text:
Definition of Snowmobile safety
Snowmobile safety: Snowmobiles pose a significant risk to children, adolescents and young adults. Head injuries are the leading cause of mortality (death) and serious morbidity (disease). They are due mainly to snowmobilers colliding, falling, or overturning. Children also are injured while being towed in a variety of conveyances by snowmobiles.
Although there is no uniform code of state laws governing the use of snowmobiles by children and youth, recommendations have been made in a Policy Statement on Snowmobiling Hazards by the American Academy of Pediatrics (Pediatrics, vol 106, pp 1142-1144, November 2000). These recommendations include but are not limited to the following points:
Last Editorial Review: 6/14/2012
Medical Dictionary Definitions A - Z
Search Medical Dictionary
From WebMD
Fitness Resources
Featured Centers
- Early Care for Your Premature Baby
- What to Eat When You Have Cancer
- When to Take More Pain Medication


text:
Expedia.com offers a selection of 15 hotels in Castelsardo. You are sure to find cheap deals and discount rates among them with our Best Price Guarantee. No matter what type of hotel strikes your fancy, we will let you search for your preferred one to accommodate your travel needs.
Expedia.com will not only save you time, but also money when booking your stay at a hotel in Castelsardo. Making reservations so you can stay within your budget is easy with us.
Expedia.com even provides you with a map so you may find hotels in the Castelsardo neighborhood you prefer.
Go ahead. Enter your travel dates to view the best offers on hotel rooms in Castelsardo, Italy now!
Looking for local hotels nearby? Check out the new hotels near me page.

subset: commoncrawl


text:
Director Jonathan Glazer promised to cut Scarlett Johansson's nude scene from new movie Under The Skin if he felt the footage was in any way gratuitous.
The pregnant star's alien character strips in a pivotal scene in the movie and Johansson admits she struggled with the idea of having to disrobe, until the filmmaker assured her he'd only use the footage if it was necessary for the storyline.
She tells Wenn, "You have to weigh the value of the risk that you're taking. Is this gratuitous? Is this a vanity project? Or is this an important part of this character's journey to self discovery? What's the game? Of course the last thing Jonathan wanted to do was take the audience out of the story and have this nudie alien shot!
"Going into this we talked about it and he said, 'I do not want that at all, so, if the value of storytelling is suddenly overshadowed by the freak factor - for lack of a better term, then I'm not gonna put it in.' So knowing the door was always kind of open and it would be there if it was right made me much more comfortable."
But the actress insists fans shouldn't think her flesh flash will be repeated: "I'm not a provocative person and it's not like I live for being photographed in the buff!
"It actually made me aware when I was doing it of all the kind of judgements and how self-conscious you can be about the way you look and your own vanity. I had to be liberated of that and it was challenging because I realised I really am self-conscious about this."


text:
The contract between Microsoft & ATVI has been renewed...unsurprisingly.
With that bein said, please do not come on here asking if we will get the DLC simultaneously.
Do not think that subscribing to Elite will "buck" the deal and get you DLC the same time or before no Premium Xbox players.
For that matter, why even subscribe to a service that only gets you a few pennies off the original price, that promotes crap matchmaking if you do subscribe, that barely allows you to play the maps you have subscribed to?
sigh* now we get to wait longer instead of doing the fair thing and releasing the dlc's for both systems. But as always money talks. -_-
just ripped up my pre order reciept not gonner bother with BO 2 now as rumour has it microsoft have also asked for a longer period of time before DLC hits PS3 ( approx 3 months )
Well this is just one more thing on a long list of reasons as to why I will not be buying Black Ops II. I just watched the E3 preview and that sealed the deal. I was originally thinking would get it if all the reviews are positive and some of the major issues are fixed but after hearing this and watching the preview I am out, time to find something new.
yeah in that case they can shove their ps3 elite sells right up their asses. hahaha
I somewhat wonder if they keep this deal going because they cannot make the maps simultaneously. In the sense, we get a port anyway, so maybe they need time to port the maps to our console. Oh well, we always end up getting the maps Beta tested anyway.
Oh yeah. They hurt themselves with that.
@least i only lost out on a fiver
I wouldn't see you as anything other than a intelligent fellow. I will state though, wait until more Multiplayer info comes out.
Who dude! It's CoD, we know the mp info for years!


text:
Alumni Bulletin - Winter 2009 - (Page 18)
SCIENCE • TECHNOLOGY • ENVIRONMENT • POLICY • SOCIETY Nelson Tansu Tim Coull Sudhakar Neti FINDING ENERGY SOLUTIONS When you turn on an incandescent light bulb, more than 95 percent of the electricity needed to power it is wasted as heat. A fluorescent lamp is about four times more efficient, but it contains mercury, which can pose environmental and health concerns. So imagine if there was a way to increase the energy efficiency of an incandescent light bulb by 10 times, from 5 percent to 50 percent—without the harmful environmental side effects of fluorescent lighting. That's what Nelson Tansu, the P.C. Rossin Assistant Professor in the department of electrical and computer engineering, and his team are working on using semiconductor technology at Lehigh's Center for Optical Technologies (COT). Solid-state lighting, a technology that relies on light-emitting diodes (LEDs), which emit light from semiconducting materials, could significantly reduce the amount of energy needed to light houses, schools, office buildings, and more. LEDs illuminate cell phones, car dashboards, and the backlighting in the liquid crystal displays in laptops and TVs, and are also used in a growing number of traffic signals and city and highway billboards. Meanwhile, Sudhakar Neti, professor of mechanical engineering and mechanics, is working on how to more effectively harness the power of the sun to generate electricity. Neti and four other Lehigh researchers recently received a $1.5 milToday, about one-third of the energy produced in the United lion grant from the U.S. Department of Energy to explore novel heat-transfer methods that will States is used to generate electricity. And about 22 percent enable solar thermal systems to store energy long enough so it can be generated on cloudy days of that electricity is used for lighting. and at night. "By greatly improving the efficiency of the 22 percent of Solar thermal systems employ panels of mirrors to concentrate sunlight and convert it into electrical power that we currently use for lighting, solid-state heat, which in turn is used to drive turbines or engines to generate electricity. Finding a solution to lighting can significantly reduce the worldwide demand for the storage problem is one of the keys to making solar energy viable on a large scale, Neti says. energy, while still providing reliable and environmentally By recognizing how inextricably linked issues involving energy and the environment are and by friendly solutions," says Nelson Tansu, who with Volkmar fostering collaboration across disciplines, the new STEPS initiative will be a boon to researchers Dierolf, an associate professor of physics and COT faculty working in these areas, Tansu says. researcher, recently received a three-year grant from the DOE "Lehigh is putting significant energy behind issues that matter," he says. to study methods of improving the efficiency of white LEDs. Tim Coull '08, a mechanical engineering graduate who is working with Tansu and Neti on The $600,000 award is being matched by $150,000 in heat transfer issues related to thermo-electric generation as he pursues a master of sciadditional funding from the commonwealth of Pennsylvania. ence degree, says of STEPS: "It's really great to have an actual program here that recThe research on potential storage solutions for solar ognizes and legitimizes the idea that all of these global issues need to be addressed. thermal systems being conducted by Sudhakar Neti's And students get to work on them. It's a visible thing." multidisciplinary group could ultimately address two of the main factors that have held solar energy back from more widespread use: cost and availability. 18 LEHIGH UMNIULLETIN AL B Why it matters
Table of Contents for the Digital Edition of Alumni Bulletin - Winter 2009
Lehigh Bulletin - Winter 2009
From the President's Desk
On Campus
Arts & Culture
Teaming Up to Tackle Global Challenges
Positively 4th Street
A Sweet Job
From the Publisher's Desk
Alumni News
Alumni Bulletin - Winter 2009
If you would like to try to load the digital publication without using Flash Player detection, please click here.


text:
Amarillo Youth Choirs Inc. will have auditions for the 2014-2015 season for Amarillo Boy Preparatory Choir and Amarillo Girl Preparatory Choir from 4:30 to 6 p.m. today and Thursday at St. Stephen United Methodist Church, 4600 S. Western St.
ACE Scholarship program at Palo Duro, Caprock and Tascosa high schools will be the topic of a program for the League of Women Voters meeting at 11:30 a.m. Sept. 3 at Red Lobster, 3311 W. Interstate 40.
League of Women Voters of Amarillo will host a Meet the Candidates reception and program Tuesday at Amarillo College Downtown Campus auditorium, 1314 S. Polk St...
The Amarillo Council of Beta Sigma Phi has named Lou Ann Garrett as the sorority's 2012-13 Woman of the Year and will honor her during a tea at First Christian Church on Nov. 3.
Do you have questions for candidates seeking the Texas Senate District 31 seat?
On Monday, the League of Women Voters of Amarillo will host a candidates reception at Sunset Center at 5:45 p.m. All the local/area Republican and Democratic candidates for...
The League of Women Voters of Amarillo will host a Candidates' Reception from 6:30 to 8 p.m. Tuesday at Amarillo National Bank Plaza II for all candidates on the ballot for Potter and Randall counties, including the 13th District Congressional race.
The death penalty, gun control and raising taxes were among topics broached Thursday at a League of Women Voters of Amarillo "Meet the Candidates Forum" for local judicial races at the Bank of America's Virgil Patterson Auditorium...
Ceta Canyon Camp & Retreat Center, 37201 Farm-to-Market Road 1721 in Happy, will host a Father & Son Retreat on Friday through Sunday. Information: (806) 488-2268.
As a native son of Amarillo and a decorated Vietnam War veteran, I was appalled and saddened at the reception I received at the League of Women Voters of Amarillo candidate forum April 28. I was there to represent my brother, a candidate for mayor, who was in France visiting his family.
Amarillo Globe-News ©2013. All Rights Reserved.


text:
Transform yourself into a head-turning sensation by slipping into this sublime Tadashi YV923L gown! Its lacy bodice is decked out in romantic floral patterns and topped off by a high, scalloped neckline. Touchably soft material drapes down from one shoulder, crosses the bodice and continues down alongside the elegant, floor-length skirt.
Find a lower price and we will match and beat it by 5%
We are an authorized retailer of all the dresses we sell. We only sell 100% authentic dresses.
Please refer to our return policy.
Free ground shipping on orders over $50.
5 out of 5 (5 Reviews)
Read More


text:
Easy Recycled Robot
Kids love robots and this craft allows them to make their own special robotic pal to include in their dreams of visiting other planets.
Empty container (we used a country time lemonade container)
Aluminum foil
Black marker (a sharpie works best since you are writing on aluminum foil)
Silver pipe cleaners
2 googly eyes
Soda bottle lids (optional as feet)
Put a few lines of glue on your empty container and wrap the container with aluminum foil. Make sure to glue the edges down. We found that this was the most frustrating part for our daughter. To make it easier use thinner, longer strips of aluminum foil or get it started and then have your child help you tuck in the ends.
Have an adult poke a hole in each side of the container for the arms.
Wrap the pipe cleaner around the marker and then remove it. This gives you a nice spiral pipe cleaner that works great as an arm. Do this with two pipe cleaners and then insert them into the holes on your container.
Glue on two googly eyes. Use the marker to draw a mouth on.
Glue the sequins on the belly of the robot to look like buttons.
Glue three bottle caps to the bottom of the container to serve as feet or wheels
This craft reprinted courtesy of AllKidsNetwork.com.
Listed under:
More Boys Crafts, Recycled Containers and Lids, Space Crafts
Tagged as:
What do you think?
Everyday Dishes & DIY (formerly Cherylstyle) is an online lifestyle media company stirring up dependable recipes and crafts for the everyday family. We collaborate, develop, test and re-test all original content in-house, working out the kinks so you don't have to! Everydaydishes.com publishes new recipes, diy projects, craft ideas and videos daily.


text:
What was the legacy of the 1871 Paris Commune at the time of the Russian Revolution of 1905? Was it understood as part of an obsolete revolutionary tradition or as something new? The French left responded equivocally even as the 1905 revolution provided a comparison, and as Lenin identified the practical lessons of 1871. Once the Bolsheviks came to power, the Commune would be given new life as a legitimizing agent for the USSR. French Communists would also adopt the Leninist reading. This article examines the "rupture" of 1905 and the transformation of the revolutionary tradition from the perspective of the French left, Parisian workers and Russian émigrés, and in light of the funeral of the former Communard Louise Michel.
Ruling elites often make pernicious national myths for instrumental purposes, creating divergent historical memories of the same events in different countries. But they tend to exploit international history disputes only when they feel insecure domestically. Societal reactions to elite mythmaking, reflected in radicalized public opinion, can reinforce history disputes. During the 1950s–1970s, China avoided history disputes with Japan to focus on geostrategic interests. Only from the early 1980s did domestic political incentives motivate Beijing to attack Japanese historical memory and promote assertive nationalism through patriotic history propaganda, which radicalized Chinese popular views about Japan. Media highlighting of Japan's historical revisionism exacerbated societal demands to settle war accounts with Japan, while factional politics within the Chinese Communist Party made it difficult for the top leaders to compromise on the bilateral "history issue."
Drawing on newspaper articles, contemporary architectural writings, diaries and archival records, this essay examines National Socialism's afterlife in the early Federal Republic in a local context. Focusing on debates that shaped the postwar reconstruction of Bremen's historic market square and its monuments, it shows how complicated the recovery of local history proved to be. The interplay of silence on issues of guilt, narratives of victimization and stories of reconstruction explains why postwar efforts to reestablish a historically grounded sense of Heimat furthered the acceptance of the Federal Republic but retained a tentative, provisional feel a decade after the war's end.
Since the instigation of the peace process in Northern Ireland, issues relating to the commemoration of the political conflict there have moved to center stage in media, political, cultural and academic discourses. This essay locates the film Bloody Sunday (Paul Greengrass, 2002) within the framework of a growing body of literature on historical trauma and memory studies in order to explicate the ways in which it discursively "works through" a historical event of traumatic magnitude. Analysis considers the film's relationship to its historical referent, memorial activity at its contemporary moment of production, and generic modes of address specific to the docudrama.
Historians are dumb witnesses to a culture wrangling with itself about its criminal past if they only narrate the sequence of historical controversies such as those that have dotted the German public landscape since the Holocaust. They need to be alive to the subterranean biblical themes flowing beneath the surface froth of events, linking past and present through the continuity of German political emotions that are necessarily collective and therefore sensitive to anxieties about accusations of collective, inherited sin. This article argues that the guilt/shame couplet so common both in public German and academic discourses about postwar Germany cannot account for the intergenerational transmission of moral pollution signified by Holocaust memory. In order to understand the dynamics of German political emotions, it is more useful to employ an alternative couplet: stigma and sacrifice.


text:
This product is already in your Wish List.
View Wish List
This product is now in your Wish List.
Frontier Natural Products, Maple Flavor, Alcohol-Free, 2 fl oz (59 ml)
By Frontier Natural Products33 Reviews
Expiration Date:?Aug 2017
Expiration Date
The length of time for the expiration date or "best used before" date depends on the type of product, as well as the brand.
Perishable items (such as flax oils or certain probiotics) generally have shorter expiration dates. Although our warehouse is fully air-conditioned, these more fragile items are put in cold storage (freezer or refrigeration unit) for maximum freshness.
Our receiving department does its best to verify and then enter the correct expiration dates for all incoming products. However, discrepancies do occur from time to time. This being said, the exceptionally high turnover at iHerb ensures that our inventory is among the freshest in the industry.
Shipping Weight:?0.5 lbs (0.23 kg)
Shipping Weight
The Shipping Weight includes the product, protective packaging material and the actual shipping box. In addition, the Shipping Weight may be adjusted for the Dimensional Weight (e.g. length, width & height) of a package. It is important to note that certain types of products (e.g. glass containers, liquids, fragile, refrigerated or ice packed) will often require protective packaging material. As such, these products will reflect a higher Shipping Weight compared to the unprotected product.
- Product Code: FRO-23081
- UPC Code: 089836230812
- Package Quantity: 2 fl oz (59 ml)
- Dimensions: 1.5 x 1.5 x 3.6 in, 0.3 lbs (0.14 kg)
Total: $17.78
Combo Discount: -$2.00
Together: $15.78
Product Overview
- All-Natural
- Kosher
- With Other Natural Flavors
Suggested Use
Add maple flavor or fudge, brownie, taffy and frosting recipes. Try a dash or two in baked beans, sweet potatoes, cookies and pies.
Glycerin, water, natural flavors (contains soy), malt extract (contains barley), caramel color.
iHerb Customer Reviews
I absolutely love the taste of maple syrup and I though that this would be a nice sugar free alternative. In a way it is, but I think that you have to pour a big quantity to get the flavour in baking. I think it evaporates easily, maybe because it is alcohol free. Click on my username for more reviews! If you thought that this review was helpful please feel free to press the 'yes' button.
I have been using a lot of real grade B maple syrup from NOW brand and this doesn't compare to the real thing ... a flavouring but not an authentic taste for me. If you have a problem with sugar then of course this will be better for you.


text:
Creamy Ranch Dip Recipe
Janice Freeman in Kewanee, Illinois shares her secret for a wonderfully thick and easy dip to dress up fresh veggies, crackers or chips—and it goes together with only three ingredients! "I just add cream cheese to the directions on the dressing envelope," she says, "and it's much better."
- 2 cups (16 ounces) sour cream
- 1 package (8 ounces) cream cheese, softened
- 1-1/2 to 2 envelopes ranch salad dressing mix
- Fresh vegetables, crackers or chips
- 1. In a small bowl, beat the sour cream, cream cheese and ranch dressing mix on medium speed until smooth. Transfer to a serving bowl; refrigerate until serving. Serve with vegetables, crackers or chips. Yield: 3 cups.
Reviews for Creamy Ranch Dip
© 2016 RDA Enthusiast Brands, LLC


text:
So Nancy Kwan will be showing up in San Jose for tonight's world premiere of the documentary on her life: To Whom It May Concern: Ka Shen's Journey; and she'll be there for the subsequent screening on Sunday at 7. There are some elders who still refer to the dress that Cantonese call a cheong-sam, and the Shanghai people call a qi pao as a "Suzy Wong". That's because of the way Kwan wore one in The World of Suzy Wong, a 1960 Hong Kong romance film by Richard Quine (Bell, Book and Candle).
This story of a Hong Kong call girl involved with an architect turned artist (William Holden) was considered the In the Mood for Love in its day; Kwan followed up this hit with the San Francisco-set Rogers and Hammerstein musical Flower Drum Song and thence to a lot of TV (Kung Fu, Hawaii 5-0) and a second career in Hong Kong cinema.
She's bound to have some stories to tell; Matt Helm fans will be happy to note that she was in The Wrecking Crew with Dino, and Sharon Tate. Though one winces to recall that her character was named "Yu-Rang". It was a different era, and Kwan survived the stereotypes well.
Also today: the 3-D Forum at 1:30 at Camera 12; the slacker comedy The Bone Man; Jarod Whaley's Hell is Other People at the Rep, 4:15; a repeat of The Real Revolutionaries, and an 11:45 pm screening of the talky caper film Little Fish, Strange Pond by Gregory Dark (ex of The Dark Brothers, of New Wave Hookers, et all) an ex-Stanford MFA now renowned as a video director.


text:
Despite the numerous and oft-touted risks, government follies, and frequent calls for recession by well-known prognosticators, the world did not end and equity markets did not decline in 2012. In fact, quite the opposite occurred; economies improved and equities returned the highest amount since 2009. Not even fixed income securities declined. If 2012 were a fairy tale, undoubtedly the year told the story of Cinderella.
For the year, the S&P 500 returned 16.0% (including dividends) and the Barclays U.S. Aggregate Bond Index returned 4.2%.
We are proud to say that we performed well in both an absolute and relative sense in 2012. Black Cypress equity portfolios were up over 18.5% in 2012, outperforming broad markets by 2.6% with less volatility. In fact, the year transpired largely as we expected.
From our December 2011 letter, Well-Positioned (dated January 17, 2012):
"In our fixed income positioning, we should experience positive returns–in both absolute and relative terms–if rates are flat or rise."
U.S. Treasuries and high quality bond rates were roughly flat in 2012. Our fixed income securities returned over 7.0%, besting the Barclays Aggregate by 3.0%. We were able to achieve this out-sized return with less duration due to our exposure to non-Treasury markets and therefore credit risk.
"While the market has risen over 14% since September of last year [2011]…our portfolio still has an 18.5% implied return."
During 2012, our equity portfolios rose almost exactly 18.5%. That was certainly a call we are happy to have gotten right, but we do not expect to be that precisely right again. Though, we are proud to say we have compounded capital at almost 18.0% per year since inception.
As we have noted in the past, we should do well on both a relative and absolute basis when markets are down, flat, or moderately up. However, when markets surge from pricey levels like they did in 2012, we should be expected to underperform on a relative basis. While this is our expectation of the way our portfolios will behave in various market environments due to the structure of our strategy, it is only an expectation. It was not the case in 2012, as we outperformed the S&P 500 by 2.6% despite its double digit gain.
We urge you to not expect the same from indices over the next several years. Mid-single digit returns, at most, is likely in our opinion, as asset returns leave us wanting. There are a few reasons for caution.
For one, current valuation levels lower expected returns. Estimates for S&P 500 earnings per share (EPS) for 2012 hover around $87. At today's closing price of 1,472.12, the S&P 500 is trading for 16.9x trailing earnings. Of course, the market's P/E must be put into context. And for today's P/E, the context makes the valuation look worse.
Profit margins are near peak so multiples trading above the long-term average on record EPS don't inspire cheapness. If we had a 17 multiple on recession-level earnings, then we could get excited. But an above average P/E on the highest earnings ever? Not so much. Last year's return was due almost entirely to multiple expansion; nominal earnings growth was essentially flat and negative if adjusted for inflation. This change in the growth rate of profits is troubling.
Since March of last year, EPS have been declining on both a nominal and real basis. Analysts are expecting $100.71 in EPS for next year, but the past two quarters' earnings actually declined. Will earnings grow almost 16% next year, as analysts expect, when sales growth rates have fallen for four straight quarters and grew just 3% in 2012? It isn't without possibility, but it does imply that profit margins will surge above 9.0% in 2013, a level that has never been reached.
Fixed income is in an even more precarious position. Government rates hover just above all-time lows. Credit spreads have narrowed and absolute yields are rock bottom. Yields on the most risky debt have plummeted thanks to Fed policy and as well as current low default rates due to lush liquidity available to finance each and every debt offering at lower and lower yields. Junk bonds for instance, which returned over 14% in 2012, now yield just 6.0%, the lowest rate ever.
It was certainly a Cinderella story in 2012, but we can't help but think that the clock is about to strike midnight and our glorious horse-drawn carriage will turn back into a pumpkin pulled by mice.
Rich equity and bond prices are not the only reason for tempering asset return expectations. Fiscal policy is about to become a drag on growth. We do not yet know how large a drag because not even tax policy has clarity. While Republicans and Democrats voted for what will now be known as Obama's tax cuts, Democrats have recently stated their desire for additional revenue. Spending cuts were absent from the fiscal cliff deal and a battle looms in March over sequestration, the debt ceiling, and how to reduce the deficit. The one known, however, is that austerity–in some amount–is coming to the U.S. in 2013 to dampen growth.
We wrote in last year's letter,
"None of the data is overly strong, but enough so to make us still believe the U.S. economy will continue to "muddle through" over the next year."
Although several prominent economists and equity managers made calls for a recession, the economy did in fact muddle through, growing approximately 2.0% in 2012.
On the bright side, the U.S. economy has much improved. Housing, once a major drag on growth, has seen price increases and building volumes surge. Auto sales are also recovering, employment continues to make gains, and measures of income have started to grow again. Gasoline and natural gas prices should remain moderate over the coming years, as the U.S. energy production resurgence continues. Banks are better capitalized and are lending again. These, coupled with loose Fed policy, should help to offset some of the fiscal tightening and keep the economy growing, albeit slowly, in 2013.
These positives won't likely lead to a banner year, but more moderate returns. Not quite the story book ending we may want, but if the slipper fits…
To our partners: thank you for the opportunity and privilege to serve you another year!


text:
Get notified about updates to this publication
Page 1
231PHYTOLOGIA BALCANICA 12 (2): 231–244, Sofia, 2006Evolution of Veronica (Plantaginaceae) on the Balkan PeninsulaDirk C. Albach Institut für Spezielle Botanik, Johannes Gutenberg-Universität Mainz, Bentzelweg 9b, 55099 Mainz, email@example.com Received: October 10, 2005 ▷ Accepted: March 30, 2006Abstract. With more than 6500 species of native seed plants on the Balkans and almost a third of them endemic, the Balkan Peninsula is known to be a place for diversification and formation of new species and an important refugium during the Ice Ages. One plant group, which exemplifies this well, is the genus Veronica (Plantaginaceae, formerly Scrophulariaceae). Four groups from this genus (V. subg. Stenocarpon; V. subg. Chamaedrys; V. subg. Pseudolysimachium, V. alpina-complex) display putative tertiary relict species, speciation within Pleistocene refugia and Pleistocene or Holocene speciation by hybridization and polyploidization on the Balkan Peninsula. I here review earlier published results for these groups and present new data. DNA sequence analyses from the nuclear ribosomal DNA (ITS) and plastid genome (trnLF region) were examined so as to shed more light on the relationship of the species from the Balkans. In addition, AFLP fingerprints were used to study V. subg. Pseudolysimachium, which exhibits limited DNA sequence divergence. Results support the distinctiveness of taxa from the Balkans as a divergent group of plants on the intra- and interspecific level. Limited resolution and support of the results further demonstrate the need for another marker system to continue the study of evolution of these plants of the Balkan Peninsula.Key words: AFLP, Balkan Peninsula, hybridization, phylogeography, polyploidy, VeronicaIntroductionThe plant-life on the Balkan Peninsula is richer than any comparable area in Europe. There are more than 6500 species of native seed plants in the Balkans (Turrill 1929; Horvat & al. 1974: 72–78; Polunin 1980: 22). Several factors are responsible for this diversity. Apart from human impact, the combination of var-ied topography and habitats at the crossroads of sev-eral major floras (Central European, Mediterranean, Anatolian, and Pontic), which had found favora-ble refugia there to survive the Ice Age, is exception-al. Almost one-third of the species is endemic to the Balkans (Turrill 1929; Horvat & al. 1974: 72–78). The Balkan Peninsula is known as an Ice Age ref-ugium for many important plant species (Willis 1996; Taberlet & al. 1998). The climate during the maxi-mum glaciation did not allow continuous forest veg-etation but was rather a steppe with scattered trees and isolated pockets of forest (Frenzel 1968). Steppe species included mainly species from Asia and led to a major influx of Asian taxa into the European flora (Frenzel 1968; Ozenda 1988). In addition to being a source for the recolonisation of Europe (Taberlet & al. 1998; Hewitt 2000; Schmitt & Seitz 2002), the Balkan Peninsula was a place for further diversification and speciation, given its richness in endemic species. The Pleistocene climate shifts have led to cycles of isolation of the plants in refugia and to their subse-quent spread in more favorable times for a more con-tinuous distribution. Whereas the more temperate el-ements were restricted to refugia in glacial times, the
Page 2
232Albach, D. & al. • Veronica on the Balkan Peninsulacold-adapted plants had likely a more or less continu-ous distribution during these times and were restrict-ed to the refugia during the interglacials. Many alpine plants might have originated in the Pleistocene in re-stricted alpine regions and had diversified owing to genetic drift (Zhang & al. 2001). Those taxa that were restricted in the Pleistocene to small refugia and after-wards expanded often did so into areas inhabited by plants from other refugia, which provided ample op-portunity for hybridization and formation of new spe-cies (Hewitt 2000).Veronica is a genus of the Plantaginaceae sensu APG (1998) and Albach & al. (2005), to which have been transferred most of the well-known genera of Scrophulariaceae from the Northern Hemisphere flo-ras. Veronica includes about 500 species (Albach & al. 2004a). It is distributed over much of the Northern Hemisphere and beyond and is ecologically highly di-verse, with species living in aquatic to dry steppe hab-itats, from sea level to high alpine regions. This diver-sity may explain the interest Veronica has evoked for a long time. Recently, molecular techniques and phylo-genetic analyses have been applied to Veronica and re-lated genera (Wagstaff & Garnock-Jones 1998; Albach & Chase 2001, Albach & al. 2004b, c). These studies have helped revolutionise our ideas about the evolu-tion of the genus and have led to a new phylogenet-ic intrageneric classification of Veronica (Albach & al. 2004a). Combined with the vast amount of informa-tion from other aspects of their biology, we have now a much better understanding of how major groups in Veronica are delimited, related, and evolved. In the present article I summarise what we currently know about the evolution of the species of Veronica na-tive to the Balkan Peninsula. Apart from a review of earlier publications, I present new sequence data on the species from V. subg. Stenocarpon and V. subg. Chamaedrys and AFLP-fingerprint data on V. subg. Pseudolysimachium.Materials and MethodsDNA Extraction, Amplification, SequencingThe total genomic DNA was extracted from herbarium material and silica gel-dried samples according to the 2x CTAB procedure of Doyle and Doyle (1987) and then washed twice with 70 % ethanol or using NucleoSpin plant DNA extraction kits (Macherey-Nagel; Düren, Germany) following the manufacturer's specifications. Nine sequences (seven for the ITS-region, two for the trnLF-region) are used here for the first time.The trnLF region was amplified with primers c and f of Taberlet & al. (1991) and includes the trnL intron, 3´ trnL-exon and the trnL-trnF spacer. ITS sequences were amplified and sequenced using the primers 17SE (Sun & al. 1994) and ITS4 (White & al. 1991) and in-clude ITS1, 5.8S rDNA and ITS2. PCR products were either separated on 1 % TBE-agarose gels; fragments corresponding to the expected size were excised and cleaned using the QIAquick™ PCR purification and gel extraction kit (Qiagen GmbH, Hilden, Germany) fol-lowing the manufacturer's protocols or cleaned using the Macherey-Nagel PCR purification kit (Macherey-Nagel; Düren, Germany) following manufacturer's specifications. Sequencing reactions (10 μl) were car-ried out using one μl of the BigDye Terminator Cycle Sequencing mix (Applied Biosystems Inc.). Both strands were sequenced on a Prism 377 automated se-quencer (Applied Biosystems Inc.). Sequences were assembled and edited using Sequence Navigator™ (Applied Biosystems Inc.) or Sequencher™4.1 (Gene Codes Corp., Ann Arbor, MI, USA). Assembled se-quences were manually aligned prior to analysis. Origin, voucher information and GenBank accession numbers for all sequences used in this study are giv-en in Table 1.AFLP FingerprintsAFLP profiles were generated following the estab-lished procedures (Vos & al. 1995) and according to the PE Applied Biosystems (1996) protocol with on-ly minor modifications (Tremetsberger & al. 2003). Genomic DNA (approx. 500 ng) was digested with MseI (New England BioLabs) and EcoRI (Promega) and ligated (T4 DNA-Ligase; Promega) to double-stranded adapters in a thermal cycler for 2hr at 37°C. Preselective amplification (5 μL reactions) was per-formed using primer pairs with a single selective nu-cleotide, MseI-C and EcoRI-A. Selective amplifica-tions were performed with the primer combinations MseI-CAT/EcoRI-ACT, MseI-CTC/EcoRI-AAG, and MseI-CAT/EcoRI-ACC. Selective amplification prod-ucts were run in a 5 % denaturing polyacrylamide gel with an internal size standard (GeneScan®-500 [ROX], PE Applied Biosystems) on an automated DNA se-
Page 3
233Phytol. Balcan. 12(2) • Sofia • 2006 quencer (ABI 377). Polyacrylamide gels run on au-tomated sequencers increase the resolution and de-crease the probability of scoring fragments of similar size as homologues. Fragments from the polyacryla-mide gel were analyzed using the ABI Prism Gene-Scan® 2.1 Analysis Software (PE Applied Biosystems) and Genographer 1.1 (Benham 1998). Peaks (i.e. frag-ments) were scored manually as present (1) or absent (0) in a readable region of bands from 50 to 500 base pairs in length and used to construct a presence/ab-sence data matrix with individual plants in rows and bands in columns. Origin and vouchers for all speci-men used in the AFLP analysis are given in Table 2.Data Analysis – sequencesData matrices were analysed separately and com-bined using maximum parsimony and maximum likelihood criteria in PAUP*4.0b10 (Swofford 2002). Potentially parsimony informative gap characters were coded as present/absent. Heuristic parsimony searches were conducted using 10 replicates, start-ing from random trees with tree bisection reconnec-tion (TBR) branch swapping and MulTrees in effect and no tree limit. Parsimony bootstrap analyses in-volved 1000 replicates with the same search param-eters as above but simple taxon addition. Maximum Table 1. Species name, origin, voucher information and GenBank accession number of accessions (ITS/trnLF).OutgroupsVeronica jacquinii Baumg. – cult. BG Bonn (Albach 70, WU – AF313000/AF513341); V. scutellata L. – Waldviertel, Austria (Dobes 7026, WU – AF509805/AF486393); V. serpyllifolia L. – Bonn, Germany (Albach 64, WU – AF313017/AF486400); V. triloba Opiz – Aphrodisias, Turkey (Albach 242, WU – AF509803/AF486366); V. triphyllos L. – Aphrodisias, Greece (Albach 244, WU – AF509795/AF486396); Data set of V. subg. Stenocarpon (Boriss.) M.M. Mart. Ort., Albach, & M.A. Fisch Paederotella pontica (Rupr.) Kem.-Nath. – Georgia (Sachokia, 01.9.1951, TBS – AF515214/AF486382); Veronica ciliata Fisch. – Qinghai, China (Miehe & al., 98-33313, GOET – AF515215/AF486385); V. densiflora Ledeb. – Altai (Staudinger s.n., SALA – AY741521/AY776282); V. erinoides Boiss. & Sprun. – Gioua Mts, Greece (Hagemann, Scholz & Schmitz 461, SALA – AY741523/n.a.); V. fruticans Jacq. – Scotland, UK (Viv Halcro VH030, K – AY144462/n.a.); V. fruticulosa L. – cult. BG Bonn (Albach 71, WU – AF313004/AF486383); V. lanosa Royle ex Benth. – Pakistan (Schickhoff 1377, GOET – AY540868/AY486442); V. lanuginosa Benth. – 13 km east of Mt Everest, Nepal (Dickore 6482, GOET – AF509793/AF486386); V. macrostemon Bunge ex Ledeb. – Altai (Staudinger AL23-18, SALA – AY741522/AY486441); V. mampodrensis Losa & P. Monts. – Velilla de Río Carrión , Spain (Martínez Ortega 713, SALA – DQ227331/ DQ227337); V. mexicana S. Watson – Mesa el Campanero, Sonora, Mexico (Fishbein 2586, TEX – DQ227332/n.a.); V. monticola Trautv. – Georgia (Ivanisvili 26.07.1983, WU – DQ227333 & DQ227334/n.a.); V. nummularia Gouan – Tosses, pr. La Molina, subida al pico pico Niu d'Aliga, Spain (Martínez Ortega 718, SALA – DQ227335/n.a.); V. saturejoides Vis. subsp. kellereri (Degen & Urum.) P. Monts. – Mt Pirin, Bulgaria (Albach 558, WU – AY144461/AY486450); V. thessalica Benth. – Olymbos, Greece (Raus & Rogl 5072, SALA – AF509792/AF513343).Data set of V. subgenus Chamaedrys (W.D.J. Koch) M.M. Mart. Ort., Albach & M.A. Fisch.Veronica arvensis L. – Stromberg, Germany (Albach 147, WU – AF313002/AF486380); V. arvensis – Richmond Park, UK (M. Sheahan 9, K – DQ227328/n.a.); V. chamaedryoides Bory & Chaub. – Peloponnes, Greece (Albach 393, WU – AF673611/AY673631); V. chamaedrys subsp. chamaedrys L. – Homefield Wood, UK (M. Fay 149, K – DQ227329/n.a.); V. chamaedrys subsp. chamaedrys – cult. RBG Kew, ex Norway (Albach 121, K – AF313003/ AF486377); V. chamaedrys subsp. micans M.A. Fisch. – Austria (Schönswetter 2567, WU – AY673616/AY673632); V. krumovii (Peev) Peev – Eastern Rhodopes, Bulgaria (Albach 484, WU – AY673612/AY673633); V. laxa Benth. – Pakistan (Dickoree 13042, GOET – AY673613/AF486378); V. magna M.A. Fisch. – Orbetia, Georgia (Albach 360, WU – AY6736152/AY673634); V. micrantha Hoffmanns. & Link – Portugal (Martínez Ortega 1754, SALA – DQ227330/ DQ227336); V. verna L. – Bad Kreuznach, Germany (Albach 149, WU – AF509789/AF486379); V. vindobonensis (M.A. Fisch.) M A. Fisch. – cult. BG Wien (M.A. Fischer s.n., WU – AY673614/AF510426). Table 2. Samples (with vouchers) used in the AFLP analysis of V. subg. Pseudolysimachium. Veronica bachofenii Heuff. – cult. Bot. Gard. Wien, ex Bot. Gart. Halle (Albach 978, MJG); V. barrelieri Schott ex Roem. & Schult. – Bulgaria (Vladimirov s.n., WU); Croatia (Schneeweiss & Schönswetter, WU); Croatia (5 individuals, Martínez Ortega 908, SALA); V. crassifolia Wierzb. ex Heuff. – Demogled-Herkulesbach, Romania (Köster s.n., WU); V. daurica Steven – cult. Bot. Gard. Jena (Albach s.n., WU); V. incana L. – cult. Bot. Gard. Bonn (Albach 155, WU); V. longifolia L. – cult. Bot. Gard. Bonn (Albach 66, WU); Kühkopf, Germany (Fay et al. s.n., K); Frey´s Is., UK (Sheahan 48, K); Altai (Tribsch 31.7.2002, WU); V. orchidea Crantz – Wien-Salmansdorf, Austria (Fischer 21.7.2000, WU); Montana, Bulgaria (Albach 540, WU); White Carpathians, Czech Republic (Köster s.n., WU ); Klausenburg, Romania (Köster s.n., WU); V. porphyriana Pavlov – Altai (Tribsch 2002, WU); V. schmidtiana Regel – Kushiro-mashi, Hokkaido, Japan (Umezawa 20130, WU); V. spicata L. – Stanner Rock, UK (5 samples, Jones s.n., K); Avon Gorge, UK (2 samples, M. Fay s.n., K); Uppsala, Sweden(Thulin 10035, UPS); Neusiedler See, Austria (Krefft s.n., WU); Brey, Germany (Albach 224, WU); Thorenburg canyon (Koester s.n., WU); cult. RBG Kew, ex Hainburg, Austria (Kew 1970-759 – Chase s.n., K); cult. Bot. Gard. Bonn (Albach 65, WU); ex Knauber, Bonn (Albach 214, WU); "Nana Alba" cult. RBG Kew (Kew 1979-6053 – Chase s.n., K); "Sarabande" cult. RBG Kew (Kew 1973-21579 – Chase s.n., K); Ukraine (2 samples, WU); V. spuria L. – Burgenland, Austria (Fischer 04.06.2000, WU).
Page 4
234Albach, D. & al. • Veronica on the Balkan Peninsulalikelihood analyses were conducted using a mod-el of sequence evolution as inferred by Modeltest 3.06 (Posada & Crandall 1998) using the Akaike Information Criterion, three to six replicates of ran-dom taxon addition starting from random trees with TBR branch swapping, MulTrees in effect and no tree limit. Likelihood bootstrap analyses for the com-bined dataset involved 500 replicates with the same search parameters as above but simple taxon addi-tion. For analyses of V. subg. Stenocarpon, sequenc-es of V. scutellata, V. serpyllifolia, V. triloba, V. triphyl-los, V. jacquinii, and V. chamaedrys were designated as outgroups. For analyses of V. subg. Chamaedrys, the same taxa were designated as outgroups ex-cept for replacing V. chamaedrys with V. thessalica. To evaluate different phylogenetic hypotheses in V. subg. Stenocarpon, the likelihood-based Shimodaira-Hasegawa test (with the same model used in the max-imum likelihood analysis and RELL optimization) and parsimony-based Templeton test were used as implemented in PAUP4.0b (Swofford 2002) with the combined data set.Data Analysis – AFLPAFLP fingerprints were examined using neighbour-joining and prin-cipal coordinate analysis (PCoA). Neighbour-joining Nei 1987) and neighbour-join-ing bootstrap analyses using the Nei-Li-distances have been con-ducted in PAUP4.0b10 (Swofford 2002). Principal coordinate analysis (PCoA) was conducted using Dice genetic distances between all pairs of accessions as implemented in R4.0 (Casgrain & Legendre 2001) and the first two axes projected in two two-dimensional graphs. (Saitou & Fig. 1. Results from the analysis of the ITS-dataset. Left, optimal tree from the maximum likelihood analysis. Right, strict consensus of 14 most parsimonious trees. Numbers above the branches indicate bootstrap support. ResultsVeronica subg. StenocarponThe ITS-dataset included 21 taxa with 745 aligned charac-ters, 91 of them potentially parsimony-informative and no indel character, whereas the trnLF-dataset with 17 taxa in-cluded 998 aligned characters, 43 of them potentially parsi-mony informative with one indel character. The combined dataset included 17 taxa and 1743 characters with 135 of them potentially parsimony-informative. Modeltest (Posada & Crandall 1998) chose the GTR + Γ-model (Γ = 0.18) for the ITS data set, the TVM + Γ-model model (Γ = 0.74) for the trnLF dataset and the GTR + I + Γ-model (Γ = 0.72) for the combined analysis as the optimal model. The 14 most parsi-monious trees of the ITS-analysis required 365 steps (Fig. 1; CI = 0.72; RI = 0.59), with the 1755 most parsimonious tree from the trnLF-analysis requiring 239 steps (not shown; CI = 0.90; RI = 0.69). The most likely tree of the maximum likelihood analysis of the ITS-dataset (Fig. 1) required six more steps under parsimony, whereas that from the analy-
Page 5
235Phytol. Balcan. 12(2) • Sofia • 2006 sis of the trnLF-dataset being one of the most par-simonious trees (ITS ML vs. MP: -ln = 2858.12 vs. 2860.88-2862.06; trnLF: -ln = 2792.57). The com-bined dataset had a single optimal parsimony tree requiring 593 steps (Fig. 2; CI = 0.79; RI = 0.72; -ln = 5583.40) and the best tree chosen by the like-lihood analysis required 594 steps under parsimo-ny (-ln = 5579.49).Veronica subg. Stenocarpon is a well sup-ported group by both the ITS-analysis and the combined analysis (Figs 1, 2; >98 % BP). Support for internal relationships within V. subg. Stenocarpon is low, with internal branch-es being short (Fig. 1). The combined analysis (Fig. 2) reveals a clade of Asian species with two Eastern Himalayan and the two Siberian species, forming each well supported (>75 % bootstrap percentage (BP)) sister-groups. Support for other internal groups is lacking. For example, V. densiflora groups in the results of the ITS-analysis (Fig. 1) with the Western Himalayan V. lanosa and the Mexican species, V. mexicana, for which a trnLF-sequences is still miss-ing and which therefore was not included in the trn-LF- and combined analysis, but the relationship in the ITS-analysis is not supported by the bootstrap analy-sis. Results from the parsimony analysis of the trn-LF dataset are largely unresolved and even some trees in which the subgenus is not monophyletic are equal-ly parsimonious (result not shown). The only clade re-trieved in this analysis is the sister-group relationship of V. ciliata and V. lanuginosa. The tree resulting from the maximum likelihood analysis of the trnLF-data-set is identical with the maximum likelihood analysis of the combined dataset (Fig. 2) and is therefore also not shown. Surprisingly, the rooting inferred by par-simony and maximum likelihood analyses differs dra-matically. Whereas parsimony analyses from both ITS and the combined dataset (Figs 1, 2) reveal a grade of Balkan-Iberian species (V. thessalica, V. saturejoides, V. mampodrensis) as sisters to the rest of the subgenus, all maximum likelihood analyses (Figs 1, 2) support a sister-group relationship of V. fruticulosa (plus V. fru-ticans in the ITS-analysis) to the rest of the subgenus. All Central Asian-Himalayan species form a clade with the exception of V. lanosa. Shimodaira-Hasegawa tests and Templeton tests were insignificant (p = 0.29 and p = 0.80, respectively). Increasing outgroup sampling did not lead to different results (Albach, unpubl.). Fig. 2. Unrooted phylogram from the maximum likelihood analysis of the combined dataset. Numbers along the branches indicate parsimony branch lengths/ parsi-mony bootstrap percentage/ maximum likelihood bootstrap percentage/ parsimony bootstrap percentage from the analysis of the trnL-F-dataset only. Asterisk indicates branch not present in the most parsimonious tree. ML – Maximum likelihood; BP – bootstrap percentage.Veronica subg. ChamaedrysThe ITS-dataset included 18 taxa with 743 aligned characters, 111 of them potentially parsimony-in-formative with four indel characters, whereas the trnLF-dataset included 16 taxa with 988 aligned characters, 48 of them potentially parsimony in-formative with two indel characters and the com-bined dataset with 16 taxa had 1731 characters and 151 of them potentially parsimony-informa-tive. Modeltest (Posada & Crandall 1998) chose the GTR + I + Γ-model (Γ = 0.38) for the ITS data set, the GTR + Γ-model (Γ = 0.45) for the trnLF dataset and the TIM + I + Γ-model (Γ = 0.63) for the combined data set as the optimal model. The ten most parsi-monious trees of the ITS-analysis required 367 steps (Fig. 3A; CI = 0.73; RI = 0.73), with the four most par-simonious tree from the trnLF-analysis requiring 223 steps (Fig. 3B; CI = 0.89; RI = 0.81). The most likely trees (Fig. 3A, B) were just one step longer under par-simony in both cases (ITS ML vs. MP: -ln = 2834.53 vs. 2835.07-2837.93; trnLF: ML vs. MP: -ln = 2618.47 vs. 2619.47). The combined dataset had a single op-timal parsimony tree requiring 593 steps (CI = 0.79; RI = 0.72; -ln = 5583.40) and the best tree chosen by the likelihood analysis required 594 steps under par-simony (Fig. 3C; -ln = 5579.49).
Page 6
236Albach, D. & al. • Veronica on the Balkan PeninsulaAll analyses support four groups in V. subg. Chamaedrys: V. arvensis, V. verna, the Asian per-ennial species, and the European perennials. Support for V. arven-sis as sister to the rest of the sub-genus is high (99 BP in combined analysis). The branching order of the other three groups varies with all three possibilities realized in at least one analysis. Monophyly of the perennials is, however, only shown in the parsimony analysis of the ITS dataset (Fig. 3A), with most analyses supporting the sis-ter-group relationship of V. ver-na and the perennial Europeans (Fig. 3B, C; 77 BP in maximum likelihood analysis of combined dataset). Within the European perennials, the Balkan endemics, V. chamaedryoides and V. krumov-ii, are consistently occupying first branches but support for relation-ships within this group is low.Veronica subg. PseudolysimachiumThe AFLP dataset consists of 42 taxa and 732 bands scored with 73 of them constant. The number of AFLP fragments per taxon ranged between 97 and 159 bands. Ploidy level is not known for the individ-uals used in this study but there was a tendency for taxa known to be diploid (2n = 34) to have fewer bands (e.g. V. porphyriana, which had the lowest number of bands) than tetraploid (2n = 68) taxa (V. spicata, which is mostly tetraploid had the highest number of bands). Intraspecific varia-tion was large in V. spicata (n = 104–159). Intraspecific variation in V. barrelieri was significant with respect to geographical origin with the Bulgarian sample hav-ing markedly fewer bands than the Croatian samples (109 vs. on average 140). No species-specific frag-ments were found for species for which individuals from more than one population was analyzed.Fig. 3. Phylograms derived from the maximum likelihood analyses of V. subg. Chamaedrys. Numbers above the branches indicate bootstrap support. D, German accession; N, Norwegian accession; UK, English accessions; X, branch not pres-ent in most parsimonious tree; A, optimal tree for the ITS-dataset. Dashed line indicates branch present in most parsimonious tree; B, optimal tree for the trnL-F-dataset; C, optimal tree for the combined dataset. Neighbour-joining Coordinate Analysis (PCoA) did not cluster samples into species specific groups but show similar results. Hybridization and polyploidy are common in V. subg. Pseudolysimachium (see below). Relationships based on hybridization are difficult to show on a phylogenet-ic tree. Therefore, only the results from the PCoA are shown (Fig. 4). The x-axis explains 17.0 %; the y-axis explains 9.0 % of the variation. Both are low values and further emphasize the complexity of the results. analysis and Principal
Page 7
237Phytol. Balcan. 12(2) • Sofia • 2006 DiscussionVeronica in EuropeBased on detailed morphological analyses (e.g., Fischer 1972, 1973b, 1975a, b, 1978, 1991; Trávnícek 1998, 2000; Martínez-Ortega & Rico 2001; Albach & Fischer 2003), we currently have a well-founded es-timate of species diversity of Veronica, especially in Europe. Molecular analyses have been contributing to this (Martínez-Ortega & al. 2004; Albach & al. in press; Albach, submitted). On the base of these anal-yses our estimate that about 80 species of Veronica, representing 10 of 13 subgenera recognized by Albach & al. (2004a), are found in Europe. About 40 species found in nine different subgenera are endem-ic to Europe. On the Balkan Peninsula, we can find about 60 species of Veronica, with 14 of them endem-ic (V. chamaedryoides, V. contandriopouli, V. crinita, V. erinoides, V. glauca, V. krumovii, V. oetaea, V. or-belica, V. orbiculata, V. rhodopea, V. sartoriana, V. sa-turejoides, V. thessalica, V. turrilliana). Some of these species have been investigated in the present study using DNA sequence and AFLP data. Furthermore, I discuss results from earlier analyses shedding more light on the evolution of Veronica on the Balkan Peninsula.Veronica subg. Stenocarpon – relicts of alpine regionsVeronica subg. Stenocarpon is probably the most sur-prising group revealed by molecular phylogenetic analysis. The morphological diversity and especially its biogeographical distribution pattern make it a fas-cinating group to study. Veronica subg. Stenocarpon includes approximately 25 species, most from Central Asia but also seven species from Europe plus the Mexican endemic representative of the genus, V. mexicana. This Mexican-Himalayan disjunction appearing in the analysis of ITS-sequences (Fig. 1), however without bootstrap support, merits further study. Among the European species most are endem-ic in the Balkans, often known only from one or very few localities, such as V. saturejoides subsp. kellere-ri (Mt Pirin), V. saturejoides subsp. munellensis (Mt Mnela), and V. thessalica (Mt Gjaliqa e Lumes; Mt Olymbos; Mt Jakupica; Sar Planina; Mt Koritnik). Veronica erinoides is known from only five locali-ties (Giona, Vardousia, Lidorikiou Ori, Parnassos, Fig. 4. First two axes of the principal coordinate analysis of V. subg. Pseudolysimachium. 13 • Phytol. Balcan. 12(2) • 2006
Page 8
238Albach, D. & al. • Veronica on the Balkan PeninsulaKillini). Veronica thessalica and V. erinoides were thought to be synonyms (e.g., Stroh 1942) but even before the results presented here (Fig. 1) a detailed morphological analysis had shown that one is hav-ing terminal and the other only lateral inflorescenc-es (Fischer 1969), a character formerly used for di-viding sections in Veronica. Molecular analyses on the genus level (Albach & Chase 2001; Albach & al. 2004c) has shown that the character is labile and not useful for delimiting natural groups in Veronica. So far, all species for which chromosome num-bers have been counted are diploid, although apart from the seven European species only one Asian spe-cies has been investigated (Albach & al. submitted). Sequence analysis using parsimony (Figs 1, 2) revealed that the Balkan endemics V. saturejoides, V. thessali-ca and V. erinoides, together with V. mampodrensis from the Iberian Peninsula constitute a paraphyletic grade of consecutive sisters to the Asian species of the subgenus, plus V. mexicana and three more European species (V. fruticans, V. fruticulosa, V. nummularia). Veronica mampodrensis has been included here for the first time in a phylogenetic analysis. Its position in V. subg. Stenocarpon is in general agreement with mor-phology, although Fischer & Fischer (1981) suggest-ed a closer relationship with V. fruticans and V. fru-ticulosa. Based on their restricted distribution areas (in comparison with other species of the subgenus), diploid ploidy level and their position in parsimony analyses, the Balkan endemic species could be consid-ered to be relictual species, in accord with the idea of the Balkan Peninsula as a refugium for plant groups since the Tertiary (Horvat & al. 1974; Willis 1996; Taberlet & al. 1998). However, support for the rela-tionships is low and, more important, maximum like-lihood analyses differ with regard to the topology of V. subg. Stenocarpon in V. fruticulosa being a sister to the rest of the subgenus (Figs 1, 2). The reason for this difference is solely a difference in rooting the tree, be-cause unrooted phylogenies from both kinds of anal-yses are identical (Fig. 2). I evaluated several reasons for the difference in rooting. For example, addition of other outgroup taxa to the dataset does not change the results (results not shown). Rather the lack of suffi-cient informative characters leads to spurious differ-ences in the results. Thus, the most parsimonious tree is not rejected as significantly worse by the likelihood-based Shimodaira-Hasegawa test, and the most like-ly tree is not rejected as significantly worse by the par-simony-based Templeton test. This lack of sufficient informative characters currently renders it impossi-ble to resolve the mysteries of the biogeographical and morphological evolution in this clade. Faster evolving DNA regions will be necessary for future analyses of the evolution of the group. Furthermore, no calibration point for molecular dat-ing is available for Veronica, and extensive rate hetero-geneity in the genus (Albach & Müller in prep.) renders the common substitution rate unusable. Therefore, I am currently unable to substantiate the claim that the first branching event in V. subg. Stenocarpon repre-sents a Tertiary event. Biogeographically, the situation resembles Ramonda (Gesneriaceae), which is likewise considered a Tertiary relict and found in the Pyrenees and the Balkan Peninsula (Meyer 1970), although, in contrast to Veronica, Gesneriaceae are of tropical ori-gin. Work is in progress studying phylogeographical patterns within the Balkan endemics V. saturejoides, V. thessalica, V. erinoides, the Iberian endemics V. num-mularia and V. mampodrensis and the more wide-spread V. fruticans, so as to investigate the hypothesis that the endemic species are relictual species that had failed to disperse out of their glacial refugia, where-as V. fruticans had managed to spread from a glacial distribution area into and close to the Alps, north to-wards to Scandinavia. Additional sequence data will be helpful to estimate the genetic variation within the Balkan endemic species in order to differentiate be-tween the hypotheses of recent bottlenecks or long persistence in refugial areas, so as to explain the limit-ed distribution of these taxa and to help protect these rare alpine species.Veronica subg. Chamaedrys – Diploid taxa marking hypothesized forest refugiaVeronica subg. Chamaedrys consists of four different groups: V. arvensis (incl. V. sartoriana = V. subsect. Microsperma (Römpp) Stroh), V. verna (most likely together with V. dillenii and V. brevistyla; = V. subsect. Microspermoides Albach1), V. laxa and V. magna ( = V. 1 Veronica subsectio Microspermoides Albach, subsect. nov. – Typus: V. verna L. Spec. Pl. 1: 14, 1753; lectotypus (ab E. Fischer 1997 designatus): LINN 26.63. Annua aut raro biennis, −30 cm, erecta, simplex. Folium sessile, pinnati- vel palmatipartitum vel –lobatum. Inflorescentia terminalis, multiflora, glandulosa. Pedicellus brevior quam calyx. Capsulae obcordatae, numquam glandulosae. Semina plana. Numerus basalium chromosomatium x = 9. Planta inutilis in Europa et Asia diffusa.
Page 9
239Phytol. Balcan. 12(2) • Sofia • 2006 subsect. Asiachamaedrys Albach2), and the European perennial species (V. Chamaedrys, V. vindobonensis, V. chamaedryoides, V. krumovii, and V. orbelica; = V. subsect. Multiflorae Benth.). Surprisingly, the peren-nial species do not form a monophyletic group, ex-cept for the parsimony analysis of the ITS dataset (Fig. 3A). Thus, a likely scenario is that first a for-merly widespread Eurasian perennial taxon split in-to a Mediterranean and an Asian taxon, and V. verna (and relatives) evolved from the Mediterranean group. Veronica verna is nowadays widespread across Eurasia and nothing is known about its place of origin to con-firm this hypothesis.Veronica subsect. Multiflorae includes prominent species of forest vegetation, mainly along the forest edges, in open places and clearings. Originally con-sidered one species, the group consists of five species and three additional intraspecific taxa. Whereas V. chamaedrys subsp. chamaedrys is a tetraploid plant, all others are diploid. They differ not only in their ploi-dy level but also in the distribution range, with the tetraploid widespread throughout Europe and the on-ly one that has reached North Europe. The diploids occur in South, especially South and Central Europe, and are mostly confined to small areas that have been assumed as Pleistocene refugia. Veronica chamaedry-oides is endemic to Greece, where it is fairly wide-spread, and found in open forests, meadows and rocky slopes up to 1900 m. Greece is commonly suggested as a Pleistocene refugium for many submediterranean taxa (e.g. Huntley & Birks 1983; Tzedakis & al. 2002). Veronica krumovii is endemic to Bulgaria. It is mor-phologically separated but its ecology and exact dis-tribution has not been studied adequately (Mirek & Fischer 1986). Although currently not restricted to the western Black Sea Coast, a suggested Pleistocene refu-gium by Huntley & Birks (1983), it could have expand-ed from there to its current distribution area. Veronica orbelica is another Bulgarian endemic. It is found in Southwest Bulgaria and is morphologically close to V. vindobonensis (Mirek & Fischer 1986). It grows in dri-er habitats and may have originated in the drier inte-rior part of Bulgaria. Unfortunately, no authenticated 2 Veronica subsectio Asiachamaedrys Albach, subsect. nov. – Typus: V. laxa Benth. Scroph. Ind.: 35, 1835; typus: K n.v. Perennis. 35−90 cm, erecta. Caulis ubique pubescens. Folia brevipetiolata, ovata, grosse dentate, plus quam 25 cm. Inflorescentia lateralis, plus quam 10 cm longa, multiflora. Calyx longior quam pedicel-lus, plus quam 4 mm. Corolla caerulea. Capsula obcordata, bre-vior quam calyx, base cuneata, glabra.material of V. orbelica was available for this study be-cause much of the material seen in nature in Bulgaria was intermediate between V. orbelica and V. vindobo-nensis. Clear delimitation of these two species will be an important task for the future.Veronica vindobonensis is the most widespread diploid taxon occurring from Northwestern Anatolia to South Germany. It is probably adapted to the dri-est types of forests within the group. Its wide distri-bution argues either for rapid spread from a refugium (possibly furthest to the north in Hungary, accord-ing to Willis & al. 2000) after the last glaciation, or a more widespread distribution even in the Pleistocene. It sometimes reaches subalpine habitats in Bulgaria (Albach pers. obs.), which might contend for the latter. Veronica chamaedrys subsp. micans is mostly known from the Austrian and German northern calcare-ous Alps and is the only taxon that can be considered truly subalpine. Its distribution is especially intrigu-ing, because it is exactly confined to the hypothesized Pleistocene refugia in the northeastern Alps (Tribsch & Schönswetter 2003). Another diploid taxon is found in South Austria and is informally called V. chamae-drys "carintho-stiriaca" (Fischer 1973a, pers. comm.). It is similar to V. chamaedrys subsp. micans occur-ring on drier habitats but is morphologically distinct (Fischer 1973b) and may have survived in a forest re-fugium in the southeastern Alps (Bennett & al. 1991). Another diploid population can be found further south in Dalmatia but has been not studied intensive-ly (Fischer unpubl.). The tetraploid V. chamaedrys has the widest distribution of all taxa. It occurs through-out Europe, with the exception of the Mediterranean islands and the Arctic. In Northern Greece it barely overlaps with V. chamaedryoides (Fischer 1991). Results from sequence analysis suggest that the southern diploid taxa of the group, V. krumovii and V. chamaedryoides, were not involved in the origin of tetraploid V. chamaedrys subsp. chamaedrys but were isolated in their Pleistocene refugia. Unfortunately, molecular dating is not possible in Veronica and there-fore the age of divergence is not inferable. Several sce-narios regarding the origin of tetraploid V. chamaedrys are possible on the base of sequence data. Veronica chamaedrys subsp. micans is very likely a parent of it, based on the high similarity of their ITS-sequenc-es (Fig. 3A.). The other parent could be an unsampled diploid taxon, possibly the "stiriaca-carinthiaca"-type or the "dalmatica"-type, based on the fact that none of
Page 10
240Albach, D. & al. • Veronica on the Balkan Peninsulathe sampled diploid taxa had the tetraploids plastid-sequence (Fig. 3B), although an autopolyploid event cannot be excluded. Another possibility to be consid-ered is the polytopic origin, supported by morpho-logical variation in the tetraploid cytotype (Fischer 1973a). More intensive sampling will be needed to re-veal the parents of tetraploid V. chamaedrys. So far, we can be only reasonably certain that the tetraploid V. chamaedrys sampled in our analysis had ancestors that lived close to the ice-sheet during the Pleistocene. Such a scenario would support Stebbins' (1984) sec-ondary-contact hypothesis, in which polyploids arise at the margin of distribution. Thus, diploid taxa sur-vived in their Pleistocene refugia and, after the ice re-treated, spread out into the formerly glaciated area and encountered other diploid survivors, with which they formed polyploid hybrids. Such kinds of neo-polyploids are known to surpass their ancestors in in-vasion potential, especially in habitats that have be-come available after the retreat of the Pleistocene ice sheets (Ehrendorfer 1965, 1980). The involvement of the subalpine V. chamaedrys subsp. micans in the or-igin of V. chamaedrys rather than a true forest spe-cies, such as V. krumovii, further supports such a sce-nario. Consequently, the Balkan endemic species of V. subsect. Multiflorae, in the sense applied here, rep-resent relicts from Pleistocene refugia, which did not contribute to the recolonization of Central and North Europe – a situation resembling that of many animals, such as the brown bears (Taberlet & Bouvet 1994), grasshoppers (Cooper & al. 1995), shrews and bank voles (Bilton & al. 1998).Veronica subg. Pseudolysimachium – speciation by hybridization Veronica subg. Pseudolysimachium is one of the most beautiful but also systematically complex groups of Veronica. It is well differentiated from other species of Veronica by its dense inflorescence, different flower morphology, pollen, and embryology, as well as a dif-ferent chromosome base number. The group contains about forty species but is notorious for its hybridiza-tion, intraspecific ploidy level changes, and phenotyp-ic variability leading to vast amounts of synonyms and subspecies or varieties. Crossing experiments by Härle (1932) have shown the wide cross-compatibility with-in a ploidy-level in the group. In recent times, studies by Fischer (1974), Fischer & Bedalov (1988), Fischer & Peev (1995), Albach & Fischer (2003) and Trávnícek (1997, 1998, 2000) have helped greatly delimit mor-phologically distinct units in Europe. However, AFLP fingerprints failed to give support to those taxa stud-ied here.Within the European members of V. subg. Pseudolysimachium, we find about 11 diploid and 6–7 tetraploid taxa. Only two of the tetraploid taxa are exclusively tetraploid, V. crassifolia and V. spicata subsp. fischeri. Among the other tetraploids we find the most common European species, V. spicata and V. longifolia ( = V. maritima sensu Trávnícek 2000). On the base of crossing experiments Graze (1933) assumed widespread hybridization in nature, which Fischer (1974) doubted on the basis of extensive her-barium revision. DNA sequences from the ITS- and trnLF-regions were unable to distinguish species be-cause of the low variability (Albach & al. 2005), so we depend on a faster evolving molecular marker. AFLP were chosen here, because they have been success-ful in detecting variation among closely related spe-cies and within species in other groups of Veronica, such as V. tenuifolia (Martínez-Ortega & al. 2004), V. alpina (Albach & al. in press) and V. cymbalaria (Albach submitted). AFLP have been useful in de-tecting patterns of hybridization in an earlier study of Veronica (V. hederifolia and V. cymbalaria; Albach submitted) and other plant taxa (e.g., Hedrén & al. 2001; Gobert & al. 2002) and thus were thought to be a reliable and cost-effective way to give some ini-tial support for hypotheses of hybridization and its importance on the Balkan Peninsula. Hybrids have commonly been analyzed together with their par-ents (e.g., Hedrén & al. 2001; Hodkinson & al. 2002; Guo & al. 2005) but their effect on the analysis has never been investigated in great detail. Results from the analysis here show large-scale incongruence with currently accepted species delimitation (Fig. 4) and no fixed species-specific fragment was found. Several reasons that do not mutually exclude each other may explain this incongruence. Hybridization may be more prevalent than discernible by morpholog-ical inspection (Fischer 1974). Polyploidy increases homoplasy in cases in which either different species share one parent or one species is of polytopic origin with genetically different parentage (Albach submit-ted). Furthermore, V. subg. Pseudolysimachium is al-ready of polyploid origin itself, which may increase the chances for homoplasy.
Page 11
241Phytol. Balcan. 12(2) • Sofia • 2006 Hybridization and polyploidy may be very com-mon in V. subg. Pseudolysimachium for historical rea-sons. Pleistocene glaciations had considerable im-pact on the vegetation of Europe. During glaciations, the climate in Europe was more continental, favoring steppe vegetation in larger parts of Europe (Mai 1995). This may have led to invasions of steppe species, such as V. subg. Pseudolysimachium, from West Siberia in-to Eastern Europe. Trávnícek (1998) hypothesized such an invasion and a subsequent retreat in more hu-mid Holocene times for V. incana. Unfortunately, such steppe species have seldom been investigated phyloge-ographically in much detail (see Franzke & al. 2004). Climatological oscillations during the Ice Ages may have led to repeated advances of steppe species and re-peated chances for hybridization. Another remarkable fact that may indicate a high chance for hybridization in this group is the prevalence of diploid cytotypes in marginal areas and relictual areas (Trávnícek 1998, 2000, pers. comm.), whereas tetraploids predominate in the central range of the species. Similar situations in other plant groups led to the hypothesis that tetraploid forms replaced diploid forms gradually during expan-sion after the Pleistocene (Ehrendorfer 1962, 1965). This situation seems on first sight in contrast to the general notion that polyploids evolve at the margins of distribution but supports Stebbins' (1984) secondary-contact hypothesis. Following this hypothesis, diploid taxa survived in their Pleistocene refugia and after the Ice Age spread and encountered other diploid survi-vors, with which they formed polyploid hybrids. Such kinds of neopolyploids are known to surpass their an-cestors in invasion potential, e.g. in the habitats that have become available after the retreat of Pleistocene ice sheets (Ehrendorfer 1965, 1980). Consequently, a phylogeographic study of V. subg. Pseudolysimachium should retrieve a patchy distribution pattern of the Pleistocene refugia in Europe for the diploids and a pattern mirroring the post-Pleistocene migration for the tetraploid cytotypes, which blurs the genealogical history of the group. Unfortunately, it was not possi-ble in this study to ascertain the ploidy level of the an-alyzed individuals. Subsequent studies will be need to confirm this factor. Despite these fallacies, some results raise doubts about the earlier taxonomic conclusions. Veronica bar-relieri is known to have diploid and tetraploid popula-tions and is found here in two widely divergent groups (Fig. 4). It is also morphologically difficult to differen-tiate and sometimes considered part of a broad V. spi-cata (Elenevsky 1971; Walters & Webb 1972) but dif-ferentiated by us in five subspecies (Albach & Fischer 2003). Therefore the species needs closer inspection. Veronica porphyriana is also occasionally included in a broader V. spicata (Elenevsky 1971) but is clearly dif-ferentiated here from the European samples (Fig. 4). Its status as a distinct species therefore seems to be val-id. Veronica longifolia is also found in two widely sepa-rated groups: the European samples in one group and the Asian sample from the Altai in another (Fig. 4). This would support Trávnícek´s (2000) hypothe-sis of two species being hidden in V. longifolia, which he termed V. maritima for the European populations and V. longifolia for the Asian samples. It should be borne in mind that the situation may be more com-plex, because only one population from Asia was ana-lyzed and no geographically intermediate sample was included. Furthermore, it is not clear whether V. longi-folia in Asia, where diploids are frequent (Albach & al. submitted), represent diploid progenitors of their mostly tetraploid European relatives, or just relative-ly unrelated diploid relatives that were not involved in the origin of the tetraploids. Future investigations in V. subg. Pseudolysimachium will be need to take the lessons from this study into consideration. 1.) DNA sequence analyses are supe-rior to anonymous markers, because hybridization renders homology assessment of markers more dif-ficult. However, even DNA sequence studies will be problematic in the presence of hybridization and mul-tiple markers will be necessary to infer a species tree. 2.) Ploidy level should be controlled in all samples. 3.) Morphological characters may be homoplastic due to character shuffling by hybridization. 4.) Ancestral genotypes are likely to be found in Asian members of the group, which is in line with the inferred Asian or-igin of the subgenus (Albach & al. 2005). The analysis of additional diploid taxa from East Europe and West Siberia will be crucial to understand the origin of the European taxa.Veronica subsect. Alpina – relict populations on the Balkan PeninsulaVeronica alpina and V. bellidioides are closely related species of V. subg. Veronica (Albach & al. in press). They are both species of subalpine meadows but dif-fer ecologically. This ecological difference is responsi-
Page 12
242Albach, D. & al. • Veronica on the Balkan Peninsulable for differences related to climate change as shown by simulation studies (Guisan & Theurillat 2000) and different phylogeographic histories (Albach & al. in press). Phylogeographic analyses of AFLP finger-print data revealed that V. alpina shows much high-er intraspecific genetic diversity than V. bellidioides (Albach & al. in press). Populations of V. bellidioides from the Balkan Peninsula (Bulgaria) form a mono-phyletic sister group to the populations from the Alps and the Pyrenees (Albach & al. in press) representing a long historic divergence. In the absence of molecu-lar dating, we are unable to estimate the divergence time, but given the absence of plastid DNA variation (Albach & al. in press), a Pleistocene origin is likely. In contrast, populations of V. alpina from the Balkan Peninsula (Bulgaria) are not well separated from oth-er populations of this species in Europe. Interestingly, however, there does not seem to be a close connection with populations from the Carpathians (Albach & al. in press). Unfortunately, populations from other lo-calities of both species on the Balkan Peninsula could not be sampled. Populations of both V. alpina and V. bellidioides may nevertheless be Pleistocene relicts of similar age that were unable to migrate north after the Ice Ages, similar to the case in V. subg. Chamaedrys (see above). However, the cases of V. alpina and V. bel-lidioides differ in that extinctions and genetic bottle-necks in surviving populations of V. bellidioides led to a clear bifurcation in the phylogenetic tree.ConclusionsThe chosen examples highlight the different respons-es of plants to the Pleistocene climate changes. Plant species probably had very different histories on the Balkan Peninsula. They may have become relict taxa of probable Tertiary origin such as species from V. subg. Stenocarpon that differentiated into morphologically clearly distinguishable species. Other groups may have become separate taxa in the Pleistocene on the verge of speciation by genetic drift, such as taxa in V. subg. Chamaedrys. Or they may actively speciate by hybrid-ization, such as taxa in V. subg. Pseudolysimachium. Despite these differences, the Balkan Peninsula rep-resents an important center of genetic diversity for all investigated groups of Veronica. Other species and species complexes of Veronica on the Balkan Peninsula, such as V. sect. Pentasepalae, are currently under investigation (Martínez Ortega, pers. comm.). Undoubtedly, they will further highlight the impor-tance of the Balkan Peninsula for the survival and di-versification of plant taxa in the Pleistocene. Acknowledgements. The author wishes to thank the Austrian Science Fund (FWF, project P-15336-Bio) for the financial support of this research. Antonio Abad (AFLP) and Marion Kever (DNA se-quencing) provided invaluable help in the lab. Finally, I am grateful to Montserrat Martínez Ortega (Univ. Salamanca), M. Fay (RBG Kew, UK), Gerald Schneeweiss (Univ. Wien), and Andreas Tribsch (Univ. Zalzburg) for providing plant material for this publication.ReferencesAlbach, D.C. & Chase, M.W. 2001. Paraphyly of Veronica (Veroniceae; Scrophularaceae): Evidence from the internal tran-scribed spacer (ITS) sequences of nuclear ribosomal DNA. – J. Pl. Res., 114: 9-18.Albach, D.C. & Fischer, M.A. 2003. AFLP- and genome size analyses: contribution to the taxonomy of Veronica subg. Pseudolysimachium sect. Pseudolysimachion (Plantaginaceae), with a key to the European taxa. – Phytol. Balcan., 9: 401-424.Albach, D.C., Jensen, S.R., Özgökce, F. & Grayer, R.J. 2005. Chemical characters for the support of phylogenetic relationships based on nuclear ribosomal and plastid DNA sequence data in Veronica. – Biochem. Syst. Ecol., 33: 1087-1106.Albach, D.C., Martínez-Ortega, M.M., Fischer, M.A. & Chase, M.W. 2004a. A new classification of the tribe Veroniceae − prob-lems and a possible solution. – Taxon, 53: 429-452.Albach, D.C., Martínez-Ortega, M.M. & Chase, M.W. 2004b. Veronica: Parallel morphological evolution and phylogeography in the Mediterranean. – Pl. Syst. Evol., 246: 177-194.Albach, D.C., Martínez-Ortega, M.M., Fischer, M.A. & Chase, M.W. 2004c. Evolution of Veroniceae: A phylogenetic perspec-tive. – Ann. Missouri Bot. Gard., 91: 275-302.Albach, D.C., Meudt, H.M. & Oxelman, B. 2005. Piecing together the "new" Plantaginaceae. – Amer. J. Bot., 92: 297-315.Albach, D.C., Schönswetter, P. & Tribsch, A. In press. Comparative phylogeography of the Veronica alpina-complex in Europe and North America. – Mol. Ecol.Angiosperm Phylogeny Group. 1998. An ordinal classification for the families of flowering plants. – Ann. Missouri Bot. Gard., 85: 531-553. Benham, J.J. 1998. Genographer, 1.1. Montana State Univ., Bozeman, Montana.Bennett, K.D., Tzedakis, P.C. & Willis, K.J. 1991. Quaternary refugia of north European trees. – J. Biogeogr., 18: 103-115.Bilton, D.T., Mirol, P.M., Mascheretti, S., Fredga, K., Zima, J. & Searle, J.B. 1998. Mediterranean Europe as an area of ende-mism for small mammals rather than a source for northwards
Page 13
243Phytol. Balcan. 12(2) • Sofia • 2006 postglacial colonization. – Proc. Roy. Soc. London, Ser. B, Bio. Sci., 265: 1219-1226.Casgrain, P. & Legendre, P. 2001 The R Package for multivariate and spatial analysis. Version 4.0 d5. User's manual. Dept. Sci. Biol., Univ. Montreal, Montreal.Cooper, S.J.B., Ibrahim, K.M. & Hewitt, G.M. 1995. Postglacial expansion and genome subdivision in the European grasshopper Chorthippus parallelus. – Mol. Ecol., 4: 49-60.Doyle, J.J. & Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. – Phytochem. Bull. Bot. Soc. Amer., 19: 11-15.Ehrendorfer, F. 1962. Cytotaxonomische Beiträge zur Genese der mitteleuropäischen Flora und Vegetation. – Ber. Deutsch. Bot. Ges., 75: 137-152.Ehrendorfer, F. 1965. Über stammesgeschichtliche Differen-zierung smuster bei den Dipsacaceae. – Ber. Deutsch. Bot. Ges., 77: 83-94.Ehrendorfer, F. 1980. Polyploidy and distribution. – In: Lewis, W.H. (ed.), Polyploidy: Biological relevance. Pp. 45-60. Plenum Publishing Corp., New York.Elenevsky, A. 1971. De Veronica spicata L. s. l. notulae systemati-cae. – Novosti Sist. Vyssh. Rast., 8: 215-227.Fischer, M.A. 1969. Veronica caespitosa Boiss., V. bombycina Boiss. & Kotschy und V. thessalica Benth. gehören nicht in die Sektion Veronicastrum Benth. – Österr. Bot. Z., 117: 54−63.Fischer, M.A. 1972. Neue Taxa, Chromosomenzahlen und Systematik von Veronica subsect. Acinifolia (Römpp) Stroh. – Österr. Bot. Z., 121: 413-437.Fischer, M.A. 1973a. Notizen zur Systematik, Chromosomenzahl und Verbreitung einiger Veronica-Sippen in Kärnten. – Carinthia, 83: 379-388.Fischer, M.A.1973b. Zur Cytotaxonomie von Veronica chamaedrys L. agg., II.: subsp. micans M. Fischer, subsp. nova, eine weitere diploide Sippe. – Österr. Bot. Z., 121: 73-79.Fischer, M.A. 1974. Beitrag zu einer systematischen Neubearbeitung der Gruppe um Pseudolysimachion spicatum (L.) Opiz ( = Veronica spicata L.). – Phyton, 16: 29-47.Fischer, M.A. 1975a. Untersuchungen über den Polyploidkomplex Veronica cymbalaria agg. (Scrophulariaceae). – Pl. Syst. Evol., 123: 97-105.Fischer, M.A. 1975b. The Veronica hederifolia group: Taxonomy, ecology, and phylogeny. – In: Walter, S.M. (ed.), European floris-tic and taxonomic studies. Conf. Rep. Pp. 48-60. Bot. Soc. British Isles, E.W. Classey Ltd, Farington.Fischer, M.A. 1978. Veronica L. – In: Davis, P.H. (ed.), Flora of Turkey and the East Aegean Islands. Vol. 6, pp. 689-753. Edinburgh Univ. Press, Edinburgh. Fischer, M.A. 1991. Veronica. – In: Strid, A. & Tan, K. (eds), Mountain Flora of Greece. Vol. 2, pp. 209-234. Edinburgh Univ. Press, Edinburgh.Fischer, M.A. & Bedalov, M. 1988. The genera Paederota and Pseudolysimachion (Scrophulariaceae) in Croatia. – Acta Bot. Croat., 47: 149-156.Fischer, M.A. & Fischer, G. 1981. Distribution patterns of Veronica species in the Balkan Peninsula. – In: Velčev, V.I. & Kožuharov, S.I. (eds), Mapping the flora of the Balkan Peninsula. Pp. 175-232. Publishing House Bulg. Acad. Sci., Sofia.Fischer, M.A. & Peev, D. 1995. Pseudolysimachion Opiz. – In: Kozhuharov, S. (ed.), Fl. Reipublicae Bulgaricae. Vol. 10, pp. 190-202. Editio Acad. "Prof. Marin Drinov", Serdicae (in Bulgarian).Franzke, A., Hurka, H., Janssen, D., Neuffer, B., Friesen, N., Markov, M. & Mummenhoff, K. 2004. Molecular signals for Late Tertiary/Early Quaternary range splits of an Eurasian steppe plant: Clausia aprica (Brassicaceae). – Mol. Ecol., 13: 2789-2795.Frenzel, B. 1968. Grundzüge der pleistozänen Vegetationsgeschichte Nord-Eurasiens. Franz Steiner Verlag, Wiesbaden.Gobert, V., Moja, S., Colson, M. & Taberlet, P. 2002. Hybridization in the section Mentha (Lamiaceae) inferred from AFLP mark-ers. – Amer. J. Bot., 89: 2017-2023.Graze, H. 1933. Die chromosomalen Verhältnisse in der Sektion Pseudolysimachia Koch der Gattung Veronica. – Jahrb. Wiss. Bot., 77: 507-559.Guisan, A., Theurillat, J.-P. 2000. Assessing alpine plant vulner-ability to climate change: a modelling perspective. – Integr. Assessm., 1: 307-320.Guo, Y.-P., Saukel, J., Mittermayr, R. & Ehrendorfer, F. 2005. AFLP analyses demonstrate genetic divergence, hybridization, and multiple polyploidization in the evolution of Achillea (Asteraceae-Anthemideae). – New Phytol., 166: 273-290.Härle, A. 1932. Die Arten und Formen der Veronica-Sektion Pseudolysimachia Koch auf Grund systematischer und experi-menteller Untersuchungen. – Bibl. Bot., 26: 1-86.Hedrén, M., Fay, M.F. & Chase, M.W. 2001. Amplified frag-ment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae). – Amer. J. Bot., 88: 1868-1880.Hewitt, G. 2000. The genetic legacy of the quaternary ice ages. – Nature, 405: 907-913.Hodkinson, T.R., Chase, M.W., Takahashi, C., Leitch, I.J., Bennett, M.D. & Renvoize, S.A. 2002. The use of DNA sequenc-ing (ITS and trnL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae). – Amer. J. Bot., 89: 279-286.Horvat, I., Glavac, V. & Ellenberg, H. 1974. Vegetation Südosteuropas. G. Fischer, Stuttgart.Huntley, B. & Birks, H.J.B. 1983. An atlas of past and present pollen maps for Europe. Cambridge Univ. Press, Cambridge.Mai, D. H. 1995. Tertiäre Vegetationsgeschichte Europas. Gustav Fischer Verlag, Jena, Stuttgart, New York.Martínez-Ortega, M.M. & Rico, E. 2001. Taxonomy of Veronica subsect. Serpyllifolia (Scrophulariaceae). – Bot. J. Linn. Soc., 135: 179-194.Martínez-Ortega, M.M., Delgado, L., Albach, D.C., Elena-Rosselló, J.A. & Rico, E. 2004. Species boundaries and phyloge-ography of Veronica subsect. Pentasepalae (Scrophularaceae) in the Western Mediterranean inferred from AFLP markers. – Syst. Bot., 29: 965-986.Meyer, F.K. 1970. Gesneriaceae als Glieder der Flora des Tertiär in Europa. – Wiss. Z. Friedrich-Schiller-Univ. Jena, Math.-Naturwiss. Reihe, 19: 401-411.
Page 14
244Albach, D. & al. • Veronica on the Balkan PeninsulaMirek, Z., & Fischer, M.A. 1986. Additions to the ecogeography of Veronica vindobonensis with special reference to Poland. – Phyton, 26: 107-129.Ozenda, P. 1988. Die Vegetation der Alpen im europäischen Gebirgsraum. Gustav Fischer Verlag, Stuttgart, New York.PE Applied Biosystems. 1996. AFLP™ Plant Mapping Protocol. Foster City, California.Polunin, O. 1980. Flowers of Greece and the Balkans. Oxford Univ. Press, Oxford.Posada, D. & Crandall, K. A. 1998. MODELTEST: testing the model of DNA substitution. – Bio-informmation, 14: 817-818.Saitou, N., & Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. – Mol. Biol. Evol., 4: 406.Schmitt, T., & Seitz, A. 2002. Postglacial distribution area expan-sion of Polyommatus coridon (Lepidoptera: Lycaenidae) from its Ponto-Mediterranean glacial refugium. – Heredity, 89: 20-26.Stebbins, G.L. 1984. Polyploidy and the distribution of the arctic-alpine flora: new evidence and a new approach. – Bot. Helv., 94: 1−13.Stroh, G. 1942. Die Gattung Veronica L. Versuch einer systema-tischen Kodifizierung der Arten (mit Ausnahme der endemischen Arten von Neuseeland). – Beih. Bot. Centralbl., 61: 384-451.Sun, Y., Skinner, D.Z., Liang, G.H. & Hulbert, S.H. 1994. Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. – Theor. Appl. Genet., 89: 26-32.Swofford, D.L. 2002. PAUP* Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.10. Sinauer Associates, Sunderland, Massachusetts. Taberlet, P. & Bouvet, J. 1994. Mitochondrial DNA polymorphism, phylogeography and conservation genetics of the brown bear Ursus arctos in Europe. – Proc. Roy. Soc. London, Ser. B, Biol. Sci., 255: 195-200.Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chlo-roplast DNA. – Pl. Mol. Biol., 17: 1105-1109.Taberlet, P., Fumagalli, L., Wust-Saucy, A.-G. & Cosson, J.-F. 1998. Comparative phylogeography and postglacial colonization routes in Europe. – Mol. Ecol., 7: 453-464.Trávnícek, B. 1997. Pseudolysimachion (W.D.J. Koch) Opiz. – In: Goliašová, K. (ed.), Flora of Slovakia. Vol. 5(2), pp. 264-298. Veda Press, Bratislava (in Slovak).Trávnícek, B. 1998. Notes on the taxonomy of Pseudolysimachion sect. Pseudolysimachion (Scrophulariaceae) in Europe. I. P. inca-num and P. spicatum. – Preslia, 70: 193-223.Trávnícek, B. 2000. Taxa of the genus Pseudolysimachion (Scrophulariaceae) in the Czech Republic. I. Identification key to the species. Geographical distribution of species of sect. Longifolia. – Preslia, 72: 411-439.Tremetsberger, K., Stuessy, T.F., Samuel, R.M., Baeza, C.M. & Fay, M.F. 2003. Genetics of colonization in Hypochaeris tenuifolia (Asteraceae, Lactuceae) on Volcán Longiquimay, Chile. – Mol. Ecol., 12: 2649-2659.Tribsch, A. & Schönswetter, P. 2003. Patterns of endemism and comparative phylogeography confirm palaeo-environmental evidence for Pleistocene refugia in the Eastern Alps. – Taxon, 52: 477-497.Turrill, W. 1929. The plant life of the Balkan Peninsula. Oxford Univ. Press, Oxford.Tzedakis, P.C., Lawson, I.T., Frogley, M.R., Hewitt, G.M. & Preece, R.C. 2002. Buffered tree population changes in a qua-ternary refugium: evolutionary implications. – Science, 297: 2044-2047.Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. 1995. AFLP: a new technique for DNA fingerprint-ing. – Nucl. Acids Res., 23:4407-4414.Wagstaff, S.J. & Garnock-Jones, P.J. 1998. Evolution and biogeog-raphy of the Hebe complex (Scrophulariaceae) inferred from ITS sequences. – New Zealand J. Bot., 36: 425−437.Walters, S.M. & Webb, D.A. 1972. Veronica L. – In: Tutin, T.G. & al. (eds), Flora Europaea. Vol. 3, pp. 242−251. Cambridge Univ. Press, Cambridge.White, T. J., Bruns, T., Lee, S. & Taylor, J. 1991. Amplification and direct sequencing of fungal ribosomal RNA genes for phy-logenetics. – In: Gelfand, D., Innis, M., Sninsky, J. & White, T. (eds), PCR Protocols: A guide to Methods and Applications. Pp. 315-322. Acad. Press, San Diego.Willis, K.J. 1996. Where did all the flowers go? The fate of tem-perate European flora during glacial periods. – Endeavour, 20: 110−114.Willis, K.J., Rudner, E. & Sümegi, P. 2000. The full-glacial forests of central and southeastern Europe. – Quatern. Res., 53: 203-213.Zhang, L., Comes, H.P., & Kadereit, J.W. 2001. Phylogeny and quaternary history of the European montane/alpin endemic Soldanella (Primulaceae) based on ITS and AFLP variation. – Amer. J. Bot., 88: 2331-2345.


text:
Comment: Mother and child health must improve
Increasing access to health services will prevent many mothers and newborn babies from dying.
Increasing access to health services will prevent many mothers and newborn babies from dying.
Bill and Melinda Gates believe poor people's live will change rapidly in the near future. M&G's Health reporter Amy Green speaks to Bill Gates.
Philanthropists Bill and Melinda Gates released their annual letter on Thursday. In it, they predict what the world would look like by 2030.
Aid may often be criticised, but it works, says the Gates Foundation.
The Bill and Melinda Gates Foundation has partnered with SA institutions to develop new medicines and vaccines for HIV, TB and malaria.
Family planning is not a luxury to everyone. Melinda Gates talks about why she has dedicated so much of her time to helping women plan their families.
An effective healthcare system will free up donor money for more desperate countries.
With World Immunisation Week (April 20 – 26) drawing attention to vaccinations, Nicky Willemse turned the spotlight on Dr Seth Berkley.
Aids kills more people prematurely in SA than another other disease – in sharp contrast to two decades ago, which set them at just 12%.
The long-acting implant contraceptive Jadelle is being made available at 50% of its original price to women in developing countries.
Bill Gates seems bent beneath a stronger gravity as he talks about the 'puzzle' that is South Africa's health system.
Meaningful progress has been made to overcome the continent's health hurdles, but more can be done through strong leadership, writes Bill Gates
If Bill Gates is excited about online education, you should be, too, suggests Anna Malczyk.


text:
T-Rex Prop. 96 inches long-36 inches wide-54 inches high. This amazing life size, movie/museum quality prop is made of fiberglass, so it can be used in or oudoors. Great effort is given to every detail from skin texture to facial expression..
our price: $ 4,150.00
Gargoyle Hanging Wall Mount
Moon Beast Plaque


text:
out in the 7th
is he injuried ???
Most Accurate MLB Experts Past 4 Years
Return to Injuries & Player Updates
Moderator: Baseball Moderators
Coppermine wrote:It's a day game after a night game, V-Mart's a catcher and the Tribe is up by 5 runs... you do the math
nbb23 wrote:Yes, he took a fastball right off his wrist. He was in some pain and was replaced. I didn't hear anything official but seemed like it would most likely be a day to day type thing.
Return to Injuries & Player Updates
Users browsing this forum: No registered users and 4 guests


text:
amikacin sulfate, Amikin (discontinued) (cont.)
Pharmacy Author:
Omudhome Ogbru, PharmD
Omudhome Ogbru, PharmD
Dr. Ogbru received his Doctorate in Pharmacy from the University of the Pacific School of Pharmacy in 1995. He completed a Pharmacy Practice Residency at the University of Arizona/University Medical Center in 1996. He was a Professor of Pharmacy Practice and a Regional Clerkship Coordinator for the University of the Pacific School of Pharmacy from 1996-99.
Medical and Pharmacy Editor:
Charles Patrick Davis, MD, PhD
Charles Patrick Davis, MD, PhD
Dr. Charles "Pat" Davis, MD, PhD, is a board certified Emergency Medicine doctor who currently practices as a consultant and staff member for hospitals. He has a PhD in Microbiology (UT at Austin), and the MD (Univ. Texas Medical Branch, Galveston). He is a Clinical Professor (retired) in the Division of Emergency Medicine, UT Health Science Center at San Antonio, and has been the Chief of Emergency Medicine at UT Medical Branch and at UTHSCSA with over 250 publications.
SIDE EFFECTS: Side effects of Amikacin are:
Amikacin carries a boxed warning of loss of hearing and kidney dysfunction for individuals treated with high doses or for longer periods.
PRESCRIPTION: Yes
GENERIC AVAILABLE: Yes
PREPARATIONS: Amikacin sulfate injection is colorless solution. It is available in 50 mg/ml and 250 mg/ml strengths in 2 ml vials. It is also available in 250 mg/ml strength in 4 ml vials.
STORAGE: Store Amikacin at room temperature between 20 C to 25 C (68 F to 77 F).
DOSING: The recommended dose for adults, children, and older infants with normal kidney function is 15 mg/kg/day divided into 2 or 3 doses; for example, 7.5 mg/kg every 12 hours or 5 mg/kg every 8 hours.
The duration of treatment is usually 7 to 10 days.
DRUG INTERACTIONS: Amikacin should not be used with amphotericin due to significant risk of loss of hearing and severe kidney dysfunction.
Amikacin should be used with great caution with medications like furosemide (Lasix), torsemide (Demadex), cyclosporine, and tacrolimus (Prograf) due to high risk of loss of hearing and kidney dysfunction.
PREGNANCY: Amikacin is harmful to the fetus and should not be used during pregnancy unless there are not safer options.
NURSING MOTHERS: It is not known whether Amikacin enters breast milk; therefore, it is best to be cautious before using it while breastfeeding.
Medically reviewed by Eni Williams, PharmD
REFERENCE: FDA Prescribing Information
Medically Reviewed by a Doctor on 9/28/2015
Report Problems to the Food and Drug Administration
You are encouraged to report negative side effects of prescription drugs to the FDA. Visit the FDA MedWatch website or call 1-800-FDA-1088.
Need help identifying pills and medications?
Back to Medications Index
- Home
Image Collection (categories)
- Allergic Skin Disorders
- Bacterial Skin Diseases
- Bites and Infestations
- Diseases of Pigment
- Fungal Skin Diseases
- Medical Anatomy and Illustrations
- Noncancerous, Precancerous & Cancerous Tumors
- Oral Health Conditions
- Papules, Scales, Plaques and Eruptions
- Scalp, Hair and Nails
- Sexually Transmitted Diseases (STDs)
- Vascular, Lymphatic and Systemic Conditions
- Viral Skin Diseases
- Additional Skin Conditions
See All Quizzes (119)
& Conditions
Topic Centers
& Living
- Medications


text:
iPhone 5 Screen
by: PlayWithAndroid | 3
by: PlayWithAndroid | 3
This is iPhone 5 fake screen app!
This free Android app allow you to trick/prank your friends into thinking you have the latest iPhone!
To develop more free apps, we have implemented some ads and our app contains "NO notification or icon ads"!!
iPhone is a trademark of Apple Computer Inc.
This app is not affiliated with or endorsed by Apple.
Tags: screen , not endorsed by disclaimer ad , papel de parede 3 screen , computer screen with apps image
from 3 reviews
Free - V1.0 - 476K
Sorry ...
This app is no longer available.


text:
Announcing Cotton Spandex Jersey!
More about this preview.
Preview scale: 21.0 x 18.0 inches

Change DPI Base size is 150
What we do and how we do it.
See last week's winners
Explore Our fabrics
Optional Filters
I'm going to use this fabric to make...
I'm looking for a fabric that is...
Receive our weekly email with news about contests, giveaways and prodution releases.
Email Address
No Thanks
© 2008-2015 Spoonflower Inc


text:
FiltersShow / Hide
- All Events
- Music
- Comedy
- Theatre & Shows
- Sport
- Cinema
- Festivals
- Days Out
- All Tickets
- Latest Tickets
- Free Events
- Special Tickets
- Disabled Access Venues
- Festival Offers
- Brit Awards 2016
- Anywhere In The UK
- Location: Anywhere in the UK
Radius: ... miles
Get an alert when new Edan dates are announced
A highly talented MC from Boston whose 'Primitive Plus' album topped many hip-hop 'Best Of' lists in 2002. But it is live where Edan shines; a witty freestyler, often trading off rhymes with his counterpart Insight and incorporating improvisation and effect manipulation into his sets, Edan has a bright future on both sides of the Atlantic.
See more info at humblemagnificent.com
Edan Tour Dates
No current UK tour dates for Edan
Alert me when new Edan tour dates are announced...
You are tracking Edan.
We will email you as soon as UK tour dates are announced.
- Be the first to know about new Edan tour dates
- Alerts are free always will be
- We hate spam and will never share your email address with anyone else
- 1,236,680 fans now rely on Ents24 to track their favourite artists and venues
Puttour dates on your web site
- Customisable, flexible tour date widget
- Super quick set-up, get the code here
News and Announcements RSS feed
No current news or announcements.
Have Your Say
- Have you seen Edan? Why not write a review?
Fans who like 'Edan' also like:
Do you represent Edan, or are you a member of their promotional team? Click here to claim this page