It’s just a box …

illumina_MiSeq_linocut

Today’s post starts with a linocut of an Illumina MiSeq Next Generation Sequencer (NGS), as part of an ongoing campaign to demystify these state-of-the-art £80,000 pound instruments. It’s just a box stuffed with clever electronics.   The problem is that tech-leaning biologists go misty-eyed at the very mention of NGS, and start to make outrageous claims for what it can do.   But how much are they actually going to change the way that we assess the state of the environment?   I approach this topic as an open-minded sceptic (see “Replaced by a robot?” and “Glass half full or glass half empty?” and other posts) but I have friends who know what buttons to press, and in what order. Thanks to them, enough of my samples have been converted into reams of NGS data for me now to be in a position to offer an opinion on their usefulness.

So here are three situations where I think that that NGS may offer advantages over “traditional” biology:

  1. reducing error / uncertainty when assessing variables with highly-contagious distributions.
    Many of the techniques under consideration measure “environmental DNA” (“eDNA”) in water samples. eDNA is DNA released into water from skin, faeces, mucus, urine and a host of other ways.   In theory, we no longer need to hunt for Great Crested Newts in ponds (a process with a high risk of “type 2 errors” – “false negatives”) but can take water samples and detect the presence of newts in the pond directly from these.  The same logic applies to lake fish, many of which move around the lake in shoals, which may be missed by sampler’s nets altogether or give false estimates of true abundance.   In both of these cases, the uncertainties in traditional methods can be reduced by increasing effort, but this comes at a cost, so methods based on eDNA show real potential (the Great Crested Newt method is already in use).
  2. Ensuring consistency when dealing with cryptic / semi-cryptic species
    I’ve written many posts about the problems associated with identifying diatoms.   We have ample evidence, now, that there are far more species than we thought 30 years ago. This, in turn, is challenging the ability to create consistent datasets when analysts spread around several different laboratories are trying to make fine distinctions between species based on a very diffuse literature.   Those of us who study diatoms now work at the very edge of what can be discriminated with the light microscope and the limited data we do now have from molecular studies suggests that there are sometimes genetic differences even when it is almost impossible to detect variation in morphology.   NGS has the potential for reducing the analytical error that results from these difficulties although, it is important to point out, many other factors (spatial and temporal) contribute to the overall variation between sites and, therefore, to our understanding of the effect of human pressures on diatom assemblages.
  3. Reducing costs
    This is one of the big benefits of NGS in the short term.   The reduction in cost is partly a result of the expenses associated with tackling the first two points by conventional means.   You can usually reduce uncertainty by increasing effort but, as resources are usually limited, this increase in effort means channelling funds that could be used more profitably elsewhere.   However, there will also be a straightforward time saving, because of the economies of scale that accompanies high-throughput NGS.   A single run of an Illumina MiSeq can process 96 samples in a few hours, whereas each would have required one to two hours for analysis by light microscope. Even when the costs of buying and maintaining the NGS machines are factored in, NGS still offers a potential cost saving over conventional methods.

It is worth asking whether these three scenarios – statistical, taxonomic and financial – really amount to better science, or whether NGS is just a more efficient means of applying the same principles (“name and count”) that underpins most ecological assessment at present.   From a manager’s perspective, less uncertainty and lower cost is a beguiling prospect.   NGS may, as a result, give greater confidence in decision making, according to the current rules. That may make for better regulation, but it does not really represent a paradigm shift in the underlying science.

The potential, nonetheless, is there. A better understanding of genetic diversity, for example, may make it easier to build emerging concepts such as ecological resilience into ecological assessment (see “Baffled by the benthos (2)” and “Making what is important measurable”). Once we have established NGS as a working method, maybe we can assess functional genes as well as just taxonomic composition?   The possibilities are endless.  The Biomonitoring 2.0 group is quick to make these claims.   But it is important to remember that, at this stage, they are no more than possibilities   So far, we are still learning to walk …

Reference

Baird, D.J. & Hajibabaei, M. (2012). Biomonitoring 2.0: a new paradigm in ecosystem assessment made possible by next-generation DNA sequencing. Molecular Ecology 21: 2039-2044.

In search of SuDS …

Fellgate_150918

The best ideas in environmental management are often the simplest. That sometimes means that they are not particularly photogenic.   I offer this as an excuse for the photograph above, in which six ecologists appear to be somewhat underwhelmed by a shallow manmade depression just on the edge of a housing estate near Jarrow.   The main topic of conversation, I have to admit, was: “why do they insist on us wearing hard hats when all we are doing is standing in a shallow depression?”   That’s health and safety for you.

The visit came as part of a seminar on Sustainable Drainage Systems (SuDS) at Northumbria University which I attended last week in order to brush up some lecture notes ready for the new academic year.   I wrote a post last year about how quickly river levels can change in urban areas (see “Fieldwork in the rain”) and the effects can sometimes be catastrophic (the infamous “Toon Monsoon” in June 2012 being a case in point).   The problem stems from the lack of permeable ground in urban areas, which means that water runs straight into drains and into rivers.

We could address problems of urban flooding by building bigger and more efficient drains and sewers but that comes at a huge price – both in financial costs and in the disruption caused as roads are dug up to allow access to the drains.   Or we could try to mimic nature and simply slow down the journey that rainwater takes once it hits a surface.   That’s the basic principle behind SuDS, achieved by providing a range of permeable surfaces, channels and collection / retention ponds, all of which can, at the same time, have aesthetic and biodiversity benefits for local communities.   The photograph above doesn’t really capture the aesthetic benefits but my camera was on the wrong setting when we visited a nice reed-lined pond as part of the same development. Sorry.

The works we were visiting were part of a scheme funded by Northumbrian Water to address flooding issues around the Fellgate estate in Tyne and Wear. That a major utility company is investing in SuDS shows how seriously these are being taken as options for flood control whilst, at the same time, illustrating a more profound point. There is a tendency for us to externalise problems such as flooding and to expect local authorities and water companies to find solutions on our behalf. Yet the problem is partly one of our own making. I can look out of my windows and see front and back gardens of neighbours that have been paved over, much reducing the amount of permeable surface into which rainwater can percolate.   Your lawn is a part of your local SuDS, whether or not you realised it.   And your gutters do not need to funnel straight into the drains either: that water could be collected and used to water the garden or wash the car.

The two pictures below show the direction we could be following: the first is a “green roof” atop one of Durham’s colleges, which uses rain that would otherwise go straight into a water course to sustain vegetation that, in turn, provides insulation for the building below. The lower image shows a “green wall” at a local branch of Marks and Spencer, fed by rainwater harvested from the roof and providing an aesthetic diversion for Newcastle shoppers, as well as playing a role in flood prevention.  I also wrote about a flood channel that doubled as an urban recreation space in the centre of Seoul (see “A brief diversion to South Korea”).

Green_roofs

Top picture: the green roof of Josephine Butler College, University of Durham; bottom picture: the green wall at Marks & Spencer, Northumberland Street, Newcastle.

One of the vaguer promises of those involved with SuDS is improvements in water quality. I am sure that these will accrue but have not seen the hard evidence to demonstrate this.   The problem that I mentioned in my post from last year is that our old “combined sewers” can overflow during floods and dump raw, untreated sewage directly into rivers.   In theory, SuDS should reduce the peaks in hydrographs during floods and, in the process, reduce the incidences when the storm sewers overflow.   In a river such as the Ouseburn, this could make a real difference to the ecology. But that’s a story for another day…

A genuinely rare diatom genus?

About ten months ago I speculated on the practicalities of producing a “red list” of British diatoms (see “A red list of endangered British diatoms”).   A couple of weeks ago, I reported on progress towards this goal (see “Why do you look for the living amongst the dead?”) and, today, I can show the first evidence for a genuinely rare freshwater diatom in Great Britain.   Great Britain, alas, rather than the United Kingdom, as we struggled to get the Irish grid references to plot on our graphs, but it is a step in the right direction.

In my post last year, I commented that one of the problems we faced was the taxonomic uncertainty, particularly as there have been so many new species described, often as a result of “splitting” taxa that older books regarded as a single entity.   One of the criteria my intern, Susannah, and I used was a stable taxonomic concept, in order to ensure that older records could be merged with our more recent records.   This does not have to be at the level of species and, indeed, the first subject of our scrutiny was a genus, Tetracyclus. It fits our bill perfectly as it is quite recognizable, despite not being very common, so we can be fairly sure that the chance of an identification error is low.

Tetracyclus

The left hand map shows all hectads (10 km2) squares from which we have at least one freshwater diatom sample in Great Britain; the right hand map shows those hectads with records of Tetracyclus spp.

As you can see from the map, there are a cluster of locations in the Scottish highlands, three in the Cheviots in Northumberland and a few in Snowdonia. Then there is a record in Pembrokeshire and another in Wiltshire.   That is 23 hectads throughout the UK for all three species, none of which, individually, is found in more than 15 hectads, and therefore each qualifies as “nationally rare” according to JNCC guidelines.   There may be a few more records out there but, at the same time, there is also a risk of false negatives – a single valve, remember, does not necessarily mean that a viable population of the organism is present at the site.   The Wiltshire record worries me, and I need to check that there has not been a transcription error as the record was transferred from notebook to database, and to ensure that the grid reference was recorded correctly.

The literature offers little help when it comes to understanding the habitat of Tetracyclus: West and Fritsch tell us that Tetracyclus lacustris “… prefers hilly districts and is often found in the plankton of mountain lakes” whilst T. rupestris “… occurs on dripping rocks in mountain areas.” Other writers also hint that it might be partly sub-aerial in distribution, which may explain why the records in our dataset only ever record it in small quantities.   Is it possible that our samples, which are mostly from submerged rocks in streams and lake littoral zones, are just picking up a few stray cells that have been washed away from their preferred habitat?   We can interpret each dot, perhaps, as indicating that the species was “present in the vicinity”.

We should also point out that one of the Snowdonian locations on the map is Llyn Perfeddau, the lake from which the first Tetracyclus lacustris specimens were described, back in 1843 by John Ralfs.   Allan Pentecost re-visited Llyn Perfeddau in 2014 but was unable to find it in his samples, which adds to the mystique that this genus and species exert.

So are these three Tetracyclus species the first bona fide candidates for a UK (or GB) red list of diatoms?   Each fulfils the criterion of “nationally rare”, being found in less than 15 hectads, albeit with the provisos set out above. However, rarity alone is not sufficient to place a species on a red list; we also need to demonstrate that it is vulnerable or endangered.   This implies knowledge of trends over time, not just patterns in space.   The default under such circumstances is to consider whether the locations where it is found are fragmented and are, themselves, threatened or vulnerable.   The restricted distribution in low nutrient waters in mountainous and northern areas does suggest that changes in land management or climate change could affect the small isolated populations that we do have, so a designation of “vulnerable” is probably appropriate.   Though I doubt that WWF will be replacing their panda logo with a diatom any time soon.

Reference

Pentecost A. (2014). In search of the Welsh Tetracyclus. The Phycologist 88: 42-43.

 

Glass half full or glass half empty?

UKeDNAWG_Bangor

I’m tapping away at the back of a conference hall during the UK eDNA working group at the University of Bangor, absorbing the latest developments in the use of molecular technologies for applied ecology. Things have moved fast over the last couple of years, with one method, for the detection of Great Crested Newt, having moved out of the research laboratory and into widespread use. Several other methods – including our own method for ecological assessment using diatoms – are drawing close to the stage where their adoption by end-users is a real possibility, and lots more good ideas were bouncing around during the sessions and during the breaks.

The fascination of much of this work lies in the interplay between the new and the old, using the sensitivity of molecular tools to push our understanding of what traditional methods have told us. At the same time, the new methods are largely underpinned by libraries of reference barcode sequences that provide the essential link between the ‘old’ and the ‘new’, and these are wholly dependent upon specialists rooted in old-school taxonomy.

Here’s the rub: if the goal is to produce methods for ecological assessment, then the point will come when the research scientists step back and the end-users start to use it.   At this point, real world exigencies take over and, with the public sector battered by funding cuts, any opportunity to save money will be grasped. What this means is that we cannot assume the same depth of ecological knowledge in the users as in the developers. This was not always the case as the Environment Agency had a cadre of extremely capable ecologists, with a deep knowledge their local rivers and broad understanding of ecology. I don’t believe that this was knowledge that they were taught, rather that they learnt it on the job, seeing streams in all of their moods as they collected samples, poring over identification guides as they named their specimens and sharing knowledge with colleagues.   The result was a depth of understanding which, in turn, they drew upon to advise colleagues involved in regulation and catchment management.

In the last few years this system has come under scrutiny as managers have searched for cost savings. Environment Agency biologists are no longer expected to collect their own samples, which are taken for them by a separate team in the misguided assumption that highly-trained biologist will be more productive if they stay in the laboratory and focus on using their specialist identification skills. Moving to molecular techniques will just continue this process.   Once the excitement of the research is over, the methods will be ripe for economies of scale; sample processing will be a task for molecular biological technicians, and the first time an ecologist encounters the sample it will be as processed output from a Next Generation Sequencing machine.

The function of the laborious process of collecting and analysing ecological samples is not just to produce the data that underpins evidence-driven catchment management. It also allows biologists to acquire the experience that lets them interpret these data sensitively and wisely.   The urge to find short-term savings has focussed on the quantifiable (how many samples can a laboratory process) and ignored the unquantifiable (how good is the advice that they offer their colleagues?).   I don’t think the full calamity of what we are seeing will hit for a few years because most of the ecology staff in the Environment Agency have done their apprenticeships in the field. Over time, however, a new generation will join the organisation for whom computer print-outs may be the closest they get to encountering river and stream life.

I am basically enthusiastic about the potential that molecular technologies can offer to the applied ecologist but we do need to be aware that implications of these approaches extend beyond issues of analytical performance characteristics and cost. This is because ecology is more about making lists of what organisms we find at a particular location (and, judging by the presentations, this seems to be the focus of most current studies) but about what those organisms do, and how they fit together to form ecosystems and food webs. OK, so you can read about Baetis rhodani in a textbook, but that is never going to be the same experience as watching it scuttle across the tray where you’ve dumped the contents of your pond net, or of observing a midge larva graze on algae under a microscope. The problem is that we cannot put a value on those experiences, in the same way that the cost of processing a sample can be calculated.

This is a theme that I’ve explored several times already (see “When a picture is worth a thousand base-pairs …”, “Simplicity is the ultimate sophistication: McEcology and the dangers of call centre ecology” and “Slow science and streamcraft”) but it bears repeating, simply because it is an issue that falls through the cracks of most objective appraisals of ecological assessment.   The research scientists tend to see purely in terms of the objective collection and interpretation of data, whilst managers look at it in terms of the cost to produce an identifiable product.   It is more than both of these: it is a profession.   The challenge now is to integrate molecular methods into the working practices of professional “frontline” ecologists, and how we prevent this professionalism being degraded to a series of technical tasks, simply because the bean counters have worked out that this is the most efficient way to distribute their scarce resources around the organisation.

Notes

Biggs, J., Ewald, N., Valentini, A., Gaboriaud, C., Dejean, T., Griffiths, R.A., Fosterd, J., Wilkinson, J.A., Arnell, A., Brothertone, P., Williams, P. & Dunna, F. (2015). Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biological Conservation 183: 19-28.

Why do you look for the living among the dead?

This post continues a theme that I’ve touched upon before: that those of us who study diatoms are Not Like Other Biologists (see “Diatoms and Dinosaurs”).   Inside every diatomist, there is a deep-seated yearning for silica that overrides the love affair with carbon that drives most biologists. Hand a diatomist a sample of living organisms and he (or she) will drop it into a pot of strong oxidising agents and then smile contentedly until the last vestiges of life have fizzed out of the cells.

I have commented before on the problems that this causes us when we are trying to understand their ecology (see “The meaning of … nothing” and “All things bright and beautiful”) and this post extends that theme, looking at problems of mapping the distribution of diatoms.  It is based on work done by my intern, Susannah Collings, who has been funded by the British Phycological Society over the summer to look into the prospects for a UK “red list” of rare and threatened diatoms (see “A “red list” of endangered British diatoms?”).   We decided to use Achnanthes oblongella, a species that is not particularly rare, is straightforward to identify and which has a fairly well-understood ecology to illustrate some of the challenges.

The database that we used for these analyses contains records from 6308 samples from over 3000 sites throughout the UK (and some sites in the Republic of Ireland too), within which there were 2180 records of Achnanthes oblongella.   In many of these, only a few valves of Achnanthes oblongella were recorded, but there were 384 samples where it constituted a third or more of all the diatoms recorded. In sixteen samples it constituted 80 per cent of all diatoms, with two samples having 98% A. oblongella.

Ach_oblongella

Achnanthes oblongella: top left: three examples of the rapheless valve; bottom left: three examples of the raphe valve (photographs: M. Bayer, RBGE; scale bar: 10 micrometres, or 1/100th of a millimetre). Right: histogram showing the relative abundance of A. oblongella in samples.

When these records are plotted onto a map using DMAP software, we see the records clustered in those parts of the country that are associated with soft, often peaty and acidic waters: Cornwall and Devon, North Wales, parts of the Pennines and Lake District, south-west Scotland and the highlands.   There are also a number of records in the New Forest and around London, the latter probably reflecting patches of heathland on the Greensand.   But, wait a minute, remember that predilection for silica that I mentioned above; how can we be sure that each of these dots on the map represents a living population of Achnanthes oblongella at that location?   The analyst never saw a living cell of the species, only parts of a dead cell.   In situations where the relative abundance is very low, can we eliminate the possibility that a few dead cells had been washed downstream and ended up in our samples?

Ach_oblongella_maps

The distribution of Achnanthes oblongella in Great Britain: a. all records; b. only records where A. oblongella constitutes > 1% of all diatoms; c. only records where A. oblongella constitutes > 2% of all diatoms.

It may seem surprising that no-one has studied how well samples of cleaned diatoms represent living populations but the problems associated with naming diatoms from live material (real or perceived … that’s a subject for another day) mean that the literature on this topic is sparse.   We compared the map produced with all data with maps produced if we just used records with more than one or two per cent of Achnanthes oblongella and, in this particular case, the broad patterns are the same, even though we are trimming away almost half of the data. I suspect that this is partly because A. oblongella is quite straightforward to identify, so the number of spurious records is relatively low. The distinctive ecology means that even where low abundances are due to washed-in cells, there is an inoculum a short distance upstream. The dots, therefore, may indicate proximity to viable populations, rather than direct evidence of their presence in situations where the percentage in the sample is low.

All this points up the great irony of compiling a red list for diatoms: red lists provide an inventory of the conservation status of species. It is supposed to catalyse action for biodiversity conservation. Yet to gather the data we need, we first have to kill the organisms that we are trying to protect, most of which have never been observed in the live state at all.   I did a quick search amongst my own books and via Google and could not find a single image of a living cell of Achannthes oblongella. The silica shell is, I am ashamed to say, the only reality that many of my fellow diatomists recognise.