes and highly copied nuclear loci such as 18S and 28S rRNA21. This approach, sometimes called "genome skimming", does not require the collection of fresh tissues from the animal. For sepiolids, the genome of E. scolopes10 provides a suitable reference to map reads and recover additional nuclear loci.
In this study, we reconstruct the evolutionary relationships of Sepiolida and performed molecular dating to estimate their divergence timeframe. We first characterized our specimens based on morphological characters, and then sequenced their DNA at low coverage to skim for mitochondrial genes and nuclear ribosomal genes, and to define a set of ultraconserved loci based on shared alignments to the reference genome of E. scolopes. Our datasets allowed us to reconstruct mitochondrial and nuclear phylogenetic trees that support a sister relationship between Rossinae and Heteroteuthinae, and the split of Sepiolinae into lineages from the Indo-Pacific and Atlantic plus Mediterranean region. We also infer that the ancestor of Sepiolinae possessed bilobed light organ hosting symbiotic bacteria, lost independently in Inioteuthis and Sepietta from Sepiolinae. Moreover, the monophyly of Heteroteuthinae confirms a unique origin of the autogenic luminescence in this group, validating its association with pelagic lifestyles in Cephalopoda. In addition, the time-calibrated phylogeny shows that the split of many sepiolids is correlated with and was plausibly driven by major biogeographic events after the late Cretaceous.
Genome skimming provides robust phylogeny
Pioneering molecular phylogenetic studies in Sepiolida that used short regions of a few mitochondrial and nuclear genes failed to resolve the relationship of major clades9,22,23. To increase the number of phylogenetically informative sites, Sanchez et al.11 sequenced and analyzed the transcriptomes of multiple species of Euprymna Steenstrup, 1887, related bobtail squids including Sepiola parva Sasaki, 1913 and Sepiola birostrata Sasaki, 1918, and several bottletail squids. They found that S. parva grouped with the Euprymna species to the exclusion of S. birostrata, and further morphological analysis led to the formal redefinition of the genus Euprymna and the reassignment of S. parva Sasaki, 1913 to Euprymna parva11. The following year, in an exhaustive study of hectocotylus structure, Bello19 proposed that Euprymna be split back into the original Euprymna Steenstrup, 1998 and a newly defined genus, Eumandya Bello 2020 that contains E. parva Sasaki, 1913 and E. pardalota Reid 2011, two taxa whose arms have two rows of suckers rather than four as in other Euprymna species. Similarly, Bello introduced a new genus, Lusepiola Bello, 2020 that has the effect of renaming Sepiola birostrata Sasaki, 1918 as Lusepiola birostrata. For clarity, we adopt the finer-grained nomenclature of Bello below, but happily note that E. parva and E. pardalota have the same abbreviations in both the notation of Sanchez et al.11 and Bello19.
Sanchez et al.11 also emphasized the need for more taxon sampling, careful species assignment, and the inclusion of more informative sites when studying this group of cephalopods. However, the distribution and lifestyle of many lineages of Sepiolida makes the collection of fresh tissue for RNA sequencing very challenging. To overcome this limitation, we sequenced the genomic DNA of several Sepiolida species at shallow coverage up to 3.6× and accessed by this way several mitochondrial and nuclear loci. Most of our samples were carefully identified at the species level based on morphological characters.
We recovered the mitochondrial genomes of the species targeted in this study and annotated the 13 protein-coding genes, 22 tRNAs, and two rRNAs (although only the conserved region of the large and small rRNA was obtained for Rondeletiola minor Naef, 1912).
Additionally, we also downloaded the complete mitochondrial genomes of S. austrinum and Idiosepius sp., and the transcriptome of E. tasmanica available in the NCBI database. The transcriptome of E. tasmanica was used to extract its complete set of mitochondrial protein-coding genes. We could reconstruct the mitochondrial gene order for all species with complete mtDNA genomes, but we observed no re-arrangement for members of Sepiolidae, and only Sepiadarium austrinum deviated from the arrangement seen in all other Sepiadariidae (Fig. S1).
To complement the mitochondrial-based evolutionary history, we also annotated several nuclear loci. As ribosomal gene clusters are present in numerous copies, they were successfully retrieved for almost all the species, except for 28S of the Sepiadariidae sp. specimen, which appeared problematic and was excluded.
By mapping reads to the reference genome of E. scolopes, we obtained 3,279,410 loci shared between at least two species and further selected 5215 loci presented in most of our Sepiolidae species, but allowed some missing data in the Euprymna + Eumandya clade. This was done because the phylogenetic relationships of the Euprymna + Eumandya species were previously described in detail in Sanchez et al.11 using transcriptome data. Out of the 5215 loci, 5164 loci had a per-site coverage ranging between two and five. After trimming and removal of regions without informative sites, 577 loci remained. These ultraconserved loci had lengths ranging between 10 and 690 base pairs (bp), with an average of 65 bp. Our alignment matrix had a length of 37,512 bp and consisted of 16,495 distinct site patterns, and variable sites between 1 and 130 bp with an average value of 7 bp. We expected a low value of variable sites because these regions are highly conserved.
We considered resolved nodes to be those with the ultrafast bootstrap support and posterior probability larger than 95% and 0.9, respectively. Only the very unresolved nodes were found based on the mito_nc matrix (Fig. 1). However, among the species in these nodes, Adinaefiola ligulata Naef, 1912 was well supported with amino acid sequences from mitochondrial genes (posterior probability of 1 and 94% bootstrap support) and partially by the ultraconserved loci (posterior probability of 1, but only 85% bootstrap support) as sister to the Sepiola clade (Figs. 2 and S2). Moreover, compared to the mito_nc matrix and with identical topology, mito_aa and UCEbob fully resolved the relationship of the Indo-Pacific and Mediterranean Sea Sepiolinae. The tree generated by the nuclear_rRNA produces a topology with most nodes unsupported (Fig. S3), suggesting these markers are too conserved for assessing the relationships among this group.
Fig. 1: Phylogeny of Sepiolida based on nucleotide sequences from the mitochondria (mt_nc matrix).
The topology of the maximum likelihood tree is shown. Numbers by the nodes indicate bootstrap support and the Bayesian posterior probabilities. Values of bootstrap support and posterior probabilities above 95% and 0.95, respectively, are not shown. (*) indicates that the node was resolved with the mito_aa and UCEbob matrices. (+) indicate that A. ligulata is sister to Sepiola using mito_aa with ultrafast bootstrap support of 94% and a posterior probability of 1. Abbreviations: IP, Indo-Pacific Ocean; MA, Mediterranean Sea, and the Atlantic Ocean.
Fig. 2: Phylogenetic tree of Sepiolida based on conserved nuclear loci (UCEbob matrix).
The topology of the maximum likelihood tree is shown. Numbers in by the nodes indicate the bootstrap support and the Bayesian posterior probability. Values of bootstrap support and posterior probabilities above 95% and 0.95, respectively, are not shown. IP, Indo-Pacific Ocean; MA, Mediterranean Sea, and the Atlantic Ocean.
Using the UCEbob matrix, the topology and supported relationships of Euprymna + Eumandya species resemble those reported in Sanchez et al.11 using transcriptome sequences, proving our protocol valid when using low coverage sequencing and when a reference genome of the closest related species is available.
The position of R. minor showed discordance between mitochondrial and nuclear datasets. Using the mitochondrial matrices, R. minor rendered the Sepietta Naef, 1912 clade paraphyletic, whereas using the UCEbob and rRNA_nc matrices, R. minor appeared sister to the Sepietta clade. These relationships were resolved in both mitochondrial and nuclear-based trees and require further investigation with more DNA markers and a wider population sampling.
Molecular systematics of Sepiolida clades
Using the complete mitochondrial genome, ribosomal nuclear genes, and ultraconserved loci, we recovered the monophyly of the two families of the order Sepiolida—Sepiadariidae and Sepiolidae9,24—and the monophyly of the three described subfamilies of the family Sepiolinae. However, contrary to what is proposed based on morphology in Young24, the Rossinae is not sister to all the remaining Sepiolidae but rather is sister to Heteroteuthinae, although this is unresolved in the UCE phylogeny. With the lack of systematic work on these subfamilies, our robust phylogenetic backbone in Sepiolida using new samples carefully identified by morphology and with museum vouchers, represents a notable advance to clarify the evolution of morphological traits in major clades within the family.
Based on morphological characters of the hectocotylus, Bello19 recently split the polyphyletic Sepiola Leach 1817 into Lusepiola, Adinaefiola, and Boletzkyola, reserving Sepiola for the S. atlantica group sensu Naef 1923. These newly defined clades are consistent with our molecular phylogeny here and in Sanchez11, who also noted the polyphyly of Sepiola in the Indo-Pacific lineage.
We find that Sepiolinae can be robustly split into two geographically distinct tribes: one that comprises species with known distribution in the Indo-Pacific region (tribe Euprymmini new tribe, defined as Sepiolinae with a closed bursa copulatrix, type genus Euprymna) and the other including all the Mediterranean and Atlantic species (tribe Sepiolini Appellof, 1989, defined here as Sepiolinae with an open bursa copulatrix, type genus Sepiola). Our molecular relationship is consistent with 13 of the 15 apomorphies used in the cladogram shown in Fig. 21 in Bello19. The other two proposed apomorphies in Bello (his apomorphic characters 4 and 6) group two IP lineages, Lusepiola and Inioteuthis, in a clade with species from the Mediterranean and Atlantic. Such relationships contradict our Euprymmini-Sepiolini sister relationship. Moreover, according to our phylogeny, apomorphy 6 of Bello, characterized by the participation of ventral and dorsal pedicels in the formation of the hectocotylus copulatory apparatus, implies that the male ancestor of Sepiolinae had a more developed hectocotylus that was simplified in the Euprymna and Eumandya clades.
Among euprymins, we confirmed the monophyly of Euprymna Steenstrup 1887 as found previously by transcriptome analysis11. We also support the monophyly of Eumandya Bello, 2020 (Figs. 1 and 2), grouping the type species E. pardalota with E. parva along with the unnamed "Type 1" Ryukyuan species of Sanchez et al.11, for which only hatchlings were available. The phylogenomic grouping of Ryukyuan "Type 1" with Eumandya suggests that when its adults are found (or hatchlings are raised to maturity), its arms will carry two rows of suckers. We also found an adult of a Ryukyuan "Type 4" (extending the notation of Sanchez et al.11 in the coastal waters of Kume Island, that groups with E. scolopes from Hawaii, suggesting a divergence based on geographic isolation in the North Pacific. We also find that Lusepiola birostrata (formerly Sepiola birostrata) is grouped with Inioteuthis japonica as sister to a clade containing Euprymna, Eumandya, and an unnamed sepioline from Port Kembla, at the northeast of Martin Island in Australian waters.
Among the sepiolins, we confirm the monophyly of Sepietta (only for nuclear-genome-based trees, see below). Adinaefiola, another genus erected by Bello19, with Sepiola ligulata Naef 1912 as its type species; was found sister to the Sepiola clade, but only in the tree based on amino acid mitochondrial sequences (mito_aa matrix) with a bootstrap value of 94% and a posterior probability of 1 (Fig. S2).
Outside the sepiolines, members of the subfamily Heteroteuthinae are the most elusive and underrepresented in studies of cephalopod systematics due to their oceanic lifestyles. The placement of several heteroteuthin remains controversial. Lindgren et al.9, with six nuclear and four mitochondrial genes downloaded from GenBank found that Sepiolina Naef, 1912 was sister to Heteroteuthis Gray, 1849 + Rossia Owen, 1834+ Stoloteuthis Verril, 1881; rendering the subfamily Heteroteuthinae polyphyletic. In contrast, our work supports the monophyly of Heteroteuthinae by including Stoloteuthis and Heteroteuthis in this subfamily, while Rossia was placed within the Rossinae (Figs. 1, 2, S2). Members of Heteroteuthinae included in this study formed a sister group to a monophyletic Rossinae (Figs. 1, 2, S2). Semirossia, however, rendered the Rossia clade paraphyletic. Further discussion about the position of Semirossia is difficult because of the lack of information about the original source of this specimen in Kawashima et al.25.
The light organ and luminescence evolution
Bobtail squids are thought to use the bioluminescence of their light organ to camouflage them from predators while foraging and swimming at night through a mechanism called counter-illumination. This has been researched extensively using E. scolopes as a model system26,27,28. Unfortunately, the limited number of sequences available and the misidentification of bobtail squids in the GenBank database11,29,30 have hindered our understanding of the light organ evolution in the whole taxon.
Our robust phylogeny and Bayesian reconstruction of ancestral bioluminescence clarify how the light organ and its luminescence have evolved in the family Sepiolidae. Members of Sepiolinae comprise neritic and benthic adults with bilobed light organs, except for two genera: Inioteuthis from the Indo-Pacific region, and the Sepietta species from the Mediterranean Sea and the Atlantic waters. The ancestor of the Sepiolinae very likely possessed a bilobed light organ that harbored luminescent symbiotic bacteria (Fig. 3). This character persisted until the ancestor of the euprymnins and sepiolins. Assuming that R. minor is sister to the Sepietta clade (as shown with the nuclear-based dataset, Fig. 2), it is clear that the bilobed light organ was lost once in Inioteuthis and Sepietta, and simplified to a rounded organ in R. minor. The alternative scenario, where R. minor renders the Sepietta clade paraphyletic (based on mitochondrial matrices, Fig. 1), is less plausible as it implies that the light organ was lost twice in the Sepietta group, once in S. obscura and then in the ancestor of S. neglecta and S. oweniana; or alternatively that it was lost in the ancestor of Sepietta-Rondelentiola followed by a reversion of this character in the lineage of Rondelentiola.
Fig. 3: Ancestral character reconstruction (ASR).
ASR of (a) the shape of the light organ and (b) the origin of luminescence in the Sepiolida. The posterior probability of each state is shown as a pie chart, mapped tree generated in BEAST (based on mito_nt matrix, see below), with the outgroups removed.
The light organ is also present in all members of Heteroteuthinae. These bobtails are pelagic as adults, and their light organ appears as a single visceral organ rather than the bilobed form found in nektobenthic Sepiolinae. In contrast to the bacteriogenic luminescence of the light organ in E. scolopes31, previous studies in H. dispar3 failed to detect symbiotic bacteria and suggested that the luminescence has an autogenic origin. Thus, it seems plausible that the monophyly of Heteroteuthinae found in our study supports the findings in Lindgren et al.9 for convergent evolution of autogenic light organs associated with pelagic lifestyle in many squid, octopus, and Vampyroteuthis Chun, 19039,32.
Divergence time of Sepiolida
The absence of fossils for this group limited our calculations of divergence time to the use of secondary calibrations. These calibrations can provide more accurate estimates depending on the type of primary calibrations that are used33. We retrieved secondary calibrations from previous estimations in Tanner et al.15, who used eleven fossil records spanning from coleoids to gastropods in transcriptome-based phylogenetic trees. Specifically, we used the time for the splits of Sepia esculenta and S. officinalis (~91 Mya), Idiosepiidae, and Sepiolida (~132 Mya) and the origin of the Decapodiformes (root age, ~174 Mya) (Fig. 4). These calibrations and our robust phylogenetic trees allow us to investigate the events that shape the divergence of some clades of the order Sepiolida (Figs. 4, S4).
Fig. 4: A chronogram of sepiolids using complete mitochondrial genes.
Red dots indicate the nodes with secondary calibrations. K-Pg, refers to the Cretaceous-Paleogene boundary and MSC, to the Messinian salinity crisis.
Sepiolida appeared before the Cretaceous-Paleogene extinction event34, during the middle Mesozoic around 94 Mya (95% HPD = 60.61–130.72). This time frame coincides with the rapid diversification of several oegopsida lineages15,35. Our molecular estimates also indicate that radiation of Sepiolidae and Sepidariidae occurred around the Cretaceous-Paleogene boundaries and is concurrent with the rapid diversification of modern marine percomorph fishes around the globe, after the extinction of Mesozoic fishes36,37.
Among the species of Sepiolinae collected in the Mediterranean Sea for this study, only Sepiola robusta Naef, 1912, and Sepiola affinis Naef, 1912 are endemic to the Mediterranean Sea38. The distribution of the other species includes the Mediterranean Sea, North Atlantic Ocean, East Atlantic Ocean, and/or up to the Gulf of Cadiz. The confidence intervals for the split between the Mediterranean-Atlantic and Indo-Pacific lineages, and their diversification, overlap during the early Eocene to the beginning of the Oligocene (Figs. 4 and S4). This time interval coincides with the end of the Tethys Sea, which separated the Indo-Pacific from the Mediterranean and Atlantic region through the Indian-Mediterranean Seaway39,40. This separation also influenced the divergence of loliginid clades, coinciding with the split between the Eastern Atlantic plus Mediterranean clade (Loligo, Afrololigo, Alloteuthis) and Indo-Pacific clade (Uroteuthis and Loliolus) (~55 Mya based on Fig. 2 in Anderson and Marian41).
Our chronogram indicates that the ancestor of Sepiolinae arose prior to the early Eocene around 46 Mya (95% HPD = 25.16–69.49) (Fig. 4), already possessing a bilobed light organ hosting luminescent bacteria (Fig. 3). We estimate that the split between S. affinis and S. intermedia occurred around 2.62 Mya (95% HPD = 0.3–7.4) (Fig. 4) during the end of the Zanclean period, when the Atlantic Ocean refilled the Mediterranean after the Messinian salinity crisis42,43. While S. affinis is a coastal species with a narrow depth limit, S. intermedia inhabits a wider range of deeper waters. It is possible that two populations of their ancestor, each adapted to a different ecological niche and diverged sympatrically in Mediterranean waters, and, after the speciation, S. intermedia extended its distribution outside the Mediterranean to the Gulf of Cadiz44.
We also estimate that the split between H. dispar Rüppell, 1844 and H. hawaiiensis (Berry, 1909) occurred around 2.4 Mya (95% HPD = 0.46–5.88), coinciding with the closure of the Isthmus of Panama around 2.8 Mya45. Surveys of these species found H. hawaiiensis in the North Pacific and H. dispar in the North Atlantic Ocean and Mediterranean Sea46. A recent speciation event might be the reason for the lack of morphological differences between the two species46. Thus, these species may be rendered as cryptic species, a phenomenon increasingly reported in oceanic cephalopods47. The sister species of this cryptic species complex, H. dagamensis Robson, 1924, appeared before, around 6 Mya, and is reported with broad distribution in the South Atlantic Ocean off South Africa, the Gulf of Mexico, North Atlantic Ocean between Ireland and Newfoundland in Canada, and the South Pacific Ocean off New Zealand48,49,50.
The origin of the Heteroteuthis ancestor of H. dispar, H. hawaiiensis, and H. dagamensis can be placed in the Pacific Ocean. After the formation of the Isthmus of Panama, the northern population of Heteroteuthis might have split into H. hawaiiensis in North Pacific and H. dispar in the Atlantic Ocean (from where it also migrated to the Mediterranean Sea). Meanwhile, the formation of the equatorial currents isolated the southern population of Heteroteuthis and gave rise to H. dagamensis. Then, H. dagamensis extended its distribution from the Southern Pacific to the South Atlantic Ocean, the North Atlantic waters, and the Gulf of Mexico. Analysis of molecular species delimitation, however, suggests that H. dagamensis includes cryptic lineages among Atlantic and New Zealand populations30.
While the origin of Heteroteuthis might also be in the Atlantic Ocean, the higher diversity of heteroteuthins in the Pacific (H. hawaiiensis, H. dagamensis, H. ryukyuensis Kubodera, Okutani and Kosuge, 2009, H. nordopacifica Kubodera and Okutani, 2011, and an unknown H. sp. KER (only known from molecular studies49)) than at the Atlantic (H. dispar and H. dagamensis), make its origin at the Atlantic less plausible. Moreover, the Atlantic Heteroteuthis were found nested within Heteroteuthinae species from the Pacific, supporting Pacific Ocean origin (Figs. 1, 4).
By sequencing the genomic DNA of sepiolids at low coverage, we recovered complete mitochondrial genomes and nuclear ribosomal genes for most of our collections. Furthermore, mapping reads to the reference genome of E. scolopes allowed us to retrieve additional nuclear-ultraconserved regions. We demonstrate that these nuclear and mitochondrial loci are useful to reconstruct robust phylogenetic trees, especially when the transcriptomes of specimens are difficult to collect, as for sepiolids inhabiting oceanic environments. Finally, our study integrated genomic DNA sequencing with confident morphological identification, which helped to reconstruct the ancestral character of the light organ and its luminescence in sepiolids, and clarify how major lineages have evolved, establishing the existence of distinct Indo-Pacific and Mediterranean-Atlantic subfamilies of Sepiolinae. Our collections and genomically anchored phylogenies will provide a reliable foundation classification of sepiolids for future studies.
Specimens and data collection
For this study, we sampled 32 different species of bobtail and bottletail squids. Samples were collected in different locations of the Japan Sea, Western North Pacific Ocean, Atlantic Ocean, Mediterranean Sea, Tasman Sea, Great Australian Bight, Banda Sea, and South Pacific Ocean. Specimens comprised members of the family Sepiadariidae and all the valid subfamilies of Sepiolidae (Sepiolinae, Heteroteuthinae, and Rossinae). For each individual, a small piece of tissue from the mantle was stored in 70% ethanol for DNA analyses and the remaining animals were stored in museums. Details of our specimens are found in Table S1.
Individuals collected off the coast of mainland Japan and the Ryukyu Archipelago were previously identified in Sanchez et al.11. Mediterranean species were identified based on general morphology and/or the hectocotylus following Bello51. We also retrieved the full mitochondrial genome of Semirossia patagonica (E. A. Smith, 1881) (AP012226) and Sepiadarium austrinum Berry, 1921 (KX657686), mitochondrial genes from the transcriptome of Euprymna tasmanica (Pfeffer, 1884) (SRR2984339), and the nuclear reads of Euprymna scolopes (PRJNA470951) from the GenBank database. For the outgroups, we retrieved the complete mitochondrial genome of Sepia esculenta Hoyle, 1885 (NC_009690), Sepia officinalis Linné, 1785 (NC_007895) and Idiosepius sp. (KF647895), the complete 18S rRNA gene of Idiosepius pygmaeus Steenstrup, 1881 (AY557477.1), and a partial region of the 28S rRNA gene of Idiosepius pygmaeus (AY293684.1). While the proximal outgroup of the Sepiolidae remains unclear (with different tree topologies found based on transcriptome sequences in Tanner et al15, Lindgren and Anderson52, and Anderson and Lindgren17), we believe the inclusion of Sepiadariidae should prevent any conflict relationships within Sepiolidae.
DNA isolation, library preparation, and data filtering
We isolated the genomic DNA of our specimens using the DNeasy Blood and Tissue kit (Qiagen). Genomic libraries were constructed using Illumina TruSeq DNA LT Sample Prep Kit. Libraries were size selected to 300 bp, pooled, and sequenced on an Illumina Hiseq 4000 (2 × 150 bp paired-end run). We generated around 1.6–3.6X coverage per species (assuming the total number of reads, the read length, and a genome size of 5 GB reported for Euprymna scolopes10 (Table S1).
Illumina reads were demultiplexed, and low-quality reads were filtered using Trim Galore v. 0.4.0 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and adapter-trimmed with cutadapt53.
Genome skimming
Bioinformatic analyses were performed on the Okinawa Institute of Science and Technology (OIST) HPC cluster and the National Institute of Genetics cluster.
Mitochondrial genes
We assembled the mitochondrial genome de novo for all our collections using NOVOplasty 4.254 with the cytochrome oxidase I gene of each species or the closest relative species available on the GenBank as a seed and the complete mitochondrial genome of Idiosepius sp. Reid and Strugnell 2018 (KF647895) as a reference. The FASTA files recovered were annotated using the standalone ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). We selected protein-coding genes per species based on the best hit of their amino-acid sequences with the amino-acid sequences retrieved from the mitochondrial genome of Idiosepius sp. using the blastp in the standalone NCBI BLAST v2.10 (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/). We used the MITOS Web Server55 to annotate ribosomal and transfer genes. The information on the gene order from each mitochondrial genome was obtained from the overall results of the MITOS Web Server and ORFfinder.
Protein coding genes were codon-based aligned using MUSCLE 3.856 implemented in AliView57 and the borders were trimmed manually. Ribosomal RNAs were aligned using R-coffee58 and poorly aligned regions were deleted through Gblocks v0.91b59. Transfer RNAs were concatenated for further analyses.
Nuclear genes
We assembled the nuclear ribosomal genes 28S and 18S using NOVOplasty. The seed used was a conserved region from multiple 18S and 28S sequences retrieved from GenBank and our alignment. The contigs retrieved were annotated in RNAmmer 1.2 Server60. Alignment and trimming were performed following the same methodology as the mitochondrial ribosomal genes.
Ultraconserved loci
We only considered 24 species sequenced with a depth of coverage higher than 2X. Illumina reads were aligned to the reference genome of Euprymna scolopes using the bwa mem algorithm from the Burrows-Wheeler Aligner (BWA) v0.7.1761 with the default mode. We filtered bam files with reads mapped with a quality of at least 40 using Samtools v1.962 and marked duplicate reads with Sambamba v0.7.163. Using these bam files, we generated a list of regions shared between at least two species using PHYLUCE64. This list was used to generate a dataset of loci (UCEbob) that are shared in a minimum of 14 species (58%) to recover enough polymorphic sites for the phylogenetic analyses (see below).
For UCEbob, we first created VCF files for each species per shared loci using freebayes v.1.3.265 with the settings: -min-coverage 2, -report-monomorphic, -limit-coverage 5, and -use-best-n-alleles 1. Then, VCF files were used to reconstruct aligned FASTA files of DNA sequences using custom scripts. For each locus, we removed sites with gaps presented in more than 50% of the species using Gblocks v0.91b59.
Apart from the UCEbob matrix, we also created different alignment matrices: (1) mito_aa: with amino acid sequences from mitochondrial genes, (2) mito_nc: with nucleotide sequences from all coding and non-coding mitochondrial genes, (3) nuclear_rRNA: with the nuclear ribosomal genes.
For each matrix, we performed maximum likelihood (ML) phylogeny in IQ-TREE66 with the best model and partition scheme selected by ModelFinder67, and 1000 replicates of ultrafast likelihood bootstrap68. Additionally, we performed a phylogenetic inference with a Bayesian approach in Exabayes v.1.569 for all matrices using a partition per gene and the GTR + Gamma model of substitution. Two independent chains of more than 1.5 × 106 generations were run in parallel, sampling every 1000 generations. The chains' convergence was validated when the average standard deviation from split frequencies was less than 0.5% and the ESS more than 200. Finally, we discarded the first 25% generations as burn-in, and the 50% majority consensus tree was summarized using the Consensus script from Exabayes.
Ancestral character reconstruction
We used BayesTraits v370 with the multistate model of discrete traits to obtain the posterior probability of the light organ shape and the luminescence origin of ancestral sepiolids. We coded the shape of the light organ as bilobed, rounded, and absent; and luminescence origin as autogenic, bacteriogenic, and absent. For taxa with no information about the state of the trait analyzed, we assume such a state is the same as its congeneric species.
We applied the reversible jump MCMC model with hyper prior exponential distribution of interval from 0 to 30, as this prior produces the best acceptance rate (average of 30%). As the input trees, we used 24335 trees generated from our divergence time analysis in BEAST (see below) after 25% of burn-in. We run BayesTraits with 1 million interactions, sampling every 1000th iteration; and plot the mean probabilities of each character in the ultrametric tree generated with the mito_nt matrix using the R package ape71.
Divergence time
Divergence time for several lineages within Sepiolida was estimated using a Bayesian approach implemented in BEAST v2.672 with nucleotides sequences of the mitochondrial gene (the mito_nc matrix). The analysis in BEAST was run with the best model of substitution for each partition selected by bmodeltest73, the Yule model of speciation, and the fast relaxed lognormal clock. We used internal calibrations estimated in Fig. S4 of Tanner et al.15 as priors with the normal distribution and the mean in real space as follows: Decapodiformes Leach, 1817 (mean: 174.22 Mya; SD: 26), Sepiida Zittel, 1895 (mean: 91.13 Mya; SD: 20; and "use originate"), and for the split of Idiosepiidae Appellof, 1898 and Sepiolida (mean: 132.37 Mya; SD: 26; and "use originate"). The analysis was run twice for 160 million generations, sampling every 5000 generations. The convergence of ESS > 200 was evaluated in Tracer v.1.7.174 and the chronogram was annotated in TreeAnnotator using the topology of the maximum likelihood tree constructed with mito_nc matrix, with a burn-in of 10% and mean node heights.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Input and output for analyses in BayesTraits, and alignments, best model and partition scheme for each matrix can be found in FigShare (https://figshare.com/s/1e0decb1d073a34fee2a). Raw reads can be found in the GenBank database under the BioProject number PRJNA640585.
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This project was funded by the Okinawa Institute of Science and Technology (OIST) to the Molecular Genetic Units. D.S.R. is also grateful for the generous support of the Marthella Foskett-Brown Chair in Biology and from the Chan-Zuckerberg BioHub. We wish to thank the staff of the OIST DNA sequencing section for the library preparation and sequencing. G.S. and M.T. wish to especially thank Amanda Reid from the Australian Museum Research Institute for her help in identifying Rossia sp., Inioteuthis japonica, and Euprymna Type 4; and for providing samples of Euprymna pardalota, Iriodioteuthis sp., Sepiolinae sp., and Heteroteuthis hawaiiensis used in this study. M.T. also wants to thanks to Professor Kat Bolstad, head of the AUT Lab for Cephalopod Ecology & Systematics at the Institute for Applied Ecology New Zealand for kindly sharing the specimens of Heteroteuthis dagamensis and Sepiadariidae sp., and to Professor Jan Strugnell from the James Cook University in Australia for Stoloteuthis sp.. F.Á.F-Á wants to thank Elena Guerrero (ICM‐CSIC) for performing the curation of some morphological vouchers in the Biological Reference Collections (CBR‐ICM) at the Institut de Ciències del Mar (ICM-CSIC), Barcelona, Spain. S.C. wishes thanks to Dr. Hiroshi Miyagai who through the Fisheries Cooperative Association of Hamasaka in Hyogo Prefecture, collected samples of Rossia sp. and to Dr. Hideo Takahara from the Fisheries and Oceans Hakodate for samples from Niigata Prefecture. J.J. and S.C. also wish to thank Ryo Minemizu from Ryo Minemizu Photo Office for the collection of Euprymna sp. Type 4. F.Á.F-Á was supported by the Irish Research Council-Government of Ireland Post-doctoral Fellowship Award (Ref. GOIPD/2019/460). M.T. is funded by a Ph.D. fellowship from the Irish Research Council and is supported by the Dr. Tony Ryan Research Fund.
These authors contributed equally: Gustavo Sanchez, Fernando Á. Fernández-Álvarez.
Graduate School of Integrated Science for Life, Hiroshima University, Higashi Hiroshima, Hiroshima, Japan
Gustavo Sanchez
Molecular Genetics Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan
Gustavo Sanchez, Chikatoshi Sugimoto, Jeffrey Jolly & Daniel S. Rokhsar
Ryan Institute and School of Natural Sciences, National University of Ireland Galway, Galway, Ireland, UK
Fernando Á. Fernández-Álvarez, Morag Taite & Louise Allcock
Department of Molecular Evolution and Development, University of Vienna, Vienna, Austria
Oleg Simakov
Department of Genetics, Evolution and Environment, Centre for Life's Origins and Evolution, University College London, London, UK
Ferdinand Marlétaz
Department of Molecular and Cell Biology, Life Sciences Addition #3200, Berkeley, CA, USA
Daniel S. Rokhsar
Chan-Zuckerberg BioHub, San Francisco, CA, USA
Fernando Á. Fernández-Álvarez
Morag Taite
Chikatoshi Sugimoto
Jeffrey Jolly
Louise Allcock
G.S. and D.S.R. conceived and designed the project. G.S. performed computational analyses with input from D.S.R. and F.M.. G.S. and F.Á.F-Á. interpreted phylogenetic trees. G.S., O.S., C.S., and J.J. collected samples from Japanese waters. F.Á.F-Á. collected and identified samples of the Atlantic Ocean and the Mediterranean Sea. M.T. and L.A. collected and sequenced samples from the South Pacific Ocean. G.S., F.Á.F-Á. and D.S.R. wrote the manuscript. All authors provided comments on the paper.
Correspondence to Daniel S. Rokhsar.
Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work.
Sanchez, G., Fernández-Álvarez, F.Á., Taite, M. et al. Phylogenomics illuminates the evolution of bobtail and bottletail squid (order Sepiolida). Commun Biol 4, 819 (2021). https://doi.org/10.1038/s42003-021-02348-y
DOI: https://doi.org/10.1038/s42003-021-02348-y
Unravelling the phylogenetic and ecological drivers of beak shape variability in cephalopods
Antoni Sánchez-Márquez
Joan Navarro
Reviews in Fish Biology and Fisheries (2022)
Two new 'bottletail squids' (Cephalopoda: Sepiadariidae: Sepioloidea) from New Zealand, with new observations on Sepioloidea pacifica (Kirk, 1882)
Jaever M. Santos
Kathrin S. R. Bolstad
Heather E. Braid
Marine Biodiversity (2022)
Communications Biology (Commun Biol) ISSN 2399-3642 (online)


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The QPR will be providing a suicide prevention training at Eastside's Caberet Theater located at 603 E. Green Street in Butler. The training will take place January 23, 2019 from 5:30-8:00 pm.
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text:
local wx = require 'wx'
local utils = require 'packagemanager-gui/utils'

local TimerStatic = { defaultWindow = nil }
setmetatable(TimerStatic, TimerStatic)

local Timer = {}
Timer.__index = Timer

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    self.running = true
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return TimerStatic

redpajama_set_name: RedPajamaGithub


text:
Dmitry Smirnov
During the last 2 years Dmitry was invited as a soloist by the Mariinsky Theater Orchestra Saint-Petersbourg, the Moscow Philharmonic State and the Chamber Orchestra of Moscow, the Lucerne Festival Strings, the Sinfonieorchester Basel, the Argovia Philharmonic and others with Concertos by Haydn, Mozart, Nielsen, Schumann, Stravinsky, Bartok, Prokofiev, Shostakovich, Bernstein, He was among the "Jeunes Etoiles" at Gstaad Menuhin Festival 2019. In the season 2022-23 his schedule also includes the performance of Mozart Violin Concerto on historical instruments with Accademia Theresia conducted by Giovanni Antonini. Past engagements include performance of chamber music with Sol Gabetta at SOLsberg Festival, the Debut recital with pianist Marco Scilironi in LAC for LuganoMusica on 21. April 2021 as well as a Debut solo recital during Lucerne Festival on 19. August 2021. Still in 2021 he releases his first CD with works by Bach and Bartok (FHR London). He is working with an ensemble "Il Pomo d'Oro'', with Heinz Holliger for "Swiss Chamber Concerts", with Giovanni Antonini for "Haydn 2032 Project". In 2018 he founded his own chamber ensemble, the "Camerata Rhein" in Basel for experimental and film music performances. In 2021 he created the string trio "Infernale" performing only modern music. Dmitry was born in St. Petersbourg, and studied at the Rimsky-Korsakov Saint-Petersburg State Conservatory (Elena Zaiceva), afterwards at the Lausanne Institute of Advanced Musical Studies (Pavel Vernikov) and at the Basel Music Academy (Rainer Schmidt). Dmitry won prizes and awards of international music competitions among which are:
ARD Musikwettbewerb – (2st Prize and GENUIN Prize 2021)
Concours international Long-Thibaud-Crespin (Paris, 3d Prize 2018 and Prize Etienne Vatelot for best interpretation of contemporary music)
Concours international de violon Tibor Varga (Sion, Switzerland 2015) - 1st Prize and audience award
Menuhin International Competition for Young Violinists (Cardiff, 2008) – 2nd Prize
International Oistrakh Competition (Moscow, 2006) – 1st Prize and Special Award
He is playing on a violin (2018) made by Philipp Bonhoeffer and a viola (2019) from the same maker. His second double CD is planned to be released in Summer 2022 with the works of Eugene Ysaye and rare salon music of the early 20th century as Chansarel, Crickboom, Godowsky and others.


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