DNA Barcoding Amphibians and Reptiles

Chapter 5 DNA Barcoding Amphibians and Reptiles Miguel Vences, Zoltán T. Nagy, Gontran Sonet, and Erik Verheyen Abstract Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecific lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a sufficient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplification strategy needs to be adjusted depending on the specific research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplification of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specific primers which then allow an easy and reliable amplification of all samples. Key words: Amphibia, Testudines, Crocodylia, Sphenodontia, Squamata, COI primers

1. Introduction In contrast to numerous other taxa, especially fishes and birds among vertebrates, DNA barcoding of amphibians and reptiles is in a very early stage. We here use the term amphibians as encompassing all Lissamphibia, i.e., frogs, salamanders, and caecilians (as of February 2012, totaling 6,922 species: 6,115 frogs, 618 salamanders, and 189 caecilians) (1). Reptiles are a paraphyletic group and we use the term here to include, all nonavian extant taxa of the Testudines, Crocodylia, Sphenodontia, and Squamata (as of February 2008, 8,734 species: 313 turtles, 23 crocodiles, 2 tuataras, and 8,396 squamates) (2). W. John Kress and David L. Erickson (eds.), DNA Barcodes: Methods and Protocols, Methods in Molecular Biology, vol. 858, DOI 10.1007/978-1-61779-591-6_5, © Springer Science+Business Media, LLC 2012

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Only a few DNA barcoding campaigns on reptiles were initiated recently, e.g., DNA barcoding of the South African reptile fauna (also see the International Barcode of Life web site; www.ibol. org). To date, the number of studies and publications dedicated to DNA barcoding of reptiles in general is very limited. Exceptions are the manageable few species of marine turtles with high conservational implications, where a good progress of DNA barcoding was recently achieved (3, 4). Related to this issue of conservational biology and genetics, DNA barcoding was recently applied to identify species targeted by bushmeat practices and to identify among others alligators and crocodiles (5, 6). In amphibians, several test cases of COI DNA barcoding have been published (7–9) and an extensive DNA barcoding program is currently being carried out on Central and South American taxa and has already led to remarkable results (10). From our own work in progress, rich data sets, taxon coverage ca. 90 and 80% respectively, on amphibians and reptiles of Madagascar are available with research continuing to achieve complete taxon coverage, while ongoing field surveys will enable us to initiate similar barcoding efforts for the frogs of the Congo basin and of Cuba. Given the critical conservation status especially of many amphibians, implementation of larger amphibian DNA barcoding programs would be very useful. They would allow to more efficiently delimit the distribution area and habitat use of endangered species also on the basis of larvae or juveniles which currently cannot be reliably identified. Integration of molecular assessment would help to accelerate the pace of species discovery and the quality of species hypotheses (11, 12). Until 2010, the vast majority of amphibian and reptile COI sequences were not produced in the framework of the global DNA barcoding initiative but they are mostly the result of phylogenetic or phylogeographic studies where COI was used as one of the genetic markers. In addition, numerous COI sequences in GenBank originated from sequencing strategies in which a stretch containing full or partial ND1 and ND2 genes, intervening tRNAs, and only a short section (100–200 bp) of the 5¢ terminus of the COI gene are obtained for phylogenetic analysis (e.g., for amphibians see refs. 13, 14). We have not considered the studies involving this fragment in the primer tables given herein. Beyond investigations on DNA barcoding and phylogeny, there are a growing number of mitogenomic studies that have yielded COI sequences. Among the ones with stronger impact or including several species are (15–21) for reptiles and (22–25) for amphibians. These studies have certainly contributed to the number of available COI sequences, but are otherwise not related to the DNA barcoding effort as such. However, the available coverage of higher taxa such as orders and families in mitogenomic studies is of crucial importance because it allows the design of primers for a variety of regions of the mitochondrial genome (26),

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DNA Barcoding Amphibians and Reptiles

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including targeted COI primers for particular taxonomic groups or species in which universal primers may fail. A common theme in amphibian and reptile DNA barcoding is that there is no single pair of primers that will guarantee a sufficient rate of success across all taxa, which means that the strategy needs to be adjusted depending on the specific research question. As far as known there are also no primers universal within major amphibian or reptile subgroups, such as salamanders, frogs, snakes, or lizards. Our experiences show that for amplifying and sequencing large numbers of samples from a restricted taxonomic group (a single species or a complex of closely related species), it is most convenient to design specific primers. If a wide array of taxa are to be screened, either usage of a primer cocktail or a hierarchical approach is advisable (first using one pair of universal primers, and subsequently using a different set of primers for samples that have failed to amplify in the first attempt). A first compilation of mitochondrial DNA primers used in amphibians was published in 1999 (27) but only included a few COI primers. Although not comprehensive, Tables 1 and 2 show a representative overview of primers and annealing temperatures used so far in studies that involved sequencing of COI in a larger number of samples of amphibians or reptiles, respectively. The specificity of primers and the targeted fragment size vary case by case, and the position of primers in the COI gene, and relative to the Folmer region (28), is shown in Figs. 1 and 2. When barcoding amphibians and reptiles, it is to be kept in mind that many species and species complexes are evolutionarily old and contain cryptic candidate species and deep conspecific lineages (refs. 7, 29; see also Note 1). This situation appears to be more commonly encountered in the tropics. In temperate regions, on the one hand, species are better studied so that discovery of new cryptic lineages happens less frequently; on the other hand these species have often expanded from glacial refuges in the Pleistocene, so that similar mitochondrial haplotypes can be encountered over vast geographic ranges and divergences within species are less deep. Altogether, DNA barcoding of amphibians and reptiles based on COI is not fundamentally different from that in other animal groups and has the same promises. Specifics to be kept in mind are mainly the old age of many species and the potential presence of very deeply diverged mitochondrial lineages within species which (a) make it necessary to have very complete COI reference databases for a successful species identification and (b) accentuate the problem of primer failure in single samples even within species or species complexes. Below we give a brief overview of laboratory methods for tissue sampling and for extracting DNA as well as amplifying and sequencing COI from amphibian and reptile specimens. These methods,

Primer name

COIf

COIa

COIa2

LCO1490

HCO2198

VF2 t1

FishF2 t1

Specificity/origin

Universal

Universal

Universal

Universal

Universal

Fishes

Fishes

5,391

5,392

6,089

5,406

6,662

6,707

6,047

52 used in cocktail (tailed)

TGTAAAACGACGGCCA F GTCGACTAATCATA AAGATATCGGCAC

50; 49–50

50; 49–50

45

45; 57

45; 57

Annealing temperature (°C)

52 used in cocktail (tailed)

R

F

R

R

F

Direction

F

GTAAAACGACGGCCA GTCAACCAACCACA AAGACATTGGCAC

TAAACTTCAGGGA CCAAAAAATCA

GGTCAACAAATCA TAAAGATATTGG

CCTGCYARYCCTA RRAARTGTTGAGG

AGTATAAGCGTCT GGGTAGTC

CCTGCAGGAGGA GGAGAYCC

Position Sequence (5¢–3¢)

Tungara frogs (Physalaemus)

(50)

(52)

(52)

(28)

Clawed frogs (Xenopus)


Poison frogs (Oophaga); Malagasy frogs (Mantellidae)

Poison frogs (Oophaga); Malagasy frogs (Mantellidae)

Tungara frogs (Physalaemus); dirt frogs (Craugastor)

(46)

(28)

Tungara frogs (Physalaemus), dirt frogs (Craugastor)

(46)

Primer reference Used for

(53)

(53)

(7, 8, 51)

(7, 8, 51)

(48)

(49, 51)

(49, 51)

Studies

Table 1 Selection of primers used for amplifying COI (fragments) in phylogenetic or phylogeographic studies of amphibians with details on taxon specificity and PCR conditions

82 M. Vences et al.

FR1d t1

VF1-d

VR1-d

LepF1

LepRI

BirdF1

BirdR1

BirdR2

“Desmognathus- 5,370 forward”

“Desmognathus- 6,005 reverse”

MVZ_201

Fishes

Fishes

Fishes

Butterflies

Butterflies

Birds

Birds

Birds

Dusky salamanders

Dusky salamanders

Arboreal salamanders (Aneides)

5,408

6,129

6,129

5,408

6,089

5,406

6,089

5,405

6,086

6,086

FishR2 t1

Fishes

TCAACAAAYCATAAA GATATTGGCACC

GTATTAAGATTTCGG TCTGTTAGAAGTAT

CGGCCACTTTACCYR TGATAATYACTCG

ACTACATGTGAGATG ATTCCGAATCCAG

ACGTGGGAGATAATT CCAAATCCTG

TTCTCCAACCACAAA GACATTGGCAC

TAAACTTCTGGATGT CCAAAAAATCA

ATTCAACCAATCATA AAGATATTGG

TAGACTTCTGGGT GGCCRAARAAYCA

TTCTCAACCAACCA CAARGAYATYGG

CAGGAAACAGCTAT GACACCTCAGGG TGTCCGAARAAYC ARAA

CAGGAAACAGCTAT GACACTTCAGGG TGACCGAAGAAT CAGAA


Primer name

Specificity/origin

F

R

F

R

R

F

R

F

R

F

NA

52

52

49–50

49–50

49–50

45 and 51

45 and 51

45 and 51

45 and 51

(7)

(58)

(58)

(57)

(57)

(57)

(56)

(56)

(55)

(55)

52 used in cocktail (54) (tailed)

R

( 8)

( 8)

( 8)

( 9)

( 9)

( 9)

( 9)

(53)

(53)

Studies


DNA Barcoding Amphibians and Reptiles (continued)

( 7)

Dusky salamanders (58) (Desmognathus)

Dusky salamanders (58) (Desmognathus)

Malagasy frogs (Mantellidae)



Various frog and salamander taxa







52 used in cocktail (52) (tailed)


R

Direction

5 83

Primer name

MVZ_202

PP6

PP7

PP8

PP9

COI-1

COI-2

COI-3

COI-4

Specificity/origin


Physalaemus

Physalaemus

Physalaemus

Physalaemus

Fire-bellied toads

Fire-bellied toads

Fire-bellied toads

Fire-bellied toads

Table 1 (continued)

5,903

6,503

6,503

5,412

6,467

6,467

6,302

6,302

6,695

CCAGCAATGTCAC AATACCAAAC

GACAGAACATAGTGG AAGTGAGCTAC

GATACGACATAGTGG AAGTGGGCTAC

CAAATCACAAAGACA TTGGCACCCT

TCATGTAATACAATG TCTAGAGA

TCTCTAGAYATTGT ATTACATGA

GTTGGAATTGCRAT GATTATTGT TGCAGA

TCTGCAACAATAAT YATYCGCAATT CCAAC

GCGTCWGGGTART CTGAATATCGTCG


F

R

R

F

R

F

R

F

R

Direction

NA

NA

NA

NA

Internal sequencing primer




NA


(38)

(38)

(38)

(38)

(50)

(50)

(50)

(50)

(7)

Fire-bellied toads (Bombina)










(38)

(38)

(38)

(38)

(48)

(48)

(48)

(48)

(7)

Studies

84 M. Vences et al.

6,176 5,908

6,707

COI-6

Cox

Coy

COI-smallF

COI-smallR

KLPf

HmCO1

CO1AXen-H

CO1h-L

CO1g-L

Fire-bellied toads

Australian Litoria frogs


Australian frogs (Litoria aurea)


Australian frogs (Litoria)

South American hylid frogs (Dendropsophus minutus)


Toads (Bufonidae)

Toads (Bufonidae)

TTCATACGTGGTAA CATTTTAGTCAAG

GGAATTATTTCCC AYGTWGTAAC

TGTATAAGCGT CTGGGTAGTC

CGTCACTCAGTA CCAAACCCCC

AAAGAACCTTTT GGTTACATGGG

CAAATACGG CCCCCATAGAT

TTGGCCTGCTA GGTTTTATTG

GGGGTAGTCAG AATAGCGTCG

TGATTCTTTGGG CATCCTGAAG

GCAGGGGTGTCC TCAATTCTAG

TGGTAATTCCTG CAGCAAGAAC

F

F

R

F

F

R

F

R

F

F

R

Direction

NA

NA

NA

NA

NA

(27)

Toads (Bufonidae) (27)

Toads (Bufonidae) (27)

(27)

(63)

(63)

(62)

(37)



Australian frogs (Litoria)


(37)

(59–61)

(59–61)

(38)

(38)

Studies

(63)

(63)

(62)

Step-down profile: (37) 60, 58, 56, 54




(59) (59)



(38)

(38)


Step-down profile: (37) 60, 58, 56, 54

NA

NA

NA

NA


Position is given relative to the complete mitochondrial genome sequence of Discoglossus galganoi (GenBank accession number: AY585339). When multiple annealing temperatures are given, it refers to alternative temperatures used in different studies for the same primer or primer combination

5,162

6,137

6,526

6,222

6,695

6,089

5,840

5,984

COI-5

Fire-bellied toads


Primer name

Specificity/origin

5 DNA Barcoding Amphibians and Reptiles 85

HCO2198

C1-J-1718

C1-J-2191

CO1a

CO1f

COIcXen

COIfXen

COIaXen

COIeXen

Universal

Universal

Universal

Vertebrata

Vertebrata

Vertebrata

Vertebrata

Vertebrata

Vertebrata

6,398

6,539

5,307

5,787

5,898

6,539

5,939

5,466

5,921

CCAGTAAATAAC GGGAATCAGTG

TGTATAAGCGTC TGGGTAGTC

CCTGCCGGAGG AGGTGACCC

TCGTTTGATCAG TATTAATCAC

CCTGCAGGAGGA GGAGAT(orY)CC

AGTATAAGCGTCT GGGTAGTC

CCCGGTAAAATTAAAA TATAAACTTC

GGAGGATTTGGAAA TTGATTAGTTCC

TAAACTTCAGGGT GACCAAAAAATCA

GGTCAACAAATCAT AAAGATATTGG

LCO1490

Universal

5,262

Primer name Position Sequence (5¢–3¢)

Specificity

R

R

F

F

F

R

R

F

R

F

Direction

47

47

47

47

45–58

45–58

42

42

42–45

42–45


(45)

(45)

(45)

(45)

(45)

(45)

(67)

(67)

(28)

(28)

Primer reference

Anolis

Anolis

Anolis

Anolis

Turtle, tortoise, iguana, skink, crocodile

Turtle, tortoise, iguana, skink, crocodile

Lizard

Lizard

Lizard, turtle, gecko

Lizard, turtle, gecko

Used for

(74)

(74)

(74)

(74)

(68–73)

(68–73)

(66)

(66)

(62–64)

(62–64)

Studies

Table 2 Primers used for amplifying COI (fragments) in phylogenetic or phylogeographic studies of reptiles with details on taxon specificity and PCR conditions

86 M. Vences et al.

FishF2_t1

FishR2_t1

FR1d_t1

M13F (221)

M13R (227)

VF1

VR1

RepCOI-F

RepCOI-R

M72

Vertebrata (COI-3 cocktail)



Universal (COI-3 cocktail)

Universal (COI-3 cocktail)

Vertebrata

Vertebrata

Squamata

Squamata

Testudines

5,946

5,921

5,256

5,921

5,262

NA

NA

5,918

5,918

5,265

TGATTCTTCGGTCACCCA GAAGTGTA

ACTTCTGGRTGKCC AAARAATCA

TNTTMTCAACNAACC ACAAAGA

TAGACTTCTGGGTGGCC AAAGAATCA

TTCTCAACCAACCACAAA GACATTGG

CAGGAAACAGCTATGAC

TGTAAAACGACGGCCAGT

[M13R]ACCTCAGGGT GTCCGAARAAYCARAA

[M13R]ACTTCAGGGT GACCGAAGAATCAGAA

[M13F]CGACTAATCAT AAAGATATCGGCAC

[M13F]CAACCAACCAC AAAGACATTGGCAC

VF2_t1


5,265


Specificity

F

R

F

R

F

R

F

R

R

F

F

Direction

48 or 55

48.5

48.5

52

NA

(69)

(77)

(77)

(55)

(55)

51.1 5×, then 56.9 (54) 30×

51.1 5×, then 56.9 (54) 30×

51.1 5×, then 56.9 (54) 30×

51.1 5×, then 56.9 (54) 30×

Side-necked turtle

Squamata

Squamata

Boelen’s python, watersnake

Boelen’s python

Crocodile

Crocodile

Crocodile

Crocodile

Crocodile

51.1 5×, then 56.9 (54) 30×

Used for Crocodile

Primer reference

51.1 5×, then 56.9 (54) 30×


(continued)

(69)

(77)

(77)

(75, 76)

(76)

(5)

(5)

(5)

(5)

(5)

(5)

Studies

5 DNA Barcoding Amphibians and Reptiles 87

L-330COI

H-610COI

H-715COI

L-turtCOIc

H-turtCOIc

L-turtCOI

H-turtCOI

H-turtCOIb

L-COIint

H-COIint

Testudines

Testudines

Testudines

Testudines

Testudines

Testudines

Testudines

Testudines

Testudines

Testudines

5,634

5,792

6,119

6,059

5,968

6,066

5,234

5,946

5,843

5,564

TAGTTAGGTCTACAG AGGCGC

TGATCAGTACTTATCAC AGCCG

GTTGCAGATGTAAAA TAGGCTCG

CCCATACGATGAA GCCTAAGAA

ACTCAGCCATCTTA CCTGTGATT

TGGTGGGCTCATAC AATAAAGC

TACCTGTGATTTTAA CCCGTTGAT

GCCAAATCCTGGTAA GATTAAGAT

GTATTTAGGTTTCGGT CAGTGAG

TACTTTTACTCCTAGCC TCCTCAG

CCTATTGATAGGACGTA GTGGAAGTG

M73

Testudines

6,342


Specificity

Table 2 (continued)

R

F

R

R

F

R

F

R

R

F

R

Direction

(79)

(79)

(79)

(79)

(79)

(78)

(78)

(78)

(69)

Primer reference

For sequencing only (79)

For sequencing only (79)

56–58

56–58

56–58

56

56

50–54

50–54

50–54

48 or 55


(78)

(78)

(79)

(78)

(78)

(78)

(78)

(78)

(69)

Studies

Yunnan box turtle

(78)

Yunnan box turtle, (74, 78) anoles

Yunnan box turtle

Yunnan box turtle

Turtle

Yunnan box turtle

Yunnan box turtle

Yunnan box turtle

Yunnan box turtle

Yunnan box turtle

Side-necked turtle

Used for

88 M. Vences et al.

CoxIH2

COIf-ot1

COIr-ot2

COIf-ot2

COIr-ot1

L7354

H7794

rTrp–1L

rCOI−1H

LCOI5973

HCOI6576

LCOI5982

HCOI6570

NA

Crocodylia

Crocodylia

Crocodylia

Crocodylia

Crocodylia

Squamata

Squamata

Squamata

Squamata

Squamata

Squamata

Squamata

Squamata

Serpentes

5,222

5,864

5,317

5,921

5,262

6,332

4,879

6,365

5,925

5,871

5,654

5,595

5,891

6,042

R

F

F

R

F

R

R

F

Direction

TCAGCCATACTACCTG TGTTCA

TGCTGGGTCGAAGAA GGTNGT

GGTATAACCGGAACA GCCCTNAGY

TAAACTTCAGGGTGA CCAAAAAATCA

GGTCAACAAATCATAAA GATATTGG

F

R

F

R

F

TAGTGGAARTGKGCTACTAC R

TAAACCARGRGCCTTCAAAG F

ATAATGGCAAATACTGCCCC

TACCAACACCTATTCTGATT

CGAAACYTAAACACTACCTT

CAGCAAGATGAAGGG AGAAGAT

CGCCGGTACAGGATGAAC

TTGGTATAGRATTGGA TCYCC

CCTAAGAAGCCAATTG ATATTATGC

GGCTACTGCCACTAA TAATCGC

CoxIL2

Crocodylia

5,478


Specificity

52

50

50

46–50

46–50

48 5×, 58 35×

48 5×, 58 35×

47–55

47–55

NA

(65)

(65)

(65)

(65)

(15)

(15)

(68)

(68)

50–46 touchdown (6)



Snake

Gecko

Gecko

Gecko

Gecko

Gecko, Komodo dragon

Gecko, Komodo dragon

Iguana, lizard

Iguana, lizard

Dwarf crocodile

Dwarf crocodile

Dwarf crocodile

Dwarf crocodile


Dwarf crocodile

Used for

Dwarf crocodile

(6)

Primer reference

(6)

50

50



(continued)

(75)

(65)

(65)

(65)

(65)

(15, 81)

(15, 81)

(68, 80)

(68, 80)

(6)

(6)

(6)

(6)

(6)

(6)

Studies

5 89

COI(−)bdeg

COI(+)b

Serpentes

Serpentes

TAAATAATATAAGCTTCT GACTGCTACCACC

ATTATTGTTGCYGCT GTRAARTAGGCTCG F

R

F

Direction

56.5

56.5–65

56.5–65


(83)

(82)

(82)

Primer reference

Snake

Snake

Snake

Used for

(83)

(82)

(82)

Studies

Position is given relative to the complete mitochondrial genome of Furcifer oustaleti (GenBank accession number: NC_008777). When multiple annealing temperatures are given it refers to alternative temperatures used in different studies for the same primer or primer combination

5,535

6,119

AAGCTTCTGACTNCTA CCACCNGC

COI(+)deg1

Serpentes

5,538


Specificity

Table 2 (continued)

90 M. Vences et al.

5


91

HmCO1/CO1AXen-H

Anura

Cox/KLPf/COI-smallF/COI-smallR/Coy COI-1/COI-2&COI-3/COI-6/COI-4/COI-5 CO1g-L/CO1h-L

Urodela

PP6&PP7/PP8&PP9 MVZ_201/MVZ_202 Desmognathus-forward/-reverse

Vertebrata

BirdF1/BirdR1/BirdR2 LepF1/LepRI VF1-d/FR1d-t1&VR1-d

universal

FishF2-t1&VF2-t1/FishR2-t1 LCO1490/HCO2198 COIf/COIa2/COIa

6000

5500

6500

« Folmer region »

Fig. 1. Some primers used to amplify COI in amphibians sorted according to their specificity (for details, see Table 1). Black triangles represent forward, empty squares reverse primers, respectively. Numbers on the axis refer to the position on the complete mitochondrial genome of Discoglossus galganoi (GenBank accession number: AY585339).

however, are straightforward and similar to those established in other vertebrates (see Note 2). We also provide an overview of selected primers that have thus far been used to amplify COI from amphibians and reptiles, and which should be helpful to design amplification strategies in future DNA barcoding studies targeting these animals. To obtain this compilation of primers, we focused on studies where the COI gene as a molecular genetic marker was targeted, in particular, the standard animal barcoding region, the so-called Folmer region (28).

2. Materials 2.1. DNA Extraction and Preservation (See Note 3)

1. For routine DNA barcoding, we recommend a salt extraction protocol. 2. Extraction buffer: 0.01 M Tris–HCl (pH 8.0), 0.1 M NaCl, 0.01 EDTA (pH 8.0) in dH2O.

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M. Vences et al.

COI(+)b/COI(+)deg1/COI(-)bdeg

Serpentes

Squamata

LCOI5973/LCOI5982/HCOI6570/HCOI6576 rTrp–1L/rCOI–1H L7354/H7794

Testudines

CoxIL2/COIr-ot2/COIf-ot2/COIr-ot1/COIfCrocodylia ot1/CoxIH2 L-turtCOIc/H-COIint/L-COIint/L-turtCOI/HturtCOI/H-turtCOIc/H-turtCOIb L-330COI/H-610COI/H-715COI M72/M73

Reptiles

RepCOI-F/RepCOI-R

Vertebrata

VF2(t1)/FR1d(t1) FishF2(t1)/FishR2(t1) F