ESSEX   BOTANY   AND   MYCOLOGY  GROUPS 

Progress with a project to locate and DNA fingerprint the Water Poplars of Southern England.

        KENNETH J ADAMS, School of Health & Biosciences, University of East London.E15 4LZ. (ken.adams@virgin.net).

What is a Water Poplar? It is the old English name (Cook, 1676) for what we now call the ‘Native’ Black Poplar or the ‘Atlantic race of the European Black Poplar’: Populus nigra betulifolia. Neither vernacular is particularly appropriate as it would seem that many if not all our trees are probably no more native than the ‘English’ Elm, Ulmus procera; now known to have been introduced to England in the first century, AD and to derive from an Italian clone to the east of Rome. More appropriately given the less embarrassing appellation Atinian Elm (Gil, L et al, 2004).  Furthermore, Populus nigra extends right across Europe into central Asia and south into N. Africa, so it’s hardly appropriate to call P. nigra sensu lato the ‘European’ Black Poplar. Hence my endeavour to promote the old English name to minimise confusion with so-called ‘Italian’ Black Poplars/Hybrid Black Poplars and other Euroamerican and Euroasian hybrids and the various American Cottonwoods’.

 There has been a growing realization Europe wide, that enormous quantities of pollen are being produced by the tens of thousands of ornamental and commercial plantings of crosses between the exotic American Eastern Cottonwood P. deltoides and P. nigra typica, and P. nigra betulifolia; together with fastigiate clones of Populus nigra typica, in particular the male ‘Italica’, and the various male P. nigra betulifolia x ‘Italica’ clones known collectively as ‘Plantierensis’. Although females of P. nigra have been shown to respond preferentially to conspecific pollen in mixed pollen clouds (Vanden Broecka et al 2006), if none alights on their stigmas, they will accept pollen from, and cross with P. x canadensis. They can also cross with ‘Italica’, and Water Poplar males can cross with the fastigiate female ‘Gigantea’. Despite the thinly scattered distribution of our WPs, this is not a significant problem in the U.K. however, as suitable conditions for germination and establishment no longer exist due to destruction of our riparian forests. It is however, a problem on the continent, where for example around 50% of the young trees along the Rhine were found to be of hybrid origin (Smulders et al 2008), and will become our problem if, as proposed recently, riparian forests are re-establised in the U.K.. In another sense, it is already a problem, in that numerous hybrid trees imported from the continent involving ‘Italica’ and ‘Gigantea’ as parents have been turning up in the U.K. Thus, if we don’t vegetatively propagate the full range of genetic variation present in our veteran WPs this will be lost when the old trees die. Genuine Water Poplar seedlings are very exacting in their environment, requiring a gravelly, weed-free substrate, that remains wet with well-oxygenated water, right through the summer, so that any seedlings that survive are likely to be the less exacting and more vigorous hybrid crosses. A few seedlings are occasionally found in the U.K., but we can only be really sure that they are the product of a genuine intraspecific cross if we bag the young female flowers and dust them when receptive with pollen from a bono fide male and grow up the seedlings in a nursery. Until we know more about our clones I would urge restraint on making artificial crosses. Suppose for example one of our strains has an extra thick cuticle that protects it against fungal attack. If we cross it with one that has a thin cuticle we could end up with compromised progeny.

 In recent decades, enthusiasts have become concerned about the apparent scarcity of Water Poplars, and are now aware of the need to conserve as much remaining genetic variation as possible, to try and avoid the sort of epidemic disease outbreak that decimated our monoclonal Ulmus procera. This has led a number of counties to extend and refine the recording of the exact location and sex of individual trees started by the late Edgar Milne-Redhead, and they have begun to collect cuttings from every individual tree, and one or two from each group of trees, and to propagate them in clone banks. This resource, combined with new highly sensitive DNA microsatellite analyses, has made it possible to begin to answer a number of outstanding questions about our U.K. Water Poplars. This analysis is based on the detection of size differences in a set of seven highly repetitive regions of DNA known as microsatellites, that are widely dispersed across the poplar genome. These small size differences, which accumulate over long periods of time, can be detected by isolating these DNA segments and running them on polyacrylamide gels. The resulting information can be used to indicate whether a tree is likely to have been derived vegetatively via a cutting, in which case the pattern of microsatellites of parent and offspring will be identical, or whether it has grown from a seed, in which case the microsatellite pattern will be a combination of the fingerprint of the parents. This technique has been adopted in the U.K. by Joan Cottrell and Stuart A’Hara at Forest Research, Roslin, and was initially applied to the WPs held in clone banks across the U.K (A’Hara et al 2009). Their results for the 250 trees sampled, suggest, that as already suspected, most of our U.K. trees have been cloned from cuttings taken from a small number of genetically unique individuals.

Joining forces with Peter Ennis and Keith Turner, who had been locating and mapping WPs in Suffolk and Essex, I began in 1997 to search for, locate, and accurately map all the veteran WP trees in Essex, and more recently throughout the London area and parts of North Wiltshire/East Gloucestershire. It quickly became apparent that using national grid references, whether derived by map or GPS, was not a sufficiently accurate way of locating or recording an individual tree, and many hours and days were spent searching for trees that others had reported. The problem has been solved by annotating the crown of each tree recorded on satellite aerial images[ using the site http://wtp2.appspot.com/wheresthepath.htm, in conjunction with PaintshopPro 5] so that now that we have begun to sample the trees for DNA fingerprinting it is possible to be certain which tree has been sampled. During 2009 a further 126 trees were sampled from the wild, completing the list of known trees in Essex and as many isolated individuals and groups of trees that we could find in the London area. These were sent to Forest Research for DNA fingerprinting.

At the outset of this work I had set myself a number of questions. Firstly, did the range of clones in clone banks represent the full range of clones that grow out there in the wild in Britain? Or had these been swapped around by nurseries from a relatively few isolates? Secondly, have most of the veteran trees in the wild been derived vegetatively from a small number of clones? - or are there any populations that appear to have been derived genuinely from seedlings? Thirdly, have we been ignoring genuine WP veterans that do not have the typical morphology described for P.nigra ssp./var. betulifolia, such as lack of hairy petioles or burring? And finally, are there any insects that are entirely specific to WPs so that we can use them to identify the species, or we likely to come unstuck if backcrosses between P. nigra betulifolia and any of the F1 P. deltoides x nigra hybrids also act as hosts to these insects.

Taking the last question first, I had been struck by my observation back in 1998, that four male trees on the cliffs at Southend were so infested with the aphid Pemphigus spyrothecae that they had up to six spiral galls on their petioles, noticeably disfiguring the canopies from some distance away, despite their complete absence from several P. x canadensis growing in the vicinity. Since that time, I have never seen a spiral gall on an obvious P. x canadensis, or any poplar species other than those obviously derived from P. nigra, such as betulifolia, ‘Italica’, ‘Gigantea’ or ‘Plantierensis’. Others however have claimed to find spiral galls on hybrids, and it occurred to me that these might be back crosses. If there is something in the genome of P. deltoides that makes the petioles of the F1 hybrid with P. nigra unattractive or unpalatable to P. spyrothecae, - what if this was backcrossed with P. nigra? Might in some cases the gene(s) responsible be lost at meiosis? I therefore collected samples from a number of trees that I came across that looked as if they might be backcrosses, or did not quite fit the typical betulifolia morphology. In addition to the seven microsatellites common to the Aigeiros (Cottonwoods and Black Poplars) subgenus, there is a shared gene that in P. deltoides has a sizeable DNA deletion, that is being used by Forest Research to detect first generation P. x canadensis hybrids. Unfortunately, although infallible for F1 hybrids, just like my hypothetical P. spyrothecae resistance gene, it will be lost at meiosis in half the offspring in a F2 backcross. So far only one of my samples has exhibited both the deltoides specific deletion and possessed spiral galls, although only a couple of galls were found on the tree. Thus it could have been a backcross in which ‘my gene’, had been lost but not the deltoides deletion. Fortunately, further genetic markers have now become available that are also specific to the deltoides genome, and it will therefore henceforth be possible to detect such a backcross. A word of caution, however, spiral galls will not be found in any numbers, if at all, on P. nigra agg. trees that are water stressed, such as those that flush late in a spring drought, or are recovering from drastic pollarding or crown reduction. Although aphids of the genus Pemphigus also occur in N. America on their various Cottonwoods, and four species have been found to be specific to P. deltoides; there are several European species which appear to be specific to P. nigra sensu lato.

The following are regarded as specific to the old world P. nigra aggregate:

                                                   Secondary hosts                           Abundance

Pemphigus spyrothecae               no secondary host                         common

Pemphigus protospirae                Berula erecta, Sium latifolium       very rare

Pemphigus bursarius                   Lettuce Root aphid                        frequent

Pemphigus populi                        Medicago, Melilotus, Lathyrus      rare

Pemphigus populinigrae              Marsh Cudweed aphid                   common?

Pemphigus phenax                       Carrot Root aphid                         common?

Pemphigus gairi                          Aethusa cynapium                        common?       

Apart from P. spyrothecae, which overwinters as an egg on the bark of its host, all the others migrate to the roots of various herbaceous plants as alternate hosts. I have never seen P. protospirae, - not surprising perhaps as it has Berula and Sium as its secondary hosts. Maybe it will turn up in the Cambridge or Norfolk fens. It is distinguished by the presence of more than the two complete turns typical of the spiral gall of P. spirothecae. P. bursarius, the pouch gall is another petiole specialist and is next in order of frequency. It occurs commonly on ‘Italica’ and occasionally on betulifolia. P. populi, which forms a globular gall on the upper surface of the midrib with an access hole below and often splits open above, is rare and I have only seen it a couple of times [on betulifolia]. The next three belong to a group which form similar linear, pouch-like galls on the mid-rib, and you have to look at the aphids inside to identity the exact species. Something I have not yet done, although the pouches which start off green, and then go red, are not uncommon. So the answer to my question would appear to be, if any of these galls appear in large numbers on a tree it must be one of the P. nigra agg. If only a few galls are present, it could either be due to water stress or the possibility of a (deltoides x typica) x (betulifolia) backcross, requiring a test for several of the new deltoides specific DNA markers to be sure.

Going back to my list of questions. Do the genetic fingerprints of veterans in the wild match those in existing clone banks, and have these veteran trees mostly been propagated from cuttings? The clone bank results referred to above suggested that the 7,000 male trees planted around Greater Manchester were probably all Clone 28, and that the 5,000 male trees in Aylesbury Vale are probably all Clone 35. Clone 28 also turned up in clone banks elsewhere, and our results suggest that at least 150 of the trees scattered over the London area derive from this clone, and a further 120 occur in Essex. The large population in the Lee Valley at Hackney and surrounding suburbs is thought to have been planted in the 1890s, and the population in Hainault Country Park in 1910. This raises the intriguing question as to the social history of these plantings. Did the nursery that propagated the enormous number of trees for Greater Manchester also sell them country wide? Possibly to finance the project? Or did they just happen to choose a stock tree from a clone that was already widespread. This clone is known to be susceptible to a strain of Poplar Scab that is decimating the Greater Manchester population. What we don’t know, is whether other clones are equally susceptible, or whether the stresses of living in a damp urban environment have caused the epidemic, and this clone just happened to be in the firing line. Until we know the answer, and since there are already large numbers of clone 28 around, it would be prudent not to plant any more of them.

Of the females, my limited results so far for N. Wilshire/East Gloucestershire suggest that the large population of morphologically distinct females occurring along the tributaries of the Isis are all likely to be of a new Clone (49) unique to this catchment. In addition our results extend the suggestion that Clone 47 of which there are at least 30-40 trees, is probably unique to the Herts./Cambs./Suffolk confluence and we can therefore confidently expect more trees of this clone among the 100 or so trees recorded for Cambs., yet to be fingerprinted. The large population of female trees of clone 32 in Suffolk extends thinly scattered right across Essex and into the London area. One male tree near Waples Mill near the R.Roding in Essex, is so far a new clone with a single representative, and might possibly derive from a genuine seedling. On the other hand the intriguing occurrence of a male tree in a hedgerow near Harlow with an identical fingerprint to a tree on an island in the Lea on the Middx. side of the river, and so far no match elsewhere, is a real social history puzzle. Thus far then, it is clear that there are more multiple plantings out there in the wild of clones that have yet to be discovered, that saturation mapping is going to be worthwhile, and that virtually all our trees are likely to have been planted from truncheons (large 5 to 6ft long cuttings) taken from existing trees.

Finally, perhaps the most exciting discovery of all, concerns the enigmatic mixed population of males and females in a line along the south-west bank of the tidal Thames from Hammersmith to Putney. They were recorded there as long ago as 1852 by J. Boswell Syme (Lousley 1976). Of the 40 odd trees I have only sampled the first two males and first two females up from the Barnes end, but intriguingly they have all four proved to be genetically unique, suggesting that they may represent a relict population originally germinating as seedlings along the gravelly shore line. The Victorian engineers obviously appreciated the binding power of their roots, and the role of their trailing branches in slowing the marginal currents, thus protecting the river bank, and they carefully constructed their stone and brick revetment around the base of the trees. As my photograph shows the revetment does not appear to have been repaired since then, suggesting that the trees were already fully grown at the time and that they must therefore be close to 200 years old. No new seedlings are likely to survive however, due to the sealing effect of the revetment and the metalling of the towpath. The role of riverine trees in stabilizing river banks is well known abroad and engineers in Australia have carried out experiments to quantify the advantages of using trees for bank protection, concluding that the presence of tree roots can increase the safety factor of an otherwise unstable bank section by 60% (Abernethy & Rutherford 2000). Even in the U.K. the company Salix [www.salixrw.com] routinely uses willow trees for this purpose. However the Port of London Authority have been advised otherwise and this, possibly the only substantial surviving population of Water Poplars grown naturally from seed in the U.K. is under imminent threat.  

In 2010/2011 I would like to sample all of this line of trees, as well as additional trees that have been discovered in London, Suffolk and Essex; the trees that have now been  located in Surrey (c.200); Cambs. (c.100), and Norfolk (c.70); and to continue to map and sample the trees in East Herts. I also have a substantial shopping list for N.Wilts and E. Gloucestershire. Fingerprinting is an expensive business however, and I need to find around £10,000 to finance the next phase of the project. So if any of our readers would like to make a donation to the project, or help to raise the cash, your assistance would be most welcome. And if you are influential and can help to save that unique row of trees along the Thames your help would be appreciated. Another aspect of the Water Poplar saga that needs financing is the experimental work that needs to be done to establish whether any of our clones are resistant to the current strain of Poplar Scab. When in 50 years time all the tropical hardwood trees are carbon sequestered as people’s front doors or expensive furniture, we may need to turn once again to the Water Poplar as a timber tree. A more extensive résumé of the plight of the world’s poplar species, how to distinguish Water Poplars morphologically, and how to grow them and sample them for DNA fingerprinting – plus tables and colour coded maps of the trees we have sampled so far, can be found on the BSBI linked website. www.bsbi.org,uk >>Recorders>>Essex>>Water Poplars.

Many thanks to all who have sent in records of tree locations, please keep them coming – and to the Essex Biodiversity Project and the Royal Parks for covering the DNA fingerprinting costs to date. I am also very grateful to Joan Cottrell for carefully reading through this article and making corrections and suggestions to improve it.

References

ABERNETHY, B. & RUTHERFORD I.D. (2000) The effect of riparian tree roots on the mass stability of riverbanks. Earth Surface    Processes and Landforms. 25 (9):921-937.

A’HARA, S. SAMUEL S. & COTTRELL, J. (2009) The role of DNA-fingerprinting in the conservation of Black Poplar. British Wildlife 21 (2): 110-115.

COOK, M. (1676). Manner of raising, ordering and improving forest trees, p. 81. London.

GIL, L. et al, (2004) English Elm is a 2,000-year-old Roman Clone. Nature 431:1053.

LOUSLEY, J.E. 1976 Flora of Surrey. David & Charles. Devon.

VANDEN BROECK, A. COTTRELL, J. QUATAERT, P. BREYNE, P. STORME, V. BOERJAN.W. & VAN SLYCKEN J. (2006) Paternity analysis of Populus nigra L. offspring in a Belgian plantation of native and exotic poplars. Ann.For.Sci. 63:783-790.