gb-2003-4-6-p6 GBJ Deposited research article <p>Ancient origin and evolution of the Indian wolf: evidence from mitochondrial DNA typing of wolves from Trans-Himalayan region and Pennisular India</p> Aggarwal K Ramesh rameshka@ccmb.res.in Ramadevi J Singh Lalji

Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

 Genome Biology 1465-6906 2003 4 6 P6 http://genomebiology.com/2003/4/6/P6 This was the first version of this article to be made available publicly. 10.1186/gb-2003-4-6-p6 22 4 2003 29 4 2003 2003 BioMed Central Ltd

A study of mitochondrial DNA diversity across three different taxonomically informative domains (cytochrome-B gene, 16S rDNA and hypervariable d-loop control region) revealed that the Himalayan wolf and the Indian Gray wolf are genetically distinct from each other as well as from all other wolves of the world

Abstract

The two wolf types found in India are represented by two isolated populations and believed to be two sub-species of Canis lupus. One of these wolf, locally called Himalayan wolf (HW) or Tibetan wolf, is found only in the upper Trans-Himalayan region from Himachal Pradesh to Leh in Kasmir and is considered to be C. lupus chanco. The other relatively larger population is of Indian Gray wolf (GW) that is found in the peninsular India and considered to be C. lupus pallipes. Both these wolves are accorded endangered species status under the Indian Wildlife Protection Act. In 1998 for the first time in India, we initiated molecular characterization studies to understand their genetic structure and taxonomic status. Since then, we have analyzed the genetic variability in 18 of the total of 21 HW samples available in Zoological parks along with representative samples of GW, wild dogs and jackals. Our study of mitochondrial DNA diversity across three different taxonomically informative domains i.e., cytochrome-B gene, 16S rDNA and hypervariable d-loop control region revealed HW to be genetically distinct from the GW as well as from all other wolves of the world, including C. lupus chanco from China. Most importantly, d-loop haplotypic diversity revealed both HW and GW from India to be significantly diverse from other wolf populations globally and showed that these represent the most ancient lineages among them. Phylogenetic analysis revealed the Indian wolves as two independent lineages in a clade distinct and basal to the clade of all wolves from outside of India. Conservative estimate of evolutionary time-span suggests more than one million years of separation and independent evolution of HW and GW. We hypothesize that Indian wolves represent a post-jackal pre-wolf ancestral radiation that migrated to India about 1-2 mya and underwent independent evolution without contamination from other wolf like canids. The study thus, suggests that Indian subcontinent had been one major center of origin and diversification of the wolf and related canids. Further, the significant degree of genetic differentiation of HW from GW and of these two from other wolves, suggest the interesting possibility of them to be new wolf species/subspecies in evolution that may need to be described possibly as C. himalayaensis and C. indica (or as C. lupus himalyaensis and C. lupus indica), respectively. Thus for the first time, the study reveals new ancient wolf lineages in India and also highlights the need to revisit the origin, evolution and dispersion of wolf populations in Asia and elsewhere. Simultaneously, it increases the conservation importance of Indian wolves warranting urgent measures for their effective protection and management, especially of the small HW population that at present is not even recognized in the canid Red List.

 Evolution Ecology

Introduction

The gray wolf, Canis lupus, is believed to be the most widely distributed terrestrial mammal that ever lived on earth at one time point 1, originally inhabiting major parts of the Northern hemisphere. From being omnipresent and abundant, it has assumed endangered species status in many countries. In most parts of Southern, Western and Northern Europe and Americas, major gray wolf populations are lost and their present distribution is highly fragmented 2. In England and Japan, it has already become extinct while in many other parts it is on the verge of extinction 3. The wolves are now restricted to few large forested areas in Eastern Europe, a few isolated mountain ranges in the Mediterranean, mountains and semi-desert areas of middle-East and the wilderness areas of North America, Russia and China, with largest concentrations in Russia followed by Canada and Alaska [cf. 4]. Therefore, serious efforts have been initiated in Europe and America for protection and conservation of the existing wolf populations in recent years.

In the last century, up to 32 sub-species of gray wolf have been described by wolf taxonomists, based on the variations in physical features, behavioral aspects and geographical distribution, many of which are now believed to be extinct 45. Status of many of these subspecies is contested eg., subspecies campestris, chanco and desertorum are suggested to be synonymous of subspecies lupus 6, making the gray wolf taxonomy highly debatable pending validation and revision. Presently, only six subspecies viz., lupus, campestris, albus, tundaram, lycaon and nubilus, are believed to be surviving globally, and only the first two being in Asia 4. In recent years, though many molecular-genetic studies were carried out to understand the origin and evolution of canidae family [78 and cf. therein; 9) and also on the population dynamics of isolated, specific gray wolf populations 3101112, similar studies to resolve the largely debatable gray wolf taxonomy at the subspecies level are generally lacking. The two detailed studies on the latter aspect based on mitochondrial genome variations (mtDNA d-loop haplotyping) show little genetic partitioning on the continental or regional scales 1314.

In Indian subcontinent, wolf is known to exist for a longtime but not much is known about its origin and evolutionary history. Generally, as per the conventional taxonomy, it is believed that the small numbers of wolves found in today's India, are two different sub-species of Canis lupus which are represented by geographically isolated broadly non-overlapping (allopatric) populations. One of these wolf populations is found only in the upper Trans-Himalayan region of India across the two Northernmost states of Himachal Pradesh and Jammu and Kasmir, which as per the available census numbers ~350 individuals 15. This Himalayan wolf (HW) population, adapted to the cold environment, is considered to be representing the extant population (direct relatives) of the relatively better known Tibetan wolf, Canis lupus chanco, which is found throughout central Asia with its range extending into Tibet, China, Manchuria and Magnolia 15. On the other hand, the second wolf population estimated to be less then 1500 http://www.wolf.org/wolves/learn/basic/populations/fall99insert.asp, is found throughout the arid /semi-arid plains of pennisular India. These wolves, commonly called Indian gray wolf (GW), are believed to represent the second sub-species in India, i.e., C. lupus pallipes that is also found in the middle-Eastern countries like Iran and Israel 16. The wolf in India are accorded schedule '1' endangered species status under the Indian Wildlife Protection Act of 1972 and of CITES, and in last few decades federal efforts were initiated for wolf protection and conservation by setting up wildlife preserves in the country and captive breeding programs in few National Zoological Parks.

Since early 1970s, to help the conservation efforts few studies were also initiated to understand the ecology and population biology of wolves, but all of these were on GW of peninsular India (Wildlife Institute of India website). The only detailed account on HW population is by Fox and Chundawat 15 and its earlier references in the wildlife surveys of snow leopard 17. There is essentially no study attempted to understand the genetics of Indian wolves and their relationship with wolves from other parts of the world. Perusal of the published literature on molecular-genetic analysis of wolf and other canids, reveals only six analyzed samples referred as Indian wolf, five of which originated from Afghanistan 9 and the source of the only one sample used in studies by Wayne group 1418 is indicated to be from India. Under this dismal scenario and ever increasing threat to the remaining small wolf populations in India, it becomes imperative to understand their genetic make-up and exact taxonomic status between themselves and with other world-wide wolf populations to have a long-term and effective plan in place for their continual survival and conservation.

Many features of mtDNA viz., high mutation rate than nuclear genome; uniparental (maternal) inheritance, high copy number and lack of recombination, makes it the biological material of choice for mirroring the evolutionary past of a species. Moreover, the mtDNA diversity can be efficiently used to infer both, relatively recent as well as ancient evolutionary events by selectively analyzing selection-pressure independent (d-loop control region) or dependent (essential functional genes like cytochrome-B) domains. These attributes made mtDNA analysis a powerful tool in the molecular systematics pursuits, which in recent years has extensively and successfully been utilized to understand the population history, phylogeography and taxonomic relationships of a large number of animal species. Our present day understanding of origin, evolution and taxonomy of wolf and related canid species is largely based on mtDNA polymorphism analysis 891418.

It is in this background; in 1998 we initiated molecular characterization studies on the highly endangered HW to understand their genetic structure and taxonomic status relative to the GW and other wolf-like canids from Pennisular India and wolf populations from rest of the world. Our detailed mitochondrial DNA polymorphism studies on a large number of HW and representative samples of GW, jackal and wild dog of India followed by comparative genomics with information available for world-wide wolf populations have led to some novel and startling findings about the origin and evolution of wolves of India. Significantly, our studies suggest that: a) both the Indian wolf populations are genetically unique within themselves and both of them from all other wolf populations recorded world-wide; b) HW is distinct from the purported similar wolf from China, C. lupus chanco, and represent the most ancient wolf lineage ever recorded and analyzed using molecular means; c) similarly, GW is distinct from the purported similar wolves from South-East Asia, C. lupus pallipes, and probably also represent the other ancient wolf lineage from India; d) each of the two wolf populations from India thus represents a case of new wolf species in evolution waiting to be re-christened; e) Indian subcontinent as one ancient center of wolf origin and diversification, wherein the two wolf populations represent wolf/canid lineages derived from an ancestral wolf radiation that underwent evolution independent from other world wolf populations for the last 1-2 mya. Thus, our results increase the biological significance of the Indian canid populations and warrants urgent and effective conservation measures for their protection and management.

Materials and Methods

Study sample

The HW population is estimated to be of ~350 animals in the wild, spread over an area of 70,000 km2 encompassing the Trans-Himalayan region of Jammu and Kashmir and Himachal Pradesh 15. Apart from these animals in the wild, there are a total of only 21 live animals in four of the Zoological Parks of India 19. Most of these animals are captive bred from few wild caught animals from the Transhimalayan region. In the present study we analyzed 18 of these animals available in the Padmaja Naidu Himalayan Zoological Park, Darjeeling, West Bengal. In addition to these animals, we analyzed wild caught representative samples of related canids from middle India viz. gray wolf (originally caught from three widely separated geographical regions in middle India), wild dog and Indian jackal, available in the Nehru Zoological Park, Hyderabad, Andhra Pradesh and Sakkarbaug Zoo, Junagarh, Gujarat (Table 1).

<p>Table 1</p>

Details of samples used and DNA sequencing analysis done in the present study

Sample/Species

N

Status

Origin

Source

Himalayan wolf (HW)

18

Captive bred from few wild-caught animals

Trans-Himalayan region of India

Padmaja Naidu, Himalayan Zoological Park, Darjeeling (West Bengal)

Gray wolf (GW)

5a

Wild caught

Pennisular India

Nehru Zoological Park, Hyderabad (Andhra Pradesh); Sakkarbaug Zoo, Junagarh (Gujarat)

Jackal (J), [C. aureus]

2

Wild caught

Pennisular India

Nehru Zoological Park, Hyderabad (Andhra Pradesh); Sakkarbaug Zoo, Junagarh (Gujarat)

Wild dog (WD) [Cuon alpinus]

2

Wild caught

Pennisular India

Nehru Zoological Park, Hyderabad (Andhra Pradesh); Sakkarbaug Zoo, Junagarh (Gujarat)

DNA sequencing Analysis done:

Mitochondrial Domain

Samples analyzed (n=number)

D-loop control region (~1300 or more bp)

HW (18); GW (5); J (2)

Partial Cytochrome-b gene (~350 bp)

HW (18); GW (3); J (2); WD (2)

Complete Cytochrome-b gene (1140 bp)

HW (2); GW (2); J (1)

16S rRNA gene (560 bp)

HW (18); GW (5); J (2); WD (2)

a : Samples originated from three different states of pennisular India

DNA isolation

From each animal, ~10 ml blood was drawn using vaccutainers having EDTA as anti-coagulant and transported from the Zoo to the laboratory at room temperature where the same were stored at 4°C till further use. The total genomic DNA was isolated from each of the samples following the lysis-freeze fracture method 20 for molecular analysis.

DNA amplification and sequencing for nucleotide diversity/ DNA polymorphisms

To understand the genetic status of HW in relation to other related canids from India and other parts of the world, nucleotide diversity was ascertained for three taxonomically informative domains of the mitochondrial genome. These included, part of cytochrome-B gene, complete cytochrome-B gene for few representative samples, hypervariable d-loop control region and 16S rDNA. In each case, the target mitochondrial sequences were PCR amplified using standard protocols on PE9600 thermocycler. The PCR reactions were set using approximately 50 ng of template genomic DNA in 15-20 μl reaction volume containing: 5 pico-moles of each primer, 150 μM dNTP, 1.5 mM MgCl2, 0.1 M KCl, 20 mM Tris-HCl, and 0.5 to 1.0 U of AmpliTaq Gold polymerase (Perkin Elmer). In general, PCR profile comprised an initial denaturation of 10 min at 95°C, followed by 35 cycles of: 94°C for 1 min., 50-55°C for 1 min., 72°C for 2 min.; and 72°C for 5 min. The PCR products were treated with exonucleaseI and shrimp alkaline phosphatase at 37°C and 80°C, 15 min each to remove the remaining primers and free nucleotides. Subsequently, the purified PCR products (~100 ng) were sequenced for both strands using the Big dye terminator ready reaction kit (Perkin Elmer). Further, to confirm sequence data, many of the samples were sequenced more than once. Sequencing PCR cycle condition were: 30 cycles of 96°C for 10 sec., 50°C for 5 sec. and 60°C for 4 min. Extended products were purified by alcohol precipitation followed by washing with 70% ethanol. The processed samples were then dissolved in loading dye and sequenced using automated DNA sequencer ABI Prism377 or ABI Prism3700. Sequences were edited using auto-assembler software package for further analysis.

Cytochrome-B gene: A ~332 bp conserved region of mitrochondrial cytochrome-B gene towards the amino-terminus of the protein was amplified and directly sequenced using primers L14841 [5'-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3'] and H15149 [5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3'] described by Kocher et al 21, for all the samples. In some cases, the PCR amplified product was gel purified using gene clean kit (BIO 101), and cloned in pMOS T vector (Amersham Pharmacia Biotech) following manufacturers details. In each case about 10 recombinant clones were randomly chosen and sequenced as above using M13 universal sequencing primers.

In addition, complete cytochrome-B gene sequence was determined for one sample each of HW, GW and Indian jackal. For the purpose, the target gene was amplified and sequenced using the conserved flanking primers L-14724 [5'-CGAAGCTTGATATGAAAAACCATCGTTG-3'], H-15915 [5'-AACTGCAGTCATCTCCGGTTTACAAGAC-3'] and internal primers L-15162 [5'-GCAAGCTTCTACCATGAGGACAAATATC-3'], L-15513 [5'-CTAGGAGACCCTGACAACTA-3'] and H-15149 [see above] 22.

D-loop control region: For each of the sample, the complete d-loop region (Figure 1), encompassing 5' hypervariable control region-1 (CR-I), repetitive region and 3' relatively conserved control region-II (CR-II) was amplified and sequenced using the primers described by Tsuda et al 9. Initially the region was amplified using primers DL-14 [5'-TGTAAAACGACGGCCAGTGCTCTTGCTCCACCATCA-3'] and DH-7 [5'-CAGGAAACAGCTATGGCCCGTGCGACTCATCTTGGC-3'], which amplified a product of ~1300 bp. To sequence the amplified products, two more internal primers were used along with the above two. The two internal primers were DH-6 [5'-CAGGAAACAGCTATGGCCCCCCTTGATTTTTATGCG-3'] and DH-10 [5'-TGTAAAACGACGGCCAGTATCACTCATCTACGACCG-3'].

<p>Figure 1</p>

Mitochondrial d-loop control region organization

Mitochondrial d-loop control region organization. Upper panel shows the sequence polymorphism seen in the repetitive domain of d-loop in canids from India (present study).

Partial d-loop sequences were also amplified and sequenced using the primers DLH-16340 [5'-CCTGAAGTAGGAACCAGATG-3'] and Thr-L15926 [5'-CAATTCCCCGGTCTTGTGAACC-3'] 14, which essentially gives ~ first 250 bases that are obtained using the primer DL-14 (that span the t-RNAPro gene, see above) in addition to about 70 additional bases corresponding to phenylalanine t-RNAPhe that is present on the left of t-RNAPro.

16S rDNA: Part of 16 sRDNA was amplified and sequenced using the primers 16Sar-L [5'-CGCCTGTTTATCAAAAACAT-3'] and 16Sbr-H [5'-CCGGTCTGAACTCAGATCACGT-3'] 23 for all the 27 samples analyzed in the study.

EMBL databse sequences for comparative analysis

The Genbank database (National Center for Biotechnology Information, USA: NCBI Home page http://www.ncbi.nlm.nih.gov) was searched for similar sequences using BLAST search. Based on the search, large number of reference mitochondrial DNA sequences described for wolf and other related canid species were retrieved from the database, for each of the analyzed domain for phylogenetic comparisons. The details of the reference sequences viz., EMBL accession number, source species, sequence length and location, and their original contributors are given in Tables 2 and 3. The 90 d-loop reference sequences retrieved for comparative analysis represented the wolf samples from all over the globe across all the continents. In comparison, there were only few wolf reference sequences specific to cyto-B gene and none for 16S ribosomal DNA. Accordingly, for these two mitochondrial domains reference sequences were from related canid genera/species.

<p>Table 2</p>

Reference mitochondrial Cytochrome-b and 16S RNA gene sequences used in the phylogenetic analysis

Species

Common name

Continent / Country

EMBL Acc.No.

Reference

Partial Cytochrome-b gene reference sequences

1

C. lupus lupus

Gray wolf

Holarctic

AF028141

8

2

C. simensis

Ethiopian wolf

Africa

AF028144

8

3

C. familiaris

Dog

U12829

41

4

Cuon alpinus

Dhole

Asia

AF028137

8

5

Arelocynus microtis

Small eared dog

Asia (Japan)

AF028135

8

6

C. aureus

Golden jackal

Old world

AF028138

8

7

C. mesomelas elongae

Black-backed jackal

Eastern Africa

AF028142

8

8

C. adustus

Side-striped jackal

Eastern Africa

AF028136

8

9

C. latrans

Coyote

North America

AF028140

8

10

Pseudalopex sechurae

Desert fox

NW S. America

AF028154

8

Complete Cytochrome-b gene reference sequences

1

C. familiaris

Dog

?

38

2

Vulpes vulpes

Fox

?

38

16S rDNA reference sequence

1

C. familiaris

Asia (Japan)

AB048590

<p>Table 3</p>

Reference mitochondrial control-region sequences obtained from EMBL database and used in the phylogenetic analysis

Sample Code

Species

Subspecies

Country/Conttinent

EMBL AccNo

D-loop (bp)

Haplotypea

Reference

D-loop region sequenced

1-lu1

C. lupus

lupus

Yugosolavia

Europe

AB007372

672

9

Complete 5'-CR-I

2-lu2

C. lupus

lupus

Yugosolavia

Europe

AB007373

672

9

Complete 5'-CR-I

3-p1

C. lupus

pallipes

Afganisathan

Asia

AB007374

672

9

Complete 5'-CR-I

4-ch1

C. lupus

chanco

Mongol, China

Asia

AB007375

673

9

Complete 5'-CR-I

5-ch2

C. lupus

chanco

Mongol, China

Asia

AB007376

673

9

Complete 5'-CR-I

6-ch3

C. lupus

chanco

Mongol, China

Asia

AB007377

673

9

Complete 5'-CR-I

7-ch4

C. lupus

chanco

Mongol, China

Asia

AB007378

673

9

Complete 5'-CR-I

8-ch5

C. lupus

chanco

Mongol, China

Asia

AB007379

673

9

Complete 5'-CR-I

9-Wo11

C. lupus

crassodon

Canada

N. America

AF098125

707

Wo-11

45*

Complete 5'-CR-I

10-Wo10

C. lupus

crassodon

Canada

N. America

AF098124

707

Wo-10

45*

Complete 5'-CR-I

11-Wo9

C. lupus

crassodon

Canada

N. America

AF098123

707

Wo-9

45*

Complete 5'-CR-I

12-Wo4

C. lupus

columbianus

Canada

N. America

AF098118

707

Wo-4

45*

Complete 5'-CR-I

13-Wo5

C. lupus

columbianus

Canada

N. America

AF098119

707

Wo-5

45*

Complete 5'-CR-I

14-Wo6

C. lupus

columbianus

Canada

N. America

AF098120

707

Wo-6

45*

Complete 5'-CR-I

15-Wo7

C. lupus

columbianus

Canada

N. America

AF098121

707

Wo-7

45*

Complete 5'-CR-I

16-Wo8

C. lupus

columbianus

Canada

N. America

AF098122

707

Wo-8

45*

Complete 5'-CR-I

17-Wo2

C. lupus

lupus

Canada

N. America

AF098116

707

Wo-2

45*

Complete 5'-CR-I

18-Wo3

C. lupus

lupus

Canada

N. America

AF098117

707

Wo-3

45*

Complete 5'-CR-I

19-Wo1

C. lupus

tundrarum

Canada

N. America

AF098115

707

Wo-1

45*

Complete 5'-CR-I

20-W27

C. lupus

China

Asia

AF008135

713

W-27

18

Complete 5'-CR-I

21-W 6

C. lupus

Romania

Europe

AF008136

712

W-6

18

Complete 5'-CR-I

22-W 1

C. lupus

Spain

Europe

AF008137

712

W-1

18

Complete 5'-CR-I

23-W 4

C. lupus

France/Italy

Europe

AF008138

712

W-4

18

Complete 5'-CR-I

24-W 5

C. lupus

Greece/Romania

Europe

AF008139

712

W-5

18

Complete 5'-CR-I

25-W12

C. lupus

India

Asia

AF008140

712

W-12

18

Complete 5'-CR-I

26-W15

C. lupus

SaudiArabia

Asia

AF008141

713

W-15

18

Complete 5'-CR-I

27-W14

C. lupus

SaudiArabia/China

Asia

AF008142

712

W-14

18

Complete 5'-CR-I

28-w 1

C. lupus

Italy-East European

Europe

AF115687

542

w-1

42

Most of 5'-CR-I

29-w 2

C. lupus

Italy-East European

Europe

AF115688

542

w-2

42

Most of 5'-CR-I

30-w 3

C. lupus

Italy-East European

Europe

AF115689

542

w-3

42

Most of 5'-CR-I

31-w 4

C. lupus

Italy-East European

Europe

AF115690

542

w-4

42

Most of 5'-CR-I

32-w 5

C. lupus

Italy-East European

Europe

AF115691

542

w-5

42

Most of 5'-CR-I

33-w 7

C. lupus

Italy-East European

Europe

AF115692

542

w-7

42

Most of 5'-CR-I

34-w 8

C. lupus

Italy-East European

Europe

AF115693

542

w-8

42

Most of 5'-CR-I

35-w 9

C. lupus

Italy-East European

Europe

AF115694

542

w-9

42

Most of 5'-CR-I

36-w 10

C. lupus

Italy-East European

Europe

AF115695

542

w-10

42

Most of 5'-CR-I

37-w 11

C. lupus

Italy-East European

Europe

AF115696

542

w-11

42

Most of 5'-CR-I

38-w 12

C. lupus

Italy-East European

Europe

AF115697

542

w-12

42

Most of 5'-CR-I

39-w 13

C. lupus

Italy-East European

Europe

AF115698

542

w-13

42

Most of 5'-CR-I

40-w 14

C. lupus

Italy-East European

Europe

AF115699

542

w-14

42

Most of 5'-CR-I

41-w 15

C. lupus

Italy-East European

Europe

AF115700

542

w-15

42

Most of 5'-CR-I

42-w 16

C. lupus

Italy-East European

Europe

AF115701

542

w-16

42

Most of 5'-CR-I

43-w 19

C. lupus

Italy-East European

Europe

AF115702

542

w-19

42

Most of 5'-CR-I

44-w 20

C. lupus

Italy-East European

Europe

AF115703

542

w-20

42

Most of 5'-CR-I

45-Do9

C. familiaris

Dog

707

Do-9

Complete 5'-CR-I

46-DogABO

C. familiaris

Dog

AB048590

737

Complete 5'-CR-I

47

ulpus vulpus

Red fox

Japan

D-83639

666

43

Complete 5'-CR-I

48

procyonoides

Raccoon dog

Japan

D-83614

667

43

Complete 5'-CR-I

49-W1

C. lupus

France/Italy/Spain

Europe

AF338803

342

W-1

46*

Part of 5'-CR-I

50-W2

C. lupus

France/Italy/Spain

Europe

AF338804

342

W-2

46*

Part of 5'-CR-I

51-W3

C. lupus

France/Italy/Spain

Europe

AF338805

342

W-3

46*

Part of 5'-CR-I

52-W4

C. lupus

Croatia

Europe

AF338806

342

W-4

46*

Part of 5'-CR-I

53-W5

C. lupus

Croatia

Europe

AF338807

342

W-5

46*

Part of 5'-CR-I

54-W6

C. lupus

Portugal

Europe

AF338808

342

W-6

46*

Part of 5'-CR-I

55-W7

C. lupus

Spain

Europe

AF338809

342

W-7

46*

Part of 5'-CR-I

56-W8

C. lupus

Romania

Europe

AF338810

342

W-8

46*

Part of 5'-CR-I

57-W9

C. lupus

Poland

Europe

AF338811

342

W-9

46*

Part of 5'-CR-I

58-W10

C. lupus

Spain

Europe

AF338812

342

W-10

46*

Part of 5'-CR-I

59-W2

C. lupus

Polland-Belarus

Europe

AF344300

257

W-2

47*

Part of 5'-CR-I

60-W3

C. lupus

Polland-Belarus

Europe

AF344301

257

W-3

47*

Part of 5'-CR-I

61-W4

C. lupus

Polland-Belarus

Europe

AF344302

257

W-4

47