DNA Barcoding and the biological species concept:

Valerio Sbordoni

Dipartimento di BiologiaUniversità di Roma “Tor Vergata”

‘The species concept is one of the oldest and most fundamental in biology. And yet it is almost universally conceded that no satisfactory definition of what constitutes a species has ever been proposed.’

Th. Dobzhansky (1935). A Critique of the Species Concept in Biology. Philosophy of Science, 2: 344-355

Who needs species?

An accurate definition of species has become a requirement not only within the frame of systematics but also in all those fields relevant to conservation, food, health, trade and the ensuing laws.

In the unavoidable progress of taxonomy and nomenclatural changes, we face such paradoxes as that of the recognition of a species status for an endemic organism, which brings as a consequence its bureaucratic cancellation from a conservation red list (e.g.: Bombina variegata- B.pachypus problem in the Habitat Directive)

It is therefore necessary to outline operational, clear-cut rules for the delimitation of species.

Bombina variegata

Bombina pachypus

Mugil cephalus

Chelon labrosus

Liza aurata

Tapes decussatus

Tapes philippinarum

Species concepts according to Mayr (1969)

•Typological

•Nominalistic

•adimensional•Biological

•pluridimensional

The Nominalistic Species Concept

“From this remarks it will be seen that I look at the term species as one arbitrarily given, for the sake of convenience, to a set of individuals closely resembling each other, and that it does not essentially differ from the term variety, which is given to less distinct and more fluctuating forms. The term variety, again, in comparison with mere individual differences, is also applied arbitrarily, for convenience’ sake.”

(C.Darwin, 1859)

Biological species concept related definitions: I

"No matter what variations occur in the individuals or the species, if they spring from the seed of one and the same plant, they are accidental variations and not such as to distinguish a species permanently; one species never springs from the seed of another nor viceversa.“

(Ray, 1686)

John Ray (1627-1705)

Biological species concept related definitions: II

«un ensemble» ou «une collection d'individus semblables ou presque semblables», «qui furent produits par d'autres individus pareils à eux»

(J.B.Lamarck, 1803)

Jean Baptiste de Lamarck

1744-1829

Biological species concept related definitions: III

“…a whole of individual organisms resembling each other more than they resemble to any other individual organism, that are able, by interfertility, to reproduce fertile individuals who reproduce themselves in the same way, so that by analogy one could assume that they all are originated by a single individual organism.” (De Candolle, 1813)

Augustin Pyramus de CANDOLLE

1778 - 1841

Biological species concept related definitions: IV-VI

"a genetic species is a group of organisms so constituted and so situated in nature that a hereditary character of anyone of these organisms may be transmitted to a descendant of any other“ (Simpson,1943)

"...the largest and most inclusive ...reproductive community of sexual and cross-fertilizing individuals which share in a common gene pool" (Dobzhansky,1950)

"groups of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups" (Mayr,1940)

Phenetic concept: Michener,1970, Sneath & Sokal, 1973, Nelson & Platnick,1981

Recognition concept: Paterson,1978

Ecological concept: Van Valen, 1976, Mayr, 1982

Evolutionary concept: Simpson,1961, Wiley,1978, etc.

Phylogenetic concept: deQuieroz and Donoghue, 1990

Phenetic concept: Michener,1970, Sneath & Sokal, 1973, Nelson & Platnick,1981

Economical concept: Ghiselin,1974

Cohesion concept: Templeton, 1989

A necessary premise

We must distinguish DNA taxonomy from DNA barcoding, where the former directlyconcerns the circumscription and delineation of species using evolutionary species concepts and the latter is a means of identifying a priori entities by sequence similarity

Vogler AP, Monaghan MT (2007) J Zool Syst Evol Res 45:1–10.

The actual demarcation of species taxa uses morphological, geographical, ecological, behavioral, and molecular information to infer the rank of isolated populations.

Mayr, E. (1995) Philos. Sci. 63, 262–277.

Do species exist in nature? Objectivity vs.conventionalism

In an adimensional perspective, in sexual organisms effectively or potentially amphigonic (including geographic, facultative parthenogenesis, etc.), species exist since they are recognized, as potential mates, by organisms themselves.

136 bird species on Arfak Mountains (Irian Jaya)

Species can, at times, be predicted

The Malgasy Orchid Angraecum sesquipedale Thouars and its pronube: the sphingid moth Xanthopan morgani praedicta Rothschild & Jordan.

Based on the exceptional depth of the Orchid nectaria, this moth, first imagined by Darwin in1862, and painted by Wallace in 1864, was effectively discovered 40 years later.

Do species exist in nature? Objectivity vs.conventionalism

In an adimensional perspective, in sexual organisms effectively or potentially amphigonic (including geographic, facultative parthenogenesis, etc.), species exist since they are recognized, as potential mates, by organisms themselves. The problems arise when considering species in a pluridimensional perspective.

Moreover:nobody would think that first RNA organisms, supposedly the originators of life, were organized in species, and similar situations can be expected to occur in other living beings.

Some critical views

1 Reality of species is now doubted by many.

2 Reproductive isolation is no longer generally recognized as the best definition of species.

3 Speciation does not require allopatry.

4 Natural selection is becoming viewed as the primary cause of speciation.??

Coyne, J.A. 1994. Evolution 48: 19-30.Mallet, J. 2001. J .Evol. Biol. 14: 887-888.

Detecting species from DNA sequences

DNA sequencing, with key sequences serving as a "barcode", has been proposed as a technology that might speed up species identification.

How this approach performs in different kinds of species?

Birds of North America 437 specimens, 263 species

Amphibia COI GenBank (89 species)

Cognato, (2006) Journal of Economic Entomology, 99:1037-1045

In summary:

DNA barcoding resolves most species, although some taxa have proved intractable

Waugh J (2007) BioEssays 29:188–197

Why?

Species reflect, in their properties, the mark of speciation, where the following factors have more or less predominant roles:

• The genetic system (polyploid speciation, stasipatric speciation, inversion polymorphism speciation, etc.)

• Natural selection promoting niche shift (sympatric speciation)

• Evolutionary time leading to divergent mutation accumulation (allopatric, peripatric speciation, etc.)

Sympatric speciation in Rhagoletis pomonella

MitochondrialCOII sequences (687 nucleotides)

Smith and Bush.1997. Mol.Phyl.Evol.7:33–43

0.01 Genetic distance value

Allopatric speciation in Dolichopoda cave crickets

D.aegilion

D.baccettii

D.schiavazzii

Hybridization experiments in Dolichopoda: relationship between hatching rate and genetic distance

between parental populations and species

ORO

KAS

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PUG

T.cavicola E.insolitus

H.cumberlandicus

CAMPST

FICMRC

ORS

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SAB

SAR

BRASIS

VLTD.bormansi D.cyrnensis

D.ligustica

D.laetitiae

D.geniculata

D.schiavazzii

D.muceddai

D.aegilion D.baccettii

D.palpata CPR

TRE

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DIAPSC

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D.linderi

D.capreensis

D.cassagnaui

D.dalensi

D.thasosensis

D.hussoni D.remyi

D.graeca D.pavesi

D.makrikapa

D.steriotisi

D.matsakisi

D.petrochilosi D.insignis

D.unicolor

D.paraskevi

D.vandeli

PNZ

Molecular phylogeny of the cave cricket genus Dolichopoda

Tree topology is congruent with the morphologically based taxonomy and clearly reflects a phylogeographic pattern

0.8<P<0.90.9<P<1.0

Allegrucci, Todisco & Sbordoni, 2005Mol. Phyl. Evol. 37: 53–164, and unpublished

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2.52.8 3.5

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5.0 5.95.9

4.44.5

5.05.1

6.7 7.0

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Genus Dolichopoda: 90 pop. samples, 33 species

1 30

25

50

75

100

015 17 19 22

25

50

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100

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50

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Divergence (%)

Within species Within genus Within family

Data from: Allegrucci, Todisco & Sbordoni, 2005. Mol. Phyl. Evol. 37: 53–164,

and unpublished

Species are the result of historical processes, such as population changes in demography, geographical location and range extent, and eventually speciation.

Phylogeographical analysis is therefore of great help to inferring these stories and understanding the real meaning of species.

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P. apolloP. apollo

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0.960.96

0.960.96

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0.910.91

1.01.0

1.01.0

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1.01.0

1.01.0

0.960.96

0.870.87

0.960.96

COI 869bpCOI 869bp50% MR Bayesian consensus50% MR Bayesian consensus

MrBayes (Huelsenbeck and Ronquist)MrBayes (Huelsenbeck and Ronquist)

Gratton, P., V.Todisco, and V.Sbordoni, in prepGratton, P., V.Todisco, and V.Sbordoni, in prep ..

Max D: 0.047Max D: 0.047

Max D: 0.024Max D: 0.024

How How manymany species in species in Parnassius?Parnassius?

COI, 931bp ML tree of 101 haplotypes

Parnassius mnemosyne

Gratton, P., Konopinski, M. & V.Sbordoni, in preparation

1.00

0.1

Parides vertumnusB. laos

0.98

0.971.00

1.00

0.590.62

1.00

1.00

1.00

1.00

1.00

1.00

0.98

0.63

1.00

1.00

1.00

0.89

0.97

1.000.52

1.00

1.00

0.98

1.00

B.crassipesB.demonius

B.daemoniusB.daemonius0.54

1.000.87

1.00

O.priamus euphorionTroides helena

1.00

B.p.pembertoniB.plutonius

B.plutoniusB.plutoniusB.plutonius

B.dasarada barataB.dasaradaB.dasaradaB.dasarada

B.d.barata

B.hedistus

B. alcinousB. a mansonensis

B. alcinous confusus

B.adamsoniiB.adamsonii

B.menciusB.mencius

B.polyeuctesB.polyeuctesB.p.polyeuctesB.p.polyeuctesB:p.lamaB.p.lamaB.p.lamaB.p.lama

B.polyeuctes

B.nevilliB.nevilliB.nevilli

B.latreillei Buthan

B.latreillei ButhanB.latreillei Buthan

B.latreillei India

B.latreillei Nepal

B.latreillei BirmaniaB.latreillei Birmania

B.latreillei Buthan

B.latreillei IndiaB.latreillei India

B.latreillei Himalaya

B.genestieri YunnanB.genestieri Yunnan

B.genestieri YunnanB.genestieri Yunnan

0.97

1.00

0.52

1.00

1.00

0.98

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1.00

B.latreillei Bhutan

B.latreillei Bhutan

B.latreillei Bhutan

B.latreillei W Bengala

B.latreillei Nepal

B.latreillei Burma

B.latreillei

B.latreillei Bhutan

B.latreillei W Bengala

B.latreillei W Bengala

B.latreillei Him.Prad.

B.genestieri Yunnan

B.genestieri Yunnan

B.genestieri Yunnan

B.genestieri Yunnan

Burma

Byasa tree

Deodati T., D.Cesaroni & V.Sbordoni, in preparation

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0.97

1.00

1.00

1.00

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B.latreillei

B.l.genestieri

p=0.045

Byasa latreilleiByasa latreillei

Byasa genestieriByasa genestieri

0 0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.17

0

50

100

150

200

250

300

350

400

450

N

Genetic distance

Within species Between Byasa species

Deodati T., D.Cesaroni & V.Sbordoni, in preparation

S. perspicillata(Savi, 1821)

S. terdigitata(Lacépède, 1788)

Genetic distance: 6.4%

Two Salamandrina cryptic species

Mattoccia M., A.Romano & V. Sbordoni Zootaxa 995: 1–19. (2005)

Photo: A.Romano

The Italian Peninsula in the Pliocene

Black circles: sampling sites of Salamandrina perspicillata.

Black stars: sampling sites of Salamandrina terdigitata.

Sites where haplotypes of both clades have been found are reported as a star inside a black circle.

M. Mattoccia, A.Romano and V. Sbordoni, in prep.

The role of hybrid zones

Rather then representing a confusing issue, secondary contact, hybrid zones stand for a great tool for the delimitation of species. In fact they contribute to the solution of the conflict between adimensional and polytypic species.

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0.2

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0.6

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-600 -400 -200 0 200 400 600 800 1000

Villani, F., A.Sansotta, M.Cherubini, D.Cesaroni and V.Sbordoni. 1999. Genetic structure of natural populations of Castanea sativa in Turkey: evidence of a hybrid zone. J. Evol. Biol., 12: 233-244.

Castanea genotypes: Trees vs.seeds. Evidence of selection in the Anatolian

hybrid zone.

D.Cesaroni, F.Villani, A.Sansotta and V.Sbordoni, unpublished

Castanea genotypes: Trees vs.seeds. No apparent selection outside the

hybrid zone.

D.Cesaroni, F.Villani, A.Sansotta and V.Sbordoni, unpublished

Towards an operational approach

The actual demarcation of species taxa uses morphological, geographical, ecological, behavioral, and molecular information to infer the rank of isolated populations.

Mayr, E. (1995) Philos. Sci. 63, 262–277.

A necessary word of warning

We must distinguish DNA taxonomy from DNA barcoding, where the former directlyconcerns the circumscription and delineation of species using evolutionary species concepts and the latter is a means of identifying a priori entities by sequence similarity

Vogler AP, Monaghan MT (2007) J Zool Syst Evol Res 45:1–10.

Towards an operational approach

An irreducible basal cluster of organisms diagnostically different from other such clusters and within which there is a parental pattern of descent - applies to both asexual and sexual organisms

(deQuieroz and Donoghue, 1990).

Towards an operational approach

Species are seen as groups of individuals which are discretely separated from other such groups in the space defined by their descriptors

V.Sbordoni, 1993: Molecular Systematics and the Multidimensional Concept of Species. Biochem. Syst. Ecol.21:39-42

Fontaneto et al. (2007). PLoS Biol

A final homage to Alfred Russel Wallace for his farsighted ideas on species and speciation

Tanysiptera hydrocharis

Aru islands, N Guinea

Tanysiptera galatea

New Guinea

Ranges of Tanysiptera hydrocharis (H1 and H2) and T. galatea according to Mayr

Thanks for your attention