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Environmental DNA: how to track biodiversity “barcodes”

Tania Louis
Tania Louis
PhD in biology and Columnist at Polytechnique Insights
Key takeaways
  • Environmental DNA (eDNA) makes it possible to study the presence of living beings in the environment without endangering species: it is a population monitoring tool.
  • Analysis of eDNA is based on the use of molecular barcodes to identify a species or category of organisms.
  • eDNA allows the study of biodiversity, targeted monitoring of certain species, estimation of species numbers and reconstruction of diets.
  • But DNA does not provide as much information as direct observation and can be moved or degraded.
  • It is crucial to optimise our study of eDNA to improve our understanding of biodiversity.

Bio­di­ver­si­ty is in the midst of a cri­sis1 and it is both impor­tant to mon­i­tor the evo­lu­tion of wild pop­u­la­tions and to dis­turb them as lit­tle as pos­si­ble. There two seem­ing­ly con­tra­dic­to­ry actions could be rec­on­ciled thanks to an increas­ing­ly use­ful source of infor­ma­tion: envi­ron­men­tal DNA (or eDNA).

Tracking organisms using their DNA

Since the 1980s, sam­ples col­lect­ed from the envi­ron­ment have made it pos­si­ble to study the invis­i­ble micro-organ­isms they con­tain by analysing their genomes. This approach, devel­oped thanks to advances in sequenc­ing tech­niques and com­put­er tools, gave rise to metage­nomics, the large-scale study of genomes.

But although micro-organ­isms are to be found just about every­where, they are not the only sources of DNA in the envi­ron­ment! Mucus, dead skin, hair, car­cass­es, fae­ces… All liv­ing beings, what­ev­er their size, leave traces of their pas­sage. And these traces can con­tain DNA that makes it pos­si­ble to track their pres­ence, which was demon­strat­ed for the first time in 2008 by a team from the Alpine Ecol­o­gy Lab­o­ra­to­ry, which iden­ti­fied the DNA of bull­frogs in var­i­ous ponds2.

eDNA is there­fore also a tool for mon­i­tor­ing pop­u­la­tions of macro-organ­isms, whose effec­tive­ness has been demon­strat­ed in many envi­ron­ments, from sed­i­ments to the seabed… and even in the air itself! Two stud­ies pub­lished at the begin­ning of 2022,34 have shown that by suck­ing air from zoos and analysing its DNA con­tent, it is pos­si­ble to iden­ti­fy dozens of species of ani­mals liv­ing in or near these zoos. This promis­ing approach opens up new pos­si­bil­i­ties but still has a num­ber of weak­ness­es. To under­stand them, we need to look in more detail at how it works.

Fig­ure tak­en from the study at Hamer­ton Zoo, show­ing the dif­fer­ent species iden­ti­fied by study­ing eDNA recov­ered from the air. The colours indi­cate the type of species (yel­low: those used to feed the zoo res­i­dents!), the size of the discs sym­bol­is­es the lev­el of detec­tion of each species.

Making environmental DNA talk

Once recov­ered from the envi­ron­ment, DNA can be analysed in three ways. The first is to per­form glob­al metage­nomics, sequenc­ing all the DNA to try to iden­ti­fy as many of the genomes present as pos­si­ble. This approach is suit­able for the study of micro-organ­isms, which are direct­ly con­tained in the sam­ple and whose DNA is there­fore well pre­served and present in large quan­ti­ties. It is less rel­e­vant for macro-organ­isms, whose DNA is rar­er and more dam­aged in the envi­ron­ment, gen­er­al­ly being found in the form of frag­ments of a few dozen to a few thou­sand nucleotides long.

The analy­sis of this type of eDNA relies on the use of mol­e­c­u­lar bar­codes, genet­ic sequences that iden­ti­fy a species or class of organ­isms. These must be short enough to be found in nat­u­ral­ly frag­ment­ed DNA. These “bar­code” sequences are specif­i­cal­ly ampli­fied by PCR and then sequenced and analysed. They can be more or less spe­cif­ic, mak­ing it pos­si­ble to mon­i­tor a sin­gle species, a group of close­ly relat­ed species or to make a more exten­sive inven­to­ry of the bio­di­ver­si­ty of an envi­ron­ment. Depend­ing on the range cho­sen, this is known as bar­cod­ing or metabar­cod­ing, which is a form of tar­get­ed metagenomics.

The analy­sis is then based on the com­par­i­son of the bar­codes obtained with those list­ed in data­bas­es. These data­bas­es are more or less well pop­u­lat­ed depend­ing on the field and this is one of the weak points of eDNA stud­ies: the rich­er the data­bas­es, the less lim­it­ed the analy­ses. The accu­mu­la­tion of new data is grad­u­al­ly chang­ing the situation!

Strengths and weaknesses of environmental DNA

Cur­rent­ly, eDNA has four main types of appli­ca­tion: the study of bio­di­ver­si­ty (cat­a­logu­ing of species, mon­i­tor­ing over time, analy­sis of bio­log­i­cal func­tions5, etc.); the tar­get­ed mon­i­tor­ing of cer­tain species (par­tic­u­lar­ly threat­ened, inva­sive or bioindi­ca­tor species6); the esti­ma­tion of the rel­a­tive abun­dance of species in a giv­en envi­ron­ment; and the recon­struc­tion of diets by analysing the DNA con­tained in excre­ment7.

In some envi­ron­ments, such as sed­i­ments and very cold envi­ron­ments, eDNA is pre­served for long peri­ods of time, mak­ing it pos­si­ble to go back in time. For exam­ple, researchers have stud­ied uni­cel­lu­lars from the Brest har­bour over a peri­od of 1,400 years, high­light­ing the impact of the Sec­ond World War and recent changes in agri­cul­tur­al prac­tices8. The record for the use of eDNA in palaeoe­col­o­gy was bro­ken at the begin­ning of Decem­ber thanks to per­mafrost sam­ples from Green­land, which made it pos­si­ble to recon­struct a palaeoe­cosys­tem of about two mil­lion years9!

eDNA also has many advan­tages for the study of present-day species. Indeed, its col­lec­tion is non-inva­sive, which avoids dis­turb­ing the envi­ron­ments stud­ied, and very sim­ple. The cor­re­spond­ing field­work requires lit­tle equip­ment and train­ing, allows access to places unsuit­able for direct obser­va­tions, can be eas­i­ly graft­ed onto expe­di­tions already planned else­where and remains decou­pled from the analy­sis work. It is much less cost­ly and restric­tive than con­ven­tion­al obser­va­tion meth­ods, allows for more sam­pling and opens up pos­si­bil­i­ties for mon­i­tor­ing on a large spa­tiotem­po­ral scale. Ana­lyt­i­cal meth­ods also lend them­selves to this expan­sion, as pool­ing the pro­cess­ing of many sam­ples gen­er­ates economies of scale.

In some envi­ron­ments, such as sed­i­ments and very cold envi­ron­ments, eDNA is pre­served for long peri­ods of time, mak­ing it pos­si­ble to go back in time.

As promis­ing as it is, how­ev­er, the use of eDNA has its lim­i­ta­tions. To begin with, and this is fun­da­men­tal even if it seems obvi­ous, detect­ing an indi­vid­u­al’s DNA is not the same as detect­ing their pres­ence. It does not tell us any­thing about its state of health, size, or stage of devel­op­ment, all of which are only acces­si­ble through direct obser­va­tion. It does not nec­es­sar­i­ly tell us where it is, either, as DNA can be trans­port­ed in the envi­ron­ment! In rivers, species can leave traces sev­er­al kilo­me­tres down­stream from their actu­al position.

More­over, not all organ­isms release DNA in a com­pa­ra­ble way into their envi­ron­ment and, depend­ing on the fragili­ty of the struc­ture con­tain­ing the DNA and the con­di­tions of the envi­ron­ment (in par­tic­u­lar pH and tem­per­a­ture), eDNA can be degrad­ed more or less rapid­ly. The absence of DNA does not nec­es­sar­i­ly mean the absence of a species. Con­verse­ly, DNA can eas­i­ly con­t­a­m­i­nate sam­ples, whether it comes from the exper­i­menters and their equip­ment or from anoth­er source. For exam­ple, restau­rants or mar­kets in coastal areas can lead to the detec­tion of eDNA from non-liv­ing fish10.

Sum­ma­ry of the steps involved in envi­ron­men­tal DNA analy­sis (green box­es) and asso­ci­at­ed lim­i­ta­tions (red inserts). Fig­ure based on a design by Paul Castag­né and Garance Casti­no, for Plan­et Vie11.

Final­ly, bio­mol­e­c­u­lar and com­put­er pro­cess­ing tech­niques gen­er­ate their own bias­es. Beyond the incom­plete­ness of the data­bas­es, PCR ampli­fi­ca­tion is not equal­ly effi­cient on all DNA sequences and, depend­ing on the bar­codes cho­sen and the way they are detect­ed, false neg­a­tives or false pos­i­tives may appear, which is dif­fi­cult to mon­i­tor on a case-by-case basis in large-scale analy­ses. This vari­abil­i­ty in ampli­fi­ca­tion, togeth­er with sam­pling bias, lim­its the suit­abil­i­ty of eDNA as a quan­tifi­ca­tion tool. Each study of this type requires metic­u­lous checks to ensure that the results obtained via eDNA are com­pa­ra­ble to those obtained by man­u­al count­ing. The final icing on the cake of com­pli­ca­tions is that the sequenc­ing itself can be a source of error.

Solv­ing these tech­ni­cal prob­lems is a cen­tral issue for teams inter­est­ed in eDNA. Sev­er­al research projects aim to reduce the uncer­tain­ties of analy­sis, notably by stan­dar­d­is­ing pro­to­cols, pop­u­lat­ing data­bas­es and iden­ti­fy­ing rel­e­vant mol­e­c­u­lar bar­codes12, which bodes well for future improvements.

As Sam Chew Chin, a PhD stu­dent study­ing fish pop­u­la­tions via eDNA, puts it, this tool can be seen as a genet­ic nose – a new way of smelling the bios­phere. It can­not detect every­thing per­fect­ly, but it opens up pos­si­bil­i­ties that, com­bined with oth­er approach­es, can only improve our under­stand­ing of biodiversity.

5Un exem­ple : https://​www​.ajspi​.com/​c​o​m​m​u​n​i​q​u​e​s​-​c​l​u​b​/​c​l​i​m​a​t​-​l​e​-​r​o​l​e​-​m​e​s​e​s​t​i​m​e​-​d​e​-​l​a​-​b​i​o​d​i​v​e​r​s​i​t​e​-​d​e​s​-​a​b​y​s​s​e​s​-​d​a​n​s​-​l​a​-​p​o​m​p​e​-​a​-​c​a​r​b​o​n​e​-​o​c​e​a​n​ique/
6Un exem­ple : https://​www6​.inrae​.fr/​s​y​n​aqua/
7Etude du régime ali­men­taire de l’apron du Rhône : https://​hal​.sci​ence/​h​a​l​-​0​2​4​62400
12Exem­ples d’un pro­jet européen http://​dnaqua​.net/​a​bout/ et d’un pro­jet cana­di­en https://​itrackd​na​.ca/​i​n​d​e​x​.​p​h​p​/​b​u​t​s​-​o​b​j​e​c​t​i​f​s​/​?​l​a​ng=fr

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