Character-based DNA barcoding for identifying conservation units in Odonates J. Rach 1, R. DeSalle 2, I.N. Sarkar 2, B. Schierwater 1,2 & H. Hadrys 1,

Character-based DNA barcoding for identifying conservation units in

Odonates

J. Rach1, R. DeSalle2, I.N. Sarkar2, B. Schierwater1,2 & H. Hadrys1, 3

1ITZ- Ecology & Evolution, TiHo Hannover, Germany

2Division of Invertebrate Zoology, American Museum of Natural History, New York,

USA3Dept. Ecology & Evolutionary Biology, Yale

University, New Haven, USA

Thank you to:

• DAWB (CBOL)/DIMACS

• Sandra Giere

• Antonia Wargel

• Janne Timm

• Linn Groeneveld

• Nadine Habekost

• Kai Kamm

• DFG & BMBF

Character-based DNA barcoding:

A rapid and reliable method for the identification of conservation units in

dragonflies

Contents

1. Introduction:

- Why barcoding dragonflies?

- Why character-based DNA barcoding?

- Which genetic marker is appropriate?

2. Methods

- Character-based DNA barcoding

3. Case studies

I. Species identification

II. Discrimination of conservation units

4. Conclusions & Future prospects

1. Introduction: Why barcoding dragonflies?

Odonata (demonstrator system):

- Small insect order

- Model organisms for ecology and evolution

- Wide range of habitat specificity (generalists / specialists)

- Fast respond to environmental changes


- Prime indicators for all types of fresh water ecosystems

Terrestrial

Aquatic

Increasing importance for conservation management


- Wing veneation: requires a lot of

experience

- Colours: Bright colours of males

fade quickly after death; females

of same genus inconspiciuous

- Ecological and behavioural patterns:

difficult and time-consuming

- Larvae: discrimination often

impossible

Identification through phenotypic traits is difficult:

♀♀


If phenotypic traits do not serve Need of genetic

approaches!

How to get DNA non-invasive:

Rapid and reliable identification of dragonflies valuable for conservation management:

Exuvia Middle leg (Hadrys et al. 1992)

1. Introduction: Why character-based DNA barcoding?

- High intraspecific genetic variability (e.g.

geographical clusters) can hinder assignment of

unknown samples to their species

- Distances between species often lower than within

species

- Thresholds cannot be defined (might lead to

overestimated biodiversity)

Distance approaches can be misleading:

1. Introduction: Why character-based DNA barcoding?

Identification at any taxonomic level

Diagnostic characters useful for DNA barcoding:

Species (n) 123 234 350A (100) A C GB (100) T A T

Population (n) 110 123 200 234 310 350B1 (25) A T C A T TB2 (25) C T C A C TB3 (25) A T T A G TB4 (25) G T A A T T

Character-based DNA barcodes for species

and single populations

1. Introduction: Which genetic marker is appropriate?

Has not been applied for Odonates before:

CO1 (cytochrome c oxidase 1) supposed to be appropriate for DNA barcoding of most animal groups:

Search for conserved primer sequences

Optimization of PCR conditions

Test for suitability

1. Introduction: Which genetic marker is appropriate?

- Sequences easy to obtain and analyse

- Detection of geographical patterns

- Identification of conservation units

ND1 (NADH dehydrogenase subunit 1) is a suitable marker:

Namibia Naukluft

Südafrika Ostafrika

Namibia Naukluft

Namibia Okavango

Südafrika

Ostafrika

Namibia Okavango

Cryptic speciation in Trithemis stictica

2. Methods: Character-based DNA barcoding

- PCR with gene specific primers

- Sequencing (MegaBACE 500)

- Alignment (MUSCLE)

- NJ tree based on Kimura-2-parameter (K2P) distances

(PAUP)

1. Standard Methods


- Search for diagnostic characters by application of

CAOS algorithm

- Development of perl scripts to assist further

analyses

- Selection of nucleotide positions for final DNA

barcodes by eye

2. Establishment of character-based DNA barcodes:


2. Establishment of character-based DNA barcodes:

0

1

10

0 1

NODE GROUP POS STATE CONF0 0 90 C 10 0 171 T 10 1 90 T 10 1 171 A 11 0 108 A 11 0 153 T 11 1 108 T 11 1 153 A 1

Nucleotide Position Taxa 90 108 153 171

A C T A TB T T T AC T A A A

I. Phylogenetic Tree

III. Find unique combinations of character states

II. Search for characteristic attributes with CAOS algorithm


- Pure (Pu): Exist in all elements of a group but not in

alternate group

Types of characteristic attributes (CAs):

- Private (Pr): Only present in some members of a

group but absent from alternate group

- Simple (s): At a single nucleotide position

- Compound (c): combination of states

sPu and sPr CAs shared by at least 80% of

members of a group were used (Filtered by

diagViewer)


1. “BarcodeFilter”: sorts out non-relevant nodes

Analyses were assisted by a set of perl scripts:

Nodes within species cluster are not relevant for barcoding species


2. “BarcodeMaker”: Convertion of “diagViewer-attributes file” into tab delimited file importable to Microsoft Excel:

NODE GROUP 19 20 21 220 00 115 1 T [1.00]15 0 A [0.89]16 0 T [1.00]16 1 C [1.00]17 117 0 G [0.81]18 1 A [0.95] G [1.00]18 0 A [1.00]


3. “BarcodeHistMaker”:Counting numbers of CAs at each nucleotide position (selection of sequence fragment with highest number of CAs:

16 1 *17 4 ****18 4 ****19 9 *********20 9 *********21 16 ****************22 1 *23 7 *******24 20 ********************25 19 *******************26 4 ****

0

5

10

15

20

25

30

35

40

45

0 50 100 150 200 250 300 350 400 450 500

Nucleotide position

Number of CAs

Case studies (Study I)

Case Study I: Species identification

842 ND1 sequences (65 species)

- Suitability of ND1 for DNA barcoding

- Applicability of the CAOS algorithm for character-based DNA barcoding


NJ tree based on K2P distances:

Overview tree:

ND1 sequence of one individual of each species

Overlap of species cluster


Results: Character-based DNA barcodes

Species (n) 201 207 213 225 243 255 273 285 294 298 306 318 324

Ce (43) G A T T T T C G G G A T A

Cs (14) A T A T A T T G A G T T A

Nf (9)A (0.67) G (0.33) T T C C T T A T A T A G

Ot (7) T T T T C T A A A G T T A

Ob (9) T A T C T T A T A G T T A

Ocoe (24) T A T T T T T T T A C C A

Oc (34) T A T T T T A T T G T C G

Oj (47) T A T T T C A C T A T T A

Tk (19) A T A A A T T T T G C A T

Unique combinations of character states at 13 selected nucleotide position



Overview tree:





Species (n) 201 207 213 225 243 255 273 285 294 298 306 318 324Aeelel (1) A T A T T T A T T G T T GAeelus (1) A T A T T T A T T G T T GAj (1) T A A T T T A T T G T T GAp (1) T A A T T T A T T G T T AAi (85) T A A T T T A T T G T T AAs (23) T A A T T T A T T G T T AAecy (1) T T A T T T T A T G T T GAegr (1) T T A T T T T A T G T T G

Family Aeshnidae: Combination of character states shared by two or more species

Additional analysis with CAOS algorithm


Results: Character-based DNA barcodes for Aeshnids

- Search for diagnostic characters within whole ND1 fragment

better resolution

Taxa/ ()=n 213 216 222 228 231 246 264 273 276 282 285 294 324 366 428 437 443

Ai (85) A T G A A A T A T T T T A T A T T

As (23) A C A G G A T A T T T T A T A T T

Aeelel (1) A T A A A G T A T T T T G T A T A

Aeelus (1) A T A A A G T A T T T A G T A T A

Anaiso (1) G T A A A A T T C T T T A T A C A

Brpr (1) A A G A A A T C T G A T A T A C A

Aj (1) A T G A A G T A T T T T G T A C T

Ap (1) A T G A A A T A T T T T A T G T C

Anatri (1) A T A A A A A A T A A G A T A T T

Aeri (1) A T A A A A T A T T G T A T G T T

Aegr (1) A A G A G A T T T T A T G C A T A

Aecy (1) A A G A G A T T T T A T G T A T A

Ae (19) A T A A A A T T T T T GG

(AX1) T A C T

Gu (9) G A A A A A T T T T T A G T G T T

Gyvill (1) G T A T G A T T T A G A G T G T T

Gyma (1) A A A A A A T C T T T A G T A T A

Corad (1) G T A A A A G T T T T A A T A T A

Corpe (1) G C A A A A T C T T A A A T A T A



Overview tree:





- Combination of character states shared by several individuals of Calopteryx splendens (cs) and of Calopteryx virgo (cv)

- No diagnostic characters found through additional analysis with CAOS algorithm

Hybridisation

Wrong identification

Recent radiation

Species (n) 201 207 213 225 243 255 273 285 294 298 306 318 324

cs (20)G T T T T G (0,8)

A (0,2)

T (0,8)

C (0,2)

C (0,8)

G (0,2)T A T T (0,8)

A (0,2)A

cv (5)G T T T T A (0,6)

G (0,4)

C (0,6)

T (0,4)

G (0,6)

C (0,4)T A T A (0,6)

T (0,4)A


Summary: Case study I

- 60 of 65 species distinguishable through

unique combinations of character states

within ND1 fragment

- ND1 suitable

- Diagnostic characters easily found by

application of the CAOS algorithm

Case studies (Study II)

Case Study II: Discrimination of conservation units

Subset of Case study I; 122 ND1 sequences (9 species)

+ 101 CO1 sequences (same 9 species)

- Suitability of CO1 for DNA barcoding

- Ability of both markers to discrimininate conservation units


NJ trees based on K2P distances

CO1 ND1ND1



unique combinations of character states at 11 selected nucleotide positions of CO1 fragment

CO1 also suitable

Species / (no. individuals=n) 105 111 162 174 180 192 207 260 263 272 279Paragomphus genei (n=6) T A T A C T A T C C ACrocothemis sanginolenta (n=9) G A C T T T T T C A ATrithemis stictica (n=14) T A T A C T T T C A ACoryphagrion grandis (n=5) T G C A C A C C T A GPseudagrion bicoerulans (n=22) A A T A T G A C T G AChlorocnemis abotti (n=15) A A T A C A A C C A AOrthetrum julia falsum (n=12) A A T A T T A T C A AOrthetrum trinacria (n=5) A T A C C A T T G A GCrocothemis erythreae (n=13) G A T T T T A T C A A


Results: Identification of populations

Combination of CO1 and ND1 to improve identification success

CO1 ND1

Population/ (no. Individuals=n) 21 210Orthetrum julia falsum / Oj32 (n=7) A TOrthetrum julia falsum / Oj16 (n=5) G A

Population/ (no. Individuals=n) 258Orthetrum julia falsum /16 (n=5) COrthetrum julia falsum /32 (n=7) T

Population/ (no. Individuals=n) 214Coryphagrion grandis/19 (n=9) TCoryphagrion grandis/22 (n=6) CX


Results: Identification of conservation units

Population/ (no. Individuals=n) 27 30 33 42 45 138 171 234 273 309 318Pseudagrion bicoerulans / Pb77 (n=6) C T A A G A C T T T CPseudagrion bicoerulans / Pb78 (n=7) C T G A A G T C T C TPseudagrion bicoerulans / Pb79 (n=4) T C T C A C T T A T CPseudagrion bicoerulans / Pb113 (n=5) C T A A G A T T T T C

Population/ (no. Individuals=n) 111 112 145 168 180 195 210 237 258 294 372 433Pseudagrion bicoerulans /77 (n=6) A T G T G G A T A T A CPseudagrion bicoerulans /78 (n=6) A T G C A A G T A C G TPseudagrion bicoerulans /79 (n=4) T A A T T G G G A C T TPseudagrion bicoerulans /113 (n=5) A T G C G G A T G T A T

CO1

ND1

Pb77Pb113

Pb78

Pb79


Results: Identification of cryptic species

Populations:

Tst94:

Tst118:

Tst119:

Pb128:

CO1

ND1

Population/ (no. Individuals=n) 159 247 295 312 319 324 336 378 408 414 432Trithemis stictica / Tst119 (n=8) T C C A T T G C T C TTrithemis stictica / Tst128 (n=6) C T T G C A A T C A C

Population/ (no. Individuals=n) 102 113 121 138 166 169 177 192 217 231 255 258Trithemis stictica /128 (n=6) A T A A A C A G T A G TTrithemis stictica /119 (n=6)* A C G T A T A A C A G TTrithemis stictica /118+94 (n=15) G G G T T T G G T G A C

* One individual of Tst119 shares a combination of character states with 6 individuals of Tst128

Popa Falls

Zebra River

Kwando Tst128

Tst119

Tst118

Tst94 = Kenya


Summary: Case study II

- All nine species distinguishable through

unique combinations of character states

within ND1 and CO1 fragments

- Both markers suitable

- Character-based DNA barcodes established

for conservation units of several species

Conclusions

Rapid

Reliable

- Application of CAOS algorithm

- Assignment of samples through a few nucleotide

positions

- Discrete characters

- Combination of ND1 and CO1 increases success

- DNA barcodes for several conservation units

Character-based approaches are:

Conclusions

Character-based DNA barcoding:

A rapid and reliable method for

the identification of conservation

units in dragonflies !

Future Prospects

Next steps:

- More species

- More individuals of some species

- Development of data base

- Character-based DNA barcodes for genera

- Application of character-based DNA barcodes

Identification of adults, exuvia and

larvae

Long-time monitoring

Thank you !!

Documents

Character-based DNA barcoding for identifying conservation units in Odonates J. Rach 1, R. DeSalle 2, I.N. Sarkar 2, B. Schierwater 1,2 & H. Hadrys 1,